To Investigate The Mechanism Of Action Of Psammosilene Tunicoides In The Treatment Of RA Based On MH7A Cell Migration,Apoptosis And NLRP3 Inflammatory Bodies

Objective:Rheumatoid arthritis（RA）is a chronic autoimmune disease with low disability rate and high morbidity.At present,the drugs for the treatment of RA have great side effects and difficult to control the disease.Traditional Chinese medicine（TCM）is considered to be a research hotspot in the treatment of RA.There are many similarities between the symptoms of RA and arthralgia syndrome in TCM;therefore,during the study process,safer and more effective RA therapeutic drugs can be carried out with expelling wind and dampness drugs as the starting point based on the theoretical guidance of arthralgia syndrome treated with TCM.Psammosilene tunicoides W.C.Wuet C.Y.Wu is widely used in Yunnan for the treatment of RA,with good clinical efficacy and long-term use basis.However,studies on the mechanism of action of Psammosilene tunicoides in the treatment of RA are lacking.Therefore,this study was guided by the theory of traditional Chinese medicine to investigate the effects of Psammosilene tunicoides on the proliferation,migration,apoptosis and NLRP3inflammatory body activation of synovial fibroblasts in rheumatoid arthritis,in order to elucidate the regulatory mechanism of Psammosilene tunicoides on MH7A cells and NLRP3 inflammatory bodies,and then provide a new theoretical basis and experimental basis for the study of the mechanism of action of Psammosilene tunicoides in the treatment of RA.METHODS:CIA mouse arthritis model in vivo and RAW264.7 and MH7A cell inflammation models induced in vitro were established.Based on the characteristics of NLRP3inflammatory bodies and MH7A cell proliferation,migration and apoptosis;to explore the mechanism of action of Psammosilene tunicoides in the treatment of RA;1.Study on the mechanism of action of Psammosilene tunicoides on joint inflammation and NLRP3 inflammatory bodies in CIA mice:to establish a mouse model of rheumatoid arthritis（CIA）;on the 32nd day after the first immunization,the mice were randomly divided into MOD,MTX,CEPT.L,CEPT.H,TSPT.L,TSPT.H and CON according to the arthritis index score（AI）,with 10 animals in each group.Each group was dosed for 21 consecutive days according to the dose.During the experiment,mice were weighed to assess the AI score;mouse paw edema was determined by volumetric method.The mice were anesthetized and sacrificed for sampling;the spleen index of the mice was measured;the serum was collected and the levels of IL-1βand IL-18 in the serum were measured by ELISA kits;and the knee joints of CIA mice were examined（HE staining,safranin O staining,immunohistochemistry）.The expression of NLRP3,ASC,caspase-1 protein and m RNA in the leg and ankle tissues of CIA mice in each group was detected by q-PCR and Western blot;2.Study on the mechanism of action of Psammosilene tunicoides on NLRP3inflammatory bodies in an in vitro cellular inflammation model.Establish LPS-induced inflammation model of RAW264.7 cells;set up CON,MOD,TSPT concentration gradient group,QA concentration gradient group,GYP concentration gradient group;carry out administration intervention;collect the cell culture supernatant and cells of each group;determine the levels of IL-1βand IL-18 in the cell supernatant by ELISA kit;determine the expression of NLRP3 and caspase-1 protein in the cells by Western blot.After screening the administered concentration of MH7A cells,a TNF-α-induced MH7A cell inflammation model was established,and CON,MOD,TSPT concentration gradient groups,QA concentration gradient groups,and GYP concentration gradient groups were set up.Cell administration was performed;cell culture supernatants and cells were collected from each group;the contents of IL-1β,IL-18,and IL-6 and the expression of m RNA in the cells were determined by ELISA and q-PCR;and the expression of NLRP3 inflammatory body-related proteins and m RNA in the cells was determined by Western blot,q-PCR,and cell immunofluorescence.3.Effect of Psammosilene tunicoides on migration,proliferation and apoptosis of MH7A cells.Set CON,TSPT concentration gradient group,QA concentration gradient group and GYP concentration gradient group.Administered intervention.Cell migration rate was measured by cell scratch assay;apoptosis was measured by flow cytometry;MH7A cell proliferation was induced by TNF-αand treated with drug intervention,cell viability was measured by MTS assay,and the proliferation of MH7A cells was measured.Results:Effect of Psammosilene tunicoides on joint inflammation and NLRP3inflammatory body expression in CIA mice.On the 32 day after the first immunization,the success rate of modeling in CIA mice was 57%;compared with MOD mice,the contents of IL-1β,IL-18 inflammatory factors in serum and spleen index were significantly decreased in MTX,CEPT.L,CEPT.H,TSPT.L and TSPT.H mice（p<0.05or p<0.01）;AI score and the degree of joint swelling were significantly decreased（p<0.01）;knee arthritic infiltration and cartilage hyperplasia were improved;the expression levels of NLRP3,ASC,caspase-1 protein and m RNA in ankle joints were significantly decreased（p<0.01）.2.Effect of Psammosilene tunicoides on NLRP3 inflammatory body expression and inflammatory factor release in an in vivo cellular inflammation model.Compared with the LPS model group,the contents of inflammatory factors IL-1βand IL-18 in RAW264.7 cells were significantly decreased in the TSPT concentration gradient,QA concentration gradient,and GYP concentration gradient groups（P<0.01）;NLRP3 and caspase-1 protein expression was significantly decreased（P<0.05,P<0.01）.Compared with the TNF-αmodel group,the contents of IL-1β,IL-6,and inflammatory IL-18 factors and the expression levels of m RNA in MH7A cells were significantly decreased in the TSPT concentration gradient group,QA concentration gradient group,and GYP concentration gradient group（P<0.01）;the expression levels of NLRP3 and ASC protein and m RNA were significantly decreased（P<0.01）;compared with the TNF-αmodel group,the QA intervention concentration was 0.05 mg.m L^-1 and GYP was 0.05 mg.m L^-1,the expression of caspase-1 protein and m RNA in MH7A cells was also significantly decreased（P<0.01）;however,the TSPT intervention concentration was 0.1 mg.m L^-1.There was no significant difference in the expression levels of caspase-1 protein and m RNA in MH7A cells（P>0.05）.3.Effect of Psammosilene tunicoides on migration,proliferation and apoptosis of MH7A cells.Compared with the normal group,the apoptosis rate of MH7A cells in the TSPT concentration gradient group,QA concentration gradient group,and GYP concentration gradient group was significantly increased（p<0.05 or p<0.01）;the migration rate was significantly decreased（p<0.05 or p<0.01）,and the migration of MH7A cells was inhibited in a concentration-dependent manner;however,the TSPT intervention concentration was 0.05 mg.At m L-1,there was no significant difference in MH7A cell mobility（p>0.05）;the proliferation level of MH7A cells was significantly inhibited in the TSPT concentration gradient,QA concentration gradient,and GYP concentration gradient groups compared with the TNF-αgroup（p<0.01）.CONCLUSION:Psammosilene tunicoides can effectively inhibit the occurrence of inflammatory reaction and the activation of NLRP3 inflammatory bodies in CIA mice and inflammatory cell models,and inhibit the proliferation,migration and anti-apoptotic ability of MH7A cells in vitro and in vivo,thus indicating that Psammosilene tunicoides may be related to the inhibition of NLRP3 inflammatory bodies activation and the inhibition of proliferation,migration and the promotion of apoptosis of rheumatoid arthritis synovial fibroblasts in the treatment of RA.This study not only reveals that NLRP3 inflammatory bodies can be used as targets for immunomodulation in RA patients in vivo,but also provides some experimental basis and theoretical basis for the study of immunomodulatory function of Psammosilene tunicoides in RA diseases.