心脏成纤维细胞释放的IL-6在阿霉素致SD乳鼠心肌细胞损伤中的作用研究

Role of IL-6 released by neonatal rat cardiac fibroblasts in doxorubicin-induced cardiomyocyte injury

目的:探讨阿霉素/多柔比星(doxorubicin,DOX)刺激后,SD乳鼠心脏组织中白细胞介素6(inter‐leukin-6,IL-6)的细胞来源,并初步探讨IL-6在DOX引起SD乳鼠心肌损伤中发挥的作用及机制。方法:原代培养SD乳鼠的心脏成纤维细胞和心肌细胞,CCK-8法检测DOX对心脏成纤维细胞和心肌细胞的毒性;ELISA检测心脏成纤维细胞和心肌细胞上清中炎症因子IL-6含量;鬼笔环肽染色观察心肌细胞骨架F-肌动蛋白的改变;Transwell小室共培养SD乳鼠成纤维细胞和心肌细胞,进行如下分组:心肌细胞对照组、DOX处理心肌细胞组、共培养细胞对照组和DOX处理共培养细胞组,Western blot检测各组心肌细胞心房钠尿肽(atrial natriuretic peptide,ANP)、脑钠尿肽(brain natriuretic peptide,BNP)和肌球蛋白重链7(myosin heavy chain 7,MYH7)的蛋白水平;使用IL-6抗体后,进行如下分组:共培养细胞对照组、DOX处理共培养细胞组、IL-6处理共培养细胞对照组和DOX+IL-6处理共培养细胞组,Western blot检测共培养组中心肌细胞ANP和MYH7的蛋白水平。结果:(1)与对照组比较,DOX显著抑制乳鼠心脏成纤维细胞增殖,亦可导致心肌细胞损伤(P<0.01);(2)DOX刺激48 h后,心脏成纤维细胞释放IL-6显著增加(P<0.01),而心肌细胞释放IL-6与对照组相比,无显著差异(P>0.05);(3)DOX刺激的心脏成纤维细胞条件培养液作用于心肌细胞后,心肌细胞骨架蛋白肌丝排列出现紊乱、断裂;(4)DOX刺激后,共培养组心肌细胞ANP、BNP和MYH7的蛋白水平增加,与单独心肌细胞组相比,具有统计学意义(P<0.01);(5)共培养组加入IL-6抗体后,再给予DOX刺激,其ANP和MYH7的表达均显著下降(P<0.05)。结论:DOX刺激原代细胞培养的SD乳鼠心脏成纤维细胞分泌产生大量的IL-6,而后通过旁分泌引起心肌细胞骨架蛋白改变、促进心肌细胞肥大,进而导致心肌损伤。

AIM:To explore the cell type which releases interleukin-6(IL-6)after doxorubicin(DOX)stimu‐lation in neonatal rat heart.Furthermore,to explored the role of IL-6 in DOX-induced cardiomyocyte injury preliminarily.METHODS:The CCK-8 assay was used to detect the toxicity of DOX in neonatal ratcardiac fibroblasts and cardiomyo‐cytes.The releases of IL-6 in neonatal rat cardiac fibroblasts and cardiomyocytes supernatants were measured by ELISA.The morphological changes of cytoskeleton of neonatal rat cardiomyocytes were observed by phalloidin staining.Cardiac fi‐broblasts and cardiomyocytes were co-cultured by Transwell chamber,and divided into 4 groups:cardiomyocytes group,cardiomyocytes treated with DOX group,fibroblasts and cardiomyocytes co-cultured group,and fibroblasts and cardiomyo‐cytes co-cultured with DOX treatment group.The protein expression of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and myosin heavy chain 7(MYH7)in cardiomyocytes was determined by Western blot.In the blocking ex‐periment of IL-6 antibody with the co-cultured fibroblasts and cardiomyocytes,the protein expression of ANP and MYH7 in cardiomyocytes was also detected by Western blot.RESULTS:Compared with control group,DOX significantly de‐creased the viability of cardiac fibroblasts and caused cardiomyocytes damage(P<0.01).DOX promoted the release of IL-6 in neonatal rat cardiac fibroblasts(P<0.01),but there was no significant difference in the release of IL-6 in neonatal rat cardiomyocytes with or without DOX treatment(P>0.05).Cardiac fibroblasts promoted disordered and broken of myocar‐dial cytoskeletal protein myofilament arrangement induced by DOX.After DOX stimulation,the protein levels of ANP,BNP and MYH7 of the neonatal rat cardiomyocytes in co-culture group were increased with statistically significant com‐pared with cardiomyocytes group(P<0.01).After adding with IL-6 antibody,the expression of ANP and MYH7 was de‐creased significantly in the neonatal rat cardiomyocytes induced by DOX(P<0.05).CONCLUSION:DOX promotes neonatal SD rat cardiac fibroblasts to release IL-6,resulting in cardiomyocyte cytoskeletal protein changes and cardiomyo‐cyte hypertrophy by paracrine signaling.