Effect And Mechanism Of Artesunate On Osteoarthritis

Objective:Artesunate(ART)is a semi-synthetic derivative of artemisinin.To explore the effects of ART on osteoarthritis both in vitro and in vivo,and futher to explore the mechanism of the effects,in order to provide new ideas for the treatment of osteoarthritis.Method:1.In vitro cell experiment exploration: To study the effect of ART on mouse osteoarthritis body and its specific mechanism from in vitro cell lines.From osteoarthritis of the latest research progress,the development of early osteoarthritis is the most important process for the cartilage cell loss increase and osteoclast activation increase of subchondral bone reconstruction,so this research mainly from the ART of cartilage inflammation process and the influence of osteoclast formation two aspects to explore the role of osteoarthritis.(1)Part of cartilage: Firstly,the inflammatory environment of chondrocytes in the development of OA was simulated under the action of INTERleukin-1 β(IL-1β)in ATDC5 cells,the safe concentration range of ART for 24,48 and 72 hours was determined by CCK-8 method.Then,different concentrations of ART within the safe range were selected to study its influence on the inflammatory process of chondrocytes and whether it changed the cell apparent morphology.(2)Part of subchondral bone: Firstly,CCK-8 assay was also performed on BMMs(bone marrow derived monocyte)cells to determine the range of safe concentration of ART on BMMs cells.Then,BMMs were induced to differentiate into osteoclasts under the stimulation of cytokines M-CSF(macrophage colony stimulating factor)and RANKL(nuclear factor-κB receptor activating factor ligand).During this process,BMMs were treated with different concentrations of non-cytotoxic ART to explore the effect of BMMs on osteoclast differentiation.2.In vivo animal experiment:Based on the cell phenotype experiment,in vivo animal experiment futher confirmed the effect of ART on osteoarthritis of mouse.Forty 8-week-old C57B6/L mice were divided into 4 groups: Sham(Sham group),V vehicle(control group),Low ART(low-dose drug group)and High ART(high-dose drug group).The latter three groups were operated to induce the instability of the medial meniscus of the right lower extremity knee joint to establish the DMM osteoarthritis model in mice.The first group was sham operation group,and only the joint capsule incision and suture of the right lower extremity knee joint were performed.Drug intervention was given from the next day after surgery.According to the body weight of mice,the low-dose group was intraperitoneally injected with 10 mg/kg ART,the high-dose group was intraperitoneally injected with 50 mg/kg ART,and the sham operation group and control group were intraperitoneally injected with the same amount of normal saline,once every other day.After 4 weeks of drug grouping,6 mice were randomly killed from 10 mice in each group,and complete knee specimens of their right lower limbs were obtained,including femur and tibia at both ends.After paraffin fixed sections,H&E staining,Safranin O staining and osteoclast specific TRAP(tartrate-resistant acid phosphatase)staining were used to observe the degree of cartilage destruction and subchondral bone reconstruction in different groups of mice.To determine the effect of ART on osteoarthritis induced by DMM in mice.3.Explore the mechanism at the protein molecular level: In this part of the study,Weastern Blot will be used to explore the possible pathways and molecular mechanisms of ART inhibiting chondrocyte inflammation and osteoclast formation from the protein expression level.For the signal transduction pathway activated by IL-1β mediated chondrocyte inflammatory response,explore the effects of ART.Similarly,explore the main pathway activated by RANKL-induced osteoclast differentiation,and explore the signal transduction pathway of ART on the activation.Results:1.In vitro cell experiments:(1)CCK-8 results showed that the safe concentration range of ART on ATDC5 cells was 10 ng/ m L IL-1β and 10 μM or less.ART had no cytotoxicity to BMMs at 20 μM and below,which was within the safe range of ART on BMMs.(2)ART inhibited IL-1β mediated chondrocyte inflammation in a concentration-dependent manner.ART inhibited RANKL-induced osteoclast formation in a concentration-dependent manner.2.In vivo animal experiments: ART can reduce cartilage and subchondral bone damage.Compared with the sham operation group,the damage of knee cartilage and subchondral bone was obvious in the control group,while the damage of knee joint was significantly reduced after ART treatment,especially in the high-dose group.3.Related mechanisms: ART inhibits IL-1β-induced MMP-13 synthesis by inhibiting MAPK(P38)signaling pathway,thus reducing cartilage inflammation;ART inhibits RANKL-induced osteoclast formation by inhibiting MAPK(JNK)and PI3K/AKT signaling pathways.Conclusion:ART inhibits IL-1β-mediated MMP-13 synthesis by inhibiting MAPK(P38)pathway,and inhibits RANKL-induced osteoclast formation by inhibiting MAPK(JNK)and PI3K/AKT pathways.In order to alleviate the osteoarthritis induced by DMM model in mice,it can reduce cartilage destruction and inhibit subchondral bone reconstruction.