基于Wnt信号通路探讨七氟烷预处理对高糖培养心肌细胞损伤的影响

Effect of sevoflurane pretreatment on injury of high glucose cultured cardiomyocytes based on Wnt signaling pathway

目的基于Wnt信号通路探讨七氟烷预处理对高糖培养心肌细胞损伤的影响。方法H9c2大鼠心肌细胞随机分为对照组、贫氧组、七氟烷组、高糖+七氟烷组、高糖+七氟烷+XAV-939组,对照组细胞采用正常糖浓度(5.56 mmol/L葡萄糖)培养基进行培养,贫氧组细胞细胞采用在正常培养基中厌氧培养,高糖组细胞贫氧处理后采用高糖(33 mmol/L)培养基进行培养,高糖+七氟烷组细胞贫氧处理后采用含2.2%七氟烷的高糖培养基培养,高糖+七氟烷+XAV-939组细胞贫氧处理后采用含2.2%七氟烷和1μm XAV-939的高糖培养基培养。5组细胞处理12 h后,采用细胞计数试剂盒(CCK-8)试剂盒检测细胞活力;采用原位缺口末端标记法(TUNEL)染色分析细胞凋亡水平;采用酶联免疫吸附试验(ELISA)试剂盒检测细胞氧化应激指标;采用ROS试剂盒检测心肌细胞ROS水平;采用蛋白质印迹法(Western blot)分析Wnt/β-连环蛋白(β-catenin)信号通路转录活性相关蛋白表达的影响。组间计量数据比较采用t检验。结果高糖+七氟烷组细胞活力(1.89±0.12)明显增加高糖组细胞(1.21±0.14),差异有统计学意义(t=4.951,P<0.05)。高糖+七氟烷组细胞SOD活性[(654.23±54.87)nmol/mg]明显高于高糖组[(398.59±51.49)nmol/mg],差异有统计学意义(t=6.021,P<0.05)。高糖+七氟烷组细胞ROS水平[(38.69±)IU/ml]明显高于高糖组[(52.67±6.46)IU/ml],差异有统计学意义(t=3.894,P<0.05)。高糖+七氟烷组TUNEL染色阳性率[(15.63±3.19)%]显著低于高糖组心肌细胞TUNEL染色阳性率[(36.35±4.15)%],差异有统计学意义(t=8.541,P<0.05)。高糖+七氟烷组Wnt3a和β-catenin表达水平(1.26±0.15、1.48±0.14)显著低于高糖组心肌细胞Wnt3a和β-catenin表达水平(2.36±0.12、2.79±0.15),差异有统计学意义(t=4.289、4.012,P<0.05)。结论心肌细胞损伤后,高糖可拮抗七氟烷对心肌细胞的保护作用,主要机制可能与是通过Wnt/β-catenin信号通路激活和氧化应激失衡相关。

Objective To investigate the effect of sevoflurane pretreatment on the injury of high glucose cultured cardiomyocytes based on Wnt signaling pathway.Methods H9C2 rat cardiomyocytes were randomly divided into control group,high glucose group,high glucose+sevoflurane group.The cells in the control group were cultured in normal glucose concentration(5.56 mmol/L glucose)medium,the cells in the high glucose group were cultured in high glucose(33 mmol/L)medium,and the cells in the high glucose+sevoflurane group were cultured in high glucose medium containing 2.2%sevoflurane,After 12 h treatment,the cell viability was detected by CCK8 kit.The level of apoptosis was analyzed by TUNEL staining.The indexes of oxidative stress were detected by ELISA kit.ROS level was analyzed by ROS kit.Wnt/β-catenin signaling pathway was analyzed by Western blot.The comparison of inter group measurement data adopts t-test.Results The cell viability of the high glucose+sevoflurane group(1.89±0.12)was significantly increased(1.21±0.14),and the difference was statistically significant(t=4.951,P<0.05).The SOD activity of cells in the high glucose+sevoflurane group[(654.23±54.87)nmol/mg]was significantly higher than that in the high glucose group[(398.59±51.49)nmol/mg],and the difference was statistically significant(t=6.021,P<0.05).The ROS level in cells of the high glucose+sevoflurane group[(38.69±)IU/ml]was significantly higher than that of the high glucose group[(52.67±6.46)IU/ml],and the difference was statistically significant(t=3.894,P<0.05).The positive rate of TUNEL staining in the high glucose+sevoflurane group[(15.63±3.19)%]was significantly lower than that in the high glucose group[(36.35±4.15)%],and the difference was statistically significant(t=8.541,P<0.05).High sugar+sevoflurane group Wnt3a andβ-catenin the expression level of(1.26±0.15,1.48±0.14)was significantly lower than that of Wnt3a and Wnt3a myocardial cells in the high glucose groupβ-catenin the expression level of(2.36±0.12,2.79±0.15)showed statistically significant differences(t=4.289,4.012,P<0.05).Conclusion After myocardial cell injury,sevoflurane can antagonize the damaging effect of high glucose on myocardial cells,and the main mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.