咳喘宁对支气管哮喘大鼠免疫调节机制的研究
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摘要
目的:在建立支气管哮喘大鼠模型基础上,通过动物实验,探讨咳喘宁对哮喘大鼠免疫调节的作用及其机制,为治疗支气管哮喘临床用药提供实验依据。
方法
第一部分:咳喘宁对支气管哮喘大鼠Th1∕Th2细胞因子的调节作用及病理形态学的影响
清洁级SD大鼠40只,雌雄各半,体重180-200g。动物随机分为5组,即正常组、模型组、咳喘宁高剂量组(简称高剂量组)、咳喘宁低剂量组(简称低剂量组)、桂龙咳喘宁对照组(简称桂龙咳喘宁组),每组8只。各治疗组均从第1次哮喘激发开始(造模第3周)至处死前每天灌胃给药,连续4周,剂量分别为:高剂量组27g/Kg,低剂量组13.5g/Kg,桂龙咳喘宁组0.41 g/Kg,正常组、模型组予0.5%的羧甲基纤维素钠液灌胃,一日一次。
参考文献,除正常组外,于实验第1天和第8天各组大鼠腹腔内注射10%卵蛋白、氢氧化铝混合液1ml,腰部两侧各取1点,每点皮下注射0.5 ml,共计2 ml,使大鼠致敏。第15天起用3%卵蛋白50 ml喷雾激发大鼠哮喘发作,雾化流量为5ml∕min,每次20min,隔日一次,共激发4周。以大鼠出现呼吸加快、口唇发绀、腹肌痉挛、点头呼吸及站立不稳等表现为激发成功。正常对照组以生理盐水代替卵蛋白进行腹腔注射及雾化吸入。
病理组织学观察:动物麻醉完全后,开胸迅速结扎右主支气管,取右肺中叶4%多聚甲醛固定,脱水、包埋、切片,苏木精-伊红HE染色,镜下观察病理学改变,采用NYD1000型计算机图象处理软件。
IL-4和IFN-γ水平的检测:动物麻醉完全后,开胸迅速结扎右主支气管,取右肺上叶,称重后移入玻璃匀浆管中,加入2倍肺组织重量的生理盐水,充分研碎,使组织匀浆化。制备好的匀浆,3500r/min离心10 min,提取上清液分装。采用双抗体夹心ABC-ELISA法测定IL-4和IFN-γ水平。用抗IL-4(IFN-γ)单克隆抗体包被酶标板,PBS洗板4次;用1%BSA(牛血清白蛋白)加入酶标板(200μl∕孔),4oC过夜;然后分别加入标本及不同浓度的标准品(100μl∕孔),室温孵育120min,形成免疫复合物连接在板上,PBS洗板4次;加入辣根过氧化物酶标记的抗IL-4单克隆抗体(100μl∕孔),室温孵育60min, PBS洗板4次;加显色剂OPD底物溶液,避光室温10-30 min,出现黄色;加终止液硫酸,混匀,5 min内颜色变深;用酶标仪在490nm处测吸光度OD值,绘制标本曲线,查出标本浓度。
数据采用均数±标准差(±s)表示,用SPSS for Windows 11.5统计软件处理,多组比较采用单因素方差分析,然后用Student-Newman-Keuls Test进行每两组间比较,显著性差异水平以0.05和0.01为标准。
第二部分:咳喘宁对支气管哮喘大鼠炎性介质NO和ET-1的影响动物分组、模型建立、用药情况以及数据统计方法同前。
病理组织学观察:动物麻醉完全后,开胸迅速结扎右主支气管,取右肺中叶4%多聚甲醛固定,脱水、包埋、切片,苏木精-伊红HE染色,镜下观察病理学改变。并参照文献测量大鼠气道壁的面积,并用气管内周长进行标准化。采用NYD1000型图像分析软件测定完整支气管管腔的内周长(Pi)、管壁面积(WA)、支气管平滑肌的面积,用Pi进行标准化,分别以WA/Pi、支气管平滑肌的面积/Pi表示支气管管壁厚度、支气管平滑肌厚度;同时测定每高倍视野(400倍)的EOS、淋巴细胞数。
NO和ET-1含量测定:动物麻醉完全后,开胸迅速结扎右主支气管,取右肺上叶,称重后移入玻璃匀浆管中,加入2倍肺组织重量的生理盐水,充分研碎,使组织匀浆化。制备好的匀浆,3500r/min离心10 min,提取上清液分装。采用硝酸还原酶法测定肺组织NO含量,采用放免法测定肺组织ET-1含量,并用蛋白含量来校正。
第三部分:咳喘宁对支气管哮喘大鼠核因子-kB(NF-kB)的影响动物分组、模型建立、用药情况以及数据统计方法同前。
气道壁EOS计数:病理组织切片制作同前,采用NYD1000型计算机图象处理软件。选择结构较完整的支气管壁由同一观察者随机选取5个高倍视野(×400)计数支气管壁中浸润EOS数,计算其平均值作为该切片的代表值。
NF-kB表达的检测:动物麻醉完全后,开胸迅速结扎右主支气管,取右肺中叶4%多聚甲醛固定24小时,脱水、石蜡包埋、连续切片,厚4μm,做免疫组织化学ABC法染色。一抗为小鼠抗大鼠P65单克隆抗体(F-6)、二抗为生物素化马抗小鼠IgG,主要操作步骤如下:(1)石蜡切片经二甲苯脱蜡及梯度酒精脱水,PBS漂洗5 min×2次;(2)3%的双氧水甲醇溶液室温孵育5~10 min,以消除内源性过氧化物酶的活性,PBS洗涤3次;(3)热修复抗原:将切片浸入0.01M枸橼酸盐缓冲液(pH6.0),微波炉加热至沸腾后断电,间隔5-10 min,反复1-2次,冷却后PBS洗涤2次(4)滴加5%正常山羊血清封闭,室温孵育20 min。倾去多余液体;(5)滴加NF-kBⅠ抗(1:100稀释)湿盒孵育,4℃冰箱内过夜,PBS洗涤5 min×2次;(6)滴加生物素标记的二抗工作液,37℃孵育45 min,PBS洗涤5 min×2次;(7)滴加试剂SABC(1:100稀释), 37℃孵育20 min,PBS洗涤5 min×2次;(8)DAB显色5~15 min,终止呈色反应;(9)苏木素衬染,常规裱片、干燥、脱水、透明、封片。在光学显微镜下观察,选择结构较完整的支气管壁由同一观察者随机选取5个高倍视野(×400)计算支气管壁中胞核NF-kB阳性的细胞百分数,取平均值作为该切片的代表值。
第四部分:咳喘宁对支气管哮喘大鼠嗜酸粒细胞凋亡和相关基因蛋白bcl-2和bax表达的影响
动物分组、模型建立、用药情况以及数据统计方法同前。组织标本制作:
动物麻醉完全后,开胸迅速结扎右主支气管,取右肺中叶4%多聚甲醛固定24小时,脱水、石蜡包埋、连续切片,厚4μm,分别做HE染色、TUNEL标记和免疫组织化学ABC法染色。
嗜酸粒细胞凋亡检测:本实验参照TUNEL试剂盒说明进行操作,主要步骤如下:(1)石蜡切片经二甲苯脱蜡及梯度酒精脱水,PBS(0.01M,pH7.4)漂洗5 min×2次;(2)蛋白酶K(2mg/L)37℃消化15 min, PBS漂洗5 min×3次;(3)DNA平衡液平衡阳性对照片2 min后,加入DNase I, 37℃反应10 min;(4)实验组和阳性对照片分别加入TUNEL工作液(1:19稀释)50μl, 37℃孵育45 min,PBS漂洗5 min×3次;(5)加入碱性磷酸酶(AP)标记的抗荧光素抗体50μl, 37℃孵育30 min,PBS漂洗5 min×3次;(6)经BufferⅢ平衡后,NBT/BCIP显色,室温下暗处显色至阳性对照显色良好时终止呈色反应;(7)缓冲甘油(PBS:甘油=1:3)封片和观察。
bcl-2和bax蛋白表达的检测:采用免疫组织化学ABC法染色(方法同前)。
结果判定及图象处理:光学显微镜下观察,凋亡的嗜酸粒细胞为蓝紫色双核或单核,位于胞核内,胞浆不着色。Bcl-2和Bax蛋白染色呈棕黄色颗粒,位于细胞浆内。每张切片高倍镜下(×400)由同一观察者随机选取5个视野,分别计算支气管壁和肺组织嗜酸粒细胞数量(HE)染色及嗜酸粒细胞凋亡指数(TUNEL法,凋亡嗜酸粒细胞占相应嗜酸粒细胞的百分比,AI),以及胞核bcl-2和bax强阳性的细胞百分数,取平均值作为该切片的代表值。
第五部分:咳喘宁对支气管哮喘大鼠肺组织IL-5 mRNA表达的影响动物分组、模型建立、用药情况以及数据统计方法同前。动物麻醉完全后,开胸迅速结扎右主支气管,取近肺门处的右肺组织约50-100mg,放入匀浆器内,加入1ml的Trizol用匀浆器匀浆,采用RT-PCR法检测各组大鼠肺组织IL-5 mRNA表达的变化。
结果
第一部分:咳喘宁对哮喘大鼠Th1∕Th2细胞因子的调节作用及病理形态学的影响
1咳喘宁对支气管哮喘大鼠支气管-肺组织病理学的影响
正常组:大鼠肺组织支气管内及周围无明显炎性细胞浸润,管壁完整,管壁和平滑肌厚度正常,组织黏膜未见充血水肿,肺泡间隔狭窄,肺泡腔清亮宽敞。
模型组:支气管壁、管腔内及血管、支气管周围有大量嗜酸性粒细胞及淋巴细胞为主的炎性细胞浸润,支气管粘膜水肿、增厚、上皮脱落、微血管渗漏、管腔内分泌物增多,基层细胞增生、平滑肌增厚,组织黏膜充血水肿,肺泡间隔变宽,肺泡腔变狭窄。
桂龙咳喘宁组:肺组织嗜酸性粒细胞及其它炎性细胞浸润略微减少,管壁和平滑肌厚度略微减少,组织充血水肿现象略微减轻。
咳喘宁低剂量组:肺组织嗜酸性粒细胞及其它炎性细胞浸润减少,管壁和平滑肌厚度减少,组织充血水肿现象减轻,肺泡腔变宽,肺泡间隔变窄。
咳喘宁高剂量组:肺组织嗜酸性粒细胞及其它炎性细胞浸润明显减少,几乎消失;管壁和平滑肌厚度明显减少,组织充血水肿现象明显减轻,肺泡腔变宽,肺泡间隔变窄。
2咳喘宁对支气管哮喘大鼠肺组织匀浆IL-4含量的影响
与正常组比较,模型组大鼠肺组织IL-4含量明显升高,具有非常显著性差异(P<0.01);与模型组比较,各治疗组均可降低IL-4含量,咳喘宁高剂量组效果更为明显,具有非常显著性差异(P<0.01);咳喘宁低剂量组和桂龙咳喘宁组,具有显著性差异(P<0.05);与桂龙咳喘宁组比较,咳喘宁高剂量组IL-4含量明显降低,具有显著性差异(P<0.05)。
3咳喘宁对支气管哮喘大鼠肺组织匀浆IFN-γ含量的影响
与正常组比较,模型组大鼠肺组织IFN-γ含量较正常组有所升高,但无显著性差异(P>0.05);与模型组比较,各治疗组均可升高IFN-γ含量,咳喘宁高剂量组效果更为明显,具有非常显著性差异(P<0.01);咳喘宁低剂量组和桂龙咳喘宁组可升高IFN-γ含量,具有显著性差异(P<0.05);与桂龙咳喘宁组比较,咳喘宁高剂量组IFN-γ含量明显增高,具有显著性差异(P<0.05)。
4咳喘宁对支气管哮喘大鼠肺组织IFN-γ∕IL-4比值的影响
与正常组比较,模型组大鼠肺组织IFN-γ∕IL-4比值明显降低,具有非常显著性差异(P<0.01);与模型组比较,各治疗组均可升高IFN-γ∕IL-4比值,具有显著性差异(P<0.05或P<0.01);咳喘宁高剂量组效果更为明显,具有非常显著性差异(P<0.01);与桂龙咳喘宁组比较,咳喘宁高剂量组IFN-γ∕IL-4比值明显增高,具有显著性差异(P<0.05)。
第二部分:咳喘宁对支气管哮喘大鼠炎性介质NO和ET-1的影响
1咳喘宁对支气管哮喘大鼠支气管-肺组织病理学的观察及定量分析:
正常组大鼠肺组织偶见炎细胞浸润,管壁完整,粘膜未见充血水肿,管壁和平滑肌厚度正常;模型组大鼠可见支气管壁及血管、支气管周围有大量嗜酸性粒细胞、淋巴细胞等炎性细胞浸润(P<0.01),支气管粘膜水肿、增厚、上皮脱落、微血管渗漏、管腔内分泌物增多,基层细胞增生、平滑肌增厚(P<0.01);与模型组比较,各治疗组嗜酸性粒细胞、淋巴细胞等炎性细胞浸润明显减少(P<0.01),肺组织充血、水肿现象减轻,管壁和平滑肌厚度明显减少(P<0.01);高低剂量组病理组织学指标改善优于桂龙咳喘宁组(P<0.05或P<0.01)。
2咳喘宁对支气管哮喘大鼠肺组织NO含量的影响
与正常组比较,模型组大鼠肺组织NO含量明显升高,具有非常显著性差异(P<0.01);与模型组比较,各治疗组均可降低NO含量,具有显著性差异(P<0.05或P<0.01);咳喘宁高剂量组效果更为明显,具有非常显著性差异(P<0.01);与桂龙咳喘宁组比较,咳喘宁高剂量组NO含量明显降低,具有显著性差异(P<0.05)。
3咳喘宁对支气管哮喘大鼠肺组织ET-1含量的影响
与正常组比较,模型组大鼠肺组织ET-1含量明显升高,具有非常显著性差异(P<0.01);与模型组比较,各治疗组均可降低ET-1含量,具有显著性差异(P<0.05或P<0.01);咳喘宁高剂量组效果更为明显,具有非常显著性差异(P<0.01);与桂龙咳喘宁组比较,咳喘宁高剂量组NO含量明显降低,具有显著性差异(P<0.05)。
第三部分:咳喘宁对支气管哮喘大鼠核因子-kB(NF-kB)的影响
1咳喘宁对支气管哮喘大鼠气道壁EOS浸润情况比较模型组大鼠的气道壁EOS计数较正常组显著增加(P<0.01),治疗组大鼠的气道壁EOS计数较模型组显著降低(P<0.05或P<0.01),以咳喘宁高低剂量组改善更为显著(P<0.01),其优于桂龙咳喘宁组(P<0.01)。
2咳喘宁对支气管哮喘大鼠肺组织NF-kB表达的影响
正常组大鼠肺组织及气道壁见少许NF-kB阳性表达细胞,模型组大鼠肺组织及气道壁中胞核表达NF-kB的细胞比例显著高于正常组,表现为不均匀的黄褐色颗粒状物(P<0.01),而治疗组肺组织及气道壁中胞核阳性细胞的比例显著低于模型组(P<0.05或P<0.01),以咳喘宁高低剂量组改善更为显著(P<0.01),其优于桂龙咳喘宁组(P<0.01)。
第四部分:咳喘宁对支气管哮喘大鼠嗜酸粒细胞凋亡和相关基因蛋白bcl-2和bax表达的影响
1咳喘宁对支气管哮喘大鼠肺组织EOS凋亡指数的影响
正常组大鼠肺组织及气道壁极少EOS浸润。模型组大鼠的气道壁EOS计数较正常组显著增加(P<0.01),治疗组大鼠的气道壁EOS计数较模型组显著降低(P<0.05或P<0.01),以咳喘宁高低剂量组改善更为显著(P<0.01),其优于桂龙咳喘宁组(P<0.01)。正常组大鼠肺组织及气道壁极少EOS凋亡,治疗组大鼠的气道壁EOS凋亡指数较模型组显著增高(P<0.05或P<0.01),以咳喘宁高剂量组改善更为显著(P<0.01),其作用优于桂龙咳喘宁组(P<0.01)。
2咳喘宁对支气管哮喘大鼠肺组织bcl-2蛋白表达的影响
正常组大鼠肺组织及气道壁见少许bcl-2阳性表达细胞,模型组大鼠肺组织及气道壁中胞浆表达bcl-2的细胞比例显著高于正常组,表现为不均匀的黄褐色颗粒状物(P<0.01),而治疗组肺组织及气道壁中胞浆阳性细胞的比例显著低于模型组(P<0.05或P<0.01),以咳喘宁高剂量组改善更为显著(P<0.01),其优于桂龙咳喘宁组(P<0.01)。
3咳喘宁对支气管哮喘大鼠肺组织bax蛋白表达的影响
正常组大鼠肺组织及气道壁见较多bax阳性表达细胞,表现为不均匀的黄褐色颗粒状物。模型组大鼠肺组织及气道壁中胞浆表达Bcl-2的细胞比例显著低于正常组,表现为较浅的黄褐色颗粒状物(P<0.01),而治疗组肺组织及气道壁中胞浆阳性细胞的比例显著高于模型组(P<0.01),以咳喘宁高剂量组改善更为显著,其优于桂龙咳喘宁组(P<0.01)。
第五部分:咳喘宁对支气管哮喘大鼠IL-5 mRNA表达的影响
哮喘模型组肺组织IL-5 mRNA表达较正常组明显增高,与模型组比较,各用药组肺组织IL-5 mRNA的表达明显降低(P<0.05或P<0.01),以咳喘宁高剂量组更为显著(P<0.01);咳喘宁高剂量组IL-5 mRNA表达含量明显低于桂龙咳喘宁组(P<0.01)。
结论
1咳喘宁能有效地改善支气管哮喘肺组织病理改变,减少肺组织EOS浸润,减轻组织充血、水肿现象,减少支气管管壁和平滑肌厚度,有效地减轻哮喘气道炎症及气道重塑。
2咳喘宁能显著降低支气管哮喘大鼠肺组织IL-4的含量,提高IFN-γ的含量,提高IFN-γ∕IL-4比值,有效地调节哮喘细胞因子网络系统,改善哮喘气道炎症。
3咳喘宁能显著降低支气管哮喘大鼠肺组织NO和ET-1的含量,减少哮喘炎性介质的分泌,改善哮喘气道炎症及气道重塑。
4咳喘宁能显著降低支气管哮喘大鼠肺组织NF-kB的表达,通过调整信号传导途径,促进EOS凋亡,改善哮喘气道炎症。
5咳喘宁能显著降低支气管哮喘大鼠肺组织bcl-2蛋白的表达,升高bax蛋白的表达,通过调整凋亡相关基因的表达,促进EOS凋亡,有效地改善哮喘气道炎症。
6咳喘宁能显著降低支气管哮喘大鼠肺组织IL-5 mRNA的表达,从转录水平降低相关细胞因子含量,减少肺内EOS浸润,促进EOS凋亡,有效地改善哮喘气道炎症。
Objective: On the basis of establishing the rat model with bronchial asthma, through the animal experiments to study the role and mechanism of Kechuanning to immuno-regulation of asthmatic rats. Which were to provide experimental evidence for the clinical medication on treating the bronchial asthma.
Methods
PartⅠ: Effects of Kechuanning on regulatory role of Th1/Th2 cytokines and patho-morphological of the asthmatic rats.
40 clean grade SD rats, half and half for either sex and were 180-200g on weights. These animals were randomly divided into five groups: namely, the normal group, the model group, high dose Kechuanning group (high dose group for short) low dose Kechuanning group (low-dose group for short), Guilong Kechuanning control group (Guilong Kechuanning group for short) with 8 rats for each group. The drugs were given through stomach-perfusion to each treatment group from the beginning of the 1st asthma provocation (third weeks after making model) to the day before killing and continued for four weeks. The dose was: 27g/Kg for the high-dose group and 13.5g/Kg for the low dose group, 0.41 g/Kg for Guilong Kechuanning group and 0.5% sodium carboxymethyl cellulose were given to the normal and model group through stomach-perfusion with one time per day.
According to the report, in addition to the normal group, in the first and eighth days after experiment, 1 ml mixture of 10% of egg protein and the aluminum hydroxide were injected intra-peritoneally to the rats from each group, one point was prepared from both sides of waist with subcutaneous injection was 0.5 ml for per point and it was 2 ml in total. All these were to sensitize rats. From 15th days, 50 ml of 3% egg protein was used for spray to provoke the rat asthma; the aerosol flow was 5ml/minutes with 20 minutes for each time, once for every other day and continued for four weeks in total. The manifestations of the provocative success were that there were fast breathing, cyanotic lips, abdominal muscle cramps, nodding breathing and unstable standing etc. In the normal group, the normal saline was used to replace the egg protein for intraperitoneal injection and spray inhalation.
Histopathological observation: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of the right lung was taken and fixed with 4% paraformaldehyde and gave the dehydration, embedding and sections, then, the hematoxylin - eosin HE staining. The pathological changes were observed microscopically and NYD1000 computer image processing software was used.
IL-4 and IFN-γlevel test: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The upper lobe of right lung was taken and put into the glass homogenate tube after weighing, adding the normal saline which were 2 times of the weight of lung tissue and gave the full levigation and make the tissue to be homogenized. Then, the prepared homogenate was given the centrifugation with 3500r/min for 10 minutes, after that, the supernatants was extracted and gave subpackage. IL-4 and IFN-γlevel were measured with the double-antibody sandwich ABC-ELISA method. The enzyme label plates were coated with IL-4 (IFN-γ) monoclonal antibody and the plates were washed with PBS for 4 times. Then, 1%BSA (bovine serum albumin) was added to enzyme label plate (200μl/ hole) and kept 4°C overnight. Later, adding the samples and the standard with different concentrations (100μl/ hole) and gave 120 minutes incubation at room temperature. Thus, it formed the immune compounds and connected with the plate, then, the plates were washed with PBS for 4 times and added horseradish peroxidase labeled anti-IL-4 monoclonal antibody (100μl/hole) and gave 60 minute’s incubation at room temperature, the plates were washed again with PBS for 4 times and added OPD substrate solution and kept in room temperature for 10-30 minutes with being away from light, there was yellow. At this point, added the sulfuric acid of stopping solutions and mixed evenly, the solution became darker in color in five minutes. Absorbance OD value was measured at 490 nm with enzyme sign analyzer and drew the specimen’s curves and searched out the specimen’s concentration.
The data was expressed with mean±standard deviation (±s) and was processed with the statistical software- SPSS for Windows 11.5. Single factor variance analysis was used to compare the each group. Then, Student-Newman-Keuls Test was used for the comparison between each two groups and the significant difference level took 0.05 and 0.01 as the standard.
Part II: the influence of Kechuanning on the inflammatory mediators NO and ET-1 of rats with bronchial asthma.
Histopathological observation: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of the right lung was taken and fixed with 4% paraformaldehyde and gave the dehydration, embedding and sections, then, the hematoxylin - eosin HE staining. The pathological changes were observed microscopically and the area of the rats airway wall were measured in the light of the literature and the intratracheal perimeter were standardized. NYD1000 image analysis software was used to measure the internal circumference (Pi)of intact bronchial lumen, bronchial wall area (WA), bronchial smooth muscle area. Pi was used for standardization. WA/Pi and the bronchial smooth muscle area/ Pi expressed the bronchial wall thickness and bronchial smooth muscle thickness respectively. At the same time, the number of EOS and the lymphocytes from each high-power field (400 times) were measured.
NO and ET-1 content test: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The upper lobe of right lung was taken and put into the glass homogenate tube after weighing, adding the normal saline which were 2 times of the weight of lung tissue and levigating it fully and making it homogenizate. Then, the prepared homogenate was centrifugated with 3500r/min for 10 minutes, after that, the supernatants was extracted and the NO content of lung tissue were measured with nitric acid reductase method. The ET-1 content of lung tissue was measured with radio immunity and was calibrated with the protein content. Animal grouping, making model, drug administration and the statistical methods of data are same as the former.
Part III: The influence of Kechuanning on the nuclear factor-kB(NF-kB) of rats with bronchial asthma.
Animal grouping, making model, drug administration and the statistical methods of data are same as the former.
EOS counts of airway wall: the method of making pathological tissue section was the same with before and NYD1000 computer image-processing software was used. A rather complete structure of the bronchial wall was selected and five high-power fields (×400) were chosen randomly, and the number of infiltrating EOS from the bronchial wall was calculated by the same observer,and the averaged value represent as the value of the section.
The measurement of NF-kB expression: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of right lung was taken and fixed 24 hours with 4% paraformaldehyde and gave the dehydration, paraffin embedding and serial sections with 4μm in thickness. ABC staining of immunohistochemistry was made .The primary antibody was mouse anti-rat P65 monoclonal antibody (F-6), the secondary antibody was the biotinylated horse anti-mouse IgG. Main operation steps are as follows: (1) through xylene dewaxing and gradient alcohol dehydration, the paraffin sections were rinsed with PBS for 5 minutes×2 times; (2) incubating it in 3% hydrogen peroxide and methanol solution for 5-10 minutes at room temperature in order to remove the activity of endogenous peroxidase, and rinsing it with PBS for 3 times; (3) heat retrieval antigen: the slice were immersed in 0.01M citrate buffer solution (pH 6.0) and after heating it to boil with the microwave oven, turning power off with 5-10 minute’s interval, repeating 1-2 times. The material was washed with PBS for 2 times after cooling; (4) dropping 5% normal goat serum for blocking and being incubated for 20 minutes at room temperature. Then, pouring the redundant liquid; (5) dropping NF-kBⅠantibody (1:100 dilution)for wet case incubation. Keeping it in 4℃refrigerator overnight and washing in PBS for 5 minutes×2 times;(6) dropping the secondary antibody working fluid with biotinated label and incubating it in 37℃for 45 minutes, then washing it in PBS for 5 minutes×2 times;(7) Dropping SABC reagents (1:100 dilution), then incubating it for 20 minutes and washing it in PBS for 5 minutes×2 times; (8) giving DAB color development for 5-15 minutes, then, stopping the color development; (9) Hematoxaylin staining, the conventional mounting slices, dryness, dehydration, vitrification and mounting. The slice was observed with the optical microscope. A rather complete structure of the bronchial wall was selected and five high-power fields (×400) were chosen by the same observer randomly, which was to count the positive cell percentage of the karyon NF-kB from the bronchial wall and took its average value as the representative value of the section.
Part IV: The influence of Kechuanning on the eosinophilic granulocyte apoptosis and bcl-2 and bax expression of the relative gene from the rats with asthma
Animal grouping, making model, drug administration and the statistical methods of data are same as the former.
Tissue sample making:
After complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of the right lung was taken and fixed for 24 hours with 4% paraformaldehyde and gave the dehydration, paraffin embedding and serial sections with 4μm in thickness, then, the hematoxylin - eosin HE staining and TUNEL marking and immunohistochemical ABC staining.
Eosinophilic granulocyte apoptosis detection: this experiment is carried out in the light of TUNEL reagent kit and the major steps as follows: (1) through xylene dewaxing and gradient alcohol dehydration, the paraffin sections were rinsed with PBS(0.01M, pH7.4) for 5 minutes×2 times (2) proteinase K (2mg/L)digested it in 37℃for 15 minutes, then rinsed in PBS for 5 minutes×3 times (3) after balancing the positive contrast with DNA equilibrium liquid for 2 minutes, added DNase I for reaction in 37℃for 10 minutes (4) TUNEL working fluid (1 : 19 dilution) 50μl were added respectively to the experimental group and positive contrast and was incubated in 37℃for 45 minutes, then, rinsing them with PBS for 5 min×3 times. (5)thus, adding 50μl anti-fluorescein antibody with alkaline phosphatase (AP) label and being incubated in 37℃for 30 minutes, then , rinsing them with PBS for 5 min×3 times. (6) Through Buffer III balance and NBT/BCIP color development, the color development in dark at room temperature got to positive and good control color development, the color reaction can be stopped (7) mounting is taken with glycerol buffer (PBS: glycerol 1:3) and observating.
The detection of bcl-2 and bax protein expression: immunohistochemical ABC staining (same as the former method) was used.
Results verdict and image processing: it was observed with the optical microscope, the apoptostic eosinophilic granulocyte was the blue-purple binuclear or mononuclear, which was located in the nuclei with no staining for the cytoplasm. Bcl-2 and Bax protein staining showed brown yellow granules and were in the cytoplasm. Five fields were chosen by the same observer randomly from each section under high-power microscope (×400), then, the number (HE) of eosinophilic granulocyte from the bronchial wall and lung tissue, the apoptostic index of staining and eosinophilic granulocyte (TUNE L method, the percentage of the apoptostic eosinophilic granulocyte that occupied in the corresponding eosinophilic granulocyte AI) and the percentage of strong positive cells of Bcl-2 and Bax nuclei were calculated respectively, the averaged value was taken as the representative value of the section.
Part V: The influence of Kechuanning on the IL-5 mRNA expression of lung tissue from the rats with bronchial asthma
Animal grouping, making model, drug administration and the statistical methods of data are same as the former.
After complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The right lung that was adjacent to the porta was taken for about 50-100mg, then puting it into the homogenizer, adding 1 ml Trizol and homogenatizating by homogenizer. The IL-5 mRNA expression changes of the lung tissue in rats from each group were detected by RT-PCR method.
Results
PartⅠ: The regulatory role and the patho-morphological effects of Kechuanning to Th1/Th2 cytokines from the asthmatic rats.
1. The pathological effects of Kechuanning to the bronchial-lung tissue of rats with bronchial asthma.
Normal groups: there was no obvious inflammatory cell infiltration from the bronchus of lung tissues and surroundings in rats with intact wall, the thickness of wall and smooth muscle were normal with no congestion and edema in the tissue mucosa, the alveolar septum was narrow and the alveolar cavity was clear and spacious.
Model group: there were many inflammatory cells infiltration in bronchial wall and lumen as well as around blood vessel and bronchus, which mainly were the eosinophilic granulocyte and lymphocyte, accompanied with bronchial mucosal edema, thickening, epithelial exfoliation, microvascular leakage, the secretion increased within lumen stromal cell hyperplasia, thickened smooth muscle, the congestive edema of tissue mucosa, widened alveolar septum and narrowed alveolar lumen.
Guilong Kechuanning groups: there were slight decrease for the eosinophilic granulocyte and other inflammatory cell infiltration from lung tissue, also, there were slight decrease for the smooth muscle wall thickness with the congestion and edema from tissue got slight reduction.
Low-dose Kechuanning group: the eosinophilic granulocyte and other inflammatory cell infiltration from lung tissue decreased, also, there were decrease for the smooth muscle wall thickness with the congestion and edema from tissue got slight reduction, the alveolar lumen widened with the narrowing of alveolar septum.
High-dose Kechuanning group: the eosinophilic granulocyte and other inflammatory cell infiltration from lung tissue decreased obviously and almost disappeared, also, there were marked decrease for the wall and smooth muscle thickness with the congestion and edema from tissue got obvious reduction, the alveolar lumen widened with the narrowing of alveolar septum.
2. The influence of Kechuanning on the homogenate IL-4 content of lung tissues from the rats with bronchial asthma.
Compared with the normal group, IL-4 content of rat lung tissues from the model group increased significantly with very significant difference (P<0.01). Compared with the model group, IL-4 content can be lowered from each treatment group and it was even more obvious for the high-dose Kechuanning group with very significant difference (P<0.01). The low-dose Kechuanning group and Guilong Kechuanning group had significant difference (P<0.05). Compared with the control group, IL-4 content from the high-dose Kechuanning group was significantly lower than the control group with significant differences (P<0.05).
3. The influence of Kechuanning on the homogenate IFN-γcontent of lung tissues from the rats with bronchial asthma.
Compared with the normal group, IFN-γcontent of rat lung tissues from model group had slight increase(P>0.05). Compared with the model group, IFN-γcontent can be increased from each treatment group and it was even more obvious for the high-dose Kechuanning group with very significant difference (P<0.01). IFN-γcontent can be elevated for the low-dose Kechuanning group and Guilong Kechuanning group with the significant difference (P < 0.05). Compared with the control group of Guilong Kechuanning, IFN-γcontent from the high-dose Kechuanning group was significantly higher than control group with significant differences (P<0.05).
4. The influence of Kechuanning on IFN-γ∕IL-4 ratio of lung tissues from the rats with bronchial asthma.
Compared with the normal group, IFN-γ∕IL-4 ratio of rat lung tissues from the model group decreased obviously with very significant difference (P<0.01). Compared with the model group, IFN-γ∕IL-4 ratio can be elevated from each treatment group with very significant difference (P<0.05). It was even more obvious for the high-dose Kechuanning group and it was with very significant difference (P<0.01). Compared with the control group, IFN-γ∕IL-4 ratio from the high-dose Kechuanning group was significantly higher than that of control group with significant differences (P<0.05).
Part II: The influence of Kechuanning on the inflammatory mediators NO and ET-1 of rats with bronchial asthma.
1. The histopathological observation and the quantitative analysis for the rats with bronchial asthma and treating with Kechuanning There were less inflammatory cell infiltration in the lung tissue of rats from normal group with intact vessel wall, there was no congestion and edema in the mucosa, the thickness of vessel wall and the smooth muscle were normal. In the rats from model group, there were plenty of eosinophilic granulocyte and lymphocyte and other inflammatory cell infiltration(P<0.01) from the bronchial wall and around blood vessel and bronchus, also, there were bronchial mucosal edema, thickening, epithelial exfoliation, microvascular leakage, the secretion increased within lumen,stromal cell hyperplasia ,smooth muscle thickening(P<0.01), Compared with the model group, there were obvious decrease for the eosinophilic granulocyte and lymphocyte and other inflammatory cell infiltration from each treatment group, the congestion and edema of the lung tissue were also mitigated and the thickness of vessel wall and the smooth muscle were reduced obviously(P<0.01).The improvement on histopathological indices from high-dose group were better than control group(P<0.05 or P<0.01).
2. The influence of Kechuanning on NO content of lung tissues from the rats with bronchial asthma.
Compared with the normal group, NO content of rat lung tissues from the model group increased obviously with very significant difference (P<0.01). Compared with the model group, NO content can be decreased from each treatment group with very significant difference (P<0.05). The result was even more obvious for the high-dose Kechuanning group and it was also with very significant difference (P<0.01). Compared with the control group of Guilong Kechuanning group, NO content from the high-dose Kechuanning group was significantly lower than that of control group with significant differences (P<0.05).
3. The influence of Kechuanning on ET-1 content of lung tissues from the rats with bronchial asthma
Compared with the normal group, ET-1 content of rat lung tissues from the model group increased obviously with very significant difference (P <0.01). Compared with the model group, ET-1 content can be decreased from each treatment group with very significant difference (P<0.05). The results were even more obvious for the high-dose Kechuanning group and it was also with very significant difference (P<0.01). Compared with the control group of Guilong Kechuanning group,ET-1 content from the high-dose Kechuanning group was significantly lower than that of control group with significant differences (P<0.05).
Part III: The influence of Kechuanning on the nuclear factor-kB (NF-kB) of rats with bronchial asthma
1. The comparison of EOS infiltration of airway wall of rats with bronchial asthma with Kechuanning.
Compared with the normal group, the EOS count in airway wall of rats from the model group increased significantly(P<0.01),compared with model group,the EOS count of airway wall in the rats from the treatment group decreased significantly(P<0.05),the improvements from the high and low dose Kechuanning groups were more significant(P<0.01) ,better than control group(P<0.01).
2. The influence of Kechuanning on the NF-kB expression of lung tissue of rats with bronchial asthma
Less positive expression of NF-kB cell can be seen in the lung tissue and airway wall of rats from normal group ,the cell proportion of karyon NF-kB expression in the lung tissue and airway wall of rats from model group were significantly higher than that of the normal group, which were manifested by the unsymmetric brown yellow particulates(P<0.01), however, in the rats from the treatment group, the proportion of positive cell of karyon in the lung tissue and airway wall were significantly lower than that of the model group(P<0.05), the improvements from the high and low dose Kechuanning groups were more significant(.P<0.01), which were better than that of Guilong Kechuanning group(P<0.01).
Part IV: The influence of Kechuanning on the eosinophilic granulocyte apoptosis and Bcl-2 and Bax expression of the relative gene from the rats with asthma
1. The influence of Kechuanning on EOS apoptosis index of lung tissue from the rats with bronchial asthma. There was little EOS infiltration in lung tissue and airway wall of the rats from normal group. Compared with the normal group, the EOS count in airway wall of rats from the model group increased significantly(P<0.01);compared with the model group , the EOS count in airway wall of rats from the treatment group decreased significantly(P<0.05), the improvements from the high and low dose Kechuanning groups were more significant(P<0.01),which were better than control group (P<0.01). There was little EOS apoptosis in lung tissue and airway wall of rats from the normal group. Compared with the model group, the EOS apoptosis index in airway wall of rats from the treatment group increased significantly (P<0.05), the improvements from the high dose Kechuanning group were more significant (P<0.01),which were better than control group (P<0.01).
2. The influence of Kechuanning on Bcl-2 proteins expression of the lung tissue of rats with the bronchial asthma
Less Bcl-2 positive expression cells can be seen in the lung tissue and airway wall of rats from normal group, the proportion of cell that expressing Bcl-2 in kytoplasm in the lung tissue and airway wall of rats from the model group were significantly higher than that of the normal group ,which were manifested by the unsymmetric brown yellow particulate(sP<0.01), however, in the rats from the treatment group, the proportion of positive kytoplasm cell in the lung tissue and airway wall were significantly lower than that of the model group(P<0.05), the improvements from the high dose Kechuanning groups were more significant(P<0.01),which were better than that of Guilong Kechuanning group(P<0.01).
3. The influence of Kechuanning on Bax proteins expression of the lung tissue of rats with the bronchial asthma.
More Bcl-2 positive expression cells can be seen in the lung tissue and airway wall of rats from normal group, which were manifested by the unsymmetric brown yellow particulates .The proportion of cell that expressing Bcl-2 in kytoplasm.
In the lung tissue and airway wall of rats from model group were significantly lower than that of the normal group, which were manifested by the shallow brown yellow particulate(sP<0.01), however, in the rats from the treatment group, the proportion of positive kytoplasm cell in the lung tissue and airway wall were significantly lower than that of the model group(P<0.05), the improvements from the high dose Kechuanning groups were more significant(P<0.01),which were better than control group (P<0.01).
Part V: The influence of Kechuanning on IL-5 mRNA expression of rats with the bronchial asthma
Compared with the normal group, IL-5 mRNA expression of lung tissue in asthmatic model group increased significantly; compared with the model group, IL-5 mRNA expression of lung tissue decreased significantly from each medication group(P<0.05),which was more significant for the high dose Kechuanning group(P<0.01), the level of IL-5 mRNA expression from the high dose Kechuanning group were much lower than control group (P<0.01).
Conclusion
1. Kechuanning could effectively improve the pathological changes of lung tissue in bronchial asthma,reduce EOS infiltration in lung tissue, decrease the tissue congestion and edema, reduce the thickness of the bronchial wall and smooth muscle, effectively reduce asthmatic airway inflammation and airway remodeling.
2. Kechuanning could significantly reduce the IL-4 content in the lung tissue of rats with bronchial asthma, increase IFN-γcontent and the ratio of IFN-γ? I L-4, adjust effectively the asthmatic cytokines network system, improve asthmatic airway inflammation.
3. Kechuanning could significantly reduce NO and ET-1 content in lung tissue of rats with bronchial asthma, reduces the secretion of inflammatory mediators in asthma, improve airway inflammation in asthma and airway remodeling.
4. Kechuanning could significantly reduce NF-kB expression of lung tissue of rats with bronchial asthma and inhibit EOS apoptosis and improve airway inflammation in asthma through adjusting the signal transmission route.
5. Kechuanning could significantly reduce bcl-2 protein expression and increase bax protein expression of lung tissue of rats with bronchial asthma and inhibit EOS apoptosis and effectively improve airway inflammation in asthma through adjusting the expression of apoptosis-related genes.
6. Kechuanning could significantly reduce the IL-5 mRNA expression of lung tissue of rats with bronchial asthma, reduce the content of related cytokines by transcription level, reduce EOS infiltration in the lungs, inhibit EOS apoptosis and effectively improve airway inflammation in asthma.
方法
第一部分:咳喘宁对支气管哮喘大鼠Th1∕Th2细胞因子的调节作用及病理形态学的影响
清洁级SD大鼠40只,雌雄各半,体重180-200g。动物随机分为5组,即正常组、模型组、咳喘宁高剂量组(简称高剂量组)、咳喘宁低剂量组(简称低剂量组)、桂龙咳喘宁对照组(简称桂龙咳喘宁组),每组8只。各治疗组均从第1次哮喘激发开始(造模第3周)至处死前每天灌胃给药,连续4周,剂量分别为:高剂量组27g/Kg,低剂量组13.5g/Kg,桂龙咳喘宁组0.41 g/Kg,正常组、模型组予0.5%的羧甲基纤维素钠液灌胃,一日一次。
参考文献,除正常组外,于实验第1天和第8天各组大鼠腹腔内注射10%卵蛋白、氢氧化铝混合液1ml,腰部两侧各取1点,每点皮下注射0.5 ml,共计2 ml,使大鼠致敏。第15天起用3%卵蛋白50 ml喷雾激发大鼠哮喘发作,雾化流量为5ml∕min,每次20min,隔日一次,共激发4周。以大鼠出现呼吸加快、口唇发绀、腹肌痉挛、点头呼吸及站立不稳等表现为激发成功。正常对照组以生理盐水代替卵蛋白进行腹腔注射及雾化吸入。
病理组织学观察:动物麻醉完全后,开胸迅速结扎右主支气管,取右肺中叶4%多聚甲醛固定,脱水、包埋、切片,苏木精-伊红HE染色,镜下观察病理学改变,采用NYD1000型计算机图象处理软件。
IL-4和IFN-γ水平的检测:动物麻醉完全后,开胸迅速结扎右主支气管,取右肺上叶,称重后移入玻璃匀浆管中,加入2倍肺组织重量的生理盐水,充分研碎,使组织匀浆化。制备好的匀浆,3500r/min离心10 min,提取上清液分装。采用双抗体夹心ABC-ELISA法测定IL-4和IFN-γ水平。用抗IL-4(IFN-γ)单克隆抗体包被酶标板,PBS洗板4次;用1%BSA(牛血清白蛋白)加入酶标板(200μl∕孔),4oC过夜;然后分别加入标本及不同浓度的标准品(100μl∕孔),室温孵育120min,形成免疫复合物连接在板上,PBS洗板4次;加入辣根过氧化物酶标记的抗IL-4单克隆抗体(100μl∕孔),室温孵育60min, PBS洗板4次;加显色剂OPD底物溶液,避光室温10-30 min,出现黄色;加终止液硫酸,混匀,5 min内颜色变深;用酶标仪在490nm处测吸光度OD值,绘制标本曲线,查出标本浓度。
数据采用均数±标准差(±s)表示,用SPSS for Windows 11.5统计软件处理,多组比较采用单因素方差分析,然后用Student-Newman-Keuls Test进行每两组间比较,显著性差异水平以0.05和0.01为标准。
第二部分:咳喘宁对支气管哮喘大鼠炎性介质NO和ET-1的影响动物分组、模型建立、用药情况以及数据统计方法同前。
病理组织学观察:动物麻醉完全后,开胸迅速结扎右主支气管,取右肺中叶4%多聚甲醛固定,脱水、包埋、切片,苏木精-伊红HE染色,镜下观察病理学改变。并参照文献测量大鼠气道壁的面积,并用气管内周长进行标准化。采用NYD1000型图像分析软件测定完整支气管管腔的内周长(Pi)、管壁面积(WA)、支气管平滑肌的面积,用Pi进行标准化,分别以WA/Pi、支气管平滑肌的面积/Pi表示支气管管壁厚度、支气管平滑肌厚度;同时测定每高倍视野(400倍)的EOS、淋巴细胞数。
NO和ET-1含量测定:动物麻醉完全后,开胸迅速结扎右主支气管,取右肺上叶,称重后移入玻璃匀浆管中,加入2倍肺组织重量的生理盐水,充分研碎,使组织匀浆化。制备好的匀浆,3500r/min离心10 min,提取上清液分装。采用硝酸还原酶法测定肺组织NO含量,采用放免法测定肺组织ET-1含量,并用蛋白含量来校正。
第三部分:咳喘宁对支气管哮喘大鼠核因子-kB(NF-kB)的影响动物分组、模型建立、用药情况以及数据统计方法同前。
气道壁EOS计数:病理组织切片制作同前,采用NYD1000型计算机图象处理软件。选择结构较完整的支气管壁由同一观察者随机选取5个高倍视野(×400)计数支气管壁中浸润EOS数,计算其平均值作为该切片的代表值。
NF-kB表达的检测:动物麻醉完全后,开胸迅速结扎右主支气管,取右肺中叶4%多聚甲醛固定24小时,脱水、石蜡包埋、连续切片,厚4μm,做免疫组织化学ABC法染色。一抗为小鼠抗大鼠P65单克隆抗体(F-6)、二抗为生物素化马抗小鼠IgG,主要操作步骤如下:(1)石蜡切片经二甲苯脱蜡及梯度酒精脱水,PBS漂洗5 min×2次;(2)3%的双氧水甲醇溶液室温孵育5~10 min,以消除内源性过氧化物酶的活性,PBS洗涤3次;(3)热修复抗原:将切片浸入0.01M枸橼酸盐缓冲液(pH6.0),微波炉加热至沸腾后断电,间隔5-10 min,反复1-2次,冷却后PBS洗涤2次(4)滴加5%正常山羊血清封闭,室温孵育20 min。倾去多余液体;(5)滴加NF-kBⅠ抗(1:100稀释)湿盒孵育,4℃冰箱内过夜,PBS洗涤5 min×2次;(6)滴加生物素标记的二抗工作液,37℃孵育45 min,PBS洗涤5 min×2次;(7)滴加试剂SABC(1:100稀释), 37℃孵育20 min,PBS洗涤5 min×2次;(8)DAB显色5~15 min,终止呈色反应;(9)苏木素衬染,常规裱片、干燥、脱水、透明、封片。在光学显微镜下观察,选择结构较完整的支气管壁由同一观察者随机选取5个高倍视野(×400)计算支气管壁中胞核NF-kB阳性的细胞百分数,取平均值作为该切片的代表值。
第四部分:咳喘宁对支气管哮喘大鼠嗜酸粒细胞凋亡和相关基因蛋白bcl-2和bax表达的影响
动物分组、模型建立、用药情况以及数据统计方法同前。组织标本制作:
动物麻醉完全后,开胸迅速结扎右主支气管,取右肺中叶4%多聚甲醛固定24小时,脱水、石蜡包埋、连续切片,厚4μm,分别做HE染色、TUNEL标记和免疫组织化学ABC法染色。
嗜酸粒细胞凋亡检测:本实验参照TUNEL试剂盒说明进行操作,主要步骤如下:(1)石蜡切片经二甲苯脱蜡及梯度酒精脱水,PBS(0.01M,pH7.4)漂洗5 min×2次;(2)蛋白酶K(2mg/L)37℃消化15 min, PBS漂洗5 min×3次;(3)DNA平衡液平衡阳性对照片2 min后,加入DNase I, 37℃反应10 min;(4)实验组和阳性对照片分别加入TUNEL工作液(1:19稀释)50μl, 37℃孵育45 min,PBS漂洗5 min×3次;(5)加入碱性磷酸酶(AP)标记的抗荧光素抗体50μl, 37℃孵育30 min,PBS漂洗5 min×3次;(6)经BufferⅢ平衡后,NBT/BCIP显色,室温下暗处显色至阳性对照显色良好时终止呈色反应;(7)缓冲甘油(PBS:甘油=1:3)封片和观察。
bcl-2和bax蛋白表达的检测:采用免疫组织化学ABC法染色(方法同前)。
结果判定及图象处理:光学显微镜下观察,凋亡的嗜酸粒细胞为蓝紫色双核或单核,位于胞核内,胞浆不着色。Bcl-2和Bax蛋白染色呈棕黄色颗粒,位于细胞浆内。每张切片高倍镜下(×400)由同一观察者随机选取5个视野,分别计算支气管壁和肺组织嗜酸粒细胞数量(HE)染色及嗜酸粒细胞凋亡指数(TUNEL法,凋亡嗜酸粒细胞占相应嗜酸粒细胞的百分比,AI),以及胞核bcl-2和bax强阳性的细胞百分数,取平均值作为该切片的代表值。
第五部分:咳喘宁对支气管哮喘大鼠肺组织IL-5 mRNA表达的影响动物分组、模型建立、用药情况以及数据统计方法同前。动物麻醉完全后,开胸迅速结扎右主支气管,取近肺门处的右肺组织约50-100mg,放入匀浆器内,加入1ml的Trizol用匀浆器匀浆,采用RT-PCR法检测各组大鼠肺组织IL-5 mRNA表达的变化。
结果
第一部分:咳喘宁对哮喘大鼠Th1∕Th2细胞因子的调节作用及病理形态学的影响
1咳喘宁对支气管哮喘大鼠支气管-肺组织病理学的影响
正常组:大鼠肺组织支气管内及周围无明显炎性细胞浸润,管壁完整,管壁和平滑肌厚度正常,组织黏膜未见充血水肿,肺泡间隔狭窄,肺泡腔清亮宽敞。
模型组:支气管壁、管腔内及血管、支气管周围有大量嗜酸性粒细胞及淋巴细胞为主的炎性细胞浸润,支气管粘膜水肿、增厚、上皮脱落、微血管渗漏、管腔内分泌物增多,基层细胞增生、平滑肌增厚,组织黏膜充血水肿,肺泡间隔变宽,肺泡腔变狭窄。
桂龙咳喘宁组:肺组织嗜酸性粒细胞及其它炎性细胞浸润略微减少,管壁和平滑肌厚度略微减少,组织充血水肿现象略微减轻。
咳喘宁低剂量组:肺组织嗜酸性粒细胞及其它炎性细胞浸润减少,管壁和平滑肌厚度减少,组织充血水肿现象减轻,肺泡腔变宽,肺泡间隔变窄。
咳喘宁高剂量组:肺组织嗜酸性粒细胞及其它炎性细胞浸润明显减少,几乎消失;管壁和平滑肌厚度明显减少,组织充血水肿现象明显减轻,肺泡腔变宽,肺泡间隔变窄。
2咳喘宁对支气管哮喘大鼠肺组织匀浆IL-4含量的影响
与正常组比较,模型组大鼠肺组织IL-4含量明显升高,具有非常显著性差异(P<0.01);与模型组比较,各治疗组均可降低IL-4含量,咳喘宁高剂量组效果更为明显,具有非常显著性差异(P<0.01);咳喘宁低剂量组和桂龙咳喘宁组,具有显著性差异(P<0.05);与桂龙咳喘宁组比较,咳喘宁高剂量组IL-4含量明显降低,具有显著性差异(P<0.05)。
3咳喘宁对支气管哮喘大鼠肺组织匀浆IFN-γ含量的影响
与正常组比较,模型组大鼠肺组织IFN-γ含量较正常组有所升高,但无显著性差异(P>0.05);与模型组比较,各治疗组均可升高IFN-γ含量,咳喘宁高剂量组效果更为明显,具有非常显著性差异(P<0.01);咳喘宁低剂量组和桂龙咳喘宁组可升高IFN-γ含量,具有显著性差异(P<0.05);与桂龙咳喘宁组比较,咳喘宁高剂量组IFN-γ含量明显增高,具有显著性差异(P<0.05)。
4咳喘宁对支气管哮喘大鼠肺组织IFN-γ∕IL-4比值的影响
与正常组比较,模型组大鼠肺组织IFN-γ∕IL-4比值明显降低,具有非常显著性差异(P<0.01);与模型组比较,各治疗组均可升高IFN-γ∕IL-4比值,具有显著性差异(P<0.05或P<0.01);咳喘宁高剂量组效果更为明显,具有非常显著性差异(P<0.01);与桂龙咳喘宁组比较,咳喘宁高剂量组IFN-γ∕IL-4比值明显增高,具有显著性差异(P<0.05)。
第二部分:咳喘宁对支气管哮喘大鼠炎性介质NO和ET-1的影响
1咳喘宁对支气管哮喘大鼠支气管-肺组织病理学的观察及定量分析:
正常组大鼠肺组织偶见炎细胞浸润,管壁完整,粘膜未见充血水肿,管壁和平滑肌厚度正常;模型组大鼠可见支气管壁及血管、支气管周围有大量嗜酸性粒细胞、淋巴细胞等炎性细胞浸润(P<0.01),支气管粘膜水肿、增厚、上皮脱落、微血管渗漏、管腔内分泌物增多,基层细胞增生、平滑肌增厚(P<0.01);与模型组比较,各治疗组嗜酸性粒细胞、淋巴细胞等炎性细胞浸润明显减少(P<0.01),肺组织充血、水肿现象减轻,管壁和平滑肌厚度明显减少(P<0.01);高低剂量组病理组织学指标改善优于桂龙咳喘宁组(P<0.05或P<0.01)。
2咳喘宁对支气管哮喘大鼠肺组织NO含量的影响
与正常组比较,模型组大鼠肺组织NO含量明显升高,具有非常显著性差异(P<0.01);与模型组比较,各治疗组均可降低NO含量,具有显著性差异(P<0.05或P<0.01);咳喘宁高剂量组效果更为明显,具有非常显著性差异(P<0.01);与桂龙咳喘宁组比较,咳喘宁高剂量组NO含量明显降低,具有显著性差异(P<0.05)。
3咳喘宁对支气管哮喘大鼠肺组织ET-1含量的影响
与正常组比较,模型组大鼠肺组织ET-1含量明显升高,具有非常显著性差异(P<0.01);与模型组比较,各治疗组均可降低ET-1含量,具有显著性差异(P<0.05或P<0.01);咳喘宁高剂量组效果更为明显,具有非常显著性差异(P<0.01);与桂龙咳喘宁组比较,咳喘宁高剂量组NO含量明显降低,具有显著性差异(P<0.05)。
第三部分:咳喘宁对支气管哮喘大鼠核因子-kB(NF-kB)的影响
1咳喘宁对支气管哮喘大鼠气道壁EOS浸润情况比较模型组大鼠的气道壁EOS计数较正常组显著增加(P<0.01),治疗组大鼠的气道壁EOS计数较模型组显著降低(P<0.05或P<0.01),以咳喘宁高低剂量组改善更为显著(P<0.01),其优于桂龙咳喘宁组(P<0.01)。
2咳喘宁对支气管哮喘大鼠肺组织NF-kB表达的影响
正常组大鼠肺组织及气道壁见少许NF-kB阳性表达细胞,模型组大鼠肺组织及气道壁中胞核表达NF-kB的细胞比例显著高于正常组,表现为不均匀的黄褐色颗粒状物(P<0.01),而治疗组肺组织及气道壁中胞核阳性细胞的比例显著低于模型组(P<0.05或P<0.01),以咳喘宁高低剂量组改善更为显著(P<0.01),其优于桂龙咳喘宁组(P<0.01)。
第四部分:咳喘宁对支气管哮喘大鼠嗜酸粒细胞凋亡和相关基因蛋白bcl-2和bax表达的影响
1咳喘宁对支气管哮喘大鼠肺组织EOS凋亡指数的影响
正常组大鼠肺组织及气道壁极少EOS浸润。模型组大鼠的气道壁EOS计数较正常组显著增加(P<0.01),治疗组大鼠的气道壁EOS计数较模型组显著降低(P<0.05或P<0.01),以咳喘宁高低剂量组改善更为显著(P<0.01),其优于桂龙咳喘宁组(P<0.01)。正常组大鼠肺组织及气道壁极少EOS凋亡,治疗组大鼠的气道壁EOS凋亡指数较模型组显著增高(P<0.05或P<0.01),以咳喘宁高剂量组改善更为显著(P<0.01),其作用优于桂龙咳喘宁组(P<0.01)。
2咳喘宁对支气管哮喘大鼠肺组织bcl-2蛋白表达的影响
正常组大鼠肺组织及气道壁见少许bcl-2阳性表达细胞,模型组大鼠肺组织及气道壁中胞浆表达bcl-2的细胞比例显著高于正常组,表现为不均匀的黄褐色颗粒状物(P<0.01),而治疗组肺组织及气道壁中胞浆阳性细胞的比例显著低于模型组(P<0.05或P<0.01),以咳喘宁高剂量组改善更为显著(P<0.01),其优于桂龙咳喘宁组(P<0.01)。
3咳喘宁对支气管哮喘大鼠肺组织bax蛋白表达的影响
正常组大鼠肺组织及气道壁见较多bax阳性表达细胞,表现为不均匀的黄褐色颗粒状物。模型组大鼠肺组织及气道壁中胞浆表达Bcl-2的细胞比例显著低于正常组,表现为较浅的黄褐色颗粒状物(P<0.01),而治疗组肺组织及气道壁中胞浆阳性细胞的比例显著高于模型组(P<0.01),以咳喘宁高剂量组改善更为显著,其优于桂龙咳喘宁组(P<0.01)。
第五部分:咳喘宁对支气管哮喘大鼠IL-5 mRNA表达的影响
哮喘模型组肺组织IL-5 mRNA表达较正常组明显增高,与模型组比较,各用药组肺组织IL-5 mRNA的表达明显降低(P<0.05或P<0.01),以咳喘宁高剂量组更为显著(P<0.01);咳喘宁高剂量组IL-5 mRNA表达含量明显低于桂龙咳喘宁组(P<0.01)。
结论
1咳喘宁能有效地改善支气管哮喘肺组织病理改变,减少肺组织EOS浸润,减轻组织充血、水肿现象,减少支气管管壁和平滑肌厚度,有效地减轻哮喘气道炎症及气道重塑。
2咳喘宁能显著降低支气管哮喘大鼠肺组织IL-4的含量,提高IFN-γ的含量,提高IFN-γ∕IL-4比值,有效地调节哮喘细胞因子网络系统,改善哮喘气道炎症。
3咳喘宁能显著降低支气管哮喘大鼠肺组织NO和ET-1的含量,减少哮喘炎性介质的分泌,改善哮喘气道炎症及气道重塑。
4咳喘宁能显著降低支气管哮喘大鼠肺组织NF-kB的表达,通过调整信号传导途径,促进EOS凋亡,改善哮喘气道炎症。
5咳喘宁能显著降低支气管哮喘大鼠肺组织bcl-2蛋白的表达,升高bax蛋白的表达,通过调整凋亡相关基因的表达,促进EOS凋亡,有效地改善哮喘气道炎症。
6咳喘宁能显著降低支气管哮喘大鼠肺组织IL-5 mRNA的表达,从转录水平降低相关细胞因子含量,减少肺内EOS浸润,促进EOS凋亡,有效地改善哮喘气道炎症。
Objective: On the basis of establishing the rat model with bronchial asthma, through the animal experiments to study the role and mechanism of Kechuanning to immuno-regulation of asthmatic rats. Which were to provide experimental evidence for the clinical medication on treating the bronchial asthma.
Methods
PartⅠ: Effects of Kechuanning on regulatory role of Th1/Th2 cytokines and patho-morphological of the asthmatic rats.
40 clean grade SD rats, half and half for either sex and were 180-200g on weights. These animals were randomly divided into five groups: namely, the normal group, the model group, high dose Kechuanning group (high dose group for short) low dose Kechuanning group (low-dose group for short), Guilong Kechuanning control group (Guilong Kechuanning group for short) with 8 rats for each group. The drugs were given through stomach-perfusion to each treatment group from the beginning of the 1st asthma provocation (third weeks after making model) to the day before killing and continued for four weeks. The dose was: 27g/Kg for the high-dose group and 13.5g/Kg for the low dose group, 0.41 g/Kg for Guilong Kechuanning group and 0.5% sodium carboxymethyl cellulose were given to the normal and model group through stomach-perfusion with one time per day.
According to the report, in addition to the normal group, in the first and eighth days after experiment, 1 ml mixture of 10% of egg protein and the aluminum hydroxide were injected intra-peritoneally to the rats from each group, one point was prepared from both sides of waist with subcutaneous injection was 0.5 ml for per point and it was 2 ml in total. All these were to sensitize rats. From 15th days, 50 ml of 3% egg protein was used for spray to provoke the rat asthma; the aerosol flow was 5ml/minutes with 20 minutes for each time, once for every other day and continued for four weeks in total. The manifestations of the provocative success were that there were fast breathing, cyanotic lips, abdominal muscle cramps, nodding breathing and unstable standing etc. In the normal group, the normal saline was used to replace the egg protein for intraperitoneal injection and spray inhalation.
Histopathological observation: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of the right lung was taken and fixed with 4% paraformaldehyde and gave the dehydration, embedding and sections, then, the hematoxylin - eosin HE staining. The pathological changes were observed microscopically and NYD1000 computer image processing software was used.
IL-4 and IFN-γlevel test: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The upper lobe of right lung was taken and put into the glass homogenate tube after weighing, adding the normal saline which were 2 times of the weight of lung tissue and gave the full levigation and make the tissue to be homogenized. Then, the prepared homogenate was given the centrifugation with 3500r/min for 10 minutes, after that, the supernatants was extracted and gave subpackage. IL-4 and IFN-γlevel were measured with the double-antibody sandwich ABC-ELISA method. The enzyme label plates were coated with IL-4 (IFN-γ) monoclonal antibody and the plates were washed with PBS for 4 times. Then, 1%BSA (bovine serum albumin) was added to enzyme label plate (200μl/ hole) and kept 4°C overnight. Later, adding the samples and the standard with different concentrations (100μl/ hole) and gave 120 minutes incubation at room temperature. Thus, it formed the immune compounds and connected with the plate, then, the plates were washed with PBS for 4 times and added horseradish peroxidase labeled anti-IL-4 monoclonal antibody (100μl/hole) and gave 60 minute’s incubation at room temperature, the plates were washed again with PBS for 4 times and added OPD substrate solution and kept in room temperature for 10-30 minutes with being away from light, there was yellow. At this point, added the sulfuric acid of stopping solutions and mixed evenly, the solution became darker in color in five minutes. Absorbance OD value was measured at 490 nm with enzyme sign analyzer and drew the specimen’s curves and searched out the specimen’s concentration.
The data was expressed with mean±standard deviation (±s) and was processed with the statistical software- SPSS for Windows 11.5. Single factor variance analysis was used to compare the each group. Then, Student-Newman-Keuls Test was used for the comparison between each two groups and the significant difference level took 0.05 and 0.01 as the standard.
Part II: the influence of Kechuanning on the inflammatory mediators NO and ET-1 of rats with bronchial asthma.
Histopathological observation: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of the right lung was taken and fixed with 4% paraformaldehyde and gave the dehydration, embedding and sections, then, the hematoxylin - eosin HE staining. The pathological changes were observed microscopically and the area of the rats airway wall were measured in the light of the literature and the intratracheal perimeter were standardized. NYD1000 image analysis software was used to measure the internal circumference (Pi)of intact bronchial lumen, bronchial wall area (WA), bronchial smooth muscle area. Pi was used for standardization. WA/Pi and the bronchial smooth muscle area/ Pi expressed the bronchial wall thickness and bronchial smooth muscle thickness respectively. At the same time, the number of EOS and the lymphocytes from each high-power field (400 times) were measured.
NO and ET-1 content test: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The upper lobe of right lung was taken and put into the glass homogenate tube after weighing, adding the normal saline which were 2 times of the weight of lung tissue and levigating it fully and making it homogenizate. Then, the prepared homogenate was centrifugated with 3500r/min for 10 minutes, after that, the supernatants was extracted and the NO content of lung tissue were measured with nitric acid reductase method. The ET-1 content of lung tissue was measured with radio immunity and was calibrated with the protein content. Animal grouping, making model, drug administration and the statistical methods of data are same as the former.
Part III: The influence of Kechuanning on the nuclear factor-kB(NF-kB) of rats with bronchial asthma.
Animal grouping, making model, drug administration and the statistical methods of data are same as the former.
EOS counts of airway wall: the method of making pathological tissue section was the same with before and NYD1000 computer image-processing software was used. A rather complete structure of the bronchial wall was selected and five high-power fields (×400) were chosen randomly, and the number of infiltrating EOS from the bronchial wall was calculated by the same observer,and the averaged value represent as the value of the section.
The measurement of NF-kB expression: after complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of right lung was taken and fixed 24 hours with 4% paraformaldehyde and gave the dehydration, paraffin embedding and serial sections with 4μm in thickness. ABC staining of immunohistochemistry was made .The primary antibody was mouse anti-rat P65 monoclonal antibody (F-6), the secondary antibody was the biotinylated horse anti-mouse IgG. Main operation steps are as follows: (1) through xylene dewaxing and gradient alcohol dehydration, the paraffin sections were rinsed with PBS for 5 minutes×2 times; (2) incubating it in 3% hydrogen peroxide and methanol solution for 5-10 minutes at room temperature in order to remove the activity of endogenous peroxidase, and rinsing it with PBS for 3 times; (3) heat retrieval antigen: the slice were immersed in 0.01M citrate buffer solution (pH 6.0) and after heating it to boil with the microwave oven, turning power off with 5-10 minute’s interval, repeating 1-2 times. The material was washed with PBS for 2 times after cooling; (4) dropping 5% normal goat serum for blocking and being incubated for 20 minutes at room temperature. Then, pouring the redundant liquid; (5) dropping NF-kBⅠantibody (1:100 dilution)for wet case incubation. Keeping it in 4℃refrigerator overnight and washing in PBS for 5 minutes×2 times;(6) dropping the secondary antibody working fluid with biotinated label and incubating it in 37℃for 45 minutes, then washing it in PBS for 5 minutes×2 times;(7) Dropping SABC reagents (1:100 dilution), then incubating it for 20 minutes and washing it in PBS for 5 minutes×2 times; (8) giving DAB color development for 5-15 minutes, then, stopping the color development; (9) Hematoxaylin staining, the conventional mounting slices, dryness, dehydration, vitrification and mounting. The slice was observed with the optical microscope. A rather complete structure of the bronchial wall was selected and five high-power fields (×400) were chosen by the same observer randomly, which was to count the positive cell percentage of the karyon NF-kB from the bronchial wall and took its average value as the representative value of the section.
Part IV: The influence of Kechuanning on the eosinophilic granulocyte apoptosis and bcl-2 and bax expression of the relative gene from the rats with asthma
Animal grouping, making model, drug administration and the statistical methods of data are same as the former.
Tissue sample making:
After complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The middle lobe of the right lung was taken and fixed for 24 hours with 4% paraformaldehyde and gave the dehydration, paraffin embedding and serial sections with 4μm in thickness, then, the hematoxylin - eosin HE staining and TUNEL marking and immunohistochemical ABC staining.
Eosinophilic granulocyte apoptosis detection: this experiment is carried out in the light of TUNEL reagent kit and the major steps as follows: (1) through xylene dewaxing and gradient alcohol dehydration, the paraffin sections were rinsed with PBS(0.01M, pH7.4) for 5 minutes×2 times (2) proteinase K (2mg/L)digested it in 37℃for 15 minutes, then rinsed in PBS for 5 minutes×3 times (3) after balancing the positive contrast with DNA equilibrium liquid for 2 minutes, added DNase I for reaction in 37℃for 10 minutes (4) TUNEL working fluid (1 : 19 dilution) 50μl were added respectively to the experimental group and positive contrast and was incubated in 37℃for 45 minutes, then, rinsing them with PBS for 5 min×3 times. (5)thus, adding 50μl anti-fluorescein antibody with alkaline phosphatase (AP) label and being incubated in 37℃for 30 minutes, then , rinsing them with PBS for 5 min×3 times. (6) Through Buffer III balance and NBT/BCIP color development, the color development in dark at room temperature got to positive and good control color development, the color reaction can be stopped (7) mounting is taken with glycerol buffer (PBS: glycerol 1:3) and observating.
The detection of bcl-2 and bax protein expression: immunohistochemical ABC staining (same as the former method) was used.
Results verdict and image processing: it was observed with the optical microscope, the apoptostic eosinophilic granulocyte was the blue-purple binuclear or mononuclear, which was located in the nuclei with no staining for the cytoplasm. Bcl-2 and Bax protein staining showed brown yellow granules and were in the cytoplasm. Five fields were chosen by the same observer randomly from each section under high-power microscope (×400), then, the number (HE) of eosinophilic granulocyte from the bronchial wall and lung tissue, the apoptostic index of staining and eosinophilic granulocyte (TUNE L method, the percentage of the apoptostic eosinophilic granulocyte that occupied in the corresponding eosinophilic granulocyte AI) and the percentage of strong positive cells of Bcl-2 and Bax nuclei were calculated respectively, the averaged value was taken as the representative value of the section.
Part V: The influence of Kechuanning on the IL-5 mRNA expression of lung tissue from the rats with bronchial asthma
Animal grouping, making model, drug administration and the statistical methods of data are same as the former.
After complete anesthesia, the thoracotomies were given to the animals and the right main bronchi were ligated rapidly. The right lung that was adjacent to the porta was taken for about 50-100mg, then puting it into the homogenizer, adding 1 ml Trizol and homogenatizating by homogenizer. The IL-5 mRNA expression changes of the lung tissue in rats from each group were detected by RT-PCR method.
Results
PartⅠ: The regulatory role and the patho-morphological effects of Kechuanning to Th1/Th2 cytokines from the asthmatic rats.
1. The pathological effects of Kechuanning to the bronchial-lung tissue of rats with bronchial asthma.
Normal groups: there was no obvious inflammatory cell infiltration from the bronchus of lung tissues and surroundings in rats with intact wall, the thickness of wall and smooth muscle were normal with no congestion and edema in the tissue mucosa, the alveolar septum was narrow and the alveolar cavity was clear and spacious.
Model group: there were many inflammatory cells infiltration in bronchial wall and lumen as well as around blood vessel and bronchus, which mainly were the eosinophilic granulocyte and lymphocyte, accompanied with bronchial mucosal edema, thickening, epithelial exfoliation, microvascular leakage, the secretion increased within lumen stromal cell hyperplasia, thickened smooth muscle, the congestive edema of tissue mucosa, widened alveolar septum and narrowed alveolar lumen.
Guilong Kechuanning groups: there were slight decrease for the eosinophilic granulocyte and other inflammatory cell infiltration from lung tissue, also, there were slight decrease for the smooth muscle wall thickness with the congestion and edema from tissue got slight reduction.
Low-dose Kechuanning group: the eosinophilic granulocyte and other inflammatory cell infiltration from lung tissue decreased, also, there were decrease for the smooth muscle wall thickness with the congestion and edema from tissue got slight reduction, the alveolar lumen widened with the narrowing of alveolar septum.
High-dose Kechuanning group: the eosinophilic granulocyte and other inflammatory cell infiltration from lung tissue decreased obviously and almost disappeared, also, there were marked decrease for the wall and smooth muscle thickness with the congestion and edema from tissue got obvious reduction, the alveolar lumen widened with the narrowing of alveolar septum.
2. The influence of Kechuanning on the homogenate IL-4 content of lung tissues from the rats with bronchial asthma.
Compared with the normal group, IL-4 content of rat lung tissues from the model group increased significantly with very significant difference (P<0.01). Compared with the model group, IL-4 content can be lowered from each treatment group and it was even more obvious for the high-dose Kechuanning group with very significant difference (P<0.01). The low-dose Kechuanning group and Guilong Kechuanning group had significant difference (P<0.05). Compared with the control group, IL-4 content from the high-dose Kechuanning group was significantly lower than the control group with significant differences (P<0.05).
3. The influence of Kechuanning on the homogenate IFN-γcontent of lung tissues from the rats with bronchial asthma.
Compared with the normal group, IFN-γcontent of rat lung tissues from model group had slight increase(P>0.05). Compared with the model group, IFN-γcontent can be increased from each treatment group and it was even more obvious for the high-dose Kechuanning group with very significant difference (P<0.01). IFN-γcontent can be elevated for the low-dose Kechuanning group and Guilong Kechuanning group with the significant difference (P < 0.05). Compared with the control group of Guilong Kechuanning, IFN-γcontent from the high-dose Kechuanning group was significantly higher than control group with significant differences (P<0.05).
4. The influence of Kechuanning on IFN-γ∕IL-4 ratio of lung tissues from the rats with bronchial asthma.
Compared with the normal group, IFN-γ∕IL-4 ratio of rat lung tissues from the model group decreased obviously with very significant difference (P<0.01). Compared with the model group, IFN-γ∕IL-4 ratio can be elevated from each treatment group with very significant difference (P<0.05). It was even more obvious for the high-dose Kechuanning group and it was with very significant difference (P<0.01). Compared with the control group, IFN-γ∕IL-4 ratio from the high-dose Kechuanning group was significantly higher than that of control group with significant differences (P<0.05).
Part II: The influence of Kechuanning on the inflammatory mediators NO and ET-1 of rats with bronchial asthma.
1. The histopathological observation and the quantitative analysis for the rats with bronchial asthma and treating with Kechuanning There were less inflammatory cell infiltration in the lung tissue of rats from normal group with intact vessel wall, there was no congestion and edema in the mucosa, the thickness of vessel wall and the smooth muscle were normal. In the rats from model group, there were plenty of eosinophilic granulocyte and lymphocyte and other inflammatory cell infiltration(P<0.01) from the bronchial wall and around blood vessel and bronchus, also, there were bronchial mucosal edema, thickening, epithelial exfoliation, microvascular leakage, the secretion increased within lumen,stromal cell hyperplasia ,smooth muscle thickening(P<0.01), Compared with the model group, there were obvious decrease for the eosinophilic granulocyte and lymphocyte and other inflammatory cell infiltration from each treatment group, the congestion and edema of the lung tissue were also mitigated and the thickness of vessel wall and the smooth muscle were reduced obviously(P<0.01).The improvement on histopathological indices from high-dose group were better than control group(P<0.05 or P<0.01).
2. The influence of Kechuanning on NO content of lung tissues from the rats with bronchial asthma.
Compared with the normal group, NO content of rat lung tissues from the model group increased obviously with very significant difference (P<0.01). Compared with the model group, NO content can be decreased from each treatment group with very significant difference (P<0.05). The result was even more obvious for the high-dose Kechuanning group and it was also with very significant difference (P<0.01). Compared with the control group of Guilong Kechuanning group, NO content from the high-dose Kechuanning group was significantly lower than that of control group with significant differences (P<0.05).
3. The influence of Kechuanning on ET-1 content of lung tissues from the rats with bronchial asthma
Compared with the normal group, ET-1 content of rat lung tissues from the model group increased obviously with very significant difference (P <0.01). Compared with the model group, ET-1 content can be decreased from each treatment group with very significant difference (P<0.05). The results were even more obvious for the high-dose Kechuanning group and it was also with very significant difference (P<0.01). Compared with the control group of Guilong Kechuanning group,ET-1 content from the high-dose Kechuanning group was significantly lower than that of control group with significant differences (P<0.05).
Part III: The influence of Kechuanning on the nuclear factor-kB (NF-kB) of rats with bronchial asthma
1. The comparison of EOS infiltration of airway wall of rats with bronchial asthma with Kechuanning.
Compared with the normal group, the EOS count in airway wall of rats from the model group increased significantly(P<0.01),compared with model group,the EOS count of airway wall in the rats from the treatment group decreased significantly(P<0.05),the improvements from the high and low dose Kechuanning groups were more significant(P<0.01) ,better than control group(P<0.01).
2. The influence of Kechuanning on the NF-kB expression of lung tissue of rats with bronchial asthma
Less positive expression of NF-kB cell can be seen in the lung tissue and airway wall of rats from normal group ,the cell proportion of karyon NF-kB expression in the lung tissue and airway wall of rats from model group were significantly higher than that of the normal group, which were manifested by the unsymmetric brown yellow particulates(P<0.01), however, in the rats from the treatment group, the proportion of positive cell of karyon in the lung tissue and airway wall were significantly lower than that of the model group(P<0.05), the improvements from the high and low dose Kechuanning groups were more significant(.P<0.01), which were better than that of Guilong Kechuanning group(P<0.01).
Part IV: The influence of Kechuanning on the eosinophilic granulocyte apoptosis and Bcl-2 and Bax expression of the relative gene from the rats with asthma
1. The influence of Kechuanning on EOS apoptosis index of lung tissue from the rats with bronchial asthma. There was little EOS infiltration in lung tissue and airway wall of the rats from normal group. Compared with the normal group, the EOS count in airway wall of rats from the model group increased significantly(P<0.01);compared with the model group , the EOS count in airway wall of rats from the treatment group decreased significantly(P<0.05), the improvements from the high and low dose Kechuanning groups were more significant(P<0.01),which were better than control group (P<0.01). There was little EOS apoptosis in lung tissue and airway wall of rats from the normal group. Compared with the model group, the EOS apoptosis index in airway wall of rats from the treatment group increased significantly (P<0.05), the improvements from the high dose Kechuanning group were more significant (P<0.01),which were better than control group (P<0.01).
2. The influence of Kechuanning on Bcl-2 proteins expression of the lung tissue of rats with the bronchial asthma
Less Bcl-2 positive expression cells can be seen in the lung tissue and airway wall of rats from normal group, the proportion of cell that expressing Bcl-2 in kytoplasm in the lung tissue and airway wall of rats from the model group were significantly higher than that of the normal group ,which were manifested by the unsymmetric brown yellow particulate(sP<0.01), however, in the rats from the treatment group, the proportion of positive kytoplasm cell in the lung tissue and airway wall were significantly lower than that of the model group(P<0.05), the improvements from the high dose Kechuanning groups were more significant(P<0.01),which were better than that of Guilong Kechuanning group(P<0.01).
3. The influence of Kechuanning on Bax proteins expression of the lung tissue of rats with the bronchial asthma.
More Bcl-2 positive expression cells can be seen in the lung tissue and airway wall of rats from normal group, which were manifested by the unsymmetric brown yellow particulates .The proportion of cell that expressing Bcl-2 in kytoplasm.
In the lung tissue and airway wall of rats from model group were significantly lower than that of the normal group, which were manifested by the shallow brown yellow particulate(sP<0.01), however, in the rats from the treatment group, the proportion of positive kytoplasm cell in the lung tissue and airway wall were significantly lower than that of the model group(P<0.05), the improvements from the high dose Kechuanning groups were more significant(P<0.01),which were better than control group (P<0.01).
Part V: The influence of Kechuanning on IL-5 mRNA expression of rats with the bronchial asthma
Compared with the normal group, IL-5 mRNA expression of lung tissue in asthmatic model group increased significantly; compared with the model group, IL-5 mRNA expression of lung tissue decreased significantly from each medication group(P<0.05),which was more significant for the high dose Kechuanning group(P<0.01), the level of IL-5 mRNA expression from the high dose Kechuanning group were much lower than control group (P<0.01).
Conclusion
1. Kechuanning could effectively improve the pathological changes of lung tissue in bronchial asthma,reduce EOS infiltration in lung tissue, decrease the tissue congestion and edema, reduce the thickness of the bronchial wall and smooth muscle, effectively reduce asthmatic airway inflammation and airway remodeling.
2. Kechuanning could significantly reduce the IL-4 content in the lung tissue of rats with bronchial asthma, increase IFN-γcontent and the ratio of IFN-γ? I L-4, adjust effectively the asthmatic cytokines network system, improve asthmatic airway inflammation.
3. Kechuanning could significantly reduce NO and ET-1 content in lung tissue of rats with bronchial asthma, reduces the secretion of inflammatory mediators in asthma, improve airway inflammation in asthma and airway remodeling.
4. Kechuanning could significantly reduce NF-kB expression of lung tissue of rats with bronchial asthma and inhibit EOS apoptosis and improve airway inflammation in asthma through adjusting the signal transmission route.
5. Kechuanning could significantly reduce bcl-2 protein expression and increase bax protein expression of lung tissue of rats with bronchial asthma and inhibit EOS apoptosis and effectively improve airway inflammation in asthma through adjusting the expression of apoptosis-related genes.
6. Kechuanning could significantly reduce the IL-5 mRNA expression of lung tissue of rats with bronchial asthma, reduce the content of related cytokines by transcription level, reduce EOS infiltration in the lungs, inhibit EOS apoptosis and effectively improve airway inflammation in asthma.
引文
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