摘要
目的:探讨新型复合水凝胶对过氧化氢(H_(2)O_(2))诱导心肌细胞氧化应激损伤的保护作用,并阐明其可能的作用机制。方法:小鼠皮下注射水凝胶100μL,1、14和28 d正常饲养后处死小鼠,取材,HE染色观察水凝胶的组织相容性。提取出生1d SD大鼠的原代心肌细胞,建立心肌细胞氧化应激损伤模型。原代心肌细胞分为对照组、H_(2)O_(2)组和水凝胶(H_(2)O_(2)+Hydrogel)组。对照组原代心肌细胞正常培养,H_(2)O_(2)组原代心肌细胞采用200μmol·L^(-1)H_(2)O_(2)溶液处理24 h;HH_(2)O_(2)+Hydrogel组原代心肌细胞采用1 g·L^(-1)的水凝胶与200μmol·L^(-1)H_(2)O_(2)共孵育24 h。采用细胞计数试剂盒8 (CCK-8)法检测各组心肌细胞活性,双氢乙啶(DHE)和2',7'-二氯荧光素二乙酸酯(DCFH-DA)检测各组心肌细胞中活性氧(ROS)水平,鬼笔环肽荧光染色标记法检测各组心肌细胞中纤维形肌动蛋白(F-actin)表达情况,免疫荧光法检测各组心肌细胞中连接蛋白43 (Cx43)和心肌肌钙蛋白T (cTnT)表达情况,TUNEL染色法检测各组心肌细胞凋亡率,Western blotting法检测各组心肌细胞中凋亡相关蛋白B细胞淋巴瘤2 (Bcl-2)和Bcl-2相关X蛋白(Bax)的表达水平。结果:HE染色,水凝胶周围组织炎性细胞浸润较少,皮下埋植的炎症反应较小。与对照组比较,H_(2)O_(2)组心肌细胞活性明显降低(P<0.05),细胞中ROS水平明显升高(P<0.05),细胞中Cx43、cTnT和F-actin蛋白表达水平明显降低(P<0.001),心肌细胞凋亡率明显升高(P<0.01),心肌细胞中Bcl-2蛋白表达水平升高(P<0.001),Bax蛋白表达水平降低(P<0.01);与H_(2)O_(2)组比较,H_(2)O_(2)+Hydrogel组心肌细胞活性明显升高(P<0.05),细胞中ROS水平明显降低(P<0.01),细胞中cTnT、Cx43和F-actin蛋白表达水平明显升高(P<0.01),心肌细胞凋亡率明显降低(P<0.001),心肌细胞中Bcl-2蛋白表达水平降低(P<0.01),Bax蛋白表达水平升高(P<0.01)。结论:水凝胶能够通过清除环境中的ROS促进心肌细胞相关蛋白的表达,抑制心肌细胞凋亡,发挥对氧化应激环境下心肌细胞的保护作用。
Objective:To investigate the protective effect of a composite hydrogel against hydrogen peroxide(H₂O₂)-induced oxidative stress injury in the cardiomyocytes,and to clarify its possible mechanism.Methods:The mice were subcutaneously injected with 100μL of hydrogel.After normal feeding for 1,14,and 28 d,the mice were sacrificed.Tissue samples were collected and subjected to HE staining to observe the histocompatibity of the hydrogel.The primary cardiomyocytes isolated from 1-day-old SD rats were used to establish an oxidative stress injury model.The primary cardiomyocyties were divided into control,H_(2)O_(2) and H_(2)O_(2)+Hydrogel groups.The primary cardiomyocytes in control group were cultured normally,the primary cardiomyocytes in H_(2)O_(2) group were treated with 200μmol·L^(-1) H_(2)O_(2) for 24 h,and the primary cardiomyocytes in H_(2)O_(2)+Hydrogel group were incubated with 1 g·L^(-1) composite hydrogel and 200μmol·L^(-1) H_(2)O_(2) for 24 h.The viabilities of cardiomyocytes in various groups were assessed by cell counting kit-8(CCK-8)method.Dihydroethidium(DHE)and 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)staining were used to assess the reactive oxygen species(ROS)levels in the cells.The expressions of filamentous actin(F-actin)in the cells in various groups were detected by phalloidin fluorescence staining;the expressions of connexin 43(Cx43)and cardiac troponin T(cTnT)proteins in the cardiomyocytes in various groups were detected by immunofluorescence method.The apoptotic rates of cardiomyocytes in various groups were assessed with TUNEL staining method.The expression levels of apoptosis-related proteins B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)in the cardiomyocytes in various groups were assessed by Western blotting method.Results:The HE staining results showed that the inflammatory cells around the implanted hydrogel were less infiltrated,and the inflammatory reaction of subcutaneous implantation was less.Compared with control group,the viability of cardiomyocytes in H_(2)O_(2) group was significantly decreased(P<0.05),the level of ROS in the cells was increased(P<0.05),the expression levels of Cx43,cTnT and F-actin proteins in the cells were decreased(P<0.001),the apoptosis rate of cardiomyocytes were decreased(P<0.01),the expression level of Bcl-2 protein in the cells was increased(P<0.001),and the expression level of Bax protein was decreased(P<0.01).Compared with H_(2)O_(2) group,the viability of cardiomyocytes was significantly increased(P<0.05),the level of ROS in the cells was decreased(P<0.01),the expression levels of cTnT,Cx43 and F-actin proteins were increased(P<0.01),the apoptotic rate of cardiomyocytes were significantly decreased(P<0.001),the expression level of Bcl-2 protein in the cells were decreased(P<0.01),and the expression level of Bax protein was increased(P<0.01).Conclusion:Hydrogel may promote the expression of cardiomyocyte-related proteins by scavenging ROS in the environment and inhibit the cardiomyocyte apoptosis to achieve the protective effect on the cardiomyocytes under oxidative stress.
作者
王岳
马宁
卢嘉骏
王成瑶
陈琳渝
任宇晨
李景武
孙红
WANG Yue;MA Ning;LU Jiajun;WANG Chengyao;CHEN Linyu;REN Yuchen;LI Jingwu;SUN Hong(Key Laboratory of Chronic Diseases,School of Basic Medical Sciences,North China University of Science and Technology,Tangshan 063210,China;Key Laboratory of Rehabilitation Engineering and Regenerative Medicine,School of Nursing and Rehabilitation,North China University of Science and Technology,Tangshan 063000,China;Key Laboratory of Molecular Oncology,Hebei Provincial People’s Hospital,Tangshan 063000,China)
出处
《吉林大学学报(医学版)》
北大核心
2025年第2期352-359,共8页
Journal of Jilin University:Medicine Edition
基金
国家自然科学基金项目(U20A20261)
河北省科技厅自然科学基金项目(H2023209021,H2024209013)
河北省教育厅大学生创新创业训练计划项目(202410081057)
河北省唐山市科技局重点研发科技项目(22150201A)。
