基于lncRNA/miRNA/NF-κB通路探讨针刀对膝骨关节炎兔的软骨保护作用机制

Protective effects of acupotomy on the cartilage in rabbits with knee osteoarthritis via modulation of the lncRNA/miR NA/NF-κB pathway

目的观察针刀对膝骨关节炎(KOA)兔膝关节软骨长链非编码RNA(lncRNA)/微小RNA(miRNA)/核因子κB(NF-κB)通路的影响,探讨针刀对KOA的软骨保护作用机制。方法将新西兰兔随机分为空白对照组、模型组与针刀组,每组9只。采用Videman固定法建立KOA兔模型,造模后针刀组给予针刀干预,1次/周,连续3周。3周后各组取材膝关节软骨组织,运用HE染色观察软骨组织的病理学改变,扫描电镜观察软骨组织损伤情况。使用RT-qPCR法分析lncRNA小核仁RNA宿主基因1(lncRNA-SNHG1)、转化生长因子β激活的lncRNA(lncRNA-ATB)、miR-16-5p、miR-223-3p及环氧化酶2(COX-2)、诱导型一氧化氮合酶(iNOS)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、NF-κB p65、核因子抑制蛋白κB(IκBα)mRNA的表达;Western blotting分析NF-κB p65、磷酸化NF-κB p65(p-NF-κB p65)、IκBα、磷酸化IκBα(p-IκBα)、COX-2、iNOS的蛋白表达;ELISA法测定IL-6、TNF-α的蛋白表达量。对lncRNA-SNHG1相对表达量与miR-16-5p相对表达量,lncRNA-ATB相对表达量与miR-223-3p相对表达量分别作相关性分析。结果光学显微镜与扫描电镜观察表明,KOA兔软骨组织有明显损伤,针刀干预后损伤有一定程度的修复。与空白对照组比较,模型组膝关节软骨组织NF-κB p65、IκBα、COX-2、iNOS、IL-6、TNF-α的mRNA表达水平及lncRNA-ATB、miR-16-5p表达水平升高(P<0.01),lncRNA-SNHG1、miR-223-3p表达水平降低(P<0.01);模型组p-NF-κB p65/NF-κB p65、p-IκBα/IκBα值及COX-2、iNOS、IL-6、TNF-α蛋白表达水平升高(P<0.01)。与模型组比较,针刀组兔膝关节软骨组织IκBα、COX-2、iNOS、IL-6、NF-κB p65、TNF-αmRNA及lncRNA-ATB、miR-16-5p表达水平降低(P<0.05或P<0.01),lncRNA-SNHG1、miR-223-3p表达水平升高(P<0.01);针刀组p-NF-κB p65/NF-κB p65、p-IκBα/IκBα值及COX-2、iNOS、IL-6、TNF-α的蛋白表达水平降低(P<0.01)。lncRNA-SNHG1相对表达量与miR-16-5p相对表达量、lncRNA-ATB相对表达量与miR-223-3p相对表达量负相关(P<0.01)。结论针刀疗法保护KOA兔膝关节软骨的作用机制可能与lncRNA-SNHG1/miR-16-5p、lncRNATB/miR-223-3p通路共同影响NF-κB信号通路有关。

Objective To observe the effect of acupotomy on the lncRNA/miRNA/NF-κB pathway of knee cartilage in rabbits with knee osteoarthritis(KOA)and explore the mechanism of the cartilage-protective effect of acupotomy on KOA.Methods New Zealand rabbits were randomly divided into control,model,and acupotomy groups,with nine rabbits in each group.The rabbit model of KOA was established by the Videman fixation method,and the acupotomy group was given acupotomy intervention once weekly for 3 weeks after modeling.Cartilage tissue of the knee joint was sampled,pathological changes in cartilage tissue were observed using hematoxylin and eosin(HE)staining,and cartilage tissue damage was observed by scanning electron microscopy.RT-qPCR was used to determine the expressions of long non-coding RNA-small nucleolar RNA host gene 1(lncRNA-SNHG1),long non-coding RNA-activated by transforming growth factorβ(lncRNA-ATB),miR-16-5p and miR-223-3p,and mRNA expressions of cyclooxygenase 2(COX-2),inducible nitric oxide synthase(iNOS),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),nuclear factorκB p65(NF-κB p65)and nuclear factor inhibitor proteinκB(IκBα);immunoblotting(WB)analysis was used for the protein expressions of NF-κB p65,phosphorylated NF-κB p65(p-NF-κB p65),IκBα,phosphorylated IκBα(p-IκBα),COX-2,and iNOS.Moreover,protein expressions of IL-6 and TNF-αwere determined using ELISA.Correlation analysis was done for lncRNA-SNHG1/miR-16-5p and lncRNA-ATB/miR-223-3p.Results Pathological and scanning electron microscopic examination showed that there was significant damage to the cartilage tissue in KOA rabbits.Compared with the control group,the mRNA expression levels of NF-κB p65,IκBα,COX-2,iNOS,IL-6,and TNF-αas well as the expression levels of lncRNA-ATB and miR-16-5p were higher(P<0.01).The lncRNA-SNHG1 and miR-223-3p expression levels were lower in the knee cartilage tissue of the model group(P<0.01).The protein expression levels of p-NF-κB p65/NF-κB p65,p-IκBα/IκBα,COX-2,iNOS,IL-6,and TNF-αwere higher in the model group(P<0.01).Compared with the model group,the mRNA expression levels of IκBα,COX-2,iNOS,IL-6(P<0.01),TNF-α,NF-κB p65(P<0.05),lncRNA-ATB,and miR-16-5p expression levels were lower(P<0.01).Moreover,lncRNA-SNHG1,miR-223-3p expression levels were higher(P<0.01)in the knee cartilage tissue of the acupotomy group.The protein expression levels of p-NF-κB p65/NF-κB p65,p-IκBα/IκBα,COX-2,iNOS,and IL-6,TNF-αwere lower in the acupotomy group(P<0.01).Negative correlations of expression levels were observed between lncRNA-SNHG1 and miR-16-5p and between lncRNA-ATB and miR-223-3p(P<0.01).Conclusion The effect of acupotomy therapy to protect knee cartilage in KOA rabbits may be related to the regulation of lncRNA-SNHG1/miR-16-5p and lncRNA-ATB/miR-223-3p pathways,which jointly inhibit the NF-κB signaling pathway.