柯里拉京对脓毒症过程中TLR4信号通路的干预研究

发布时间：2018-05-11 13:26

本文选题：柯里拉京 + RAW264.7细胞　；参考：《华中科技大学》2014年博士论文

【摘要】：目的细胞模型上研究柯里拉京对TLR4信号通路的作用及其机制。方法：脂多糖(LPS)刺激RAW264.7细胞造成炎症模型。予以柯里拉京进行干预。CCK8法检测细胞存活率；实时荧光定量聚合酶链反应检测各组TLR4, MyD88, TRIF, TRAF6的mRNA的表达水平；Western blot测定各组TLR4信号通路中各信号分子蛋白的表达；ELISA法检测细胞上清中的IL-10, IL-6;结果：1.CCK8法检测的柯里拉京IC50值是0.25mg/ml;2.柯里拉京干预后,TLR4信号通路中TLR4, MyD88, TRIF, TRAF6的mRNA和蛋白水平,模型组显著高于正常组(p＜0.01),而柯里拉京处理组显著低于模型组(p＜0.01)；3.柯里拉京干预后,TLR4信号通路中产生的促炎因子IL-6, IL-1β,模型组显著高于正常组(p＜0.01),而柯里拉京处理组显著低于模型组(p＜0.01)；结论：在细胞模型上,柯里拉京可通过抑制TLR4信号通路中TLR4, MyD88, TRIF, TRAF6的表达,从而抑制促炎因子IL-6,IL-1β的释放。目的：细胞模型上研究柯里拉京对TLR4-MyD88信号通路的作用及其机制。方法：采用小分子RNA干扰技术沉默TICAM1的表达,脂多糖(LPS)刺激RAW264.7细胞造成炎症模型。予以柯里拉京进行干预。实时荧光定量聚合酶链反应检测各组TICAMl的表达,及柯里拉京干预后TLR4, TRAF6, MyD88的mRNA的表达水平；结果：1.实时荧光定量聚合酶链反应检测TICAM1siRNA转染细胞后TICAM1mRNA表达水平,与正常组相比siRNA表达量明显降低(P0.01);2.柯里拉京干预后,TLR4-MyD88信号通路中TLR4,MyD88, TRAF6的mRNA模型组显著高于正常组(P＜0.01),而柯里拉京处理组显著低于模型组(P＜0.01)；结论：在细胞模型上,柯里拉京可抑制TLR4-MyD88信号通路中TLR4, MyD88, TRIF, TRAF6的表达, 目的：动物模型上研究柯里拉京对TLR4信号通路的作用及其机制。方法：脂多糖(LPS)腹腔注射Balb/c雄性小鼠造成脓毒症模型。予以柯里拉京进行干预。HE染色检测小鼠肝,脾,肺,肾,结肠及脑组织的病理改变；实时荧光定量聚合酶链反应检测各组TLR4,MyD88,TRIF,TRAF6, HMGB1的mRNA的表达水平；Western blot测定各组TLR4信号通路中各信号分子蛋白的表达；ELISA法检测细胞上清中的IL-1β,IL-6;结果：1.HE染色病理切片示,模型组小鼠均有不同程度的病理损伤,而柯里拉京处理组,能一定程度上改善各个脏器的炎症,坏死,水肿等情况；2.柯里拉京干预后,TLR4信号通路中TLR4,MyD88,TRIF,TRAF6, HMGB1的mRNA和蛋白水平,模型组显著高于正常组(p0.01),而柯里拉京处理组显著低于模型组(p0.01)；3.柯里拉京干预后,TLR4信号通路中产生的促炎因子IL-6,IL-1β,模型组显著高于正常组(p0.01),而柯里拉京处理组显著低于模型组(p0.01)；结论：在动物模型上,柯里拉京可通过抑制TLR4信号通路中TLR4,MyD88,TRIF,TRAF6, HMGB1的表达,从而抑制促炎因子IL-6,IL-1β的释放。
[Abstract]:Objective to study the effect of Coriragin on TLR4 signaling pathway and its mechanism. Methods: lipopolysaccharide (LPS) stimulated RAW264.7 cells to establish inflammatory model. The cell survival rate was measured by Corilagine intervention. CCK8 method. The expression level of mRNA in TLR4, MyD88, TRIFand TRAF6 was detected by real-time fluorescence quantitative polymerase chain reaction and the expression of each signal molecule protein in TLR4 signal pathway in each group was detected by Western blot. Results: 1. The IC50 value of Coriragin detected by CCK8 method was 0.25 mg / ml ~ (2), and the expression of IL-10 and IL-6 in cell supernatant was detected by Elisa. The mRNA and protein levels of TLR4, MyD88, trif, TRAF6 in the model group were significantly higher than those in the normal group (P < 0.01), but significantly lower in the Coriragin treated group than in the model group (P < 0.01). IL-6 and IL-1 尾 produced in TLR4 signaling pathway after Corilagine intervention were significantly higher in the model group than in the normal group (p < 0.01), but significantly lower in the Coriragin treated group than in the model group. Conclusion: in the cell model, the level of IL-6 and IL-1 尾 in the model group is significantly lower than that in the model group. Coriragin can inhibit the release of IL-6 and IL-1 尾 by inhibiting the expression of TLR4, MyD88, trif, TRAF6 in TLR4 signaling pathway. Aim: to study the effect of Coriragin on TLR4-MyD88 signaling pathway and its mechanism. Methods: small molecular RNA interference technique was used to silence the expression of TICAM1, and lipopolysaccharide (LPS) stimulated RAW264.7 cells to create inflammatory model. Coriragin was given intervention. The expression of TICAMl and the mRNA levels of TLR4, TRAF6 and MyD88 in each group were detected by real-time fluorescence quantitative polymerase chain reaction, and the results showed that the expression level of TLR4, TRAF6 and MyD88 was 1: 1. Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression of TICAM1mRNA in TICAM1siRNA transfected cells. Compared with the control group, the expression of siRNA was significantly lower than that of the control group. The expression of TLR4-MyD88 in mRNA model group was significantly higher than that in normal group (P < 0.01), while that in Coriragin treatment group was significantly lower than that in model group (P < 0.01). Conclusion: in cell model, Coriragin can inhibit the expression of TLR4, MyD88, trif, TRAF6 in TLR4-MyD88 signaling pathway. Aim: to study the effect and mechanism of Coriragin on TLR4 signaling pathway in animal models. Methods: Balb/c male mice were injected intraperitoneally with lipopolysaccharide (LPS) to establish septic model. The pathological changes of liver, spleen, lung, kidney, colon and brain of mice were detected by HE staining. Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the expression level of TLR4, MyD88-TRIFTRAF6, and the level of mRNA in HMGB1. The expression of signal molecular proteins in the TLR4 signaling pathway was detected by Western blot and the expression of IL-1 尾 -IL-6 in the supernatant was detected by Elisa. Model group mice have different degrees of pathological injury, while Corilagine treatment group, to some extent, can improve the inflammation, necrosis, edema and other conditions of various organs. 2. The levels of mRNA and protein of TRAF6, HMGB1 in TLR4 / TLR4 signaling pathway were significantly higher in model group than in normal group (P 0.01), but significantly lower in Coriragin treatment group than in model group. IL-6 and IL-1 尾 produced in TLR4 signaling pathway were significantly higher in the model group than in the normal group (P 0.01), while that in the Coriragin treated group was significantly lower than that in the model group. Conclusion: in the animal model, the expression of IL-6 and IL-1 尾 in the TLR4 signaling pathway was significantly lower than that in the control group. Coriragin could inhibit the release of IL-6 and IL-1 尾 by inhibiting the expression of TRIFF6, HMGB1 in the TLR4 signaling pathway.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R459.7

【共引文献】