基于HPLC指纹图谱和网络药理学对淡附片质量标志物的预测分析及动物实验探讨其治疗心肾阳虚型慢性心力衰竭的作用机制OA

Objective:This paper aims to establish the HPLC fingerprint of ACONITI LATERALIS RADIX PRA-EPARATA piece and determination methods of ten major constituents including benzoyl neoaconitine,benzoyl aconitine,benzoyl neoaconitine,daidzin,glycitin,daidzein,genistin,genistein,glycyrrhizic acid,and glycyrrhizin,predict its quality marker(Q-Marker),and investigate the molecular mechanism of ACONITI LATERALIS RADIX PRAEPARATA piece in the treatment of chronic heart failure(CHF)of Xinshenyangxu(心肾阳虚)type based on network pharmacology and experimental studies.Methods:The quality of medicinal materials was comprehensively evaluated by HPLC,and the"Chinese Chromatographic Fingerprint Similarity Evaluation System"(2012 Edition)was used to establish the HPLC fingerprint of ACONITI LATERALIS RADIX PRAEPARATA piece.The common characteristic peaks were identified and assigned,and the differences between different batches of samples were evaluated.A network of"components,tar-gets,and pathways"for ACONITI LATERALIS RADIX PRAEPARATA piece was constructed through network pharma-cology,and its Q-Marker was analyzed.Potential targets and signaling pathways for the treatment of CHF of Xinshenyan-gxu type with ACONITI LATERALIS RADIX PRAEPARATA piece were identified through network pharmacology analy-sis.Experimental rats were randomly divided into normal control group,model control group,ACONITI LATERALIS RA-DIX PRAEPARATA piece(0.7,1.4,and 2.8 g/kg)groups,and Xinbao pills(0.2 g/kg)group.The CHF rat model with Xinshenyangxu type was constructed using a combination of doxorubicin and hydrocortisone.After 10 weeks of ad-ministration,hemodynamic measurements were performed.Blood samples were collected for the detection of serum levels of brain natriuretic peptide(BNP),cardiac troponin I(cTnI),and inflammatory factors.The myocardial histomorpholo-gy and mitochondrial ultrastructure were observed by Masson,uranyl acetate,and lead citrate staining,and collagen vol-ume fraction was calculated.Flow cytometry was applied to detect the relative content and M1/M2 polarization of cardiac macrophages.The mRNA expression levels of macrophage polarization markers[Cd86,Cd206,arginase 1(Arg1),and inducible nitric oxide synthase(iNos)],cardiac fibrosis markers[type I collagen(Col I),Col III,and α-smooth muscle actin(αSma)],interleukin-17A(Il17a),interleukin-17 receptor A(Il17ra),nuclear transcription factor kappa B(Nfκb)activator 1(Act1),and tumor necrosis factor receptor associated factor 6(Traf6)in cardiac tissue were deter-mined by quantitative real-time PCR.Western blot was utilized to detect the protein expression levels of Coll I,Coll III,α-SMA,IL-17A,IL-17RA,Act1,TRAF6,nuclear NF-κBp65,cytoplasmic NF-κBp65,and phosphorylated p38 mitogen activated protein kinase(p-p38MAPK)in cardiac tissue.Results:The HPLC fingerprint of ACONITI LATERALIS RA-DIX PRAEPARATA piece was established,and a total of 26 common chromatographic peaks were identified.Through comparison with reference standards,10 chemical components were determined,including benzoyl neoaconitine,benzoyl aconitine,benzoyl neoaconitine,daidzin,glycitin,daidzein,genistin,genistein,glycyrrhizic acid,and glycyrrhizin.The similarity evaluation results ranged from 0.839 to 0.984.The network pharmacology method predicted and screened three components of benzoyl neoaconitine,glycyrrhizic acid,and daidzin as Q-Marker of ACONITI LATERALIS RADIX PRAEPARATA piece.The key targets of ACONITI LATERALIS RADIX PRAEPARATA piece in treating CHF of Xin-shenyangxu type were tumor necrosis factor(TNF),epidermal growth factor receptor(EGFR),heat shock protein 90A type 1(HSP90AA1),monoamine oxidase A(MAOA),etc.Through GO and KEGG pathway enrichment analysis,it was found that signaling pathways such as IL-17 and cyclic adenosine monophosphate(cAMP)were closely related to CHF of Xinshenyangxu type.Animal experiments manifested that compared with the model group,the ACONITI LATERALIS RADIX PRAEPARATA piece groups at 0.7,1.4,and 2.8 g/kg,as well as the Xinbao pills group at 0.2 g/kg effectively ameliorated the cardiac function of rats with CHF of Xinshenyangxu type,reduced serum contents of BNP,cTnI,IL-1β,IL-6,IL-8,IL-17,IL-18,and TNF-α,down-regulated the protein and mRNA expression levels of cardiac tissue Coll I,Coll III,and α-SMA,and prominently increased serum levels of IL-4 and IL-10.It ameliorated the degree of cardiac patholog-ical damage,reduced collagen volume fraction,alleviated mitochondrial damage,lowered the ratio of F4/80+CD86+M1 cells and the Cd86 and iNos mRNA expressions in cardiac tissue,and increased the ratio of F4/80+CD206+M2 cells and the Cd206 and Arg1 mRNA expressions.In addition,ACONITI LATERALIS RADIX PRAEPARATA piece significantly reduced the mRNA expressions of Il17a,Il17ra,Act1,and Traf6,as well as the protein expressions of IL-17A,IL-17RA,Act1,TRAF6,nuclear NF-κBp65,and p-p38MAPK in cardiac tissue,and elevated the protein expression of cytoplasmic NF-κBp65(P<0.05 or P<0.01).Conclusion:The established HPLC fingerprint,multi-component content determina-tion,and Q-marker prediction method for ACONITI LATERALIS RADIX PRAEPARATA piece are simple and accurate,with good repeatability,which can be used for quality evaluation of ACONITI LATERALIS RADIX PRAEPARATA piece.Based on network pharmacology research,three components including benzoyl neoaconitine,glycyrrhetinic acid,and daidzin were found to serve as its Q-markers.This study preliminarily revealed a certain relationship between the ac-tive ingredients of ACONITI LATERALIS RADIX PRAEPARATA piece and key proteins involved in CHF of Xinsheny-angxu type,and the therapeutic effect was exerted through a comprehensive regulatory network involving multiple targets and pathways,with the mechanism likely related to the inhibition of IL-17 signaling pathway and the generation of inflam-matory factors.