牛磺脱氧胆酸抑制内质网应激介导的细胞凋亡保护糖尿病肝损伤的研究

Endoplasmic reticulum stress (ERS)-mediated apoptosis is involved in the pathogenesis of diabetic liver injury (DLI). The anti-ERS treatment has become the new choice of treatment of diabetic complications. Our previous studies showed ERS and apoptosis existed in type 1 diabetic mouse liver and diabetes resulted in fibrosis. However, the relationship between ERS and the pathogenesis of DLI in type 1 diabetic mouse model, and the effects of ERS regulation on DLI and hepatocytes incubation with high glucose have not been reported previously. The present study was undertaken to define whether ERS inbibitor tauroursodeoxycholic acid (TUDCA) can inhibite diabetic hepatic cell death, liver dysfuntion and fibrosis via suppression of ERS in type 1 diabetes. We will evaluate the ERS signal pathway and apoptosis with intervention of tauroursodeoxycholic acid in type 1 diabetic mouse liver and cultured human hepato carcinoma cell line HepG2. The methods include Western blotting, immunohistochemistry, ultrasonography, cell culture, biochemistry and immunology. The study could provide important theoretical support for prevention and treatment of DLI. The study also might open up a new way for the drugs in research and development for DLI.

内质网应激（ERS）介导的凋亡途径在糖尿病肝损伤（DLI）发病中的作用引起人们重视，抗ERS成为治疗糖尿病并发症的新选择。课题组前期研究证实1型糖尿病小鼠肝脏中存在ERS及细胞凋亡、且纤维化增强，但在1型糖尿病小鼠模型中ERS与DLI发病机制的研究、调控ERS对模型DLI的发病及高糖环境下对肝细胞的影响尚未明确。本研究采用免疫印迹法、超声检查、免疫组化法、细胞培养、生化及免疫等方法，使用1型糖尿病小鼠肝脏及体外培养的人肝癌细胞系HepG2细胞，在内质网应激抑制剂牛磺脱氧胆酸（TUDCA）干预下进行ERS信号通路及细胞凋亡的研究，探讨TUDCA是否通过抑制ERS预防糖尿病诱导的肝细胞凋亡，是否抑制肝功障碍及肝纤维化，阐明TUDCA保护DLI的机制。该研究将为DLI的预防和治疗措施选择提供重要理论支持，将为研发治疗DLI药物开辟一条新途径。

本课题探讨内质网应激抑制剂牛磺脱氧胆酸（TUDCA）是否通过抑制内质网应激（ERS）保护糖尿病诱导的肝细胞凋亡及其机制。在1型糖尿病FVB小鼠模型造模成功后2周及2个月，处死小鼠并留取肝脏及血清，使用生化法检测小鼠血清肝脏功能及血脂标志物，使用苦味酸-天狼星红染色法进行肝脏纤维化检查，使用免疫印迹法、实时荧光定量PCR法检测ERS的信号通路，使用TUNEL法、免疫印迹法、实时荧光定量PCR法检测凋亡细胞数、CHOP、cleaved caspase-3及cleaved caspase-12等凋亡标志物，利用1型糖尿病小鼠肝脏及体外培养的人肝癌细胞系HepG2细胞进行TUDCA保护糖尿病肝损伤（DLI）机制的研究。结果显示TUDCA可改善糖尿病小鼠的体重、血糖、血脂及肝脏功能。凋亡肝细胞数、CHOP、cleaved caspase-3及cleaved caspase-12等蛋白凋亡标志物在造模成功后2周小鼠肝脏中明显升高，caspase 3和CHOP的mRNA表达量亦显著增高，而在TUDCA处理的小鼠中则显著改善。与凋亡检测结果平行，ERS标志物GRP78、GRP94、cleaved ATF6及p-eIF2α蛋白在2周小鼠肝脏中显著上调，GRP78、GRP94及ATF6的mRNA表达量亦明显增加，而在TUDCA处理的小鼠中可显著改善。HepG2细胞中TUDCA可完全抑制高糖、内质网应激激活剂新霉素诱导的ERS及ERS介导的肝细胞凋亡。体内及体外实验结果显示，TUDCA保护DLI的机制至少包括抑制氧化应激及炎症反应。该研究将为DLI的预防和治疗措施选择提供重要的理论支持，将为研发治疗糖尿病并发症的药物开辟一条新途径。