【摘要】：非酒精性脂肪性肝病(NAFLD)目前已成為全世界流行的常見慢性肝病,它的病因和發(fā)病機制至今仍不完全清楚。研究已經(jīng)證明,炎癥信號和促炎癥細胞因子在NAFLD的發(fā)生機制中發(fā)揮重要作用。以往研究表明,TNF-α、IL-1β、高遷移率組box (HMGB)1和Toll樣受體(TLR)4是NAFLD發(fā)生機制中的重要炎癥介質(zhì),而且,巨噬細胞移動抑制因子(MIF)和核因子(NF)-kB均與HMGB1/TLR4信號途徑密切相關(guān)。另有研究發(fā)現(xiàn),HMGB1/MIF的釋放與缺血和機械損傷中的免疫反應(yīng)所導致的肝細胞損害之間具有相關(guān)性。我們猜測,HMGB1/MIF和HMGB1/TLR4信號途徑均與NAFLD的發(fā)病和治療機制相關(guān)。本研究檢測MIF、HMGB1、NF-kB和TLR4在高脂飼料喂食的NAFLD大鼠血清和肝組織中的表達,并研究它們的表達與大鼠肝功能損害、血清和肝臟脂質(zhì)代謝障礙以及肝臟組織學變化之間的關(guān)系,然后分別觀察荔枝核提取物和甜菜堿治療后,高脂誘導的NAFLD大鼠HMGB1/MIF和HMGB1/TLR4信號表達的變化,從而探討NAFLD的發(fā)病機制以及荔枝核提取物和甜菜堿治療NAFLD的機制。 第一部分：高脂誘導的非酒精性脂肪性肝病大鼠脂質(zhì)水平與炎癥因子表達的相關(guān)性 40只大鼠隨機分為對照組(n=20)和模型(n=20)。對照組大鼠喂食標準飼料,模型組大鼠喂食高脂飼料,共12周,誘導大鼠NAFLD模型。兩組大鼠肝臟進行NAFLD組織學評分、MIF、NF-kB和TLR4免疫組化染色評分,檢測血清ALT、 AST、TC、TG、LDL-C、HDL-C和FFA水平、肝臟TC和TG含量、MIF、HMGB1、 NF-kB和TLR4血清、肝臟mRNA和蛋白表達水平。另外,評價模型組大鼠血清轉(zhuǎn)氨酶、血清和肝臟脂質(zhì)水平與MIF、NF-kB和TLR4免疫組化染色陽性細胞百分比、MIF、HMGB1、NF-kB和TLR4的血清、肝臟mRNA和蛋白表達水平之間的相關(guān)性,以及NAFLD氵舌動度評分與MIF、NF-kB和TLR4免疫組化評分之間的相關(guān)性。結(jié)果顯示,與對照組相比,模型組大鼠肝組織可見明顯的脂肪變性、炎癥和氣球樣變,伴血清ALT、AST、TC、TG、LDL-C、FFA水平、肝臟TC和TG含量、脂肪變性、炎癥和氣球樣變評分、MIF、NF-kB和TLR4免疫組化染色評分、MIF、 HMGB1、NF-kB和TLR4血清、肝臟mRNA和蛋白表達水平顯著增高,以及血清HDL-C水平顯著降低(P0.05或P0.01)。而且,模型組大鼠血清轉(zhuǎn)氨酶、血清和肝臟脂質(zhì)含量與炎癥因子MIF、HMGB1、NF-kB和TLR4表達水平之間,以及肝組織NAFLD評分與MIF、NF-kB和TLR4免疫組化染色評分之間均具有顯著或非常顯著相關(guān)性(P0.05或P0.01)。實驗結(jié)果提示,與TNF-α和IL-1p相似,MIF、 HMGB1、NF-kB和TLR4在高脂誘導的大鼠NAFLD的發(fā)生機制中也發(fā)揮重要作用,并且提示抑制炎癥因子MIF、HMGB1、NF-kB或TLR4可能改善高脂誘導的NAFLD中肝功能損害、血清和肝臟脂質(zhì)代謝異常等。 第二部分：荔枝核提取物通過抑制HMGB1/MIF釋放從而對抗高脂飼料誘導的大鼠肝損害 34只大鼠隨機分為四組：對照(n=6)、模型組(n=8)、低劑量LSE組(n=10)和高劑量LSE組(n=10)。除對照組外,其余全部大鼠喂食高脂飼料,誘導非酒精性脂肪性肝病(NAFLD)模型。高脂飼料喂食12周后,在第13-16周,低劑量(250mg/kg)和高劑量(500mg/kg)LSE組大鼠每日灌胃給予LSE溶液。檢查肝臟組織學變化,檢測肝功能和脂質(zhì)代謝指標,用ELISA法檢測血清MIF、HMGB1、Toll樣受體(TLR)4、腫瘤壞死因子(TNF)-α和白細胞介素(IL)-1β水平。分別用RT-PCR和Western blotting檢測MIF、HMGB1、TLR4和TNF-α的mRNA表達和蛋白水平。結(jié)果顯示,LSE治療后,與模型組相比,低劑量和高劑量LSE組肝臟組織學變化、肝功能和脂質(zhì)代謝顯著改善(P0.05)。同時,與模型組相比,LSE組MIF、HMGB1、 TLR4和TNF-α血清水平、mRNA和/或蛋白表達顯著降低(P0.05)。并且高劑量LSE組顯著優(yōu)于低劑量LSE組(P0.05)。實驗結(jié)果提示,LSE能保護高脂飼料誘導的NAFLD大鼠對抗肝臟損害,并與劑量相關(guān),其可能的機制與抑制HMGB1/MIF釋放有關(guān)。 第三部分：甜菜堿抑制非酒精性脂肪性肝病大鼠高遷移率組Box1和Toll樣受體4表達 40只大鼠隨機分為對照組、模型組、低劑量甜菜堿組和高劑量甜菜堿組。模型組、低劑量和高劑量甜菜堿組大鼠喂食高脂飼料12周,誘導大鼠NAFLD模型。然后,低劑量甜菜堿組和高劑量甜菜堿組大鼠分別以每日200mg/kg和400mg/kg劑量灌胃給予甜菜堿溶液,共4周。觀察肝臟組織學變化。檢測血清ALT、AST、 TC、TG、HDL-C、LDL-C、FFA、HMGB1、NF-kB和TLR4水平、肝臟TC和TG含量。并對肝組織HMGB1、NF-kB和TLR4mRNA和蛋白的表達水平進行檢測。結(jié)果顯示,與對照組相比,模型組大鼠發(fā)生嚴重肝損害,伴血清ALT、AST、TC、 TG、LDL-C、FFA、HMGB1、NF-kB和TLR4水平、肝臟TC和TG含量、肝組織脂肪變性、炎癥和壞死評分、HMGB1、NF-kB和TLR4mRNA和蛋白的相對表達水平顯著增高以及血清HDL-C顯著降低(P0.05)。然而,經(jīng)甜菜堿治療后,與模型組相比,上述指標全部顯著改善。并且高劑量甜菜堿組顯著優(yōu)于低劑量甜菜堿組(P0.05)。實驗結(jié)果提示,甜菜堿對高脂飼料誘導的大鼠NAFLD發(fā)揮有效的保護作用,改善NAFLD大鼠肝功能,其機制與抑制HMGB1/TLR4信號途徑有關(guān),并且與劑量相關(guān)。
[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.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R575.5

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本文編號：2160651