| Rheumatoid arthritis(RA)is a systemic autoimmune disease that can lead to joint inflammation and progressive joint destruction.RA patients have a higher mortality rate compared with the general population,and evidence suggests that this may be largely attributable to an increased incidence of Cardiovascular disease(CVD)in RA.Activity of RA can release a lot of promoting inflammatory cell factor,causing blood fat metabolic disorder,chronic inflammation in RA patients with dyslipidemia,atherosclerosis(AS)the key factor for increased risk,increased incidence of CVD.Studies have shown that IL-1,IL-6,Tumor necrosis factor-α(TNF-α),etc.can affect the activity of lipoprotein lipase and abnormal its expression,and accelerate the progress of lipid metabolism disorders and AS.In this study,the blood lipid levels of RA patients were measured,and the relationship between blood lipid levels and inflammatory indicators such as Erythrocyte sedimentation rate(ESR),C-reactive proteinhs(CRP),Platelets(PLT),proinflammatory cytokines(IL-1,IL-6,TNF-α)were analyzed.It is suggested that patients with RA have abnormal lipid metabolism,and there is a correlation between inflammatory indicators,pro-inflammatory cytokines and lipid levels in patients with RA.Active control of RA disease activity may reduce the risk of arteriosclerosis cardiovascular disease(ASCVD)in patients with RA and improve the long-term prognosis.In addition,Kruppel-like factor7(KLF7)is a member of the KLF family and plays an important role in a variety of biological processes.However,its exact role in RA remains unclear.The aim of this study is to investigate the role of KLF7 in Fibroblast-like synoviocytes(FLSs).Some studies have shown that KLF7 expression is significantly up-regulated in the synovial tissue of rats with Adjuvant-induced arthritis(AIA).Some researchers have found that KLF7 knockdown may inhibit the phosphorylation of NF-κB,p65and JNK,Inhibition of cell proliferation and down-regulating the expression of pro-inflammatory factors Interleukin(IL)-28,IL-1,IL-17A and matrix Metalloproteinases(MMP)-1,MMP3,MMP13.This study confirmed that miR-9a-5p binds to KLF7 3’UTR and negatively regulates KLF7 expression in TNF-α-stimulated FLSs.miR-9a-5p is involved in the pathogenesis of RA by targeting KLF7,activating NF-κB and JNK pathways in FLSs stimulated by TNF-α,promoting cell proliferation,causing the release of pro-inflammatory cytokines and the expression of MMP.Part One Characteristics of lipid metabolism in patients with rheum-atoid arthritisObjective:By measuring the blood lipid levels of RA patients and healthy subjects,and analyzing the correlation of blood lipid levels with inflammatory indicators(ESR,CRP,PLT)and cytokines(IL-1,IL-6,TNF-α)in RA patients,the risk of CVD in RA patients was preliminarily explored.To provide a theoretical basis for the management of blood lipids in RA patients and the prevention and treatment of RA complicated with ASCVD.Methods:1.Ninety-nine hospitalized RA patients were selected according to ACR/EULAR2009 classification criteria and scoring system,and 38 healthy people were selected as control group.There was no significant difference in gender,age and body mass index(BMI)between RA patients and normal controls.2.TC,TG,HDL-C and LDL-C were detected by Japan Hitachi Blood biochemical detection analyzer,blood routine was detected by automatic blood cell analyzer,ESR and CRP were detected by Wei’s method and immune turbidimetric method,IL-1,IL-6 and TNF-αwere detected by electric luminescence method.The levels of TC,TG,HDL-C and LDL-C were compared between the RA group and the normal control group,and the correlation between PLT,ESR,CRP,IL-1,IL-6 and TNF-αin the RA group was analyzed.3.SPSS26.0 for Windows statistical software package was used for analysis.When in accordance with normal distribution,the measuremen-t data was expressed as mean±standard deviation(±s),the comparison between the two groups was performed by t or t’test,the comparison between the count data groups was performed by x~2 test,and the correlation analysis was per-formed by linear correlation analysis.The difference was statistically signi-ficant when P<0.05.Results:1.Description of basic informationA total of 99 RA patients(18 males and 81 females)aged from 25 to 85years[mean(55.72±12.02)years],with a disease duration of 1 month to 37years[mean(9.25±5.62)years]and a body mass index of 17.66 to 28.45kg/m2[mean(23.59±2.91)kg/m2].The normal control group consisted of 38 cases(29 females and 9 males),aged from 28 to 78 years,with an average age of(52.16±11.74)years,body mass index of 17.65 to 28.51kg/m2,with an average body mass index of(23.62±2.79)kg/m2.There was no significant difference in age,gender and BMI between the RA group and the normal control group(P>0.05).2.Comparison of serum lipid levels between RA patients and normal controlsCompared with the normal control group,the TG level of RA patients showed an increasing trend,and the HDL-C level showed a decreasing trend,and the difference was statistically significant(P<0.05),suggesting that the lipid metabolism in RA patients was abnormal.There were no significant differences in LDL-C and TC levels between the two groups(P>0.05).3.Correlation analysis of inflammatory indexes,cytokines and blood lipid levels in RA patientsLinear correlation analysis showed that PLT was positively correlated with TC(r=0.262,P<0.05),TG(r=0.283,P<0.05),LDL-C(r=0.319,P<0.05),and negatively correlated with HDL-C(r=-0.370,P<0.05).ESR was posi-tively correlated with TC(r=0.280,P<0.05),TG(r=0.319,P<0.05),LDL-C(r=0.221,P<0.05),and negatively correlated with HDL-C(r=-0.306,P<0.05).CRP was positively correlated with TC(r=0.341,P<0.05),TG(r=0.505,P<0.05),LDL-C(r=0.565,P<0.05),and negatively correlated with HDL-C(r=-0.407,P<0.05).Linear correlation analysis showed that IL-1 was positively correlated with TC(r=0.263,P<0.05)and LDL-C(r=0.328,P<0.05),negatively corre-lated with HDL-C(r=-0.297,P<0.05),and had no significant correlation with TG(P>0.05).IL-6 was positively correlated with TG(r=0.282,P<0.05)and LDL-C(r=0.339,P<0.05),negatively correlated with HDL-C(r=-0.347,P<0.05),and had no significant correlation with TC(P>0.05).TNF-αwas positively correlated with TC(r=0.253,P<0.05),TG(r=0.354,P<0.05),LDL-C(r=0.244,P<0.05),and negatively correlated with HDL-C(r=-0.369,P<0.05).Summary:1.The TG level in RA patients showed an increasing trend,while the HDL-C level showed a decreasing trend,indicating abnormal lipid metabolism in RA patients.2.There was a certain correlation between inflammatory indexes,cytokines and lipid levels in RA patients.Part Two To investigate the mechanism of KLF7 in RA at the animal and cellular levelsObjective:1.To detect the expression of KLF7 in the synovial tissue of AIA rats.2.To explore the pathogenesis of RA by studying the proliferation and inflammatory response of RA-FLSs induced by KLF7.Methods:1.Experimental animals:Twelve male Sprague Dawley(SD)rats,weighing 160-180g,were randomly divided into Control group and AIA group,with 6 rats in each group.After 28 days of feeding,the rats were sacrificed and their blood and synovial tissue of the right ankle joint were preserved.2.The foot swelling of AIA rats was observed,the arthritis score was performed,the swelling volume of the foot was measured,and the synovial tissue of the joint was stained with hematoxylin and eosin.3.Quantitative Real-time PCR(q RT-PCR)and Western blot(WB)were used to detect the expression of KLF7 in rat synovial tissues.4.FLSs were randomly divided into Control group,TNF-αgroup,TNF-α+NC si RNA group,TNF-α+KLF7si RNA-1 group and TNF-α+KLF7-si RNA-2 group.The m RNA and protein levels of KLF7 in each group were detected by q RT-PCR and WB.The proliferation of FLSs in each group was detected by MTT assay.The levels of IL-1β,IL-6 and IL-17A in the supernatant of FLSs were detected by ELISA.The expression levels of MMP-1,MMP-3 and MMP-13 in FLSs were detected by Western blot.5.FLSs were randomly divided into Control group,TNF-αgroup,TNF-α+NC si RNA group and TNF-α+KLF7 si RNA group.The expression levels of p65,p-p65,ERK1/2,p-ERK1/2,JNK,p-JNK,p38 and p-p38 were detected by WB.6.Dual luciferase reporter gene assay was used to detect the correlation between miR-9a-5p and KLF7,and then the rescue test of miR-9a-5p and KLF7 was performed.FLSs were randomly divided into TNF-α+NC inhibitor group,TNF-α+miR-9a-5p inhibitor group,TNF-α+miR-9a-5p inhibitor+NC inhibitor group and TNF-α+miR-9a-5p group inhibitor+KLF7 inhibitor group.The proliferation of FLSs in each group was detected by MTT assay.The expression levels of KLF7,p65,p-p65,JNK,p-JNK and MMP-13 were detected by Western blotting.The levels of IL-6 and IL-17A were measured by ELISA.7.Statistical analysis:Graph Pad Prism 8.0 software was used for statistical analysis,and the data were expressed as mean±standard deviation(SD).T-test,one-way analysis of variance or two-way analysis of variance were used for comparison.The difference was statistically significant when P<0.05.Results:1.The expression levels of KLF7 m RNA and protein in the synovial tissue of the AIA model group were significantly higher than those of the control group(P<0.01).2.Compared with the control group,the expression levels of KLF7m RNA and protein in TNF-αgroup were significantly increased(P<0.01);Compared with TNF-α+NC-si RNA group,KLF7-si RNA-1 group and KLF7-si RNA-2 group had significantly increased m RNA and protein expression of KLF7(P<0.01).3.Compared with the control group,the proliferation of FLSs in TNF-αgroup and TNF-α+NC-si RNA group was significantly enhanced(P<0.05).Compared with TNF-α+NC-si RNA group,the proliferation of FLSs in KLF7-si RNA-1 and KLF7-si RNA-2 groups was significantly decreased(24 h P<0.05,48 h and 72 h P<0.01).4.Compared with the control group,the levels of IL-1β,IL-6 and IL-17A in TNF-αgroup were significantly increased(P<0.01);Compared with TNF-α+NC-si RNA group,the levels of IL-1β,IL-6 and IL-17A in KLF7-si RNA-1 and KLF7-si RNA-2 groups were significantly decreased(P<0.05 or P<0.01).Compared with the control group,the protein expression levels of MMP-1,MMP-3 and MMP-13 in the TNF-αgroup were significantly increased(P<0.01).Compared with TNF-α+NC si RNA group,the protein expression levels of MMP-1,MMP-3 and MMP-13 in TNF-α+KLF7 si RNA-1and TNF-α+KLF7 si RNA-2 groups were significantly decreased(P<0.01).5.Compared with the control group,the protein expression levels of p-p65,p-ERK1/2,p-JNK and p-p38 in TNF-αgroup were significantly increased(P<0.01).Compared with the TNF-α+NC-si RNA group,the protein expression levels of p-p65 and p-JNK in the TNF-α+KLF7 si RNA group were significantly decreased(P<0.01).6.Dual luciferase reporter gene assay confirmed the targeted regulatory relationship between miR-9a-5p and KLF7;Compared with the NC mimic group,the protein expression level of KLF7 in the miR-9a-5p mimic group was significantly decreased(P<0.01).Compared with the NC inhibitor group,the protein expression level of KLF7 in the miR-9a-5p mimic group was significantly increased(P<0.01).Compared with the TNF-α+NC inhibitor group,the proliferation of FLSs in the TNF-α+miR-9a-5p inhibitor group was significantly enhanced(P<0.05 or P<0.01).Compared with the TNF-α+miR-9a-5p inhibitor+NC inhibitor group,the proliferation of FLSs in the TNF-α+miR-9a-5p inhibitor+KLF7 inhibitor group was significantly enhanced(P<0.05 or P<0.01).Compared with the TNF-α+NC inhibitor group,the expres-sion of p-p65,p-JNK,IL-6,IL-17A and MMP-13 in the TNF-α+miR-9a-5p inhibitor group was significantly increased(P<0.01).Compared with TNF-α+miR-9a-5p inhibitor+NC inhibitor group,the expressions of p-p65,p-JNK,IL-6,IL-17A and MMP-13 in TNF-α+miR-9a-5p inhibitor+KLF7inhibitor group were significantly increased(P<0.05 or P<0.01).Conclusions:1.High expression of KLF7 in synovial tissues and TNF-α-stimulated FLSs of AIA rats.2.KLF7 induced TNF-αstimulated FLSs proliferation and promoted synovial hyperplasia.3.Knockdown of KLF7 reduced the release of inflammatory factors and the expression of MMPs in FLSs stimulated by TNF-α.4.KLF7 caused the activation of NF-κB and JNK pathways,leading to the inflammatory response of FLSs stimulated by TNF-α.5.MiR-9a-5p regulates the proliferation and inflammatory response of FLSs by targeting KLF7.Conclusions:1.There are abnormal lipid metabolism in RA patients2.Inflammatory indicators and pro-inflammatory cytokines in RA patients have a certain correlation with lipid levels,and active improvement of RA disease activities may improve the lipid metabolism disorder of RA.3.KLF7 causes the activation of NF-κB and JNK pathways,leading to cell proliferation and inflammatory response in RA-FLSs.4.MiR-9a-5p regulated the proliferation and inflammatory response of FLSs stimulated by TNF-αby targeting KLF7. |
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