| Objective:Toobserve and investigate the clinical result of Qinjiao Dihuang Tongbi Decoction in treating rheumatoid arthritis(RA)in active phaseby randomized controlled clinical trial(RCT)and to analyze its mechanism by network pharmacology approach.To build collagen-induced arthritis(CIA)rats models of rheumatoid arthritis,the mechanism of Qinjiao Dihuang Tongbi Decoction in treating RA in activephase was explored andanalysedthrough gene transcription,proteinexpression and regulation of related signal pathways.Method:1.Clinical Result of Qinjiao Dihuang Tongbi Decoction in treating RA in active phase:According to the diagnostic criteria of RAin active phase and inclusion-exclusion criteria,RCT sample size was calculated.A total of 72 patients were recruited.Following the principle of randomization,they were divided into group A and group B.Group A was the control group and was treated by celecoxib and methotrexate.Group B was the experimental group,treated by Qinjiao Dihuang Tongbi Decoction+celecoxib+methotrexate.All patients were given drugs continuously for 12 weeks.To observe the variation of general clinical symptoms,laboratory examinations(ESR,CRP,RF,anti-CCP)and compareVAS score,DAS28 score,ACR20/50/70 improvement rate,Traditional Chinese medicine(TCM)syndrome score and TCM effective rate before and after treatment,the clinical result was estimated.Meanwhile,the safety of Qinjiao Dihuang Tongbi Decoction was monitored.2.Analyzing of Mechanism of Qinjiao Dihuang Tongbi Decoction in treating RA in active phase based on network pharmacology approach:The effectivechemical components of Qinjiao Dihuang Tongbi Decoction were screened and the targets were obtained by databases and data platforms.Thedisease-related targets of RA were searched.Deep comparison was carried out between disease-related targets of RA and effectivechemical components targets to construct "drug-effective component-target-RA" relationship network and PPI protein interaction network and key interactiontarget proteins were screened.By GO function enrichment analysis and KEGG conduction signal pathway enrichment analysis,the relevant signal pathway was predicted and the results were preliminarily proved by downstream target protein detection.3.Exploring the mechanism of Qinjiao Dihuang Tongbi Decoction through gene transcription,proteinexpression and regulation of related signal pathwaysin CIA rats models:To build CIArats models of RA,SD rats were divided into blank control group(Ctrl group),CIA model group(CIA group),MTX positive control group(Group A)and MTX+Qinjiao Dihuang Tongbi Decoction group(Group B).10 rats were in each group.All rats were given drugs continuously for 28 days.The variation of general state,arthritis index(AI)scores of rats and inflammatory changes of synovial membrane and articular cartilage by pathological sections of rat joints were observed.qRT-PCR and Western blot methods were used to detect the expression oflL-1β,IL-6,TNF-α,TLR4,MyD88 mRNA and the expression of TLR4,MyD88,p-NF-κλB(p65),NF-κB(p65)proteins in rat synovial tissues.Results:1.Clinical Result of Qinjiao Dihuang Tongbi Decoction in the treatment of RA in active phase:(1)From January 2019 to September 2019,a total of 72 patients of RAin active phase were recruited according to inclusion-exclusion criteria in the department of rheumatology of affiliated hospital of Nanjing University of Traditional Chinese Medicine.Four cases withdrew in the study course.68 cases were enrolled finally.35 cases were in Group A,13 males and 22 females,with an average age of 46.7±7.5 years old.33 case were in Group B,10 males and 23 females,with an average age of 48.1±6.3 years old.No significant differences were found inbaseline data of two groups(P>0.05).Before treatment,there were no significant differences between the two groups in common clinical symptoms such as joint tenderness,joint swelling and morning stiffness time and laboratory examinations(ESR,CRP,RF,anti-CCP)as well as VAS score,DAS28 score and TCM symptom score(P>0.05).(2)After treatment,the common clinical symptom,slaboratory examinations,VAS score and DAS28 score in Group B were significantly better than those in Group A(P<0.01).After treatment,the common clinical symptom,slaboratory examinations,VAS score and DAS28 score in each group was significantly better than before treatment(P<0.01).However,no significant differences were found in ACR20/50/70 improvement rate in two groups(P>0.05).(3)By comparison of TCM syndrome scores,Group B was significantly lower than Group A after treatment(P<0.01).After treatment,each group’s TCM syndrome score was significantlylower than before(P<0.01).After treatment,the effective rate of TCM was significantly different between two groups.Group A was 88.6 percent while Group B was 96.7 percent.Group B was better than Group A(P<0.05).(4)The safety monitoring of Qinjiao Dihuang Tongbi Decoction showed that the adverse reactions in Group A were significantly increased thanGroup B(P<0.05).2.Analyzing of Mechanism of Qinjiao Dihuang Tongbi Decoction in treating RA in active phase based on network pharmacology approach:(1)There are 68 effectivechemical components in Qinjiao Dihuang Tongbi Decoction,including 9 in Guizhi,11 in Baishao,21 in Fangfeng,4 in Qinjiao,5 in Shengdihuangt,24 in Jinyinhua and6 in Qingfengteng(some traditional Chinese medicines contain several effectivechemical components at the same time).345 targets were obtained according to the effective components.655 RA disease-related targets were retrieved.The Venn diagram of Qinjiao Dihuang Tongbi Decoction-RAwas constructed,and 110 key interaction targets in overlapping regions were found.By using the Cytoscape3.6.1 software,drug-effective component-target-RA relationship network and PPI protein interaction networkwere constructed and IL6 and TNF-αwere found to be key interaction target proteins.In consideration of the functional enrichment analysis resultsof GO-BP,GO-CC and GO-MF,it is predicted that the therapeutic effect of Qinjiao Dihuang Tongbi Decoction acted on the immune response process,involving cell microstructure and directly related to the function of cytokines.From the result of KEGG signal pathwayenrichment analysis,it is predicted that Qinjiao Dihuang Tongbi Decoction may intervene NF-κB signal pathway and regulate downstream target protein to achieve the purpose of treating RA in active phase.(2)By detecting the levels of downstream target protein of NF-κB signal pathway in serum samples of two groups before and after treatment,prediction results were preliminarily proved.The levels of pro-inflammatory cytokines were significantly reduced(P<0.01),the levels of anti-inflammatory cytokines were significantly increased(P<0.01),and NF-κB signal pathway was inhibited.3.Exploringthe mechanism of Qinjiao Dihuang Tongbi Decoction through gene transcription,proteinexpression and regulation of related signal pathwaysin CIA rats models:(1)General state of rats:In CIA group,the weight of rats were obviously reduced,the spirit were depressed,the hair were dried and yellow,and the lower limb activity weregradually reduced.The general state of rats in Groups A and B were better than those in CIA group,while Group B rats were more obvious.(2)AI scores of rats:Compared with CIA group,AI scores of Group Afrom 1 to 3 weeks and Group Bfrom 1 to 2 weeks did not significantly decreased(P>0.05);however AI scores of Group Afrom 4 to 6 weeks and Group Bfrom 3 to 6 weeks decreased significantly(P<0.05).Compared with Group A,AI score of Group B from1 to 3 weeks did not significantly decreased(P>0.05),however AI score of Group B from 4 to 6 weeks decreased significantly(P<0.05).(3)Pathological sections of ratjoints stained with safranine O and solid green:Compared with Crtl group,the cartilage cell structure in CIA group was disordered,and safranine staining on the joint surface was almost completely lost.InGroup A the articular cartilage of ratswas slightly damaged,it was significantly better than that in CIA group,and the articular cartilage damage score was significantly decreased(P<0.05).In Group B the articular cartilage of rats was relatively complete,and the articular cartilage damage score was significantly decreased than that of CIA group and Group A(P<0.05).(4)Gene transcription and protein expression level:Compared with Crtl group,the expressions of IL-1 β,IL-6,TNF-amRNA and TLR4,MyD88 mRNA and TLR4,MyD88,p-NF-κB(p65)protein in CIA group were significantly increased(P<0.05).After 4 weeks of therapy,compared with CIA group,the expressions of IL-1β,IL-6 TNF-α mRNA and TLR4,MyD88 mRNA and TLR4,MyD88,p-NF-κB(p65)protein in Group A and Group A were significantly decreased(P<0.05).Compared with Group A,the expression of the same mRNA and protein in Group Bwas significantly decreased(P<0.05).However,in each group,the variation of NF-κB(p65)expression was not significant(P>0.05).Conclusion:(1)Qinjiao Dihuang Tongbi Decoction can significantly improve the clinical results in treating RA in active phase tested by RCT,reduce the toxic and side effects of western medicine,and has a synergistic effect.(2)Through the study of network pharmacology,Qinjiao Dihuang Tongbi Decoction may intervene NF-κB signal pathway and regulate downstream target protein to achieve the purpose of treating RA in active phase.The prediction results were preliminarily proved by detecting downstream target proteins(cytokines).(3)CIA rats models of RA werebuilded and animal experiments were carried out in vivo.It was confirmed that Qinjiao Dihuang Tongbi Decoctioncan achieve the purpose of treating RA in active phase by regulating NF-κB signal pathwaythrough gross observation,histopathology and molecular biology methods. |
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