| Rheumatoid arthritis(RA)is a systemic autoimmune disease characterised by chronic,symmetrical and multiple joint inflammations,which is manifested by localised joint inflammation,synovial hyperplasia and cartilage and bone destruction.The pathology of RA is a combination of genetic,immune and infectious factors,and the excessive proliferation,migration and invasion of fibroblast-like synoviocytes(FLSs)is an important factor in the destruction of cartilage and bone in RA.Therefore,FLSs have become potential target cells for the study of RA pathogenesis and treatment.The expression of solute carrier family 3 member 2(SLC3A2)was found to be significantly increased in RA-FLSs.SLC3A2 is a co-receptor for integrin β3,which activates integrin β3 and amplifies its downstream signaling.Whether SLC3A2 coactivates integrin β3 and its downstream pathway to promote the migratory phenotype of RA-FLSs has not been reported to date.In the current treatment of RA,traditional Chinese medicine,with its high efficiency and low toxicity,has been shown to have unparalleled advantages in the treatment of RA.This formula has been clinically used for the treatment of RA for many years and has benefited thousands of RA patients.Our group found that the expression of SLC3A2 in the peripheral blood of RA patients was higher than that of the normal group by LC-MS/MS proteomics technology,and showed a decreasing trend after the treatment with YSTB.This suggests that SLC3A2 may be a biomarker for RA and may be one of the key targets for the therapeutic effect of YSTB.ObjectiveTherefore,the aim of this study is to investigate whether Yishen Tongbi Decoction can improve the invasive phenotype of RA-FLSs and inhibit synovial hyperplasia by regulating the cell migration signaling pathway downstream of integrin β3 through"SLC3A2/integrinβ3".In order to provide theoretical and scientific basis for the treatment of RA with traditional Chinese medicine and the advantages of traditional Chinese medicine in regulating the synovial environment.Methods1.Experimental study of clinical synovial samplesSynovial tissues were collected from five patients with osteoarthritis(OA)and RA who were hospitalized for joint replacement surgery from August 2020 to May 2021 at the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine.The expression levels of SLC3A2,integrin β3,p-FAK,FAK,Src and p-Src in the synovial membrane of OA and RA patients were measured by IHC and Western Blot,and the differences between the two groups were compared.2.In vivo experimentSPF-grade male DBA/1 mice were assigned to the nromal control(NC),CIA model,YSTB-H,YSTB-L and methotrexate(MTX)groups.The mice in the YSTB-H and YSTB-L groups were gavaged with 20.6g/kg/d and 10.3g/kg/d of the raw drug for 30 days,respectively,and the mice in the MTX group were gavaged with MTX(1mg/kg·q3w)for 100 μL of liquid.Mice in the NC and CIA model groups were given equal amounts of saline gavage daily.Body weight,arthritis index(AI)score and paw swelling thickness were observed and recorded at 3-day intervals during the administration.On day 51,both hind limbs were removed and the knee and ankle joints were scanned by microcomputed tomography(micro-CT)and scored for BS,BS/BV,BMD.HE and safranin o staining were used to observe the structural changes in the knee and ankle joints and to score the pathology.IHC was used to detect the localization and expression of SLC3A2,integrin β3,p-FAK and p-Src in the synovial membrane of the ankle joints.3.In vitro experiment3.1 Human fibroblast-like synoviocytes(HFLS)and human fibroblast-like synoviocytes of Rheumatoid arthritis(HFLS-RA)were used for this experiment.EdU staining was used to compare the proliferation ability between the two cell lines.Wound healing assay and Transwell migration assay were used to compare the migration ability between the two cell lines.Immunofluorescence(IF)was used to detect the co-localization of SLC3A2 and integrin β3 in the two groups.RT-qPCR was used to compare the gene expression levels of SLC3A2,integrin β3,FAK and Src between the two groups.WB was used to detect the protein expressions of SLC3A2,integrin β3,p-FAK,FAK,Src and p-Src between the two groups.3.2 HFLS-RA was transfected with lentiviral shRNA vector to knock down the expression of SLC3A2,and the changes of proliferation and migration ability of HFLS-RA were observed by EdU staining and Transwell migration assay,respectively.RT-qPCR for identification of knockdown efficiency.The effects of the knockdown of SLC3A2 on the expression of integrin β3 and its downstream proteins such as p-FAK,FAK,Src and p-Src were detected by IF and WB methods.3.3 The CCK8(Cell Counting Kit-8)method was used to screen the safe dose of HFLS-RA for the intervention of YSTB;the gradient dose of YSTB lyophilized powder was used to interfere with HFLS-RA,and the changes of HFLS-RA proliferation and migration ability were observed by EdU staining and Transwell migration assay,respectively.IF and WB methods were used to detect the effects of YSTB on the expression of SLC3A2,integrin β3,p-FAK,FAK,Src and p-Src.Results1.Experimental study of clinical synovial samplesThe expression of SLC3A2 and integrin β3 was higher in the RA group than in the OA group by IHC staining,and both showed positive staining mainly in the cell membrane and cytoplasm.The WB results showed that the expression of SLC3A2 and integrin β3 in the synovial tissue of RA patients was higher than that in the OA group,and the difference was statistically significant(P<0.01,P<0.05);there was no difference between the two groups in the expression of FAK and Src proteins(P>0.05);the expression of p-FAK and p-Src proteins was higher in RA patients,and the difference was statistically significant(P<0.01).The differences were statistically significant(P<0.01).2.In vivo experimentYishen Tongbi Decoction(YSTB)significantly improved ankle joint swelling and limb mobility in CIA mice.Compared with the NC group,foot swelling and AI scores of CIA mice increased significantly from day 27(P<0.01,P<0.05).After YSTB and MTX intervention,there were significant differences starting from day 42 and 45(P<0.01,P<0.05),with YSTB showing faster efficacy than MTX.HE staining showed that compared with the CIA group,YSTB-H and MTX groups could significantly inhibit inflammatory cell infiltration,synovial hyperplasia,cartilage and bone destruction(P<0.01).Safranin Ofast green staining showed that YSTB-H and MTX groups significantly improved cartilage erosion in CIA mice,with YSTB-H showing better efficacy.Micro-CT scanning and bone microstructure analysis showed that YSTB-H group could significantly alleviate bone destruction in knee and ankle joints of CIA mice,and bone volume(BV),bone surface area to bone volume ratio(BS/BV)and bone density(BMD)were improved to varying degrees(P<0.05).In addition,IHC staining showed that in the synovial hyperplasia site of the ankle joint of CIA mice,a large amount of SLC3A2,integrin β3,p-FAK,and p-Src positive staining were observed in the cell membrane and cytoplasm.After YSTB-H intervention,there was no significant synovial hyperplasia,and the degree of positive staining of these proteins in synovial cells was lower.MTX had no significant effect on the down-regulation of the expression of these proteins.3.In vitro experiment3.1 EdU staining showed that the proliferation ability of HFLS-RA was higher than that of HFLS(P<0.01);wound healing and Transwell migration assays showed that the migration ability of HFLS-RA cells was significantly higher than that of HFLS(P<0.05,P<0.01).IF staining showed that fluorescence intensity of SLC3A2,integrin β3 were higher and co-localization level was significant in HFLS-RA cells compared with HFLS.qPCR results showed that gene expression levels of SLC3 A2,integrin β3,FAK were higher in HFLS-RA cells than in HFLS cells,and the difference was significant(P<0.05,P<0.01);WB showed that the expression levels of SLC3A2,integrin β3,p-FAK and p-Src proteins were higher in HFLS-RA cells than in HFLS cells.3.2 After transfection of HFLS-RA cells with shRNA lentiviral vector for 72h,as verified by fluorescence microscopy,qPCR and WB,it was seen that about 80%of HFLS-RA cells showed green fluorescence under the microscope,and the expression of SLC3A2 at both gene level and protein level was significantly reduced(P<0.05);EdU staining results showed that the proliferation ability of HFLS-RA cells was decreased after knocking down SLC3A2(P<0.01);Transwell migration assay showed that knockdown of SLC3A2 affected the migration ability of HFLS-RA(P<0.01);IF and WB results showed that knockdown of SLC3A2 did not have a significant effect on integrin β3(P>0.05),but inhibited its downstream signaling molecules.The expression of p-FAK and p-Src,signaling molecules mediating cell migration,were inhibited(P<0.01,P<0.05).3.3 The safe concentration range for the intervention of YSTB in HFLS-RA cells was screened by CCK-8.Two gradient concentrations of YSTB,100 μg/mL(YSTB-L)and 200μg/mL(YSTB-H),were selected for the intervention of HFLS-RA with HFLS as the control group,and the results of EdU staining showed that YSTB-H significantly inhibited the proliferation of HFLS-RA cells(P<0.05).Transwell migration assay showed that YSTB-H reduced the migration ability of HFLS-RA(P<0.01);IF and WB results showed that YSTB-H not only reduced the expression of SLC3A2 in HFLS-RA(P<0.05),but also inhibited the expression of integrin β3 and its downstream p-FAK and p-Src protein expression(P<0.05).Conclusion1.The protein expression of SLC3A2,integrin β3 and its downstream signaling molecules p-FAK and p-Src,which mediate cell migration,were significantly higher in the synovial tissues of RA patients than in OA patients,indicating that SLC3A2/integrin β3 and downstream proteins are indeed closely related to the transformation of the migratory phenotype of RA-FLSs.2.In vivo experiments showed that the expression of SLC3A2 and integrin β3 in large amounts in the hyperplastic synovial membrane of CIA mice further demonstrated their role in the abnormal synovial membrane proliferation and phenotypic transformation,and that YSTB could reduce synovial membrane proliferation and improve cartilage and bone destruction by inhibiting the conduction of this intracellular signaling pathway.3.In vitro experiments confirmed that SLC3A2 could regulate the signaling pathway downstream of integrin β3 that mediates cell migration,and further clarified the mechanism of action of YSTB in inhibiting the migration of RA-FLSs,i.e.reducing the expression of SLC3A2,integrin β3 and downstream proteins. |
