非酒精性脂肪性肝炎性别间差异与MyD88依赖性IL-6信号通路的关系

发布时间：2018-06-01 23:42

本文选题：非酒精性脂肪性肝炎 + 性别间差异　；参考：《吉林大学》2015年博士论文

【摘要】：第一部分MCD饮食诱导C57BL/6小鼠非酒精性脂肪性肝炎的性别间差异及其与MyD88依赖性IL-6信号通路的关系目的：建立蛋氨酸-胆碱缺乏（methionine-choline-deficient，MCD）饮食诱导C57BL/6小鼠非酒精性脂肪性肝炎的动物模型，观察此模型的性别间差异，探讨该性别间差异与MyD88依赖性IL-6信号通路的关系。方法：健康14周龄雄性C57BL/6小鼠26只，随机分为2组：（1）雄性MCD组：予以MCD饮食；（2）雄性对照组：予以MCD对照饮食。健康14周龄雌性C57BL/6小鼠26只，随机分为2组：（1）雌性MCD组：予以MCD饮食；（2）雌性对照组：予以MCD对照饮食。饲养4周后处死小鼠。自动生化分析仪检测血清丙氨酸氨基转移酶（alanine aminotransferase， ALT）、天门冬氨酸氨基转移酶（aspartate aminotransferase，AST）、γ-谷氨酰转肽酶（γ-Glutamyltransferase，γ-GT）水平。肝左叶部分肝组织行苏木精—伊红（hematoxylin-eosin,HE）、锇酸染色观察肝脏病理组织学改变，并进行非酒精性脂肪性肝病活动度积分（non-alcoholic fatty liver disease activity score，NAS）评定。同时应用实时荧光定量RT-PCR方法测定肝组织髓样分化因子88（myeloid differentiation primaryresponse gene88，MyD88）、白细胞介素-6（interleukin-6，IL-6）mRNA水平，应用Western Blot蛋白印迹分析法测定肝组织MyD88、IL-6蛋白的表达水平。结果：雄性MCD组和雌性MCD组小鼠肝小叶各区均可见肝细胞脂肪变性，为以大泡性为主的混合性脂肪变性，汇管区及小叶内可见灶状炎性细胞浸润，以淋巴细胞浸润为主。其中，雄性MCD组小鼠炎性细胞浸润明显,还可见肝细胞气球样变性。而雌性MCD组小鼠炎性细胞浸润较少，未见肝细胞气球样变性。对照组小鼠肝组织形态正常，未见肝细胞脂肪变性和炎性细胞浸润。与各自对照组相比，雄性MCD组和雌性MCD组小鼠脂肪变性、炎症评分、NAS积分及血清ALT、AST、γ-GT水平显著升高（P＜0.05）。与雌性MCD组比较，雄性MCD组小鼠脂肪变性评分无显著性差异（P＞0.05），但炎症评分、NAS积分、血清ALT、AST、γ-GT水平显著升高（P＜0.05），以ALT、AST升高为著。与各自对照组相比，雄性MCD组和雌性MCD组小鼠肝组织MyD88、IL-6mRNA及其蛋白表达水平明显升高（P＜0.05）。与雌性MCD组比较，雄性MCD组小鼠肝组织MyD88、IL-6mRNA及其蛋白表达水平明显升高（P＜0.05）。结论： MCD饮食诱导非酒精性脂肪性肝炎C57BL/6雄性小鼠肝脏病理损伤较雌性小鼠更为严重，炎性细胞浸润及肝细胞气球样变性更加显著，病理组织学评分显著高于以同样方法建立的雌性小鼠模型。MCD饮食诱导非酒精性脂肪性肝炎雄性小鼠的血清生化学改变较雌性小鼠更加显著，其中ALT、AST升高较γ-GT更为明显。MCD饮食诱导非酒精性脂肪性肝炎雄性小鼠肝组织MyD88、IL-6mRNA及蛋白表达水平均较雌性小鼠更加显著，提示雄性及雌性小鼠模型在性别间的差异与MyD88依赖性IL-6信号通路的激活相关。第二部分性别干预对MCD饮食诱导非酒精性脂肪性肝炎的影响及其与MyD88依赖性IL-6信号通路的关系目的：建立MCD饮食诱导C57BL/6雄性小鼠非酒精性脂肪性肝炎的动物模型，通过手术去势、手术去势联合补充外源性激素等方式，探讨不同的性别干预方式对非酒精性脂肪性肝炎的影响，以及其与MyD88依赖性IL-6信号通路的关系。方法：健康14周龄雄性C57BL/6小鼠65只，随机分为5组：（1）雄性对照组：给予MCD对照饮食。（2）雄性MCD组：给予MCD饮食。（3）手术去势MCD组：给予MCD饮食，并行手术切除睾丸。（4）手术去势MCD+雄激素组：给予MCD饮食，并行手术切除睾丸及每日皮下注射丙酸睾酮5mg/kg。（5）手术去势MCD+雌激素组：给予MCD饮食，并行手术切除睾丸及每日皮下注射苯甲酸雌二醇0.5mg/kg。饲养4周后处死小鼠。酶联免疫吸附测定(enzyme-linkedimmunosorbent assay，ELISA)血清雌二醇、睾酮水平。自动生化分析仪测定血清ALT、AST、γ-GT水平。肝左叶部分肝组织行HE、锇酸染色观察肝脏病理组织学变化，并进行NAS积分评定。同时应用实时荧光定量RT-PCR方法测定肝组织MyD88、IL-6mRNA水平，应用Western Blot蛋白印迹分析法检测肝组织MyD88、IL-6蛋白的表达水平。结果：手术去势可使小鼠血清睾酮水平显著下降（P＜0.05）；补充外源性雄激素后，血清睾酮水平显著升高（P＜0.05），恢复至未去势水平；而补充外源性雌激素，可使小鼠血清雌二醇水平显著升高（P＜0.05）。与雄性MCD组相比，手术去势MCD组小鼠肝细胞脂肪变性、炎性细胞浸润及肝细胞气球样变性减轻，其NAS积分及血清ALT、AST水平也明显降低（P＜0.05），血清γ-GT水平略降低（P＞0.05）；手术去势MCD+雄激素组小鼠肝组织可见大量肝细胞脂肪变性，并有较多炎性细胞浸润，偶可见肝细胞气球样变。与雄性MCD组、手术去势MCD组相比，其NAS积分及血清ALT、AST、γ-GT水平均无显著性差异（P＞0.05）；而手术去势MCD+雌激素组小鼠肝组织中可见部分肝细胞脂肪变性，并有少量炎细胞浸润，未见肝细胞气球样变。与雄性MCD组、手术去势MCD组、手术去势MCD+雄激素组相比，该组小鼠NAS积分和血清ALT、AST水平均明显降低（P＜0.05）；但血清γ-GT仅在与雄性MCD组相比时具有显著性差异（P＜0.05）。与雄性MCD组比较，手术去势MCD组小鼠肝组织MyD88、IL-6mRNA及其蛋白表达水平明显降低（P＜0.05）。与雄性MCD组、手术去势MCD组相比，手术去势MCD+雄激素组小鼠肝组织MyD88、IL-6mRNA及其蛋白表达无明显降低或升高（P＞0.05）。而手术去势MCD+雌激素组小鼠肝组织MyD88、IL-6mRNA及其蛋白表达水平最低，并且与雄性MCD组、手术去势MCD组、手术去势MCD+雄激素组比较，均有显著性差异（P＜0.05）。结论：采用手术去势或补充外源性雌激素进行性别干预后，可明显改善MCD饮食诱导C57BL/6雄性小鼠非酒精性脂肪性肝炎的病理损伤，其中手术去势联合外源性雌激素组小鼠肝组织炎性细胞浸润及脂肪变性最轻，NAS积分最低。手术去势或补充外源性雌激素改善了肝脏病理损伤，也使血清ALT、AST水平较性别干预前显著降低。单纯手术干预对γ-GT影响不大，，但当手术联合应用外源性雌激素时，γ-GT也表现为显著下降。采用手术去势或补充外源性雌激素进行性别干预后，雄性C57BL/6小鼠肝组织MyD88、IL-6mRNA及蛋白的表达明显下降，其中在手术去势联合应用外源性雌激素组小鼠下降更为明显。提示性别干预促进MCD饮食诱导C57BL/6雄性小鼠非酒精性脂肪性肝炎的恢复与其抑制MyD88依赖性IL-6信号通路相关。
[Abstract]:Part one: gender differences in MCD induced diet induced nonalcoholic steatohepatitis in C57BL/6 mice and their relationship with MyD88 dependent IL-6 signaling pathway
Objective: to establish an animal model of nonalcoholic steatohepatitis in C57BL/6 mice induced by methionine-choline-deficient (MCD) diet, and to observe the gender differences in this model and to explore the relationship between the gender differences and the MyD88 dependent IL-6 signaling pathway.
Methods: 26 healthy 14 week male C57BL/6 mice were randomly divided into 2 groups: (1) male MCD group: MCD diet, and (2) male control group: MCD control diet. 26 healthy 14 week old female C57BL/6 mice were randomly divided into 2 groups: (1) female MCD group: MCD diet; (2) female control group: MCD control diet. Feeding for 4 weeks after feeding. Dead mice. The serum alanine aminotransferase (alanine aminotransferase, ALT), aspartate aminotransferase (aspartate aminotransferase, AST), gamma glutamyl transaminopeptidase (gamma -Glutamyltransferase, gamma -GT) were detected by automatic biochemical analyzer. The liver left lobe of the liver was treated with hematoxylin Yi Hong (hematoxylin-eosin, HE), osmium acid staining. Liver histopathological changes were observed and the non-alcoholic fatty liver disease activity score, NAS was evaluated by the liver pathological changes, and the real time fluorescent quantitative RT-PCR method was used to determine the liver tissue myeloid differentiation factor 88 (myeloid differentiation primaryresponse gene88,), and interleukin (IL). Interleukin-6, IL-6) mRNA level, Western Blot protein blot analysis was used to detect the expression level of MyD88 and IL-6 protein in liver tissue.
Results: the hepatocyte fatty degeneration was found in all areas of the hepatic lobules in the male MCD group and the female MCD group, and it was a mixed fatty degeneration with large bullae. The infiltration of inflammatory cells in the tubular area and the lobule was visible, mainly with lymphocyte infiltration. Among them, the inflammatory cells in the male MCD group were obviously infiltrated, and the balloon like degeneration of the hepatocytes was also seen. In the female MCD group, the inflammatory cells in the mice were less infiltrated and no hepatocyte balloon like degeneration was found. The normal liver tissue of the control group was normal, and no fatty degeneration of hepatocytes and inflammatory cell infiltration were found in the control group.
Compared with the control group, the adipose degeneration, the score of inflammation, the score of NAS and the level of ALT, AST, and gamma -GT in the male MCD group and the female MCD group were significantly higher (P < 0.05). Compared with the female MCD group, there was no significant difference in the fat denaturation score of the male MCD group (P > 0.05), but the inflammatory score, NAS integral, serum ALT, and gamma levels were significantly higher than those in the female MCD group. 0.05), with ALT, AST rising.
Compared with the control group, the expression level of MyD88, IL-6mRNA and protein in the liver tissues of the male MCD group and the female MCD group increased significantly (P < 0.05). Compared with the female MCD group, the level of MyD88, IL-6mRNA and protein expression in the liver tissue of the male MCD group was significantly increased (P < 0.05).
Conclusion: MCD diet induced the liver pathological damage in the C57BL/6 male mice of nonalcoholic steatosis hepatitis more serious than that of female mice. Inflammatory cell infiltration and balloon like degeneration of liver cells were more significant. The histopathology score was significantly higher than that of the female mice model established by the same method of.MCD diet to induce nonalcoholic steatohepatitis. The serum biochemical changes in mice were more significant than those of female mice. The increase of ALT and AST was more obvious than that of gamma -GT in.MCD diet induced MyD88, IL-6mRNA and protein expression levels were more significant than that of female mice, suggesting the difference between sex and MyD88 dependent I in the male and female mice model. The activation of the L-6 signaling pathway is related.
The second part is the effect of gender intervention on MCD induced nonalcoholic steatohepatitis and its relationship with MyD88 dependent IL-6 signaling pathway.
Objective: to establish an animal model of nonalcoholic steatohepatitis in C57BL/6 male mice induced by MCD diet, and to explore the effects of different sex intervention on nonalcoholic steatohepatitis by surgical castration, operation castration combined with exogenous hormones and the relationship between the effect of different sex intervention on nonalcoholic steatohepatitis and the MyD88 dependent IL-6 signaling pathway.
Methods: 65 healthy 14 week male C57BL/6 mice were randomly divided into 5 groups: (1) the male control group was given the MCD control diet. (2) the male MCD group was given the MCD diet. (3) the operation castrated MCD group was given the MCD diet, and the testicles were excised in parallel. (4) the operation castrated MCD+ male IP group was given MCD diet, operation excision testis and subcutaneous daily subcutaneous Testosterone propionate 5mg/kg. (5) operation ovariectomized MCD+ estrogen group: MCD diet, parallel surgical resection of testicles and daily subcutaneous injection of estradiol benzoate 0.5mg/kg. to death mice. Enzyme linked immunosorbent assay (enzyme-linkedimmunosorbent assay, ELISA) serum estradiol, testosterone level. Automatic biochemical analyzer for serum determination of serum The level of ALT, AST and gamma -GT. The liver left lobe liver tissue was performed HE, the hepatic pathological changes were observed by osmium acid staining, and the NAS integral was evaluated. The MyD88, IL-6mRNA level of liver tissue was measured by real-time quantitative fluorescence quantitative RT-PCR method, and the expression level of liver tissue MyD88 and IL-6 protein was detected by the Western Blot Western blot analysis.
Results: the serum testosterone level of mice decreased significantly (P < 0.05). After supplementation of exogenous androgen, serum testosterone level increased significantly (P < 0.05) and recovered to the level of non castration, while supplementation of exogenous estrogen could significantly increase the level of serum estradiol in mice (P < 0.05).
Compared with the male MCD group, the hepatocyte fatty degeneration, inflammatory cell infiltration and hepatocyte balloon like degeneration were reduced in the operation castrated MCD group, and the NAS score and the serum ALT, AST level were also significantly decreased (P < 0.05), the serum level of gamma -GT decreased slightly (P > 0.05), and the liver tissue of the operation castrated MCD+ androgen group mice showed a large number of hepatocyte fatty degeneration, and There were many inflammatory cells infiltrating, and even the hepatocyte balloon like change. Compared with the male MCD group and the operation castrated MCD group, the NAS score and the serum ALT, AST, and gamma -GT levels were not significantly different (P > 0.05), while the liver tissue of the operation castrated MCD+ estrogen group of the surgical ovariectomized mice showed a partial hepatic steatosis, with a small number of inflammatory cells infiltrating and no liver fine. Compared with the male MCD group, the operation castration group MCD, the operation castrated MCD+ androgen group, the NAS scores and the serum ALT and AST levels of the mice were significantly decreased (P < 0.05), but the serum gamma -GT was significantly different from the male MCD group (P < 0.05).
Compared with the male MCD group, the expression level of MyD88, IL-6mRNA and protein in the liver tissue of the operation castrated MCD group was significantly lower (P < 0.05). Compared with the male MCD group and the operation castrated MCD group, the expression of MyD88, IL-6mRNA and protein expression in the hepatic tissue of the operation castrated MCD+ androgen group was not significantly reduced or increased (P > 0.05). The expression level of MyD88, IL-6mRNA and protein in the liver tissues of mice was the lowest, and was significantly different from that of the male MCD group, the operation castrated MCD group and the operation castrated MCD+ androgen group (P < 0.05).
Conclusion: the operation castration or supplementation of exogenous estrogen for the sex dry prognosis can obviously improve the pathological damage of non alcoholic steatohepatitis in the C57BL/6 male mice induced by MCD diet. The operation castration combined with exogenous estrogen group is the lightest of inflammatory cell infiltration and fatty degeneration in the liver tissue of the mice, the NAS score is the lowest. Supplementation of exogenous estrogen improved pathological injury of liver and decreased the level of serum ALT and AST significantly before the sex intervention. Simple operation intervention had little effect on gamma -GT, but when the operation combined with exogenous estrogen, gamma -GT also showed a significant decline. The prognosis of sex dry with surgical ovariectomy or supplementation of exogenous estrogen was performed in male C57. The expression of MyD88, IL-6mRNA and protein in the liver tissue of BL/6 mice decreased significantly, and the decrease in the combined use of exogenous estrogen group in the operation castration was more obvious. It suggested that the sex intervention to promote the recovery of non alcoholic steatohepatitis in C57BL/6 male mice induced by MCD diet was related to the inhibition of MyD88 dependent IL-6 signaling pathway.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R575.5

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