人参皂苷Rg1对四氯化碳所致小鼠急性肝衰竭模型作用机制研究

发布时间：2018-02-27 19:05

本文关键词： 人参皂苷Rg1 细胞凋亡内质网应激肝衰竭　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:急性肝衰竭病情危重、病死率极高,目前仍是临床危重症,除了人工肝替代治疗、肝移植外,尚缺乏临床治疗特效药物,因此探究临床治疗药物非常必要。课题组前期研究发现人参皂苷Rg1(G-Rg1)对小鼠急性肝衰竭模型具有预防效果,且其可能与抗肝细胞凋亡和抗炎作用有关,但G-Rg1的具体作用机制、靶点仍不清楚,需要进一步研究。因此拟建立四氯化碳(CCl4)诱导的急性肝衰竭小鼠模型,进一步研究G-Rg1对急性肝衰竭模型的作用机制,验证G-Rg1对ALF小鼠模型的有效性,初步探讨G-Rg1对CCl4诱导的ALF小鼠模型抗肝细胞凋亡的影响及相关机制。方法:采用随机数字法将40只健康成年C57/BL雄性小鼠分为生理盐水对照(NS)组,G-Rg1空白对照(G-Rg1)组,CCl4模型(CCl4)组,G-Rg1预防治疗(CCl4+G-Rg1)组。建模12h后采集各组小鼠血清和肝脏组织,用试剂盒法检测血清丙氨酸转氨酶(alanine transaminase,ALT)、天冬氨酸转氨酶(aspartate aminotransferase,AST)、总胆红素(total bilirubin,Tbil)水平;HE染色评价肝脏组织病理学改变;定量PCR法检测肝脏葡萄糖调节蛋白78(GRP78)、C/EBP家族同源蛋白(CHOP)的表达量;Western blot检测GRP78、CHOP、半胱氨酸蛋白酶12(caspase12)、半胱氨酸蛋白酶3(caspase3)的表达情况;免疫组织化学法分析GRP78、caspase3的表达情况;TUNEL法检测C57/BL小鼠肝脏组织细胞凋亡情况。结果:1.CCl4+G-Rg1组血清ALT、AST、TBil水平低于CCl4组,差异具有统计学意义(P0.05)。NS组、G-Rg1组、CCl4组、CCl4+G-Rg1组ALT值分别为(50.12±9.25)U/L、(40.48±6.38)U/L、(980.66±110.29)U/L、(691.30±108.06)U/L;AST值分别为(9.69±2.78)U/L、(9.40±3.84)U/L、(319.44±89.32)U/L、(195.40±15.41)U/L;TBil值分别为(0.46±0.13)mg/d L、(0.48±0.08)mg/d L、(1.56±0.12)mg/d L、(1.09±0.11)mg/d L。2.模型构建完成后HE染色示CCl4组肝脏出现明显变性坏死并可见肝细胞坏死灶,呈典型的CCl4所致肝细胞病理表现。经G-Rg1预防治疗后的CCl4+G-Rg1组肝脏组织变性坏死程度较CCl4组明显减轻。3.CCl4+G-Rg1组GRP78、CHOP的m RNA相对表达量均较CCl4组下降,差异具有统计学意义(F=34.4、F=44.1,P0.05)。4.Western bolt结果显示CCl4+G-Rg1组与CCl4组相比,caspase3、GRP78、caspase12、CHOP蛋白相对表达量均有不同程度降低,差异均有统计学意义(F=270.58、F=34.73、F=92.38、F=31.63,P0.05)。5.免疫组织化学法检测GRP78、caspase3表达情况,光镜下观察结果显示CCl4组较NS组胞浆内棕黄色颗粒明显增多。CCl4+G-Rg1组较CCl4组胞浆内棕色颗粒减少。6.TUNEL结果显示CCl4+G-Rg1组棕色颗粒物质较CCl4组明显减少(F=330.9,P0.05),提示G-Rg1预防治疗可以减轻CCl4引起的肝细胞凋亡。结论:G-Rg1预防用药可抑制CCl4诱导的小鼠急性肝衰竭的炎症反应,同时可减轻肝细胞坏死和凋亡。其作用机制可能通过抑制内质网应激相关信号分子,改善肝细胞内质网应激进而减轻肝细胞凋亡。结合课题组之前的研究结果提示G-Rg1可能通过多个作用靶点抑制肝脏炎症反应和肝细胞凋亡,实现保护肝脏功能作用。
[Abstract]:Objective: acute liver failure (ARF) is in critical condition and has a high mortality rate. It is still a clinical critical disease. In addition to artificial liver replacement therapy and liver transplantation, there is still a lack of effective drugs for the treatment of acute liver failure. Therefore, it is very necessary to explore the clinical therapeutic drugs. Our previous study found that ginsenoside Rg1 G-Rg1) has a preventive effect on acute liver failure in mice, and it may be related to the anti-hepatocyte apoptosis and anti-inflammatory effect, but the specific mechanism of G-Rg1. The target point is still unclear and needs further study. Therefore, we intend to establish an acute liver failure model induced by CCl4), to further study the mechanism of G-Rg1 on acute liver failure model, and to verify the effectiveness of G-Rg1 on ALF mouse model. To investigate the effect of G-Rg1 on anti-hepatocyte apoptosis in ALF mice induced by CCl4 and its related mechanism. Methods: 40 healthy adult C57rBL male mice were randomly divided into normal saline control group and G-Rg1 blank control group. The serum and liver tissues of each group were collected after 12 hours of modeling. Serum alanine transaminase (alt), aspartate aminotransferase (AST), total bilirubin (Tbilirubin) were detected by Kit method. The expression of Glucose-regulated protein 78 (GRP78) was detected by quantitative PCR, and the expression of GRP78, caspase12 and cysteine protease 3caspase3 was detected by Western blot. Immunohistochemical method was used to analyze the expression of GRP78 caspase3 and Tunel method was used to detect apoptosis in liver tissue of C57 / BL mice. Results 1. The level of serum alt ASTTBil in CCl4 G-Rg1 group was lower than that in CCl4 group. The ALT values of G-Rg1 group in CCl4 group were 50.12 卤9.25 UL / L = 40.48 卤6.38 U / L + 980.66 卤110.29 U / L = 9.69 卤108.06 U / L = 9.69 卤2.78 U / L = 9.69 卤2.78 渭 L / L 9.40 卤3.84 渭 L / L ~ 319.44 卤89.32 U / L / L ~ (195.40 卤15.41) U / L / L = 0.46 卤0.13 mg / d / L, respectively. The liver necrosis was observed after liver necrosis was observed in the liver necrosis group (P < 0.05 卤0.12 mg / L 1.56 卤0.12 mg / L 1.56 卤0.12 mg / L 1.11 mg / d). The degree of degeneration and necrosis of liver tissue in CCl4 G-Rg1 group was significantly decreased than that in CCl4 group. 3. The relative expression of m RNA in GRP78 chop in CCl4 G-Rg1 group was lower than that in CCl4 group. The results of Western bolt showed that the relative expression of caspase3, GRP78, caspase12CHOP protein in the CCl4 G-Rg1 group was lower than that in the CCl4 group, and the difference was statistically significant in F270.58F34.73F74.73P0.05P0.05. the expression of GRP78caspase3 was detected by immunohistochemical method. The results of light microscopy showed that the brown granules in cytoplasm of CCl4 group were significantly higher than those of NS group. The brown granules in cytoplasm of CCl4 G-Rg1 group were less than those of CCl4 group. 6. Tunel results showed that the brown granule substance of CCl4 G-Rg1 group was significantly lower than that of CCl4 group, suggesting that G-Rg1 preventive therapy was possible. Conclusion the prophylaxis of CCl4 can inhibit the inflammatory response induced by CCl4 in mice with acute liver failure. At the same time, it can reduce hepatocyte necrosis and apoptosis. Its mechanism may be by inhibiting endoplasmic reticulum stress-related signaling molecules, In combination with previous research results, G-Rg1 may protect liver function by inhibiting liver inflammation and hepatocyte apoptosis through multiple targets.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.3;R-332

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