High Glucose Environment Induced Ferroptosis In Steatosis Hepatocyte By Promoting Iron Overload And GSH Reduction

Objectives: Non-alcoholic fatty liver disease(NAFLD)and type 2 diabetes(T2DM)are common conditions that regularly co-exist and can act synergistically to drive adverse outcomes.The presence of both NAFLD and T2 DM increases the likelihood of the development of complications of diabetes(including both macro-and microvascular complications)as well as augmenting the risk of more severe NAFLD,including cirrhosis,hepatocellular carcinoma and death.The liver constitutes a key organ in systemic metabolism,contributing substantially to the development of insulin resistance and type 2 diabetes mellitus(T2DM).The mechanisms underlying these processes are not entirely understood,but involve hepatic fat accumulation,alterations of energy metabolism and inflammatory signals derived from various cell types including immune cells.Lipotoxins,mitochondrial function,cytokines and adipocytokines have been proposed to play a major part in both NAFLD and T2 DM.Ferroptosis is a regulated form of cell death driven by loss of activity of the lipid repair enzyme glutathione peroxidase 4(GPX4)and subsequent accumulation of lipid-based reactive oxygen species,particularly lipid hydroperoxides.This form of iron-dependent cell death is distinct from other cell death modalities in genetic,biochemic,and morphologic,including apoptosis,unregulated necrosis,and necroptosis.It has been reported that iron chelators as well as a few genes related to the lipid peroxide removal system such as cystine/glutamic acid transporter(x CT)and glutathione peroxidase 4(GPX4)are involved in the suppression of Ferroptosis.Some studies have suggested that the high glucose status,will affect iron metabolism,and the level of Ferritin in patients with type 2 diabetes generally increases.Whether high glucose status can affect liver iron metabolism in NAFLD patients and its mechanism is not known.Therefore,it is of great significance to study whether the Ferroptosis pathway is involved in high glucose-induced steatosis of hepatocytes.Methods:1.Establishment of L02 cell steatosis model by palmitic acid intervention;2.CCK-8 was used to detect the effect of PA and high glucose combined with PA on the activity of L02 cells,and the effect of FER-1 on the activity of L02 cells in dif Ferent intervention groups.3.To clarify the effect of PA and high glucose combined with PA on iron metabolism in L02 cells,we detected intracellular iron ion concentration and m RNA and protein relative expression level of Tf R1,and further explored the effect of FER-1 on intracellular iron metabolism after intervention.4.The GSH kit and the MDA kit were used to detect glutathione and lipid peroxidation in L02 cells under different intervention conditions,respectively;5.RT-PCR and Western Blotting was used to detect the relative expression of GPX4 and XCT on m RNA and protein levels in L02 cells under different interventions,and determine whether the iron death pathway-related factors are involved in high glucose-induced steatosis liver cell injury;6.ROS immunofluorescence staining was applied to detect the effect of different interventions on the accumulation of ROS in L02 cells;7.RT-PCR was used to detect the effect of high glucose and FER-1 on the relative expression of TNFα,IL-6,IL-1β and other proinflammatory cytokines in steatosis hepatocytes.Results: 1.After the establishment of L02 steatosis model,the formation of red lipid droplets was observed with the oil red staining microscope after the intervention of PA,while the number of lipid droplets increased significantly after the intervention of high glucose and PA.The intracellular triglyceride(TG)content was detected under different interventions.The content of TG in the cells of Glu + PA group was higher than that of PA group which suggested that high glucose can promote TG accumulation and lipid droplets formation in hepatocytes.2.Cell activity decreased after PA intervention in L02 cells,and cell death was not inhibited after adding FER-1;The activity of cells in Glu + PA group was significantly lower than that in PA group,and FER-1 could inhibit cell death which indicated that FER-1 can inhibit the damage of hepatocytes caused by high glucose to a certain extent.3.The expression level of iron ion and trans Ferrin Tf R1 in steatotic liver cells under the intervention of high glucose were significantly increased.4.GLU + PA group had higher MDA content and lower GSH content than PA group.The results were statistically significant.5.In the process of high glucose-induced steatosis hepatocyte injury,the relative expression levels of iron death-related factors GPX4 and x CT at the m RNA and protein levels showed a decrease.6.Immunofluorescence results show that highglucose intervention can promote the accumulation of ROS in steatosis hepatocytes 7.RT-PCR was used to detect the effect of high glucose and fer-1 on the relative expression of TNF α,IL-6,IL-1 β and other proinflammatory cytokines in steatosis hepatocytes.Conclusions: The results showed that in vitro experiments,palmitic acid intervention alone cannot induce Ferroptosis in hepatocytes,while fatty degeneration liver cells under high glucose intervention show characteristic changes in Ferroptosis such as iron overload,decreased GPX4 expression,and ROS accumulation,and it can be inhibited by FER-1,suggesting that high glucose may induce steatotic liver cell damage through the iron death pathway.Proinflammatory cytokines such as TNFα,IL6,and IL1-β were highly expressed in a high glucose state,and can be significantly inhibited by adding FER-1.High glucose may induce iron death by inducing an inflammatory response in liver cells.The iron death pathway plays an important role in the pathogenesis of diabetes combined with NAFLD,and it is expected to become a target for its prevention and treatment in the future.