丙酮酸乙酯对四氯化碳诱导大鼠肝纤维化保护作用的研究

发布时间：2018-01-08 00:19

本文关键词：丙酮酸乙酯对四氯化碳诱导大鼠肝纤维化保护作用的研究　出处：《成都医学院》2016年硕士论文　论文类型：学位论文

【摘要】：肝纤维化(liver fibrosis)是机体对各种刺激损伤修复的动态过程,是细胞外基质(ECM)产生和降解失平衡所致细胞外基质过度沉积的一种状态。基质金属酶(MMPs)及其抑制剂(TIMPs)的平衡影响ECM的降解。作为肝脏纤维发生的主要效应细胞,肝星状细胞(HSCs)与肝内其他宿主细胞如肝细胞、肝巨噬细胞、内皮细胞等相互作用促进纤维化的发生、发展。HSCs处于激活状态以及维持存活需要一些信号调控。这些信号受多因子调控,既有来自HSCs自分泌,也有旁分泌。HSC中,TOLL样受体4(TLR4)诱导炎症趋化分子和粘附分子的表达,核转录因子(NF-κB)通过各种刺激和细胞应激而激活,调控炎症反应,因此他们在慢性肝病中发挥主要作用。丙酮酸乙酯(ethyl pyruvate,EP)具有抗炎性能在各种损伤模型中发挥保护作用。目的通过皮下注射四氯化碳(CCl4)建立肝纤维化模型,观察EP干预后不同时间点大鼠肝纤维化进展,以及肝脏星状细胞激活标记物(α-SMA)、TLR4、NF-κB、MMPs及TIMPs和胶原纤维1、3、4的表达变化,初步探讨EP抑制肝纤维化中星状细胞激活以及加速胶原降解,从而逆转肝纤维化的机理。方法健康成年SD大鼠,随机分为三组:对照组(Control)、四氯化碳模型组(CCl4)、丙酮酸乙酯干预组(CCl4+EP)。采用皮下注射CCl4,每周两次,共8周,制备纤维化模型;EP干预处理,每天一次;于2、4、6、8周四个时间点中最后一次注射CCl4的48h后,分别取大鼠肝脏样本。统计第8w腹水状况。HE、MASSON和ISHAK评分染色用于评定肝脏病理变化及纤维化程度。Q-PCR用于检测TLR4、NF-κB、MMPs、TIMPs和胶原纤维1、3、4mRNA的表达变化,ELISA检测肝组织中α-SMA、TLR4、MMP2、TIMP2、胶原蛋白4的含量变化;免疫组化用于检测α-SMA、TLR4、NF-κB、MMPs、TIMPs和胶原纤维1、4阳性细胞的表达变化以及细胞定位。结果1.大鼠皮下注射CCl4后,每天进行腹腔注射(40mg/kg)的EP,在2、4、6、8周4个时间点EP注射后12h取材,EP明显改善大鼠饮食和精神状态,体重相对于CCl4组有所增加,毛发光泽,皮肤损伤较小,且短期内可愈合。EP处理后大鼠血清ALT、AST水平明显下降,肝功能改善。2.CCl4注射后,肝组织病理变化明显,肝细胞水肿、脂肪变、假小叶形成等肝组织结构紊乱。EP干预后肝组织损伤明显减轻,胶原纤维面积减少。3.CCl4诱导肝纤维化中,肝星状细胞大量激活,EP干预后肝星状细胞激活标记物α-SMA明显减少。4.免疫组化结果显示α-SMA,MyD88在窦周隙中阳性表达,TLR4、NF-κB、MMP2、MMP9,TIMP1、TIMP2在肝组织中阳性表达。5.Q-PCR结果显示,与对照组相比,α-SMA、TLR4、NF-κB、HMGB1、IL6、MMPs、TIMPs和胶原纤维1、3、4mRNA的表达显著增加,应用EP后表达明显减少(P0.05),MMPs/TIMPs比率上升。但2W时间点α-SMA、MMPs、TIMPs和胶原纤维1、3、4mRNA无统计学意义。6.ELISA结果显示,α-SMA、TLR4、MMP2、TIMP2、胶原蛋白IV的含量与Q-PCR结果相一致。结论1、每天腹腔注射丙酮酸乙酯对CCl4引起的肝损伤具有保护作用。2、EP抑制CCl4所致的肝星状细胞激活。3、EP可能通过抑制TLR4/NF-κB信号通路,抑制HSCs激活、减轻炎症反应从而发挥抗纤维化作用。4、EP上调MMPs/TIMPs比率加速胶原纤维降解有关。
[Abstract]:Hepatic fibrosis (liver fibrosis) is the body of various stimulating dynamic process of injury and repair of the extracellular matrix (ECM) is a state of imbalance and degradation caused by excessive deposition of extracellular matrix. The matrix metalloproteinases (MMPs) and its inhibitor (TIMPs) balance influence the degradation of ECM. The main effect as liver fiber the cells, hepatic stellate cells (HSCs) in the liver and other host cells such as hepatocytes, hepatic macrophages, endothelial cell interactions promote fibrosis, development of.HSCs is activated and the survival need some signal regulation. These signals is affected by many factors, both from HSCs have autocrine, paracrine.HSC, TOLL like receptor 4 (TLR4) expression induced by chemokines and adhesion molecules, nuclear transcription factor (NF- K B) is activated by various stimuli and cell stress, regulate inflammation, so they are chronic Play a major role in liver disease. Ethyl pyruvate (ethyl pyruvate EP) has anti-inflammatory properties in a variety of injury play a protective role in the model. By subcutaneous injection of carbon tetrachloride (CCl4) to establish the model of hepatic fibrosis, observe the effect of EP on liver fibrosis in rats at different time points in the exhibition, and hepatic stellate cell activation markers (alpha -SMA), TLR4, NF- K B, expression of MMPs and TIMPs and collagen 1,3,4, preliminary study on inhibition of EP activation of hepatic stellate cells in liver fibrosis and accelerate collagen degradation mechanism, thus reversing hepatic fibrosis. Methods healthy years SD rats were randomly divided into three groups: control group, CCl4 model group (Control) (CCl4), ethyl pyruvate intervention group (CCl4+EP). By subcutaneous injection of CCl4, two times a week, a total of 8 weeks, the preparation of EP fibrosis model; intervention treatment, once a day; at 2,4,6,8 Thursday at the time of the last injection of CCl4 48H After the rats were taken liver samples. The statistical 8W ascites condition.HE, is used to evaluate the pathological changes of liver fibrosis and.Q-PCR for the detection of TLR4 MASSON and ISHAK NF- staining score, kappa B, MMPs, expression of TIMPs and collagen 1,3,4mRNA, detection of ELISA in liver tissues of TLR4, alpha -SMA, MMP2, TIMP2, collagen 4 content; immunohistochemistry for the detection of alpha -SMA, TLR4, NF- kappa B, MMPs, expression of TIMPs and collagen fibers of 1,4 positive cells and cellular localization. Results 1. rats by subcutaneous injection of CCl4 after intraperitoneal injection every day (40mg/kg) of EP, 2,4,6,8 in the 4 week time point after EP injection 12h based EP, significantly improve the rat diet and mental state, body weight increased compared with CCl4 group, shiny hair, skin damage is small, and the short term serum ALT healing in rats after.EP treatment, the AST level decreased significantly after.2.CCl4 injection can improve the liver function, liver disease Physical changes obviously, liver cell edema, fatty degeneration, pseudolobule liver tissue injury significantly reduced liver tissue structure disorder after.EP intervention, collagen fiber area decreased.3.CCl4 induced liver fibrosis, a large number of activated hepatic stellate cells, EP after the intervention of hepatic stellate cell activation markers alpha -SMA significantly reduced.4. immunohistochemical results show a -SMA, MyD88 in the perisinusoidal space in the positive expression of TLR4, NF-, MMP2, MMP9, B, TIMP1, TIMP2 showed positive expression of.5.Q-PCR in liver tissue, compared with the control group, a -SMA, TLR4, HMGB1, NF- kappa B, IL6, MMPs, TIMPs and 1,3,4mRNA expression of collagen fibers significantly increased after application of EP expression was significantly reduced (P0.05), MMPs/TIMPs ratio increased. But the 2W time point alpha -SMA MMPs, TIMPs and collagen 1,3,4mRNA was not statistically significant.6.ELISA results showed that alpha -SMA, TLR4, MMP2, TIMP2, consistent with the results of Q-PCR content of collagen IV. Conclusion: 1, each Intraperitoneal liver injury caused by CCl4 injection of ethyl pyruvate on the protective effect of.2 and EP in hepatic stellate cells inhibits CCl4 induced activation of.3, EP may inhibit TLR4/NF- B signaling pathway, inhibiting the activation of HSCs, relieve the inflammation and anti fibrotic effect of.4 EP MMPs raised /TIMPs ratio accelerated degradation of collagen fibers.

【学位授予单位】：成都医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.2

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