| Purpose:To evaluate the clinical efficacy of Yiqi Yangyin Tongluo Recipe in the treatment of rheumatoid arthritis of Qi and Yin deficiency type.Using the research method of network pharmacology,predict the possible targets and key pathways involved in the treatment of rheumatoid arthritis by Yiqi Yangyin Tongluo Recipe.To observe the effect of Yiqi Yangyin Tongluo Recipe on CIA rats,and to explore the mechanism of Yiqi Yangyin Tongluo Recipe in the treatment of rheumatoid arthritis based on the regulation of miR-155 on NF-κB signaling pathway.Material and method:1.Clinical retrospective study of Yiqi Yangyin Tongluo Recipe in the treatment of rheumatoid arthritis of Qi and Yin deficiency type:In the inpatient medical record system of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,the relevant clinical data of rheumatoid arthritis patients with Qi and Yin deficiency type who were hospitalized in the Department of Rheumatology in the past two years(January 1,2019 to December 31,2020)were screened.According to whether the actual treatment plan was combined with Yiqi Yangyin Tongluo Recipe during hospitalization,the patients were divided into a combination treatment group and a western medicine group.Finally,108 cases in the combination treatment group and 64 cases in the western medicine group were obtained,with a total of 172 cases.The general data,joint symptom indexes(morning stiffness time,number of joint tenderness,number of joint swelling),laboratory indexes(ESR,CRP),GH score,DAS28 score,individual score of TCM syndrome and TCM syndrome were observed and compared between the two groups of patients.To evaluate the efficacy of Yiqi Yangyin Tongluo Recipe in the treatment of rheumatoid arthritis of Qi and Yin deficiency type.The blood,urine and stool routines,liver and kidney functions of the two groups were monitored,and the occurrence of adverse reactions was recorded in detail.2.Network pharmacology study of Yiqi Yangyin Tongluo Recipe in the treatment of rheumatoid arthritis:Use TCMSP and BATMAN-TCM databases to screen the effective chemical components and potential protein targets of Yiqi Yangyin Tongluo Recipe,and convert them into official gene names through Uniprot database;Screening of rheumatoid arthritis-related disease targets in Gene Cards and OMIM databases.The intersection targets of Yiqi Yangyin Tongluo Recipe and rheumatoid arthritis were imported into the STRING 11.0 database platform to construct a PPI network diagram.The key targets of Yiqi Yangyin Tongluo Recipe in the treatment of rheumatoid arthritis were screened out by Cytoscape 3.7.2 software.The key targets were subjected to GO functional enrichment analysis and KEGG pathway enrichment analysis to construct a "drug-component-target-pathway map".3.Based on the regulation effect of miR-155 on NF-κB signaling pathway to explore the mechanism of Yiqi Yangyin Tongluo Recipe in the treatment of rheumatoid arthritis:Fifty-six SPF female SD rats were randomly divided into 7 groups with 8 rats in each group,namely blank control group,model group,western medicine group,low-dose traditional Chinese medicine group,middle-dose traditional Chinese medicine group,high-dose traditional Chinese medicine group,and combined medicine group.After 1 week of adaptive feeding,except for the blank control group,all of them were modeled into CIA rats.The blank control group and the model group were given normal saline once a day by gavage;the western medicine group was given methotrexate suspension once a week by gavage;the low-dose,medium-dose and high-dose traditional Chinese medicine groups were given different doses of Yiqi Yangyin Tongluo Recipe by gavage every day;the combination group was given daily Medium-dose Yiqi Yangyin Tongluo Recipe was administered by gavage,combined with methotrexate suspension once a week.Each group was given continuous administration for 6 weeks,and fasting was started after the last gavage,and the animals were sacrificed the next day.During the experiment,the general conditions of the rats were observed and recorded,and the degree of joint swelling and arthritis index of the rats were measured.Histopathological changes of ankle joints in CIA rats were observed by HE staining.Serum IL-1β and IL-8 levels of CIA rats were detected by ELISA.Western blot was used to detect the protein expressions of IκBα,p-IκBα,NF-κBp65 and p-NF-κBp65 in the synovial tissue of CIA rats.RT-qPCR was used to detect the levels of IκBα mRNA,NF-κBp65 mRNA and miR-155 in the synovial tissue of CIA rats.Results:1.Clinical retrospective study of Yiqi Yangyin Tongluo Recipe in the treatment of rheumatoid arthritis of Qi and Yin deficiency type:There was no significant difference in general data such as gender,age,course of disease,and length of hospitalization between the two groups(P>0.05),which were comparable.After treatment,the morning stiffness time,the number of joint tenderness and the number of joint swelling in the two groups were significantly decreased compared with those before treatment(P<0.01),and the improvement in the combined treatment group was better than that in the western medicine group(P<0.05).After treatment,ESR and CRP in the combined treatment group were lower than those in the western medicine group,but the difference was not statistically significant(P>0.05).The GH scores in both groups after treatment were significantly decreased(P < 0.001),and the combined treatment group had more advantages in improving GH scores(P<0.001).After treatment,the DAS28 scores of both groups were significantly decreased(P<0.001),and the decrease was more obvious in the combined treatment group(P<0.01).The overall efficacy level of the combined treatment group was better than that of the western medicine group(P<0.05),and the total effective rate was higher than that of the western medicine group.After treatment,the total scores of TCM syndromes in both groups were significantly decreased(P<0.001),and the decrease was more obvious in the combined treatment group(P<0.001).In the individual scores of each syndrome,the combined treatment group was significantly better than the western medicine group in improving joint swelling,shortness of breath,fatigue,muscle pain,dry mouth,spontaneous sweating,and heat in the hands,feet and heart(P<0.05).The efficacy of TCM syndromes in the combined treatment group was obviously superior(P<0.001),and the total effective rate was higher than that in the western medicine group.After treatment,the total incidence of adverse reactions in the combined treatment group was slightly higher than that in the western medicine group,but the overall incidence of adverse reactions in the two groups was comparable(P>0.05).2.Network pharmacology study of Yiqi Yangyin Tongluo Recipe in the treatment of rheumatoid arthritis:Through database screening,68 active ingredients and 415 potential protein targets of Yiqi Yangyin Tongluo Recipe were obtained.There are a total of 1284 disease targets related to rheumatoid arthritis,which are intersected with the targets of Yiqi Yangyin Tongluo Recipe,and 138 common targets are obtained.Screen the top 30 key targets for enrichment analysis,the GO functional enrichment analysis included three items.BP is mainly enriched in biological processes such as positive regulation of cell migration,response to lipopolysaccharide,regulation of smooth muscle cell proliferation,cellular response to chemical stress and response to cytokines.CC is mainly enriched in cellular components such as endoplasmic reticulum lumen,cyst lumen,membrane rafts,transcriptional repression complexes and Ficolin-1-rich granule lumen.MF is mainly enriched in molecular functions such as cytokine activity,protease binding,MAP kinase activity,R-SMAD binding and integrin binding.The results of KEGG enrichment mainly involved IL-17 signaling pathway,NF-κB signaling pathway and JAK-STAT signaling pathway.3.Based on the regulation effect of miR-155 on NF-κB signaling pathway to explore the mechanism of Yiqi Yangyin Tongluo Recipe in the treatment of rheumatoid arthritis:In model group,synovial cell proliferation,pannus formation,inflammatory cell infiltration,AI score,joint swelling,IL-1β and IL-8 were significantly increased(P<0.01).However,western medicine group,medium dose Chinese medicine group,high dose Chinese medicine group and combination group could reduce synovial cell proliferation and inflammatory cell infiltration,AI score,joint swelling,Il-1β and IL-8 expression to varying degrees(P<0.05).The histopathological morphology and AI score,joint swelling,IL-1β and IL-8 levels were significantly decreased in the combined treatment group(P<0.05).WB results showed that the IκBα protein in the model group was significantly decreased(P<0.001),while the other 6 groups could restore the inhibition of IκBα protein(P<0.001).Among them,the western medicine group,the middle-dose traditional Chinese medicine group and the high-dose traditional Chinese medicine group had similar up-regulation effects on IκBα protein expression(P>0.05),and the combined medicine group had the most significant increase in IκBα protein expression(P<0.001).The protein expression of p-IκBα,NF-κBp65,and p-NF-κBp65 in the model group was significantly increased(P<0.001),while the expressions of p-IκBα,NF-κBp65,and p-NF-κBp65 were significantly decreased in the other 6 groups(P<0.01).Among them,the western medicine group,the middle-dose traditional Chinese medicine group and the high-dose traditional Chinese medicine group had similar inhibitory effects on the expression of p-IκBα and p-NF-κBp65(P>0.05).The protein expressions of p-IκBα,NF-κBp65 and p-NF-κBp65 were down-regulated the most in the combined medicine group(P<0.001).RT-qPCR results showed that the mRNA expression of IκBα in the model group was significantly decreased(P<0.05),while the expression of IκBα mRNA was significantly increased in the western medicine group,the middle-dose traditional Chinese medicine group,the high-dose traditional Chinese medicine group and the combined medicine group(P<0.001).Among them,the western medicine group and the high-dose traditional Chinese medicine group had similar expression of IκBα mRNA(P>0.05),and the combined medicine group had the most significant increase in IκBα mRNA expression(P<0.05).The NF-κBp65mRNA in the model group was significantly increased(P<0.01),while the western medicine group,the middle-dose traditional Chinese medicine group,the high-dose traditional Chinese medicine group and the combined medicine group could significantly reduce the expression of NF-κBp65 mRNA(P<0.001).Among them,the western medicine group,the middle-dose traditional Chinese medicine group and the high-dose traditional Chinese medicine group had similar down-regulation levels of NF-κBp65 mRNA expression(P>0.05),and the combined medicine group had the most significant inhibition on NF-κBp65 mRNA(P<0.05).The level of miR-155 in the model group was abnormally high(P<0.01),while the western medicine group,the middle-dose traditional Chinese medicine group,the high-dose traditional Chinese medicine group and the combined medicine group could inhibit the abnormal expression of miR-155(P<0.001).Among them,the level of down-regulation of miR-155 in the western medicine group was comparable to that in the middle-dose traditional Chinese medicine group and the high-dose traditional Chinese medicine group(P>0.05),and the combined medicine group had the most significant inhibition of miR-155 expression(P<0.05).Conclusion:1.Yiqi Yangyin Tongluo Recipe combined with western medicine can significantly improve joint symptoms,reduce systemic inflammation,reduce disease activity and TCM syndrome scores in patients with RA due to deficiency of both Qi and Yin,confirming the clinical effectiveness and safety of Yiqi Yangyin Tongluo Recipe.2.Yiqi Yangyin Tongluo Recipe intervenes in RA’s pathophysiological processes such as cell proliferation,inflammatory response,oxidative stress,and bone metabolism through multi-component,multi-target,and multi-pathway,and then plays a role in the treatment of RA.3.Yiqi Yangyin Tongluo Recipe can improve histopathological damage of ankle joint in CIA rats,reduce the AI score and joint swelling degree,inhibit the release of pro-inflammatory cytokines such as IL-1β and IL-8,hinder the phosphorylation expression of IκBα and NF-κBp65,and reduce the expression levels of miR-155.It is suggested that Yiqi Yangyin Tongluo Recipe may inhibit the expression of miR-155 and affect the activation of NF-κB signaling pathway,thereby reducing synovial inflammation and joint damage in RA. |
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