Study On The Mechanism Of Ferrostatin-1 Inhibiting The Activation Of Human Hepatic Stellate Cells Induced By Sodium Arsenite By Regulating Ferroptosis

Objective:1)To investigate the effects of different doses of sodium arsenite（NaAsO₂）on ferroptosis in human hepatic stellate cells（LX-2）.2)Effect of Ferrostatin-1combined with NaAsO₂exposure on fibrosis indices of LX-2 cells.Methods:1)IC₅₀were determined by CCK-8 method and divided into control,5μmol/L,10μmol/L and 15μmol/L NaAsO_2groups;2)The morphological changes of cells in different NaAsO_2groups were observed by microscope;3)Oil red staining observe the distribution of lipid droplets in different groups;4)Transmission electron microscopy（TEM）observe the cell mitochondria ultrastructure;5)Ferro Orange fluorescence probe was used to detect Fe²⁺fluorescence intensity in different groups of cells;6)Detection of lipid peroxide products（lipid ROS）by BODIPY 581/591 C11 probe;7)The contents of glutathione（GSH）and glutathione peroxidase 4（GPX4）in cells were detected by enzyme labeling;8)real-time quantitative PCR（RT-PCR）were determined theα-SMA,collagen1,SLC7A11and GPX4 m RNA expression levels;9)Western-Blot were detecte theα-SMA,SLC7A11and GPX4 protein expression levels;10)Fer-1 and NaAsO₂interfered with LX-2 cells for24h to detect the changes of ferroptosis and fibrosis indexes before and after Fer-1intervention.Results:1)With the increase of NaAsO₂dose,the proliferation rate of LX-2cells decreased gradually,IC₅₀was 21.44μmol/L,R²=0.91;2)In nature conditions,LX-2morphology was saturated,with tentacles extending outward in the shape of stars and the growth mode was clinged.As the dose of NaAsO₂increased,the number of cells gradually decreased,cell antennae began to shrink and gradually turned round,cells shrank and ruptured,intercellular space widened;3)Oil red staining showed that there were less lipid droplets in LX-2 cells under normal conditions,but NaAsO₂treatment resulted in lipid loss in LX-2 cells;4)TEM showed that the mitochondrial structure of LX-2 cells in the control group was double-layer membrane,smooth closed-loop,and mitochondrial ridge was clearly visible.With the increase of NaAsO₂dose,mitochondrial membrane integrity was damaged,mitochondrial ridge was broken and disappeared;5)The fluorescence intensity of Fe²⁺in LX-2 cells increased with the increase of NaAsO₂dose.Compared with the control group,Fe²⁺levels in 5 and 15μmol/L NaAsO₂infected groups were increased（P<0.05）;6)The green fluorescence of lipid ROS was not observed in the control group and lipid ROS was gradually enhanced with the increase of the dose of NaAsO₂;7)The content of GSH in LX-2 cells was decreased by NaAsO₂dose（P<0.05）,MDA content increased after NaAsO₂treatment（P<0.05）;8)RT-PCR results showed that different doses of NaAsO₂increasedα-SMA and Collagen1 m RNA expression levels（P<0.05）.However,ferroptosis indicators SLC7A11 and GPX4 m RNA expression levels were decreased with the increase of NaAsO₂dose（P<0.05）.9)Western-Blot analysis showed that theα-SMA protein expression levels increased（P<0.05）but SLC7A11 and GPX4protein expression levels decreased with the increase of dose（P<0.05）;10)CCK-8showed that compared with 15μmol/L NaAsO_2group,Fer-1+NaAsO₂treated LX-2 cells proliferation rate was increased（P<0.05）.Compared with the 15μmol/L NaAsO_2group,Fe²⁺levels in Fer-1 co-intervention group showed a decreasing trend（P<0.05）.Compared with 15μmol/L NaAsO_2group,the level of lipid ROS in Fer-1 co-intervention group showed a decreasing trend.Compared with 15μmol/L NaAsO_2group,the content of GSH in Fer-1 co-intervention group was increased（P<0.05）.RT-PCR and Westen-Blot showed that compared with 15μmol/L NaAsO_2group,Fer-1group SLC7A11 and GPX4m RNA and protein expression were increased（P<0.05）.α-SMA and collagen1m RNA and protein expression levels of were decreased（P<0.05）.Conclusion:NaAsO₂increased the fibrosis index and ferroptosis level of LX-2 cells.There was a negative correlation between fibrosis indexes（α-SMA,collagenⅠ）and ferroptosis indexes（SLC7A11,GPX4）.The co-exposure of Fer-1 and NaAsO₂decreased the expression level of fibrosis index in LX-2 cells.