Artemisinin Relieves Osteoarthritis By Activating Autophagy Through Reducing TNFSF11 Expression And Inhibiting PI3K/AKT/mTOR Signaling In Cartilage

Objective: To analyze the regulatory relationship between Artemisinin(AT)and tumor necrosis factor(ligand)superfamily member 11(TNFSF11),and to explore the targeting of artemisinin to TNFSF11.The effects of artemisinin on important regulatory factors in cartilage degeneration and PI3K/AKT/mTOR signaling pathway verify the protective effect of artemisinin on Osteoarthritis(OA).Methods: Gene sequencing and bioinformatics analysis were used to explore the targeted regulation of AT on TNFSF11 and PI3K/AKT/mTOR signaling pathway.Realtime fluorescent quantitative polymerase chain reaction(qRT-PCR)and Western blot were used to detect the expression of TNFSF11 in OA chondrocytes.TNFSF11 overexpression group was established by plasmid transfection into OA chondrocytes,and the effect of plasmid transfection was detected by qRT-PCR,Western blot and immunofluorescence staining.PI3K/AKT/mTOR signaling pathway activator MHY1485 was used to study the relationship between AT and PI3K/AKT/mTOR signaling pathway.Western blot,qRT-PCR and immunofluorescence staining were used to evaluate the mRNA of TNFSF11 and PI3K/AKT/ mTOR signaling pathway in cartilage of each group in mRNA and protein expression levels.Western blot and qRTPCR were used to evaluate the expression levels of autophagy-related mRNA and protein in each group.Destabilization of medial meniscus(DMM)rat OA model was established.HE staining,Safranin O-fast green staining and immunohistochemical staining were used to verify the effect of AT in rats.Results: CCK-8 assay showed that 20μM was the optimal concentration of AT in the range of 0-20μM.Western blot and qRT-PCR showed that AT significantly inhibited the inflammatory cytokines IL-1β,IL-6 and TNF-α(p<0.05)and MMP3 and MMP13(p<0.01),but had no significant effect on ADAMTS5 mRNA level.The results of differentially expressed genes analysis showed that AT treatment effectively downregulated the gene expression of TNFSF11 and inhibited the PI3K-AKT-mTOR pathway.The results of immunofluorescence staining,qPCR and immunohistochemistry confirmed that TNFSF11 was up-regulated in IL-1β-induced OA and down-regulated after AT treatment(p<0.01).The results of Western blot,qRTPCR and immunofluorescence staining showed that AT could promote the expression of ATG-5,ATG-7,beclin-1 and LC-3(p< 0.05).Western blot and qRT-PCR results showed that overexpression of TNFSF11 by plasmid transfection promoted the phosphorylation of substrates of PI3K/AKT/mTOR signaling pathway,promoted the expression of inflammatory mediators,and inhibited the expression of autophagyrelated proteins(p<0.05),and immunofluorescence showed similar results.The results of HE and Safranin-O-fast green staining showed that AT could alleviate the degeneration of knee joint in rat OA model.Conclusion:1.AT could alleviate IL-1β-induced chondrocyte damage and joint degeneration in rat models with DMM.2.TNFSF11 is highly expressed in IL-1β-induced chondrocytes and OA models of DMM rats,and it can activate PI3K/AKT/mTOR signaling pathway,which can inhibit chondrocyte autophagy,promote inflammatory response and degradation of cartilage matrix.3.By inhibiting TNFSF11 gene expression and PI3K/AKT/mTOR signaling pathway,AT plays a protective role in promoting chondrocytes autophagy,inhibiting inflammatory response and preventing cartilage matrix degradation,and is expected to become a potential therapeutic drug for OA.