温胆汤含药血清调控NRF2/GPX4信号通路抑制谷氨酸诱导HT22铁死亡的作用及其机制OA

Objective:To explore the effect and underlying mechanism of Wendan Decoction(温胆汤)-containing serum in inhibiting glutamate(GLU)-induced ferroptosis in HT22 cells.Methods:Fifty SPF rats were randomly divided into three groups administered Wendan Decoction at doses of 40,30,and 20 g/kg,once daily for 7 consecutive days.Two hours after the final gavage on day 7,the rats were anesthetized by intraperitoneal injection of pentobarbital sodium,and blood was collected from the abdominal aorta to prepare Wendan Decoction-containing serum.GLU-induced ferroptosis in HT22 cells was used as the cellular model,and the cells were divided into six groups:normal control,model control,Wen-dan Decoction-containing serum groups(20,30,and 40 g/kg),and NRF2 inhibitor group(2 μmol/L ML385+10%Wendan Decoction-containing serum,30 g/kg).The CCK-8 assay was used to detect the optimal modeling concentration of GLU for HT22 cells.The biochemical assays were used to detect the intracellular levels of ferrous ion(Fe2+),glutathi-one(GSH),and malondialdehyde(MDA).Intracellular reactive oxygen species(ROS)clusters were detected via DCFH-DA fluorescent staining.Western blot and RT-qPCR were used to detect the relative protein and mRNA expres-sion levels of ferroptosis-related markers:nuclear factor erythroid 2-related factor 2(NRF2),heme oxygenase-1(HO-1),glutathione peroxidase 4(GPX4),and acyl-CoA synthetase long chain family member 4(ACSL4).Results:Based on the CCK-8 assay,5 mmol/L GLU for 24 h was selected as the modeling condition.Compared with the normal control group,the model control group showed significantly increased levels of MDA,ROS,and Fe2+,and significantly decreased GSH levels(P<0.05).The mRNA and protein expression levels of Nrf2,Ho1,and Gpx4 were significantly downregulat-ed(P<0.05),while those of the ferroptosis marker Acsl4 were significantly upregulated(P<0.01).Compared with the model control group,all Wendan Decoction-containing serum groups showed significantly reduced levels of MDA,ROS,and Fe2+(P<0.01),and significantly increased GSH levels(P<0.01).Protein expression of NRF2,HO-1,and GPX4 was significantly upregulated(P<0.01),while ACSL4 protein expression was significantly downregulated(P<0.01).In the 20 g/kg group,Ho1 and Gpx4 mRNA levels were significantly upregulated(P<0.05).In the 30 g/kg group,Nrf2,Ho1,and Gpx4 mRNA levels were significantly upregulated(P<0.05),and Acsl4 mRNA significantly downregulated(P<0.01).In the 40 g/kg group,Nrf2 and Ho1 mRNA levels were significantly upregulated(P<0.05),and Acsl4 mR-NA significantly downregulated(P<0.01).To verify the mechanism,HT22 cells were co-treated with 30 g/kg Wendan Decoction-containing serum and NRF2 inhibitor ML385.Compared with the 30 g/kg group alone,the ML385 group showed significantly downregulated mRNA and protein levels of Nrf2,Gpx4,and Ho1,and significantly upregulated mRNA and protein levels of Acsl4(P<0.05 or P<0.01).Conclusion:Wendan Decoction-containing serum can significantly in-hibit GLU-induced ferroptosis in neuronal HT22 cells,potentially by activating the NRF2/GPX4 signaling pathway.