基于TXNIP信号通路调控的艳山姜挥发油对高糖诱导血管内皮细胞损伤的保护作用  

Essential Oil from Alpiniae Zerumbet Fructus Alleviates High Glucose-induced HUVEC Injury via TXNIP Signaling Pathway

作　　者：张雯[1] 张嫩玲[1] 张彦燕[1] 陈妍[1] 付凌云[1] 潘迪[1] 沈祥春[1] ZHANG Wen;ZHANG Nen-ling;ZHANG Yan-yan;CHEN Yan;FU Ling-yun;PAN Di;SHEN Xiang-chun(Key Laboratory of Optimal Utilization of Natural Medicinal Resources,Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学天然药物资源优效利用重点实验室,贵阳550025

出　　处：《中国实验方剂学杂志》2021年第13期21-27,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81760725,U1812403-4-4);贵州省科技基金重点项目(黔科合基础[2020]1Z069);贵州省科技厅社发公关项目(黔科合支撑[2017]2840,黔科合支撑[2018]2767);贵州医科大学药学国际科技合作基地项目(黔科合平台人才[2017]5802)。

摘　　要：目的:研究艳山姜挥发油(EOAZF)对高浓度葡萄糖(HG)诱导人脐静脉内皮细胞(HUVECs)损伤的保护作用,为研究EOAZF改善糖尿病诱导的心血管疾病提供实验依据。方法:该项目研究分为正常组,模型组(25 mmol·L^(-1)葡萄糖组),阳性药组(100 mg·L^(-1)维生素C),EOAZF低、中、高浓度组(0.25,1,4μg·L^(-1))。通过建立HG诱导HUVECs损伤模型,检测不同给药组内皮细胞中的丙二醛(MDA),一氧化氮(NO)和内皮素1(ET-1)的分泌量,评价EOAZF对高糖诱导HUVECs损伤的保护作用;采用AnnexinV-FITC/PI双染实验以及DCFH-DA荧光探针标记,考察EOAZF对HG诱导HUVECs的凋亡水平和活性氧(ROS)产生的影响;采用蛋白免疫印迹法(Western blot),实时荧光定量聚合酶链式反应(Real-time PCR),检测硫氧还原蛋白相互结合蛋白(TXNIP)与硫氧还原蛋白(Trx-1)蛋白表达和转录水平,采用胰岛素二硫键还原法测定细胞内总Trx-1的酶活性。结果:细胞形态学结果显示,与正常组比较,模型组HUVECs增殖速度明显降低,细胞形态受损;与模型组比较,0.25,1,4μg·L^(-1)EOAZF对HG诱导损伤的HUVECs具有保护作用,且呈浓度依赖性;与正常组比较,模型组显著上调HUVECs内MDA和ET-1分泌量(P<0.05),下调NO的分泌(P<0.01);与模型组比较,不同浓度EOAZF均能改善HG诱导的MDA,ET-1和NO的分泌,其中4μg·L^(-1)EOAZF能明显降低HG诱导的HUVECs细胞MDA和ET-1的分泌量(P<0.05),明显上调HUVECs细胞NO的分泌量(P<0.05);细胞凋亡和ROS检测结果显示,与正常组比较,模型组细胞凋亡和ROS水平均显著上调(P<0.01);与模型组比较,4μg·L^(-1)EOAZF明显降低HG诱导的HUVECs中ROS水平和细胞凋亡率(P<0.05);免疫印迹实验结果和Trx-1活性检测结果显示,与正常组比较,造模组细胞中TXNIP的mRNA和蛋白水平显著上调(P<0.01),Trx-1的活性显著降低(P<0.01);与模型组比较,4μg·L^(-1)EOAZF明显下调HG诱导的TXNIP的mRNA与蛋白的表达上调(P<0.05),同时增强细胞内总TrxObjective:To study the protective effect of essential oil from Alpiniae Zerumbet Fructus(EOAZF)against high glucose(HG)-induced injury of human umbilical vein endothelial cells(HUVECs)in vitro,so as to provide experimental evidence for the treatment of diabetes-induced cardiovascular diseases with EOAZF.Method:The cells were divided into the normal group,model group(25 mmol·L^(-1)glucose),positive control group(100 mg·L^(-1)vitamin C),and the low-(0.25μg·L^(-1)),medium-(1μg·L^(-1)),and high-dose(4μg·L^(-1))EOAZF groups.The HUVECs were damaged by HG.The secretion amounts of malondialdehyde(MDA),nitric oxide(NO),and endothelin-1(ET-1)in HUVECs of different groups were measured to assess the protective effect of EOAZF against HG-induced injury.The effects of EOAZF on the apoptosis and reactive oxygen species(ROS)generation of HUVECs damaged by HG were detected by Annexin V-fluorescein isothiocyanate/propidium iodide(Annexin V-FITC/PI)staining and dichloro-dihydro-fluorescein diacetate(DCFH-DA)assay.The protein and mRNA expression levels of thioredoxin interacting protein(TXNIP)and thioredoxin 1(Trx-1)were determined by Western blot and Real-time polymerase chain reaction(Real-time PCR),followed by the measurement of total intracellular Trx-1 activity with insulin disulfide reduction method.Result:The comparison with the control group revealed that the proliferation of HUVECs in the model group was significantly inhibited and their shape was damaged.Compared with the model group,EOAZF protected HUVECs against HG-induced injury in a concentration-dependent manner.The secretion amounts of MDA and ET-1(P<0.05)in the model group were increased in contrast to those in the control group,while the NO level was decreased(P<0.01).Compared with the model group,EOAZF at all the three concentrations,especially at4μg·L^(-1),obviously reduced the secretion of MDA and ET-1(P<0.05),but elevated NO after HG induction(P<0.05).The cell apoptosis assay and ROS detection results demonstrated that the apoptosis and ROS level in

关 键 词：艳山姜挥发油 高糖 人脐静脉内皮细胞(HUVECs) 硫氧还原蛋白相互结合蛋白(TXNIP)信号通路 

分 类 号：R2-0[医药卫生—中医学] R22