Protective Effect Of Ma Huang Gancao Decoction On PAMs Inflammatory Damage And Anti-PRRSV Infection And Screening Of Active Ingredients

Mahuang Gancao Decoction(MGD)is derived from the Jingui Yaolue(Synopsis of Prescriptions of the Golden Chamber).Clinical studies have shown that Mahuang Gancao decoction has the effects of relieving cough and asthma and resolving phlegm,but the complexity of its active components limits its further application.In this study,network pharmacology,vitro experiments,and metabolomics methods were used to explore the protective effect of Mahuang Gancao Decoction on porcine alveolar macrophages(PAMs),the target cells of respiratory diseases and to screen the effective active ingredients of the decoction.The main research contents and results are as follows:1.PAMs cells were used as target cells to study the protective effect of MGD on PAMs from the aspects of anti-inflammatory and anti-virus.MTT assay was used to detect the safe concentration of MGD on PAMs.LPS established the inflammatory model of PAMs.The nitric oxide(NO)content secreted by PAMs and the mRNA expression levels of inflammatory factors(IL-6,TNF-α,IL-1β)were detected before and after MGD intervention.Then,the cells were treated with different concentrations of Mahuang Liquorice decoction and porcine reproductive and respiratory disorder syndrome virus(PRRSV)for 2,4,and 6 hours in three ways: the drug was added before the virus,the virus was added before the drug,and drug and virus were added at the same time.MTT assay was used to detect the inhibition rate of PRRSV to select the best mode of action.Normal PAMs were used as the control group.The effects of MGD on the number and morphology of PAMs infected with PRRSV were observed by neutral red staining and trypan blue staining,and the expression of PRRSV mRNA was detected by RT-qPCR.The apoptosis of PAMs cells was detected by flow cytometry.RT-qPCR was used to detect the mRNA expression of immune factors(IL-6,TNF-α,IL-1β).2.Liquid mass combination(LC-MS)technique was used to detect the components of Mahuang Gancao Decoction,and MGD active components and swine pneumonia and PRRSV targets were selected by combing TCMSP,Swiss target predication and Gene Cards databases.The common target of illness was obtained by Venn interaction analysis.Construction of disease target network by Cytoscape;Cytoscape plug-ins Cyto Hubba and Cyto NAC were used to screen the core targets of the constructed protein interaction network using String data platform.David database and Weisheng online mapping website were used to analyze the biological function and pathway enrichment of the target,so as to predict the anti-PRDC mechanism of ephedrine Glycyrrhiza decoction.3.Liquid chromatography-mass spectrometry(LC-MS/MS)was used to collect cell metabolites,and non-target analysis was performed on metabolic data.Combined with principal component analysis(PCA)and orthogonal partial least squares discrimination(OPLS-DA)pattern recognition methods,the primary metabolites of model group(LPS),control group(Con)and MGD groups were screened,and the metabolic pathways were analyzed.The findings were as follows:1.The results of the MGD anti-inflammatory test in vitro showed that LPS treatment promoted the secretion of NO and inflammatory factors in PAMs cells and induced the inflammation of PAMs cells.After MGD intervention,compared with the LPS group,MGD reduced NO secretion,and down-regulated the mRNA expression levels of IL-6,TNF-α,and IL-1β in PAMs.These results indicate that MGD has an excellent antiinflammatory and protective effect on PAMs cells.The study of MGD against PRRSV virus found that the inhibition rate of MGD against PRRSV was better when the drug was added for 4 hours before adding the virus.The inhibition rate of the PRRSV virus was more than 85% when the drug was more than80μg/mL.Under this mode of action,compared with the control group,the number of viable cells in the PRRSV group decreased,the morphology changed,and the viral mRNA content was higher.Flow cytometry showed that PRRSV infection caused apoptosis of PAMs cells,which was conducive to virus replication,and MGD intervention reversed this situation to a certain extent.RT-qPCR results showed that PRRSV increased the mRNA levels of IL-6,TNF-α,and IL-1β,and MGD inhibited the up-regulation of inflammatory factors.2.Through network pharmacological analysis,89 anti-inflammatory and antiPRRSV components of MGD were predicted,including wheat flavone,apigenin,luteolin,etc.PPI protein interaction network showed that the core targets were TNF、ALB、SRC、STAT3、MAPK3,etc.;GO and KEGG enrichment analysis showed that the biological functions were mainly inflammatory response and response to oxygen-containing compounds,and the regulation of PI3K-Akt,MAPK,and other signaling pathways to play an anti-inflammatory and anti-PRRSV role.3.After analyzing the metabolome data,PCA results showed that the metabolome data of the LPS group and control group could be significantly separated under the positive and negative ion modes,and the metabolome data of different doses of MGD groups had a better control tropism,which supported the protective effect of MGD on PAMs cells.After OPLS-DA screening and comparison with NIST and Mass Bank databases,19 metabolites were identified.Enrichment analysis showed that it was mainly involved in the biosynthesis of unsaturated fatty acids,fatty acid biosynthesis,pentose,and glucuronic acid conversion pathway,The different metabolites were primarily related to the metabolism of fatty acids and other substances.In conclusion,MGD has anti-inflammatory and antiviral protective effects on PAMs,network pharmacology predicted that the protective effect of MGD on PAMs was related to wheat flavone,apigenin,luteolin and other active ingredients.Metabolomics analysis showed that MGD’s protective effect on PAMs was mainly through unsaturated fatty acid biosynthesis,fatty acid biosynthesis and pentose glucuronic acid conversion.This study provides theoretical reference for the active ingredients of mahuang gancao decoction and anti-PRRSV drug development.