艳山姜挥发油调控KLF4的表达对高糖诱导血管平滑肌细胞自噬的作用机制  

Effects of essential oil from Fructus Alpiniae zerumbet on high glucose induced autophagy in vascular smooth muscle cells by regulating KLF4 expression

作　　者：敖睿颖 陈虹雨 吴江 张闯 何丽 尚雪祎 白德祚 王宇庭 AO Ruiying;CHEN Hongyu;WU Jiang;ZHANG Chuang;HE Li;SHANG Xueyi;BAI Dezuo;WANG Yuting(School of Nursing,Guizhou Medical University,Guian New Area 561113,Guizhou,China;Department of Basic Medical Sciences,Bijie Medical College,Bijie 551700,Guizhou,China;Key Laboratory of Optimal Utilization of Natural Medicine Resources,Guizhou Medical University,Guian New Area 561113,Guizhou,China;Department of Pharmacy,Qianxi People's Hospital,Qianxi 551500,Guizhou,China)

机构地区：[1]贵州医科大学护理学院,贵州贵安新区561113 [2]毕节医学高等专科学校基础医学系,贵州毕节551700 [3]贵州医科大学天然药物资源优效利用重点实验室,贵州贵安新区561113 [4]黔西市人民医院药剂科,贵州黔西551500

出　　处：《贵州医科大学学报》2025年第2期191-198,共8页Journal of Guizhou Medical University

基　　金：国家自然科学基金项目(82060775);毕节市科学技术局项目(毕科合[2023]22)。

摘　　要：目的探讨艳山姜挥发油(essential oil of fructus alpiniae zerumbet,EOFAZ)基于肠道富集Krüppellike因子(krüppel-like factor 4,KLF4)信号,对高糖(high glucose,HG)诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)自噬流障碍的作用。方法取VSMCs,以35 mmol/L葡萄糖诱导不同时间分为0 h组、12 h组、24 h组、48 h组、72 h组及96 h组,采用MTT法检测细胞存活率,Western blot检测微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)相关蛋白LC3Ⅱ、LC3Ⅰ、自噬接头蛋白1(sequestosome-1,p62)及KLF4的表达水平;联用自噬抑制剂氯喹(chloroquine,CQ)分析EOFAZ对自噬的作用,将大鼠VSMCs分为Control组(5.5 mmol/L葡萄糖)、HG组(35 mmol/L葡萄糖)、低剂量EOFAZ组(35 mmol/L葡萄糖+1μg/L EOAFZ)、高剂量EOFAZ组(35 mmol/L葡萄糖+4μg/L EOFAZ)、CQ组(35 mmol/L葡萄糖+10μmol/L CQ)及CQ+EOFAZ组(35 mmol/L葡萄糖+10μmol/L CQ+4μg/L EOFAZ);采用Western blot检测LC3Ⅱ、LC3Ⅰ、p62及KLF4的表达水平,细胞免疫荧光法检测p62的表达水平;采用siRNA技术敲低大鼠VSMCs内KLF4的表达,分为si NC组(空载体阴性对照)、si NC+HG组(si NC+35 mmol/L葡萄糖)、si NC+低剂量EOFAZ组(35 mmol/L葡萄糖+1μg/L EOFAZ)、si NC+高剂量EOFAZ组(35 mmol/L葡萄糖+4μg/L EOFAZ)、敲低KLF4(siRNA KLF4,siKLF4)组、si KLF4+HG组(si KLF4+35 mmol/L葡萄糖)、si KLF4+低剂量EOFAZ组(si KLF4+35 mmol/L葡萄糖+1μg/L EOFAZ)和si KLF4+高剂量EOFAZ组(si KLF4+35 mmol/L葡萄糖+4μg/L EOFAZ),Western blot法检测p62和KLF4的蛋白表达水平。结果35 mmol/L葡萄糖孵育24 h、48 h、72 h及96 h使VSMCs细胞增殖呈时间依赖性增加(P<0.05),LC3Ⅱ/LC3Ⅰ、p62及KLF4的蛋白表达水平上调(P<0.01);与HG组比较,低、高剂量EOFAZ组可以不同程度下调,LC3Ⅱ/LC3Ⅰ、p62以及KLF4的蛋白表达水平(P<0.01)和p62免疫荧光水平;与CQ组比较,CQ+EOFAZ组可下调,LC3Ⅱ/LC3Ⅰ、p62以及KLF4的蛋白表达水平(P<0.01)和p62免疫荧光水平;与si KLF4+HGObjective To investigate the role of essential oil of Fructus Alpiniae zerumbet(EOFAZ)based on intestinal enrichment of Krüppel-like factor 4(KLF4)signaling on high glucose(HG)-induced impairment of autophagic flow in vascular smooth muscle cells(VSMCs).Methods VSMCs were induced by 35 mmol/L glucose and divided into 0h group,12 h group,24 h group,48 h group,72 h group,and 96 h group.The cell viability was detected by MTT assay.The expression levels of microtubule-associated protein light chain 3(LC3)of LC3Ⅱand LC3Ⅰ,sequestosome-1(p62)and KLF4 were detected by Western blot.Autophagy inhibitor chloroquine(CQ)was used to analyze the effect of EOFAZ on autophagy.VSMCs were divided into the control group(5.5 mmol/L glucose),HG group(35 mmol/L glucose),low-dose EOFAZ group(35 mmol/L glucose+1μg/L EOAFZ),high-dose EOFAZ group(35 mmol/L glucose+4μg/L EOFAZ),CQ group(35 mmol/L glucose+10μmol/L CQ),and CQ+EOFAZ group(35 mmol/L glucose+10μmol/L CQ+4μg/L EOFAZ).The expression levels of LC3Ⅱ,LC3Ⅰ,p62,and KLF4 were detected by Western blot,and the expression level of p62 was detected by immunofluorescence.The expression of KLF4 in VSMCs was knocked down by siRNA technology.The experiment was divided into si NC group(empty vector negative control),si NC+HG group(si NC+35 mmol/L glucose),si NC+low dose EOFAZ group(35 mmol/L glucose+1μg/L EOFAZ),si NC+high dose EOFAZ group(35 mmol/L glucose+4μg/L EOFAZ),knockdown KLF4(si RNA KLF4,si KLF4)group,si KLF4+HG group(si KLF4+35 mmol/L glucose),si KLF4+low-dose EOFAZ group(si KLF4+35 mmol/L glucose+1μg/L EOFAZ),and si KLF4+high-dose EOFAZ group(si KLF4+35 mmol/L glucose+4μg/L EOFAZ).The protein expression levels of p62 and KLF4 were detected by Western blot.Results VSMCs were incubated with 35 mmol/L glucose for 24 h,48 h,72 h,and 96 h,the cell proliferation was increased in a time-dependent manner(P<0.05),and the protein expression levels of LC3Ⅱ/LC3Ⅰ,p62,and KLF4 were up-regulated(P<0.01).Compared with HG group,the protein expression levels of LC3Ⅱ/LC3Ⅰ,p62,a

关 键 词：肠道富集Krüppellike因子 艳山姜挥发油 血管平滑肌细胞 自噬流障碍 氯喹 

分 类 号：R965[医药卫生—药理学] R972[医药卫生—药学]