Effect Of Fushan Rheumatism External Prescription On The Expression Of Notch1/Jagged1 Pathway In CIA Rats

Objective:The purpose of the experiment is to investigate the effect of Fusan Rheumatism External Prescription(FS-med)on the expression of Notch1 / Jagged1 pathway in collagen-induced arthritis(CIA)rats.Methods:Thirty-six female Wistar with SPF grade rats were randomly divided into three groups:the healthy group(Normal,n=12),the CIA model group(CIA,n=12),and Fu shan Rheumatism External Prescription treatment group(FS-med,n=12).The CIA and FS-med group rats were given bovine type Ⅱ collagen and complete(incomplete)Freund’s adjuvant to establish collagen-induced arthritis(CIA)model.After successful modeling,Normal group and CIA group rats were externally coated with empty infusion paste,the rats in FS-med group were coated with the ointment of Fu shan Rheumatism External Prescription,2 times/d,which was used for 6 weeks.The weight,joint diameter and arthritis scores of the rats were measured and recorded weekly.After 6 weeks,all the rats were killed after blood was taken from the abdominal aorta,and the left and right ankles of the rats were removed,the left ankles were frozen with liquid nitrogen and stored in a refrigerator at-80°C for later use.The right ankles were fixed with 4% paraformaldehyde.The expressions of IFN-γ,IL-4 and IL-6 in serum were detected by ELISA.The pathological changes of rat ankle tissues were detected by HE staining and Masson staining.The m RNA and protein of Notch1,Jagged1 and Hes1 in the joints of rats were respectively detected by qRT-PCR and Western Blot,and positive signals of Notch1,Jagged1 and Hes1 in the joints of rats were detected by immunohistochemistry,and optical density values were analyzed.Results:1.The results of foot appearance,body weight,joint diameter and arthritis scores of rats in different experimental groups showed that on the 14 th day after modeling(before administration),compared with Normal group,the CIA group and FS-med group rat’s posterior ankle joint to toe joint was significantly swollen,weight loss,left ankle diameter and arthritis scores were significantly increased(P < 0.01),suggesting the model was successfully established.From day 21 to day 56(1-6 weeks after administration),the CIA group rats’ hind feet continued to swell and their joints were deformed,the body weight increased slowly.Compared with the CIA group,the swelling degree of ankle and toes in the FS-med group was significantly alleviated during this process,the body weight increased slowly,the diameter of left ankle and the arthritis scores decreased,and the effect became more significant over time(P < 0.05).2.The histopathological results HE staining and Masson staining of synovial joints in rats of different experimental groups showed that in Normal group,the ankle joint structure is complete,the articular cartilage surface is smooth,the cells are arranged neatly,the tide line is complete,and the distribution of cartilage cells is regular,neither hyperplasia nor degeneration,the synovium is smooth,without hyperplasia,the joint space is good,no inflammation and edema are observed,and no obvious pathological changes are observed.Compared with Normal group,CIA group had rough and missing ankle articular surface,degeneration and destaining of surface cartilage,obvious fibrosis,large amount of synovial tissue hyperplasia,pannus formation,articular space narrowing,inflammatory cell infiltration,and severe damage of cartilage and bone.Compared with the CIA group,the morphology of articular cartilage in the FS-med group was improved,and the articular surface was still uneven,but the proliferation of fibrous tissues and synovial cells were reduced,accompanied by a small amount of lymphocyte infiltration,the joint space was significantly improved,and the destruction of bone tissue was controlled.3.The levels of cytokines in serum of rats in different experimental groups showed that,compared with the Normal group,the expression of IFN-γ and IL-6 in the CIA group was significantly increased(P < 0.01),and the expression of IL-4 was significantly decreased(P < 0.01).Compared with the CIA group,the expression of IFN-γ and IL-6 in the FS-med group was significantly decreased(P < 0.01),and the expression of IL-4 was significantly increased(P < 0.05).4.The expression of Notch1 / Jagged1 pathway related genes in joint of rats in different experimental groups showed that the expressions of Notch1,Jagged1 and Hes1 m RNA and protein in the CIA group were significantly higher than those in the Normal group(P < 0.01).Compared with the CIA group,the expressions of Notch1,Jagged1 and Hes1 m RNA and protein in the joints of rats in the FS-med group were significantly decreased(P < 0.01).5.Immunohistochemical detection results of Notch1,Jagged1 and Hes1 in rat joints in different experimental groups showed that Notch1,Jagged1 and Hes1 were positively expressed in different experimental groups of rats.Compared with the Normal group,Notch1,Jagged1 and Hes1 in the CIA group were strongly positive,and AOD value was significantly increased(P < 0.01).Compared with the CIA group,the positive expressions of Notch1,Jagged1 and Hes1 in the FS-med group were decreased,and AOD value was significantly decreased(P < 0.01),which further indicates that FS-med can significantly inhibit the activation of Notch1 / Jagged1 signaling pathway in CIA rats.Conclusion:This study confirmed that FS-med has a good therapeutic effect on CIA rats,and the Notch1 / Jagged1 signaling pathway is involved in the occurrence and development of RA.FS-med may achieve the purpose of treating RA by inhibiting the activation of Notch1 /Jagged1 signaling pathway and regulating the balance of Th1 / Th2 cytokines.This study is of great significance for understanding the mechanism of FS-med in the treatment of RA.Targeted inhibition of the activation of Notch1 / Jagged1 signal may become a new approach for the treatment of RA.