脾虚证物质能量代谢基因差异表达及其生物信息分析
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摘要
研究背景和目的：
中医“脾主运化”、脾为“后天之本,气血生化之源”之说,揭示脾对于饮食物具有消化吸收和输转的功能,同时脾通过营养物质在体内的代谢,为人体出生以后生长发育及生命活动提供能量和物质基础。物质能量代谢是生命活动的物质基础,它包括消化吸收、中间代谢和排泄三个阶段,中间代谢是机体内代谢的主体,包括糖、脂、蛋白质、核酸、微量元素和能量代谢等(本论文物质代谢特指糖、脂、蛋白质、核酸和微量元素代谢)。目前已有大量的研究证实脾虚动物存在消化吸收和营养代谢障碍,在中间代谢方面表现为糖原含量降低；血脂含量和生物膜结构异常；氨基酸和蛋白含量降低；组织DNA和RNA含量降低；线粒体结构异常,ATP含量降低；微量元素含量异常等,可见,脾与物质能量代谢在病理上也表现出高度的一致。
目前脾虚证与物质能量代谢相关性研究主要以对某一代谢相关生化指标的测定为主,缺乏对脾虚证机体整个物质能量代谢的同步研究。基因芯片技术作为一个“组学”研究方法,与中医的“整体观”有很多相似之处,它可以反映机体某一状态所有基因的表达情况。因此,在本世纪初,基因芯片技术就已经广泛用于脾虚证动物模型的研究,但是由于我国利用基因芯片技术研究脾虚证的起步相对较晚,基因芯片数据的分析存在一些明显不足,如对芯片数据的预处理不够,对差异表达基因缺乏统计学检验,并且对于差异表达基因的功能注释也过于单一,最终导致对芯片数据的解读浮于表面,未能达到预期目的。随着分子生物学和生物信息学的发展,一些专业的生物信息学软件已经广泛用于基因芯片数据的分析,这大大提高了基因芯片数据的解读能力,并且随着大量基因芯片数据的积累,使得广泛利用现有基因芯片数据来解读脾虚证的发病机制比少量基因芯片实验本身更为重要。
对此,在课题组应用基因芯片研究慢性浅表性胃炎脾气虚证(下称脾虚证)基因表达谱的工作基础上,本文利用BRB ArrayTools和IPA软件对慢性浅表性胃炎(下称慢性胃炎)脾虚证患者(分别以健康志愿者和脾胃湿热证患者作为对照)和非幽门螺杆菌(Helicobacter pylori, Hp)感染脾虚证患者(与Hp感染脾胃湿热证患者相比)基因芯片数据进行深入挖掘,通过对前者物质能量代谢相关差异表达基因分析,探讨脾虚证与物质能量代谢的相关性,从机体整个物质能量代谢水平认识脾的生理功能和脾虚证的发病机制；通过对后者差异表达基因分析,探讨Hp的致病机制,同时认识“慢性胃炎脾胃湿热证患者Hp感染率明显比脾虚证患者高”这一临床现象的遗传背景,为中医分型和Hp相关疾病的治疗提供理论依据。此外,论文还汇总了近10年气虚证或体质人和脾虚大鼠模型差异表达基因,通过对脾虚证患者、气虚证或体质人和脾虚大鼠模型物质能量代谢相关差异表达基因的相似性比较,探讨脾虚证基因表达谱的特征,并筛选出脾虚证特征性代谢基因和代谢途径,从基因组学的角度为中医脾基础理论的现代科学诠释和脾虚证临床辨证分型提供参考,并为脾虚证的病理生理研究提供线索。
材料与方法：
一、材料
(一)基因芯片数据来源
慢性胃炎脾虚证患者与健康志愿者比较和脾虚证患者与脾胃湿热证患者比较的基因芯片原始数据,以及慢性胃炎非Hp感染脾虚证患者与Hp感染脾胃湿热证患者比较的基因芯片原始数据全部来自于课题组脾虚证发病机理研究的前期数据,并已有相关论文发表。包括肺气虚证、气阴两虚证、气虚体质在内的气虚证或体质人差异表达基因全部来源于公开发表的学术论文和学位论文。包括不同建模方法和组织来源的脾虚大鼠模型差异表达基因全部来源于公开发表的学术论文和学位论文。
(二)用于脾虚证与脾胃湿热证比较的差异表达基因验证的荧光定量PCR实验病例来源
1.诊断标准及辨证分型标准
慢性胃炎西医诊断和辨证分型标准以及Hp诊断标准全部采用课题组原实验设计标准(王颖芳,广州中医药大学2006届博士学位论文),具体如下：
(1)慢性胃炎诊断标准参照《中药新药临床研究指导原则》。
(2)脾气虚证辨证标准主症：①舌质淡、舌体胖或有齿印,苔薄白；②胃纳减少或食欲差；③腹胀；④大便溏或腹泻。次症：①消瘦；②体倦乏力；③脉细弱。判断：a)主症①必备。b)兼具主症②、③、④之二；或兼具主症②、③、④之一,同时兼具2个以上次症,诊断可成立。
(3)脾胃湿热证辨证标准主症：①舌苔黄腻；②胸闷；③胃脘痞满或胀痛；④食欲不振。次症：①口苦而黏；②口渴少饮,或喜热饮；③大便溏,或有黏液；④恶心；⑤身困乏力；⑥脉濡缓,或滑。判断：a)主症①必备。b)兼具主症②、③、④之
二,或兼具主症②、③、④之一,同时兼具2个以上次症；或兼具3个以上次症,诊断可成立。
(4)Hp诊断标准
患者近一个月内未服用抗生素类药物。病理切片染色与快速尿素酶试验二者均为阳性即为Hp感染。两者均为阴性即为非Hp感染。
2.纳入及排除标准
同时符合慢性胃炎非Hp感染脾虚证或Hp感染脾胃湿热证,并同意签署由学术道德委员会批准的《知情同意书》的患者纳入。年龄在18岁以下或65岁以上,妊娠或哺乳妇女,过敏体质者,或存在消化道其它器质性病变、心、脑、肝、肾、造血系统等严重原发性疾病及精神性疾病等患者被排除。
二、方法
(一)脾虚证患者差异表达基因分析
慢性胃炎脾虚证患者与健康志愿者比较和脾虚证患者与脾胃湿热证患者比较的基因芯片原始数据,以及慢性胃炎非Hp感染脾虚证患者与Hp感染脾胃湿热证患者比较的基因芯片原始数据,采用下述方法进行分析处理：
1.基因芯片数据的预处理和数据筛选
对Cy5和Cy3信号值进行均一化处理,消除两标记体系间的系统误差。参与均一化处理的有效基因点的条件：(1)该基因点的Cy3和Cy5信号值都大于200,或者其中之一大于800；(2)该基因点的Cy5信号值/Cy3信号值的比值在0.1-10之间。均一化系数计算方法：计算每个有效基因点Cy5信号值/Cy3信号值的比值,求出其相应的自然对数值r=ln(Cy5/Cy3),算出全部有效基因点r值的平均值R,那么实验的均一化系数就等于R的倒数即(1/R)。将所有基因点的Cy3信号值乘上均一化系数,得出调整后的Cy3*,即Cy3*=Cy3×(1/R),并将所有小于200的Cy3*值以200取代,所有小于200的Cy5信号值用200替代。将Cy5和Cy3*信号值输入芯片分析软件BRBArrayTools4.1.0Beta2进行数据筛选,包括对数转换、中位值标准化和基因筛选。其中某个基因被保留用于后续分析的基因筛选条件是：该基因的Cy5/Cy3*比值必须有超过20%的样本大于1.5,且不能有超过50%的数据缺失。对筛选后的数据进行功能注释和进一步分析。
2.GO和KEGG基因集分析
对筛选后的芯片数据进行GO和KEGG基因集分析。基因芯片分析时,将健康志愿者或脾胃湿热证患者作为对照组,将脾虚证患者作为实验组。Hp分析时,将非Hp感染脾虚证患者作为对照组,Hp感染脾胃湿热证患者作为实验组。采用配对t检验的方法,显著性检验方法为LS/KS排列检验和Efron-Tibshirani's基因集分析最大均值检验(GSA maxmean test), P<0.005。
3.芯片间分类对比分析
(1)差异表达基因筛选
对筛选后的数据进行芯片间的分类对比分析。基因芯片分析时,实验组和对照组设置同上。采用配对t检验的统计方法,将同时满足P<0.05或p<0.01或p<0.001和表达倍数>2的基因设定为差异表达基因。对表达倍数<1的基因数据进行负倒数转换,因此倍数为正的基因是实验组上调基因,负值表示实验组下调基因,分别标记为“↑”和“↓”。
(2)差异表达基因生物信息分析
将差异表达基因ID和表达倍数导入IPA8.6在线软件对差异表达基因进行注释、相互作用网络等生物学功能分析。对于脾虚证分别与健康志愿者和脾胃湿热证患者比较的差异表达基因根据BRB Array Tools软件的GO功能分析中的生物学过程和KEGG通路以及IPA在线软件的生物学功能分类和基因的生物学注释四方面信息综合确定物质能量代谢相关基因,分析脾虚证与物质能量代谢的相关性；对于非Hp感染脾虚证差异表达基因,通过差异表达基因功能注释和相互作用网络分析,探讨Hp的致病机制,为中医分型和Hp相关疾病的治疗提供理论依据。
(3)基于差异表达基因的聚类分析
利用BRB Array Tools软件的聚类功能,选择基因中心化、一减相关系数和平均连接簇设置,以选定的差异表达基因作为分析对象(Hp分析时选定所有差异表达基因,其他两组芯片分析选定物质能量代谢相关差异表达基因),对样本和基因进行层级聚类分析。
(二)脾虚证患者、气虚证或体质人和脾虚大鼠模型物质能量代谢相关差异表达基因相似性比较
1.脾虚证患者两组芯片的物质能量代谢基因之间相似性比较
对脾虚证患者与健康志愿者比较的物质能量代谢相关差异表达基因和脾虚证与脾胃湿热证患者比较的物质能量代谢相关差异表达基因进行相似性比较,分析脾虚证患者差异表达基因的特征,并筛选出脾虚证患者特征性的基因和代谢途径,进一步探讨脾虚证与物质能量代谢的关系。
2.脾虚证患者物质能量代谢基因和气虚证或体质人物质能量代谢基因相似性比较
检索并汇总文献报道的气虚证或体质人差异表达基因,按照上述物质能量代谢相关基因的界定方法,筛选出物质能量代谢相关差异表达基因,并与脾虚证患者物质能量代谢差异表达基因进行比较,探讨脾虚证患者和气虚证或体质人在物质能量代谢基因差异表达上的异同,从而确定物质能量代谢异常是脾虚证患者特有的病理现象还是气虚证所共有的病理机制。
3.脾虚证患者物质能量代谢基因和脾虚大鼠模型物质能量代谢基因相似性比较
检索并汇总文献报道的脾虚大鼠模型差异表达基因,按照上述方法2,进行分析,探讨脾虚证特有的代谢基因和途径。
(三)脾虚证与脾胃湿热证比较的差异表达基因荧光定量PCR验证
1.胃黏膜组织的采集
在广东省中医院收集慢性胃炎非Hp感染脾虚证和Hp感染脾胃湿热证患者各4例,胃镜下钳取胃窦黏膜活检组织200mg左右,液氮保存备用。
2.荧光定量PCR实验
论文对慢性胃炎非Hp感染脾虚证与Hp感染脾胃湿热证比较的差异表达基因HLA-DRB1、SCGN、COX7B和SULT1A4四个基因进行定量PCR验证。实验采用SYBR Green I的方法,利用primer express2.0软件设计引物,并按照相关试剂盒说明书提取总RNA、进行荧光定量RT-PCR反应,采用ΔΔCt的方法计算实验组与对照组之间的相对含量。
结果：
一、脾虚证患者差异表达基因
(一)脾虚证与健康志愿者比较的物质能量代谢相关差异表达基因
(1)GO分析获得287个有显著性意义的基因集,其中细胞成分49个、分子功能60个,生物学过程178个。KEGG分析获得11个有显著性意义的代谢通路。GO生物学过程和KEGG通路显示,脾虚证患者在涉及脂、蛋白质、核酸和糖代谢的相关基因表达异常。(2)分类对比分析获得15个物质能量代谢相关差异表达基因,占总差异表达基因的75%(15/20),其中下调基因14个,酶基因或有酶活性的基因11个(73%),它们参与机体脂、蛋白质、核酸和糖代谢过程。与脂类代谢相关的基因有ACAA2↓和CYP20A1↓,表现为脂肪酸分解和胆固醇转化降低；与蛋白质代谢相关的基因有RPS28↓、B3GNT1↓、GCNT1↓、PPP1R3C↓、UBXN1↓、UBE2D2↓、ASL↓、 ASS1↓和PCYOX1L↓、ALDH9A1↓,表现为蛋白质合成和糖基化和磷酸化修饰降低,蛋白质泛素化降解异常,参与尿素循环、自主神经等生物学过程的氨基酸代谢降低；与核酸代谢相关的基因有RMI1↓、SMARCD3↓和PARP1↑,表现为DNA复制和转录降低,DNA损伤修复增加；与糖类代谢相关的基因有B3GNT1↓、GCNT1↓和PPP1R3C↓,表现为聚糖和糖原合成降低。(3)基于物质能量代谢基因差异表达的样本聚类分析显示脾虚证患者与健康志愿者不能分开。
(二)脾虚证与脾胃湿热证比较的物质能量代谢相关差异表达基因
(1)GO分析获得373个有显著性意义的基因集,其中细胞成分58个、分子功能59个,生物学过程256个。这些生物学过程包括脂、蛋白质、核酸、糖代谢、微量元素和能量代谢等。KEGG分析获得2个有显著性意义的代谢通路,即核糖体和核黄素代谢。(2)分类对比分析获得56个物质能量代谢相关差异表达基因,占总差异表达基因的71%(56/79),其中下调基因45个、酶或具有酶活性的基因30个、转运蛋白基因5个,它们参与机体脂、蛋白质、核酸、糖、微量元素和能量代谢过程。与脂代谢相关的基因有ACADVL↓、LRP11↑、SULT1A4↓、CRLS1↑、GPCPD1↓、PIGL↓、 B3GNT1↓、ST8SIA4↓和FUT9↑,表现为脂肪酸p氧化降低、胆固醇摄取增加和代谢转化降低、磷脂和糖脂代谢异常；与蛋白质代谢相关的基因有ASRGL1↓、AARSD1↓、 EBNA1BP2↓、PUM2↑、MRPL52↓、C120RF65↓、PSMB8↓、PSME2↓、UBA7↓、RNF11↑、 FBX044↓、ZFYVE26↓、CHMP2A↓、SSR4↓、SNX4↑、RAB3B↓、RABL2A↓、GOLGA2↓、 KDELR1↓、PHPT1↓、ACPP↓、PTPRF↓、CRKL↓、HDAC7↓、ADPRHL2↓、B3GNT1↓、 ST8SIA4↓、DDOST↓和FUT9↑,主要表现为蛋白质的生物合成降低、蛋白质泛素化减弱、靶向输送和翻译后磷酸化和糖基化修饰降低；与核酸代谢相关的基因有TOP2A↓、 SF3A3↓、CREB3↓、CRTC2↓、NR1D2↑、MED6↓、GTF2IRD1↓、C1ORF83↓、ZNF773↓、ZMYND11↑、DFFB↓、FLJ35220↓和ADPRHL2↓,主要表现为DNA复制和转录减弱,DNA损伤修复增强；与糖代谢相关的基因有AGL↑、PTPRF↓、B3GNT1↓、FUT9↑、 ST8SIA4↓、SULT1A4↓、DDOST↓和PIGL↓,主要表现为糖原的分解增加和糖复合物的合成降低；与微量元素代谢相关的基因有COMMD1↓、FTL↓、SLC39A6↑、 CHRFAM7A↓、SCGN↑和S100A6↓,表现为铜离子和铁离子代谢下降、锌离子代谢增加、钙离子代谢异常；与能量代谢相关的基因有AK3↓和COX7B↓,表现能量代谢障碍。(3)基于物质能量代谢基因差异表达的样本聚类分析能够将脾虚证和脾胃湿热证患者分开。
(三)非Hp感染脾虚证与Hp感染脾胃湿热证比较的差异表达基因
(1)GO分析获得703个有显著性意义的基因集,其中细胞成分114个、分子功能114个,生物学过程475个。KEGG分析获得29个有显著性意义的代谢通路。GO生物学过程和KEGG通路分析结果一致显示,Hp感染会使宿主在涉及蛋白质代谢、信号通路、免疫炎症反应、细胞骨架等方面的基因差异表达。(2)分类对比分析获得34个有生物学功能注释的差异表达基因,它们参与蛋白质代谢、免疫和炎症反应、信号通路、基因转录、微量元素代谢等过程。(3)差异表达基因相互作用网络分析发现82%的基因被IPA软件归类到三个与基因表达、癌症进展、抗原递呈和免疫反应等相关的相互作用网络。这些结果显示Hp感染能够刺激颗粒富集的胞质结构(Particle-rich Cytoplasmic Structure PaCS)的形成,改变细胞基因表达过程,逃逸宿主防御机制,增加免疫和炎症反应,激活NFKB和Wnt/β-catenin信号通路,扰乱金属离子平衡,诱导癌症发生。(4)差异表达基因聚类分析能够将非Hp感染脾虚证患者和Hp感染脾胃湿热证患者分开。
二、脾虚证患者、气虚证或体质人和脾虚大鼠模型物质能量代谢相关差异表达基因相似性比较
(一)脾虚证患者两组芯片的物质能量代谢基因之间相似性比较
1.两组芯片数据的GO分析都显示,脾虚证患者在涉及脂、蛋白质、核酸和糖代谢的相关基因表达异常,并且以参与蛋白质代谢的基因最多；KEGG分析都显示,脾虚证患者在参与蛋白质代谢通路的相关基因表达异常。
2.两组芯片的分类对比分析显示,脾虚证与健康志愿者比较的物质能量代谢差异表达基因相对较少,主要涉及脂类、蛋白质、核酸和糖类代谢,而脾虚证与脾胃湿热证比较的物质能量代谢差异表达基因较多,主要涉及脂类、蛋白质、核酸、糖类、微量元素和能量代谢,并且两组芯片在具体的差异表达基因名称上也存在明显不同。尽管如此,两组芯片更多的存在很多共同点,表现在：(1)脾虚证患者在两组芯片中都存在大量物质能量代谢基因表达异常,占总差异表达基因的70%以上,并且大部分为酶基因或具有酶活性基因,总体以表达下调为主。(2)B3GNT1在两组基因芯片数据中都一致下调。(3)在代谢途径上,脾虚证患者在两者芯片中都表现为胆固醇代谢转化和脂肪酸p氧化过程降低,核糖体形成障碍,蛋白质翻译后磷酸化和糖基化修饰降低,蛋白质泛素化降解途径降低,DNA复制和转录水平低下,DNA损伤修复加强。同时,脾虚证患者胃肠道组织糖原合成降低,分解增强,并且还存在聚糖合成障碍。
3.基于物质能量代谢基因差异表达的样本聚类分析显示,脾虚证患者与健康志愿者不能很好的分开,而脾虚证患者与脾胃湿热证患者则能很好的分开。
(二)脾虚证患者物质能量代谢基因和气虚证或体质人物质能量代谢基因相似性比较
脾虚证患者与其他气虚证或体质人物质能量代谢相关差异表达基因存在明显差异,具体表现在：(1)脾虚证患者,除与气虚体质人差异表达基因存在很多相似之处(差异表达基因都以下调为主,但是脾虚证患者在导致物质能量代谢障碍方面更加明显)之外,与其他气虚证或体质人在差异表达基因的种类、表达趋势和涉及的代谢途径上都明显不同；(2)UBA、ST、RAB和S100是脾虚证患者和气虚体质人的共有基因,可能与脾虚证密切相关,而RP、PTP和UBE2是脾虚证患者与其他气虚证或体质人的共有基因,可能与气虚证密切相关；(3)在代谢途径上,脾虚证患者参与糖、脂、蛋白质和核酸代谢的相关基因表达下调,而气虚证或体质人差异表达基因参与的代谢过程相对较少,并且相关基因以上调为主；(4)蛋白质泛素化异常是气虚证的共性,但脾虚证患者和气虚体质人表现降低,其他气虚证或体质人表现增强；(5)脾虚证患者和气虚证或体质人都存在DNA损伤,这可能是气虚证共有的病理现象。
(三)脾虚证患者物质能量代谢基因和脾虚大鼠模型物质能量代谢基因相似性比较
1.脾虚证患者和脾虚大鼠模型在差异表达基因的数量上存在明显差异,在表达趋势上,脾虚证患者差异表达基因总体以下调为主,脾虚大鼠模型差异表达基因除海马组织外,都以表达上调为主。这些结果说明脾虚证患者和脾虚大鼠模型在病理上可能存在明显不同。2.脾虚证患者和大鼠模型之间重复出现的代谢相关差异表达基因有13个：B3GNT1、CYP、SLC39A、SNX、FUT、SLC2A、GCNT、ACAD、LDH、RP、 PPP、S100和PTPR,其中RP和PTPR下调与气虚证患者共有基因分析结果一致；(1)这些基因表达产物,除RP外,全为酶和运载蛋白,与脾主运化的认识一致；(2)这些基因涉及糖、脂、蛋白质和微量元素代谢过程,具体表现为葡萄糖运输和分解、糖原合成与分解以及聚糖的合成紊乱；脂肪酸和胆固醇代谢异常；核糖体形成障碍、蛋白质翻译后磷酸化修饰降低和糖基化异常以及细胞内靶向输送增加；锌离子代谢增加、钙离子代谢降低。这些结果说明,不同建模方法构建的脾虚大鼠模型虽然都不能完全反映脾虚证的所有特征,但是都能从某一方面部分反映脾虚证的特征,其重复出现的代谢相关差异表达基因可能就是脾虚证的重要特征。
三、脾虚证与脾胃湿热证对照的差异表达基因荧光定量PCR验证
慢性胃炎非Hp感染脾虚证与Hp感染脾胃湿热证比较的差异表达基因HLA-DRB1、SCGN、COX7B和SULT1A4四个基因(后三个基因也是脾虚证与脾胃湿热证差异表达基因)的定量PCR结果与基因芯片实验结果一致,但是定量PCR结果整体比基因芯片结果偏低。此外,之前课题组对脾虚证患者与健康志愿者差异表达基因ACAA2进行了验证,定量PCR结果与基因芯片结果一致下调,定量PCR结果同样比基因芯片结果偏低。
结论：
1.脾虚证患者与其他气虚证不同,主要通过代谢相关基因,尤其是酶基因的表达下调,从而出现营养代谢障碍,具体表现为：(1)胆固醇代谢转化和脂肪酸β氧化过程降低；(2)核糖体形成障碍,蛋白质翻译后磷酸化和糖基化修饰降低,蛋白质泛素化降解途径降低；(3)DNA复制和转录水平低下,DNA损伤修复加强；(4)脾虚证患者胃肠道组织糖原合成降低,分解增强,并且还存在聚糖合成障碍。这些结果描绘出了脾虚证患者宏观物质能量代谢草图,支持《内经》中脾病理生理涉及物质能量代谢的论述,为脾藏象科学本质的现代研究和诠释提供了线索。同时,为从脾论治代谢性疾病研究提供了理论依据和学术指引。
2.基于基因芯片数据分析获得的物质能量代谢相关差异表达基因与文献报道的临床代谢相关生化指标的测定结果相互印证,共同揭示了脾的病理生理主要涉及饮食物的消化吸收和体内代谢两方面。
3.B3GNT1、ST、RAB、CYP、SLC39A、SNX、FUT、SLC2A、GCNT、ACAD、 LDH、RP、PPP1、S100和PTPR可能与脾虚证密切相关,而RP、PTP和UBE2可能与气虚证密切相关,这些基因为脾虚证的进一步研究提供了线索。
4.基因表达谱分析显示,慢性胃炎脾虚证与脾胃湿热证之间临床表现差异较大,而慢性胃炎脾虚证与健康人之间临床表现差异不明显,这为中医疾病分型提供了理论依据。
5.Hp感染会刺激PaCS的形成,改变细胞基因表达过程,逃离宿主防御机制,增加炎症和免疫反应,激活NFκB和Wnt/β-catenin信号通路,干扰金属离子的平衡,诱导肿瘤的发生。
Research background and objective
The theory of "Spleen governing transportation and transformation"and "the spleen is the origin of the acquired constitution and source of qi-blood" in the Chinese medicine illustrates the spleen has a function in ingesta digest, absorb and transport, and provides energy and substance foundation for human growth, development and vital movement by metabolism of nutritive substance.Substance and energy metabolism is foundation of vital movement. It includes three stage of digest and absorb, intermdediate metabolism and excrete.Intermdediate metabolism is the main body of metabolism, and includes carbohydrate, lipide, protein, nucleic acid, trace element and energy metabolism.At present, many research results showed splenic asthenia animal existed the disorder of digest, absorb and nutrition metabolism.Thus, it is also similar between patho-manifestations of spleen and metabolism.
In relativity research between splenasthenic syndrome and metabolism, it is a common method to determine biochemical indicator related to certain metabolism, but this method can not simultaneously study whole substance and energy metabolism for splenasthenic syndrome patients.Microarray technology is a omics resesrch method, and able to reflect organism all gene expression in certain state, which is very similar to the holism of Chinese medicine.So, at the beginning of this century, microarray has been extensive utilized to research animal model of splenasthenic syndrome.But because of a late beginning in China, microarray data analysis exist some problems, such as not pretreatment for microarray data, not statistics test for differentially expressed gene, and simple function annotation. These problems cause microarray data not to well explain disease mechanism.Follow the development of molecule biology and bioinformatics, some professional anlysis softwares raise the readout ability of microarray data. Moreover, many microarray data have been accumulated in recently years.So,it is more important to mine reported microarray data of splenasthenic syndrome by using bioinformics software than microarray experiment in study pathogenesy of splenasthenic syndrome.
Based on the work foundation of gene expression profiling for splenasthenic syndrome with chronic superficial gastritis in our research groups, at the present study, microarray data of chronic superficial gastritis (below named chronic gastristis) splenasthenic-splenogastric hygropyrexia syndrome and splenasthenic syndrome-healthy volunteer, and Helicobacter pylori (H. pylori, Hp)-uninfected splenasthenic-Hp-infected splenogastric hygropyrexia syndrome were mined and bioinformatics analysis by BRB Array Tools and IPA software.For the former, differentially expressed genes related to substance and energy metabolism were analyzed to approach the correlation of splenasthenic syndrome and substance and energy metabolism, and the physiological function of spleen and pathogenesy of splenasthenic syndrome based on whole substances and energy metabolism level of organism; For the latter, differentially expressed genes were analyzed to approach pathogenic mechanism of Hp infection and understood genetic background of clinical outcome that Hp infection rate of splenogastric hygropyrexia syndrome with chronic gastritis was higher than splenasthenic syndrome, and provided theoretical basis for classification of syndrome of Chinese medicine and therapia of Hp-related diseases.In addition, the microarray data from patients of syndrome or constitution of deficiency of QI and rat models of splenasthenic syndrome were also collected.Differentially expressed genes related to substance and energy metabolism from patients of splenasthenic syndrome, and that of rat models of splenasthenic syndrome and the peoples of syndrome or constitution of deficiency of QI were similarly compared to approach marked gene and metabolic pathway of splenasthenic syndrome, and provide some references for scientic annonation of basic theory of Chinese medicine spleen and differentiation of symptoms and classification of syndrome of splenasthenic syndrome, and provide some clue for pathophysiology research of splenasthenic syndrome.
Materials and methods
1.Materials
(1)Microarray data origin
All microarray raw data from chronic gastritis patients of splenasthenic-splenogastric hygropyrexia syndrome, splenasthenic syndrome-healthy volunteer and Hp-infected splenogastric hygropyrexia syndrome-Hp-uninfected splenasthenic syndrome were from early data studying pathogenesy of splenasthenic syndrome in our group, and some papers have been published based on these data. All differentially expressed genes from patients of syndrome or constitution of deficiency of QI including insufficiency of lung-QI, deficiency of both vital energy and yin, deficiency of vital energy, and rat models of splenasthenic syndrome including different modeling methods and tissue origin were from open publishing academic and degree papers.
(2) Case origin of fluorescent quantitation PCR verification of differentially expressed genes from microarray data of splenasthenic-splenogastric hygropyrexia syndrome
1) Diagnostic and differentiation of symptoms and signs for classification of syndrome criteria
All criteria of diagnose and classification symptons were from previosly criteria (WANG,Ying-fang, doctor thesis of Guangzhou university of Chinese medicine,2006),as followed:
Diagnostic criteria of chronic gastritis refer to the Guide Principle of Clinical Research of Chinese Material Medica and New Drug.
Differentiation of symptoms and signs criteria of splenasthenic syndrome Main sign:①body of the tongue light and big or has imprints of the teeth, moss thin and white;②appetite decrease;③abdominal distension;④loose stool or diarrhoea. Secondary sign:①emaciated;②weary and hypodynamia;③pulses extenuate.Decision:a) Main sign①necessary, b) Including the two of main sign②,③and④; Or including one of main sign②,③and④,and including over two of secondary sign.
Differentiation of symptoms and signs criteria of splenogastric hygropyrexia syndrome Main sign:①coated tongue yellow freasy;②chest distress;③gastric cavity painful abdominal mass or gas pains;④poor appetite.Secondary sign:①bitter taste and sticky of mouth;②thirsty and few drink, or hot drink preference;③oose stool or including mucus;④nauseated;⑤body exhausted and debilitation;⑥pulses slow or slide.Decision:a) Main sign①necessary, b) Including the two of main sign②,③and④;or including one of main sign②,③and④,and including over two of secondary sign; or including over three of secondary sign.
Diagnostic criteria of Hp infection Patients donot take medicine of antibiotics in recent one month.Both pathological section-staining and fast urease test were positive, which were Hp infected, otherwise were Hp uninfected.
2) Receive and remove criteria
The patients who meet criteria of chronic gastritis, splenasthenic syndrome and Hp uninfection or meet chronic gastritis, splenogastric hygropyrexia syndrome and Hp infection were received. The patients whose age less18or over65,pregnancy or breast-feed woman, hypersensitiveness constitution, with other digestive canal disease or serious primary disease and psychiosis, were removed.
2.Methods
(1)The analysis of differentially expressed genes related to substance and energy metabolism from patients of splenasthenic syndrome
All microarray raw data from chronic gastritis patients of splenasthenic-splenogastric hygropyrexia syndrome, splenasthenic syndrome-healthy volunteer and Hp-infected splenogastric hygropyrexia syndrome-Hp-uninfected splenasthenic syndrome were analyzed as follows:
①Microarray raw data preprocessing and filting
These gene spots, in which Cy5and Cy3signal intensity values exceeded200or one of them exceeded800,and the ratio of Cy5and Cy3was between0.1-10.0, were used to calculate the natural logarithm for their ratios of Cy5and Cy3(r=lnCy5/Cy3).Subsequently, the mean value (R) of all r value was obtained.All Cy3signal intensity value was corrected by product of Cy3and1/R to decrease systemic error of Cy5and Cy3fluorescently labeled system. All corrected Cy3and Cy5signal intensity values lessed200were substituted with200. The corrected Cy3and Cy5signal intensity values were uploaded to BRB Array Tools4.1.0Beta2software to analyze. Intensity filers excluded the spot in which both intensities are below200.If only one intensity was below200, this intensity was increased to200. A log base2transformation was applied to intensities before the arrays were normalized by using median over entire array. Gene filters were used to exclude corresponding spots for an entire gene from all arrays. Exclude a gene under any of the following conditions: minimum fold-change less than20%of expression data values have at least a1.5-fold change in either direction from the gene's median value and percent missing exceeded50%. Filtered data were annonated and further analyzed.
②GO and KEGG gene sets analysis
Filtered data were used to analysis of GO and KEGG. Healthy volunteers and patients of splenogastric hygropyrexia syndrome were acted as contrast groups, and splenic asthenia patients as experiment groups (In Hp analysis, contrast and experiment groups were exchanged).Statistics analysis were performed by paired t-test, LS/KS permutation test, and Efron-Tibshirani's GSA maxmean test,and P<0.005.
③Class comparison of between groups of arrays
Filtered data were used to class comparison between groups of arrays, which was used to identify differentially expressed gene among classes of samples.The patients of splenogastric hygropyrexia syndrome, healthy volunteer and Hp-uninfected splenasthenic syndrome were used for the baseline gene expression. Statistics analysis was performed by paired t-test with random variance model and the found gene list was determined by significance at0.05,0.01and0.001levels of univariate tests, respectively. Negatively reciprocal transformation was done for the genes with fold change of expression less than1. The genes which matched p<0.01or0.05or0.001and fold change exceeded2were defined as differentially expressed genes.UP-regulated and down-regulated genes were signed as "↑" and "↓"
④efinition and analysis of genes related to substrance and energy metabolism
The differentially expressed genes containing the gene identifiers along with the corresponding fold changes were uploaded into the Ingenuity Pathways Analysis (IPA) website (version8.6)(www.ingenuity.com) to annotate bio-functions, construct and visualize molecular interaction networks. For only splenasthenic syndrome, the metabolism genes were definited by GO annonation and KEGG analysis of BRB Array Tools, biofunction classification and annonation of IPA website software to approach the correlation of splenasthenic syndrome and metabolism.For differentially expressed genes of Hp-uninfected splenasthenic syndrome,to approach pathogenic mechanism of Hp
infection and provided theoretical basis for classification of syndrome of Chinese medicine and therapia of Hp-related diseases.
⑤Clustering analysis of differentially expressed genes related metabolism
Hierarchical clustering of genes and samples for differentially expressed genes were performed by BRB ArrayTools software.Analysis settings included center the genes, one minus correlation metric for genes or centered correlation for samples, and average linkage.
(2) The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic syndrome, peoples of syndrome or constitution of deficiency of QI and rat models of splenasthenic syndrome
①The similar comparison of genes of substance and energy metabolism from microarray data of between two patients of splenasthenic syndrome
Differentially expressed genes related metabolism from patients of splenasthenic syndrome-healthy volunteers and that of splenasthenic-splenogastric hygropyrexia syndrome were compared to analyze characteristic of gene expression profiling for splenasthenic syndrome, and filted its characteristic genes and pathways, and further approach the relationship of splenasthenic syndrome and metabolism.
②The similar comparison of genes of substance and energy metabolism from patients of splenasthenic syndrome and that of peoples of syndrome or constitution of deficiency of QI
Differentially expressed genes from patients of syndrome or constitution of deficiency of QI were collected from publishing paper, and differentially expressed genes related to metabolism were definited according to above mentioned and selected to contrasting anlysis with that of patients of splenasthenic syndrome.The differentance of them were analyzed to decide that metabolism abnormality existed in only splenasthenic syndrome or all syndrome of deficiency of QI.
③The similar comparison of genes of substance and energy metabolism from patients of splenasthenic syndrome and that of rat models of splenasthenic syndrome
Differentially expressed genes from rat models of splenasthenic syndrome were collected from publishing paper, and were analyzed according to above mentioned method
③to approach characteristic metabolic genes and pathways of splenasthenic syndrome.
(3)Fluorescent quantitation PCR verification of differentially expressed genes from splenasthenic-splenogastric hygropyrexia syndrome
1)Gastric mucosa collection
Four patients who meet criteria of chronic gastritis, splenasthenic syndrome and Hp-uninfection, and4patients who meet criteria of chronic gastritis, splenogastric hygropyrexia syndrome and Hp-infection were collected in Guangdong province hospital of Chinese medicine.Their gastric mucosas were taken200mg in gastroscope, and were preserved in liquid nitrogen.
(3)Fluorescent quantitation PCR of differentially expressed genes
HLA-DRB1,SCGN, COX7B and SULT1A4, which were from differentially expressed genes of Hp-uninfected splenasthenic syndrome-and Hp-infected splenogastric hygropyrexia syndrome and the latter three genes were also differentially expressed gene of splenasthenic-splenogastric hygropyrexia syndrome, were used to quantitation PCR experiment by the method of SYBR Green I.The primers were designed using primer express2.0software.Total RNA extraction and fluorescent quantitation PCR were performed according to direction of kits.Relative amount of experiment and control group was calculated by the method of△△Ct.
Results
1.Differentially expressed genes related to substrance and energy metabolism from the patients of splenasthenic syndrome
(1)Differentially expressed genes from patients of splenasthenic syndrome-healthy volunteers
①287gene sets including49cellular components,60molecular functions and178biological processes were obtained by GO analysis and11pathways were obtained by KEGG analysis.GO biological process and KEGG pathway showed genes related to metabolism of lipid, protein, nucleic acid, carbohydrate and trace element were differentially expressed in the patients of splenasthenic syndrome;②Fifteen differentially expressed genes related to metabolism were obtained by class comparison between groups of arrays, they occupied75%(15/20) of total differentially expressed genes,14genes were down-regulated and11gene productions have enzyme activities,they participated in the metabolism process related to lipid, protein, nucleic acid and carbohydrate.ACAA2↓and CYP20A1↓were participated in lipid metabolism, and showed fatty acid degradation and cholesterol metabolic transformation decreased;RPS28↓,UBXN1↓,UBE2D2↓,ASL↓, ASS1↓,PCYOX1L↓,ALDH9A1↓,B3GNT1↓,GCNT1↓and PPP1R3C↓were participated in protein metabolism, and showed that protein biosynthesis and modification of glycosylation and phosphorylation decreased,protein ubiquitination disturbed, amino acid metabolism related in urea cycle and autonomic nerve decreased, protein; RMI1↓, SMARCD3↓and PARP1↑were participated in nucleic acid metabolism, and showed that DNA duplication and transcription decreased, DNA damage repair increased; B3GNT11↓, GCNT1↓and PPP1R3C↓were participated in carbohydrate metabolism, and showed that biosynthesis of glycan and glycogen decreased.③Clustering analysis of differentially expressed genes related metabolism showed patients of splenasthenic syndrome and healthy volunteers could not be separated.
(2) Differentially expressed genes from patients of splenasthenic-splenogastric hygropyrexia syndrome
①373gene sets including58cellular components,59molecular functions and256biological processes were obtained by GO analysis.These GO biological process included metabolism of lipid, protein, nucleic acid, carbohydrate, trace element and energy. Two pathways obtained by KEGG analysis were ribosome and lactoflavin metabolism.② Fifty-six differentially expressed genes related to metabolism were obtained by class comparison between groups of arrays, they occupied71%(56/79) of total differentially expressed genes,45genes were down-regulated and30gene productions have enzyme activities, they participated in the metabolism process related to lipid, protein, nucleic acid, carbohydrate, trace element and energy metabolism.ACADVL↓, LRP11↑,SULT1A4↓, CRLS1↑,GPCPD1↓, PIGL↓, B3GNT1↓, ST8SIA4↓and FUT9↑were participated in lipid metabplism, and showed that (3-oxidation of fatty acid cut down, cholesterol intake increased and its metabolic transformation decreased, phospholipid and glycolipid metabolism were abnormal;ASRGL1↓, AARSD1↓,EBNA1BP2↓, PUM2↑, MRPL52↓, C120RF65↓, PSMB8↓, PSME2↓, UBA7↓, RNF11↑,FBXO44↓,ZFYVE26↓,CHMP2A↓, SSR4↓,SNX4↑,RAB3B↓,RABL2A↓,GOLGA2↓, KDELR1↓,PHPT1↓,ACPP↓,PTPRF↓, CRKL↓,HDAC7↓,ADPRHL2↓,B3GNT1↓, ST8SIA4↓,DDOST↓and FUT9↑were participated in protein metabolism, and showed that protein biosynthesis, ubiquitination and targeted transport decreased, posttranslation modification of phosphorylation and glycosylation decreased;TOP2A↓, SF3A3↓,CREB3↓,CRTC2↓,NR1D2↑, MED6↓, GTF2IRD1↓, C1ORF83↓, ZNF773↓,ZMYND11↑,DFFB↓,FLJ35220↓and ADPRHL2↓, were participated in nucleic acid metabolism, and showed that DNA duplication and transcription decreased, DNA damage repair increased; AGL↑, PTPRF↓, B3GNT1↓, FUT9↑,ST8SIA4↓,SULT1A4↓,DDOST↓and PIGL↓were participated in carbohydrate metabolism, and showed that glycogen degradation increased and glycoconjugate biosynthesis decreased;COMMD1↓,FTL↓,SLC39A6↑,CHRFAM7A↓,SCGN↑and S100A6↓were participated in trace metabolism, and showed copper and ferri ion metabolism decreased, zinc ion metabolism increased, calcium ion metabolism were abnormal;AK3↓and COX7B↓were participated in energy metabolism, and showed energy metabolism blocked.③Clustering analysis of differentially expressed genes related metabolism showed patients of splenasthenic syndrome and those of splenogastric hygropyrexia syndrome could be separated.
(3)Differentially expressed genes from patients of Hp-uninfected splenasthenic-Hp-infected splenogastric hygropyrexia syndrome
①703gene sets including114cellular components,114molecular functions and475biological processes were obtained by GO analysis and29pathways obtained by KEGG analysis.The GO biological process and KEGG pathway showed that Hp infection caused host differential expression of genes related to protein metabolism, signal pathway, immunity and inflammation reaction, cystoskeleton etc.②Thirty-four annonated differentially expressed genes which obtained by class comparison between groups of arrays, were participated in protein metabolism, inflammatory and immunological reaction, signal transduction, gene transcription, trace element metabolism, and so on.The82%of these genes (28/34) were categorized in three molecular interaction networks involved in gene expression, cancer progress, antigen presentation and inflammatory response.Taken together, these data indicated that Hp infection could alter cellular gene expression processes, escape host defense mechanism, increase inflammatory and immune responses, activate NF-κB and Wnt/β-catenin signaling pathway, disturb metal ion homeostasis,and induce carcinogenesis.③Clustering analysis of differentially expressed genes showed patients of Hp-uninfected splenasthenic syndrome and those of Hp-infected splenogastric hygropyrexia syndrome could be separated.
2.The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic syndrome, peoples of syndrome or constitution of deficiency of Ql and rat models of splenasthenic syndrome
(1)The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic-splenogastric hygropyrexia syndrome and patients of splenasthenic syndrome-healthy volunteer
①The GO biological process from the two microarray data showed genes related to metabolism of lipid, protein, nucleic acid, carbohydrate and trace element were differentially expressed in the patients of splenasthenic syndrome;KEGG pathway from the two microarray data showed that genes related to protein metabolism were differentially expressed.
②The results of class comparison between groups of arrays showed, differentially expressed genes related to substrance and energy metabolism from patients of splenasthenic-splenogastric hygropyrexia syndrome and patients of splenasthenic syndrome-healthy volunteer showed obviously difference.The dissimilarity was the numbers and metabolic process of differentially expressed genes.The former was more than the latter, metabolic processes included lipid, protein, nucleic acid, carbohydrate, trace element and energy. The latter was participated in lipid, protein, nucleic acid and carbohydrate metabolism. Moreover, differentially expressed gene name was also difference.The similarity was followed as (1) Many genes related to substrance and energy metabolism were differentially expressed in the two microarray data, and they occupated over70%of total differentially expressed genes, and most of these genes were enzyme genes or enzyme activity gene, and their expressions were down-regulated in patients of splenasthenic syndrome;(2)B3GNT1expression was down-regulated in the two microarray data;(3) In the metabolic pathway, the results of two microarray data from patients of splenasthenic syndrome showed cholesterol metabolic transformation and β-oxidation of fatty acid decreased, ribosome assembly blocked, protein modification of phosphorylation and glycosylation decreased, protein ubiquitation decreased, DNA duplication and transcription decreased,DNA damage repair increased, tissue glycogen biosyntheis decreased and degradation increased, and glycan biosynthesis decreased.
③Clustering analysis of differentially expressed genes related metabolism showed patients of splenasthenic syndrome and those of splenogastric hygropyrexia syndrome could be separated, while patients of splenasthenic syndrome and healthy volunteers could not be separated.
(2) The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic syndrome and syndrome or constitution of deficiency of QI
Differentially expressed genes related to substrance and energy metabolism from patients of splenasthenic syndrome and syndrome or constitution of deficiency of QI showed obviously difference.(1)Differentially expressed genes related to metabolism from patients of splenasthenic syndrome were similar to that of constitution of deficiency of QI in expression tendency and metabolic pathway, and both existed metabolic block, but patients of splenasthenic syndrome more obvious.For these results, patients of other syndrome or constitution of deficiency of QI had completely different results;(2) ST, RAB and S100might be mark genes of splenasthenic syndrome, while RP, PTP and UBE2might be mark genes of syndrome of deficiency of QI;(3)In metabolic pathway, differentially expressed genes related to lipid, carbohydrate, protein and nucleic acid metabolism from patients of splenasthenic syndrome were down-regulated, while that of syndrome or constitution of deficiency of QI were up-regulated and participated in less metabolic process;(4)Protein ubiqutination abnormality was commonness of syndrome of deficiency of QI,but splenasthenic syndrome and constitution of deficiency of QI decreased, other syndrome or constitution of deficiency of QI increased;(5) DNA damage might be common pathological phenomenon of syndrome and constitution of deficiency of QI.
3.The similar comparison of differentially expressed genes related to metabolism from patients and animal models of splenasthenic syndrome
Differentially expressed genes related to metabolism from patients and animal models of splenasthenic syndrome had obvious differentence in numbers and expression tendency. But thirteen differentially expressed genes were repeatedly appeared in microarray results of patients and animal models of splenasthenic syndrome, they were B3GNT1,CYP, SLC39A, SNX, FUT, SLC2A, GCNT, ACAD, LDH, RP, PPP1, S100and PTPR. That RP and PTPR were down-regulated also appeared in syndrome of deficiency of QI;(1)Except for RP, all these genes production were enzyme and transporter;(2) These genes were participated in lipid, carbohydrate, protein and tace element metabolism, and showed that glucose transport and degradation, glycogen synthesis and degradation, and glycan synthesis, were disordered;Fatty acid and cholesterol metabolism were abnormal; Ribosome assembly blocked, protein modification of phosphorylation decreased and that of glycosylation was abnormal, and targeted transport increased; Zinc ion metabolism increased and calcium ion metabolism increased.
3.Fluorescent quantitation PCR verification of differentially expressed genes from splenasthenic-splenogastric hygropyrexia syndrome
The results of fluorescent quantitation PCR for HLA-DRB1,SCGN,COX7B and SULT1A4, which were from differentially expressed genes of Hp-uninfected splenasthenic syndrome-and Hp-infected splenogastric hygropyrexia syndrome and the latter three genes were also differentially expressed gene of splenasthenic-splenogastric hygropyrexia syndrome,and that of microarray experiment were similar, but the ratio of the former was lower than the latter. ACAA2of differentially expressed genes from splenasthenic syndrome-healthy volunteers was also verified by our research groups previously.
Conclusions
1.It was different to other syndrome of deficiency of QI that the patients of splenasthenic syndrome showed nutrition dysmetabolism by down-regulation of metabolic genes, especially enzyme genes.Dysmetabolism showed that cholesterol metabolic transformation and β-oxidation of fatty acid decreased, ribosome assembly was blocked, protein modification of phosphorylation and glycosylation decreased, protein ubiquitation decreased, DNA duplication and transcription decreased, DNA damage repair increased, tissue glycogen biosyntheis decreased and degradation increased, and glycan biosynthesis decreased. All of these results might be the one of important reason causing nutrition dysmetabolism of splenasthenic syndrome.All these results acted out macro-metabolism sketch of patients of splenasthenic syndrome, and supported discussion related to metabolism for pathophysiology of spleen in Neijing, which provided some clues for modern research and annotation of scientic essence of spleen picture.At the same time, the macro-metabolism sketch of patients of splenasthenic syndrome well revealed molecular pathophysiology background of spleen, which provided theoretical basis and academic guide for metabolic disease research based on the treatment from spleen.
2.The differentially expressed genes related to metabolism from analysis of gene microarray data and the determination results of biochemical indicator related to metabolism from literature report were confirmed each other and revealed pathophysiology of spleen mainly included digest and absorb,and metabolism of substance.
3.The B3GNT1,ST, RAB,CYP, SLC39A, SNX, FUT, SLC2A, GCNT, ACAD, LDH, RP, PPP1, S100and PTPR, might be closely related to the occurrence of splenasthenic syndrome, while RP, PTP and UBE2might be closely related to the occurrence of syndrome of deficiency of QI.All of these genes provided some clues for research of splenasthenic syndrome.
4.The analysis of gene expression profiling showed the clinical manifestation between splenasthenic and splenogastric hygropyrexia syndrome was more obvious, while between splenasthenic syndrome and healthy volunteers was not obvious,which provided theoretical basis for classification of cases of disease in Chinese medicine.
5.Hp infection could affect host on protein metabolism, inflammatory and immunological reaction, signal transduction, gene transcription, trace element metabolism, and so on, which provided theoretical basis for classification of syndrome of Chinese medicine and therapia of Hp-related diseases.
中医“脾主运化”、脾为“后天之本,气血生化之源”之说,揭示脾对于饮食物具有消化吸收和输转的功能,同时脾通过营养物质在体内的代谢,为人体出生以后生长发育及生命活动提供能量和物质基础。物质能量代谢是生命活动的物质基础,它包括消化吸收、中间代谢和排泄三个阶段,中间代谢是机体内代谢的主体,包括糖、脂、蛋白质、核酸、微量元素和能量代谢等(本论文物质代谢特指糖、脂、蛋白质、核酸和微量元素代谢)。目前已有大量的研究证实脾虚动物存在消化吸收和营养代谢障碍,在中间代谢方面表现为糖原含量降低；血脂含量和生物膜结构异常；氨基酸和蛋白含量降低；组织DNA和RNA含量降低；线粒体结构异常,ATP含量降低；微量元素含量异常等,可见,脾与物质能量代谢在病理上也表现出高度的一致。
目前脾虚证与物质能量代谢相关性研究主要以对某一代谢相关生化指标的测定为主,缺乏对脾虚证机体整个物质能量代谢的同步研究。基因芯片技术作为一个“组学”研究方法,与中医的“整体观”有很多相似之处,它可以反映机体某一状态所有基因的表达情况。因此,在本世纪初,基因芯片技术就已经广泛用于脾虚证动物模型的研究,但是由于我国利用基因芯片技术研究脾虚证的起步相对较晚,基因芯片数据的分析存在一些明显不足,如对芯片数据的预处理不够,对差异表达基因缺乏统计学检验,并且对于差异表达基因的功能注释也过于单一,最终导致对芯片数据的解读浮于表面,未能达到预期目的。随着分子生物学和生物信息学的发展,一些专业的生物信息学软件已经广泛用于基因芯片数据的分析,这大大提高了基因芯片数据的解读能力,并且随着大量基因芯片数据的积累,使得广泛利用现有基因芯片数据来解读脾虚证的发病机制比少量基因芯片实验本身更为重要。
对此,在课题组应用基因芯片研究慢性浅表性胃炎脾气虚证(下称脾虚证)基因表达谱的工作基础上,本文利用BRB ArrayTools和IPA软件对慢性浅表性胃炎(下称慢性胃炎)脾虚证患者(分别以健康志愿者和脾胃湿热证患者作为对照)和非幽门螺杆菌(Helicobacter pylori, Hp)感染脾虚证患者(与Hp感染脾胃湿热证患者相比)基因芯片数据进行深入挖掘,通过对前者物质能量代谢相关差异表达基因分析,探讨脾虚证与物质能量代谢的相关性,从机体整个物质能量代谢水平认识脾的生理功能和脾虚证的发病机制；通过对后者差异表达基因分析,探讨Hp的致病机制,同时认识“慢性胃炎脾胃湿热证患者Hp感染率明显比脾虚证患者高”这一临床现象的遗传背景,为中医分型和Hp相关疾病的治疗提供理论依据。此外,论文还汇总了近10年气虚证或体质人和脾虚大鼠模型差异表达基因,通过对脾虚证患者、气虚证或体质人和脾虚大鼠模型物质能量代谢相关差异表达基因的相似性比较,探讨脾虚证基因表达谱的特征,并筛选出脾虚证特征性代谢基因和代谢途径,从基因组学的角度为中医脾基础理论的现代科学诠释和脾虚证临床辨证分型提供参考,并为脾虚证的病理生理研究提供线索。
材料与方法：
一、材料
(一)基因芯片数据来源
慢性胃炎脾虚证患者与健康志愿者比较和脾虚证患者与脾胃湿热证患者比较的基因芯片原始数据,以及慢性胃炎非Hp感染脾虚证患者与Hp感染脾胃湿热证患者比较的基因芯片原始数据全部来自于课题组脾虚证发病机理研究的前期数据,并已有相关论文发表。包括肺气虚证、气阴两虚证、气虚体质在内的气虚证或体质人差异表达基因全部来源于公开发表的学术论文和学位论文。包括不同建模方法和组织来源的脾虚大鼠模型差异表达基因全部来源于公开发表的学术论文和学位论文。
(二)用于脾虚证与脾胃湿热证比较的差异表达基因验证的荧光定量PCR实验病例来源
1.诊断标准及辨证分型标准
慢性胃炎西医诊断和辨证分型标准以及Hp诊断标准全部采用课题组原实验设计标准(王颖芳,广州中医药大学2006届博士学位论文),具体如下：
(1)慢性胃炎诊断标准参照《中药新药临床研究指导原则》。
(2)脾气虚证辨证标准主症：①舌质淡、舌体胖或有齿印,苔薄白；②胃纳减少或食欲差；③腹胀；④大便溏或腹泻。次症：①消瘦；②体倦乏力；③脉细弱。判断：a)主症①必备。b)兼具主症②、③、④之二；或兼具主症②、③、④之一,同时兼具2个以上次症,诊断可成立。
(3)脾胃湿热证辨证标准主症：①舌苔黄腻；②胸闷；③胃脘痞满或胀痛；④食欲不振。次症：①口苦而黏；②口渴少饮,或喜热饮；③大便溏,或有黏液；④恶心；⑤身困乏力；⑥脉濡缓,或滑。判断：a)主症①必备。b)兼具主症②、③、④之
二,或兼具主症②、③、④之一,同时兼具2个以上次症；或兼具3个以上次症,诊断可成立。
(4)Hp诊断标准
患者近一个月内未服用抗生素类药物。病理切片染色与快速尿素酶试验二者均为阳性即为Hp感染。两者均为阴性即为非Hp感染。
2.纳入及排除标准
同时符合慢性胃炎非Hp感染脾虚证或Hp感染脾胃湿热证,并同意签署由学术道德委员会批准的《知情同意书》的患者纳入。年龄在18岁以下或65岁以上,妊娠或哺乳妇女,过敏体质者,或存在消化道其它器质性病变、心、脑、肝、肾、造血系统等严重原发性疾病及精神性疾病等患者被排除。
二、方法
(一)脾虚证患者差异表达基因分析
慢性胃炎脾虚证患者与健康志愿者比较和脾虚证患者与脾胃湿热证患者比较的基因芯片原始数据,以及慢性胃炎非Hp感染脾虚证患者与Hp感染脾胃湿热证患者比较的基因芯片原始数据,采用下述方法进行分析处理：
1.基因芯片数据的预处理和数据筛选
对Cy5和Cy3信号值进行均一化处理,消除两标记体系间的系统误差。参与均一化处理的有效基因点的条件：(1)该基因点的Cy3和Cy5信号值都大于200,或者其中之一大于800；(2)该基因点的Cy5信号值/Cy3信号值的比值在0.1-10之间。均一化系数计算方法：计算每个有效基因点Cy5信号值/Cy3信号值的比值,求出其相应的自然对数值r=ln(Cy5/Cy3),算出全部有效基因点r值的平均值R,那么实验的均一化系数就等于R的倒数即(1/R)。将所有基因点的Cy3信号值乘上均一化系数,得出调整后的Cy3*,即Cy3*=Cy3×(1/R),并将所有小于200的Cy3*值以200取代,所有小于200的Cy5信号值用200替代。将Cy5和Cy3*信号值输入芯片分析软件BRBArrayTools4.1.0Beta2进行数据筛选,包括对数转换、中位值标准化和基因筛选。其中某个基因被保留用于后续分析的基因筛选条件是：该基因的Cy5/Cy3*比值必须有超过20%的样本大于1.5,且不能有超过50%的数据缺失。对筛选后的数据进行功能注释和进一步分析。
2.GO和KEGG基因集分析
对筛选后的芯片数据进行GO和KEGG基因集分析。基因芯片分析时,将健康志愿者或脾胃湿热证患者作为对照组,将脾虚证患者作为实验组。Hp分析时,将非Hp感染脾虚证患者作为对照组,Hp感染脾胃湿热证患者作为实验组。采用配对t检验的方法,显著性检验方法为LS/KS排列检验和Efron-Tibshirani's基因集分析最大均值检验(GSA maxmean test), P<0.005。
3.芯片间分类对比分析
(1)差异表达基因筛选
对筛选后的数据进行芯片间的分类对比分析。基因芯片分析时,实验组和对照组设置同上。采用配对t检验的统计方法,将同时满足P<0.05或p<0.01或p<0.001和表达倍数>2的基因设定为差异表达基因。对表达倍数<1的基因数据进行负倒数转换,因此倍数为正的基因是实验组上调基因,负值表示实验组下调基因,分别标记为“↑”和“↓”。
(2)差异表达基因生物信息分析
将差异表达基因ID和表达倍数导入IPA8.6在线软件对差异表达基因进行注释、相互作用网络等生物学功能分析。对于脾虚证分别与健康志愿者和脾胃湿热证患者比较的差异表达基因根据BRB Array Tools软件的GO功能分析中的生物学过程和KEGG通路以及IPA在线软件的生物学功能分类和基因的生物学注释四方面信息综合确定物质能量代谢相关基因,分析脾虚证与物质能量代谢的相关性；对于非Hp感染脾虚证差异表达基因,通过差异表达基因功能注释和相互作用网络分析,探讨Hp的致病机制,为中医分型和Hp相关疾病的治疗提供理论依据。
(3)基于差异表达基因的聚类分析
利用BRB Array Tools软件的聚类功能,选择基因中心化、一减相关系数和平均连接簇设置,以选定的差异表达基因作为分析对象(Hp分析时选定所有差异表达基因,其他两组芯片分析选定物质能量代谢相关差异表达基因),对样本和基因进行层级聚类分析。
(二)脾虚证患者、气虚证或体质人和脾虚大鼠模型物质能量代谢相关差异表达基因相似性比较
1.脾虚证患者两组芯片的物质能量代谢基因之间相似性比较
对脾虚证患者与健康志愿者比较的物质能量代谢相关差异表达基因和脾虚证与脾胃湿热证患者比较的物质能量代谢相关差异表达基因进行相似性比较,分析脾虚证患者差异表达基因的特征,并筛选出脾虚证患者特征性的基因和代谢途径,进一步探讨脾虚证与物质能量代谢的关系。
2.脾虚证患者物质能量代谢基因和气虚证或体质人物质能量代谢基因相似性比较
检索并汇总文献报道的气虚证或体质人差异表达基因,按照上述物质能量代谢相关基因的界定方法,筛选出物质能量代谢相关差异表达基因,并与脾虚证患者物质能量代谢差异表达基因进行比较,探讨脾虚证患者和气虚证或体质人在物质能量代谢基因差异表达上的异同,从而确定物质能量代谢异常是脾虚证患者特有的病理现象还是气虚证所共有的病理机制。
3.脾虚证患者物质能量代谢基因和脾虚大鼠模型物质能量代谢基因相似性比较
检索并汇总文献报道的脾虚大鼠模型差异表达基因,按照上述方法2,进行分析,探讨脾虚证特有的代谢基因和途径。
(三)脾虚证与脾胃湿热证比较的差异表达基因荧光定量PCR验证
1.胃黏膜组织的采集
在广东省中医院收集慢性胃炎非Hp感染脾虚证和Hp感染脾胃湿热证患者各4例,胃镜下钳取胃窦黏膜活检组织200mg左右,液氮保存备用。
2.荧光定量PCR实验
论文对慢性胃炎非Hp感染脾虚证与Hp感染脾胃湿热证比较的差异表达基因HLA-DRB1、SCGN、COX7B和SULT1A4四个基因进行定量PCR验证。实验采用SYBR Green I的方法,利用primer express2.0软件设计引物,并按照相关试剂盒说明书提取总RNA、进行荧光定量RT-PCR反应,采用ΔΔCt的方法计算实验组与对照组之间的相对含量。
结果：
一、脾虚证患者差异表达基因
(一)脾虚证与健康志愿者比较的物质能量代谢相关差异表达基因
(1)GO分析获得287个有显著性意义的基因集,其中细胞成分49个、分子功能60个,生物学过程178个。KEGG分析获得11个有显著性意义的代谢通路。GO生物学过程和KEGG通路显示,脾虚证患者在涉及脂、蛋白质、核酸和糖代谢的相关基因表达异常。(2)分类对比分析获得15个物质能量代谢相关差异表达基因,占总差异表达基因的75%(15/20),其中下调基因14个,酶基因或有酶活性的基因11个(73%),它们参与机体脂、蛋白质、核酸和糖代谢过程。与脂类代谢相关的基因有ACAA2↓和CYP20A1↓,表现为脂肪酸分解和胆固醇转化降低；与蛋白质代谢相关的基因有RPS28↓、B3GNT1↓、GCNT1↓、PPP1R3C↓、UBXN1↓、UBE2D2↓、ASL↓、 ASS1↓和PCYOX1L↓、ALDH9A1↓,表现为蛋白质合成和糖基化和磷酸化修饰降低,蛋白质泛素化降解异常,参与尿素循环、自主神经等生物学过程的氨基酸代谢降低；与核酸代谢相关的基因有RMI1↓、SMARCD3↓和PARP1↑,表现为DNA复制和转录降低,DNA损伤修复增加；与糖类代谢相关的基因有B3GNT1↓、GCNT1↓和PPP1R3C↓,表现为聚糖和糖原合成降低。(3)基于物质能量代谢基因差异表达的样本聚类分析显示脾虚证患者与健康志愿者不能分开。
(二)脾虚证与脾胃湿热证比较的物质能量代谢相关差异表达基因
(1)GO分析获得373个有显著性意义的基因集,其中细胞成分58个、分子功能59个,生物学过程256个。这些生物学过程包括脂、蛋白质、核酸、糖代谢、微量元素和能量代谢等。KEGG分析获得2个有显著性意义的代谢通路,即核糖体和核黄素代谢。(2)分类对比分析获得56个物质能量代谢相关差异表达基因,占总差异表达基因的71%(56/79),其中下调基因45个、酶或具有酶活性的基因30个、转运蛋白基因5个,它们参与机体脂、蛋白质、核酸、糖、微量元素和能量代谢过程。与脂代谢相关的基因有ACADVL↓、LRP11↑、SULT1A4↓、CRLS1↑、GPCPD1↓、PIGL↓、 B3GNT1↓、ST8SIA4↓和FUT9↑,表现为脂肪酸p氧化降低、胆固醇摄取增加和代谢转化降低、磷脂和糖脂代谢异常；与蛋白质代谢相关的基因有ASRGL1↓、AARSD1↓、 EBNA1BP2↓、PUM2↑、MRPL52↓、C120RF65↓、PSMB8↓、PSME2↓、UBA7↓、RNF11↑、 FBX044↓、ZFYVE26↓、CHMP2A↓、SSR4↓、SNX4↑、RAB3B↓、RABL2A↓、GOLGA2↓、 KDELR1↓、PHPT1↓、ACPP↓、PTPRF↓、CRKL↓、HDAC7↓、ADPRHL2↓、B3GNT1↓、 ST8SIA4↓、DDOST↓和FUT9↑,主要表现为蛋白质的生物合成降低、蛋白质泛素化减弱、靶向输送和翻译后磷酸化和糖基化修饰降低；与核酸代谢相关的基因有TOP2A↓、 SF3A3↓、CREB3↓、CRTC2↓、NR1D2↑、MED6↓、GTF2IRD1↓、C1ORF83↓、ZNF773↓、ZMYND11↑、DFFB↓、FLJ35220↓和ADPRHL2↓,主要表现为DNA复制和转录减弱,DNA损伤修复增强；与糖代谢相关的基因有AGL↑、PTPRF↓、B3GNT1↓、FUT9↑、 ST8SIA4↓、SULT1A4↓、DDOST↓和PIGL↓,主要表现为糖原的分解增加和糖复合物的合成降低；与微量元素代谢相关的基因有COMMD1↓、FTL↓、SLC39A6↑、 CHRFAM7A↓、SCGN↑和S100A6↓,表现为铜离子和铁离子代谢下降、锌离子代谢增加、钙离子代谢异常；与能量代谢相关的基因有AK3↓和COX7B↓,表现能量代谢障碍。(3)基于物质能量代谢基因差异表达的样本聚类分析能够将脾虚证和脾胃湿热证患者分开。
(三)非Hp感染脾虚证与Hp感染脾胃湿热证比较的差异表达基因
(1)GO分析获得703个有显著性意义的基因集,其中细胞成分114个、分子功能114个,生物学过程475个。KEGG分析获得29个有显著性意义的代谢通路。GO生物学过程和KEGG通路分析结果一致显示,Hp感染会使宿主在涉及蛋白质代谢、信号通路、免疫炎症反应、细胞骨架等方面的基因差异表达。(2)分类对比分析获得34个有生物学功能注释的差异表达基因,它们参与蛋白质代谢、免疫和炎症反应、信号通路、基因转录、微量元素代谢等过程。(3)差异表达基因相互作用网络分析发现82%的基因被IPA软件归类到三个与基因表达、癌症进展、抗原递呈和免疫反应等相关的相互作用网络。这些结果显示Hp感染能够刺激颗粒富集的胞质结构(Particle-rich Cytoplasmic Structure PaCS)的形成,改变细胞基因表达过程,逃逸宿主防御机制,增加免疫和炎症反应,激活NFKB和Wnt/β-catenin信号通路,扰乱金属离子平衡,诱导癌症发生。(4)差异表达基因聚类分析能够将非Hp感染脾虚证患者和Hp感染脾胃湿热证患者分开。
二、脾虚证患者、气虚证或体质人和脾虚大鼠模型物质能量代谢相关差异表达基因相似性比较
(一)脾虚证患者两组芯片的物质能量代谢基因之间相似性比较
1.两组芯片数据的GO分析都显示,脾虚证患者在涉及脂、蛋白质、核酸和糖代谢的相关基因表达异常,并且以参与蛋白质代谢的基因最多；KEGG分析都显示,脾虚证患者在参与蛋白质代谢通路的相关基因表达异常。
2.两组芯片的分类对比分析显示,脾虚证与健康志愿者比较的物质能量代谢差异表达基因相对较少,主要涉及脂类、蛋白质、核酸和糖类代谢,而脾虚证与脾胃湿热证比较的物质能量代谢差异表达基因较多,主要涉及脂类、蛋白质、核酸、糖类、微量元素和能量代谢,并且两组芯片在具体的差异表达基因名称上也存在明显不同。尽管如此,两组芯片更多的存在很多共同点,表现在：(1)脾虚证患者在两组芯片中都存在大量物质能量代谢基因表达异常,占总差异表达基因的70%以上,并且大部分为酶基因或具有酶活性基因,总体以表达下调为主。(2)B3GNT1在两组基因芯片数据中都一致下调。(3)在代谢途径上,脾虚证患者在两者芯片中都表现为胆固醇代谢转化和脂肪酸p氧化过程降低,核糖体形成障碍,蛋白质翻译后磷酸化和糖基化修饰降低,蛋白质泛素化降解途径降低,DNA复制和转录水平低下,DNA损伤修复加强。同时,脾虚证患者胃肠道组织糖原合成降低,分解增强,并且还存在聚糖合成障碍。
3.基于物质能量代谢基因差异表达的样本聚类分析显示,脾虚证患者与健康志愿者不能很好的分开,而脾虚证患者与脾胃湿热证患者则能很好的分开。
(二)脾虚证患者物质能量代谢基因和气虚证或体质人物质能量代谢基因相似性比较
脾虚证患者与其他气虚证或体质人物质能量代谢相关差异表达基因存在明显差异,具体表现在：(1)脾虚证患者,除与气虚体质人差异表达基因存在很多相似之处(差异表达基因都以下调为主,但是脾虚证患者在导致物质能量代谢障碍方面更加明显)之外,与其他气虚证或体质人在差异表达基因的种类、表达趋势和涉及的代谢途径上都明显不同；(2)UBA、ST、RAB和S100是脾虚证患者和气虚体质人的共有基因,可能与脾虚证密切相关,而RP、PTP和UBE2是脾虚证患者与其他气虚证或体质人的共有基因,可能与气虚证密切相关；(3)在代谢途径上,脾虚证患者参与糖、脂、蛋白质和核酸代谢的相关基因表达下调,而气虚证或体质人差异表达基因参与的代谢过程相对较少,并且相关基因以上调为主；(4)蛋白质泛素化异常是气虚证的共性,但脾虚证患者和气虚体质人表现降低,其他气虚证或体质人表现增强；(5)脾虚证患者和气虚证或体质人都存在DNA损伤,这可能是气虚证共有的病理现象。
(三)脾虚证患者物质能量代谢基因和脾虚大鼠模型物质能量代谢基因相似性比较
1.脾虚证患者和脾虚大鼠模型在差异表达基因的数量上存在明显差异,在表达趋势上,脾虚证患者差异表达基因总体以下调为主,脾虚大鼠模型差异表达基因除海马组织外,都以表达上调为主。这些结果说明脾虚证患者和脾虚大鼠模型在病理上可能存在明显不同。2.脾虚证患者和大鼠模型之间重复出现的代谢相关差异表达基因有13个：B3GNT1、CYP、SLC39A、SNX、FUT、SLC2A、GCNT、ACAD、LDH、RP、 PPP、S100和PTPR,其中RP和PTPR下调与气虚证患者共有基因分析结果一致；(1)这些基因表达产物,除RP外,全为酶和运载蛋白,与脾主运化的认识一致；(2)这些基因涉及糖、脂、蛋白质和微量元素代谢过程,具体表现为葡萄糖运输和分解、糖原合成与分解以及聚糖的合成紊乱；脂肪酸和胆固醇代谢异常；核糖体形成障碍、蛋白质翻译后磷酸化修饰降低和糖基化异常以及细胞内靶向输送增加；锌离子代谢增加、钙离子代谢降低。这些结果说明,不同建模方法构建的脾虚大鼠模型虽然都不能完全反映脾虚证的所有特征,但是都能从某一方面部分反映脾虚证的特征,其重复出现的代谢相关差异表达基因可能就是脾虚证的重要特征。
三、脾虚证与脾胃湿热证对照的差异表达基因荧光定量PCR验证
慢性胃炎非Hp感染脾虚证与Hp感染脾胃湿热证比较的差异表达基因HLA-DRB1、SCGN、COX7B和SULT1A4四个基因(后三个基因也是脾虚证与脾胃湿热证差异表达基因)的定量PCR结果与基因芯片实验结果一致,但是定量PCR结果整体比基因芯片结果偏低。此外,之前课题组对脾虚证患者与健康志愿者差异表达基因ACAA2进行了验证,定量PCR结果与基因芯片结果一致下调,定量PCR结果同样比基因芯片结果偏低。
结论：
1.脾虚证患者与其他气虚证不同,主要通过代谢相关基因,尤其是酶基因的表达下调,从而出现营养代谢障碍,具体表现为：(1)胆固醇代谢转化和脂肪酸β氧化过程降低；(2)核糖体形成障碍,蛋白质翻译后磷酸化和糖基化修饰降低,蛋白质泛素化降解途径降低；(3)DNA复制和转录水平低下,DNA损伤修复加强；(4)脾虚证患者胃肠道组织糖原合成降低,分解增强,并且还存在聚糖合成障碍。这些结果描绘出了脾虚证患者宏观物质能量代谢草图,支持《内经》中脾病理生理涉及物质能量代谢的论述,为脾藏象科学本质的现代研究和诠释提供了线索。同时,为从脾论治代谢性疾病研究提供了理论依据和学术指引。
2.基于基因芯片数据分析获得的物质能量代谢相关差异表达基因与文献报道的临床代谢相关生化指标的测定结果相互印证,共同揭示了脾的病理生理主要涉及饮食物的消化吸收和体内代谢两方面。
3.B3GNT1、ST、RAB、CYP、SLC39A、SNX、FUT、SLC2A、GCNT、ACAD、 LDH、RP、PPP1、S100和PTPR可能与脾虚证密切相关,而RP、PTP和UBE2可能与气虚证密切相关,这些基因为脾虚证的进一步研究提供了线索。
4.基因表达谱分析显示,慢性胃炎脾虚证与脾胃湿热证之间临床表现差异较大,而慢性胃炎脾虚证与健康人之间临床表现差异不明显,这为中医疾病分型提供了理论依据。
5.Hp感染会刺激PaCS的形成,改变细胞基因表达过程,逃离宿主防御机制,增加炎症和免疫反应,激活NFκB和Wnt/β-catenin信号通路,干扰金属离子的平衡,诱导肿瘤的发生。
Research background and objective
The theory of "Spleen governing transportation and transformation"and "the spleen is the origin of the acquired constitution and source of qi-blood" in the Chinese medicine illustrates the spleen has a function in ingesta digest, absorb and transport, and provides energy and substance foundation for human growth, development and vital movement by metabolism of nutritive substance.Substance and energy metabolism is foundation of vital movement. It includes three stage of digest and absorb, intermdediate metabolism and excrete.Intermdediate metabolism is the main body of metabolism, and includes carbohydrate, lipide, protein, nucleic acid, trace element and energy metabolism.At present, many research results showed splenic asthenia animal existed the disorder of digest, absorb and nutrition metabolism.Thus, it is also similar between patho-manifestations of spleen and metabolism.
In relativity research between splenasthenic syndrome and metabolism, it is a common method to determine biochemical indicator related to certain metabolism, but this method can not simultaneously study whole substance and energy metabolism for splenasthenic syndrome patients.Microarray technology is a omics resesrch method, and able to reflect organism all gene expression in certain state, which is very similar to the holism of Chinese medicine.So, at the beginning of this century, microarray has been extensive utilized to research animal model of splenasthenic syndrome.But because of a late beginning in China, microarray data analysis exist some problems, such as not pretreatment for microarray data, not statistics test for differentially expressed gene, and simple function annotation. These problems cause microarray data not to well explain disease mechanism.Follow the development of molecule biology and bioinformatics, some professional anlysis softwares raise the readout ability of microarray data. Moreover, many microarray data have been accumulated in recently years.So,it is more important to mine reported microarray data of splenasthenic syndrome by using bioinformics software than microarray experiment in study pathogenesy of splenasthenic syndrome.
Based on the work foundation of gene expression profiling for splenasthenic syndrome with chronic superficial gastritis in our research groups, at the present study, microarray data of chronic superficial gastritis (below named chronic gastristis) splenasthenic-splenogastric hygropyrexia syndrome and splenasthenic syndrome-healthy volunteer, and Helicobacter pylori (H. pylori, Hp)-uninfected splenasthenic-Hp-infected splenogastric hygropyrexia syndrome were mined and bioinformatics analysis by BRB Array Tools and IPA software.For the former, differentially expressed genes related to substance and energy metabolism were analyzed to approach the correlation of splenasthenic syndrome and substance and energy metabolism, and the physiological function of spleen and pathogenesy of splenasthenic syndrome based on whole substances and energy metabolism level of organism; For the latter, differentially expressed genes were analyzed to approach pathogenic mechanism of Hp infection and understood genetic background of clinical outcome that Hp infection rate of splenogastric hygropyrexia syndrome with chronic gastritis was higher than splenasthenic syndrome, and provided theoretical basis for classification of syndrome of Chinese medicine and therapia of Hp-related diseases.In addition, the microarray data from patients of syndrome or constitution of deficiency of QI and rat models of splenasthenic syndrome were also collected.Differentially expressed genes related to substance and energy metabolism from patients of splenasthenic syndrome, and that of rat models of splenasthenic syndrome and the peoples of syndrome or constitution of deficiency of QI were similarly compared to approach marked gene and metabolic pathway of splenasthenic syndrome, and provide some references for scientic annonation of basic theory of Chinese medicine spleen and differentiation of symptoms and classification of syndrome of splenasthenic syndrome, and provide some clue for pathophysiology research of splenasthenic syndrome.
Materials and methods
1.Materials
(1)Microarray data origin
All microarray raw data from chronic gastritis patients of splenasthenic-splenogastric hygropyrexia syndrome, splenasthenic syndrome-healthy volunteer and Hp-infected splenogastric hygropyrexia syndrome-Hp-uninfected splenasthenic syndrome were from early data studying pathogenesy of splenasthenic syndrome in our group, and some papers have been published based on these data. All differentially expressed genes from patients of syndrome or constitution of deficiency of QI including insufficiency of lung-QI, deficiency of both vital energy and yin, deficiency of vital energy, and rat models of splenasthenic syndrome including different modeling methods and tissue origin were from open publishing academic and degree papers.
(2) Case origin of fluorescent quantitation PCR verification of differentially expressed genes from microarray data of splenasthenic-splenogastric hygropyrexia syndrome
1) Diagnostic and differentiation of symptoms and signs for classification of syndrome criteria
All criteria of diagnose and classification symptons were from previosly criteria (WANG,Ying-fang, doctor thesis of Guangzhou university of Chinese medicine,2006),as followed:
Diagnostic criteria of chronic gastritis refer to the Guide Principle of Clinical Research of Chinese Material Medica and New Drug.
Differentiation of symptoms and signs criteria of splenasthenic syndrome Main sign:①body of the tongue light and big or has imprints of the teeth, moss thin and white;②appetite decrease;③abdominal distension;④loose stool or diarrhoea. Secondary sign:①emaciated;②weary and hypodynamia;③pulses extenuate.Decision:a) Main sign①necessary, b) Including the two of main sign②,③and④; Or including one of main sign②,③and④,and including over two of secondary sign.
Differentiation of symptoms and signs criteria of splenogastric hygropyrexia syndrome Main sign:①coated tongue yellow freasy;②chest distress;③gastric cavity painful abdominal mass or gas pains;④poor appetite.Secondary sign:①bitter taste and sticky of mouth;②thirsty and few drink, or hot drink preference;③oose stool or including mucus;④nauseated;⑤body exhausted and debilitation;⑥pulses slow or slide.Decision:a) Main sign①necessary, b) Including the two of main sign②,③and④;or including one of main sign②,③and④,and including over two of secondary sign; or including over three of secondary sign.
Diagnostic criteria of Hp infection Patients donot take medicine of antibiotics in recent one month.Both pathological section-staining and fast urease test were positive, which were Hp infected, otherwise were Hp uninfected.
2) Receive and remove criteria
The patients who meet criteria of chronic gastritis, splenasthenic syndrome and Hp uninfection or meet chronic gastritis, splenogastric hygropyrexia syndrome and Hp infection were received. The patients whose age less18or over65,pregnancy or breast-feed woman, hypersensitiveness constitution, with other digestive canal disease or serious primary disease and psychiosis, were removed.
2.Methods
(1)The analysis of differentially expressed genes related to substance and energy metabolism from patients of splenasthenic syndrome
All microarray raw data from chronic gastritis patients of splenasthenic-splenogastric hygropyrexia syndrome, splenasthenic syndrome-healthy volunteer and Hp-infected splenogastric hygropyrexia syndrome-Hp-uninfected splenasthenic syndrome were analyzed as follows:
①Microarray raw data preprocessing and filting
These gene spots, in which Cy5and Cy3signal intensity values exceeded200or one of them exceeded800,and the ratio of Cy5and Cy3was between0.1-10.0, were used to calculate the natural logarithm for their ratios of Cy5and Cy3(r=lnCy5/Cy3).Subsequently, the mean value (R) of all r value was obtained.All Cy3signal intensity value was corrected by product of Cy3and1/R to decrease systemic error of Cy5and Cy3fluorescently labeled system. All corrected Cy3and Cy5signal intensity values lessed200were substituted with200. The corrected Cy3and Cy5signal intensity values were uploaded to BRB Array Tools4.1.0Beta2software to analyze. Intensity filers excluded the spot in which both intensities are below200.If only one intensity was below200, this intensity was increased to200. A log base2transformation was applied to intensities before the arrays were normalized by using median over entire array. Gene filters were used to exclude corresponding spots for an entire gene from all arrays. Exclude a gene under any of the following conditions: minimum fold-change less than20%of expression data values have at least a1.5-fold change in either direction from the gene's median value and percent missing exceeded50%. Filtered data were annonated and further analyzed.
②GO and KEGG gene sets analysis
Filtered data were used to analysis of GO and KEGG. Healthy volunteers and patients of splenogastric hygropyrexia syndrome were acted as contrast groups, and splenic asthenia patients as experiment groups (In Hp analysis, contrast and experiment groups were exchanged).Statistics analysis were performed by paired t-test, LS/KS permutation test, and Efron-Tibshirani's GSA maxmean test,and P<0.005.
③Class comparison of between groups of arrays
Filtered data were used to class comparison between groups of arrays, which was used to identify differentially expressed gene among classes of samples.The patients of splenogastric hygropyrexia syndrome, healthy volunteer and Hp-uninfected splenasthenic syndrome were used for the baseline gene expression. Statistics analysis was performed by paired t-test with random variance model and the found gene list was determined by significance at0.05,0.01and0.001levels of univariate tests, respectively. Negatively reciprocal transformation was done for the genes with fold change of expression less than1. The genes which matched p<0.01or0.05or0.001and fold change exceeded2were defined as differentially expressed genes.UP-regulated and down-regulated genes were signed as "↑" and "↓"
④efinition and analysis of genes related to substrance and energy metabolism
The differentially expressed genes containing the gene identifiers along with the corresponding fold changes were uploaded into the Ingenuity Pathways Analysis (IPA) website (version8.6)(www.ingenuity.com) to annotate bio-functions, construct and visualize molecular interaction networks. For only splenasthenic syndrome, the metabolism genes were definited by GO annonation and KEGG analysis of BRB Array Tools, biofunction classification and annonation of IPA website software to approach the correlation of splenasthenic syndrome and metabolism.For differentially expressed genes of Hp-uninfected splenasthenic syndrome,to approach pathogenic mechanism of Hp
infection and provided theoretical basis for classification of syndrome of Chinese medicine and therapia of Hp-related diseases.
⑤Clustering analysis of differentially expressed genes related metabolism
Hierarchical clustering of genes and samples for differentially expressed genes were performed by BRB ArrayTools software.Analysis settings included center the genes, one minus correlation metric for genes or centered correlation for samples, and average linkage.
(2) The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic syndrome, peoples of syndrome or constitution of deficiency of QI and rat models of splenasthenic syndrome
①The similar comparison of genes of substance and energy metabolism from microarray data of between two patients of splenasthenic syndrome
Differentially expressed genes related metabolism from patients of splenasthenic syndrome-healthy volunteers and that of splenasthenic-splenogastric hygropyrexia syndrome were compared to analyze characteristic of gene expression profiling for splenasthenic syndrome, and filted its characteristic genes and pathways, and further approach the relationship of splenasthenic syndrome and metabolism.
②The similar comparison of genes of substance and energy metabolism from patients of splenasthenic syndrome and that of peoples of syndrome or constitution of deficiency of QI
Differentially expressed genes from patients of syndrome or constitution of deficiency of QI were collected from publishing paper, and differentially expressed genes related to metabolism were definited according to above mentioned and selected to contrasting anlysis with that of patients of splenasthenic syndrome.The differentance of them were analyzed to decide that metabolism abnormality existed in only splenasthenic syndrome or all syndrome of deficiency of QI.
③The similar comparison of genes of substance and energy metabolism from patients of splenasthenic syndrome and that of rat models of splenasthenic syndrome
Differentially expressed genes from rat models of splenasthenic syndrome were collected from publishing paper, and were analyzed according to above mentioned method
③to approach characteristic metabolic genes and pathways of splenasthenic syndrome.
(3)Fluorescent quantitation PCR verification of differentially expressed genes from splenasthenic-splenogastric hygropyrexia syndrome
1)Gastric mucosa collection
Four patients who meet criteria of chronic gastritis, splenasthenic syndrome and Hp-uninfection, and4patients who meet criteria of chronic gastritis, splenogastric hygropyrexia syndrome and Hp-infection were collected in Guangdong province hospital of Chinese medicine.Their gastric mucosas were taken200mg in gastroscope, and were preserved in liquid nitrogen.
(3)Fluorescent quantitation PCR of differentially expressed genes
HLA-DRB1,SCGN, COX7B and SULT1A4, which were from differentially expressed genes of Hp-uninfected splenasthenic syndrome-and Hp-infected splenogastric hygropyrexia syndrome and the latter three genes were also differentially expressed gene of splenasthenic-splenogastric hygropyrexia syndrome, were used to quantitation PCR experiment by the method of SYBR Green I.The primers were designed using primer express2.0software.Total RNA extraction and fluorescent quantitation PCR were performed according to direction of kits.Relative amount of experiment and control group was calculated by the method of△△Ct.
Results
1.Differentially expressed genes related to substrance and energy metabolism from the patients of splenasthenic syndrome
(1)Differentially expressed genes from patients of splenasthenic syndrome-healthy volunteers
①287gene sets including49cellular components,60molecular functions and178biological processes were obtained by GO analysis and11pathways were obtained by KEGG analysis.GO biological process and KEGG pathway showed genes related to metabolism of lipid, protein, nucleic acid, carbohydrate and trace element were differentially expressed in the patients of splenasthenic syndrome;②Fifteen differentially expressed genes related to metabolism were obtained by class comparison between groups of arrays, they occupied75%(15/20) of total differentially expressed genes,14genes were down-regulated and11gene productions have enzyme activities,they participated in the metabolism process related to lipid, protein, nucleic acid and carbohydrate.ACAA2↓and CYP20A1↓were participated in lipid metabolism, and showed fatty acid degradation and cholesterol metabolic transformation decreased;RPS28↓,UBXN1↓,UBE2D2↓,ASL↓, ASS1↓,PCYOX1L↓,ALDH9A1↓,B3GNT1↓,GCNT1↓and PPP1R3C↓were participated in protein metabolism, and showed that protein biosynthesis and modification of glycosylation and phosphorylation decreased,protein ubiquitination disturbed, amino acid metabolism related in urea cycle and autonomic nerve decreased, protein; RMI1↓, SMARCD3↓and PARP1↑were participated in nucleic acid metabolism, and showed that DNA duplication and transcription decreased, DNA damage repair increased; B3GNT11↓, GCNT1↓and PPP1R3C↓were participated in carbohydrate metabolism, and showed that biosynthesis of glycan and glycogen decreased.③Clustering analysis of differentially expressed genes related metabolism showed patients of splenasthenic syndrome and healthy volunteers could not be separated.
(2) Differentially expressed genes from patients of splenasthenic-splenogastric hygropyrexia syndrome
①373gene sets including58cellular components,59molecular functions and256biological processes were obtained by GO analysis.These GO biological process included metabolism of lipid, protein, nucleic acid, carbohydrate, trace element and energy. Two pathways obtained by KEGG analysis were ribosome and lactoflavin metabolism.② Fifty-six differentially expressed genes related to metabolism were obtained by class comparison between groups of arrays, they occupied71%(56/79) of total differentially expressed genes,45genes were down-regulated and30gene productions have enzyme activities, they participated in the metabolism process related to lipid, protein, nucleic acid, carbohydrate, trace element and energy metabolism.ACADVL↓, LRP11↑,SULT1A4↓, CRLS1↑,GPCPD1↓, PIGL↓, B3GNT1↓, ST8SIA4↓and FUT9↑were participated in lipid metabplism, and showed that (3-oxidation of fatty acid cut down, cholesterol intake increased and its metabolic transformation decreased, phospholipid and glycolipid metabolism were abnormal;ASRGL1↓, AARSD1↓,EBNA1BP2↓, PUM2↑, MRPL52↓, C120RF65↓, PSMB8↓, PSME2↓, UBA7↓, RNF11↑,FBXO44↓,ZFYVE26↓,CHMP2A↓, SSR4↓,SNX4↑,RAB3B↓,RABL2A↓,GOLGA2↓, KDELR1↓,PHPT1↓,ACPP↓,PTPRF↓, CRKL↓,HDAC7↓,ADPRHL2↓,B3GNT1↓, ST8SIA4↓,DDOST↓and FUT9↑were participated in protein metabolism, and showed that protein biosynthesis, ubiquitination and targeted transport decreased, posttranslation modification of phosphorylation and glycosylation decreased;TOP2A↓, SF3A3↓,CREB3↓,CRTC2↓,NR1D2↑, MED6↓, GTF2IRD1↓, C1ORF83↓, ZNF773↓,ZMYND11↑,DFFB↓,FLJ35220↓and ADPRHL2↓, were participated in nucleic acid metabolism, and showed that DNA duplication and transcription decreased, DNA damage repair increased; AGL↑, PTPRF↓, B3GNT1↓, FUT9↑,ST8SIA4↓,SULT1A4↓,DDOST↓and PIGL↓were participated in carbohydrate metabolism, and showed that glycogen degradation increased and glycoconjugate biosynthesis decreased;COMMD1↓,FTL↓,SLC39A6↑,CHRFAM7A↓,SCGN↑and S100A6↓were participated in trace metabolism, and showed copper and ferri ion metabolism decreased, zinc ion metabolism increased, calcium ion metabolism were abnormal;AK3↓and COX7B↓were participated in energy metabolism, and showed energy metabolism blocked.③Clustering analysis of differentially expressed genes related metabolism showed patients of splenasthenic syndrome and those of splenogastric hygropyrexia syndrome could be separated.
(3)Differentially expressed genes from patients of Hp-uninfected splenasthenic-Hp-infected splenogastric hygropyrexia syndrome
①703gene sets including114cellular components,114molecular functions and475biological processes were obtained by GO analysis and29pathways obtained by KEGG analysis.The GO biological process and KEGG pathway showed that Hp infection caused host differential expression of genes related to protein metabolism, signal pathway, immunity and inflammation reaction, cystoskeleton etc.②Thirty-four annonated differentially expressed genes which obtained by class comparison between groups of arrays, were participated in protein metabolism, inflammatory and immunological reaction, signal transduction, gene transcription, trace element metabolism, and so on.The82%of these genes (28/34) were categorized in three molecular interaction networks involved in gene expression, cancer progress, antigen presentation and inflammatory response.Taken together, these data indicated that Hp infection could alter cellular gene expression processes, escape host defense mechanism, increase inflammatory and immune responses, activate NF-κB and Wnt/β-catenin signaling pathway, disturb metal ion homeostasis,and induce carcinogenesis.③Clustering analysis of differentially expressed genes showed patients of Hp-uninfected splenasthenic syndrome and those of Hp-infected splenogastric hygropyrexia syndrome could be separated.
2.The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic syndrome, peoples of syndrome or constitution of deficiency of Ql and rat models of splenasthenic syndrome
(1)The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic-splenogastric hygropyrexia syndrome and patients of splenasthenic syndrome-healthy volunteer
①The GO biological process from the two microarray data showed genes related to metabolism of lipid, protein, nucleic acid, carbohydrate and trace element were differentially expressed in the patients of splenasthenic syndrome;KEGG pathway from the two microarray data showed that genes related to protein metabolism were differentially expressed.
②The results of class comparison between groups of arrays showed, differentially expressed genes related to substrance and energy metabolism from patients of splenasthenic-splenogastric hygropyrexia syndrome and patients of splenasthenic syndrome-healthy volunteer showed obviously difference.The dissimilarity was the numbers and metabolic process of differentially expressed genes.The former was more than the latter, metabolic processes included lipid, protein, nucleic acid, carbohydrate, trace element and energy. The latter was participated in lipid, protein, nucleic acid and carbohydrate metabolism. Moreover, differentially expressed gene name was also difference.The similarity was followed as (1) Many genes related to substrance and energy metabolism were differentially expressed in the two microarray data, and they occupated over70%of total differentially expressed genes, and most of these genes were enzyme genes or enzyme activity gene, and their expressions were down-regulated in patients of splenasthenic syndrome;(2)B3GNT1expression was down-regulated in the two microarray data;(3) In the metabolic pathway, the results of two microarray data from patients of splenasthenic syndrome showed cholesterol metabolic transformation and β-oxidation of fatty acid decreased, ribosome assembly blocked, protein modification of phosphorylation and glycosylation decreased, protein ubiquitation decreased, DNA duplication and transcription decreased,DNA damage repair increased, tissue glycogen biosyntheis decreased and degradation increased, and glycan biosynthesis decreased.
③Clustering analysis of differentially expressed genes related metabolism showed patients of splenasthenic syndrome and those of splenogastric hygropyrexia syndrome could be separated, while patients of splenasthenic syndrome and healthy volunteers could not be separated.
(2) The similar comparison of differentially expressed genes related to metabolism from patients of splenasthenic syndrome and syndrome or constitution of deficiency of QI
Differentially expressed genes related to substrance and energy metabolism from patients of splenasthenic syndrome and syndrome or constitution of deficiency of QI showed obviously difference.(1)Differentially expressed genes related to metabolism from patients of splenasthenic syndrome were similar to that of constitution of deficiency of QI in expression tendency and metabolic pathway, and both existed metabolic block, but patients of splenasthenic syndrome more obvious.For these results, patients of other syndrome or constitution of deficiency of QI had completely different results;(2) ST, RAB and S100might be mark genes of splenasthenic syndrome, while RP, PTP and UBE2might be mark genes of syndrome of deficiency of QI;(3)In metabolic pathway, differentially expressed genes related to lipid, carbohydrate, protein and nucleic acid metabolism from patients of splenasthenic syndrome were down-regulated, while that of syndrome or constitution of deficiency of QI were up-regulated and participated in less metabolic process;(4)Protein ubiqutination abnormality was commonness of syndrome of deficiency of QI,but splenasthenic syndrome and constitution of deficiency of QI decreased, other syndrome or constitution of deficiency of QI increased;(5) DNA damage might be common pathological phenomenon of syndrome and constitution of deficiency of QI.
3.The similar comparison of differentially expressed genes related to metabolism from patients and animal models of splenasthenic syndrome
Differentially expressed genes related to metabolism from patients and animal models of splenasthenic syndrome had obvious differentence in numbers and expression tendency. But thirteen differentially expressed genes were repeatedly appeared in microarray results of patients and animal models of splenasthenic syndrome, they were B3GNT1,CYP, SLC39A, SNX, FUT, SLC2A, GCNT, ACAD, LDH, RP, PPP1, S100and PTPR. That RP and PTPR were down-regulated also appeared in syndrome of deficiency of QI;(1)Except for RP, all these genes production were enzyme and transporter;(2) These genes were participated in lipid, carbohydrate, protein and tace element metabolism, and showed that glucose transport and degradation, glycogen synthesis and degradation, and glycan synthesis, were disordered;Fatty acid and cholesterol metabolism were abnormal; Ribosome assembly blocked, protein modification of phosphorylation decreased and that of glycosylation was abnormal, and targeted transport increased; Zinc ion metabolism increased and calcium ion metabolism increased.
3.Fluorescent quantitation PCR verification of differentially expressed genes from splenasthenic-splenogastric hygropyrexia syndrome
The results of fluorescent quantitation PCR for HLA-DRB1,SCGN,COX7B and SULT1A4, which were from differentially expressed genes of Hp-uninfected splenasthenic syndrome-and Hp-infected splenogastric hygropyrexia syndrome and the latter three genes were also differentially expressed gene of splenasthenic-splenogastric hygropyrexia syndrome,and that of microarray experiment were similar, but the ratio of the former was lower than the latter. ACAA2of differentially expressed genes from splenasthenic syndrome-healthy volunteers was also verified by our research groups previously.
Conclusions
1.It was different to other syndrome of deficiency of QI that the patients of splenasthenic syndrome showed nutrition dysmetabolism by down-regulation of metabolic genes, especially enzyme genes.Dysmetabolism showed that cholesterol metabolic transformation and β-oxidation of fatty acid decreased, ribosome assembly was blocked, protein modification of phosphorylation and glycosylation decreased, protein ubiquitation decreased, DNA duplication and transcription decreased, DNA damage repair increased, tissue glycogen biosyntheis decreased and degradation increased, and glycan biosynthesis decreased. All of these results might be the one of important reason causing nutrition dysmetabolism of splenasthenic syndrome.All these results acted out macro-metabolism sketch of patients of splenasthenic syndrome, and supported discussion related to metabolism for pathophysiology of spleen in Neijing, which provided some clues for modern research and annotation of scientic essence of spleen picture.At the same time, the macro-metabolism sketch of patients of splenasthenic syndrome well revealed molecular pathophysiology background of spleen, which provided theoretical basis and academic guide for metabolic disease research based on the treatment from spleen.
2.The differentially expressed genes related to metabolism from analysis of gene microarray data and the determination results of biochemical indicator related to metabolism from literature report were confirmed each other and revealed pathophysiology of spleen mainly included digest and absorb,and metabolism of substance.
3.The B3GNT1,ST, RAB,CYP, SLC39A, SNX, FUT, SLC2A, GCNT, ACAD, LDH, RP, PPP1, S100and PTPR, might be closely related to the occurrence of splenasthenic syndrome, while RP, PTP and UBE2might be closely related to the occurrence of syndrome of deficiency of QI.All of these genes provided some clues for research of splenasthenic syndrome.
4.The analysis of gene expression profiling showed the clinical manifestation between splenasthenic and splenogastric hygropyrexia syndrome was more obvious, while between splenasthenic syndrome and healthy volunteers was not obvious,which provided theoretical basis for classification of cases of disease in Chinese medicine.
5.Hp infection could affect host on protein metabolism, inflammatory and immunological reaction, signal transduction, gene transcription, trace element metabolism, and so on, which provided theoretical basis for classification of syndrome of Chinese medicine and therapia of Hp-related diseases.
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