The Anti-inflammatory Mechanisms Of Wantong Jingu Tablet Against Rheumatoid Arthritis As Well As The 16S RDNA High-throughput Sequencing And Metabolomics Analysis

Rheumatoid arthritis(RA)is a chronic autoimmune disease with the characteristics of high disability rate,long treatment period,poor prognosis and easy relapse,which often brings heavy burden to the patients' families and society.Fibroblast-like synovial cells(RA-FLSs)are an important component of joint synovial tissues,the excessive proliferation of which can promote the release of a large number of pro-inflammatory factors from immune cells,and then activate inflammatory responses and aggravate pathology progression in RA.Besides,the pro-inflammatory factors in joint cavity will further activate RA-FLSs.Therefore,inhibiting the proliferation of RA-FLSs cells or reducing the levels of pro-inflammatory factors in joints may be an effective approach for RA treatment.Currently,drug therapy is still the major therapeutic strategy for RA.As the application of chemically synthesized drugs with high price and side effects,it is essential to design safer?more effective and inexpensive drugs to treat RA.Recently,traditional Chinese medicine(TCM)has received more and more attention on RA treatment due to its remarkable curative actions and fewer side effects.Furthermore,TCM exert therapeutic roles in RA mediated by multi-components,multi-pathways and multi-targets.Wantong Jingu Tablet(WJT),characterized by “dispel wind and disperse cold,clear collaterals and relieve pain”,is a combined medicine containing 25 TCM.It is mainly used for rheumatism,rheumatoid arthritis and other bone diseases.On the other hands,the unclear mechanisms of WJT hamper its expansion and application at home and abroad.Therefore,it is necessary to systematically study the functions and mechanisms of WJT in RA treatment.Firstly,the anti-inflammatory actions of WJT on RA were investigated relative to the collagen-induced rheumatoid arthritis of rats.Secondly,the corresponding mechanisms of WJT were illuminated based on the characteristics of RA-FLSs.Thirdly,the anti-inflammatory gene targets were identified using high-throughput sequencing.Finally,the effects of WJT on gut microbiota and metabolic profile in serum were conducted via 16 S rDNA high-throughput sequencing and metabolomics in order to screen for the microorganism targets and metabolic targets.Therefore,the work provided experimental evidence for the clinical application of WJT in RA treatment,promoted its expansion and popularity,as well as shed a novel light on the therapeutic mechanisms of TCM on RA.Part ?.Anti-inflammatory actions of Wantong Jingu Tablet on RA.Firstly,acute toxicity tests,routine blood tests,as well as functional analyses for liver,spleen and kidney were conducted to determine the safe dose of WJT in rats.Secondly,the toe swelling,the levels of TNF-?,IL-1? and IL-6 in joints,the joint pathology,and the protein expression level of Vimentin,the specific marker of FLSs,in synovial tissues were measured to evaluate the effectivity of CIA rat model.Finally,after 14 days and 28 days of continuous intragastric administration,we evaluated the swelling degrees in toes,tested the pathologies within inflammatory joints,and measured the levels of TNF-?,IL-1? and IL-6 in both joint cavity and tissues in order to investigate the anti-inflammatory actions of WJT on RA.The results have shown that 150,300 and 600 mg/kg were the safe dose of WJT,as well as the effectivity of CIA rat model was above 99.00%.The toe swelling of CIA rats were not significant alteration among different groups after continuous administration for 14 days.However,the toe swelling of CIA rats with continuous administration using 300 mg/kg and 600 mg/kg were remarkably reduced on 28 days.The histopathological results showed that the pathological state of joints in CIA rats within different groups,to some extent,have been reversed,particularly the CIA rats with 600 mg/kg administration for 28 days.The results of pro-inflammatory factors in articular cavity showed that the different concentrations of WJT were responsible for the anti-inflammation after administration,especially the 600 mg/kg treatment for 28 days with the better results.The levels of TNF-? and IL-1? in synovial tissues were markedly decreased after continuous treatment for 14 days.Meanwhile,the levels of TNF-?,IL-1? and IL-6 were dominantly reduced in synovial tissues after continuous administration for 28 days.All above results indicated that WJT exerted antiinflammatory roles in RA,which was positively associated with administration dose and time.Part ? .Anti-inflammatory mechanisms of Wantong Jingu Tablets in RA.The anti-inflammatory mechanisms were further investigated in vivo and in vitro due to the anti-inflammatory functions of WJT.Firstly,the RA-FLSs morphology,the activity of lactate dehydrogenase(LDH),Ed U nucleic acid labeling,and immunohistochemistry(IHC)were used to calculate the cells number in vitro as well as measure the vimentin expression level in synovial tissues,the aim of which was to investigate the effects of WJT on RA-FLSs proliferation.Secondly,the western blotting analysis,immunofluorescence and IHC were applied to measure protein expression levels of MEK,p MEK,ERK1/2 and p ERK1/2 in RA-FLSs and synovial tissues,respectively.Meanwhile,the plasmid transfection of ERK1/2 was conducted to the roles of MEK/ERK signaling pathway during anti-inflammatory progression.Finally,the activity of caspase-3/9 proteinase as well as the protein expression levels of cleavedcaspase-3 and cleaved-PARP in RA-FLSs were conducted to determine whether WJT was involved in anti-inflammatory progression mediated by inducing apoptosis,which was further confirmed by the protein expression levels of Bax,Bcl-2,caspase-3 and cleaved-caspase-3 in synovial tissues.The results suggested that the morphology of RA-FLSs among different groups has changed with different degrees under treatment of WJT for 24 h and 48 h,particularly the remarkable alteration in RA-FLSs incubated with 3 mg/ml and 4 mg/ml WJT for 48 h.The cells number was not changed when RA-FLSs were treated with WJT for 24 h,while 2 mg/ml,3 mg/ml,and 4 mg/ml WJT could significantly suppress cellular proliferation after 48 h.Besides,the protein expression levels of MEK,p MEK,ERK1/2 and p ERK1/2 were markedly induced after WJT treatment for 24 h and 48 h,particularly the higher significant difference mediated by 3 mg/ml,and 4 mg/ml WJT treatment for 48 h.The RA-FLSs with overexpressed ERK1/2 using plasmid have presented the strong proliferative capacity,which was significantly reversed by 3 mg/ml WJT.Meanwhile,the protein expression levels of MEK,p MEK,ERK1/2 and p ERK1/2 were decreased by WJT.Furthermore,4 mg/ml WJT could significantly upregulated the activity of caspase-3/9 proteinase after 24 h treatment,as well as both 3 mg/ml and 4 mg/ml WJT dominantly promote the activity of caspase-3/9 proteinase after 48 h treatment.We also found that 3 mg/ml and 4 mg/ml WJT markedly upregulated the protein expression of cleaved-caspase-3,and 2 mg/ml,3 mg/ml,and 4 mg/ml WJT remarkably increased the protein expression of cleaved-PARP after 24 h and 48 h treatment.Besides,WJT could contribute to protein expression of Bax and cleavedcaspase-3,as well as hamper expression of Bcl-2 and caspase-3 in synovial tissues.All above results indicated that WJT performed the anti-inflammatory functions mediated by inhibiting RA-FLSs proliferation and inducing apoptosis.Part ?.Identification of anti-inflammatory targets for Wantong Jingu Tablets against RA via RNA high-throughput sequencing.According to the effects of WJT on RA-FLSs,the high-throughput sequencing was performed for the RA-FLSs.In the work,two different approaches,DESeq2 difference analysis and STEM time sequencing analysis combined with WGCNA,were applied to accurately identify the anti-inflammatory targets.The DESeq2 was used to screen for differentially expressed genes(DEGs)among different groups,and subsequently the enrichment analysis as well as protein-protein interaction(PPI)analysis were conducted for the DEGs,which was to identify the corresponding candidate hub genes.The combination analysis of STEM time sequencing with WGCNA was used to identify the DEGs characterized by the same expression trend with time sequencing between WJT treated and untreated groups.And then we would further screen for the DEGs with the same expressing patterns.Besides,enrichment analysis,weighted-coexpression network analysis and PPI network analysis were applied to identify the corresponding candidate hub genes according to the connectivity degrees of nodes.Finally,the q RTPCR and western blotting analyses were performed to confirm the expression of candidate hub genes,and then identify the anti-inflammatory targets.The results showed that a total of 184 DEGs were identified from DESeq2,and the genes were significantly associated with cell cycle and homologous recombination.Besides,BRCA1,ATR,SMC3 and BUB1 have been identified as candidate hub genes according to PPI.On the other hands,a total of 111 DEGs with the same expression trend and pattern were identified by combination analysis of STEM with WGCNA.The genes were significantly enriched in cell cycle and RNA transport.Furthermore,PNN,SMC3,EIF3 A,BUB1,ATR and ORC4 have been considered as candidate hub genes according to WGCNA network and PPI network.Additionally,TTK,STAG2 and THOC1 were served as the candidate hub genes due to the essential correlation with cell cycle.Therefore,there were 10 candidate hub genes,namely BRCA1,ORC4,TTK,BUB1,SMC3,ATR,PNN,EIF3 A,STAG2 and THOC1.The results of q RT-PCR and western blotting analyses have shown that the expression patterns of SMC3 and BUB1 were consistent with sequencing data,which indicated that SMC3 and BUB1 might be the anti-inflammatory targets for WJT.Part ?.Identification of anti-inflammatory targets of Wantong Jingu Tablets against RA based on 16 S high-throughput sequencing and metabolomicsAccording to the anti-inflammation of WJT against CIA rats,the 16 S highthroughput sequencing and metabolomics were performed for the faeces and the serum in CIA rats,which was used to screen for gut microbiota targets and metabolic targets for WJT.The operating unit clustering and species diversity analysis were conducted to investigate the effects of WJT against the microbiota community.And then the difference analysis for the gut microbiota was performed to identify the difference microorganism community with the higher abundance.Besides,according to the sample correlation and multivariate statistical analysis,we aimed to illuminate the influence of WJT for the metabolic profile in serum.Identification of difference metabolites,filtration of functional enrichment and differential expression analysis were conducted to select the reliable difference metabolites.Finally,the combination analysis of 16 S high-throughput sequencing with metabolomics was performed to study the correlation of gut microbiota with metabolites,as well as further confirm the microorganism targets and metabolic targets.The results showed that WJT could alter the composition of gut microbiota according to the 16 S high-throughput sequencing.At phylum level,the relative abundances of Bacteroidetes,Tenericutes and Deferribacteres were significantly reversed.Besides,the ratio of Bacteroidetes to Firmicutes was also normalized.At genus level,the relative abundance of Vibrio,Macrococcus and Vagococcus were reduced to the normal levels.Meanwhile,we found that WJT could also alter the metabolic profile in serum of CIA rats according to metabolomics,of which Serotonin,Glutathione disulfide,N-acetylneuraminic acid,Naphthalene,and Thromboxane B2 were tended to be reversed by WJT.Additionally,we also found that Vibrio,Macrococcus and Vagococcus were included in the top10 difference microorganism,and there were 5 metabolites significantly associated with the 3 bacterial colonies.Meanwhile,only one metabolite,Tubulysin B,was the endogenous metabolite among the 5 metabolites due to the false positives during the progression of metabolite identification.All above results indicated that WJT conducted anti-inflammatory functions might mediated by regulation of gut microbiota and metabolic profiles in serum.Besides,Vibrio,Macrococcus and Vagococcus might be the microorganism targets for anti-inflammation of WJT.And Tubulysin B might be the metabolic targets for anti-inflammation of WJT...