梓醇缓解内质网应激抑制非酒精性脂肪肝细胞凋亡

Catalpol inhibits cell apoptosis through alleviating endoplasmic reticulum stress in nonalcoholic fatty liver disease

探讨梓醇对非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)状态下肝细胞凋亡的保护作用及其机制。体内实验采用高脂饮食(high fat diet,HFD)诱导小鼠建立NAFLD模型,实验过程和动物福利均遵循江汉大学动物伦理委员会的规定;体外实验采用棕榈酸(palmitate,PA)诱导人肝癌细胞系HepG2建立脂毒性模型。结果显示,梓醇显著降低HFD小鼠血清中总甘油三脂(total glyceride,TG)、总胆固醇(total cholesterol,TC)、谷丙转氨酶(alanine aminotransferase,ALT)和谷草转氨酶(aspartate transaminase,AST)含量;TUNEL染色和流式细胞术显示梓醇显著抑制了肝细胞凋亡;Western blot结果显示梓醇显著降低内质网应激标志性蛋白如结合免疫球蛋白(binding immunoglobulin protein,BiP)、磷酸化的PKR样内质网激酶(phosphorylated PKR-like endoplasmic reticulum kinase,p-PERK)、肌醇需求酶1α(inositol-requiring enzyme 1α,IRE1α)和活化转录因子6(activating transcription factor 6,ATF6)的蛋白表达水平以及应激性凋亡蛋白C/EBP同源蛋白(C/EBP homology protein,CHOP)、磷酸化的c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinase,p-JNK)、剪切的半胱氨酸天冬氨酸蛋白酶(cysteinyl aspartate specific proteinase,caspase)-12、-9和-3的蛋白表达水平。此外,HepG2细胞经内质网应激激动剂衣霉素(tunicamycin,TM)处理后,显著降低了梓醇对内质网应激相关蛋白BiP、p-PERK、IRE1α、ATF6和凋亡相关蛋白CHOP、p-JNK、Bcl-2、Bax、cleaved caspase(-12、-9和-3)表达水平的抑制作用,证明梓醇通过缓解内质网应激抑制肝细胞凋亡。以上结果表明,梓醇能够通过缓解内质网应激抑制肝细胞凋亡,从而发挥保护肝损伤的作用。

This study was designed to explore the protective effect and underlying mechanism of catalpol on hepatocyte apoptosis in nonalcoholic fatty liver disease(NAFLD).High fat diet(HFD)was used to establish NAFLD model in the in vivo experiment,and the procedures of the experiments and animal care protocol were approved by the Animal Care and Use Committee of Jianghan University.Human liver cancer cell line HepG2 was treated with palmitate(PA)to establish a lipid toxicity model in the in vitro experiments.The results showed that catalpol significantly decreased the contents of serum total glyceride(TG),total cholesterol(TC),alanine aminotransferase(ALT),and aspartate transaminase(AST)in HFD-fed mice.Results of TUNEL staining and flow cytometry analyses revealed that catalpol significantly inhibited hepatocytes apoptosis in HFD-fed mice and PA-treated HepG2 cells.Moreover,catalpol treatment significantly reduced the endoplasmic reticulum stress-related protein expression levels of binding immunoglobulin protein(BiP),phosphorylated PKR-like endoplasmic reticulum kinase(p-PERK),inositol-requiring kinase 1α(IRE1α),and transcriptional factor activating transcription factor 6(ATF6),and apoptosis-related protein expression levels of C/EBP homology protein(CHOP),phosphorylated c-Jun N-terminal kinase(p-JNK),and cleaved cysteinyl aspartate specific proteinases(caspases)-12,-9,and-3 in HFD-fed mice and PA-treated HepG2 cells.Furthermore,endoplasmic reticulum stress agonist tunicamycin(TM)significantly reversed the inhibitory effect of catalpol on protein expression levels of BiP,p-PERK,IRE1α,and ATF6,subsequently the inhibitory effect of catalpol on expression levels of CHOP,p-JNK,Bcl-2,Bax,and cleaved caspases(-12,-9,and-3)was also attenuated in PA-treated HepG2 cells.Taken together,these findings demonstrated that catalpol could inhibit hepatocytes apoptosis and had a significant protective effect on liver injury,and its mechanism might be related to the relief of endoplasmic reticulum stress.