| Part ? Literature reviewThe research progress on the relationship between synovial angiogenesis and lncRNA HOTAIR in RA and the understanding and treatment of RA by "treating arthralgia from collaterals" and the influence of traditional Chinese medicine(TCM)on the prognosis of RA were discussed in detail in this part.Part ? Clinical studyObjective To analyze the prognosis of the patients,investigate the long-term effectiveness of Wenjing Tongluo method and the affecting factors of the prognosis of RA by following up the patients with cold and dampness syndrome of RA treated by Wenjing Tongluo method.Methods 84 patients who had participated in our previous study(2013-ZYLC-010)were followed up.Disease activity,TCM syndrome score and HAQ were evaluated.The diagnosis,treatment,medication,complications and joint exercise situations were also recorded during the follow-up period.Logistic regression was used to analyze the factors related to the prognosis of RA.Results The scores of ESR,RF,TJC,VAS,DAS28,TCM syndrome score and HAQ in the treatment group were lower than those in the control group(P<0.05).There was no significant difference in CRP,SJC and stiffness time between the two groups.There was no significant difference in past medical history,new onset diseases,BMI and bad living habits between the two groups,but the application of oral and external treatment of TCM in the treatment group was more than that in the control group.Regular outpatient service,reexamination and joint exercise situations were better than those in the control group(P<0.05).Logistic regression test showed that the treatment of TCM and joint exercise with Wenjing Tongluo method entered the regression equation(P<0.05),which showed protective significance for the clinical remission of RA and the maintenance of low disease activity.Conclusion Wenjing Tongluo method can improve the compliance of patients with RA and play a positive role in the clinicial outcome of RA.Treatment of TCM and joint exercise represented by Wenjing Tongluo method can improve the long-term prognosis of patients with the cold and dampness syndrome.Part ? Experimental studyExperiment 1.Objective To construct and screen HFLS-RA stably overexpressing HOTAIR and its negative control,and to provide experimental materials for the further study of Wenjing Tongluo method.Methods The overexpression HOTAIR plasmid was constructed by construction,packaging,purification and titer determination of lentivirus vector.HFLS-RA were transfected by MOI and screened by puromycin for 2 weeks.Finally,the expression of HOTAIR in the stable strain was verified by qPCR.Results The titer of the lentivirus was 1.54×108IU/mL.After transfection and screening,the fluorescence rate of EGFP in the stable transformation strains was over 98%under microscope,and the puromycin resistance was obvious.There was no fluorescence expression in the blank group.The results of qPCR showed that the relative expression of HOTAIR in the overexpression group was significantly higher than that in the negative control group and blank group(P<0.05).Conclusion Overexpression of HOTAIR and its negative control stable strains were successfully constructed.Experiment two.Objective To investigate the role of HOTAIR in the pathogenesis of RA and the intervention mechanism of Wenjing Tongluo method on synovial angiogenesis in RA,and to enrich the theoretical basis of Wenjing Tongluo method in the treatment of RA with cold and dampness syndrome.Methods The models of HOTAIR(+)and HOTA1R-NC cells induced by IL-1? were divided into blank group,induction group,TwHF group and modified Shaogan Fuzi decoction(SGFZ)with small,middle and high dose groups.CCK-8 assay was used to detect cell proliferation,qPCR and WB were used to detect the relative expression and protein expression of HIF-la,VEGF,VEGFR-2,Ang-1 and Ang-2 mRNA in the cells.The contents of HIF-la,VEGF,VEGFR-2,Ang-1 and Ang-2 in the supernatant of cell culture were detected by ELISA.Results After induction,the proliferation rate of HOTAIR(+)group was higher than that of HOTAIR-NC group,compared with the induction group,the cells in each dose treatment group showed inhibitory effect on cell proliferation.The effect of high dose group was similar to that of TwHF group though the difference was not significant(P>0.05).The results of qPCR showed that with the increase of HOTAIR level,the expression levels of HIF-1a,VEGFA,VEGFR-2,Ang-1 and Ang-2 mRNA in the induction group were significantly higher than those in the control group(P<0.05),HOTAIR(+)showed more significance than HOTAIR-NC,and the expression of each factor decreased after treatment.There was no significant difference in the expression of angiogenesis related factors mRNA and HOTAIR in HOTAIR-NC cells except Ang-2 in a dose-dependent manner in the treatment group,the expression of angiogenesis factors mRNA and HO TAIR were not significantly different between the two groups(P>0.05)The results of protein expression showed that the expression of HIF-1?,VEGFR-2 and Ang-2 protein in HOTAIR(+)induced group was significantly higher than that in HOTAIR-NC group(P<0.05),but the expression of Ang-2 in HOTAIR-NC was significantly higher than that in induced group(P<0.05).The expression of Ang-2 protein was significantly inhibited in the two high dose groups(P<0.05),and the inhibitory effect of each TCM treatment group on the expression level of Ang-2 protein was more obvious.The result showed a positive correlation between the serum concentration and the depressing of Ang-2(P<0.05).The level of VEGFA in NC group was higher than that in overexpression group(P<0.05),but the levels of VEGFR-2,Ang-1 and Ang-2 induced by IL-1? were lower than those in HOTAIR(+)group,which was more significant in Ang-1 group(P<0.05).After stimulation with IL-1?,the concentrations of HIF-1?,VEGFA,VEGFR-2,Ang-1 and Ang-2 in the supernatant of the induction group were significantly higher than those of the blank group(P<0.05).The levels of each factor in each treatment group were decreased after drug serum intervention(P<0.05),and the curative effect of each TCM intervention group was basically dose-dependent.The higher the dose.,the more obvious the improvement,and the contents of VEGFA,VEGFR-2 and Ang-1 in the supernatant of HOTAIR-NC group were lower than those in overexpression group after stimulation(P<0.05).Conclusion HOTAIR may promote the proliferation of RA synovial fibroblasts and affect the prognosis of RA by promoting the expression of HIF-VEGF-Ang axis-related angiogenesis factors.Modified Shaogan Fuzi decoction,as the representative of Wenjing Tongluo method,may downregulate the expression of intracellular HOTAIR.It can inhibit the proliferation of HIF-VEGF-Ang axis-related angiogenesis factor and RA synovial fibroblasts in order to control the condition of RA and improve the prognosis of the disease,among which the intervention of high-dose drugs is the most significant. |
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