艳山姜挥发油对四氧嘧啶诱导的胰岛细胞损伤保护作用及机制  

Effect of essential oil from Fructus Alpiniae Zerumbet on alloxan-inducedpancreatic islet cell injury and its mechanism

作　　者：文波 罗芳 付凌云 徐旖旎 陶玲 张甜 林浩恒 沈祥春 WEN Bo;LUO Fang;FU Lingyun;XU Yini;TAO Ling;ZHANG Tian;LIN Haoheng;SHEN Xiangchun(School of Pharmaceutical Sciences,Key Laboratory of Optimal Utilizaiton of Natural Medicine Resources&the High Educational Key Laboratory of Guizhou Province for Natural Medicinal Pharmacology and Druggability,Guiyang 550025,Guizhou,China;Department of Neurology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550001,Guizhou,China)

机构地区：[1]贵州医科大学药学院天然药物资源优效利用重点实验室&贵州省高等学校天然药理与成药性评价重点实验室,贵州贵阳550025 [2]贵州医科大学附属医院神经内科,贵州贵阳550001

出　　处：《贵州医科大学学报》2023年第7期767-773,共7页Journal of Guizhou Medical University

基　　金：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(QZYY-2022-032);国家自然科学基金项目(81760725);国家自然科学基金委-贵州喀斯特中心项目子课题(U1812403-4-4);贵州省科技计划项目(黔科合基础-ZK〔2022〕一般005);贵州省科技计划项目(黔科合基础〔2020〕1Z069)。

摘　　要：目的探讨艳山姜挥发油(EOFAZ)对四氧嘧啶诱导大鼠胰岛细胞(NIT-1)损伤的保护作用及机制。方法体外培养NIT-1细胞至对数生长期,测定不同浓度四氧嘧啶(0、1、4、10、20、40、60、80及100 mmol/L)下细胞存活率,取存活率较高的浓度进行后续实验;细胞分为对照组、模型组、EOFAZ低、中、高剂量组(分别为50、100及200μg/L),采用MTT法检测各组细胞存活率,生化法检测丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)含量,ELISA法检测乳酸脱氢酶(LDH)含量和胰岛素分泌量,Western blot法检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2关联X蛋白(Bax)以及半胱氨酸-天冬氨酸特异性蛋白酶9(Caspase-9)蛋白的表达;机制研究进一步设置EOFAZ组(200μg/L)、Z-VAD-FMK组(10μmol/L)以及EOFAZ+Z-VAD-FMK组(200μg/L EOFAZ+10μmol/L Z-VAD-FMK),采用Western blot法检测Caspase-9蛋白的表达。结果选择40 mmol/L四氧嘧啶(存活率70.7%)作为诱导细胞最佳损伤浓度,并复制NIT-1细胞损伤模型;与对照组比较,模型组细胞存活率降低、MDA和LDH释放量增加、SOD、GSH-PX释放和胰岛素分泌量减少、Bcl-2蛋白表达下调、Bax和Caspase-9蛋白表达上调(P<0.05);与模型组比较,EOFAZ各剂量组细胞存活率升高、胰岛素分泌增加(P<0.05),EOFAZ中、高剂量组MDA和LDH释放降低、SOD和GSH-PX释放量增加、Bcl-2蛋白表达上调、Bax和Caspase-9蛋白表达下调、Bax/Bcl-2降低(P<0.05);与EOFAZ组、Z-VAD-FMK组比较,EOFAZ+Z-VAD-FMK组Caspase 9蛋白表达均下调(P<0.05)。结论EOFAZ可改善四氧嘧啶诱导的NIT-1胰岛细胞损伤,其机制与抑制细胞凋亡有关。Objective To investigate the protective effect of essential oil from Fructus alpiniae zerumbet(EOFAZ)on alloxan-induced rat pancreatic islet cell(NIT-1)injury and its mechanism.Methods NIT-1 cells were cultured in vitro until logarithmic growth phase,and cell survival rate was measured under different concentrations(0,1,4,10,20,40,60,80,and 100 mmol/L)of Alloxan treatment.The concentration with a high survival rate was taken for subsequent experiments.Cells were divided into a control group,a model group and EOFAZ groups including high dose(200μg/L),medium dose(100μg/L)and low dose(50μg/L).MTT assay was used to examine cell survival rate.Biochemical methods were used to detect the contents of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX).ELISA was used to detect the contents of lactate dehydrogenase(LDH)and insulin secretion.Western blot was used to detect the expression of B-lymphoblastoma 2(Bcl-2),Bcl-2 associated X protein(Bax)and cysteine aspartate-specific protease-9(Caspase-9).For mechanism study,cells were further divided into a EOFAZ group(200μg/L),a Z-VAD-FMK group(10μmol/L)and EOFAZ plus Z-VAD-FMK group(200μg/L EOFAZ plus 10μmol/L Z-VAD-FMK).Caspase-9 protein expression was detected by Western blot.Results Alloxan at 40 mmol/L concentration which kept 70.7%cell survival rate was selected as the optimal concentration to induce cell damage.NIT-1 cells were treated with Alloxan at 40 mmol/L to replicate cell damage model.When compared with control group,model group showed decreased cell survival rate,increased MDA and LDH release,decreased SOD,decreased GSH-PX release and insulin secretion,downregulated Bcl-2 protein expression,and upregulated Bax and Caspase-9 protein expression(P<0.05).When compared with model group,the cell survival rates and insulin secretion in all EOFAZ groups were increased(P<0.05).When compared with model group,the release of MDA and LDH were decreased,while the release of SOD and GSH-PX were increased,and the expression of Bcl-2 protein wa

关 键 词：艳山姜挥发油 四氧嘧啶 糖尿病 NIT-1 胰岛细胞 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]