| 1 ObjectiveWe investigated the impacts of jianpi qingre tongluo prescription(JQP)on the circ0001566/NF-κB/VEGF,combination on self-perception of patient(SPP)and immune inflammation in rheumatoid arthritis(RA)patients with damp-heat obstruction syndrome.2 Method2.1 Study 1:Based on retrospective data mining,the study on the change of SPP in RA patients with damp-heat obstruction syndrome and the intervention of Jianpi Qingre Tongluo PrescriptionThe method of self-control test was used to collect the peripheral blood mononuclear cells(PBMCs)of patients with RA with damp-heat obstruction syndrome and healthy people before and after treatment with Jianpi Qingre Tongluo Prescription.Collect the SPP scale and laboratory indexes of RA patients with damp-heat obstruction syndrome;Real-time quantitative PCR(RT-qPCR)was used to detect the expression of circ0001566,and enzyme-linked immunosorbent assay(ELISA)was used to detect interleukin-6(IL-6),vascular endothelial growth factor(VEGF),interleukin-10(IL-10).Correlation analysis,association rule analysis and logistic regression were used to analyze the correlation between SPP and circ0001566,immune inflammation and VEGF;The receiver operating characteristic curve(ROC)was used to analyze the potential relationship between circ0001566 and RA;Association rules were used to analyze the correlation between JQP and SPP,circ0001566,immune inflammation,and the improvement of VEGF.2.2 Study 2:Based on over expression study on the effect of Jianpi Qingre Tongluo Prescription containing serum on the inflammatory response of RA fibroblast-like synovial cells by circ0001566/NF-κB/VEGF combinationPrepare the medicated serum of jianpi qingre tongluo prescription(JQP),esTab.lish the co-culture cell model of RA-PBMCs and RA fibroblast-like synovial cells(FLS),construct pcDNA3.1-circ0001566/si-circ0001566 and transfect it into RA-FLSs;Cell counting kit 8(CCK-8)was used to detect cell viability and screen the best concentration of drug-containing serum;The expression of circ0001566 was detected by RT-qPCR;Detection the expression of IL-1β、IL-4、IL-6 and IL-10 by ELISA;Detection the expression of IKBα、P65 and phosphorylation(p)-IKBα、p-P65 protein and VEGF by western blot(WB).Through the recovery experiment,on the basis of the intervention of JQP drug-containing serum,the overexpression plasmid circ0001566 was transfected to observe the changes of inflammation indexes,NF-κB protein expression and VEGF.3 Results3.1 Study 1:Based on retrospective data mining,the study on the change of the self-perception of RA patients with damp-heat obstruction syndrome and the intervention of Jianpi Qingre Tongluo Prescription(1)The changes of SPP,immune inflammation index and VEGF in RA patients with damp-heat obstruction syndrome:the SPP indexes of RA patients with damp-heat obstruction syndrome:the score of damp-heat obstruction syndrome,SAS,SDS,VAS,DAS28,disease activity score increased significantly,and the score of BP,SF,PF,RP,GH,VT,MH decreased abnormally;Immune inflammatory indexes:RF,ESR,CRP,anti-CCP were significantly higher than the normal value,and the expression of VEGF was also significantly higher.IgA,IgG,IgM,C3,C4 were not significantly abnormal.(2)ROC curve analysis of circ0001566 in RA with damp-heat obstruction syndrome:the expression of circ0001566 in PBMCs of RA with damp-heat obstruction syndrome patients was significantly up-regulated(P<0.05);ROC curve results showed that AUC=80.03%,P<0.001,95%CI:0.69-0.92,sensitivity=88%,specificity=70%,cut off value=1.78,circ0001566 can be utilized as a molecular marker to help in RA diagnosis,according to this information.(3)Changes of cytokines in RA with damp-heat obstruction syndrome:pro-inflammatory factor IL-6 expression rose whereas pro-inflammatory factor IL-10 expression dropped(P<0.05).(4)Correlation analysis of SPP and circ0001566,immune inflammation indexes,cytokines and VEGF in RA with damp-heat obstruction syndrome:SPP has significant correlation with immune inflammation index:through correlation analysis,circ0001566 has positive correlation with RF and VEGF(P<0.05);There was significant correlation between SPP and immune inflammation indexes in RA patients with damp-heat obstruction syndrome:SAS was positively correlated with circ0001566 and RF(P<0.05);SDS was positively correlated with ESR and IL-6(P<0.05);The score of damp-heat obstruction syndrome was positively correlated with circ0001566,RF and VEGF(P<0.05);Disease activity was positively correlated with RF(P<0.05);VAS was positively correlated with circ0001566 and RF(P<0.05);BP was negatively correlated with CRP(P<0.05);PF was negatively correlated with ESR(P<0.05);MH was negatively correlated with circ0001566 and C4(P<0.05);GH was negatively correlated with circ0001566 and RF(P<0.05).(5)Analysis of association rules between SPP and circ0001566,immune inflammation indexes,cytokine and VEGF of RA with damp-heat obstruction syndrome:the results of association rule analysis showed a significant connection between the rise in DAS28 value and the rise in ESR and IL-6,a strong correlation between the rise in VEGF and the decline in RE,a strong correlation between the rise in SDS value and the rise in RF,circ0001566,VEGF,and CRP,and a strong correlation between the rise in VAS value and the rise in VEGF,the increase of damp-heat obstruction syndrome integral was strongly correlated with anti-CCP,VEGF The increase of IL-6 is strongly correlated,with support greater than 60%,rule support greater than or equal to 50%,confidence greater than 70%,and improvement greater than 1.(6)Logistic regression analysis of SPP and circ0001566,immune inflammation indexes,cytokines and VEGF in RA with damp-heat obstruction syndrome:SAS is associated with IL-10,IgG,SDS and RF,BP and anti-CCP,SF and CRP(P<0.05),so IL-10,IgG,RF,anti-CCP and CRP are risk factors of SPP.(7)Effects of jianpi qingre tongluo prescription on SPP,immune inflammation indexes and VEGF of RA with damp-heat obstruction syndrome:Compared with RA with damp-heat obstruction syndrome patients before treatment,after treatment,SPP index of Jianpi Qingre Tongluo Prescription:damp-heat obstruction syndrome score,SAS,SDS,VAS,DAS28,disease activity score significantly decreased(P<0.05),RE,BP,SF,PF,RP,GH,VT,MH score increased(P<0.05);The indexes of immune inflammation:ESR,CRP,RF,IgA,IgG,IgM,C3,C4 decreased(P<0.05).(8)Effects of jianpi qingre tongluo prescription on circ0001566 and cytokines in RA with damp-heat obstruction syndrome:Compared with RA with damp-heat obstruction syndrome patients before treatment,the expression of circ0001566 in patients after treatment with Jianpi Qingre Tongluo Prescription was significantly lower(P<0.05);IL-6 expression was dramatically downregulated,while IL-10 expression was significantly upregulated(P<0.05).(9)Analysis of the association rules between jianpi qingre tongluo prescription and the improvement of RA with damp-heat obstruction syndrome SPP,circ0001566,immune inflammation indexes,cytokines,VEGF:JQP has strong correlation with the improvement of RF,ESR,anti-CCP,CRP,IL-6,C3,IgG and other immune inflammation indexes,and JQP has strong correlation with the improvement of damp-heat obstruction syndrome integral,SAS,DAS28,VAS;The above support is greater than 70%,the confidence is greater than 70%,the rule support is greater than 50%,and the improvement is greater than 1(P<0.05).3.2 Study 2:Based on small interference/over expression study on the effect of Jianpi Qingre Tongluo Prescription containing serum on the inflammatory response of RA fibroblast-like synovial cells by circ0001566/NF-KB/VEGF combination(1)The ideal co-culture model for RA-PBMCs and RA-FLSs is 3:1,and the ideal culture period is 48 hours.(2)20%of the serum containing Jianpi Qingre Tongluo Prescription is the best concentration and the best action time is 48 hours.(3)Effects of RA-PBMCs on RA-FLSs:compared with normal group,the expression of circ0001566,IL-1β,IL-6 and VEGF in RA-FLSs increased significantly(P<0.05),while the expression of IL-4 and IL-10 decreased significantly(P<0.001).Compared with RA-FLSs,after stimulation of RA-PBMCs,the expression of circ0001566,IL-1β,IL-6 and VEGF in RA-FLSs increased significantly(P<0.01),while the expression of IL-4 and IL-10 decreased significantly(P<0.001).(4)Effects of circ0001566 on RA-FLSs cell viability,cytokines,VEGF,NF-κB signal pathway:the expression of circ0001566 increased significantly after overexpression of circ0001566,and decreased significantly after interference with circ0001566(P<0.001);Compared with pc-DNA3.1-NC or si-NC group,overexpression of circ0001566 enhanced the cell viability of RA-FLSs(P<0.001),IL-1β,IL-6 was significantly up-regulated,IL-4 and IL-10 were significantly down-regulated(P<0.001);The expression of p-IKBα p-P65 protein and VEGF increased(P<0.001);The interference of circ0001566 reduced the cell viability of RA-FLSs(P<0.001),IL-1β,IL-6 was significantly down-regulated,IL-4 and IL-10 were significantly up-regulated(P<0.001),The expression of p-IKBα,p-P65 protein and VEGF decreased(P<0.001).(5)Effects of NF-κB on RA-FLSs cell viability,cytokines and VEGF:After adding NF-κB signal pathway activator(LPS),the expression of p-IKBα,p-P65 was significantly increased(P<0.001),the cell viability of RA-FLSs was significantly increased(P<0.001).The expression of IL-1β and IL-6 grown massively(P<0.01),the expression of IL-4 and IL-10 decreased significantly(P<0.001),and the expression of VEGF also grown massively(P<0.001).Add LPS after silent circ0001566,the activation of NF-κB signal pathway can partially reverse the decrease of cell viability,the increase of IL-4 and IL-10 expression and the reduce of IL-1β and IL-6 by interference with circ0001566(P<0.01).(6)Effects of VEGF on RA-FLSs cell viability,cytokines,NF-κB signal pathway:compared with si-VEGF-NC group,interference with VEGF significantly reduced the cell viability of RA-FLSs(P<0.001),IL-1β,IL-6 was significantly down-regulated,IL-4 and IL-10 were significantly up-regulated(P<0.001),there was no significant change in the expression of p-IKBα and p-P65(P>0.05).The interference of VEGF after overexpression of circ0001566 can partially reverse the effect of overexpression of circ0001566 on cytokines and cell viability(P<0.001).(7)Effects of jianpi qingre tongluo prescription drug-containing serum on circ0001566,NF-κB signal pathway,VEGF and cytokines:compared with the normal control group,JQP drug-containing serum significantly reduced the expression of circ0001566 in RA-FLSs(P<0.001).After overexpression of circ0001566,adding JQP drug-containing serum can reduce the expression of circ0001566(P<0.001).CCK8 results showed that compared with the normal control group,JQP drug-containing serum significantly reduced the activity of RA-FLSs cells,and after overexpression of circ0001566,the activity of RA-FLSs cells increased,Adding JQP drug-containing serum can reverse some of the increased cell viability(P<0.05).Compared with the normal control group,JQP drug-containing serum significantly reduced the expression of IL-1β and IL-6,IL-4 and IL-10 were increased(P<0.001);However,after overexpression of circ0001566 and addition of JQP drug-containing serum,the expression of IL-6 and IL-1β decreased,and the expression of IL-4 and IL-10 increased(P<0.05).JQP medicated serum reduces the expression of p-P65,p-IKBα protein and VEGF in RA-PBMCs+RA-FLSs co-cultured cells(P<0.01);Overexpression of circ0001566,when JQP drug-containing serum was added,p-P65,p-IKBα protein,and VEGF expression were all reduced(P<0.05).4 Conclusion(1)RA with damp-heat obstruction syndrome is accompanied by poor SPP,which is manifested by the increase of damp-heat obstruction syndrome score,SAS,SDS,VAS,DAS28 score,the decrease of BP,SF,PF,RP,GH,VT,MH score,the up-regulation of circ0001566 expression,the increase of immune inflammation index,and the abnormality of VEGF.The SPP were significantly correlated with circ0001566,immune inflammation index and VEGF.(2)Jianpi qingre tongluo prescription can improve the expression of circ0001566,immune inflammation,cytokines and VEGF in RA with damp-heat obstruction syndrome,and also significantly improve SPP.(3)Jianpi qingre tongluo prescription can reduce the expression of circ0001566 and inhibit NF-κB signal pathway activation,regulate the level of VEGF,reduce inflammatory reaction,and improve SPP of RA patients with damp-heat obstruction syndrome. |
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