Study On The Related Mechanism Of Xiaodian Decoction In The Treatment Of Children With Henoch-Schonlein Purpura Based On NLRP3 Inflammasome

BackgroundHenoch-Schonlein Purpura(HSP)is the most common systemic vasculitis in children,and renal damage directly affects the prognosis of HSP.There are limitations in Western medical treatment.How to treat HSP early and reduce renal damage is a pressing clinical challenge.The NLRP3 inflammasomes can be activated by multiple pathogens and danger signals and hence have become a hot spot in immune research in recent years.NLRP3 inflammasomes are closely related to a variety of systemic vasculitis diseases.However,there is no report on the relationship between Henoch Schonlein purpura and NLRP3 inflammasome.ObjectivesThrough clinical experimental research,to explore the correlation between HenochSchonlein purpura and NLRP3 inflammasome in children,to observe the effect of Xiaodian Decoction on the activation of NLRP3 inflammasome;Detect Treg cells and the immune function of lymphocytes in peripheral blood,to explore the relevant mechanism of Xiaodian Decoction on Henoch Schonlein purpura in children from the perspective of immunology,and analyze the possible target.Based on network pharmacology,to analyze the targets of traditional Chinese medicine Xiaodian Decoction,and its connection with HSP and NLRP3 inflammasome-related pathways,and to provide a theoretical and experimental basis for the clinical application of Xiaodian Decoction.MethodsFrom January 2018 to December 2020,26 children with Henoch-Schonlein purpura hospitalized in the Pediatrics Department of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine were taken as the research objects,and the observed cases were compared before and after treatment by prospective self-control method,and another 15 healthy children were taken as healthy control group;In the treatment group children with Henoch-Schonlein purpura were treated with oral Chinese medicine Xiaodian Decoction for 2 weeks on the basis of routine treatment.The healthy control group was not given drugs.Statistical analysis of general sociological data of the HSP group and the healthy control group was performed after clinical observation and records,and the traditional Chinese medicine(TCM)syndrome scores were analyzed before and after the HSP group.Peripheral venous blood was collected from children with HSP before and after treatment and healthy controls for relevant experiments and statistical analysis.1.Three experimental assays were used to investigate the expression of NLRP3 inflammasomes in children with HSP.(i)RT-PCR assay:to measure the expression of ASC,Caspase-1,NLRP3,and NF-κB in the HSP pre-group and healthy control group,to initially investigate whether there was any difference in the expression of NLRP3 inflammasomes in children with HSP and healthy controls;(ii)Western-blot assay:to determine the expression of NLRP3 inflammasomes key proteins ASC,Caspase-1,and NLRP3 in the HSP pre-group,HSP post-group,and healthy control group;(iii)ELISA assay:to measure the serum concentrations of inflammatory-related factors IL-18 and IL-1β downstream of NLRP3 inflammasomes in the three groups.2.Flow cytometry was performed to detect Treg cells and the immune function of lymphocytes.The effect of Xiaodian Decoction on lymphocyte immune function was analyzed,as well as the correlation between NLRP3 inflammasome and lymphocyte immunity.The proportion of Treg cells in serum of children with Henoch-Schonlein purpura before and after treatment and healthy control group was detected by flow cytometry,Total lymphocyte count(Lym),total T cells(CD3+),T cells,Th cells(CD3+CD4+),CD4+T cell count(Th Cells),Ts cells(CD3+CD8+),CD8+T cell count(TC Cells),Th lymphocyte/Ts cells(Th/Ts),NK cells(CD3/16+56),NK lymphocyte count,B cells(CD3/CD19),B lymphocyte count(B cells).3.Network pharmacology research:Based on bioinformatics,network pharmacological research using SwissTargetPrediction,TCMSP and other databases was applied to screen the effective components and targets of the traditional Chinese medicine Xiaodian Decoction.By searching databases such as Drugbank,the targets of Henoch-Schonlein purpura are obtained and the shared targets of Xiaodian Decoction and Henoch-Schonlein purpura are screened and integrated.The String platform wasutilized to build a protein-protein interaction(PPI)network of shared targets to predict potential targets.Finally,focused on the shared targets,Gene ontology(GO)and Kyoto encyclopaedia of genes and genomes(KEGG)enrichment analysis were performed using R software.Results1.Analysis of general sociological data and TCM syndrome scores:there was little difference in average age and sex ratio between the HSP group and the control group,and the differences were not statistically significant(P>0.05).Among them,nearly half of the HSP children had onset in winter,with the highest proportion of patients with simple skin purpura and the least proportion of patients with joint symptoms.The highest TCM syndrome scores were detected in the HSP pre-group and gradually reduced after 1 week and 2 weeks of treatment,suggesting that the treatment with Xiaodian Decoction could alleviate the condition of HSP.2.Detection of NLRP3 inflammasomes:RT-PCR results showed that the differences in the expression levels of ASC and NF-κB between the HSP pre-treatment group and the healthy control group were statistically significant(P<0.05),and there was no statistical difference in the expression levels of Caspase-1 and NLRP3 between the two groups.Western-blot assay of NLRP3 inflammasomes in the three groups showed that the relative protein expression of NLRP3,ASC,and Caspase-1,the constituent proteins of NLRP3 inflammasomes,were increased in children with HSP compared with healthy controls,and the intervention of Xiaodian Decoction down-regulated NLRP3,ASC,and Caspase-1 levels(P<0.05).ELISA detected the levels of inflammatory factors downstream of NLRP3 inflammasomes and the results showed that IL-18 expression was significantly higher in the serum of children with HSP than that in the healthy controls,and the Xiaodian Decoction intervention down-regulated the expression of IL-18(P<0.05).And the difference of IL-1β expression in the healthy control group and HSP group before and after treatment was not statistically significant(P>0.05).The above results suggest that the relative expression of NLRP3,ASC,and Caspase-1,the constituent proteins of NLRP3 inflammasomes were increased in children with HSP compared with healthy controls,and the expression of NLRP3 inflammasomes was down-regulated after the intervention of Xiaodian Decoction,showing a positive correlation with IL-18 and TCM syndrome scores.3.Flow cytometry of Treg cells and other immune function of lymphocytes:compared with healthy controls,the expression of Treg cells,Th cells,and NK cells(%)was downregulated in the HSP pre-group,while the expression of total lymphocytes(Lym),T lymphocytes absolute value T cells,CD4+T lymphocytes,CD8+T lymphocytes,B cells(%),and B lymphocytes absolute number was upregulated(P<0.05).The intervention of Xiaodian Decoction significantly upregulated the expression of Treg cells and NK cells(%)and downregulated the expression of Lym,T cells,CD4+T lymphocytes,CD8+T lymphocytes,B cells(%),and the absolute number of B lymphocytes compared with the HSP pre-group(P<0.05).There was no statistical difference in the expression of Th cells before and after treatment(P>0.05).The results suggested that the expression of NLRP3 inflammasomes was positively correlated with Lym,T lymphocytes absolute value T cells,CD4+T lymphocytes,CD8+T lymphocytes,and B cells,and negatively correlated with the expression of Treg and NK cells.4.The analysis of network pharmacology revealed that Xiaodian Decoction has a variety of active natural ingredients,and its effective active ingredients for the treatment of HSP may be quercetin and falcarindiol.The potential mechanism may be to interfere with the inflammatory response and regulate the immune function by affecting many cytokines,inflammatory factors,and immune cells.The cytokines involved mainly include IL-1β,IL-6,TNF,NOS3,VEGFA,etc.The signaling pathways involved mainly included:NOD-like receptor signaling path way,the IL-17 signaling pathway,MAPK/ERK signaling pathway,TNF signaling pathway,Th17 cell differentiation pathway,Toll-like receptor signaling pathway,PI3K-Akt signaling pathway,and JAK-STAT signaling pathway.Xiaodian Decoction has complex regulatory network of multi-component,multi-target and multichannel in the treatment of HSP.It play a role in the treatment of HSP,may affect NLRP3 inflammasome and regulate IL-6 and IL-1βthrough nod like receptor pathway.Conclusions1.The relative protein expression of NLRP3 inflammasomes in peripheral blood of children with HSP was higher than that of healthy controls.The intervention of Xiaodian Decoction could down-regulate the expression of NLRP3 inflammasomes in children with HSP,reduced the TCM syndrome score,and alleviate the symptoms.2.Children with HSP exhibited lymphocyte immune dysfunction,and the intervention of Xiaodian Decoction could up-regulate the expression of Treg cells and NK cells,downregulate the expression of total lymphocytes,the absolute value of T lymphocytes,CD4+T lymphocytes,CD8+T lymphocytes,and B cells,playing an immunomodulatory role and reducing the inflammatory response.3.Mechanism of Xiaodian Decoction in the treatment of children with HSP may interfere with the assembly and activation of NLRP3 inflammasomes and activate some downstream inflammatory factors through the NOD-like receptor pathway.