三棱丸方对克罗恩病肠纤维化的影响及机制研究

发布时间：2018-01-08 17:03

本文关键词：三棱丸方对克罗恩病肠纤维化的影响及机制研究　出处：《南京中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：目的使用克罗恩病肠纤维化大鼠模型观察三棱丸方对大鼠肠纤维化的影响,观察三棱丸在转化生长因子β1(TGF-β1)所诱导的肠上皮细胞(IEC-6)上皮间质转化(EMT)过程中对纤维化相关因子及PPAR-γ表达的影响,并探讨三棱丸方通过TGFβ1/Smads信号转导途径干预肠纤维化的EMT的机制。方法体内实验部分:将48只SD大鼠随机均分为空白组、模型组、罗格列酮对照组、三棱丸方高、中、低剂量治疗组,组除空白组外,后五组采每周一次给予5%的三硝基苯磺酸(trinitrobenzene sulphonic acid TNBS),1Omg 于第 1 天,15mg 于第 8 天,20mg 于第 15 天,25mg于第22天,30mg于第29天,分别配成50%乙醇溶液0.8ml 一一次性灌肠,逐渐增量灌肠法诱导肠纤维化模型;从造模开始,大鼠同时每日分别以10.Og/kg,5.0g/kg,2.5g/kg不同浓度的三棱丸方水煎剂灌胃,罗格列酮组以8mg/kg浓度罗格列酮灌胃,每天一次;模型组、正常组给予等量生理盐水灌胃。期间观察各组大鼠的一般情况,35天后收集结肠组织,用于病理学评价,并检测结肠组织中TGF-β1、E-cadherin、α-SMA、FN、CTGF及PPARy水平。体外实验部分:以TGF-β1诱导IEC-6细胞形成的EMT模型为研究对象,用Western blot法检测高、中、低剂量的三棱丸方含药血清及罗格列酮作用于1EC-6细胞后PPARγ、Smad2/3及pSmad3蛋白的表达,RT-PCR法检测E-cadherin、α-SMA的mRNA的表达;免疫荧光法观察三棱丸含药血清及罗格列酮对PPARγ、pSmad3入核水平的影响,并检测加入PPARγ拮抗剂GW9662后,上述相关指标发生的变化。结果体内实验研究:与模型组比较,三棱丸方中、高剂量组的疾病活动指数(DAI)评分、结肠大体评分、组织学纤维化评分均明显低于模型组(P0.05),Masson染色显示三棱丸方高剂量组胶原纤维含量明显低于模型组;E-cadherin、PPARγ含量均较模型组明显增加(P0.05),TGF-[β1、FN、α-SMA水平均明显降低(P0.05)。体外实验研究:与10%正常大鼠血清组比较,三棱丸含药血清组能够抑制α-SMA,促进E-cadherin、PPARγ的mRNA的表达;提高E-cadherin、PPAR-γ,降低pSmad3、α-SMA的蛋白水平;免疫荧光结果显示三棱丸含药血清组促进PPARγ入核,抑制pSmad3的入核;加入PPARy拮抗剂GW9662后,逆转了上述指标的变化趋势。结论三棱丸方能改善大鼠CD肠纤维化模型的纤维化程度,对IEC-6细胞的EMT有抑制的作用;其治疗克罗恩病肠纤维化的机制可能是和激活PPAR-γ来抑制TGF-β1/Smads的信号通路相关。
[Abstract]:The purpose of the use of Crohn's disease intestinal fibrosis rat model to observe the effect of sanlengwan Decoction on intestinal fibrosis in rats, observe the sanlengwan in transforming growth factor beta 1 (TGF- beta 1) induced by intestinal epithelial cells (IEC-6) of epithelial mesenchymal transition (EMT) process of fibrosis related factors and PPAR- expression. And to explore the mechanism of TGF beta sanlengwan through 1/Smads signal transduction pathway intervention of intestinal fibrosis in vivo EMT. Methods: 48 SD rats were randomly divided into control group, model group, rosiglitazone group, Sanleng pill high, low dose treatment group, group except the blank group, after five group each week for 5% three trinitrobenzene sulfonic acid (trinitrobenzene sulphonic acid TNBS 1Omg), on the first day, 15mg on the eighth day, 20mg on the fifteenth day, 25mg on the twenty-second day, 30mg on the twenty-ninth day, respectively, with 50% 0.8ml ethanol solution of a disposable enema, gradually increase The amount of enema induced intestinal fibrosis model; from the start of the model, the rats of the day were 10.Og/kg, 5.0g/kg, 2.5g/kg different concentrations of sanlengwan Decoction orally, rosiglitazone group with 8mg/kg concentration of rosiglitazone orally, once a day; the model group and normal group were given equal volume of saline. During the period of observation the situation of the rats were collected 35 days after colon tissue for pathological evaluation, and detection of colonic tissue TGF- beta 1, E-cadherin, FN, alpha -SMA, CTGF and PPARy. In vitro experiment: TGF- beta 1 induced IEC-6 cells to form EMT model as the research object, using the Western blot assay. In the low dose of sanlengwan medicated serum and rosiglitazone on 1EC-6 cell by PPAR, expression of Smad2/3 and pSmad3 protein, E-cadherin RT-PCR detection method, the expression of alpha -SMA mRNA; immunofluorescence observation of serum and Roger sanlengwan containing columns Ketone of PPAR gamma, pSmad3 effect into the level of the nucleus, and detected by PPAR gamma antagonist GW9662, change the relevant indicators. The experimental results of in vitro: compared with model group, three pills in high dose group disease activity index (DAI) score, colonic general score, histological fibrosis scores were significantly lower than the model group (P0.05), Masson staining showed that the three pill high dose group collagen fiber content was significantly lower than the model group; E-cadherin, PPAR gamma content increased than model group (P0.05), TGF-[beta 1, FN, alpha -SMA levels were decreased significantly (P0.05). In vitro study: compared with 10% normal rat serum group, sanlengwan medicated serum can inhibit alpha -SMA, promote the expression of PPAR E-cadherin and mRNA E-cadherin gamma; improve, reduce pSmad3, PPAR- gamma, alpha -SMA protein level; immunofluorescence results showed sanlengwan containing serum promote PPAR gamma nuclear entry, suppression PSmad3 into the nucleus; adding PPARy antagonist, GW9662, reversed the trend of the index. The degree of fibrosis conclusion sanlengwan decoction can improve the intestinal fibrosis in rats with CD model, on IEC-6 cell EMT has inhibiting effect; the treatment of Crohn's disease and intestinal fibrosis mechanism may be the activation of signal transduction pathways to PPAR- gamma inhibition of TGF- beta 1/Smads.

【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

【相似文献】