Mechanism of mTORC2 deficiency ameliorating osteoarthritis in mice by regulating pathogenic CD4+T cell ferroptosis
Objective To explore the mechanism by which mTORC2 deficiency improves mouse osteoarthri-tis(OA)by regulating pathogenic CD4+T cell ferroptosis.Methods Sixty mice were randomly divided into con-trol,OA,mTORC2-/-+OA,mTORC2-/-+OA+Erastin(n=16).The right knee OA model was surgically constructed using normal and mTORC2-/-mice.Ferroptosis was induced by intraperitoneal injection of 40 mg/kg Erastin.The lev-els of cartilage degeneration,joint inflammation and ferroptosis were detected,and T-bet and RORγt were detected by immunostaining to evaluate the Th1 and Th 17 levels of pathogenic CD4+T cells.Results The expression level of mTORC2 in knee tissue of OA group(0.69±0.08)was higher than that of control group(0.16±0.03)(t=24.813,P=0.001).The OARSI score of the OA group(4.17±0.59)was significantly higher than that of the control group(1.05±0.13)(t=20.657,P=0.001),and the OARSI score of the mTORC2-/-+OA group(1.82±0.23)was significantly lower than that of the OA group(t=16.844,P=0.001).The OARSI score of mTORC2-/-+OA+Erastin group(3.81±0.43)was significantly higher than that of mTORC2-/-+OA group(t=16.323,P=0.001).Compared with control group,IL-1β(22.75 μg/mL±2.06 μg/mL)and IL-6(31.26 μg/mL±2.97 μg/mL)were increased in OA group(t=31.237,P=0.001;t=30.390,P=0.001);IL-1 β(10.54 μg/mL±1.25 μg/mL)and IL-6(13.56 μg/mL±1.29 μg/mL)in mTORC2-/-+OA group were significantly lower than those in OA group(t=20.269,P=0.001;t=21.865,P=0.001).The levels of IL-1 β(19.86 μg/mL±2.13 μg/mL)and IL-6(27.12 μg/mL±3.04 μg/mL)in mTORC2-/-+OA+Erastin group were significantly higher than those in mTORC2-/-+OA group(t=15.095,P=0.001;t=16.425,P=0.001).The relative expression level of GPX4 protein in OA group(0.11±0.02)was lower than that in control group(0.78±0.07),while the relative expression level of ACSL4 in OA group(0.65±0.07)was higher than that in control group(0.19±0.03)(t=36.813,P=0.001;t=24.160,P=0.001),the relative expression level of GPX4 protein in mTORC2-/-+OA group(0.63±0.07)was higher than that in OA group,while the relative expression level of ACSL4(0.30±0.04)was lower than that in OA group(t=28.571,P=0.001;t=17.365,P=0.001),the relative expression level of GPX4 protein in mTORC2-/-+OA+Erastin group(0.23±0.03)was lower than that in mTORC2-/-+OA group.The relative expression level of ACSL4 protein(0.57±0.06)was higher than that of mTORC2-/+OA group(t=21.009,P=0.001;t=14.977,P=0.001).The levels of Th1 and Th17 cells in OA group(25.36％±1.67％,21.42％±1.85％)were significantly higher than those in control group(2.63％±0.26％,1.92％±0.28％)(t=53.795,P=0.001;t=41.752,P=0.001),Th1 and Th17 cell levels in mTORC2/+OA group(9.35％±1.02％,8.41％±0.96％)were significantly lower than those in OA group(t=32.726,P=0.001;t=24.968,P=0.001),the levels of Th1 and Th17 cells in mTORC2-/-+OA+Erastin group(22.54％±1.79％,19.06％±1.74％)were significantly higher than those in mTORC2-/-+OA group(t=25.609,P=0.001;t=21.437,P=0.001).Conclusion mTORC2 suppresses pathogenic CD4+T cells by regulating ferroptosis and improves OA inflammation in mice.
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