葛根素通过AMPK/ASMase激活自噬减轻阿霉素诱导的心肌细胞毒性

Puerarin Alleviates Doxorubicin-induced Cardiomyocyte Toxicity by Activation of Autophagy Through AMPK/ASMase Pathway

目的探讨葛根素减轻阿霉素诱导的心肌细胞毒性的机制。方法将对数生长期的细胞分为正常对照组、模型组、阳性对照卡托普利组(1 mmol·L^(-1))及葛根素低(20 mmol·L^(-1))、中(40 mmol·L^(-1))、高(80 mmol·L^(-1))剂量组。除正常对照组外每组加入阿霉素5 mmol·L^(-1)共同孵育。通过CCK-8法和乳酸脱氢酶(LDH)试剂盒检测细胞活力,ROS探针检测ROS产生水平;HBAD-mcherry-EGFP-LC3腺病毒转染检测自噬流;Western Blot法检测Beclin-1、LC3、p62、p-AMPKα、AMPKα蛋白表达水平;溶酶体探针检测溶酶体功能;免疫荧光检测酸性鞘磷脂酶(ASMase)和溶酶体相关膜蛋白1(LAMP1)相对荧光强度和共定位程度;分子对接分析预测葛根素与ASMase的结合能力。基因富集分析揭示差异表达基因。结果与正常对照组比较,模型组细胞活力降低(P<0.01),LDH和ROS的释放水平升高(P<0.05,P<0.01),自噬小体数量增加(P<0.01)而自噬溶酶体数量减少(P<0.05),Beclin-1蛋白表达及LC3-II/LC3-I比值下降(P<0.01),p62蛋白表达升高(P<0.01),溶酶体荧光强度降低(P<0.01),ASMase的荧光强度升高(P<0.01),ASMase与LAMP1的免疫荧光共定位现象增加(P<0.01),p-AMPKα/AMPKα蛋白表达比值下降(P<0.05)。与模型组比较,葛根素高剂量组细胞活力回升(P<0.05),葛根素中、高剂量组LDH水平呈现下降趋势(P<0.05),葛根素低、中、高剂量组ROS水平呈现下降趋势(P<0.01),葛根素高剂量组自噬小体数量减少(P<0.01),葛根素低、中、高剂量组自噬溶酶体数量增多(P<0.05,P<0.01),葛根素高剂量组Beclin-1蛋白表达比值上升(P<0.05),LC3-II/LC3-I蛋白表达比值上升,p62蛋白表达下降(P<0.01),葛根素低、中、高剂量组溶酶体荧光强度升高(P<0.05,P<0.01),葛根素中、高剂量组ASMase荧光强度降低(P<0.05),ASMase与LAMP1的免疫荧光共定位现象减少(P<0.01),p-AMPKα/AMPKα蛋白表达比值上升(P<0.01)。葛根素与ASMase分子对接分析结合能是-8.6 kcal·mol^(-1)。基因富集分析显示阿霉素诱导的心脏毒性模型差异基因与细胞凋亡、自噬和溶酶体功能有关。结论葛根素可以通过AMPK/ASMase调控自噬,恢复自噬通量,减轻阿霉素诱导的心肌细胞毒性,保护心肌细胞。

Objective To explore the mechanism by which puerarin alleviates the cardiotoxicity induced by doxorubicin in myocardial cells.Methods Cells in the logarithmic growth phase were divided into normal control group,model group,low-(20 mmol·L^(-1)),medium-(40 mmol·L^(-1))and high-(80 mmol·L^(-1))dose puerarin groups,and positive control group(captopril,1 mmol·L^(-1)).Except for the normal control group,the other groups were co-incubated with 5 mmol·L^(-1) doxorubicin.Cell viability was assessed using CCK-8 and lactate dehydrogenase(LDH)assays.ROS levels were detected using a ROS probe.Autophagy flux was detected by transfection with HBAD-mcherry-EGFP-LC3 adenovirus.Western Blot was used to measure the protein expression levels of Beclin-1,LC3,p62,p-AMPKα,and AMPKα.Lysosomal function was assessed using a lysosomal probe.Immunofluorescence was used to detect the relative intensity and co-localization of ASMase and LAMP1.Molecular docking analysis was performed to predict the binding capacity of PUE with ASMase.Differential gene expression was analyzed by gene set enrichment analysis.Results Compared to the normal control group,the model group showed reduced cell viability(P<0.01),increased release levels of LDH and ROS(P<0.05,P<0.01),increased number of autophagosomes(P<0.01),and decreased number of autophagic lysosomes(P<0.05).Beclin-1 protein expression and LC3-II/LC3-I ratio decreased(P<0.01),but p62 protein expression increased(P<0.01).Fluorescence intensity of lysosome decreased(P<0.01),whereas fluorescence intensity of ASMase increased(P<0.01).Immunofluorescence co-localization of ASMase and LAMP1 increased(P<0.01),the ratio of p-AMPKα/AMPKαdecreased(P<0.05).Compared to the model group,the high-dose puerarin group showed a rebound in cell viability(P<0.05).The medium-and high-dose puerarin groups showed a decreasing trend in LDH level(P<0.05),and all puerarin groups showed a decreasing trend in ROS level(P<0.01).The number of autophagosomes in high-dose puerarin group reduced(P<0.01).The number of autophagic lysosomes in all puerarin groups increased(P<0.05,P<0.01).The high-dose puerarin group showed increased expression of Beclin-1(P<0.05)and LC3-II/LC3-I ratio,and decreased p62 expression(P<0.01).All puerarin groups showed increased lysosomal fluorescence intensity(P<0.05,P<0.01).The medium-and high-dose puerarin groups showed a decrease in ASMase fluorescence intensity(P<0.05),a reduction in the immunofluorescence colocalization of ASMase with LAMP1(P<0.01),and an increase in the p-AMPKα/AMPKαratio(P<0.01).Molecular docking analysis discovered puerarin showed a binding energy of-8.6 kcal·mol-1 with ASMase.Gene enrichment analysis indicated that the differentially expressed genes in the doxorubicin cardiotoxicity model were related to apoptosis,autophagy,and lysosomal function.Conclusion Puerarin can alleviate doxorubicin-induced cardiotoxicity in myocardial cells and protect myocardial cells by regulating autophagy through AMPK/ASMase,as well as restoring autophagic flux.