Effect Of Prangenidin On Ameliorating Rheumatoid Arthritis Synovial Inflammation

Background:Rheumatoid arthritis(RA)is a chronic systemic autoimmune disease of unknown etiology.It is characterized by progressive and aggressive destruction of bone,cartilage,and synovial tissue at the joint site,with persistent synovial inflammation,formation of vascular opacities,joint destruction,and adjacent bone erosion.Synovitis is the pathological basis of RA.Fibroblast-like synoviocytes(FLSs)are the core target cells with an immune role in RA.In the inflammatory environment of RA synovium,FLSs undergo apoptotic abnormalities,and synovial overgrowth and further accumulation and adhesion to bone and cartilage aggravate joint destruction.Similarly,the“tumor-like”abnormal proliferation of RA FLSs leads to excessive cell activation,enhanced migration and invasion ability,and the production of a large number of proteases,cytokines and adhesion molecules,forming a positive feedback regulation that further promotes the synovial inflammatory response and contributes to cartilage destruction,further activating osteoclasts to cause bone destruction and exacerbating the progression of RA disease.With regard to the treatment of RA,there is no effective therapy to date,and the currently available therapies are mainly aimed at reducing joint inflammation,preventing irreversible bone destruction,and maintaining joint function as much as possible.Chinese medicine has the benefits of multi-component,multi-target,and low toxic side effects,which can be an ideal strategy for the treatment of RA.“Fangfeng”has an extensive and long history of use in the treatment of rheumatic diseases,showing good efficacy and is an ideal herbal medicine for RA treatment,but its specific therapeutic mechanism is still unknown.Systems biology and pharmacology-based network pharmacology is a powerful approach to evaluate drug metabolism and efficacy,analyze specific pathways of drug treatment for diseases and explain the molecular mechanisms of drug action.Combined with molecular docking techniques,the binding activity and mechanisms of drugs to key targets of diseases can be evaluated and widely applied to drug screening and development.In this way,we explored the therapeutic mechanism of“Fangfeng”for RA,screened out potential active ingredients for RA treatment and important pathways for their action,further investigated the possible mechanisms involved through basic biochemical experiments on the core ingredient Prangenidin and the core pathway PI3K/AKT,and validated the therapeutic effect of Prangenidin on arthritis through animal experiments,providing some theoretical support and experimental basis for the treatment of RA by“Fangfeng”and its active ingredient Prangenidin.Objective:1.Investigating the potential molecular biological mechanism of“Fangfeng”in treating RA through a pharmacology-based strategy;2.To study the regulatory effects of Prangenidin on FLSs activation,migration,invasion,and apoptosis;3.To explore the molecular mechanism involved in the regulation of MH7A cells activation,migration,invasion,and apoptosis by Prangenidin;4.To observe the therapeutic effect of Prangenidin on collagen-induced arthritis(CIA)mice.Methods:1.A network pharmacology-based approach for exploring the bioactive phytochemicals in“Fangfeng”and its therapeutic mechanism for rheumatoid arthritisThe bioactive phytochemicals of“Fangfeng”and their corresponding targets were screened by using multiple databases of Chinese medicine,evaluating phytochemicalsrelated parameters such as pharmacology,and toxicology,etc.,and disease-related targets of RA were screened through GeneCard,OMIM,DisGeNET,and DrugBank databases.The PPI network was constructed for the intersection targets through the STRING database and Cytoscape software.Next,gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis as well as the validation with multiple Gene Expression Omnibus(GEO)database were performed on the“Fangfeng”targets to explore the mechanism of“Fangfeng”in RA treatment.The network of“IngredientsPotential target genes-Signaling pathways”was established with Cytoscape and further screened to identify the core ingredients and key targets.Finally,molecular docking studies were performed on the core components and key targets to verify the binding affinity of the interaction.2.Effect of Prangenidin activation,migration,invasion,and apoptosis of FLSsMH7A cells were cultured to logarithmic growth phase,and MH7A cells were intervened with different concentrations of Prangenidin and methotrexate(MTX),and TNFα.The effect of drugs on cell viability was detected by CCK-8 method;the protein and mRNA expression levels of inflammatory factors(IL-1β,IL-6,IL-8)and matrix metalloproteinases(MMPs)(MMP-1,MMP-3)in cell culture supernatants and total cell RNA extracts were detected by ELISA and RT-qPCR,respectively;the migration and invasion ability of cells in vitro was detected by scratch healing assay and Transwell migration and invasion assay;apoptosis was analyzed by flow cytometry.3.Molecular mechanism for regulation of FLSs activation,migration,invasion,and apoptosis by PrangenidinMH7A cells were cultured to logarithmic growth phase,and MH7A cells were intervened with Prangenidin and PI3K inhibitor LY294002,as well as TNF-α.The expression levels of signaling pathway proteins p-PI3K,PI3K,p-AKT,and AKT,as well as apoptosisrelated proteins Bax,Bcl-xL,and Cleaved caspase 3 were detected by western blotting;the expression levels of inflammatory factors(IL-1β,IL-6,IL-8)and MMPs(MMP-1,MMP-3)in MH7A cells after the intervention of PI3K inhibitor LY294002 were detected by RT-qPCR;The migration and invasion ability of MH7A cells after PI3K inhibitor LY294002 intervention were detected by migration and invasion assays;the apoptosis of MH7A cells after PI3K inhibitor LY294002 intervention was analyzed by flow cytometry.4.The therapeutic effect of Prangenidin on CIA miceA classical mice CIA model was constructed by twice immunization method on DBA/1 mice.Twenty DBA/1 mice were randomly divided into four groups:control group,model group,Prangenidin group(60mg/kg/d),and MTX group(2mg/kg/3d).CIA models were constructed for the three groups of mice except the control group.Booster immunization was performed on day 21 of the initial immunization,and the administration of the drug by group was started for 28 consecutive days.The therapeutic effects of Prangenidin on RA model CIA mice were investigated by dynamically observing and recording the general condition,paw swelling,and polyarthritis index score of mice,and detecting the expression levels of serum inflammatory factors IL-1β,IL-6,and TNF-α and assessing the H&E staining pathology score of synovial tissue at the end of the experimental cycle.Results:1.A network pharmacology-based approach for exploring the bioactive phytochemicals in“Fangfeng”and its therapeutic mechanism for rheumatoid arthritisA total of 18 bioactive phytochemicals and 80 target genes were identified from“Fangfeng”,of which 66 intersected with the screened RA disease target genes and were considered as potential target genes.“Fangfeng”core ingredients such as wogonin,betasitosterol,5-O-methylvisamminol,decursin,and divaricatol and core targets such as PTGS2,RELA,AKT1,MAPK14,and JUN were obtained for further analysis.The underlying mechanism of“Fangfeng”in treating RA might be achieved by regulating pathways such as PI3K/AKT signaling pathway,IL-17 signaling pathway,apoptosis signaling pathway,TNF signaling pathway,and multiple biological processes to exert anti-inflammatory and immunomodulatory effects.Molecular docking confirmed that all core ingredients and key targets had great docking activity.2.Effect of Prangenidin activation,migration,invasion,and apoptosis of FLSs(1)The results of CCK-8 experiments showed that after MH7A cells were treated for 24h,cell viability was significantly inhibited in the MTX and Prangenidin(160,120,80,60μM)groups compared with the control group(P<0.05 or P<0.01),while Prangenidin(40,20μM)group showed no significant decrease in cell viability(P>0.05);after 48 h of drug treatment,the MTX and Prangenidin(160,120,80μM)groups significantly inhibited cell activity in a dose-dependent manner compared to the control group(P<0.01),however,no significant decrease in cell viability was observed in the Prangenidin(60,40,20μM)group(P>0.05).(2)The results of ELISA experiments showed that IL-1β,IL-6,and MMP-1 protein expression levels of MH7A cells in each group were significantly increased after TNF-αinduction compared with the control group(P<0.05 or P<0.01);while compared with the TNF-α group,different concentrations of Prangenidin and MTX both significantly reversed the TNF-α-induced upregulation of IL-1β,IL-6,and MMP-1 protein expression levels(P<0.01),and showed a certain degree of concentration dependence.(3)The results of RT-qPCR experiments showed that after TNF-α induction,compared with the control group,IL-1β,IL-8,and MMP-1 in the TNF-α+PG 60 group and IL-6 and MMP3 in the TNF-α+MTX group were not significantly upregulated(P>0.05),while the mRNA expression levels of inflammatory factors(IL-1β,IL-6,IL-8)and MMPs(MMP-1,MMP-3)in the remaining groups of MH7A cells showed significantly different levels of upregulation(P<0.05 or P<0.01);compared with the TNF-α group,both different concentrations of Prangenidin and MTX significantly downregulated TNF-α-induced elevated levels of inflammatory factors and MMPs mRNA expression(P<0.01),which also showed a degree of concentration dependence.(4)The results of migration and invasion assay showed that different concentrations of Prangenidin and MTX could significantly inhibit the migratory ability of MH7A cells compared with control group(P<0.05 orP<0.01);while compared with MTX group,24h scratch assay showed no significant difference in the effect of different concentrations of Prangenidin on the migratory ability of MH7A cells(P>0.05),and 24 h migration assay and 48h scratch assay showed that 60μM Prangenidin and MTX inhibited the migratory ability of MH7A cells with comparable potency(P>0.05),but both were better than 20μM and 40μM Prangenidin on the migratory ability of MH7A cells(P<0.01).48h invasion assay showed that compared with the control group,different concentrations of Prangenidin and MTX both significantly inhibited the in vitro invasion ability of MH7A cells(P<0.01);and compared with the MTX group,the inhibitory potency of 40μM and 60μM Prangenidin and MTX on the invasive ability of MH7A cells was comparable(P>0.05),but both were superior to the inhibitory effect of 20μM Prangenidin on the invasive ability of MH7A cells(P<0.01).The results of apoptosis assay showed that both 40μM and 60μM Prangenidin and MTX significantly induced increased apoptosis in MH7A cells compared with the control group(P<0.05 or P<0.01)in a dose-dependent manner,while compared with the MTX group,60μM Prangenidin had a stronger induction of apoptosis in MH7A cells(P<0.05).3.Molecular mechanism for regulation of FLSs activation,migration,invasion,and apoptosis by Prangenidin(1)Western blotting results showed that PI3K and AKT phosphorylation levels were significantly upregulated in MH7A cells after TNF-α induction(P<0.01),and Prangenidin intervention was able to significantly downregulate the elevated PI3K and AKT phosphorylation levels after induction by TNF-α(P<0.01).LY294002,Prangenidin,and combined interventions were all able to significantly downregulate Bcl-xL(P<0.01)and upregulate Bax and Cleaved caspase 3 levels of protein expression(P<0.01).(2)The results of RT-qPCR experiments showed that LY294002 could significantly inhibit the significant upregulation of mRNA expression levels of inflammatory factors(IL-1β,IL6,IL-8)and MMPs(MMP-1,MMP-3)induced by TNF-α(P<0.01).(3)The results of migration and invasion assays showed that PI3K inhibitor LY294002 significantly inhibited the migration and invasion of MH7A cells(P<0.01).The results of apoptosis assay showed that Prangenidin,LY294002,and combined intervention all significantly induced increased apoptosis in MH7A cells(P<0.05 or P<0.01).4.The therapeutic effect of Prangenidin on CIA mice(1)On day 21 of the initial immunization,some mice already showed joint redness and swelling,and 3-5 days after the booster immunization,mice successively developed the disease,reaching a peak on day 37,with joint redness and swelling,and severe cases showed restricted movement,tonicity,and even ulceration.At the end of the experimental cycle,compared with the control group,the model group mice still had obvious redness and swelling of the foot and paw,and the redness and swelling were less in the Prangenidin and MTX groups.(2)After 8 days of administration,the Prangenidin group started to differ significantly from the model group(P<0.05);on day 37,both Prangenidin and MTX treatment groups differed significantly from the model group(P<0.05 or P<0.01).This difference was further amplified with longer dosing cycles(P<0.01).(3)Compared with the control group,serum IL-1β,IL-6,and TNF-α expression levels were significantly upregulated in the model group mice(P<0.01),while there was no significant difference between the Prangenidin and MTX groups(P>0.05),and the Prangenidin and MTX groups could significantly downregulate the expression levels of serum IL-1β,IL-6,and TNF-α in CIA mice(P<0.01).(4)Prangenidin and MTX improved blurring joint and synovial structure,disorganized synovial alignment,bone and cartilage erosion,inflammatory cell infiltration,thickened synovial lining layer,and lamellar neovascularization formation in CIA mice;it reduced the arthritis,synovitis,and cartilage erosion pathology scores in CIA mice(P<0.01).Conclusion:1.A network pharmacology-based strategy was used to verify that the treatment of RA with“Fangfeng”is multi-component,multi-target,and multi-pathway.2.Prangenidin significantly down-regulated the expression levels of IL-1β,IL-6,IL-8,MMP-1,and MMP-3 in MH7A cells,inhibited the migration and invasion of MH7A cells,and was able to induce increased apoptosis in MH7A cells.3.Prangenidin achieves regulation of MH7A cells activation,migration,invasion,and apoptosis through PI3K/AKT signaling pathway.It was suggested that Prangenidin could regulate the inflammatory and abnormal biological behavior of FLSs through multiple pathways.4.Prangenidin could effectively alleviate the symptoms of arthritis in CIA mice,reduce arthritis,synovitis and cartilage erosion scores,and decrease arthritis,synovial inflammation,bone and cartilage destruction by reducing the abnormal biological behavior of synovial tissue and serve as a therapeutic role.