沙格列汀通过降低整合素avβ5的表达缓解糖尿病合并非酒精性脂肪肝

Saxagliptin alleviates non-alcoholic fatty liver disease with comorbid diabetes through reducing the expression of integrin avβ5 hereby

目的观察沙格列汀对2型糖尿病(T2DM)合并非酒精性脂肪肝(NAFLD)大鼠血清及肝组织中整合素avβ5表达及肝窦毛细血管化的影响,探讨糖尿病合并NAFLD的发病机制以及沙格列汀对糖尿病合并NAFLD的可能保护机制.方法利用高脂饮食联合小剂量链脲佐菌素腹腔注射建立T2DM合并NAFLD大鼠模型.将10周龄雄性SPF级Wistar大鼠50只分为5组(每组各10只):正常对照组、单纯NAFLD组、糖尿病合并NAFLD模型对照组、整合素avβ5抑制剂干预组、沙格列汀干预组.avβ5抑制剂干预组通过尾静脉注射整合素avβ5抑制剂慢病毒质粒(109 TU/ml,20μl/d),沙格列汀干预组给予沙格列汀溶液(10 mg·kg^-1·d^-1)灌胃6周,对照组给予同体积生理盐水灌胃.实验结束后检测各组大鼠空腹血糖、胰岛素、血脂、肝功能、肝脏系数及病理变化,透射电镜检测肝脏超微结构,免疫组化及Western blotting检测整合素avβ5、玻连蛋白(Vn)在肝组织中的蛋白表达水平,采用RT?PCR检测整合素avβ5及Vn的mRNA表达水平,ELISA法检测大鼠血清整合素avβ5的水平.2组间比较采用t检验.结果(1)与模型组相比,沙格列汀能明显降低大鼠血清整合素avβ5的表达水平(0.47±0.06比0.96±0.12,t=3.36,P<0.05);(2)糖尿病合并NAFLD组大鼠肝组织中整合素avβ5和Vn蛋白的表达水平均明显高于正常对照组,而使用沙格列汀干预治疗后其表达水平均明显下降(t=3.16~9.86,均P<0.05),avβ5和Vn mRNA的表达结果与其蛋白表达结果一致(t=3.50~8.33,均P<0.05);(3)透射电镜结果显示:模型组大鼠肝窦内皮细胞基底膜明显增厚,呈连续的、无窗孔的基底膜,肝细胞胞质中内质网增生或扩张,肿胀的线粒体嵴减少或溶解消失,Disse氏间隙增宽,且间隙内可见大量胶原纤维增生,使用整合素avβ5或沙格列汀干预治疗后,基底膜逐渐恢复为有窗孔或不连续或变薄的基底膜,扩张的内质网和肿胀的线粒体明显改善;(4)与模型组相比,整合素avβ5或沙格列汀干预组肝脏系数、谷草转氨酶、谷丙转氨酶、空腹胰岛素、稳态模型评估胰岛素抵抗指数均明显降低(t=6.11~9.7,均P<0.05).结论沙格列汀通过抑制整合素avβ5的表达及其诱导的肝窦毛细血管化而发挥保护糖尿病合并非酒精性脂肪肝的作用.

Objective To observe the effect of saxagliptin on the expression of integrin avβ5 in serum and liver tissues of type 2 diabetes mellitus (T2DM) with non-alcoholic fatty liver disease (NAFLD) and the sinusoidal capillarization, and to explore the pathogenesis of diabetes mellitus complicated with fatty liver and the possible protective mechanism of saxagliptin on diabetes with fatty liver. Methods Rats models of T2DM with NAFLD were established by feeding on a high-fat diet combined with low-dose streptozotocin (STZ) intraperitoneal injection. Fifty male SPF Wistar rats (10 in each group) at 10 weeks of age were divided into 5 groups: normal control (NC), NAFLD, T2DM with NAFLD model control group, integrin avβ5 inhibitor intervention group and saxagliptin intervention group. The rats of the avβ5 inhibitor intervention group were injected with the integrin avβ5 inhibitor lentiviral plasmid (109 TU/ml, 20 μl/day) via the tail vein. The rats of saxagliptin intervention group were treated with saxagliptin (daily dose of 10 mg/kg) gavage for 6 weeks, and the rats of the control group were given the same volume of normal saline. After the treatment for 6 weeks, fasting blood glucose, serum insulin, blood lipids, liver function, liver index and pathological changes were evaluated in all the rats, and the expressions of integrin avβ5 and Vn in the liver tissue were detected with immunohistochemistry and western blotting. Ultrastructure of the liver was detected by transmission electron microscopy. The expression levels of integrin avβ5 and Vn mRNA were detected by RT-PCR. The serum levels of integrin avβ5 in the rat were detected by ELISA. T test was used for comparison between groups. Results (1) Compared with the model group, saxagliptin significantly reduced the serum level of integrin avβ5 in the rats (0.47±0.06 vs 0.96±0.12, t=3.36, all P<0.05);(2) The protein expression levels of integrin avβ5 and Vn in the liver tissue of diabetic rats with fatty liver were significantly higher than those in the normal control group, but the levels were significantly decreased after the treatment with saxagliptin (t=3.16-9.86, all P<0.05). The mRNA expression levels of integrin avβ5 and Vn were consistent with those protein expression (t=3.50-8.33, all P<0.05);(3) Transmission electron microscopy results show: in the model group, the basement membrane of sinusoidal endothelial cells is thickened, continuous and windowless. Moreover, the endoplasmic reticulum in the cytoplasm of hepatocytes proliferates or expands, the mitochondrial sputum decreases or dissolves, and the Disse′s gap widens. And a large amount of collagen fibrosis can be seen in the gap. The basement membrane gradually returns to a basement membrane with a window or discontinuity or thinning, and the expanded endoplasmic reticulum and swollen mitochondria are significantly improved after the treatment with integrin avβ5 inhibitor or saxagliptin;(4) Compared with the model group, the levels of liver coefficient, aspartate aminotransferase, alanine aminotransferase, fasting insulin, and homeostatic model assessment for insulin resistance were significantly decreased in the integrin avβ5 inhibitor or sax intervention group (t=6.11-9.7, all P<0.05). Conclusions Saxagliptin protects diabetes with nonalcoholic fatty liver disease by inhibiting the expression of integrin avβ5 in serum or liver tissue of the rats and integrin avβ5-induced sinusoidal capillarization.