| Objective:To observe the clinical efficacy and safety of Honeysuckle Compound on rheumatoid arthritis(RA)with damp heat and blood stasis syndrome in active stage,and to explore the effect of galuteolin(Galu)on fibroblast-like-cells induced by TNF-α in rheumatoid arthritis.We hope to analyze some potential mechanisms of Honeysuckle Compound in alleviating active RA from a molecular perspective,and to inherit and explore the experience of famous traditional Chinese medicine better,so as to lay a foundation for further exploring the mechanism of action of honeysuckle compound.Method:LClinical trial:60 cases of active rheumatoid arthritis with damp heat and blood stasis syndrome were collected.We chose the method of clinical randomized controlled trials to collect the basic data of patients,four diagnostic information,laboratory examination and other data,Objective To observe the clinical efficacy of Honeysuckle Compound in the treatment of active rheumatoid arthritis with damp heat and blood stasis syndrome.The TCM syndrome score,disease activity score(DAS28 score),rheumatoid factor(RF),erythrocyte sedimentation rate(ESR)and other indicators were mainly observed.According to the TCM syndrome efficacy standard and ACR20/50/70 standard,the overall efficacy of disease treatment can be evaluated.2.Basic experiment:Use TNF-α to stimulate RA-FLS that pretreated with different concentrations of Galu to observe the effect of different concentrations of Galu on the proliferation of RA-FLS cells stimulated by TNF-α,and the optimal concentration of was selected for follow-up experiment;the effects of Galu on the proliferation,apoptosis and inflammation of RA-FLS were observed to explore the possible way of Galu.In the study,CCK-8 colorimetric method was used to detect cell proliferation;TUNEL method was used to detect cell apoptosis;Western blot was used to detect the expression of IKKα,p-p65,p65,P-I κB,I-κ B,cleaved-caspase-3,Caspase-3,Bcl-2 and Bax;qRT-PCR was used to detect HO-1;ELISA was used to detect the content of proinflammatory cytokines IL-1 β,IL-6,IL-8 and MMP1.Results:1.Clinical part:57 patients completed 12 weeks observation in this clinical trial,including 41 female patients and 16 male patients.No statistically significant differences(P>0.05)were found between the two groups regarding gender,age,disease duration,TCM syndrome score before treatment,and baseline of DAS28 before treatment.1.1 in terms of TCM syndrome efficacy,10 cases were in clinical remission in the treatment group,15 cases were in marked effect,2 cases were effective,and 2 cases were ineffective;4 cases were in clinical remission in the control group,9 cases were marked effect,9 cases were effective,and 6 cases were ineffective.Because the class of effect is a hierarchical variable,the rank sum test was used to analyze the difference in efficacy between the two groups,and we found that the average rank order of the treatment group was 34.98,while that of the control group was 22.80,with higher rank order indicating better efficacy,z=-2.918,P=0.004<0.05,that is,there was a difference in efficacy between the two groups,and the treatment group had better efficacy.1.2 ACR20/50/70 in terms of efficacy,16 patients fulfilled ACR20 criteria in the treatment group,7 patients fulfilled acr50 criteria and 1 patient fulfilled acr70 criteria,12 patients fulfilled ACR20 criteria in the control group,9 patients fulfilled acr50 criteria and no patient fulfilled acr70 criteria.Because ACR efficacy was a hierarchical variable,the rank sum test was used to analyze the difference in ACR efficacy between the two groups,which showed that the mean rank order was 29.41 in the treatment group and 28.57 in the control group,z=-0.208,P=0.835>0.05,that is,there was no difference in ACR efficacy between the two groups.1.3 aspects of TCM syndrome scores,after 12 weeks of treatment,both the treatment group and the control group can significantly reduce the TCM syndrome scores in patients,and the efficacy of the treatment group is better than that of the control group.We subsequently further compared the individual scores of each symptom and found that both the treatment and control group could significantly reduce the joint pain,joint swelling,joint tenderness and morning stiffness scores of the patients,and the treatment group could also significantly reduce the flexion extension disadvantage,joint fever,pain night and tongue bleak syndrome scores.The efficacy in the treatment group was significantly better than that in the control group in terms of joint fever,night pain,and bleak tongue,whereas the difference in efficacy between the two groups did not reach statistical significance in the five points of joint pain,joint swelling,joint tenderness,unfavorable flexion and extension,and morning stiffness.1.4 after 12 weeks of treatment,DAS28 scores of patients were significantly reduced in both treatment and control groups,but there was no statistical difference between the two groups,i.e.,efficacy was comparable in both groups in reducing DAS28 scores.1.5 in terms of ESR/RF,both the treatment group and the control group could significantly reduce the ESR,RF of the patients,the efficacy of the treatment group was superior to the control group in terms of reducing the ESR level,and there was no statistical difference between the two groups in terms of improving the RF.2.Experimental section2.1 Galu significantly inhibited the proliferation of RA-FLS cells in a dose-dependent manner.2.2 Galu can upregulate the expression levels of HO-1,cleaved-caspase-3 and Bax anddecrease the expression level of Bcl-2,which significantly promotes the apoptosis rate of RA-FLS cells.Moreover,HO-1 may be involved in the pro apoptotic process of Galu and is a downstream target in Galu induced apoptosis in RA-FLS cells.2.3 Galu also significantly reduced the levels of pro-inflammatory factors IL-1β,IL-6,IL-8,and MMP1 in RA-FLS cells and had a favorable inhibitory effect on TNF-a-induced inflammation in RA-FLS cells;moreover,HO-1 may be a downstream target of Galu in suppressing inflammation in RA-FLS cells.2.4 Galu significantly suppressed the levels of IKKβ,p-p65 and P-IκB,while silencing HO-1 upregulated the expression of the above proteins in RA-FLS.The results suggest that Galu may inhibit the IKKβ/NF-κB pathway by activating HO-1 to suppress TNF-α-induced proliferation and inflammation of RA-FLS cells.Conclusions:1.Combined use of Honeysuckle Compound can better alleviate the clinical symptoms of patients with RA damp heat and blood stasis Syndrome in the active phase,reduce the levels of related inflammatory indicators,and improve the degree of alleviation of the syndrome in traditional Chinese medicine.2.Galu,a monomer of the important drug of honeysuckle compound,may inhibit synoviocyte proliferation and inflammation and promote synoviocyte apoptosis in RA by activating HO-1 expression and inhibiting IKKβ/NF-κB signaling pathway. |
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