Protective effects of Tripterygium glycoside on IL-1β-induced inflammation and apoptosis of rat chondrocytes via microRNA-216a-5p/TLR4/NF-κB axis

Abstract

Background This study is designed to fill the research gap concerning the efficacy of Tripterygium glycoside (TG) on Interleukin-1β (IL-1β)-induced inflammation and injury in chondrocytes. Methods Chondrocytes were isolated from Sprague–Dawley rats. After the treatment with IL-1β and TG and transfection, the viability and apoptosis of chondrocytes were determined via Cell Counting Kit-8 (CCK-8) assay and flow cytometry. The levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-8 were determined by enzyme-linked immunosorbent assay (ELISA). Relative expression levels of potential microRNAs (miRNAs, miRs) that may target toll-like receptor 4 (TLR4), as well as apoptosis- and TLR4/nuclear factor-κB (TLR4/NF-κB) pathway-associated factors were quantified using quantitative real-time (qRT) PCR and western blot. The targeting relationship between miR-216a-5p and TLR4 was predicted by TargetScan and further confirmed by dual-luciferase reporter assay. Results The viability was reduced yet the apoptosis and inflammation were promoted in IL-1β-treated chondrocytes, where upregulation of Bax, Cleaved caspase 3, TLR4, Myeloid differentiation factor 88 (MyD88), phosphorylation of P65 and IκBα yet downregulation of Bcl-2 and IκBα were evidenced. Strikingly, the above changes were reversed by TG. TG also offset the effects of IL-1β on repressing the expression of miR-216a-5p, the miRNA targeting TLR4. Additionally, TLR4 overexpression neutralized the impacts of TG upon viability, apoptosis, and TLR4 expression in IL-1β-treated chondrocytes, while all these effects induced by TLR4 overexpression could be restored by miR-216a-5p. Conclusions TG protects chondrocytes against IL-1β-induced inflammation and apoptosis via miR-216a-5p/TLR4/NF-κB axis.