| BackgroundRheumatoid arthritis(RA)is a chronic systemic inflammatory autoimmune disease characterised by diffuse polyarthritis and inflammatory factor infiltration,characterised by symmetrical synovitis,which can cause irreversible joint destruction.Epidemiology shows that it affects approximately 1%of the population worldwide,with a slightly lower rate of 0.32%to 0.36%in China,and is currently one of the leading causes of workforce loss and disability in the population.Activated cells of all types stimulate the release of a large number of inflammatory factors,mainly IL-6,IL-8 and TNF-α,which are an important part of the pathogenesis of RA.SOCS can negatively regulate the JAK/STAT pathway,which has an important protective role in RA,and can modulate the expression of various inflammatory factors directly or through the JAK/STAT pathway to alleviate inflammatory damage.The role of epigenetic alterations at the RNA level,especially N6-methyladenosine(m6A)modifications,have received much attention.m6A is the most prevalent endogenous base modification of mRNA involved in a variety of immune diseases.m6A has been less studied in the development of RA and needs to be further explored.RA belongs to the category of "paralysis","calendar joints" or "crane knee wind".The theory of damp-heat stasis and obstruction has formed a theoretical system based on previous authors,and clinical treatment is mostly remarkable.Previous studies have confirmed that the treatment can interfere with the development by inhibiting the release of pro-inflammatory factors,inhibiting TNF-a-induced FLS proliferation and revascularization,etc.Therefore,the present study will further explore the mechanism of action of the clearing of heat and invigorating blood formula on the basis of epigenetics.ObjectiveA cross-sectional study was conducted to investigate the clinical distribution characteristics of TCM evidence of active RA and the correlation with serum inflammatory factors,providing a theoretical basis for clinical use;by observing the effects of Qingre Huoxue Decoction on the general condition,pathological and imaging changes and the expression of inflammatory factors(IL-6,IL-8,TNF-α)and key proteins(METTL3,SOCS2,JAK2,STAT3)in CIA rats,the mechanism of Qingre Huoxue Decoction on the regulation of JAK/STAT pathway by METTL3-mediated m6A modification of SOCS2 was explored,The effect of the formula on the expression of key proteins(METTL3,SOCS2,JAK2,STAT3),was explored to explore the mechanism of formula in the prevention and treatment of active RA,which is based on the METTL3-mediated m6A modification of the SOCS2-regulated JAK/STAT pathway.Methods1.A cross-sectional study was conducted to investigate the clinical distribution characteristics of TCM evidence of active RA and the correlation with serum inflammatory factors,providing a theoretical basis for clinical use;by observing the effects of Qingre Huoxue Decoction on the general condition,pathological and imaging changes and the expression of inflammatory factors(IL-6,IL-8,TNF-α)and key proteins(METTL3,SOCS2,JAK2,STAT3)in CIA rats,the mechanism of action of Qingre Huoxue Decoction on the regulation of JAK/STAT pathway by METTL3-mediated m6A modification of SOCS2 was explored,The effect of the formula on the expression of key proteins(METTL3,SOCS2,JAK2,STAT3),etc.was explored to explore the mechanism of action of the formula in the prevention and treatment of active RA,which is based on the METTL3-mediated m6A modification of the SOCS2-regulated JAK/STAT pathway.2.Experiment 2 50 SD rats were randomly divided into normal control group(Con),model group(Mod),Chinese medicine group(QRHXF),Western medicine group(MTX),and combined medicine group(Q+M).The CIA rat model was constructed by mixing type II collagen with incomplete Fosjoe in equal proportions to make a moulding agent.From the first immunization onwards,the rats were given Qinghe and Blood Activation Formula,MTX,Qinghe and Blood Activation Formula combined with MTX and saline by gavage,and the intervention cycle was 28 days,and the dosage was adjusted according to 1ml/100g body weight.After 28 days of modeling,the rats were stained with HE and Micro-CT to observe the local pathological and imaging changes of the synovial membrane and joints of CIA rats.3.Experiment 3,on the basis of experiment 2,the expression levels of JAK mRNA and STAT mRNA in the ankle joint of each group were further detected by QRT-PCR;the protein contents of JAK and STAT in the ankle joint of each group were detected by Western Blot.4.In Experiment 4,on the basis of Experiment 3,Western Blot and QRT-PCR were used to detect the protein and gene expression of SOCS2 and METTL3 in the ankle joint of each group,and LCMS/MS was used to detect the m6A content in the total RNA of the ankle joint of each group,and SELECT-QPCR was used to detect the m6A expression of the SOCS2 locus in the ankle joint of each group.The level of m6A expression was measured by SELECT-QPCR.Results1.Experiment 1 Among the 273 patients with active RA in this study,the most common type of evidence was damp-heat paralysis and obstruction in 86 cases(31.50%of the total number);IL-6,IL-8 and TNF-α were all significantly elevated in 273 RA patients,and the differences in the expression of IL-6 and TNF-αbetween the groups of Chinese medicine evidence were statistically significant(P<0.05,P<0.05).Among them,the elevation was most obvious in the group with damp-heat paralysis obstruction evidence.2.The results of Experiment 2 showed that compared with the model group,the Qingre Huoxue Decoction could enhance the general condition of CIA rats,reduce joint redness and swelling,and lower the AI score;in terms of pathology,it could maintain the normal morphology of synovial membrane and ankle joint tissue cells,reduce vascular proliferation and inflammatory cell infiltration;in terms of imaging,it could maintain the structural integrity of ankle joint,reduce synovial proliferation and delay the destruction of ankle joint cartilage;in terms of serum inflammatory In terms of serum inflammatory factors,it can inhibit the expression of inflammatory factors such as IL-6,IL-8 and TNF-α(P<0.05,P<0.01,P<0.05).3.The results of Experiment 3 showed that Clear Heat and Blood Activation Formula could effectively inhibit the expression of JAK2 protein(P<0.05)compared with the model group,and had some inhibitory effect on STAT3 protein but not statistically significant;it could effectively inhibit the gene expression of JAK2 and STAT3(P<0.05,P<0.05).4.Experiment 4 found that compared with the model group,the blood-clearing and activating formula was effective in inhibiting m6A alterations in total RNA of the ankle joint of CIA rats(P<0.05),inhibiting METTL3 protein and gene expression(P<0.01,P<0.05),up-regulating SOCS2 protein and gene expression(P<0.01,P<0.05)and decreasing m6A alterations at the SOCS2 locus(P<0.05).Conclusions1.The inflammatory factors IL-6,IL-8 and TNF-α are elevated in all groups,and are more obvious in damp-heat paralysis.2.In animal experiments,it was found that Qinghehe and Blood Activation Formula could effectively prevent the development of RA.Its mechanism of action may be related to the fact that Qinghe and Blood Activation Formula has enough to effectively inhibit the effect of METTL3 protein,reduce the m6A level of total RNA in the ankle joint of CIA rats and the m6A alteration of SOCS2 site,effectively maintain the stability of SOCS2,thus inhibit the activation of JAK/STAT pathway and reduce the the expression levels of inflammatory factors IL-6,IL-8,and TNF-α,and finally exerting inflammatory protection and alleviating bone destruction.Therefore,this study provides an active exploration of the correlation between the TCM evidence of RA and inflammatory factors,as well as the mechanism of action of the formula for clearing heat and activating blood to prevent and treat RA,especially in terms of epigenetics. |
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