Mechanisms Of Acrylamide-induced Mitochondrial ROS Release Causing HSC-T6 Cell Death

Acrylamide（AA）,an important product of the Melad reaction in thermally processed foods,has reproductive toxicity,neurotoxicity,hepatotoxicity,and potential carcinogenicity.The liver is the main site of AA metabolism and the main target organ of AA attack,and the hepatotoxicity of AA in heat-processed foods has caused widespread concern.AA-induced hepatotoxicity is closely related to oxidative stress,and mitochondrial oxidative stress induced by mitochondrial ROS（mt ROS）is the main source of oxidative stress in the organism.Oxidative stress is directly involved in cell death such as ferroptosis and pyroptosis.Ferroptosis is dependent on iron ions and is characterized by dysregulation of lipid peroxidation and antioxidant systems.Oxidative stress opens the classical pathway of pyroptosis by activating inflammatory vesicles and inducing the release of inflammatory factors.The aim of this study was to investigate the mechanism of AA on the release of mt ROS and the induction of ferroptosis and pyroptosis in rat hepatic stellate cells（HSC-T6 cells）by establishing an AA-stained HSC-T6 cells model to further elucidate the role of mt ROS on ferroptosis and pyroptosis,and to lay a research foundation for the ultimate uncovering of AA hepatotoxicity mechanism.The main research content and results of this thesis are as follows:（1）AA impaired mitochondrial function and released mt ROS in HSC-T6 cells.The HSC-T6 cells model was established by AA（0,0.5,1,2 m M）.By measuring the changes in LDH,ALT and AST levels,it was concluded that the values of LDH,ALT and AST in HSC-T6 cells treated with 2 m M AA were 1.4,2.9 and 3.5 times higher than those in the control group,respectively.This could determine that AA caused damage to the cells.The expression level of membrane pore protein VDAC1 in HSC-T6 cells was detected by Western Blot,and the mitochondrial MMP and ATP levels were measured,while the mitochondrial structure was observed by transmission electron microscopy,and the results were that AA induced elevated VDAC1 protein expression,decreased MMP and ATP levels,and impaired mitochondrial structure.This concluded that AA caused structural and functional damage to mitochondria.By detecting the mt ROS release level,it was determined that AA increased mt ROS release.（2）AA-induced mt ROS caused imbalance of XCT-GSH-GPX4 antioxidant signaling pathway,which led to ferroptosis in HSC-T6 cells.The expression levels of key proteins for ferroptosis in HSC-T6 cells were examined by Western Blot,and GSH,MDA and Fe²⁺were measured in HSC-T6 cells.It was concluded that AA affects the expression of key proteins,increases the level content of MDA and Fe²⁺levels,and down-regulates GSH levels,and it was determined that AA affects the XCT-GSH-GPX4 antioxidant signaling pathway in HSC-T6 cells.Ferroptosis inhibitor Fer-1（5 mΜ）was used for validation.The expression levels of key proteins of ferroptosis in HSC-T6 cells were examined by Western Blot,and GSH,MDA and Fe²⁺were measured to determine that AA induced ferroptosis in HSC-T6 cells by causing an imbalance in the XCT-GSH-GPX4 antioxidant signaling pathway.The key protein expression of ferroptosis was detected by adding 50μM mt ROS scavenger Mito-TEMPO,and it was concluded that Mito-TEMPO restored the XCT-GSH-GPX4antioxidant system and determined that AA-induced mt ROS release from HSC-T6cells promoted the occurrence of ferroptosis.（3）AA-induced mt ROS release activates NLRP3-caspase1-GSDMD pathway to cause pyroptosis in HSC-T6 cells.The assay of SOD enzyme activity and ROS release levels concluded that AA induced an increase in ROS release and SOD enzyme activity was inhibited.It was determined that AA induced oxidative stress.The expression of NLRP3-caspase1-GSDMD pathway-related proteins was examined by Western Blot,and the levels of downstream inflammatory factors were detected by the kit,and AA upregulation of NLRP3,GSDMD,caspase1 protein expression and IL-18 and IL-1βinflammatory factor levels were obtained.This identified that AA activation of NLRP3-caspase1-GSDMD signaling pathway caused pyroptosis in HSC-T6 cells.By adding the mt ROS scavenger Mito-TEMPO to detect the expression of key proteins of pyroptosis,it was concluded that Mito-TEMPO inhibited the NLRP3-caspase1-GSDMD signaling pathway,and it was determined that AA induced mt ROS release in HSC-T6 cells to promote the onset of pyroptosis.In summary,AA induced mt ROS release by inducing mitochondrial dysfunction.AA-induced mt ROS induced ferroptosis in HSC-T6 cells by imbalance of XCT-GSH-GPX4 antioxidant signaling pathway.It also induced pyroptosis in HSC-T6 cells through activation of NLRP3-caspase1-GSDMD signaling pathway.