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

• 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
• 2:贵州医科大学附属医院神经内科

摘要(Abstract)：

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(200 μg/L), medium dose(100 μg/L) and low dose(50 μg/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(200 μg/L), a Z-VAD-FMK group(10 μmol/L) and EOFAZ plus Z-VAD-FMK group(200 μg/L EOFAZ plus 10 μmol/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.

关键词(KeyWords)： 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

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 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

DOI: 10.19367/j.cnki.2096-8388.2023.07.004

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