基于单细胞数据挖掘、转录组学及网络药理学探讨暖心康防治慢性心力衰竭的机制

Mechanism of Nuanxinkang(暖心康)Preventing Chronic Heart Failure Based on scRNA-Seq Data Mining,Transcriptomics and Network Pharmacology

目的医院院内制剂暖心康在治疗慢性心力衰竭中取得良好效果,但其潜在的防治慢性心衰的具体分子机制尚不十分明确,拟通过单细胞数据挖掘、转录组学、网络药理学及动物实验等探讨暖心康防治慢性心力衰竭的机制。方法随机将40只C57/BL6雄性小鼠分成4组,假手术组(Sham)10只、模型组(TAC)10只、生理盐水组(Vehicle)10只、暖心康组(NXK)10只。动物模型制作采用主动脉弓缩窄术,先后予小动物超声心功能检测、HE染色、Masson染色、钙黄绿染色、实时荧光定量PCR(qPCR)验证暖心康防治慢性心衰。再予GEO数据库下载人源单细胞数据集(GSE95140)进行下游分析以得到慢性心衰差异基因,将暖心康药物-慢性心力衰竭疾病进行网络药理分析得到相应的药物-疾病靶点,联合转录组学所得关键靶基因进行网络互作,得到三者交集基因及相关GO条目和通路,将得到的预测基因予WB进行验证。结果在各项验证中TAC组与Vehicle组数据比较无统计学意义。而与Sham组相比,心脏体质量及质量联合超声显示TAC组、Vehicle组的HW/BW值明显升高(P<0.01),而EF值、FS值明显降低(P<0.01),HE、Masson染色、钙黄绿染色显示TAC组、Vehicle组心肌纤维化明显、心肌细胞排列紊乱及心肌细胞变大(P<0.01),qPCR显示TAC组、Vehicle组ANP、BNP、Myh7、Collagen I、Collagen III等基因的表达升高(P<0.01);与TAC组、Vehicle组比较,NXK组HW/BW值降低(P<0.05),而EF值、FS值均明显升高(P<0.01),HE、Masson染色、钙黄绿染色显示NXK组心肌纤维化、心肌细胞排列及心肌细胞大小改善明显(P<0.01),qPCR显示TAC组、Vehicle组ANP、BNP、MYH7、Collagen I、Collagen III等基因的表达降低(P<0.01);经单细胞数据挖掘所得差异基因、课题组前期心衰小鼠转录组数据所得差异基因及暖心康网络药理所得潜在治疗慢性心衰靶基因三者进行网络互作分析得到3个治疗慢性心力衰竭的核心基因,分别为SLC8A1、RYR2、NFIB;桑基图显示GO富集所得条目与钙流、跨膜转运、能量代谢、氧化磷酸化、氧化应激、凋亡等生物过程相关。WB验证实验显示,与Sham组相比,TAC组、Vehicle组RYR2、p-RYR2(ser2808)、Calpain1、Calpain2、NF-κB p65的蛋白表达量明显升高(P<0.01);与TAC、Vehicle组相比,NXK组RYR2、p-RYR2(ser2808)、Calpain1、Calpain2、NF-κB p65的蛋白表达量降低(P<0.01)。结论暖心康可改善慢性心力衰竭心室重构,其可能通过RYR2调控钙泄漏激活钙蛋白酶(Calpain1、Calpain2),进而激活NF-κB通路来发挥作用。

Objective Nuanxinkang(暖心康)has achieved good results in the treatment of chronic heart failure,but the specific molecular mechanism of its potential prevention to chronic heart failure is not very clear.This article used some technologies such as single-cell data mining,transcriptomics and network pharmacology and animal experiments to explore the mechanism of Nuanxinkang in preventing chronic heart failure.Methods Forty C57/BL6 male mice were randomly divided into four groups,10 in the sham operation group(Sham group),10 in the model group(TAC group),10 in the vehicle group(Vehicle group)and on in the Nuanxinkang group(NXK group).The animal model was made using transverse aortic constriction,which was successively verified by small animal echocardiographic function test,HE staining,Masson staining,calcein staining and real-time fluorescent quantitative PCR(qPCR).And we used the single-cell data,transcriptomics data and Nuanxinkang network pharmacology to predict key target genes and related GO terms.And then we validated it at the animal level with WB.Results In this animal experiment verification,we found that there was no statistical significance in the data comparison between the TAC group and the Vehicle group.Compared with that of the Sham group,the heart weight and quality combined ultrasound showed that the ration of HW/BW of the TAC group and the Vehicle group was increased significantly(P<0.01),while the values of EF and FS were decreased significantly(P<0.01).HE,Masson staining and calcium yellow green staining showed obvious myocardial fibrosis,disordered arrangement of myocardial cells and enlargement of myocardial cells in the TAC group and the Vehicle group(P<0.01).QPCR showed the expressions of ANP,BNP,MYH7,collagen I and collagen III were significantly increased(P<0.01).Compared with that of the TAC group and the Vehicle group,the ration of HW/BW of the NXK group were decreased(P<0.05),while the values of EF and FS were increased significantly(P<0.01).HE,Masson staining and calcium yellow green staining showed that the myocardial fibrosis,cardiomyocyte arrangement and cardiomyocyte size of the NXK group were significantly improved(P<0.01).QPCR showed the expressions of ANP,BNP,MYH7,collagen I and collagen III and other genes were decreased(P<0.01).Through the network interaction analysis of the differential genes obtained from single-cell data mining,the differential genes obtained from the transcriptome data of mice with heart failure in the early stage of the research team and the potential target genes for the treatment of chronic heart failure obtained from the network pharmacology of Nuanxinkang,three core genes for the treatment of chronic heart failure were obtained,which were slc8a1,RYR2 and NFIB respectively.Sanggi diagram showed that the items obtained from GO enrichment were related to biological processes such as calcium flow,transmembrane transport,energy metabolism,oxidative phosphorylation,oxidative stress and apoptosis.WB validation experiment showed that compared with those of the Sham group,the protein expressions of RYR2,p-RYR2(ser2808),calpain1,calpain2 and NF-κB p65 were increased significantly(P<0.01)in the TAC group and the Vehicle group.Compared with those of the TAC group and the Vehicle group,the protein expressions of RYR2,p-RYR2(ser2808),calpain1,calpain2 and NF-κB p65 were decreased(P<0.01).Conclusion Nuanxinkang could regulate calcium leakage by RYR2,and then make the expressions of Calpain1 and Calpain2 increase,and then activate the NF-κB pathway to improve ventricular remodeling.