基于Nrf2/GPX4信号通路探讨三水白虎汤对膝骨关节炎软骨细胞铁死亡的影响OA

Objective:To investigate effect of Sanshui Baihu decoction on ferroptosis in osteoarthritis(OA)chondrocytes.Its regulatory mechanism on nuclear factor-E2 related factor 2(Nrf2)/glutathione peroxidase 4(GPX4)pathway.Methods:Drug-containing serum of Sanshui Baihu(0,0.5,1 g/d)was prepared.OA chondrocyte model was established by induction with interleukin-1β(IL-1β).The cell experiments were divided into following groups:normal control model,Sanshui Baihu decoction low-dose,Sanshui Baihu decoction high-dose,and Sanshui Baihu decoctionhigh-dose and inhibitor ml385 groups.The cell proliferative activity was detected by Calcein-AM/PI staining.The cell apoptosis rate was detected by TUNEL staining.Thecytokine levels were measured by enzyme-linked immunosorbent assay.The mitochondrial membrane potential was assessed using JC-1.Theintracellular reac-tive oxygen species(ROS)levels were detected by DHE staining.The intracellular iron levels were measured by Fer-roOrange staining.Lipid peroxidation levels were evaluated using BODIPY581/591 C11 staining.The expressions of DRP1 and MFN2 were examined by immunofluorescence.The protein expressions of Nrf2,GPX4,and ACSL4 were determined by Western blot.Ubiquitination of GPX4 was detected by co-immunoprecipitation.Results:In IL-1β-in-duced OA chondrocyte model,Sanshui Baihu decoction could significantly increase the cell proliferation,decrease the apoptosis rate,elevate the superoxide dismutase(SOD)levels,reduce the levels of tumor necrosis factor-α(TNF-α),IL-1β,and malondialdehyde(MDA),enhance the mitochondrial membrane potential,and lower the levels of ROS,i-ron,and lipid peroxidation.It could also significantly decrease DRP1 expression,increase MFN2 expression,reduce ACSL4 expression and GPX4 ubiquitination,and upregulate Nrf2 and GPX4 expression.The protective effects of SSBH were markedly reversed by ml385.All differences were statistically significant(P<0.05).Conclusion:Sanshui Baihu decoc-tioncould effectively alleviate IL-1β-induced inflammatory response,oxidative stress,and the apoptosis in chondrocytes.Its mechanism may be closely related to activating Nrf2/GPX4 signaling pathway,thereby regulating iron metabolism homeo-stasis,inhibiting lipid peroxidation,improving mitochondrial dynamics,and ultimately suppressing chondrocyte ferroptosis.