| 1 ObjectiveThis study aims to explore the mechanism of the circ-CBLB/mi R-486-5p functional axis in inflammation polarization of rheumatoid arthritis(RA)and abnormal proliferation of rheumatoid arthritis synovial fibroblasts(RA-FLS)through clinical research and in vitro experiments,and investigate the intervention effect of the“nourishing the spleen,resolving dampness,and regulating the meridians”Chinese medicine Xinfeng capsule(XFC)developed based on the theoretical foundation of“Consolidate the source”of Xin’An medicine in the treatment of RA.2 Methods2.1 Clinical researchThe 30 healthy individuals(HC group)were recruited from the Physical Examination Center of AHZYY,and the 60 RA patients were recruited from the First Affiliated Hospital of AZY.They were randomly assigned into two groups:a control group of 30subjects and an observation group of 30 subjects.Immune-inflammatory indicators were measured by relevant instruments,inflammatory cytokines were detected by enzyme-linked immunosorbent assay(ELISA),macrophage markers were detected by flow cytometry(FCM),pathway gene expression levels were measured by real-time fluorescence quantification(RT-q PCR).The disease activity was evaluated by VAS and DAS28 scores,and joint symptoms and spleen deficiency syndrome were assessed by classification and quantitative methods.The potential mechanism of XFC in improving RA was analyzed by protein-protein interaction(PPI)and common target enrichment(GO,KEGG)using network pharmacology.Clinical efficacy was evaluated by ACR20/50/70,and changes in clinical efficacy,immune-inflammatory indicators,cytokines,inflammatory polarization markers,pathway gene levels,disease activity-related indicators,joint symptoms,and spleen deficiency syndrome were observed after XFC treatment.The predictive effect of circ-CBLB and mi R-486-5p on RA disease activity was evaluated by ROC curve.2.2 In vitro researchDual-luciferase targeting validation of the binding relationship between circ-CBLB/mi R-486-5p.Cell viability was detected by CCK8 assay,cell cycle and apoptosis were detected by FCM,cell colony formation ability was detected by plate cloning,relevant gene expression was detected by RT-q PCR,and related cell cytokines were detected by ELISA.Firstly,cell grouping was conducted:(1)control group;(2)model group;(3)overexpression group;(4)overexpression NC group;(5)interference group;(6)interference NC group;(7)hedging against group,to investigate the effects of circ-CBLB/mi R-486-5p functional axis on proliferation and apoptosis of RA-FLS.Then,the best serum intervention concentration of XFC was screened through CCK8 assay in XFC-fed rats,and cell grouping was conducted:(1)control group;(2)model group;(3)XFC group;(4)overexpression NC group;(5)overexpression group;(6)overexpression+XFC group,to explore whether XFC drug-containing serum can affect the proliferation and apoptosis of RA-FLS by regulating the circ-CBLB/mi R-486-5p functional axis.3 Results3.1 Clinical results3.1.1 Changes in immune inflammatory indicators in RA patientsThe RA group had significantly higher levels of immune inflammation than the HC group(P<0.01)and significantly lower levels of anti-inflammatory factors than the HC group(P<0.01).3.1.2 Changes in pathway gene expression in RA patientsCompared with the HC group,the expression level of circ-CBLB in the RA group significantly decreased(P<0.01),and the expression level of mi R-486-5p significantly increased(P<0.01).3.1.3 Changes in macrophage polarization markers expression levels in RA patientsCompared with the HC group,the expression levels of M1 macrophage markers were significantly increased(P<0.01)and the expression levels of M2 were significantly decreased(P<0.01)in the RA group.3.1.4 Correlation between pathway genes,inflammation polarization markers,and immune inflammation in RA patientsThe expression level of circ-CBLB in RA patients was positively correlated with CD14~+CD163~+and negatively correlated with mi R-486-5p and CD14~+CD86~+(P<0.01);the expression level of mi R-486-5p was negatively correlated with CD14~+CD163~+and positively correlated with CD14~+CD86~+(P<0.01);CD14~+CD86~+was negatively correlated with CD14~+CD163~+(P<0.01);ESR was negatively correlated with circ-CBLB and positively correlated with mi R-486-5p,CD14~+CD86~+,and CRP(P<0.01);CRP was negatively correlated with circ-CBLB and CD14~+CD163~+and positively correlated with CD14~+CD86~+and ESR(P<0.05,P<0.01).3.1.5 Correlation between pathway gene expression levels and joint symptoms and spleen deficiency syndrome scores in RA patientsThe expression level of circ-CBLB in RA patients was negatively correlated with joint tenderness,morning stiffness,shortness of breath,and postprandial abdominal distension scores(P<0.05,P<0.01);the expression level of mi R-486-5p was positively correlated with joint tenderness and loss of appetite scores(P<0.05).3.1.6 Relationship between pathway gene expression levels and RA disease activity-related indicators in RA patientsThe low expression level of circ-CBLB and high expression level of mi R-486-5p have certain predictive value for RA disease activity.3.1.7 Intersection targets of XFC and RAThere were 16 kinds of astragalus,28 kinds of Tripterygium wilfordii,5 kinds of Coix seed,and 10 kinds of centipede,with predicted target genes of 290 for astragalus,282for Tripterygium wilfordii,30 for Coix seed,and 35 for centipede.There were 4165 RA disease target genes,and 175 genes were common targets.3.1.8 PPI network construction and screening of inflammation polarization-related targetsA core target network was obtained consisting of 172 nodes and 3788 edges.3.1.9 KEGG and GO enrichment analysis of intersection targets of XFC and RAKEGG and GO enrichment analysis showed that GO BP was enriched in cell response to nitrogen compounds and hormone response,GO MF was enriched in DNA-binding transcription factors and RNA polymerase II,GO CC was enriched in membrane microdomains and plasma membrane rafts,and KEGG pathways mainly involved tumor necrosis factor in the immune system,chemokines,and osteoclast differentiation-related signaling pathways.3.1.10 Comparison of clinical efficacy between the two groups of RA patientsThe observation group had a significantly higher ACR70 improvement rate(66.7%)than the control group(26.7%)(P<0.01).3.1.11 Comparison of changes in immune inflammation-related indicators between the two groupsCompared with before treatment,the levels of ESR,CRP,RF,and Anti-CCP-Ab were all decreased in both groups(P<0.01);after treatment,the observation group had a more significant decrease in ESR,CRP,RF,and Anti-CCP-Ab levels(P<0.01).3.1.12 Comparison of cytokine-related indicators between the two groupsCompared with before treatment,the levels of anti-inflammatory cytokines IL-4 and IL-10 were increased in both groups after treatment(P<0.05,P<0.01),while the levels of pro-inflammatory cytokines IL-6 and TNF-αwere decreased(P<0.01).Compared with the control group,the levels of IL-4 and IL-10 were significantly increased in the observation group(P<0.01),while the levels of IL-6 and TNF-αwere significantly decreased(P<0.05).3.1.13 Comparison of pathway gene-related indicators between the two groupsCompared with before treatment,the expression of mi R-486-5p was significantly decreased(P<0.01),while the expression of circ-CBLB was significantly increased(P<0.01)in both groups after treatment.Compared with the control group,the expression of mi R-486-5p was more significantly decreased and the expression of circ-CBLB was more significantly increased in the observation group(P<0.01).3.1.14 Comparison of macrophage polarization markers between the two groupsCompared with before treatment,the proportion of CD14~+CD86~+expression was significantly decreased(P<0.05,P<0.01),while the proportion of CD14~+CD163~+expression was significantly increased(P<0.01)in both groups after treatment.Compared with the control group,the proportion of CD14~+CD86~+expression was more significantly decreased and the proportion of CD14~+CD163~+expression was more significantly increased in the observation group(P<0.01).3.1.15 Comparison of disease activity-related indicators between the two groupsCompared with before treatment,the VAS and DAS28 scores were significantly decreased in both groups after treatment(P<0.01).Compared with the control group,the observation group showed a more significant reduction in VAS and DAS28 scores after treatment(P<0.01).3.1.16 Comparison of joint symptoms and spleen deficiency syndrome scores between the two groupsCompared with before treatment,the joint symptom scores were significantly reduced in the control group(P<0.01),while there was no significant difference in the spleen deficiency syndrome scores(P>0.05).Both the joint symptom scores and spleen deficiency syndrome scores were significantly reduced in the observation group(P<0.01).Compared with the control group,the observation group showed a more significant reduction in both joint symptom scores and spleen deficiency syndrome scores after treatment(P<0.01).3.2 In vitro study results3.2.1 Dual-Luciferase Reporter AssayThe dual-luciferase reporter assay showed that circ-CBLB has binding sites with the 3’UTR of mi R-486-5p.3.2.2 Effect of circ-CBLB on RA-FLS cell viabilityCompared with the model group at the same time,the overexpression group showed a decrease in cell viability(P<0.01),while the interference group showed an increase(P<0.01).3.2.3 Effect of circ-CBLB on RA-FLS cell apoptosisCompared with the model group,the overexpression group showed an increase in apoptosis rate(P<0.01),while the interference group showed a decrease(P<0.01).3.2.4 Effect of circ-CBLB on RA-FLS cell cycleCompared with the model group,the proportion of cells in S+G2 phase increased in the overexpression group(P<0.01),and decreased in the interference group(P<0.01).3.2.5 Effect of circ-CBLB on RA-FLS colony formation abilityCompared with the model group,the overexpression group showed a decrease in colony formation rate(P<0.01),while the interference group showed an increase(P<0.01).3.2.6 Effect of circ-CBLB on mi R-486-5pCompared with the model group,the overexpression group showed a decrease in mi R-486-5p expression level(P<0.01),while the interference group showed an increase(P<0.01).3.2.7 Effect of circ-CBLB on inflammatory cytokines of cellsCompared with the model group,the expression levels of anti-inflammatory cytokines IL-4 and IL-10 were upregulated(P<0.05,P<0.01)and those of pro-inflammatory cytokines IL-6 and TNF-αwere downregulated(P<0.05,P<0.01)in the overexpression group.The TNF-αlevel was upregulated in the interference group(P<0.01).3.2.8 Determination of the optimal serum concentration of XFCThe optimal serum concentration of XFC for RA-FLS intervention was 20.0%,and the optimal intervention time was 72 hours.3.2.9 Effect of XFC-containing serum on the viability of RA-FLS cells under circ-CBLB overexpressionCompared with the model group at the same time,the cell viability of the XFC group,overexpression group,and overexpression+XFC group decreased(P<0.01).Compared with the overexpression group,the cell viability of the XFC group and overexpression+XFC group also decreased(P<0.01).3.2.10 Effect of XFC-containing serum on the apoptosis of RA-FLS cells under circ-CBLB overexpressionCompared with the model group,the apoptosis rate increased in the XFC group,overexpression group,and overexpression+XFC group(P<0.01).Compared with the overexpression group,the apoptosis rate increased in the control group,XFC group,and overexpression+XFC group,and decreased in the remaining group(P<0.01).3.2.11 Effect of XFC-containing serum on the cell cycle of RA-FLS cells under circ-CBLB overexpressionCompared with the model group,the proportion of cells in S+G2 phase increased in the overexpression group,XFC group,and overexpression+XFC group(P<0.01).Compared with the overexpression group,the proportion of cells in S+G2 phase increased in the XFC group and overexpression+XFC group,and decreased in the remaining group(P<0.01).3.2.12 Effect of XFC containing serum on circ-CBLB and mi R-486-5p under overexpression of circ-CBLBIn terms of circ-CBLB,compared with the model group,the gene levels in the XFC group,overexpression group,and overexpression+XFC group increased(P<0.01);Compared with the overexpression group,the overexpression group showed an increase in gene levels,while the remaining groups showed a decrease(P<0.01);Compared with the overexpression group,the other groups showed a decrease(P<0.01);In terms of mi R-486-5p,compared with the model group,the gene levels in the XFC group,overexpression group,and overexpression+XFC group decreased(P<0.01);Compared with the overexpression group,the gene levels in the control group,administration group,and overexpression administration group decreased,while the other groups all increased(P<0.05,P<0.01).3.2.13 Effect of XFC medicated serum on cellular inflammatory factorsCompared with the model group,the levels of anti-inflammatory factors IL-4 and IL-10in the XFC group,overexpression group,and overexpression+XFC group increased(P<0.05,P<0.01),and the pro-inflammatory factors IL-6 and TNF-αincreased The content decreased(P<0.01);Compared with the overexpression group,there was no statistically significant difference in IL-4,IL-6,and IL-10 levels in the XFC group(P>0.05),with TNF-αThe content decreased(P<0.01);The overexpression group showed an increase in IL-4 and IL-10 levels(P<0.01),as well as IL-6 and TNF levels-αThe content decreased(P<0.05,P<0.01).4 Conclusion4.1 XFC can improve the polarization state of inflammation and spleen deficiency syndrome,and improve clinical efficacy.XFC can balance the polarization state of inflammation in patients with RA,and improve the immune inflammation,disease activity,joint symptoms,and spleen deficiency syndrome,thereby significantly improving the clinical efficacy of RA treatment.It can also regulate the expression of circ-CBLB,mi R-486-5p,and M1/M2macrophage polarization markers.4.2 XFC-containing serum can regulate RA-FLS through circ-CBLB/mi R-486-5p.XFC-containing serum can regulate the circ-CBLB/mi R-486-5p functional axis,increase the expression of circ-CBLB,inhibit the expression of mi R-486-5p,and show better results than circ-CBLB’s positive expression in suppression of RA-FLS cell viability,increasing apoptosis rate,prolonging cell cycle,improving anti-inflammatory factors,and reducing pro-inflammatory factors. |
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