基于miRNA-30b-5p/TLR4/NF-κB轴探究针灸改善大鼠膝骨关节炎损伤的实验研究

Experimental study on improvement of knee osteoarthritis injury in rats by acupuncture based on miRNA-30b-5p/TLR4/NF-κB axis

目的:探究基于miRNA-30b-5p/TLR4/NF-κB轴针灸治疗膝骨关节炎(KOA)损伤大鼠的疗效。方法:40只大鼠随机分为5组(n=8):正常组、模型组、单一针灸治疗组、针灸+miRNA-30b-5p-mimics联合治疗组以及针灸+siRNA-TLR4联合治疗组。经过4周的治疗后,采用HE染色观察膝关节软骨组织病理变化;ELISA法检测各组大鼠滑膜液中TNF-α、IL-1β、IL-6和MMP-13的分泌水平;qRT-PCR检测软骨组织中miRNA-30b-5p、TLR4和NF-κB的表达水平;Western blot检测软骨组织中TLR4、NF-κB、IL-6和MMP-13蛋白水平的表达;TUNEL染色检测各组大鼠膝骨关节软骨中软骨细胞凋亡情况;双荧光素酶报告试验检测miRNA-30b-5p与TLR4的靶向关系。结果:与模型组比较,单一针灸及联合治疗显著改善了KOA大鼠软骨组织的病理学变化;显著下调了滑膜液中TNF-α、IL-1β、IL-6和MMP-13的分泌水平(P<0.05,P<0.01);显著抑制软骨组织中TLR4、NF-κB、IL-6和MMP-13的表达水平(P<0.05,P<0.01);同时促进miRNA-30b-5p的表达(P<0.01);显著抑制了膝关节软骨中软骨细胞的凋亡。此外,双荧光素酶报告试验证实TLR4为miRNA-30b-5p的靶基因。结论:针灸能显著改善KOA大鼠膝关节软骨组织的病理学变化,缓解炎症反应,抑制膝骨关节中软骨细胞的凋亡,这可能与其对miRNA-30b-5p/TLR4/NF-κB轴的调控密切相关。

Objective:To investigate the curative effect of acupuncture on knee osteoarthritis injury rats based on miRNA-30b-5p/TLR4/NF-κB axis.Methods:40 rats were randomly divided into 5 groups(n=8):normal group,model group,single acupuncture treatment group,acupuncture+miRNA-30b-5p-mimics combined treatment group and acupuncture+siRNA-TLR4 combined treatment group.After 4 weeks of treatment,HE staining was used to observe the pathological changes of the cartilage tissues in the knee.ELISA was used to detect the secretion levels of TNF-α,IL-1β,IL-6 and MMP-13 in synovial fluid of rats in each group.The mRNA expression of miRNA-30b-5p,TLR4 and NF-κB in cartilage tissues were detected by qRT-PCR.Western blot were used to detect the protein level of TLR4,NF-κB,IL-6 and MMP-13.The apoptosis of chondrocytes in the knee joint of rats in each group were detected by TUNEL staining.The targeted relationship between miRNA-30b-5p and TLR4 was detected by dual-luciferase reporter assay.Results:Compared with the model group,single acupuncture and combined treatment significantly improved the pathological changes of cartilage in KOA rats,significantly reduced the secretion levels of TNF-α,IL-1β,IL-6 and MMP-13 in synovial fluid and expression levels of TLR4,NF-κB,IL-6 and MMP-13 in cartilage tissue(P<0.05,P<0.01),significantly increased the expression of miRNA-30b-5p and inhibited the apoptosis of chondrocytes in knee joint(P<0.01).In addition,the dual-luciferase reporter assay confirmed that TLR4 was the target gene of miRNA-30b-5p.Conclusion:Acupuncture can significantly improve the pathological changes of KOA rats,relieve the inflammatory reaction and inhibit the apoptosis of chondrocytes in knee joint cartilage,which may be closely related to the regulation of miRNA-30b-5p/TLR4/NF-κB axis.