Study On The Mechanism Of Shen’ge Formula And Its Components Against Heart Failure

Objectives:Through the network pharmacology to explore the mechanism of the ingredients of Shen’ge formula(SGF)in heart failure(HF)treatment,observe and verify the effect of SGF and its main components on abdominal aortic constriction in rats with HF and doxorubicin-induced cardiomyocyte apoptosis,then explore the efficacy and mechanism of SGF and its main components in treating HF.Methods:1.The composition and content of SGF and Shen’ge lyophilized powder(SGP)were determined by high performance liquid chromatography-tandem mass spectrometry.2.The mechanism of SGF against HF was analyzed from network pharmacology.Through the database,target genes related to drug active ingredients and diseases were identified,protein interactions were analyzed,gene enrichment analysis was conducted,component-target-signaling pathway network was constructed,and the chemical structure of core genes was obtained,and then the model was verified and evaluated by molecular docking technology.3.To observe the effect and mechanism of SGF on the heart of rats with abdominal aortic constriction.Experiments were divided into control group(Sham),abdominal aorta model(AAC),bisoprolol group(BSP)and SGF group(SGF).The drug SGF was analyzed by mass spectrometry.After 8 and 24 weeks of intragastric administration,all groups were performed with echocardiography and then cardiac extraction was been performed.Paraffin section of HE staining and immunofluorescence observations of the expression of α-MHC and VEGFR2 through myocardial tissue,q PCR detection of the m RNA expression of α-MHC,β-MHC,VEGF,β-MHC,VEGF,Hif-1α,NF-κB,Caspase-3,p53,Bax,and Bcl-2.Meanwhile,the expressions of p-p53,p53,p-Akt,Akt,VEGFR2,and Hif-1α were detected by Western Blot.4.The mechanism of SGF and ginsenoside Rg2 on doxorubicin(DOX)-induced apoptosis of H9c2 was observed at the cellular level.The effect of SGF on cell migration was detected by scratch test.Cell survival rate was detected by CCK-8 method,while cell apoptosis was observed by Hoechst and TUNEL staining,and the expressions of p-p53,p53,p-Akt,and Akt were detected by Western Blot.Results:1.The results showed that ginsenoside Rb1,Rb3,RC and RD could be detected in the power of SGF and SGP.The content of ginsenoside Rb1 in SGF was higher than in SGP,while the content of ginsenoside Rb1 was the highest in the sample.2.Results of network pharmacological experiments:we identified 17 bioactive components of ginseng and 107 targets associated with HF treatment.Network analysis revealed AKT serine/threonine kinase 1(AKT1),RELA proto-oncogene(MAPK8),interleukin 1 beta(IL-1β),and Jun proto-oncogenes(JUN)as hub genes.Additionally,molecular docking simulation showed that most bioactive components in ginseng had good binding activity with the predicted hub genes.Aposiopolamine showed a strong binding affinity to AKT1.Kaempferol showed strong binding to IL-1β and JUN while dianthramine bound to MAPK8.Functional enrichment analysis revealed that the potential mechanisms underlying ginseng treatment of HF mainly focused on fluid shear stress,neuroactive ligand-receptor interaction,and calcium signaling pathways.3.Results of animal experiments:Compared with the sham operation group,ejection fraction and shortening fraction in the abdominal aortic coarctation model group were significantly decreased(P<0.01);compared with model group,ejection fraction and shortening fraction in bisoprolol group and SGF group were also significantly increased(P<0.05);compared with model group,the LVIDs of bisoprolol group and SGF group were significantly decreased(P<0.05),and the values of LVIDD,LVVVOLs and LVVOLd in SGF group were significantly decreased(P<0.05).HE staining showed that both SGF and Bisoprolol could make the rats’ myocardial cells more closely arranged,reduce the cell space and reduce the degree of myocardial cell hypertrophy in the abdominal aorta.Immunofluorescence showed that SGF and bisoprolol could reduce the expression of α-SMA and promote the expression of VEGFR2 in rat myocardium induced by abdominal aortic coarctation.Compared with model group,SGF significantly decreased the m RNA expressions of β-MHC,Caspase-3,p53 and Bax(P<0.01),increased the m RNA expressions of Bcl-2(P<0.01),α-MHC,VEGF and HIF-1α(P<0.05),and decreased the m RNA expression of NF-κB(P>0.05).Western Blot results showed that SGF could significantly reduce the expressions of p-p53 and p53(P< 0.01),and increase the expressions of p-Akt,Akt,VEGFR2 and HIF-1α(P<0.01)in the expression of myocardial total protein.In the expression of myocardial nuclear protein,SGF promoted Hif-1α(P<0.05)and inhibited the expression of p53(P<0.01)protein.4.Results of cell experiments:CCK-8 method and scratch test showed that SGF could promote the proliferation of myocardial cells(P<0.01).Hoechst and TUNEL staining showed that SGF and Ginsenoside Rg2 could reduce DOX-induced apoptosis of cardiomyocytes(P<0.01),and ROS detection showed that SGF and Ginsenoside Rg2 could improve DOX-induced oxidative stress response(P<0.01).CCK-8 assay showed that SGF and ginsenoside Rg2 could significantly improve the survival rate of cardiomyocytes induced by DOX(P<0.01),and LY294002 could significantly inhibit the protective effect of SGF on apoptosis of cardiomyocytes induced by DOX(P<0.01).Western Blot showed that SGF down-regulated the expression of DOX-induced p-p53(P<0.01)and p53(P<0.05)proteins,and promoted the phosphorylation of Akt(P<0.01).Ginsenoside Rg2 also significantly decreased the expression of p53(P<0.01)protein and promoted the phosphorylation of Akt(P<0.01).Nuclear protein results showed that SGF could significantly reduce the expression of p53(P<0.05)protein.Conclusions:1.The network pharmacology showed that the main active components of ginseng had good binding properties with the hub target genes of anti-HF.2.SGF could significantly improve the cardiac function of rats with pressure load HF,and this was related to the inhibition of myocardial cell apoptosis,hypertrophy and fibrosis,and the promotion of vascular growth.SGF exerted an anti-heart failure effect by increasing Hif-1α and reducing p53 to the transport to the nucleus.3.SGF and ginsenoside Rg2 could inhibit doxorubicin-induced apoptosis of H9c2 cells through PI3K/Akt pathway.SGF might inhibit the apoptosis by reducing p53 to the transport to the nucleus.4.The possible mechanism of SGF and its components against HF was related to the anti-apoptosis,hypertrophy,fibrosis and inflammation of myocardium and the promotion of blood vessel growth.