高脂诱导的大鼠非酒精性脂肪性肝病的发病机制和治疗的实验研究

发布时间：2018-08-02 20:34

【摘要】：非酒精性脂肪性肝病(NAFLD)目前已成为全世界流行的常见慢性肝病,它的病因和发病机制至今仍不完全清楚。研究已经证明,炎症信号和促炎症细胞因子在NAFLD的发生机制中发挥重要作用。以往研究表明,TNF-α、IL-1β、高迁移率组box (HMGB)1和Toll样受体(TLR)4是NAFLD发生机制中的重要炎症介质,而且,巨噬细胞移动抑制因子(MIF)和核因子(NF)-kB均与HMGB1/TLR4信号途径密切相关。另有研究发现,HMGB1/MIF的释放与缺血和机械损伤中的免疫反应所导致的肝细胞损害之间具有相关性。我们猜测,HMGB1/MIF和HMGB1/TLR4信号途径均与NAFLD的发病和治疗机制相关。本研究检测MIF、HMGB1、NF-kB和TLR4在高脂饲料喂食的NAFLD大鼠血清和肝组织中的表达,并研究它们的表达与大鼠肝功能损害、血清和肝脏脂质代谢障碍以及肝脏组织学变化之间的关系,然后分别观察荔枝核提取物和甜菜碱治疗后,高脂诱导的NAFLD大鼠HMGB1/MIF和HMGB1/TLR4信号表达的变化,从而探讨NAFLD的发病机制以及荔枝核提取物和甜菜碱治疗NAFLD的机制。第一部分：高脂诱导的非酒精性脂肪性肝病大鼠脂质水平与炎症因子表达的相关性 40只大鼠随机分为对照组(n=20)和模型(n=20)。对照组大鼠喂食标准饲料,模型组大鼠喂食高脂饲料,共12周,诱导大鼠NAFLD模型。两组大鼠肝脏进行NAFLD组织学评分、MIF、NF-kB和TLR4免疫组化染色评分,检测血清ALT、 AST、TC、TG、LDL-C、HDL-C和FFA水平、肝脏TC和TG含量、MIF、HMGB1、 NF-kB和TLR4血清、肝脏mRNA和蛋白表达水平。另外,评价模型组大鼠血清转氨酶、血清和肝脏脂质水平与MIF、NF-kB和TLR4免疫组化染色阳性细胞百分比、MIF、HMGB1、NF-kB和TLR4的血清、肝脏mRNA和蛋白表达水平之间的相关性,以及NAFLD氵舌动度评分与MIF、NF-kB和TLR4免疫组化评分之间的相关性。结果显示,与对照组相比,模型组大鼠肝组织可见明显的脂肪变性、炎症和气球样变,伴血清ALT、AST、TC、TG、LDL-C、FFA水平、肝脏TC和TG含量、脂肪变性、炎症和气球样变评分、MIF、NF-kB和TLR4免疫组化染色评分、MIF、 HMGB1、NF-kB和TLR4血清、肝脏mRNA和蛋白表达水平显著增高,以及血清HDL-C水平显著降低(P0.05或P0.01)。而且,模型组大鼠血清转氨酶、血清和肝脏脂质含量与炎症因子MIF、HMGB1、NF-kB和TLR4表达水平之间,以及肝组织NAFLD评分与MIF、NF-kB和TLR4免疫组化染色评分之间均具有显著或非常显著相关性(P0.05或P0.01)。实验结果提示,与TNF-α和IL-1p相似,MIF、 HMGB1、NF-kB和TLR4在高脂诱导的大鼠NAFLD的发生机制中也发挥重要作用,并且提示抑制炎症因子MIF、HMGB1、NF-kB或TLR4可能改善高脂诱导的NAFLD中肝功能损害、血清和肝脏脂质代谢异常等。第二部分：荔枝核提取物通过抑制HMGB1/MIF释放从而对抗高脂饲料诱导的大鼠肝损害 34只大鼠随机分为四组：对照(n=6)、模型组(n=8)、低剂量LSE组(n=10)和高剂量LSE组(n=10)。除对照组外,其余全部大鼠喂食高脂饲料,诱导非酒精性脂肪性肝病(NAFLD)模型。高脂饲料喂食12周后,在第13-16周,低剂量(250mg/kg)和高剂量(500mg/kg)LSE组大鼠每日灌胃给予LSE溶液。检查肝脏组织学变化,检测肝功能和脂质代谢指标,用ELISA法检测血清MIF、HMGB1、Toll样受体(TLR)4、肿瘤坏死因子(TNF)-α和白细胞介素(IL)-1β水平。分别用RT-PCR和Western blotting检测MIF、HMGB1、TLR4和TNF-α的mRNA表达和蛋白水平。结果显示,LSE治疗后,与模型组相比,低剂量和高剂量LSE组肝脏组织学变化、肝功能和脂质代谢显著改善(P0.05)。同时,与模型组相比,LSE组MIF、HMGB1、 TLR4和TNF-α血清水平、mRNA和/或蛋白表达显著降低(P0.05)。并且高剂量LSE组显著优于低剂量LSE组(P0.05)。实验结果提示,LSE能保护高脂饲料诱导的NAFLD大鼠对抗肝脏损害,并与剂量相关,其可能的机制与抑制HMGB1/MIF释放有关。第三部分：甜菜碱抑制非酒精性脂肪性肝病大鼠高迁移率组Box1和Toll样受体4表达 40只大鼠随机分为对照组、模型组、低剂量甜菜碱组和高剂量甜菜碱组。模型组、低剂量和高剂量甜菜碱组大鼠喂食高脂饲料12周,诱导大鼠NAFLD模型。然后,低剂量甜菜碱组和高剂量甜菜碱组大鼠分别以每日200mg/kg和400mg/kg剂量灌胃给予甜菜碱溶液,共4周。观察肝脏组织学变化。检测血清ALT、AST、 TC、TG、HDL-C、LDL-C、FFA、HMGB1、NF-kB和TLR4水平、肝脏TC和TG含量。并对肝组织HMGB1、NF-kB和TLR4mRNA和蛋白的表达水平进行检测。结果显示,与对照组相比,模型组大鼠发生严重肝损害,伴血清ALT、AST、TC、 TG、LDL-C、FFA、HMGB1、NF-kB和TLR4水平、肝脏TC和TG含量、肝组织脂肪变性、炎症和坏死评分、HMGB1、NF-kB和TLR4mRNA和蛋白的相对表达水平显著增高以及血清HDL-C显著降低(P0.05)。然而,经甜菜碱治疗后,与模型组相比,上述指标全部显著改善。并且高剂量甜菜碱组显著优于低剂量甜菜碱组(P0.05)。实验结果提示,甜菜碱对高脂饲料诱导的大鼠NAFLD发挥有效的保护作用,改善NAFLD大鼠肝功能,其机制与抑制HMGB1/TLR4信号途径有关,并且与剂量相关。
[Abstract]:Nonalcoholic fatty liver disease (NAFLD) has now become a common chronic liver disease in the world. Its etiology and pathogenesis are still not completely clear. Studies have shown that inflammatory signals and proinflammatory cytokines play an important role in the pathogenesis of NAFLD. Previous studies have shown that TNF- alpha, IL-1 beta, and high mobility group box (HMGB) 1 And Toll like receptor (TLR) 4 is an important inflammatory mediator in the pathogenesis of NAFLD, and the macrophage migration inhibitory factor (MIF) and nuclear factor (NF) -kB are closely related to the HMGB1/TLR4 signaling pathway. Further studies have found the correlation between the release of HMGB1/MIF and the liver cell damage caused by ischemia and the immune response in mechanical damage. We conjecture that both HMGB1/MIF and HMGB1/TLR4 signaling pathways are associated with the pathogenesis of NAFLD and the mechanism of treatment. This study examined the expression of MIF, HMGB1, NF-kB and TLR4 in the serum and liver tissues of NAFLD rats fed with high fat diet, and studied their expression with the liver function impairment in rats, lipid metabolism disorders in blood and liver, and liver histology. The changes in the expression of HMGB1/MIF and HMGB1/TLR4 in high fat NAFLD rats after the treatment of the extracts of litchi and betaine were then observed, and the mechanism of the pathogenesis of NAFLD and the treatment of NAFLD by the extracts of litchi and betaine were discussed.
Part I: correlation between lipid level and inflammatory factors in rats with high-fat induced nonalcoholic fatty liver disease
40 rats were randomly divided into the control group (n=20) and the model (n=20). The rats in the control group were fed the standard feed. The rats in the model group were fed with high fat diet for 12 weeks, and the rat NAFLD model was induced. The liver of the two rats was evaluated by NAFLD histology, MIF, NF-kB and TLR4 immunohistochemical staining scores, and the serum ALT, AST, TC, TG, LDL-C, LDL-C, and liver were detected. C and TG content, MIF, HMGB1, NF-kB and TLR4 serum, and the level of liver mRNA and protein expression. In addition, evaluate the correlation of serum transaminase, serum and liver lipid levels with MIF, NF-kB and TLR4 immunohistochemical staining positive cells, MIF, HMGB1, liver and protein expression levels, as well as the correlation between the liver and protein expression levels. The correlation between the tongue movement score and the MIF, NF-kB and TLR4 immunohistochemical scores showed that the liver tissues of the model group showed significant fatty degeneration, inflammation and balloon like changes, with serum ALT, AST, TC, TG, LDL-C, FFA levels, the liver TC and TG content, fatty degeneration, inflammation and balloon like change scores, MIF, MIF, and immunity The serum levels of MIF, HMGB1, NF-kB and TLR4 were significantly higher in the liver, and the level of mRNA and protein expression in the liver was significantly increased, and the level of serum HDL-C was significantly decreased (P0.05 or P0.01). F, NF-kB and TLR4 immunohistochemical staining scores have significant or very significant correlation (P0.05 or P0.01). Experimental results suggest that MIF, HMGB1, NF-kB, and TLR4 are also important in the pathogenesis of high-fat rat NAFLD. High fat induced liver damage in NAFLD, abnormal lipid metabolism in serum and liver.
The second part: the extract of Litchi chinensis seed can inhibit the release of HMGB1/MIF to resist liver injury induced by high-fat diet in rats.
34 rats were randomly divided into four groups: control (n=6), model group (n=8), low dose LSE group (n=10) and high dose LSE group (n=10). In addition to the control group, the rest of the rats fed high fat diet and induced non-alcoholic fatty liver disease (NAFLD) model. After feeding high fat feed for 12 weeks, the low dose (250mg/kg) and high dose (500mg/kg) LSE group were large in the 13-16 week. Rats were given LSE solution every day. The changes in liver histology were examined, liver function and lipid metabolism were detected. The serum levels of MIF, HMGB1, Toll like receptor (TLR) 4, tumor necrosis factor (TNF) - alpha and interleukin (IL) -1 beta were detected by ELISA. The expression and protein levels were detected by RT-PCR and Western blotting, respectively. The results showed that after LSE treatment, the liver tissue changes and liver function and lipid metabolism were significantly improved in the low and high dose LSE groups (P0.05). Compared with the model group, the serum levels of MIF, HMGB1, TLR4 and TNF- alpha in the LSE group were significantly lower (P0.05). And the high dose LSE group was significantly better than the low dose LSE group. (P0.05). The results suggest that LSE can protect the NAFLD rats induced by high fat diet against the liver damage, and it is related to the dose. The possible mechanism is related to the inhibition of the release of HMGB1/MIF.
The third part: Betaine inhibits the expression of Box1 and Toll like receptor 4 in the high mobility group of rats with non-alcoholic fatty liver disease.
40 rats were randomly divided into control group, model group, low dose betaine group and high dose betaine group. Model group, low dose and high dose betaine group were fed with high fat diet for 12 weeks, and NAFLD model was induced in rats. Then, low dose betaine group and high dose betaine group were given 200mg/kg and 400mg/kg dose daily to sweeten the stomach, respectively. ALT, AST, TC, TG, HDL-C, LDL-C, FFA, HMGB1, NF-kB and TLR4 levels, liver TC and TG content were detected. TG, LDL-C, FFA, HMGB1, NF-kB and TLR4 levels, liver TC and TG content, hepatic steatosis, inflammation and necrosis score, the relative expression level of HMGB1, NF-kB and TLR4mRNA and protein, and the significant decrease in serum HDL-C. The betaine group was significantly better than the low dose betaine group (P0.05). The results suggest that betaine has an effective protective effect on the rat NAFLD induced by high fat diet and improves the liver function of NAFLD rats. The mechanism is related to the inhibition of the HMGB1/TLR4 signal pathway and is related to the dose.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.5

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