Study On Pharmacodynamic Material Basis Of Lingguizhugan Decoction In The Treatment Of Heart Failure

Lingguizhugan Decoction(LGZGD)is first proposed by Zhang Zhongjing as one of the classic prescriptions in ancient China in Shang Han Za Bing Lun.With the effects of invigorating the spleen,reducing dampness,and resolving phlegm,LGZGD was used to treat phlegm of insufficiency of spleen-YANG.It is effective in the treatment of heart failure.Pharmacological studies have shown that LGZGD can significantly improve the symptoms of heart failure.However,which substances in LGZGD play a role in the treatment of heart failure? Not clear at this time.Objective: This study aims to clarify the pharmacodynamic material basis of LGZGD in the treatment of heart failure through a multi-method integration strategy.Methods: In this study,the pharmacodynamic material basis of LGZGD in the treatment of heart failure was studied by integrating various methods,including in vivo substance identification,heart tissue study by untargeted metabolomics,targets screening method,molecular docking,in vitro cell experiment.1.In vivo substances identification.An ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS/MS)method was used to identify prototype substances in serum,urine and feces samples from normal mice and mice with doxorubicin-induced heart failure model after intragastric administration with LGZGD.2.Untargeted metabolomics.Mice were divided into five groups: normal group,doxorubicin-induced heart failure group,low,middle,and high concentration groups(L-lgzgt group,M-lgzgt group and H-lgzgt group).Heart tissue samples from each group were homogenized in methanol-water(4:1,v/v),and the supernatant was collected for assay after high-speed centrifugation.Untargeted metabolomic analysis of heart tissue was performed by UHPLC-Q-TOF-MS/MS,and raw mass spectrometry data were processed by Progenesis QI and SIMCA software.3.Targets screening.The targets corresponding to the prototype substances entering the body of a mouse in LGZGD were screened by the TCMSP database,Pub Chem database,and Swiss target prediction database;Targets of heart failure disease were screened by the Drugbank database and TTD database;The targets corresponding to endogenous differential metabolites were screened by untargeted metabolomics through KEGG database,Cytoscape software,HMDB database,and Swiss target prediction database.The above three targets were intersected to screen the consensus targets of substances in LGZGD for the treatment of heart failure.The corresponding substances were screened in LGZGD according to the common target.4.Molecular docking study.Protein structures of consensus targets were obtained through Uni Prot database and PDB database;The chemical structures of substances in LGZGD were obtained through the Pub Chem database.The two were subjected to molecular docking by Autodock Vina software.5.In vitro cell experiment.19 substances from LGZGD were selected,including liquiritin,cinnamaldehyde,cinnamic acid,glycyrrhetinic acid,poricoic acid B,coumarin,kaempferol,ferulic acid,daidzein,catechin,caffeic acid,pachymic acid,atractyloside I,atractyloside III,dehydrotrametenolic acid,dehydrotumulosic acid,dehydropachymic acid,poricoic acid AM,and geniposide.The antagonistic effects of low,middle and high concentrations of these substances on H9c2 cell injury induced by doxorubicin were studied in our experiment.Result: After giving LGZGD,42 prototype substances were identified in serum,urine,and feces samples of normal mice and heart failure mice.Subsequently,a total of 55 endogenous differential metabolites were identified in the study by untargeted metabolomics of heart tissue in mouse.Then,the target screening method screened 33 targets of LGZGD in the treatment of heart failure and 25 corresponding substances in LGZGD.Moreover,molecular docking technology further screened out 22 substances of LGZGD in the treatment of heart failure.Cell experiments showed that cinnamaldehyde,glycyrrhetinic acid,kaempferol,daidzein,caffeic acid,and catechin could significantly improve the survival rate of H9c2 cells.Conclusions: In this study,22 pharmacodynamic substances in LGZGD were screened by integrating in vivo substances identification,untargeted metabolomics,target screening,and molecular docking.The protective effects of cinnamaldehyde,glycyrrhetinic acid,kaempferol,daidzein,caffeic acid,and catechin on H9c2 cells were confirmed for the first time in vitro cell experiment.