甲基丁香酚保护胰岛细胞免受缺氧/复氧损伤的作用机制研究  

Machanism of methyleugenoc in protecting islet cells from hypoxia/reoxygenation injury

作　　者：李晴雯 杨慧芳 张季 王梦琴 张佳思 孙凯伦 王志恒 宫念樵[1] Li Qingwen;Yang Huifang;Zhang Ji;Wang Mengqin;Zhang Jiasi;Sun Kailun;Wang Zhiheng;Gong Nianqiao(Institute of Organ Transplantation,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology Key Laboratory of Organ Transplantation,Ministry of Education NHC Key Laboratory of Organ Transplantation Key Laboratory of Organ Transplantation,Chinese Academy of Medical Sciences,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院器官移植研究所,器官移植教育部重点实验室,国家卫生健康委员会器官移植重点实验室,中国医学科学院器官移植重点实验室,武汉430030

出　　处：《中华器官移植杂志》2023年第4期229-236,共8页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金(81873623和81570678);国家科技部"973"项目子课题(2013CB530803)。

摘　　要：目的探讨甲基丁香酚对胰岛缺血/再灌注损伤的保护作用及其可能机制。方法选取6~8周雄性BALB/c小鼠分离纯化胰岛,将其分为正常对照(Normal)组(普通培养,不予任何处理)、缺氧/复氧(H/R)组(予H/R处理)、H/R+二甲基亚砜(DMSO)组[予二甲基亚砜(dimethyl sulfoxide,DMSO)和H/R处理];H/R+甲基丁香酚(Me)组(予以甲基丁香酚和H/R处理)。使用吖啶橙/碘化丙啶双染,对每组胰岛细胞进行活力检测,酶联免疫吸附法(ELISA)测定胰岛细胞功能,即胰岛素分泌情况。选取小鼠的胰岛β细胞系Min6细胞,通过CCK8检测甲基丁香酚在不同的浓度梯度下对正常培养和经H/R处理后的胰岛细胞的增殖活性影响。再将Min6细胞按照处理方式不同分为正常对照(Normal)组、缺氧/复氧(H/R)组、H/R+二甲基亚砜(DMSO)组和H/R+甲基丁香酚(Me)组,分组定义同小鼠原代胰岛。通过流式细胞术和Hoechst 33342核染色检测各组Min6细胞凋亡率,Western blot检测各组p-JNK、p-p38、JNK、p38、Bcl-2、Bax等蛋白的表达情况。所得数据用单因素ANOVA或t检验进行统计学分析。结果H/R组原代胰岛细胞死亡细胞占比为(29.47±2.65)%,较Normal组的(7.63±1.53)%明显上升,组间差异有统计学意义(P<0.001)。H/R+Me组胰岛死亡细胞占比为(20.63±3.07)%,较H/R组和H/R+DMSO组的(29.47±2.65)%和(30.13±1.50)%下降,组间比较,差异均有统计学意义(P<0.05和P<0.01)。在高糖刺激下,H/R+Me组胰岛的胰岛素分泌水平为(1.76±0.08)mg/L,较H/R组和H/R+DMSO组的(1.24±0.14)mg/L和(1.27±0.05)mg/L上升,差异均有统计学意义(P<0.01)。正常的Min6细胞经甲基丁香酚处理后,在一定浓度范围(0~40μmol/L)内,对细胞的活性无明显影响。经H/R处理的Min6细胞,予甲基丁香酚(5μmol/L)后,细胞活性较未添加甲基丁香酚组增加(1.19±0.03和1.00±0),差异有统计学意义(P<0.01)。与H/R组和H/R+DMSO组相比,H/R+Me组细胞的总凋亡率下降(Hoechst 33342染色:14.50%±1.0Objective To explore the protective effect of methyl eugenol(Me)on islet ischemia/reperfusion(I/R)injury and elucidate its underlying mechanism.Methods The islets were isolated and purified from 6-8 week male BALB/c mice and divided into four groups of normal control(normal culture without any treatment),hypoxia/reoxygenation(H/R treatment),H/R+dimethyl sulfoxide(DMSO dosing plus H/R treatment)and H/R+Me(Me dosing plus H/R).Viability of islet cells in each group was detected by acridine orange(AO)/propidium iodide(PI)double stain.Function of islet cells(insulin secretion)was measured by enzyme-linked immunosorbent assay(ELISA).Murine isletβMin6 cells were selected for detecting the effect of Me on the proliferative activity of normal cultured and H/R treated islet cells under different concentration gradients by CCK8.Then Min6 cells were divided into four groups of normal,H/R,H/R+DMSO and H/R+Me.The definition of group was the same as that of primary murine islets.Flow cytometry and Hoechst 33342 nuclear stain were utilized for detecting cell apoptotic rate in each group.The protein expressions of p-JNK,p-p38,JNK,p38,Bcl-2 and Bax were detected by Western blot.And the data were processed by one-way ANOVA or t test.Results The proportion of dead islet cells in H/R group was(29.47±2.65)%and it was significantly lower than that in normal group(7.63±1.53)%.And the inter-group differences were statistically significant(P<0.001).The proportion of dead islet cells was(20.63±3.07)%in H/R+Me group.It was higher than that in H/R group(29.47±2.65)%and in H/R+DMSO group(30.13±1.50)%and inter-group difference was statistically significant(P<0.05&P<0.01).Under the stimulation of high glucose,the insulin secretion level of islet in H/R+Me group was(1.76+0.08)mg/L,which was higher than that in H/R group and H/R+DMSD group(1.24±0.14)mg/L and(1.27±0.05)mg/L,and the difference was statistically significant[(1.76±0.08)vs.(1.24±0.14)mg/L;(1.76±0.08)vs.(1.27±0.05)mg/L,P<0.01].There was no significant effect on cell viabili

关 键 词：胰岛 甲基丁香酚 凋亡 

分 类 号：R965[医药卫生—药理学]