Study On The Mechanism Of Curcumin Improving Osteoarthritis By Regulating Autophagy Of Chondrocytes Through SIRT3/HIF-1α Signaling Axis

Objective:This study investigated whether curcumin could improve osteoarthritis by regulating chondrocyte autophagy through the Silence Information Regulator 3（SIRT3）/Hypoxia Inducible Factor-1α（HIF-1α）signaling axis.Methods:1.Animal experiment:Male Sprague-Dawley（SD）rats were used for the study.The knee osteoarthritis model was established by injection of the chemical iodoacetic acid into the right knee joint cavity of rats.The rats were random Ly divided into a blank control group,a sham-operated group,an osteoarthritis model group and a curcumin-treated group.The knee joint of the blank control group was left untreated,the sham-operated group was injected with 50μL saline in the joint cavity,and the model and treatment groups were given 3 mg/50μL iodoacetic acid solution in the knee joint cavity.Rats in the treatment group were given 5m L of curcumin solution（150mg/kg）by gavage daily.The model group was given 5m L of sodium carboxymethylcellulose（0.5%）as a solvent by gavage daily.The blank control group and sham-operated group were given the same amount of drinking water by gavage for a total of 2 weeks.Rats were random Ly selected for gait testing before and after modelling and after 2 weeks of curcumin treatment.After completion of treatment,the rats were observed for the general condition of the cartilage in the knee joint,the morphological changes of the cartilage（HE and red-fixed green staining）and the expression of Microtubule-Associated Protein1 Light Chain 3（LC3）,autophagy genes Beclin1 and SIRT3,and HIF-1αprotein.2.Cell experiment:Human chondrocytes C28/I2 were divided into control group,model group and treatment group.The hypoxia model was established by adding Cobalt Chloride（Co Cl₂）;the treatment group was treated with curcumin and Co Cl₂for 24 hours.The inhibitor group was treated with SIRT3 inhibitor for 2 hours followed by curcumin and Co Cl₂for 24 hours.Cell proliferation/toxicity assay（Cell Counting Kit-8,CCK8）and cell proliferation imaging analysis（5-ethynyl-2’-deoxyuridine,5-Ethynyl-2’deoxyuridine,Ed U）were used to detect the proliferation viability of C28/I2 cells.Changes in autophagic vesicles were observed by transmission electron microscopy.Autophagic proteins LC3 and P62 were detected by immunofluorescence and Western blot.SIRT3 and HIF-1αprotein expression were detected by Western blot.The treatment group was then treated with SIRT3 inhibitor for 2 hours first as an inhibitor group and HIF-1αprotein expression was detected by Western blot.Results:1.Animal experiment:The Catwalk analysis showed that the print length,print width and the mean paw to ground pressure indexes of the hind feet of the rats decreased after modelling compared to the control group.The indexes increased after curcumin treatment compared to the model group,and the results were significantly different.In terms of gross morphology,the cartilage surface of the model group was rougher and grayer than that of the control group,with small defects visible locally.the cartilage surface of the curcumin-treated group was relatively unsmooth and the colour was between that of the control group and the model group.Pathological staining revealed a rougher articular surface,reduced cartilage layer thickness,more obvious defects and fissures,and a reduced number of cells with disorganized arrangement in the model group compared to the control group.In the treated group,the histomorphology improved compared to the control group.Osteoarthritis Research Society International（OARSI）scores were significantly higher in the model group than in the control group and lower in the treatment group than in the model group.Autophagy proteins LC3 and Beclin1 were significantly reduced in the model group and more abundant in the treatment group than in the control group.SIRT3 was less expressed in the model group and more expressed in the treatment group than in the control group,and its downstream HIF-1αprotein trend was opposite to that of SIRT3.2.Cell experiment:The proliferative activity of chondrocytes was reduced in the hypoxia model group and higher in the treatment group than in the model group.On the autophagy level,compared with the control group,the number of autophagic vesicles was reduced,LC3 immunofluorescence was weakened and P62 fluorescence was enhanced in the model group;in the treatment group,autophagic vesicles were increased,LC3 immunofluorescence was stronger than in the model group,and P62fluorescence was weaker than in the model group.WB showed that compared with the control group,LC3 and SIRT3 protein expression was reduced and P62 and HIF-1αexpression was increased in the model group;in the treatment group,LC3 In the treatment group,the expression of LC3 and SIRT3 protein was increased compared with the model group,and the expression of P62 and HIF-1αwas decreased compared with the model group.The expression of HIF-1αincreased after adding SIRT3 inhibitor.Conclusion:1.Curcumin improved gait abnormalities in knee osteoarthritis rats,effectively reduced pathological damage to knee cartilage tissue,upregulated SIRT3/HIF-1αsignalling axis protein and alleviated cartilage autophagic damage,thereby delaying articular cartilage degeneration.2.Chondrocytes have reduced proliferative activity and impaired autophagy under hypoxia.Curcumin alleviates the autophagic damage caused by chondrocyte hypoxia,and its mechanism of action may be related to activation of the SIRT3/HIF-1αsignalling axis.