Study On The Synergistic Effect Of Methotrexate Combined With Erzhi Pills On Bone Protection In Collagen-induced Arthritis Rats And Related Mechanisms

1 BackgroundRheumatoid arthritis(RA)is a chronic systemic autoimmune disease for which pathological change are synovitis,synovial cell proliferation,inflammatory cell infiltration,pannus formation,and progressive destruction of cartilage and bone.The main syptoms of RA are joint stiffness,pain,symmetric polyarthritis,and even systemic osteoporosis.In the development of RA,the osteoblast(Osteoblast,OBs)cells which mediate thereduction of bone formation and the osteoclast cells(Osteoclast,OCs)which mediate the increasing of bone resorption are important in the imbalance of bone metabolism.The studies have shown that Wnt/?-catenin signaling pathway plays the key role in the bone metabolism through mediating the formation,differentiation and maturation of OBs,regulating osteoprotegerin(OPG)levels of OBs to competitively inhibit the differentiation and function of OCs.The studies have shown that the bone formation-related markers expresses lower in mesenchymal stem cells lacking/?-catenin gene which is the key gene of Wnt signaling pathway in early embryonic development.Function changes of LRP5 which is one of the main receptor of mediating Wnt signaling pathway on cytomembrane can impact the effect of Wnt/?-catenin signaling pathway on OBs.One studies have shown that the OBs of mice which were knockouted LRP5 gene differentiation is inhibitedTraditional Chinese medicine holds that the main etiology and pathogenesis of abnormal bone metabolism happens in RA is "kidney difficiency" which impacts the balance between OBs and OCs and causes the abnormal bone remodeling.ErZhi Pill(EZP)is a famous Traditional Chinese formula which consists two herbs,Ligustrum lucidum Ait.and Eclipta prostrate L.It has been used to treat osteoporosis by TCM practitioners in clinic.Studies have shown that EZP,through activation of the Wnt3a/LRP5/?-catenin signaling pathway,had potential anti-Osteoporosis effects by preventing the degradation of the alveolar trabecular microarchitecture and alveolar bone loss in ovariectomized rats.For western medicine,methotrexate(MTX)is the first-line drug in the treatment of RA.In addition,several studies have also reported that MTX could improve the expression and activity of OB-related proteins,such as BALP.It significantly inhibits the inflammatory response and bone destruction.However,its mechanism is still not clear and the individual differences were reported when using MTX to treat RA.Hence,this thesis aimed to investigate whether the combination of MTX and EZP(MTX+EZP)can enhance the therapeutic effect in the treatment of RA and its possible mechanism.2 ObjectiveInvestigate the synergistic effect of MTX+EZP in the treatment of RA by promoting bone formation and evaluate the mechanism of Wnt1/LRP5/?-catenin signaling pathway.3 Methods3.1 Predicting the potential mechanism of MTX+EZP in treatment of rheumatoid arthritis via network pharmacologyThe ETCM,Pubchem,and NCBI Gene databases were employed to find the chemical components contained in EZP and the human-derived target proteins and human-derived RA related genes that corresponds to EZP and MTX,respectively.The interaction network among the EZP target protein,MTX target protein and RA genes,the functions of the network nodes,and the related signal pathways were furtherly analyzed using IPA biological network platform to predict a superior biological pathway for MTX+EZP in the treatment of RA.3.2 Investigate the synergistic effect of joint bone protection and mechanism of MTX+EZP on CIA ratsThe HPLC method was employed to analyze the contents of specnuezhenide in EZP.CIA rat model was established and then treated with MTX,EZP and MTX+EZP,respectively for 28 days by gavage.The rthritis scores were recorded each 3 days.H&E staining and Micro-CT scanning were used to evaluate the effect of MTX+EZP on the pathological changes and bone erosion of ankle joints in CIA rats.The serum bone metabolism markers,tartrate resistant acid phosphatase(TRACP)and bone alkalin phosphatase(BALP)protein,were detected by ELISA.RT-PCR was employed to investigate the mRNA expression levels of OBs differentiation and function-related genes,Wntl,LRP5,?-catenin,Runx2,BGP,BALP and OCs differentiation and function-related genes,OPG,RANKL,RANK,c-Fos and NFATcl,in rat joints.3.3 Explore the Wntl/LRP5/?-catenin signaling pathway in the role of MTX combined EZP effect on OBsThe osteoblast cell line UMR-106 was used and were devided into Control serum group(Control),CIA model serum group(CIA),MTX dosed serum CIA group(MTX),EZP dosed serum CIA group(EZP)and MTX+EZP dosed serum CIA group(MTX+EZP).The cells wereintervened by 15%dosed serum,respectively.After 48 hours,the effect on the proliferation rate of OBs was detected by CCK-8 assay.In addition,mRNA expression levels of Wntl,LRP5,Runx2,BGP and BALP in OBs were assessed by RT-PCR.4 Results4.1 Predicting the potential mechanism of MTX+EZP in treatment of rheumatoid arthritis via network pharmacologyA total of 62 chemical components of EZP were retrieved from the ETCM database in which 21 compounds are from Ligustrum lucidum Ait.and 41 compounds are from Eclipta prostrate L.PubChem database reported 1454 EZP target proteins and 102 MTX target proteins.A total of 1148 RA human related genes were obtained from the Gene database.After analyzing the IPA signaling pathway module,EZP and MTX target proteins were involved in 328 signaling pathways and RA was related to 374 signaling pathways.The signal pathways of EZP and MTX target protein participation were weighted.The top 14 shared signaling associated with cellular immune response and cytokine signaling were obtained in this study.Wnt/?-catenin signaling pathway was found to be the first shared signal pathwayand the focus of further study..The result suggested that EZP and MTX might reduce RA progression through the Wnt/?-catenin signaling pathway.4.2 The synergistic effect of joint bone protection and mechanism of MTX+EZP on CIA ratsAfter analyzing of the HPLC method,EZP which usd in our experiment are satisfied the requirements of Chinese pharmacopoeia and can be used in next experiments.After 28 days intervention,the ankle joint of rats in the CIA group experienced severe swelling and degeneration when compared with the Control group.Compared with the CIA group,the degree of swelling of the ankle joint and below in the MTX group,EZP group and MTX+EZP group showed different degrees of elimination.MTX group and MTX+EZP group presented a statistically significant difference in joint hindlimb joint score and ankle joint pathology score(P<0.05);compared with the MTX group,the MTX+EZP group joint score and ankle joint pathology score decreased(P<0.05).The Micro-CT results showed that the BS value and BS/BV value of the ankle joint in the CIA group were significantly increased(P<0.05),and the BV value was significantly reduced(P<0.05)from Control group.of When compared with CIA group,the MTX+EZP group was the only one that showed significantly decreased in the BV value(P<0.05).the BV values of the MTX group,EZP group and MTX+EZP group were all increased significantly(P<0.05),and BS/BV value was significantly reduced in the MTX group and the MTX+EZP group(P<0.05).Furthermore,compared with MTX group,BS/BV value in MTX+EZP group was significantly improved(P<0.05).ELISA results suggested that,compared with the Control group,the BALP levels in rats serum were significantly lower(P<0.05)while TRACP levels increased in the CIA group.The serum levels of BALP in MTX,EZP and MTX+EZP group were significantly increased(P<0.05),while the TRACP levels dropped(P<0.05),when compared with CIA group.Compared with MTX group,BALP level in MTX+EZP group was significantly enhanced(P<0.05).RT-PCR results were presented as following:comparing with the Control group,the mRNA expressions of Wntl,LRP5,?-catenin,Runx2,BALP,and BGP in the CIA group weresignificantly down regulated(P<0.05)?The expressions of Wntl,LRP5,?-catenin,Runx2,BALP and BGP genes in MTX,EZP and MTX+EZP group were significantly increased(P<0.05 or)when compared with the CIA group excludes the Runx2 mRNA expression in EZP group.Compared with the MTX group,the mRNA expression levels of the above six genes in the MTX+EZP group were significantly increased(P<0.05).Furthermore,the RT-PCR results of OCs differentiation pathways presented that compared with the Control group,the OPG gene level in the CIA group was significantly reduced(P<0.01)and the expressions of RANKL,RANK,c-Fos,and NFATc1 were significantlyincreased(P<0.05).Compared with CIA group,OPG gene expression of MTX group and MTX+EZP group increased significantly(P<0.05),while RANKL,RANK,c-Fos and NFATcl decreased significantly(P<0.05).4.3 The effect of MTX+EZP on OBs differentiation and function based on Wntl/LRP5/?-catenin signaling pathwayThe OBs were intervented dosed serum for 48h.CCK-8 detection showed that the proliferation rate of OBs in the CIA group was significantly reduced when compared with the Control group(P<0.05).Compared with the CIA group,the proliferation rates of OBs in the MTX and MTX+EZP groups were significantly higher(P<0.05).However,there was no significant difference between the MTX group and the MTX+EZP group.Results from RT-PCR presented that the mRNA expressions of Wntl,LRP5,Runx2,and BGP in the CIA group were significantly reduced compared with the Control group(P<0.05).Compared with the CIA group,the mRNA expressions of Wntl and LRP5 mRNA in theMTX group,EZP group and MTX+EZP group were significantly up-regulated(P<0.05).The mRNA expression levels of Runx2 and BGP in MTX+EZP group increased significantly when compared with CIA group(P<0.05)while there was no statistical difference showed in MTX group,EZP group with CIA group,respectively.When compared with the MTX group,the mRNA expression levels of Wntl and LRP5 were also significantly increased in the MTX+EZP group(P<0.05).5 ConclusionIn conclusion,MTX+EZP have a synergistic effect on promoting bone formation through Wntl/LRP5/?-catenin signaling pathway in the treatment of RA.