Study On Mechanism Of Shentong Zhuyu Decoction For Rheumatoid Arthritis Based On P38MAPK Signaling Pathway

Rheumatoid arthritis(RA)is an autoimmune disease characterized by chronic synovial inflammation of the joints.RA is one of the important diseases that threaten health.It has a high prevalence and disability rate in China.At present,the etiology of RA is not yet clear.The patient's joints are continuously eroded by inflammation,resulting in multiple joint deformities.The condition was progressive and irreversible,often accompanied by damage to extra-articular organs,leading to increased mortality.However,the existing treatment methods mainly focus on delaying the progress of the lesion,and it cannot be cured completely.The main drugs used for treatment have more adverse reactions and are expensive.It brings difficulties to long-term medication.Shentong Zhuyu Decoction has a definite curative effect on this disease with low side effects.It can be applied to various types of RA through flexible tailoring.It is now widely used in the clinical treatment of RA.Because its treatment mechanism is not yet clear,this study intends to observe the therapeutic effect of Shentong Zhuyu Decoction on collagen induced arthritis(CIA)rats and on RA fibroblast-like synoviocytes(RA-FLS),and explore its possible mechanism based on p38 MAPK signaling pathway.Part? Therapeutic effect of Shentong Zhuyu Decoction on CIA ratsObjective: Observe the therapeutic effect of Shentong Zhuyu Decoction on CIA rats and verify its effect on inflammation and joint synovium.Methods: 24 SD rats,6 rats were randomly selected as the normal group,and the remaining rats were constructed as CIA model.After successful modeling,they were randomly divided into three groups: model group,p38inhibitor(SB203580)group,and Shentong Zhuyu Decoction group.Then we conducted a 28-day drug intervention.During this time,we observed the general condition of rats,the degree of joint swelling,and scored joint inflammation.24 hours after the last administration,abdominal aortic blood was taken to detect the contents of CRP,RF,TNF-? and IL-1?,and the ankle joints were taken for HE staining to observe the pathological changes.Results: 1.Compared with the normal group,the rats in the model group began to show symptoms of RA after 7 days of modeling,the activity level was reduced,and the weight gain was slow.Compared with the model group,rats in p38 inhibitor group and Shentong Zhuyu Decoction group had mild symptoms of RA,slightly higher activity,and faster weight gain,P<0.05;2.Compared with the normal group,the model group's joint swelling index and arthritis index increased.Compared with the model group,the arthritis index and joint swelling index in p38 inhibitor group and Shentong Zhuyu Decoction group decreased,and their peak values also decreased,P<0.05;3.Compared with the normal group,the CRP,RF,TNF-?,and IL-1? levels in the model group increased.Compared with the model group,the above markers in the p38 inhibitor group and Shentong Zhuyu Decoction group were all reduced,P<0.01;4.Compared with the normal group,the ankle synovium of rats in the model group was hyperplasia,the inflammatory cells infiltrated obviously.Compared with the model group,the above changes in the ankle joints of rats in p38 inhibitor group and Shentong Zhuyu Decoction group were significantly improved,and the joint pathology score was reduced,P<0.05.Summary: Shentong Zhuyu Decoction can alleviate arthritis symptoms and serum indexes of CIA rats,inhibit joint inflammation and synovial hyperplasia.Part? The Effect of Shentong Zhuyu Decoction on RA-FLSObjective: To observe the regulatory effects of Shentong Zhuyu Decoction on the proliferation,invasion and apoptosis of RA-FLS.Methods: Normal rats FLS and CIA rats FLS(RA-FLS)were isolated and cultured.The pre-experiment was used to determine the intervention concentration and duration of p38 inhibitor(SB203580),PPAR? agonist(GW1929)and Shentong Zhuyu Decoction-containing serum for use in formal trials.Normal FLS was set as the normal group,RA-FLS was set as the model group,p38 inhibitor group,PPAR? agonist group and Shentong Zhuyu Decoction group.Each group conducted drug intervention separately.After 48 hours of culture,flow cytometry was used to detect the cell cycle change and apoptosis rate of each group;CCK-8 kit was used to detect the cell proliferation ability of each group;and Transwell invasion test was used to detect the cell invasion ability of each group.Results: 1.Compared with the normal group,the proportion of cells in the S phase of the model group increased significantly,and the proportion of cells in the G1 phase decreased significantly,P<0.01.Compared with the model group,the proportion of cells in the G1 phase increased and the proportion of cells in the S phase decreased in the p38 inhibitor group,PPAR?agonist group,and Shentong Zhuyu Decoction group,P<0.01;2.Compared with the normal group,the cell proliferation capacity of the model group was significantly enhanced,P<0.01.Compared with the model group,the cell proliferation ability of the p38 inhibitor group,PPAR? agonist group,and Shentong Zhuyu Decoction group were all reduced,P<0.01;3.Compared with the normal group,the number of die-penetrating cells in the model group increased significantly,and the invasion ability was enhanced,P<0.01.Compared with the model group,the number of die-penetrating cells in the p38 inhibitor group,PPAR? agonist group,and Shentong Zhuyu Tang group were all reduced,and the invasion ability was inhibited,P<0.01.4.Compared with the normal group,the apoptosis rate of the model group was significantly reduced,P<0.01.Compared with the model group,the apoptosis rate in the p38 inhibitor group,PPAR? agonist group,and Shentong Zhuyu Decoction group were all increased,P<0.01.Summary: Shentong Zhuyu Decoction can inhibit the abnormal proliferation,invasion of RA-FLS and induce its apoptosis.Part ? The Mechanism of Regulating RA-FLS by Shentong Zhuyu DecoctionObjective: To explore the mechanism of Shentong Zhuyu Decoction in regulating RA-FLS.Methods: Cell separation,culture,grouping and drug intervention are the same as the second part.Real-time PCR was used to detect the m RNA expressions of p38 MAPK,PPAR?,CTGF,Cyclin-D1 and caspase-3 in each group of cells;The proteins expressions of p38 MAPK,p-p38 MAPK,PPAR?,CTGF,Cyclin-D1 and cleared-caspase-3 in each group were detected by Western Blot.Results: 1.There was no difference in the expression of total p38 MAPK m RNA and protein among the groups.Compared with the normal group,the expression of p-p38 MAPK protein,CTGF m RNA and protein in the model group increased,and the expression of PPAR? m RNA and protein decreased,P<0.01.Compared with the model group,the expression of p-p38 MAPK protein,CTGF m RNA and protein in P38 inhibitor group,PPAR? agonist group and Shentong Zhuyu decoction group were all decreased,and the expression of PPAR? m RNA and protein was increased,P<0.05.2.Compared with the normal group,the expression of Cyclin-D1 m RNA and protein in the model group increased,P<0.01.Compared with the model group,the expressions of Cyclin-D1 m RNA and protein in the P38 inhibitor group,PPAR? agonist group,and Shentong Zhuyu Tang group were all reduced,P<0.05.3.Compared with the normal group,the expression of caspase-3m RNA and cleaved-caspase-3 protein in the model group decreased,P<0.01.Compared with the model group,the expressions of caspase-3 m RNA and cleared-caspase-3 protein in the P38 inhibitor group,PPAR? agonist group,and Shentong Zhuyu Tang group were all increased,P<0.05.Summary: 1.The abnormal activation of p38 MAPK / PPAR? / CTGF signaling pathway increases the expression of Cyclin-D1,and inhibits the expression of cleaved-caspase-3.This may be the molecular mechanism that causes the proliferation and invasion ability of RA-FLS cells to increase and the apoptosis ability to decrease.2.Shentong Zhuyu Decoction can inhibit the activity of p38MAPK/PPAR?/CTGF signaling pathway,down-regulate the expression of Cyclin-D1,and up-regulate the expression of cleaved-caspase-3.3.Shentong Zhuyu Decoction can inhibit the proliferation and invasion ability of RA-FLS and induce the apoptosis of RA-FLS.Its mechanism may be closely related to the regulation of p38 MAPK / PPAR? / CTGF signaling pathway by Shentong Zhuyu Decoction.Conclusion: Shentong Zhuyu Decoction can alleviate the symptoms of arthritis and lower the serum index,reduce inflammation and synovial hyperplasia of joint in CIA rats,which may be related to its ability to inhibit the abnormal proliferation and invasion of RA-FLS and induce its apoptosis.The mechanism may be closely related to the regulation of p38MAPK/PPAR?/CTGF signaling pathway by Shentong Zhuyu Decoction.