多柔比星对心肌细胞焦亡及Caspase-3/GSDME信号通路的影响

Effect of doxorubicin on pyroptosis of cardiomyocytes and its relationship with Caspase-3/GSDME pathway

目的观察多柔比星(Dox)对心肌细胞焦亡及Caspase-3/焦孔素E(GSDME)信号通路的影响。方法将大鼠心肌细胞H9c2分为4组,空白对照组加入培养基;Dox组加入4μmol/L Dox处理8 h;GSDME转染对照组转染GSDME siRNA对照无干扰序列,GSDME干扰组转染GSDME siRNA,24 h后加入Dox培养8 h。采用碘化丙啶(PI)染色法观察细胞焦亡情况(PI阳性细胞率),分别用比色法、ELISA法检测细胞损伤后释放入上清液中的乳酸脱氢酶(LDH)、白细胞介素1β(IL-1β)、IL-18,Western blotting法检测Caspase-3/GSDME信号通路相关蛋白Cleaved Caspase-3、GSDME-N(GSDME被Cleaved Caspase-3剪切形成具有细胞成孔作用的片段)及炎症相关蛋白IL-1β、IL-18。结果与空白对照组比较,Dox组、GSDME转染对照组PI阳性细胞率增高,细胞中LDH、IL-1β、IL-18释放量增高,细胞Cleaved Caspase-3、GSDME-N、IL-1β、IL-18蛋白表达增高(P均<0.05)。与Dox组比较,GSDME干扰组PI阳性细胞率及细胞中LDH、IL-1β、IL-18释放量和GSDME-N蛋白表达降低(P均<0.05),GSDME转染对照组各指标差异无统计学意义。结论Dox可以通过激活Caspase-3/GSDME信号通路诱导心肌细胞焦亡,导致心肌损伤。

Objective To observe the effect of doxorubicin(Dox) on pyroptosis of cardiomyocytes,and to explore its relationship with Caspase-3/GSMDE(GSDME) pathway.Methods Rat cardiomyocytes H9c2 were divided into four groups.The cells in the blank control group were added with culture medium;the cells in the Dox group were treated with 4 μmol/L Dox for 8 h;the cells in the GSDME transfection control group were transfected with GSDME siRNA control without interference sequence;the cells in the GSDME interference group were transfected with GSDME siRNA and then were added with Dox and cultured for 8 h.Propidium iodide(PI) staining method was used to observe pyroptosis(PI-positive cell rate),and colorimetry and ELISA were used to detect LDH,IL-1β,IL-18 released into the supernatant after cell injury,and Western blotting was used to detect Cleaved Caspase-3 and GSDME-N(GSDME was cleaved by Cleaved Caspase-3 to form a cell pore-forming fragment) and inflammation-related proteins IL-1β and IL-18.Results Compared with the blank control group,the rate of PI positive cells increased,the release of LDH,IL-1β,and IL-18 in the cells increased,and the expression of Cleaved Caspase-3,GSDME-N,IL-1β,and IL-18 protein increased in the Dox group and the GSDME transfection control group(all P<0.05).Compared with the Dox group,the rate of PI positive cells,the release of LDH,IL-1β,IL-18 and the expression of GSDME-N protein in the GSDME interference group decreased(all P<0.05),and there were no statistical differences in the indicators of the GSDME transfection control group.Conclusion Dox can induce pyroptosis of cardiomyocytes through Caspase-3/GSDME pathway,and lead to myocardial injury.