熊脱氧胆酸减轻内质网应激介导的胰岛β细胞凋亡的机制

The attenuating effect of ursodeoxycholic acid on endoplasmic reticulum stress-mediated pancreatic β-cell apoptosis in streptozotocin-induced diabetic rats

目的探讨熊脱氧胆酸（Ursodeoxycholic acid，UDCA）通过减轻内质网应激保护链脲佐菌素诱导的糖尿病大鼠胰岛β细胞凋亡的作用。方法链脲佐菌素（50mg／ks）一次性腹腔注射建立糖尿病大鼠模型（n=40），并将14只造模成功大鼠按随机数字表法随机分为糖尿病组7只，UDCA组7只，另取正常对照组10只。自造模成功第10天开始以UDCA（40mg·kg^-1·d^-1）给大鼠灌胃30d。饲养期间观察大鼠血糖。处死后留取大鼠血清和组织标本，测定空腹胰岛素，TUNEL检测胰岛β细胞凋亡的形态学改变。胰腺组织总RNA采用定制基因芯片对89条凋亡相关基因的表达进行检测，以RT—PCR和Western印迹法验证相关基因的表达。结果糖尿病大鼠血糖在给予UDCA治疗后逐渐下降，但仍然未降到正常水平。糖尿病大鼠空腹胰岛素降低，给予UDCA治疗后空腹胰岛素水平有所增加。TUNEL显示糖尿病组大鼠予UDCA治疗后减少了由链脲佐菌素引起的胰岛β细胞凋亡（P〈0．01）。在89条基因中，糖尿病组上调基因42条，下调基因46条。UDCA可以逆转部分基因的影响。与对照组相比，糖尿病组大鼠βax、Caspase-3、βip、CHOP mRNA和CHOP蛋白显著上调（P〈0．05），而抗凋亡蛋白βcl-2 mRNA显著下调（P〈0．05），给予UDCA治疗后这些参数均有明显改善（P〈0．05）。结论熊脱氧胆酸可能通过减轻内质网应激达到保护链脲佐菌素诱导的糖尿病大鼠胰岛β细胞凋亡的作用。

Objective To clarify the protective effect of ursodeoxycholic acid （UDCA） on endoplasmic reticulum stress-mediated apoptosis in pancreatic β-cell of streptozotocin （ STZ ）-induced diabetic rats. Methods Rats （ n = 40 ） received a single injection STZ （ 50 mg/kg） intra-peritoneally and formed a β-cell injury model. Weight-matched normal rats（ the control group, n = 10） were injected with the buffer alone. STZ-treated rats with persistent random blood glucose higher than 16.7 mmol/L for 1 week were considered as diabetic status （ n = 14）, then divided randomly into STZ-induced diabetes mellitus （ DM ） group （ n = 7 ） and UDCA-treated DM group （ n = 7 ）. UDCA（40 mg· kg^-1 · d^-1 ）was administered daily by intragastric intubations throughout the experimental period （30 d）. During the entire experiment, blood glucose in all rats was assessed. By the end of the experiment, all rats were sacrificed with the pancreas removed and the blood sample collected immediately. Fasting insulin levels were assayed by radioimmunoassay. The morphological changes of pancreatic β-cells apoptosis were determined by TUNEL assay. RNA in pancreas was abstracted and microarray containing 89 pieces of apoptosis related genes was applied. The related gene expressions were detected by RT-PCR and Western blot. Results The concentration of blood glucose in diabetic rats was gradually decreased after UDCA treatment, but at the end of the experiment it was still higher than that in the normal control group. The treatment with UDCA raised the fasting insulin level in diabetic rats, but this concentration was significantly lower as compared to the control group. Based on TUNEL stained tissue sections, the percentage of β-cell apoptosis of UDCA-treated DM group was significantly lower than that of STZ-induced diabetic group（P〈0. 05）. Among 89 genes, 42 genes up-regulated and 46 genes down-regulated in diabetic rats, some of which were ameliorated by UDCA treatment. The expressions of Caspase-3, Bax, Bip, and CHOP mRNA in pancreas of DM group were significantly up-regulated as compared with those in the control group （ P 〈 0. 05 ） ; while the expression of Bcl-2 mRNA was markedly down-regulated（ P〈0. 05 ）. However, these parameters in the UDCA-treated animals showed a marked improvement. Conclusion Ursodeoxycholic acid seems to protect pancreatic β-cell from apoptosis in STZ-induced diabetes by attenuating the severity of endoplasmic reticulum stress.