艳山姜挥发油调控CaMKⅡ信号改善糖尿病诱导视网膜Müller细胞自噬障碍  

Protection effect of essential oil from Fructus Alpiniae zerumbet on the autophagy dysfunction of retinal Müller cells induced by diabetes via CaMKⅡsignaling pathways

作　　者：陈冰[1,2] 杨红 杨惠 陈永鑫[2] 甘诗泉 文波 蒋朝晖 沈祥春[2] 李悦[1] CHEN Bing;YANG Hong;YANG Hui;CHEN Yong-xin;GAN Shi-quan;WEN Bo;JIANG Zhao-hui;SHEN Xiang-chun;LI Yue(Department of Pharmacy,Guiyang Maternal and Child Health Care Hospital,Guiyang 550003;Key Laboratory of Optimal Utilization of Natural Medicine Resources,School of Pharmaceutical Sciences,Guizhou Medical University,Guiyang 550025;Department of Clinical Laboratory,Guiyang First people’s Hospital,Guiyang 550002;College of Clinical Medicine,Jining Medical Institute,Ji’ning Shandong 272067)

机构地区：[1]贵阳市妇幼保健院药学部,贵阳550003 [2]贵州医科大学天然药物资源优效利用重点实验室,贵阳550025 [3]贵阳市第一人民医院检验科,贵阳550002 [4]济宁医学院临床医学院,山东济宁272067

出　　处：《中南药学》2023年第10期2616-2621,共6页Central South Pharmacy

基　　金：贵州省科技计划项目(黔科合基础-ZK[2022]一般005;黔科合基础-ZK[2021]一般566);贵阳市卫生健康局科学技术计划项目(【2022】筑健卫科技合同字第005号);国家自然科学基金项目(No.82060772);贵州省中医药管理局中医药、民族医药科学技术研究课题项目(No.QZYY-2022-032)。

摘　　要：目的探讨艳山姜挥发油(EOFAZ)对糖尿病诱导视网膜Müller细胞功能障碍的保护作用及机制。方法视网膜Müller细胞使用不同剂量的EOFAZ进行预处理1 h后,采用高糖(30 mmol·L^(-1))孵育48 h,Western blot法检测细胞中自噬信号Beclin1、LC3Ⅱ/Ⅰ、P62蛋白的表达水平;qRT-PCR分析细胞中P62 mRNA、Beclin1 mRNA的转录水平,免疫荧光实验分析LC3Ⅱ/Ⅰ、P62的表达。并进一步使用钙离子螯合剂BAPTA-AM及CaMKⅡ特异性抑制剂KN93孵育细胞,检测自噬信号LC3Ⅱ/Ⅰ、Beclin1、P62蛋白表达水平。使用自噬抑制剂氯喹(CQ)孵育细胞,检测细胞中CaMKⅡ及自噬信号LC3Ⅱ/Ⅰ、P62蛋白的表达。结果与高糖组相比,EOFAZ可明显上调Beclin1、LC3Ⅱ/Ⅰ、P62的表达水平;使用BAPTA-AM及KN93孵育细胞与EOFAZ的作用类似。同时观察到EOFAZ与BAPTA-AM或KN93联合应用后,与EOFAZ单独作用的结果无明显差异。当EOFAZ与CQ共同孵育细胞后,EOFAZ对自噬相关蛋白下调的改善作用可被抑制,对CaMKⅡ的磷酸化水平无明显影响。结论EOFAZ对高糖诱导的视网膜Müller细胞自噬障碍具有明显的改善作用,其作用可能是通过抑制CaMKⅡ信号发挥的。Objective To determine the protective effect and mechanism of essential oil from Fructus Alpiniae zerumbet(EOFAZ)on retinal Müller cell dysfunction induced by diabetes.Methods Retinal Müller cells were treated with high glucose for 48 hours,and different doses of EOFAZ were used as intervention.Expressions of Beclin1,LC3Ⅱ/Ⅰand P62 protein were detected by Western blot.Expression of P62 mRNA and Beclin1 mRNA in the cells was analyzed by qRT-PCR.Expression of LC3Ⅱ/Ⅰand P62 was analyzed by immuno-fluorescence.After calcium chelating agent BAPTA-AM and CaMKⅡspecific inhibitor KN93 were used as intervention.The expression of Beclin1,LC3Ⅱ/Ⅰ,and P62 protein were analyzed by Western blot.Autophagy inhibitor chloroquine(CQ)was used to detect the expression of CaMKⅡ,LC3Ⅱ/Ⅰ,and P62.Results Compared with the high glucose group,EOFAZ significantly up-regulated the level of Beclin1,LC3Ⅱ/Ⅰand P62;the effect of BAPTA-AM and KN93 on the cells was similar to that of EOFAZ.Combined EOFAZ with BAPTA-AM or KN93,there was no significant difference in the results of EOFAZ alone.When cells were treated with CQ,there was no significant effect on the phosphorylation of CaMKⅡ.Conclusion EOFAZ can significantly improve the autophagy disorder of retinal Müller cells induced by high glucose,which may be related to the inhibition of CaMKⅡsignals.

关 键 词：艳山姜挥发油 糖尿病 视网膜MÜLLER细胞 自噬障碍 CaMKⅡ 

分 类 号：R285[医药卫生—中药学]