Mechanistic Study Of Puhuang Regulating Microglia-mediated Neuropathic Pain Based On Network Pharmacology

Objective Exploring the key elements and mechanism of Puhuang in the treatment of neuropathic pain is done using network pharmacology and cellular experimental verification.Method Step 1: network pharmacological analysis and molecular docking.First of all,the TCMSP database was searched for the active components of Typha typha and their primary targets of action,as well as obtaining targets related to neuropathic pain from Gene Cards and OMIM databases.Getting the intersection target of the two by using the Venny platform,and then construct the protein-protein interaction network of their shared target using the STRING platform and Cytascape software.Secondly,DAVID and Bioconductor databases are used to carry out biological process enrichment analysis and pathway enrichment analysis,and the "active component-target-pathway" network is constructed by using Cytoscape software.Finally,Auto Dock Vina software is used to complete molecular docking to verify the binding degree of active ingredients and key action targets.Step 2: culture BV2 cells for validation in vitro.First,the effect of quercetin(Que)concentration on BV2 cells was screened by CCK-8method;Then BV2 cells were randomly divided into normal group,LPS group and(high,medium and low)dose Que+LPS group according to CCK-8 results.The normal group only added culture medium as control;The concentration of LPS in LPS group was 1 ug/ml;The cells of high,medium and low dose Que groups were pre-protected with 100 u M,50 u M and25 u M Que respectively for 2h,and then added with 1ug/ml LPS for 24 h.Real-time quantitative PCR(RT-q PCR)and ELISA were used to detect the key targets IL-6,TNF-α,IL-17 and CCL2.Results The results of network pharmacology analysis showed that a total of 6 active ingredients of Typhae were obtained,among which quercetin was the component with the largest number of target points;There were 77 common targets of pollen Typhae and neuropathic pain,and the PPI network results showed that IL-6 was the core target;The results of enrichment analysis involved 123 biological processes and 163 signaling pathways,including cytokine receptor binding and IL-17 signaling pathway with IL-6 as the main factor;Molecular docking results showed that quercetin,the core component,had high binding activity with VEGFA.According to the results of network pharmacology analysis,quercetin was selected to act on lipopolysaccharide(LPS)-induced mouse BV2 microglial inflammation model in vitro to preliminarily verify the core targets and pathways.The following results were obtained: compared with the control group,the LPS group significantly upregulated IL-6,IL-17,TNF-α And CCL2 m RNA levels,the differences were statistically significant(P<0.01);Compared with LPS group,The relative expression levels of IL-6,TNF-α and CCL2 m RNA were significantly decreased in a dose-dependent manner,and the differences were statistically significant(P<0.05,P<0.01);Compared with the LPS group,the relative expression of IL-17 m RNA in the medium and high dose experimental groups were significantly decreased,and the differences were statistically significant(P<0.01).ELISA results showed that compared with the control group,the relative concentrations of IL-17,TNF-α and IL-6 were significantly increased,in LPS group(P<0.01).Compared with LPS group,TNF-α in(low,medium,and high)dose experimental groups.The relative concentrations of IL-6 were significantly decreased,and the differences were statistically significant(P<0.05,P<0.01);Compared with the LPS group,the relative concentration of IL-17 in the medium and high dose experimental groups was significantly reduced,and the difference was statistically significant(P<0.05,P<0.01).Conclusion 1.The main active ingredient of Puhuang in relieving NP was quercetin and the main target was IL-6,which provided a theoretical basis for the mechanism of Puhuang in treating neuropathic pain;2.Through RT-q PCR and ELISA experiments,it was preliminarily verified that quercetin,an effective component of typha typha,can significantly reduce the expression and secretion of inflammatory factors in BV2 cells induced by LPS,which provides a new idea for the mechanism of typha typha in treating neuropathic pain.