To Explore The Immune Mechanism Of Qingre Huoxue Decoction In The Treatment Of RA And The Rule Of TCM Syndrome Based On TNF-α Expression

Objective1 Through clinical retrospective study,the correlation between the expression level of TNF-α and other RA-related laboratory indicators and TCM syndromes was discussed,in order to provide certain guidance for the clinical diagnosis and treatment of RA and TCM syndromes research.2 Through animal experiments,from the perspective of T cell DNA methylation,the changes of TNF-α expression and its relationship with DNA methylation after the intervention of Qingre Huoxue Decoction in CIA mice were investigated,and the epigenetic mechanism of TCM treatment of RA was explored.Method1 Clinical researchThe case data of active RA patients with TNF-α results in cytokine assay items were collected from January 1,2018 to December 31,2022 in the rheumatology department of Guang ’anmen Hospital,including the basic information of the patients and clinical and laboratory examinations at the same time.A database was established to analyze the correlation between TNF-α and experimental indexes and TCM syndrome.SPSS 26.0 software and Graphpad Prism 9.5.0 software were used for statistical analysis.2 Animal experimentTwenty-five C57 mice were randomly divided into 5 groups with 5 mice in each group:normal control group,model group,Qingre Huoxue Decoction group,methotrexate group,Qingre Huoxue Decoction+methotrexate group.Except for the normal control group,CIA mouse model was established in the other 4 groups,and enhanced immunization was given on the 21st day,and intragastric administration was started on the second immunization injection day until the end of the experiment.Normal control group and CIA model group were given normal saline intragastric administration according to the dosage of drug group.From the beginning of feeding to the end of the experiment,the behavior,diet,body weight and joint swelling of the 5 groups of mice were recorded.After the experiment,the expression of TNF-α in serum was detected by ELISA,the proportion of CD3+T lymphocytes in spleen was detected by flow cytometry,and the changes of TNF-α methylation in mouse spleen T cells were detected by Massarray technology.The correlation between TNF-α expression and DNA methylation was comprehensively analyzed.Results1 Clinical research（1）General information:A total of 534 patients were included,including 452 females and 82 males,with a male-to-female ratio of 1:5.1.The maximum age was 88 years old,the minimum was 16 years old,and the average age was 54.11±13.64 years old.The course of disease ranges from 1 month to 602 months.According to the normal value range of TNF-α≤8.1pg/ml,they were divided into normal group and abnormal group,with the abnormal group accounting for 59.55%.There was statistical significance in age between the two groups（P<0.05）,and the abnormal group was older.There were no significant differences in gender and course of disease between the two groups（P>0.05）.（2）Joint lesions and scores:The maximum number of joint tenderness was 25,the minimum number of joint swelling was 21,the minimum number of joint swelling was 0.There was statistical significance in the number of joint tenderness between normal group and abnormal group（P<0.05）,but there was no statistical significance in the number of joint swelling between normal group and abnormal group（P>0.05）.The maximum VAS score was 70,the minimum was 10,and the mean value was 39.23±14.60.DAS28（CRP）scores ranged from 2.6 to 7.26,with an average score of 4.38±1.34.There was statistical significance in VAS score between the two groups（P<0.05）,but no statistical significance in DAS28 score between the two groups（P>0.05）.（3）Laboratory index evaluation:There were statistically significant differences in ESR and CRP between the normal group and the abnormal group.Compared with the normal group,the ESR level in the abnormal group was increased（P<0.05）,and the CRP in the abnormal group was also increased（P<0.05）.The difference between the two groups in RF and anti-CCP antibodies was statistically significant.Compared with the normal group,the positive rate of RF in the abnormal group was significantly increased（P<0.001）,and the anti-CCP level in the abnormal group was significantly increased（P<0.05）.（4）Comparison of TCM syndrome:Among the 534 patients included,the top 3 TCM syndrome types were damp-heat obstruction type（55%）,qi and Yin deficiency type（11%）,phlegm-stasis obstruction type（10%）.The proportion of blood stasis blocking collaterals was the smallest,3%.There was no significant difference in age and course of disease among all syndrome types（P>0.05）.The results showed that the expression level of TNF-α in 8 TCM syndrome types in abnormal group was higher than that in normal group,the difference was statistically significant（P<0.05）.The comparison of TNF-α expression levels among different TCM syndrome types among abnormal groups showed that there were statistically significant differences between blood stasis type,liver and kidney deficiency type,wind-dampness obstruction type,cold-dampness obstruction type and damp-heat obstruction type（P<0.05）.（5）The Correlation between TNF-α expression level and laboratory indicators and clinical data:There was no statistical significance in correlation between TNF-αexpression level and ESR,CRP,CCP,SJC and disease course（P>0.05）.TNF-α had a weak positive correlation with RF level（r=0.118）,TJC（r=0.13）,VAS score（r=0.09）,and DAS28 score（r=0.136）.There was a weak positive correlation with age（r=0.153）.2 Animal experiments（1）General information:Compared with the normal control group,the joints of CIA mice showed redness,swelling,pain,stiffness and other signs consistent with the characteristics of active RA,and the weight growth rate slowed down.The body weight of mice increased slowly after the second dose of immunization,that is,after drug intervention.Compared with model group,drug intervention can reduce the influence of modeling on body weight of CIA mice and increase their body weight.The toe volume of mice in each group on day 0,day 25 and day 45 was measured by drainage method.Compared with the normal control group,the toe volume in the model group was significantly increased,and increased rapidly after the second immunization,with statistical significance（P<0.05）.Compared with the model group,the increase of joint swelling in the drug intervention group was slower,and there was no statistical significance among the three groups（P>0.05）.One week after the second immunization,the 4 groups of CIA mice outside the normal group began to show joint redness and swelling,and the AI score was also improved,especially the model group without drug intervention had the highest AI score.The AI score peaked on the 16th day after the second immunization.At this time,the AI score of the model group was 10±1.41,and the difference was statistically significant compared with the normal control group（P<0.05）.AI score was 7.8±0.84 in the TCM group,8.2±0.84 in the MTX group and 8±0.71 in the TCM group,and there was no statistical significance among the three groups（P>0.05）.（2）CD3+T lymphocyte population ratio Results:Results:The proportion of CD3+T lymphocytes in model group was significantly higher than that in normal control group,the difference was statistically significant（P<0.05）.The proportion of CD3+T lymphocytes in Qingre Huoxue Decoction,MTX group and Qingre Huoxue Decoction+MTX group decreased after drug intervention.There was no significant difference among the three groups（P>0.05）.（3）Expression level of TNF-α:Compared with normal control group,the expression level of TNF-α in model group was increased,and the difference was statistically significant（P<0.01）.Compared with model group,the expression levels of TNF-α in Qingre Huoxue Decoction group,MTX group and Qingre Huoxue Decoction+MTX group were decreased to different degrees,with statistical significance（P<0.05）,but there was no statistical significance among the three groups（P>0.05）.（4）MassARRAY DNA methylation quantitative detection results:In 25 samples,12 CpG sites were detected.Except for the unmethylated CpG₁1 site,methylation was detected in all other CpG sites.Compared with normal control group,the level of TNF-α methylation in model group was significantly decreased,and the difference was statistically significant（P<0.01）.Compared with model group,the TNF a methylation level of Qingre Huoxue Decoction group,MTX group,Qingre Huoxue Decoction+MTX group was increased in different degrees（P<0.01,P<0.05,P<0.01）.There was no significant difference among the three groups（P>0.05）.（5）Correlation analysis between the degree of TNF-α methylation and serum expression level:There was a statistically significant correlation between the degree of TNF-α methylation and the expression level of serum TNF-α（P<0.05）,and a low negative correlation between the degree of TNF-α methylation and the expression level of serum TNF-α（r2=0.38,P<0.05）.Conclusion1 Clinical studies have shown that there is a positive correlation between TNF-αand the evaluation index of disease activity in RA.TNF-α plays an important role in the diagnosis,treatment and prognosis of RA by participating in the pathological evolution process of RA.In terms of the distribution of syndrome types,damp-heat obstruction type is still the main syndrome type of RA in active stage.There are differences in the expression level of TNF-α among TCM syndrome types.Compared with solid syndrome,the expression level of deficiency syndrome is lower,and the level of TNF-α expression may provide certain reference for the clinical differentiation and staging of RA syndrome.2 Animal experiments showed that the expression level of TNF-α and the proportion of CD3+T lymphocytes in the serum of CIA mice were higher than those in the normal control group.After treatment with Qingre Huoxue Decoction,the expression level and the proportion of T lymphocytes decreased,which reflected the immune regulation effect of Qingre Huoxue Decoction in RA.In the methylation degree,compared with the normal control group,the methylation degree of CIA model group was significantly decreased,and the methylation degree of TNF-α was increased after the intervention of Qingre Huoxue Decoction.Correlation analysis showed a low negative correlation between the level of TNF-α methylation and the expression level of serum TNF-α,indicating that DNA methylation plays an important role in the pathogenesis of RA,and its hypomethylation may be one of the epigenetic manifestations of RA pathogenesis.Qingre Huoxue Decoction can improve the joint symptoms of CIA mice,and its mechanism may be related to the regulation of TNF-αgene promoter region of hypomethylation and inhibit the expression of TNF-α.