HMGCS2、PPARγ在炎症性肠病患者肠粘膜的表达与活性
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摘要
研究背景与目的
溃疡性结肠炎(ulcerative colitis, UC)是一种慢性非特异性结肠炎症,病变主要累计结肠粘膜和粘膜下层,范围多自结肠远端开始。UC和克罗恩病(Crohn's disease, CD)均属炎症性肠病(inflammatory bowel disease, IBD)之范畴。
过去欧洲和北美人群中IBD发病率较高,亚洲人群发病率较低,然而近年来IBD在亚洲人群中的发病呈现明显增高趋势.IBD的发病机制仍然不甚清楚,目前研究认为可能与遗传易感人群在持续肠道感染、肠黏膜屏障缺陷和环境改变等多因素作用下引起的肠道免疫系统异常反应相关。
国内队列研究显示,吸烟、阑尾切除、肠道感染影响IBD的发生；饮茶、母乳喂养为UC的保护因素；高学历与城市人群中IBD较多；副结核分枝杆菌感染可能与CD有关。近年来研究表明肠黏膜屏障功能异常与IBD发病关系密切.肠腔表面积很大,由上皮细胞紧密连接而成,长期暴露于各种食物成分、共生菌及病原菌.肠道上皮有重要的屏障功能,在多数情况下,上皮细胞对各种损伤稳定应答,炎症处于限制状态.IBD发生时,肠黏膜屏障功能障碍,黏膜通透性增高,导致肠腔内细菌、抗原等物质移位至黏膜固有层而激活免疫细胞,诱导黏膜过度免疫反应的发生,继而进一步破坏肠黏膜屏障,加重黏膜异常免疫反应,从而导致IBD的发生。
一般情况下UC和CD可以根据临床表现、实验室和放射学检查、内镜和组织学特征综合分析鉴别并不困难。对于结肠炎症性肠病一时难以区别UC与CD者,临床可以诊断为IBD类型待定(type unclassified,IBDU),观察病情变化,但是如果结肠广泛病变,并且内镜、病理表现不典型时,UC和CD有时难以鉴别；如果IBD内镜下炎症表现较轻,无典型内镜下改变,还需要与肠道感染性疾病相鉴别。CD、肠结核(intestinal tuberculosis, ITB)、肠道淋巴瘤(intestinal lymphoma, IL)、肠型白塞氏病(intestinal Beheet's disease, BD)因为内镜下存在诸多相似的地方,这几个疾病之间的鉴别也是临床的一个难题。
目前,临床上尚未有简单易行的实验室方法来鉴别诊断IBD：血常规、血浆蛋白、电解质、ESR、C-反应蛋白,有条件的单位也可以做粪便钙卫蛋白、乳铁蛋白、α1抗胰蛋白酶等检查,但是这些检查的都无特异性。影像学检查包括胃肠钡剂造影、腹部超声、CT、MRI等对IBD的诊断有所帮助。
我们课题组前期运用蛋白质组学技术,筛选出UC与正常人肠道黏膜差异蛋白若干,其中就包括3-羟基-3-甲基戊二酰辅酶A合成酶2(3-hydroxy-3-methylglutaryl-CoA synthase 2, HMGCS2)
PPARr是一种细胞核转录因子,即过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor, PPAR)的一员,根据结构的不同,PPAR可分为α、β(或δ)和γ三种类型,其基因分别位于人类22,6和3号染色体上,通常PPARα高表达具有丰富线粒体和β氧化活性的组织,如肝、肠粘膜、肾脏皮质和心脏,而在一些其它组织也发现了PPARα的低水平表达；PPARγ则于脂肪组织、膀胱及肠道中有高水平表达；而PPARβ几乎在所有组织中均有低水平的表达。PPARγ通过调节相关基因的表达,在脂肪形成、糖脂代谢,以及在免疫系统中发挥重要作用,并与多种疾病如糖尿病、肥胖、高血压、脑缺血、帕金森病、肾脏疾病、癌症等的发生、发展有关。目前国内外有关PPARγ与IBD的动物实验研究有一定的报道,绝大部分研究结果支持PPARγ是IBD的保护因子,但也有持有相反的观点,认为预先用PPARγ激动剂处理过的动物模型后会加重动物的肠道炎症程度,引起更多的溃疡、腺管丧失和水肿。虽然体外动物实验有不少关于PPARγ与IBD的报道,但是目前仅有少量实验评价了PPARγ受体在UC和CD患者中的表达及作用。
HMGCS2即3-羟基-3-甲基戊二酰辅酶A合成酶2,调节酮体的生成的关键酶,在肝脏组织及有些肝外组织如骨骼肌,心脏,胰腺、睾丸、结肠中均有表达,HMGCS2在这些组织中表达的作用有待于进一步阐明。然而在组织中,HMGCS2可以氧化脂肪酸,HMGCS2表达可以阻止乙酰辅酶A的沉积,反过来又可以减弱脂肪酸的氧化速率。结肠的生酮作用与HMGCS2表达有关,后者取决于肠道生成的丁酸盐量,在健康的肠上皮细胞,丁酸盐刺激细胞的增殖,然而,在该组织中的肿瘤细胞系它又可以减少增殖和诱导细胞分化和凋亡,国外有学者提出用于解释“丁酸盐矛盾”,该假说认为：健康的肠粘膜有效地分解丁酸盐,从而导致细胞内丁酸盐浓度的下降,因而阻止增殖的能力下降,在结肠癌细胞系中分解丁酸盐的能力缺失,在这种背景下有学者提出HMGCS2的诱导缺乏导致了脂肪酸的beta氧化减弱。丁酸盐是结肠粘膜的主要燃料来源,而且有证据显示在溃疡性结肠炎中氧化受损,在活动期和静止期的UC中丁酸盐氧化生成C02和酮体的量明显低于健康对照组,并且这种减少与疾病的状态有关。丁酸盐的氧化缺陷反映了UC患者的可变又明确的代谢缺陷,丁酸盐的氧化减少可以解释在结肠炎症的分布特点,特别是结肠末端的发病频率。一般认为脂肪酸(n-丁酸盐)的氧化缺陷是结肠粘膜能量缺乏的一种表现。HMGCS2的诱导缺乏导致了脂肪酸(n-丁酸盐)的beta氧化减弱,UC患者存在丁酸盐氧化缺陷,由于丁酸盐氧化生成酮体和C02,我们设想作为酮体生成的关键酶HMGCS2在UC患者粘膜中也存在下调。HMGCS2在UC、CD患者肠粘膜中的表达及其作用至今没有什么研究报道。
因此,本次实验的目的是希望通过对UC、CD及正常健康对照组中进行HMGCS2、PPARγ分子标志的系统检测,观察相对完整的HMGCS2、PPARγ在这两种疾病中的表达情况,从而推测：①它们是否能作为鉴别UC、CD的标志物；②它们是否与肠道能量代谢障碍及菌群失调相关；③通过对UC、CD及健康对照组比较,分析其IBD发生发展的分子学基础。
材料和方法
1.完全随机收集73例UC、42例CD、89例健康对照者的肠粘膜,比较UC组和CD组之间一般临床特点和检查资料的差异,包括患者性别、年龄、病变累及部位、活动分期、临床分度、内镜检查特点、发病时间等。
2.用免疫组织化学染色技术观察HMGCS2、PPARγ在上述各组的表达情况.。
3.统计分析：采用SPSS 13.0统计软件进行分析,用Independent-Samples T Test比较测定组和组之间患者年龄的差异；用R*C表资料的X2检验比较各组间蛋白表达阳性率的差异性；利用两独立样本非参数检验、多个独立样本非参数检验、Mann-Whitney U test比较各组之间性别、年龄、病变部位、分期、临床分度、内镜分级等与蛋白染色表达的差异,所有分析结果取双侧P值,P<0.05认为差异具有统计学意义。
结果：
1.73例UC中其中男46例.女27例,男：女=1.70：1,年龄13—77岁,平均年龄为(40.07±14.39)岁,发病的高峰年龄是30-49岁,其中发生在直肠的28例(38.4%),发生在直乙状结肠9例(12.3%),发生在全结肠的25例(34.2%).42例CD患者有29例发生在回末和小肠(69.0%)。
2.HMGCS2、PPARγ在73例UC患者肠粘膜中阳性表达分别为48例(65.8%)和36例(49.3%)；在42例CD患者肠粘膜中阳性表达分别为37例(88.1%)和30例(71.4%)；在89例正常对照组肠粘膜阳性表达占82例(92.1%)和66例(74.2%)。HMGCS2、PPARγ在UC患者中肠粘膜与正常健康对照组或CD患者肠粘膜比较明显下降(P<0.01),但是在CD患者及健康对照组肠粘膜的HMGCS2、PPARγ表达没有显著性差异(HMGCS2:P=0.521、PPARγ::P=0.742)。
3. HMGCS2、PPARγ在UC活动期患者的肠粘膜表达显著小于缓解期肠粘膜的表达,差异具有统计学意义(HMGCS2:P=0.002、PPARy:P=0.008)。
4. HMGCS2、PPARγ在UC患者肠粘膜的表达与患者性别、年龄、发病部位、临床分度等无明显相关性。PPARγ蛋白的表达与UC患者的内镜下分级无明显相关性,但是HMGCS2蛋白的表达与内镜分级有关,内镜分级较低也就是疾病炎症较轻时该蛋白表达相对较高。
结论：
本研究发现HMGCS2、PPARγ不是IBD特异性蛋白指标,也存在于健康对照组等肠粘膜中,对于鉴别UC、CD具有一定的意义,有可能作为UC严重程度的一个指标。HMGCS是酮体生成的关键酶,PPARγ除参与炎症因子和炎症相关转录因子的调节作用外,还有可能与HMGCS类似通过丁酸盐与肠道能量代谢及肠道菌群失调而起作用参与IBD的发生发展。
Background and aims
Ulcerative colitis (ulcerative colitis, UC) is a chronic non-specific colitis, lesions were accumulated in mucosa and submucosa of colon, lesions often start with remote colon. UC and Crohn's disease (Crohn's disease, CD) are area of inflammatory bowel disease (inflammatory bowel disease, IBD)
Europe and North America over the past presented higher incidence of IBD population, the Asian population incidence is relatively low, but in recent years in the Asian population in IBD showed significantly higher incidence trends. IBD pathogenesis is still not very clear, current studies suggest that may be related to genetic susceptibility and populations in intestinal infections, and intestinal mucosa barrier defect under the effect of environmental factors such as changes caused by abnormal intestinal immune system response.
The domestic formation studies demonstrated that smoking, the appendix excision, the enteral infection affect the occurrence of IBD; Drinks the tea, breast feeding is the protection factor of UC; highly educated and urban populations are more IBD; Vice Mycobacterium tuberculosis infection may be related with the CD. Recent studies show that intestinal barrier dysfunction was exceptionally close with the IBD morbidity. The enteric cavity surface area is very big, becomes by the epithelial cell zonula occludens, exposes for a long time in each kind of dietary ingredient the paragenesis fungus and the disease germ. The intestinal tract epidermis has the important barrier function, In the most situations, the epithelial cell to each kind of damage stable reply, the inflammation is at the limit condition. When IBD occurs, the intestines mucous membrane barrier dysfunction, the mucous membrane permeability advances, causes in the enteric cavity to activate the immune cell material shifting and so on bacterium、antigen to the mucous membrane inherent level, the induction mucous membrane excessive immune response occurrence, subsequently further destroys the intestines mucous membrane barrier, the aggravation mucous membrane unusual immune response, thus causes the occurrence of IBD.
UC and CD under normal circumstances can be the basis of clinical manifestations, laboratory and radiological, endoscopic and histological features of a comprehensive analysis is not difficult to identify. Distinguishes UC and CD with difficulty for a while regarding the colon inflammation enteropathy, clinical may diagnose is the IBD type undetermined (type unclassified, IBDU), observe the patient's condition. However, if extensive colon lesions, and endoscopic and pathologic findings are not typical, sometimes UC and CD with difficulty distinguish; If the IBD endoscopic inflammation is light, without typical endoscopic changes, but also need to be differentiated from intestinal infectious diseases. CD、intestinal tuberculosis.intestinal lymphoma.intestinal Behcet's disease because there are many similar endoscopic places, these diseases Between the clinical identification is a problem
Currently, the clinical laboratory yet simple approach to the differential diagnosis of IBD:blood, plasma proteins, electrolytes, ESR, C-reactive protein, the unit can also do conditional fecal calprotectin, lactoferrin, al antitrypsin and other tests, but these tests are nonspecific. Imaging studies, including gastrointestinal barium, abdominal ultrasound, CT, MRI and other help for the diagnosis of IBD.
Our group use proteomics technology to screen out the differences between UC and normal intestinal mucosa of several proteins, including 3-hydroxy -3--methylglutaryl coenzyme A synthase 2.
PPARy is a research hotspot in recent years, is a nuclear transcription factor, that is a member of peroxisome proliferator-activated receptor, according to the different structure, PPAR can be divided intoα,β(orδ) and y are three types, the genes were located on human chromosome 22,6 and 3. Usually rich in mitochondria with high expression of PPARa andβ-oxidation activity of the organization, such as liver, intestinal mucosa, kidney cortex and heart, while in some other organizations also found a low level of expression of PPARa; PPARy is in adipose tissue, bladder and bowel in High expression; and PPARβin almost all organizations have a low level of expression. PPARy through the adjustment related gene's expression, playing an important role in the formation of fat, glucose and lipid metabolism, the immune system, and involvement of occurrence and development with a variety of diseases such as diabetes, obesity, hypertension, cerebral ischemia, Parkinson's disease, kidney disease, cancer. PPARy at home and abroad on animal studies with IBD have some reports, most studies support the protective factor PPARy is the IBD, but also hold the opposite view that the pre-treated with PPARy agonists in animal models will Increase the animal's intestinal inflammation caused more ulcers, glandular loss, and edema. Although many animal experiments in vitro with IBD reported on PPARy, but currently only a small amount of experimental evaluation of the PPARy receptor in patients with UC and CD expression and function.
HMGCS2,the 3-hydroxy-3-methylglutaryl coenzyme A synthase 2, the formation of ketone bodies regulating the key enzyme in the liver and some extrahepatic tissues such as skeletal muscle, heart, pancreas, testes, colon in both Expression, HMGCS2 expressed in the role of these organizations need to be further elucidated. However, in the organization, HMGCS2 can be oxidized fatty acids, HMGCS2 expression can prevent the deposition of acetyl coenzyme A, which in turn can be reduced fatty acid oxidation rate. Colon's ketogenesis and HMGCS2 express related, the latter depends on the gut-generated acid salt, in healthy intestinal epithelial cells, butyrate stimulation of cell proliferation, however, in the organization of the tumor cell lines it Reduce proliferation and induce cell differentiation and apoptosis. The overseas some scholars proposed that uses in explaining" the butyrate is contradictory", this hypothesis thought:The healthy intestines mucous membrane decomposes the butyrate effectively, thus causes in the cell the butyric acid salinity drop. thus prevents ability drop which multiplies, decomposes the butyrate in the colon cancer cell line anergia, under this situation some scholars proposed that the HMGCS2 induction caused the fatty acid beta oxidation to be weaken deficient. Colon butyrate is the main fuel source, and there is evidence that oxidative damage in ulcerative colitis, in active and quiescent UC CO2 in the oxidation of butyrate and ketone bodies was significantly lower than in healthy controls Group, and this decrease is related with the disease. The butyrate oxidized flaw had reflected the UC patients' invariable and clear metabolism flaw. reducing the oxidation of butyrate can be explained by the distribution in the colonic inflammation, in particular, the incidence of frequency end of the colon. Generally believed that the fatty acids (n-butyrate) oxidation defect is the lack of a colon energy performance. Because the lack of HMGCS2 induction, the beta oxidation of the fatty acid (the n- butyrate) is weaken. UC patients have defects in butyrate oxidation, due to butyrate oxidation of ketone bodies and CO2, we assume, as a key enzyme in ketone bodies generated in HMGCS2 mucosa of UC patients also reduced. HMGCS2 in UC, CD patients and its role in the expression of the intestinal mucosa so far no studies have reported.
Therefore, the purpose of this experiment is to by UC, CD and normal healthy control group were HMGCS2, PPARy system detects molecular markers to observe the relatively complete HMGCS2, PPARy in both the expression of disease, which suggested:(a) whether they can be as a maker to distinguish UC from CD (b) whether they are associated with Energy metabolization obstacle and dysbacteriosis; (c) through the UC, CD and healthy control group, the development of IBD analyzed the molecular basis.
Materials and Methods
1. A total of 204 colorectal specimens were collected randomly from individuals who underwent endoscopic resection under total colonoscopy. The specimens consisted of 73 case of UCs,42 CDs and 89 healthy controls.compared between the UC group and CD group general information on clinical features and check the differences, including sex, age, lesions involving the location, Activities staging, clinical indexing, Endoscopic features, time of onset etc.
2.1mmunohistochemical staining observed HMGCS2, PPARy in the expression of each group
3. Statistical analysis:SPSS 13.0 statistical software for analysis, with the Independent-Samples T Test Determination of the age group and differences between-groups of patients; R* C table using theχ2 test to compare group data between the positive expression rate; Using non-parametric test of two independent samples、K independent samples of non-parametric test and Mann-Whitney U test comparing the groups of gender, age, lesion site, stage, degree of clinical, endoscopic grading related with protein dyeing and expression differences All results obtained bilateral P value, P<0.05 considered significant difference.
Results
1.73 UC patients consisted of 46 males and 27 females, male:female= 1.70:1, age 13-77 years, mean age (40.07±14.39) years, peak age of onset is 30-49 years, of which 28 cases occurred in the rectum (38.4%),9 cases occurred in the rectum, sigmoid colon (12.3%), The whole colon in 25 patients (34.2%).42 cases of CD patients,29 cases occurred in the terminal ileum and small intestine (69.0%).
2. HMGCS2, PPARy in 73 patients with positive expression in intestinal mucosa of UC patients were 48 cases (65.8%) and 36 patients (49.3%); in 42 patients with positive expression in intestinal mucosa of CD patients were 37 cases (88.1%) and 30 patients (71.4%); in 89 cases of normal mucosa positive control group accounted for 82 cases (92.1%) and 66 patients (74.2%). HMGCS2, PPARy in the colonic mucosa in UC patients and normal healthy controls and CD patients significantly decreased compared intestinal mucosa, the difference was statistically significant (P<0.01), but in CD patients and healthy controls intestinal mucosa HMGCS2, PPARy expression was not Significant difference, not statistically significant (P-0.521 and P=0.742).
3. HMGCS2, PPARy in intestinal mucosa of patients with active UC was significantly less than the expression of intestinal mucosa in remission, the difference was statistically significant。(HMGCS2:P=0.002. PPARy:P=0.008)
4. HMGCS2, PPARy expression in the intestinal mucosa of UC patients with gender, age, disease location, clinical, no significant correlation was found. The expression of PPARy protein in patients with endoscopic grade UC was no significant correlation, but HMGCS2 protein expression and endoscopic grading, endoscopy is low grade inflammation in mild disease is relatively high when the protein.
Conclusions
The study found HMGCS2, PPARy is not IBD specific protein targets, but also in healthy controls and other mucosa, for the identification of UC, CD has a certain significance, may be used as an indicator of the severity of UC. HMGCS is a key enzyme in ketone body formation, PPARy addition to factors involved in inflammation and inflammation-related regulation of transcription factors, there may be similar with the HMGCS by butyrate and intestinal energy metabolism and the role of intestinal flora in IBD The occurrence and development
溃疡性结肠炎(ulcerative colitis, UC)是一种慢性非特异性结肠炎症,病变主要累计结肠粘膜和粘膜下层,范围多自结肠远端开始。UC和克罗恩病(Crohn's disease, CD)均属炎症性肠病(inflammatory bowel disease, IBD)之范畴。
过去欧洲和北美人群中IBD发病率较高,亚洲人群发病率较低,然而近年来IBD在亚洲人群中的发病呈现明显增高趋势.IBD的发病机制仍然不甚清楚,目前研究认为可能与遗传易感人群在持续肠道感染、肠黏膜屏障缺陷和环境改变等多因素作用下引起的肠道免疫系统异常反应相关。
国内队列研究显示,吸烟、阑尾切除、肠道感染影响IBD的发生；饮茶、母乳喂养为UC的保护因素；高学历与城市人群中IBD较多；副结核分枝杆菌感染可能与CD有关。近年来研究表明肠黏膜屏障功能异常与IBD发病关系密切.肠腔表面积很大,由上皮细胞紧密连接而成,长期暴露于各种食物成分、共生菌及病原菌.肠道上皮有重要的屏障功能,在多数情况下,上皮细胞对各种损伤稳定应答,炎症处于限制状态.IBD发生时,肠黏膜屏障功能障碍,黏膜通透性增高,导致肠腔内细菌、抗原等物质移位至黏膜固有层而激活免疫细胞,诱导黏膜过度免疫反应的发生,继而进一步破坏肠黏膜屏障,加重黏膜异常免疫反应,从而导致IBD的发生。
一般情况下UC和CD可以根据临床表现、实验室和放射学检查、内镜和组织学特征综合分析鉴别并不困难。对于结肠炎症性肠病一时难以区别UC与CD者,临床可以诊断为IBD类型待定(type unclassified,IBDU),观察病情变化,但是如果结肠广泛病变,并且内镜、病理表现不典型时,UC和CD有时难以鉴别；如果IBD内镜下炎症表现较轻,无典型内镜下改变,还需要与肠道感染性疾病相鉴别。CD、肠结核(intestinal tuberculosis, ITB)、肠道淋巴瘤(intestinal lymphoma, IL)、肠型白塞氏病(intestinal Beheet's disease, BD)因为内镜下存在诸多相似的地方,这几个疾病之间的鉴别也是临床的一个难题。
目前,临床上尚未有简单易行的实验室方法来鉴别诊断IBD：血常规、血浆蛋白、电解质、ESR、C-反应蛋白,有条件的单位也可以做粪便钙卫蛋白、乳铁蛋白、α1抗胰蛋白酶等检查,但是这些检查的都无特异性。影像学检查包括胃肠钡剂造影、腹部超声、CT、MRI等对IBD的诊断有所帮助。
我们课题组前期运用蛋白质组学技术,筛选出UC与正常人肠道黏膜差异蛋白若干,其中就包括3-羟基-3-甲基戊二酰辅酶A合成酶2(3-hydroxy-3-methylglutaryl-CoA synthase 2, HMGCS2)
PPARr是一种细胞核转录因子,即过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor, PPAR)的一员,根据结构的不同,PPAR可分为α、β(或δ)和γ三种类型,其基因分别位于人类22,6和3号染色体上,通常PPARα高表达具有丰富线粒体和β氧化活性的组织,如肝、肠粘膜、肾脏皮质和心脏,而在一些其它组织也发现了PPARα的低水平表达；PPARγ则于脂肪组织、膀胱及肠道中有高水平表达；而PPARβ几乎在所有组织中均有低水平的表达。PPARγ通过调节相关基因的表达,在脂肪形成、糖脂代谢,以及在免疫系统中发挥重要作用,并与多种疾病如糖尿病、肥胖、高血压、脑缺血、帕金森病、肾脏疾病、癌症等的发生、发展有关。目前国内外有关PPARγ与IBD的动物实验研究有一定的报道,绝大部分研究结果支持PPARγ是IBD的保护因子,但也有持有相反的观点,认为预先用PPARγ激动剂处理过的动物模型后会加重动物的肠道炎症程度,引起更多的溃疡、腺管丧失和水肿。虽然体外动物实验有不少关于PPARγ与IBD的报道,但是目前仅有少量实验评价了PPARγ受体在UC和CD患者中的表达及作用。
HMGCS2即3-羟基-3-甲基戊二酰辅酶A合成酶2,调节酮体的生成的关键酶,在肝脏组织及有些肝外组织如骨骼肌,心脏,胰腺、睾丸、结肠中均有表达,HMGCS2在这些组织中表达的作用有待于进一步阐明。然而在组织中,HMGCS2可以氧化脂肪酸,HMGCS2表达可以阻止乙酰辅酶A的沉积,反过来又可以减弱脂肪酸的氧化速率。结肠的生酮作用与HMGCS2表达有关,后者取决于肠道生成的丁酸盐量,在健康的肠上皮细胞,丁酸盐刺激细胞的增殖,然而,在该组织中的肿瘤细胞系它又可以减少增殖和诱导细胞分化和凋亡,国外有学者提出用于解释“丁酸盐矛盾”,该假说认为：健康的肠粘膜有效地分解丁酸盐,从而导致细胞内丁酸盐浓度的下降,因而阻止增殖的能力下降,在结肠癌细胞系中分解丁酸盐的能力缺失,在这种背景下有学者提出HMGCS2的诱导缺乏导致了脂肪酸的beta氧化减弱。丁酸盐是结肠粘膜的主要燃料来源,而且有证据显示在溃疡性结肠炎中氧化受损,在活动期和静止期的UC中丁酸盐氧化生成C02和酮体的量明显低于健康对照组,并且这种减少与疾病的状态有关。丁酸盐的氧化缺陷反映了UC患者的可变又明确的代谢缺陷,丁酸盐的氧化减少可以解释在结肠炎症的分布特点,特别是结肠末端的发病频率。一般认为脂肪酸(n-丁酸盐)的氧化缺陷是结肠粘膜能量缺乏的一种表现。HMGCS2的诱导缺乏导致了脂肪酸(n-丁酸盐)的beta氧化减弱,UC患者存在丁酸盐氧化缺陷,由于丁酸盐氧化生成酮体和C02,我们设想作为酮体生成的关键酶HMGCS2在UC患者粘膜中也存在下调。HMGCS2在UC、CD患者肠粘膜中的表达及其作用至今没有什么研究报道。
因此,本次实验的目的是希望通过对UC、CD及正常健康对照组中进行HMGCS2、PPARγ分子标志的系统检测,观察相对完整的HMGCS2、PPARγ在这两种疾病中的表达情况,从而推测：①它们是否能作为鉴别UC、CD的标志物；②它们是否与肠道能量代谢障碍及菌群失调相关；③通过对UC、CD及健康对照组比较,分析其IBD发生发展的分子学基础。
材料和方法
1.完全随机收集73例UC、42例CD、89例健康对照者的肠粘膜,比较UC组和CD组之间一般临床特点和检查资料的差异,包括患者性别、年龄、病变累及部位、活动分期、临床分度、内镜检查特点、发病时间等。
2.用免疫组织化学染色技术观察HMGCS2、PPARγ在上述各组的表达情况.。
3.统计分析：采用SPSS 13.0统计软件进行分析,用Independent-Samples T Test比较测定组和组之间患者年龄的差异；用R*C表资料的X2检验比较各组间蛋白表达阳性率的差异性；利用两独立样本非参数检验、多个独立样本非参数检验、Mann-Whitney U test比较各组之间性别、年龄、病变部位、分期、临床分度、内镜分级等与蛋白染色表达的差异,所有分析结果取双侧P值,P<0.05认为差异具有统计学意义。
结果：
1.73例UC中其中男46例.女27例,男：女=1.70：1,年龄13—77岁,平均年龄为(40.07±14.39)岁,发病的高峰年龄是30-49岁,其中发生在直肠的28例(38.4%),发生在直乙状结肠9例(12.3%),发生在全结肠的25例(34.2%).42例CD患者有29例发生在回末和小肠(69.0%)。
2.HMGCS2、PPARγ在73例UC患者肠粘膜中阳性表达分别为48例(65.8%)和36例(49.3%)；在42例CD患者肠粘膜中阳性表达分别为37例(88.1%)和30例(71.4%)；在89例正常对照组肠粘膜阳性表达占82例(92.1%)和66例(74.2%)。HMGCS2、PPARγ在UC患者中肠粘膜与正常健康对照组或CD患者肠粘膜比较明显下降(P<0.01),但是在CD患者及健康对照组肠粘膜的HMGCS2、PPARγ表达没有显著性差异(HMGCS2:P=0.521、PPARγ::P=0.742)。
3. HMGCS2、PPARγ在UC活动期患者的肠粘膜表达显著小于缓解期肠粘膜的表达,差异具有统计学意义(HMGCS2:P=0.002、PPARy:P=0.008)。
4. HMGCS2、PPARγ在UC患者肠粘膜的表达与患者性别、年龄、发病部位、临床分度等无明显相关性。PPARγ蛋白的表达与UC患者的内镜下分级无明显相关性,但是HMGCS2蛋白的表达与内镜分级有关,内镜分级较低也就是疾病炎症较轻时该蛋白表达相对较高。
结论：
本研究发现HMGCS2、PPARγ不是IBD特异性蛋白指标,也存在于健康对照组等肠粘膜中,对于鉴别UC、CD具有一定的意义,有可能作为UC严重程度的一个指标。HMGCS是酮体生成的关键酶,PPARγ除参与炎症因子和炎症相关转录因子的调节作用外,还有可能与HMGCS类似通过丁酸盐与肠道能量代谢及肠道菌群失调而起作用参与IBD的发生发展。
Background and aims
Ulcerative colitis (ulcerative colitis, UC) is a chronic non-specific colitis, lesions were accumulated in mucosa and submucosa of colon, lesions often start with remote colon. UC and Crohn's disease (Crohn's disease, CD) are area of inflammatory bowel disease (inflammatory bowel disease, IBD)
Europe and North America over the past presented higher incidence of IBD population, the Asian population incidence is relatively low, but in recent years in the Asian population in IBD showed significantly higher incidence trends. IBD pathogenesis is still not very clear, current studies suggest that may be related to genetic susceptibility and populations in intestinal infections, and intestinal mucosa barrier defect under the effect of environmental factors such as changes caused by abnormal intestinal immune system response.
The domestic formation studies demonstrated that smoking, the appendix excision, the enteral infection affect the occurrence of IBD; Drinks the tea, breast feeding is the protection factor of UC; highly educated and urban populations are more IBD; Vice Mycobacterium tuberculosis infection may be related with the CD. Recent studies show that intestinal barrier dysfunction was exceptionally close with the IBD morbidity. The enteric cavity surface area is very big, becomes by the epithelial cell zonula occludens, exposes for a long time in each kind of dietary ingredient the paragenesis fungus and the disease germ. The intestinal tract epidermis has the important barrier function, In the most situations, the epithelial cell to each kind of damage stable reply, the inflammation is at the limit condition. When IBD occurs, the intestines mucous membrane barrier dysfunction, the mucous membrane permeability advances, causes in the enteric cavity to activate the immune cell material shifting and so on bacterium、antigen to the mucous membrane inherent level, the induction mucous membrane excessive immune response occurrence, subsequently further destroys the intestines mucous membrane barrier, the aggravation mucous membrane unusual immune response, thus causes the occurrence of IBD.
UC and CD under normal circumstances can be the basis of clinical manifestations, laboratory and radiological, endoscopic and histological features of a comprehensive analysis is not difficult to identify. Distinguishes UC and CD with difficulty for a while regarding the colon inflammation enteropathy, clinical may diagnose is the IBD type undetermined (type unclassified, IBDU), observe the patient's condition. However, if extensive colon lesions, and endoscopic and pathologic findings are not typical, sometimes UC and CD with difficulty distinguish; If the IBD endoscopic inflammation is light, without typical endoscopic changes, but also need to be differentiated from intestinal infectious diseases. CD、intestinal tuberculosis.intestinal lymphoma.intestinal Behcet's disease because there are many similar endoscopic places, these diseases Between the clinical identification is a problem
Currently, the clinical laboratory yet simple approach to the differential diagnosis of IBD:blood, plasma proteins, electrolytes, ESR, C-reactive protein, the unit can also do conditional fecal calprotectin, lactoferrin, al antitrypsin and other tests, but these tests are nonspecific. Imaging studies, including gastrointestinal barium, abdominal ultrasound, CT, MRI and other help for the diagnosis of IBD.
Our group use proteomics technology to screen out the differences between UC and normal intestinal mucosa of several proteins, including 3-hydroxy -3--methylglutaryl coenzyme A synthase 2.
PPARy is a research hotspot in recent years, is a nuclear transcription factor, that is a member of peroxisome proliferator-activated receptor, according to the different structure, PPAR can be divided intoα,β(orδ) and y are three types, the genes were located on human chromosome 22,6 and 3. Usually rich in mitochondria with high expression of PPARa andβ-oxidation activity of the organization, such as liver, intestinal mucosa, kidney cortex and heart, while in some other organizations also found a low level of expression of PPARa; PPARy is in adipose tissue, bladder and bowel in High expression; and PPARβin almost all organizations have a low level of expression. PPARy through the adjustment related gene's expression, playing an important role in the formation of fat, glucose and lipid metabolism, the immune system, and involvement of occurrence and development with a variety of diseases such as diabetes, obesity, hypertension, cerebral ischemia, Parkinson's disease, kidney disease, cancer. PPARy at home and abroad on animal studies with IBD have some reports, most studies support the protective factor PPARy is the IBD, but also hold the opposite view that the pre-treated with PPARy agonists in animal models will Increase the animal's intestinal inflammation caused more ulcers, glandular loss, and edema. Although many animal experiments in vitro with IBD reported on PPARy, but currently only a small amount of experimental evaluation of the PPARy receptor in patients with UC and CD expression and function.
HMGCS2,the 3-hydroxy-3-methylglutaryl coenzyme A synthase 2, the formation of ketone bodies regulating the key enzyme in the liver and some extrahepatic tissues such as skeletal muscle, heart, pancreas, testes, colon in both Expression, HMGCS2 expressed in the role of these organizations need to be further elucidated. However, in the organization, HMGCS2 can be oxidized fatty acids, HMGCS2 expression can prevent the deposition of acetyl coenzyme A, which in turn can be reduced fatty acid oxidation rate. Colon's ketogenesis and HMGCS2 express related, the latter depends on the gut-generated acid salt, in healthy intestinal epithelial cells, butyrate stimulation of cell proliferation, however, in the organization of the tumor cell lines it Reduce proliferation and induce cell differentiation and apoptosis. The overseas some scholars proposed that uses in explaining" the butyrate is contradictory", this hypothesis thought:The healthy intestines mucous membrane decomposes the butyrate effectively, thus causes in the cell the butyric acid salinity drop. thus prevents ability drop which multiplies, decomposes the butyrate in the colon cancer cell line anergia, under this situation some scholars proposed that the HMGCS2 induction caused the fatty acid beta oxidation to be weaken deficient. Colon butyrate is the main fuel source, and there is evidence that oxidative damage in ulcerative colitis, in active and quiescent UC CO2 in the oxidation of butyrate and ketone bodies was significantly lower than in healthy controls Group, and this decrease is related with the disease. The butyrate oxidized flaw had reflected the UC patients' invariable and clear metabolism flaw. reducing the oxidation of butyrate can be explained by the distribution in the colonic inflammation, in particular, the incidence of frequency end of the colon. Generally believed that the fatty acids (n-butyrate) oxidation defect is the lack of a colon energy performance. Because the lack of HMGCS2 induction, the beta oxidation of the fatty acid (the n- butyrate) is weaken. UC patients have defects in butyrate oxidation, due to butyrate oxidation of ketone bodies and CO2, we assume, as a key enzyme in ketone bodies generated in HMGCS2 mucosa of UC patients also reduced. HMGCS2 in UC, CD patients and its role in the expression of the intestinal mucosa so far no studies have reported.
Therefore, the purpose of this experiment is to by UC, CD and normal healthy control group were HMGCS2, PPARy system detects molecular markers to observe the relatively complete HMGCS2, PPARy in both the expression of disease, which suggested:(a) whether they can be as a maker to distinguish UC from CD (b) whether they are associated with Energy metabolization obstacle and dysbacteriosis; (c) through the UC, CD and healthy control group, the development of IBD analyzed the molecular basis.
Materials and Methods
1. A total of 204 colorectal specimens were collected randomly from individuals who underwent endoscopic resection under total colonoscopy. The specimens consisted of 73 case of UCs,42 CDs and 89 healthy controls.compared between the UC group and CD group general information on clinical features and check the differences, including sex, age, lesions involving the location, Activities staging, clinical indexing, Endoscopic features, time of onset etc.
2.1mmunohistochemical staining observed HMGCS2, PPARy in the expression of each group
3. Statistical analysis:SPSS 13.0 statistical software for analysis, with the Independent-Samples T Test Determination of the age group and differences between-groups of patients; R* C table using theχ2 test to compare group data between the positive expression rate; Using non-parametric test of two independent samples、K independent samples of non-parametric test and Mann-Whitney U test comparing the groups of gender, age, lesion site, stage, degree of clinical, endoscopic grading related with protein dyeing and expression differences All results obtained bilateral P value, P<0.05 considered significant difference.
Results
1.73 UC patients consisted of 46 males and 27 females, male:female= 1.70:1, age 13-77 years, mean age (40.07±14.39) years, peak age of onset is 30-49 years, of which 28 cases occurred in the rectum (38.4%),9 cases occurred in the rectum, sigmoid colon (12.3%), The whole colon in 25 patients (34.2%).42 cases of CD patients,29 cases occurred in the terminal ileum and small intestine (69.0%).
2. HMGCS2, PPARy in 73 patients with positive expression in intestinal mucosa of UC patients were 48 cases (65.8%) and 36 patients (49.3%); in 42 patients with positive expression in intestinal mucosa of CD patients were 37 cases (88.1%) and 30 patients (71.4%); in 89 cases of normal mucosa positive control group accounted for 82 cases (92.1%) and 66 patients (74.2%). HMGCS2, PPARy in the colonic mucosa in UC patients and normal healthy controls and CD patients significantly decreased compared intestinal mucosa, the difference was statistically significant (P<0.01), but in CD patients and healthy controls intestinal mucosa HMGCS2, PPARy expression was not Significant difference, not statistically significant (P-0.521 and P=0.742).
3. HMGCS2, PPARy in intestinal mucosa of patients with active UC was significantly less than the expression of intestinal mucosa in remission, the difference was statistically significant。(HMGCS2:P=0.002. PPARy:P=0.008)
4. HMGCS2, PPARy expression in the intestinal mucosa of UC patients with gender, age, disease location, clinical, no significant correlation was found. The expression of PPARy protein in patients with endoscopic grade UC was no significant correlation, but HMGCS2 protein expression and endoscopic grading, endoscopy is low grade inflammation in mild disease is relatively high when the protein.
Conclusions
The study found HMGCS2, PPARy is not IBD specific protein targets, but also in healthy controls and other mucosa, for the identification of UC, CD has a certain significance, may be used as an indicator of the severity of UC. HMGCS is a key enzyme in ketone body formation, PPARy addition to factors involved in inflammation and inflammation-related regulation of transcription factors, there may be similar with the HMGCS by butyrate and intestinal energy metabolism and the role of intestinal flora in IBD The occurrence and development
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