不同复温时点的热打击人骨骼肌细胞损伤情况观察及其机制

Effect and mechanism of heat stress at different rewarming time on damage of human skeletal muscle cells

目的观察不同复温时点的热打击人骨骼肌细胞(HSKMC)损伤情况,并探讨其可能的机制。方法将对数生长期的HSKMC分为热打击组和对照组,对照组正常培养,热打击组建立热打击细胞损伤模型后置于37℃、5%CO_(2)细胞培养箱中复温,复温时间分别为0、6、12、24 h。采用CCK-8法检测细胞存活率,透射电镜下观察细胞超微结构,Western blotting法检测瞬时感受器电位香草酸受体亚型4(TRPV4)蛋白相对表达量,实时荧光定量PCR法检测TRPV4 mRNA相对表达量。将对数生长期的HSKMC分为HS组、GSK+HS组、HC+HS组和NC组,NC组正常培养,其他三组建立热打击细胞损伤模型,复温时间均为12 h,GSK+HS组、HC+HS组在热打击前0.5 h分别给予TRPV4激动剂GSK1016790A 500 nmol、TRPV4抑制剂HC-0670475μmol,采用流式细胞术检测细胞内Ca^(2+)浓度。结果与对照组比较,复温0、6、12、24 h的热打击组细胞存活率均降低(P均<0.05);随着复温时间的延长,热打击组细胞存活率依次降低,两两比较P均<0.05。与对照组比较,复温0、6 h的热打击组细胞即可出现超微结构改变,主要表现为细胞肿胀、体积变大,细胞质空泡化,线粒体明显肿胀、脱颗粒、双嵴消失,细胞核周间隙增宽,细胞核凝聚、固缩,部分包膜连续性中断;复温12、24 h的热打击组细胞出现核仁溶解消失,细胞核碎裂、染色质边集,细胞质结构崩解、呈颗粒状。与对照组比较,复温0、6、12、24 h的热打击组细胞TRPV4 mRNA及蛋白相对表达量均升高(P均<0.05)。NC组、HC+HS组、HS组、GSK+HS组细胞内Ca^(2+)浓度依次升高,组间两两比较P均<0.05。结论热打击会造成HSKMC存活率降低及结构损伤,热打击后复温时间越长对细胞的损伤越大,其机制可能与促进TRPV4表达及其介导的Ca^(2+)内流有关。

Objective To explore the the effect and possible mechanism of heat stress at different rewarming time on the damage of human skeletal muscle cells(HSKMC).Methods HSKMC in the logarithmic growth phase were divided into the heat stress group and control group.Cells in the control group were cultured normally,and the heat stress models were established in the heat stress group and then were rewarmed in 37℃and 5%CO_(2) cell incubator for 0,6,12 and 24 h,respectively.The cell survival rate was detected by CCK-8,the cell ultrastructure was observed by transmission electron microscopy,TRPV4 protein was detected by Western blotting,and TRPV4 mRNA was detected by real-time fluorescence quantitative PCR.HSKMC in the logarithmic growth phase were randomly divided into the HS group,GSK+HS group,HC+HS group,and NC group.Cells in the NC group were cultured normally,and the heat stress models were established in the other three groups,and the rewarming time was 12 h.Cells in the GSK+HS group and HC+HS group were given TRPV4 agonist GSK1016790A(500 nmol)and TRPV4 inhibitor HC-067047(5μmol)at 0.5 h before heat stress,and Ca^(2+) level was detected by flow cytometry.Results Compared with the control group,the survival rate of cells in heat stress group decreased after rewarming for 0,6,12 and 24 h(P<0.05).With the extension of rewarming time,the survival rate of cells in the heat stress group decreased successively,and significant difference was found between these two groups(all P<0.05).Compared with the control group,ultrastructural changes were observed in the cells in the heat stress group after rewarming for 0 and 6 h,which were mainly manifested as cell swelling,enlarged volume,cytoplasmic vacuolation,obvious mitochondrial swelling,degranulation,double ridge loss,widening of perinuclear space,nucleus condensation and contraction,and interruption of some envelope continuity.After rewarming for 12 and 24 h,the cells in the heat stress group even showed nucleolysis and disappearance,nucleus fragmentation,chromatin boundary aggregation,and cytoplasmic structure disintegration and granulation.Compared with the control group,the relative expression levels of TRPV4 mRNA and protein in heat stress group increased after rewarming for 0,6,12 and 24 h(all P<0.05).The levels of intracellular Ca^(2+) in the NC group,HC+HS group,HS group and GSK+HS group increased successively,and significant difference was found between groups(all P<0.05).Conclusions Heat stress can reduce HSKMC survival rate and damage HSKMC structure.The longer rewarming time after heat stress,the greater the damage to HSKMC cells.The mechanism may be related to the promotion of TRPV4 expression and its mediated Ca^(2+) influx.