Study On The Regulation Of Autophagy In Rats With Collagen-induced Arthritis Induced By Jinwu Jiangu Capsule

Objective1.The research status of jinwu jiangu prescription was analyzed by literature.2.By constructing Collagen-induced Arthritis(CIA)rat model,the joint swelling rate,joint inflammation index,joint pathological changes and autophagy-related gene expression of CIA model rats were observed,and the mechanism of action of miao drug jinwu jiangu capsule on autophagy of CIA joint synovial cells was investigated.Method1.Literature research:By searching the electronic literature in CNKI,VIP,Wanfang and CBM,the research status of jinwu jiangu prescription was analyzed by selecting the literature related to jinwu jiangu prescription.2.Experimental research:A total of 72 Wistar females were randomly divided into 6 groups(12 in each group):the blank group,the model group,the leflumide group,and the high,medium and low dose groups.In addition to the blank control group,all the other 5 groups of rats were constructed CIA model,and gavage treatment was started on the 7th day after immunization,followed by continuous gavage treatment for 28 days.The joint swelling rate of the rats was monitored weekly before and after administration,respectively,and arthritis score(AI value)was calculated.After the last administration,chloral hydrate was given abdominal anesthesia,skin was cut in groin,femoral vein and artery were separated,vein was cut and blood was collected,and the rats were sacrificed with severed head after blood collection.HE staining was used to observe the pathomorph of synovial membrane.Western blot(WB)was used to detect the expressions of Beclinl and LC3 proteins from the nuclear protein extracts of synovial tissues.The gene expressions of Beclinl,LC3,ATG5,ATG7 and ATG14 were detected by rt-qpcr,and the expression of serum cytokines IL-10,IL-12,IL-17,IL-23 and HIF-la were detected by enzyme linked immunosorbent assay(ELISA).Results1.Literature research results:Miao drug jinwu jiangu prescription can improve the symptoms of RA patients,down-regulate inflammatory indicators,inhibit the autophagy level of ra-fls,reduce the toxic and side effects of anti-rheumatic drugs to improve the condition,improve bone metabolism,and improve the quality of life of patients.2.The CIA model was successfully replicated,and the joint swelling rate and AI score of rats in the model control group were significantly increased compared with the blank control group(P<0.05).After treatment,the joint swelling rate and AI score of each treatment group were reduced to different degrees compared with that of the model group,with statistical significance in each group(P<0.05 or P<0.01).The low dose group of jinwu jiangu capsule decreased less than the other groups.3.HE staining pathological results:Synovial tissue in the model control group showed synovial cell proliferation,diffuse infiltration of a large number of lymphocytes,and pannus formation.There was no significant improvement in synovial tissue pathology between the low-dose group and the model control group.A small amount of neovascularization and mild synovial hyperplasia,a small amount of inflammatory cell infiltration and pannus formation were observed in the high-dose group,medium-dose group and leflumide group.4.WB detection results:The protein expression levels of LC3 and beclin-1 in synovial tissues of CIA rats in model control group were increased,and the difference was statistically significant compared with that of blank control group(P<0.05).Compared with the model control group,the protein expression levels of LC3 and Beclinl in each treatment group decreased to different degrees,which was statistically significant(P<0.05).Compared with leflunomide group,there was no significant difference in protein expression of LC3 and Beclinl in the medium dose group(P>0.01).5.RT-qPCR test results:The expressions of LC3,Beclin1,ATG5,ATG7 and ATG14 mRNA in synovial tissues of CIA rats were all increased,and the differences were statistically significant(P<0.05);Compared with the model control group,the mRNA expression levels of LC3,Beclinl,ATG5,ATG7 and ATG14 in each treatment group all decreased to different degrees,showing statistical significance(P<0.05);Compared with leflunomide group,there was no significant difference in mRNA expression of LC3,Beclinl,ATG5,ATG7 and ATG14 in the medium dose group(P>0.01).6.ELISA test results:The contents of serum IL-23,IL-17,IL-12 and HIF-1a in the model control group were all higher than that in the blank control group,and the contents of IL-10 were lower than that in the blank control group,with statistical significance(P<0.05);After treatment,the contents of IL-23,IL-17,IL-12 and HIF-la in each group decreased to different degrees compared with that in the model control group,with statistical significance(P<0.05),while the contents of IL-10 increased compared with that in the model control group,with statistical significance(P<0.05);Compared with leflunomide group,the contents of IL-23,IL-17,IL-12,HIF-1a and IL-10 in each group after treatment were not statistically significant(P>0.01).ConclusionJinwu jiangu capsule can reduce the score index of CIA rats’ arthritis and improve the synovial pathomorphology.The expression of LC3,Beclin1,ATG5,ATG7,ATG14 and LC3,Beclinl in synovial tissues was decreased,the secretion of serum pro-inflammatory factors IL-23,IL-17,IL-12 and HIF-1a was down-regulated,the secretion of IL-10 anti-inflammatory factors was up-regulated,and the inflammatory response of joint synovium in CIA rats was inhibited.