Regulation Of Formononetin On Activation,Proliferation And Migration Of Human Rheumatoid Arthritis Fibroblast-like Synoviocytes

BackgroundRheumatoid arthritis(RA)is a common autoimmune disease with chronic synovial inflammation,pannus formation and joint destruction as its basic pathology.The pathogenesis of RA involves activation of a variety of cells and pathways.Among them,fibroblast-like synoviocytes(FLS)are the main effector cells in the development of the disease.RA FLS has multiple characteristics of transformed cell,which is manifested as promoting inflammatory response,abnormal proliferation and invasive behavior,thus participating in multiple links of RA pathogenesis.Activation,proliferation and migration of RA FLS are key steps to maintain chronic synovial inflammation and joint destruction.Therefore,targeting the pathogenic behavior of RA FLS may be an important treatment strategy to RA.RA belongs to the category of Bi disease in Chinese traditional medicine.The study team proposed that invasion of wind-dampness and blood stasis lead to damage of tendons andoones is the pathogenesis of rheumatoid arthritis and carried out a series researches on Duanteng Yimu formula.The Duanteng Yimu formula consists of Periploca forrestii Schltr,Celastrus orbiculatus,Dipsaci Radix and Herba Leonuri.Clinical studies had shown that Duanteng Yimu formula could relieve clinical symptoms and imaging changes in RA patients.In order to clarify the active ingredients and mechanism of Duanteng Yimu formula,some important active ingredients of the formula were used in vitro and in vivo studies according to the results of network pharmacology analyses.Formononetin is one of the important effective components of Periploca forrestii Schltr,which has been proved to have potent anti-inflammatory and anticancer pharmacological effects,but its effects on RA and fibroblastoid synovial cells have not been reported.Our team preliminarily found that foronetin was a potential therapeutic agent for RA model animals.Combined with the results of network pharmacology and molecular docking,it is suggested that formononetin may work on RA by modulating PI3K/AKT signaling pathway and its downstream proteins HIF-1α and VEGF-a.In this study,we used collagene-induced arthritis(CIA)mice and human rheumatoid arthritis fibroblast-like synoviocytes(HFLS-RA)to investigate the of formononetin on RA,and reveal the underlying mechanisms at the same time.This study will provide theoretical support and experimental basis for formononetin treatment of RA,and provides basis for the clinical promotion and application of of Duanteng Yimu formula.Part 1 Therapeutic effect of formononetin on CIA miceObjectivesTo explore the therapeutic effect of formononetin on collagen-induced arthritis(CIA)mice.MethodsThirty SPF DBA/1 male mice aged 7-8 weeks were randomly divided into blank(NC)group,model(CIA)group,methotrexate(MTX)group,foroneanthin low-dose(FMNL)group and foroneanthin high-dose(FMNH)group,with 6 mice in each group.Except for the normal group,the CIA mouse model was replicated by secondary immunization.After secondary immunization,FMNL group and FMNH group were given 50 mg/kg and 100 mg/kg intragastric administration once a day for 30 days,respectively.Blank group and model group were given equal volume of corn oil solvent.MTX group was administered by oral gavage of MTX once every three days and administered by corn oil other days for a period of 30 days.Mice were observed for weight,arthritis index and paw thickness.Hematoxylin-eosin(HE),safranin O/fast green staining and microcomputed tomography(micro-CT)were performed to observed pathological changes,and semi-quantitative evaluation methods and bone mineral density analysis were also conducted.The supernatant serum was separated for biochemistry testing for Aspartate transaminase(AST),Alanine transaminase(ALT),Creatinine(Cr),and Blood urea nitrogen(BUN).Immunohistochemistry was performed to detect the expression of phosphorylated phosphatidylinositol-3-kinase(p-PI3K),phosphorylated protein kinase B(p-AKT),hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor-α(VEGF-α).HE staining was used to observed pathological changes of liver and kidney.ResultsIn this study,AI scores of all CIA mice were over 4 points between Day 24 to 30 after primary immunization.The success rate of the model was 100%for the 24 mice.CIA mice showed redness and swelling ankle and activity disorder,that following obvious weight loss(P<0.001),significantly increased AI score and foot swelling thickness(P<0.001).Compared with CIA group,the degree of ankle redness and swelling in FMNH and MTX groups was significantly reduced,the AI score and paw thickness were significantly decreased(P<0.001,P<0.01),and the weight was recovered(P<0.01).As for FMNL group,paw thickness was alleviated with reducing AI score(P<0.001,P<0.05),but the impact on weight recovery was not apparent(P>0.05).HE staining showed obvious inflammatory cell infiltration in the joints of CIA group(P<0.001),and the number of synovial vessels increased significantly(P<0.001).The inflammatory cell infiltration in MTX and FMNH groups was significantly reduced(P<0.05),the inflammatory infiltration in FMNL group was slightly relieved(P>0.05).Number of synovial vessels in MTX,FMNL and FMNH groups was significantly reduced(P<0.01 or P<0.001).Safranin O-fast green staining showed cartilage erosion and bone destruction in CIA group(P<0.001),and bone destruction was significantly in remission in MTX and FMNH groups(P<0.05),while bone destruction was partially relieved in FMNL group(P>0.05).The cartilage erosion in FMNH group was significantly improved(P<0.05),and the degree of cartilage erosion in MTX and FMNL groups was slightly alleviated(P>0.05).Compared with blank group,micro CT images showed obvious focal erosion,rough bone surface and loss of integrity in CIA group,what’s more part of the joint fusion was tetanoid and bone destruction scores increased(P<0.001)with decreased bone mineral density(P<0.001).After the administration of MTX and FMN,bone destruction score in the FMNH and MTX groups was significantly decreased(P<0.05)with increased bone mineral density(P<0.05),which were wtih a bit of rough bone surface and focal erosion.Immunohistochemical results showed that serious joint destruction in CIA group with obvious synovial invasion in joint space and large brown-stained areas.Compared with CIA group,intervention groups with a small amount of synovial invasion in joint space,and only a small amount of brown area in the synovial invasion.Compared with CIA group,there were no significant changes in serum AST,ALT,BUN and CR in MTX group,FMNL group and FMNH group(P>0.05).At the same time,HE staining of liver and kidney pathological sections showed no obvious abnormalities.ConclusionFormononetin can obviously relieve joint swelling symptoms,regain weight,moderate joint synovial inflammation,destruction and other pathological changes in CIA mice,and has no obvious hepatorenal toxic side effects.In a word,formononetin has protective effect on CIA mice.Part 2 Inhibition of TNF-α induced activation,proliferation and migration of HFLS-RA by formononetinObjectivesStudy on the inhibitory effect of formononetin on the activation,proliferation and migration of HFLS-RA.MethodsCells from passages 4-8 were used for vitro experiments.Immunofluorescence staining was conducted for vimentin to identify HFLS-RA cells.CCK8 assays was used to detect the effect of formononetin on the viability of HFLS-RA cells.RT-qPCR was used to detected mRNA expressions of IL-6,IL-1β and IL-8.EdU cell proliferation assay kit was used to analyze cell proliferation rate.Flow cytometric analyses were carried out to detect the cell cycle and calculated the ratio of cells in each phase.Cell immunofluorescence was performed to analysed the expression of CyclinD1 and F-actin.Transwell assay and scratch assay were conducted to observed the migration ability of HFLS-RA induced by TNF-α.ResultsHFLS-RA cells highly expressed Vimentin,a surface marker of RA FLS.Compared with the blank group,cell viability of HFLS-RA was decreased in all FMN dosage groups,cell viability was not inhibited by concentrations range of 10-40 μM in 24/48/72h(P>0.05).Under TNF-a(20ng/mL)stimulation,mRNA levels of IL-6,IL-8 and IL-1β in HFLS-RA were decreased after FMN intervention(P<0.01 or P<0.001).Moreover,FMN could significantly down-regulate the proliferation rate of EdU cells of HFLS-RA induced by TNF-α(P<0.05 or P<0.01 or P<0.001).Compared with TNF-α group,FMN could induce cell cycle arrest in RA FLSs to a certain extent,and the proportion of cells in G0/G1 phase increased to varying degrees,while the proportion of cells in S phase decreased to varying degrees(P>0.05).FMN could down-regulate the fluorescence intensity of CyclinD1 in TNF-α-induced HFLS-RA cells(P<0.05).In cell migration experiment,FMN significantly inhibited the vertical and horizontal migration ability of HFLS-RA cells induced by TNF-α(P<0.001 or P<0.05),and inhibited the expression level of F-actin(P<0.05).ConclusionThe results showed that the intervention of FMN can down-regulate the mRNA levels of IL-6,IL-8 and IL-1β in HFLS-RA cells,reduce the proliferation rate of HFLS-RA cells,effectively inhibit the expression of CyclinD1 in a dose-dependent manner,and have a certain tendency to block the G0/G1 cell cycle.At the same time,FMN inhibited the vertical and horizontal migration of TNF-α-induced cells and decreased the expression of actin F-actin.Part 3 Formononetin inhibits HFLS-RA activation,proliferation and migration by mediating PI3K/AKT signaling pathwayObjectivesTo study the molecular signaling mechanism of regulating HFLS-RA activation,proliferation and migration by formononetin.MethodsCells from passages 4-8 were used for vitro experiments.The levels of PI3K,Akt and their phosphorylated proteins,HIF-1α and VEGF-α were detected by Western blotting.After co-treatment with the PI3K activator 740Y-P,experiments on the effect of FMN on cell activation,proliferation,migration,and protein expression were conducted to verify the molecular mechanisms.The mRNA expression of inflammatory factors was detected by RT-qPCR,cell proliferation ability was detected by EdU cell proliferation assay kit,cell cycle was detected by flow cytometry,CyclinD1 expression level was detected by cellular immunofluorescence,vertical and horizontal migration ability was detected by Transwell assay and scratch assay.ResultsFMN could down-regulate PI3K,P-PI3K,P-Akt,HIF-1α and VEGF-α proteins expression induced by TNF-α(P<0.01 or P<0.001).However,740Y-P can partially reverse the down-regulation effect of FMN on PI3K,p-PI3K,p-AKT,HIF-1α and VEGF-α proteins.Meanwhile,RT-qPCR results showed that 740Y-P could significantly reverse FMN downregulation of the expression levels of IL-6 and IL-8,EdU proliferation and Transwell migrated cell number of HFLS-RA cells(P<0.05,P<0.01 or P<0.001).To a certain extent,740Y-P weakened FMN inhibition of down-regulating IL-1β expression level,G0/G1 cycle arrest,CyclinDl protein expression,the migration rates of scratch assay(P>0.05).ConclusionsFormononetin inhibits the activation,proliferation and migration of HFLS-RA cells by mediating PI3K/AKT signaling pathway and downstream proteins HIF-la and VEGF-α,which provides treatment for joint in CIA mice.In this study,it was found that formononetin,an important effective component of Duanteng Yimu formula,had potential anti-rheumatic effects.Formononetin could improved joint symptoms and pathological changes of CIA mice without hepatotoxicity and renotoxicity.At the same time,it regulated the activation,proliferation and migration of HFLS-RA cells,so as to inhibit the pro-inflammatory activity and invasive behavior of the cells.Western blotting and immunohistochemistry experiments revealed that these effects were associated with the suppression of the PI3K/AKT signaling pathway and downregulation of HIF-la and VEGF-α.