Study On The Mechanism Of Jiawei Fengfang Pills In The Treatment Of Mastitis Based On Network Pharmacology

Objective: The network pharmacology method was used to study the mechanism of the hospital preparation Jiawei Fengfang Pill in treating mastitis,and to find effective compounds or compound groups in Fengfang Pill for treating mastitis.Based on the results of network pharmacology research,baicalin was used as a standard to determine the content of flavonoids-compound groups predicted by network pharmacology.The anti-inflammatory and antibacterial effects of the mastitis model of Staphylococcus aureus in mice were verified by histopathology,and the key targets of the mechanism were verified by fluorescent quantitative PCR.Methods: 1.In the traditional Chinese medicine database TCMSP,TCM @ Tanwan and CNKI,the main compounds in Jiawei Fengfang pill were collected to predict their targets;The targets of mastitis drugs were collected from the databases of Dis Ge NET,CTD,Drugbank and Genecards,and the common targets were found and imported into the database of Metascape for functional enrichment analysis of key targets.2.The content of total flavonoids was determined by ultraviolet spectrophotometry with baicalin as standard substance,and the extraction method of total flavonoids from 10 herbs of Jiawei Fengfang pills was optimized by orthogonal experiment.3.A mouse mastitis model was established by injecting bacteria.The success of the model was evaluated based on clinical symptom scores and histopathological criteria.4.Mice were divided into groups for administration.The clinical symptoms and histopathological characteristics of mice were observed.The key targets and compounds were verified by fluorescence quantitative PCR.Results: 1.Results of network pharmacological analysis: The main pathways of Jiawei Fengfang Pill for mastitis were inflammatory responce,response to molecule of bacterial origin pathway,and the key targets are ALB,TP53,PIR3 CA,MAPK1,SRC,MAPK3,JUN,VEGFA,IL-8.EGFR,TNF,and TLR.The compounds are mainly baicalin,kaempferol,quercetin,luteolin,galangin,isorhamnetin,and homoplanin.These compounds are flavonoids.2.Optimization of the extraction process of Jiawei Fengfang Pills: Baicalin was used as the standard to determine the content of flavonoids.The extraction process was optimized as 6 times of 70% ethanol,heating and refluxing for 3 times,and the highest content of total flavonoids was extracted one hour each time,and the extraction method was reliable and accurate.3.Establishment of a mouse model of Staphylococcus aureus mastitis and pharmacodynamics observation: According to HE staining results,it is preferable to inject bacteria to make the mold.At the same time,the position of needle entering breast tissue should be strictly controlled.According to the clinical pathological score and histopathological observation,we found that the best therapeutic effect was the alcohol extraction of Jiawei Fengfang pill.Experimental verification results: The alcohol extraction of Jiawei Fengfang pill can significantly inhibit the expression of TLR2,activate the expression of ALB,effectively reduce the inflammatory damage of Staphylococcus aureus mastitis,reduce the secretion of inflammatory cytokines,and show a significant anti-inflammatory effect in the occurrence of Staphylococcus aureus mastitis.Conclusion: The optimized alcohol extraction process of Jiawei Fengfang pill can effectively treat the mastitis caused by Staphylococcus aureus,inhibit TLR2,activate the inflammatory response,response to molecular of bacterial origin pathway of ALB.It also can effectively enhance the killing effect of breast tissue and neutrophils on Staphylococcus aureus.