利拉鲁肽通过调节 microRNA－375对胰岛细胞凋亡的影响

摘要

目的：观察利拉鲁肽通过微小 RNA－375（microRNA－375，miR－375）对 db／db 小鼠胰岛细胞凋亡的影响，探讨其可能作用机制，为临床应用提供药效学依据。方法：20只8周龄雄性 db／m 小鼠为正常对照组，皮下注射等量的生理盐水。40只8周龄雄性 db／db 小鼠随机分为2组，每组20只：糖尿病对照组的 db／db 小鼠皮下注射等量生理盐水；利拉鲁肽组的 db／db 小鼠皮下注射利拉鲁肽300μg・ kg－1・ d－1。给药8周后，检测各组小鼠体重（BW）、空腹血糖（FBG）、空腹胰岛素（FINS）、甘油三酯（TG）、总胆固醇（TC）及低密度脂蛋白胆固醇（LDL－C）含量，并进行腹腔注射葡萄糖耐量实验（IPGTT）和胰岛素耐量实验（ITT）；苏木精－伊红（HE）染色检测胰岛组织病理学变化；原位末端转移酶标记技术（TUNEL）检测胰岛凋亡情况；Western blot 法检测胰岛凋亡相关蛋白 caspase－3、Bcl－2及 Bax 的蛋白水平；实时荧光定量 PCR 检测胰岛 miR－375的表达水平。小鼠胰岛β细胞系 MIN－6分为对照组（等量溶媒孵育）、miRNA－375 mimic 组和 miRNA－375 mimic ＋利拉鲁肽组，MTT 实验检测细胞活力，Western blot法检测各组细胞 caspase－3、 Bcl－2及 Bax 的蛋白水平。结果：整体实验中，与对照组相比，利拉鲁肽组 BW、FBG、FINS、TC、TG 及 LDL－C 含量明显降低（P ＜0．05）；胰岛数量较模型组增多，体积较大，细胞结构明显改善；胰岛细胞凋亡减少；利拉鲁肽组的 Bcl－2表达明显增高，caspase－3和 Bax 的表达显著下降（P ＜0．05）；胰岛组织 miR－375的水平显著降低（P ＜0．01）。细胞实验中，经 miRNA－375 mimic 处理24 h 后，MIN－6细胞活力显著降低，Bcl－2表达减少，而 caspase－3和 Bax 的蛋白水平显著增加（P ＜0．05），给予利拉鲁肽组治疗后，MIN－6细胞活力明显上升，Bcl－2表达明显增高，caspase－3和 Bax 的蛋白水平显著下降（P ＜0．05）。结论：利拉鲁肽可以抑制糖尿病胰岛β细胞的凋亡，其机制可能与调控胰岛组织中 miR－375的表达有关。%AIM: To observe the anti-apoptosis effect of liraglutide on the islet through microRNA-375 (miR-375) for providing additional pharmacodynamic evidence for its clinical application.METHODS: For in vivo study, C57BL/KsJ-db/m mice aged 8 weeks served as normal control group.A total of 40 male genetically diabetic C57BL/KsJ-db/db mice at the same age were randomly divided into diabetic control group (the db/db mice were injected subcutaneous-ly with equivalent amount of saline) and liraglutide group (the db/db mice were injected subcutaneously with liraglutide at dose of 300 μg・ kg-1・ d-1 ).After 8 weeks of administration, body weight (BW) was measured and blood was collected for detection of fasting blood glucose (FBG), fasting blood insulin (FINS), triglyceride (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C).Before sacrifice, intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were conducted.The histopathological features in the islet tissue were examined with HE staining.The apoptosis in the islet tissue was detected by TUNEL staining.The protein levels of caspase-3, Bcl-2 and Bax were deter-mined by Western blot.The level of miR-375 in the islet tissue was detected by qPCR.For in vitro study, the MIN-6 cells were cultured and divided into control group (incubated with equivalent amount of solvent), miR-375 mimic group and miR-375 mimic +liraglutide group.The cell viability was examined by MTT assay.The protein levels of caspase-3, Bcl-2 and Bax were detected by Western blot.RESULTS: In the in vivo study, compared with control group, the levels of BW, FBG, FINS, TC, TG and LDL-C were decreased significantly in liraglutide group.The islet apoptosis was reduced by the administration of liraglutide.The expression of Bcl-2 was up-regulated significantly, while the protein levels of caspase-3 and Bax were down-regulated significantly in liraglutide group.The level of miR-375 was decreased significantly.In the in vitro study, the cell viability was decreased in miR-375 mimic group and increased in miR-375 mimic +liraglutide group. Moreover, the expression of Bcl-2 was decreased and the protein levels of caspase-3 and Bax were increased with the incu-bation of miR-375 mimic, while the expression of Bcl-2 was increased and the protein levels of caspase-3 and Bax were de-creased with the co-incubation of miR-375 mimic and liraglutide.CONCLUSION: Liraglutide attenuates islet apotosis, and the mechanism may be associated with its effects of reducing the elevated level of miR-375 in islet tissues.