基于PINK1/Parkin/铁死亡信号探讨艳山姜挥发油防治糖尿病肾病的作用机制

Mechanism of Essential Oil from Fructus Alpiniae Zerumbet Alleviates in Prevention and Treatment Diabetic Nephropathy Through PINK1/Parkin/Ferroptosis Signal Pathway

目的 基于PINK1/Parkin/铁死亡信号探究艳山姜挥发油(essential oil from Fructus Alpiniae Zerumbe,EOFAZ)调控糖尿病肾病的作用机制。方法 高糖高脂饲料饲养联合腹腔注射链脲佐菌素建立2型糖尿病(diabetes mellitus,DM)小鼠模型,给予EOFAZ进行干预,实验分组为正常组、DM组、DM+EOFAZ低剂量组(90 mg·kg^(-1))、DM+EOFAZ高剂量组(180mg·kg^(-1))、 DM+铁死亡抑制剂组(Ferrostatin-1, 5mg·kg^(-1))和EOFAZ毒性组(180mg·kg^(-1))。DM+EOFAZ组、DM+Ferrostatin-1组分别灌胃EOFAZ和Ferrostatin-1,其余组以生理盐水灌胃,每日灌胃1次,持续8周后,分析血糖指标。HE、PAS染色对肾组织进行形态学观察;Western blotting分析线粒体自噬关键蛋白PINK1及Parkin的表达;Western blotting及免疫组织化学分析铁死亡标志蛋白GPX4及COX2的表达;组织铁试剂盒检测组织的铁含量;生化试剂盒分析肾组织中SOD、GSH及MDA的水平。结果 与DM组比较,给予EOFAZ后可明显改善肾组织病理学变化;显著上调PINK1、Parkin及GPX4的表达,下调COX2的表达;降低肾组织铁含量;增加SOD活性及GSH含量,降低MDA水平。其中EOFAZ毒性组中各指标与正常组相当,无明显差异。结论 艳山姜挥发油可改善DM诱导肾组织损伤,其作用机制可能是通过调控PINK1/Parkin/铁死亡信号。

OBJECTIVE To explore the mechanism of diabetes nephropathy regulated by essential oil from Fructus Alpiniae Zerumbet(EOFAZ)based on PINK1/Parkin/ferroptosis Signal.METHODS Diabetes mellitus(DM)mice model was established by feeding high sugar and high fat diet(HFSD)and intraperitoneal injection of streptozotocin.The mice were given EOFAZ for intervention.Mice were randomly divided into normal group,DM group,DM+EOFAZ low dose group(90 mg·kg^(-1)),DM+EOFAZ high dose group(180 mg·kg^(-1)),DM+ferroptosis inhibitor group(Ferrostatin-1,5 mg·kg^(-1)),EOFAZ toxicity group(180 mg·kg^(-1)).The DM+EOFAZ group and DM+Ferrostatin-1 group were given EOFAZ and Ferrostatin-1,respectively.At the same time,the normal group and DM group were given equal volume of physiological saline.The mice in each group were orally administered once a day for 8 weeks,and their blood were collected to measured fasting blood glucose.HE and PAS staining were used to observe the morphology of renal tissue;Western blotting was used to detecte the expression of mitochondrial autophagy key proteins PINK1 and Parkin.Western blotting and immunohistochemistry analysis were used to investigate the expression of ferroptosis marker proteins GPX4 and COX2.The iron assay kit was used to detect the iron content of the renal tissue.ELISA kit was used to analyze the levels of SOD,GSH,and MDA in renal tissue.RESULTS Compared with DM group,EOFAZ could significantly ameliorate the renal histopathology changes,upregulate the expression of PINK1,Parkin,and GPX4,downregulate the expression of COX2,reduce iron content in renal tissue,increase SOD activity and GSH content,and reduce MDA levels.And there were no significant difference between the normal control group and EOFAZ toxicity group in those results.CONCLUSION EOFAZ can ameliorate diabetes nephropathy,and its mechanism may be through regulating PINK1/Parkin/ferroptosis signal.