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 was upregulated,but the expression levels of Bax and Caspase-9 proteins were downregulated,and Bax/Bcl-2 was decreased in medium-and high-dose EOFAZ groups(P<0.05).When compared with EOFAZ and Z-VAD-FMK groups,Caspase-9 protein expression was downregulated in EOFAZ plus Z-VAD-FMK group(P<0.05).Conclusion EOFAZ may effectively ameliorate condition of NIT-1 cell injury-induced by Alloxan,and its mechanism is related to the inhibition of apoptosis.
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)
出处
《贵州医科大学学报》
CAS
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)。
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
essential oil of Alpinia zerumbet
alloxan
diabetic mellitus
NIT-1
pancreatic islet cells
apoptosis
