| BackgroundRheumatoid arthritis(RA)is a multifactorial systemic,inflammatory autoimmune disease characterized by major pathological features including synovitis and bone destruction.Osteoclast imbalance in the bone microenvironment,apoptosis of osteoblasts and overactivation of osteoclasts are important links in the occurrence and development of RA bone destruction.Therefore,the dynamic balance regulation mechanism between osteoblasts(mediating bone formation)and osteoclasts(mediating bone resorption)in the bone microenvironment is studied in depth,and then the therapeutic target of maintaining the balance of osteoclasts in the bone microenvironment is of great significance for inhibiting the destruction of RA bone.Increasing research evidence suggests that traditional Chinese medicine has a good clinical effect in the treatment of RA and inhibition of bone destruction,but the specific mechanism of action needs to be further clarified.The early stage of the research group innovatively proposed that "see bone damage rather than sole responsibility for the kidney","damp heat stasis" is the core pathogenesis of active RA,but also the pathological key to RA bone destruction,for this core pathogenesis determined to clear heat and wet blood is the treatment of active RA treatment method,Qingre Huoxue Recipe is an effective Chinese medicine compound.Clinical studies have shown that Qingre Huoxue Recipe can reduce the activity of RA disease,improve the quality of life of patients,and inhibit the occurrence of bone destruction.Network pharmacological analysis shows that the Qingre Huoxue Recipe can play a role in treating RA and inhibiting bone destruction through multiple targets and multiple pathways.Therefore,we further think,what is the active ingredient that inhibits the destruction of RA bone by the Qingre Huoxue Recipe?What is the regulatory mechanism of the Qingre Huoxue Recipe on the bone microenvironment?What are the pathways and targets for regulating the balance of osteoclasts?Long noncoding RNAs,which have been studied in depth in recent years,offer the possibility of solving this problem.The previous research of the research group found that miR-214 was significantly upregulated in the serum exosomes of RA patients,and the expression of miR-214 could be reduced by Qingre Huoxue Recipe,thereby reducing disease activity and inhibiting bone destruction.By bioinformatics analysis,LncRNA MEG3 is partially complementary to miRNA-214,and ATF4 is a downstream target gene regulated by miR-214.Therefore,we make the following scientific hypothesis:LncRNA MEG3 and miR-214 interact to inhibit the expression of ATF4,so that the bone microenvironment becomes osteoclast imbalance,resulting in RA bone destruction,and the Qingre Huoxue Recipe regulates the bone microenvironment osteoclast balance through the LncRNA MEG3/miR-214/ATF4 axis,promotes bone formation,reduces bone resorption,and then inhibits RA bone destruction.Part One:Qingre Huoxue Recipe liquid chromatography mass spectrometry ObjectiveThrough the identification and analysis of the pharmacodynamic active ingredients of the Qingre Huoxue Recipe by high performance liquid chromatography technology,the material basis for the effectiveness of the Qingre Huoxue Recipe is clarified.MethodRespectively,the herbs of the Qingre Huoxue Recipe are precisely weighed,and the filtrate is obtained by water extraction,concentrated and fixed volume,and the microporous membrane is filtered.At the same time,the standard is precisely weighed,the solution is prepared,the microporous membrane is filtered,and both are injected into a high performance liquid chromatograph for detection.Set the detection conditions,use the electrospray ion source(ESI),positive and negative ion mode to collect data,through the sample solution and control solution comparison analysis,acquisition and analysis of Qingre Huoxue Recipe cube peak ion chromatogram.ResultAfter obtaining the Qingre Huoxue Recipe Atlas,the peak comparison was carried out with the standard product,and finally 9 components were successfully separated from the Qingxin Active Blood Formula,and successfully compared with the standard,namely chlorogenic acid,neocogyn,Cangshusu,berberine hydrochloride,paeoniflorin,tanzanone IIA,qingtenoid,mullein isoflavone glucoside,curcumin.Through literature review,the active ingredient in the Qingre Huoxue Recipe can regulate the proliferation and differentiation of osteoclasts.Conclusion(One)The Qingre Huoxue Recipe has a variety of active ingredients that can regulate the proliferation and differentiation of osteoclasts.Part Two:Therapeutic effect of Qingre Huoxue Recipe on bone destruction in CIA ratsObjectiveTo study the intervention effect of the Qingre Huoxue Recipe on bone destruction in CIA rats,using the lncRNA MEG3/miR-214/ATF4 axis as the entry point,the mechanism of action of the Qingre Huoxue Recipe formula to regulate the bone microenvironment into osteoclast balance inhibition of bone destruction in CIA rats was elucidated.Method1 CIA rat model was constructed,which was divided into normal group,model group,low-dose group(8.5g/kg),medium dose group(17g/kg),high-dose group(34g/kg)and methotrexate group(0.2mg/kg).The intervention effect of clearing blood on bone destruction in CIA rats was evaluated by measuring rat body weight,toe volume,arthritis clinical integrals,ankle pathology tablets,and ankle and knee micro-CT,and the safety of Qingre Huoxue Recipe was assessed by liver and kidney pathology.2 The serum IL-6 and TNF-α of rats were detected by ELISA,and the expression of LncRNA MEG3,miR-214,ATF4 and mRNA markers of bone formation and bone resorption in rat bone tissues was detected by RT-qPCR.Results1 General situation of rats:Compared with the model group,the general conditions of rats in different dose groups and MTX groups of Qingre Huoxue Recipe were improved;rats in different dose groups and MTX groups of Qingre Huoxue Recipe were more likely to gain weight(p<0.05),and after 7 days of molding,the volume of toes increased slowly,the decrease was obvious after 21 days(p<0.05),and the clinical score of arthritis decreased significantly(p<0.05),of which the high dose effect of the Qingre Huoxue Recipe was the most significant.2 Evaluation of joint bone destruction in rats:Compared with the model group,the different dose groups and MTX groups of Qingre Huoxue Recipe were able to increase the joint space and reduce the degree of joint surface collapse(H&E staining,Micro-CT);reduce the number of osteoclasts(p<0.05);improve bone microstructure,improve BV,Tb.N,Tb.Th,BV/TV,and reduce BS,BS/BV,SMI,Tb.Sp(P<0.01),in which the high-dose intervention of Qingre Huoxue Recipe was significant.The results of liver and kidney pathology show that it is safe to Qingre Huoxue Recipe.3 LncRNAMEG3/miR-214/ATF4 axis,inflammatory factors and bone metabolism marker expression:ELISA test results showed that compared with the model group,the different dose groups and MTX groups of the Qingre Huoxue Recipe could reduce the levels of serum inflammatory factors IL-6 and TNF-α(P<0.01);the RT-qPCR test results showed that compared with the model group,the different dose groups and MTX groups of the qing fever active blood party were able to downregulate the expression of LncRNA MEG3 mRNA(P<0.01)and reduce the expression of miR-214 mRNA(P<0.01),increased the expression of ATF4 mRNA(P<0.01),decreased the bone formation markers BALP,PINP,RUNX2,and bone resorption markers TRAP,CTX-1,MMP-9 levels(P<0.01),and increased the ratio of PINP/CTX-1(P<0.01),promotes bone formation and reduces bone resorption.Conclusion(Two)1 The Qingre Huoxue Recipe can effectively improve the joint arthritis status of CIA rats and inhibit bone destruction.2 By downregulating the expression of LncRNA MEG3,reducing the expression of miR-214,increasing the expression of ATF4,improving the ratio of PINP/CTX-1,regulating the balance of osteoclasts in the bone microenvironment,promoting bone formation,reducing bone resorption,and thereby inhibiting bone destruction in CIA rats.Part Three:The mechanism of Qingre Huoxue Recipe is adjusted intoosteoclasts by the LncRNA MEG3/miR-214/ATF4 axisObjectiveTo verify the targeted relationship between LncRNA MEG3,miR-214 and ATF4,and to elucidate the role and mechanism of in vitro intervention of osteoclast co-culture system of Qingre Huoxue Recipe.Method1 Mc3T3-E1 cells were used as research objects to detect cell proliferation by cell transfection of LncRNA MEG3 knockdown/overexpression vectors,cell proliferation was detected by CCK-8,fluorescence in situ hybridization was used to detect the localization of LncRNAMEG3 in cells,RNA pull down experiments and RIP experiments were used to verify the targeted relationship between LncRNA MEG3,miR-214,ANDF4,and RT-qPCR,WB and immunofluorescence were detected for lncRNA MEG3,Expression of mRNA and proteins of miR-214,ATF4 and osteogenesis markers;2 Using the Transwell chamber,a co-culture system of osteoclasts(MC3T3-E1 cells and mouse bone marrow monocytes)was constructed,and the lyophilized powder was given to Qingre Huoxue Recipe.Identification of osteoblasts and osteoclasts by alizarin red staining and TRAP staining,screening of the optimal dose of Qingre Huoxue Recipe by CCK-8,and detection of the expression of osteoblasts and osteoclasts of osteoblasts and osteoclasts of osteoblasts and osteoclasts of osteoclasts meg3,miR-214,ATF4 and osteogenesis markers of osteogenesis and osteoclast by RT-qPCR,WB and immunofluorescence,respectively;Results1 Fluorescence in situ hybridization experiments showed that lncRNA MEG3 was mainly expressed in the cytoplasm;the results of RNA pull down experiments and RIP experiments showed that lncRNA MEG3 could be directly bound to miR-214,and ATF4 was the downstream target gene of miR-214;RT-qPCR,WB and immunofluorescence test results showed that after knocking down lncRNA MEG3,miR-214 could be significantly reduced The expression of mRNA(P<0.0001)increases the expression of ATF4 mRNA and protein(P<0.05),and the expression of bone formation markers BALP,PINP,RUNX2 mRNA and protein(P<0.05).2 The induction of osteoblasts and osteoclasts was successful by alizarin red and TRAP staining,the optimal dose of CCK-8 screening of the Qingre Huoxue Recipe was 5 μg/mL,and the results of RT-qPCR,WB and immunofluorescence tests showed that knocking down lncRNA MEG3 and the optimal dose intervention of the Qingre Huoxue Recipe could reduce the expression of osteoblast miR-214 mRNA in the co-culture system(P<0.001)and increase ATF4 The expression of mRNA and protein(P<0.05)and the expression of bone formation markers BALP,PINP,RUNX2 mRNA and protein(P<0.05)were improved,and the expression of osteoclast miR-214 mRNA(P<0.05)in co-culture system was reduced after knocking down lncRNA MEG3 and the optimal dose of antipyretic blood,and the expression of ATF4 mRNA and protein(P<0.001)could be reduced,and the bone resorption marker TRAP could be reduced Expression of MMP-9,MMP-3,CTX-1 mRNA and protein(P<0.05).Conclusion(Three)1 Knocking down LncRNA MEG3 can reduce the expression of miR-214,improve the expression of ATF4,and promote the proliferation and differentiation of osteoblasts.2 Qingre Huoxue Recipe can regulate the proliferation and differentiation of osteoclasts through LncRNA MEG3/miR-214/ATF4,promote bone formation and reduce bone resorption.Part Four:Clinical verification of the regulatory mechanism for the prevention and treatment of RA bone destruction by the Qingre Huoxue Recipe ObjectiveTo verify the regulatory mechanism of the Qingre Huoxue Recipe by regulating the bone microenvironment through the LncRNA MEG3/miR-214/ATF4 axis into osteoclast balance and inhibiting the destruction of RA bone.MethodIn this part of the experiment,real-time PCR(Real-time)method was used to detect the expression of LncRNA MEG3,miR-214,ATF4,bone formation and bone resorption marker mRNA in healthy people and RA patients before and after aQingre Huoxue Recipe.Results1 Compared with the healthy control group,the expression levels of LncRNA MEG3 and miR-214 mRNA in patients with damp heat stasis were significantly increased(p<0.001)and atF4 mRNA expression levels were significantly reduced(p<0.001).2 Compared with the healthy control group,the bone formation markers BALP,PINP,RUNX2,and bone resorption markers TRAP,CTX-1,and MMP-9 were elevated(P<0.01)in patients with damp heat stasis ra,but the ratio of PINP/CTX-1 was reduced(P<0.01).3 Compared with the pre-treatment group,the expression levels of LncRNA MEG3 and miR-214 mRNA were significantly reduced(p<0.001)and the expression level of ATF4 mRNA was significantly increased(p<0.001).4 Compared with the pre-treatment group of the Qingre Huoxue Recipe,the bone formation markers BALP,PINP,RUNX2 and bone resorption markers TRAP,CTX-1,MMP-9 were lowered(P<0.01),but the ratio of PINP/CTX-1 was significantly higher(P<0.01)。Conclusion(Four)1 lncRNA MEG3/miR-214/ATF4 axis abnormalities may mediate osteoclast equilibrium in the bone microenvironment,which in turn leads to the occurrence and development of RA bone destruction.2 Qingre Huoxue Recipe can lower the lncRNA MEG3,reduce the expression of miR-214,increase the expression of ATF4,regulate the bone microenvironment into osteoclast balance,promote bone formation,reduce bone resorption,and then inhibit the destruction of RA bone.ConclusionThrough the LncRNA MEG3/miR-214/ATF4 axis,the Qingre Huoxue Recipe regulates the osteoclast balance in the bone microenvironment,promotes bone formation,reduces bone resorption,and thus inhibits RA bone destruction. |
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