杨桃根活性成分对高糖诱导H9c2心肌细胞损伤的改善作用及机制
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篇名: 杨桃根活性成分对高糖诱导H9c2心肌细胞损伤的改善作用及机制
TITLE: Improvement Effect and Its Mechanism of Active Components of Averrhoa carambola on High Glucose-induced Injury of H 9c2 Myocardial Cell
摘要: 目的:研究杨桃根活性成分2-十二烷基-6-甲氧基-2,5-二烯-1,4-环己二酮(DMDD)对高糖诱导H9c2心肌细胞损伤的改善作用及机制。方法:将H9c2心肌细胞分为正常组、高糖组、渗透压对照组和DMDD高、中、低浓度组(8、4、2μmol/L)。正常组和高糖组细胞分别加入低糖(含5.5mmol/L葡萄糖,下同)、高糖DMEM培养基(含33.3mmol/L葡萄糖,下同)培养48h;渗透压对照组细胞加入含27.5mmol/L甘露醇的低糖DMEM培养基培养48h;DMDD各浓度组细胞先加入高糖DMEM培养基培养24h,再加入含相应浓度DMDD的高糖DMEM培养基继续培养24h。各组细胞培养结束后,采用MTT法检测细胞存活率;采用相应试剂盒检测细胞上清液中活性氧簇(ROS)、谷胱甘肽过氧化物酶(GSH-Px)、乳酸脱氢酶(LDH)的活性;采用酶联免疫吸附法(ELISA)检测细胞上清液中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)的水平;采用吖啶橙/溴化乙锭(AO/EB)染色法检测细胞凋亡情况;采用Westernblot法检测细胞中凋亡相关蛋白[活化的胱天蛋白酶3(cleavedcaspase-3)、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)]的表达水平以及核因子κB(NF-κB)/NF-κB抑制蛋白α(IκBα)信号通路相关蛋白(NF-κBp65、IκBα)的磷酸化水平。结果:与正常组比较,高糖组细胞存活率、GSH-Px活性、Bcl-2蛋白表达水平均显著降低(P<0.01);ROS、LDH活性,TNF-α、IL-6水平,Bax、cleavedcaspase-3蛋白表达水平和NF-κBp65、IκBα蛋白磷酸化水平均显著升高(P<0.01);细胞呈橙黄色荧光,且形态模糊的细胞明显增多,表现出明显的凋亡状态。渗透压对照组细胞上述指标与正常组比较差异无统计学意义。与高糖组比较,DMDD各浓度组细胞上述指标活性或水平(低浓度组细胞存活率、LDH活性除外)均显著逆转(P<0.05或P<0.01);细胞中橙黄色荧光明显减少,细胞形态较完整。结论:DMDD可改善高糖诱导的H9c2心肌细胞损伤;其作用机制可能与抑制氧化应激和炎症反应、调控凋亡相关蛋白和NF-κB/IκBα通路相关蛋白的表达有关。
ABSTRACT: OBJECTIVE:To study the imp rovement effects of 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione(DMDD) from Averrhoa carambola on H 9c2 myocardial cell injury induced by high glucose and its mechanism. METHODS :H9c2 myocardial cells were divided into normal group ,high glucose group ,osmotic pressure control group ,DMDD high ,medium and low concentration groups (8,4,2 μmol/L). Normal group and high glucose group were treated with low glucose DMEM medium (containing 5.5 mmol/L glucose ,similarly hereinafter ) and high glucose DMEM medium (containing 33.3 mmol/L glucose , similarly hereinafter )for 48 h,respectively. The cells in osmotic pressure control group were cultured in low glucose DMEM medium containing 27.5 mmol/L mannitol for 48 h. In DMDD groups ,cells were cultured in high glucose DMEM medium for 24 h, and then in high glucose DMEM medium containing corresponding concentration of DMDD for 24 h. At the end of cell culture ,MTT metho d was used to detect the cell survival rate. The activities of ROS , GSH-Px and LDH in cellsupernatant were detected by using related kits. ELISA assay was used to detect the levels of TNF-α and IL-6 in cell supernatant. Cell apoptosis was d etected by acridine orange/ethidium bromide (AO/EB)staining. Western blot assay was used to detect the expression of apoptosis related proteins (cleaved caspase- 3,Bcl-2,Bax)and the phosphorylation level of nuclear factor κB (NF-κB)/NF-κB suppressor protein α(IκBα)signaling pathway related proteins (NF-κB p65,IκBα). RESULTS :Compared with the normal group ,survival rate ,the activity of GSH-Px and protein expression of Bcl- 2 in high glucose groups were decreased significantly(P<0.01);the activities of ROS and LDH ,the levels of TNF-α and IL-6,the protein expression of Bax and cleaved caspase-3,and the phosphorylation level of NF-κB p65 and IκBα were increased significantly(P<0.01);the cells showed orange yellow fluorescence ,and the number of cells with fuzzy morphology increased significantly ,showing an obvious apoptotic state. There was no statistical significance in above indexes of osmotic pressure control group compared with normal group. Compared with high glucose group ,the activities or levels of above indexes (except for cell survival rate an LDH activity in low concentration group )were reversed significantly in DMDD groups (P<0.05 or P<0.01);the orange yellow fluorescence in the cells decreased significantly ,and the cell morphology was relatively complete. CONCLUSIONS :DMDD can significantly improve H9c2 myocardial cell injury induced by high glucose ;the mechanism of which may be associated with suppressing oxidative stress and inflammatory response ,regulating the expression of apoptosis related protein and NF-κB/IκBα pathway related protein.
期刊: 2021年第32卷第15期
作者: 高雨桐,吴娅妮,陈铭,张晓琳,韦秀莎,韦晓洁,黄仁彬
AUTHORS: GAO Yutong ,WU Yani,CHEN Ming,ZHANG Xiaolin ,WEI Xiusha ,WEI Xiaojie ,HUANG Renbin
关键字: 2-十二烷基-6-甲氧基-2,5-二烯-1,4-环己二酮;糖尿病心肌病;H9c2心肌细胞;凋亡;NF-κB/IκBα信号通路
KEYWORDS: 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione;Diabetic cardiomyopathy ;H9c2 myocardial cell ;Apoptosis;
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