Mechanism Of Nrf2-mediated Autophagy Promoting SLC7A11 Cell Membrane Localization And Alleviating Acute Liver Injury

Background:Acute liver injury is an abnormal liver function caused by various factors such as drugs,alcohol,and viral infections.Long term liver injury can lead to liver fibrosis and even hepatocellular carcinoma.Therefore,controlling the occurrence and development of liver injury has significant clinical significance for the treatment of liver diseases.Ferroptosis is a non apoptotic and iron dependent form of cell death,which is related to the accumulation of lipid hydroperoxides and membrane lipid peroxidation.The emergence of ferroptosis provides a new direction for the treatment of patients with acute liver injury.Previous studies have found that nuclear factor NF-E2 related factor2/Nrf2 plays an important role in resisting ferroptosis and alleviating liver injury caused by acetaminophen.Some scholars have also found that autophagy is a survival protection mechanism of the body,which can affect the development of ferroptosis and liver injury.However,the mechanism of Nrf2 and its induced autophagy synergistically regulate ferroptosis and alleviate acute liver injury is currently unclear.Objectives:This study aimed to investigate the intrinsic link between acute liver injury and ferroptosis,and clarify the molecular mechanism of Nrf2 and autophagy interactively regulating ferroptosis to alleviate acute liver injury,so as to provide a theoretical and experimental basis for the treatment strategies of acute liver injury in clinical practice.Methods:1.CCl₄ was used to construct an acute liver injury model in rats,and after SFN administration treatment,the changes of ALT,AST,ROS,GSH/GSSG,Fe²⁺,Caspase-3 in serum or liver tissues were detected;mitochondrial morphology was observed by Transmission electron microscopy（TEM）;pathological morphology of rat liver was observed by HE staining;The expression of Nrf2,autophagy-related proteins（Microtubule-associated protein 1 light chain 3/LC3、SQSTM1/P62）and ferroptosis-related proteins（Solute carrier family 7 member 11/SLC7A11、Glutathione peroxidase4/GPX-4）were detected by Western blot;the expression of SLC7A11 and GPX-4 were detected by immunohistochemistry.2.Nrf2 knockout rats were constructed.CCl₄ was used to construct acute liver injury model in rats,after SFN administration treatment,the changes of ALT,AST,ROS,GSH/GSSG,Fe²⁺,Caspase-3 in serum or liver tissues were detected;mitochondrial morphology was observed by TEM;pathological morphology of rat liver was observed by HE staining;The expression of Nrf2,LC3,P62,SLC7A11,GPX-4were detected by Western blot;the expression of SLC7A11 and GPX-4 were detected by immunohistochemistry.3.CCl₄ was used to construct acute liver injury model in rats,after SFN and chloroquine（CQ）administration treatment,the changes of ALT,AST,ROS,GSH/GSSG,Fe²⁺,Caspase-3 in serum or liver tissues were detected;mitochondrial morphology was observed by TEM;pathological morphology of rat liver was observed by HE staining;The expression of Nrf2,LC3,P62,SLC7A11,GPX-4 were detected by Western blot;the expression of SLC7A11 and GPX-4 were detected by immunohistochemistry.The co-localization of SLC7A11 and Lamp2 was detected by Immunofluorescence.4.H₂O₂ was used to construct L02 and BRL hepatocyte injury models,after SFN administration treatment,the changes of Cell viability,ROS,GSH/GSSG ratio,Fe²⁺,Caspase-3,Lip-ROS were detected and mitochondrial morphology was observed by TEM.After treatment with ferroptosis inhibitor,apoptosis inhibitor,necrosis inhibitor,and autophagy inhibitor,the Cell viability was measured to determine the cell death form;The expression of Nrf2,LC3,P62,SLC7A11,GPX-4 were detected by Western blot.Autophagy double-labeled adenovirus assay was used to detect autophagic flow.After overexpression of Nrf2,Western blot was used to detect the expression of autophagic markers.Chromatin immunoprecipitation was used to detect binding of Nrf2 and the DNA of Atg5.5.After H₂O₂,ML385 or si Nrf2,SFN administration treatment,the changes of Cell viability,ROS,GSH/GSSG ratio,Fe²⁺,Caspase-3 were detected.The expression of Nrf2,LC3,P62,SLC7A11,GPX-4 were detected by Western blot.6.After H₂O₂,CQ or si LC3,SFN administration treatment,the changes of Cell viability,ROS,GSH/GSSG ratio,Fe²⁺,Caspase-3 were detected.The expression of Nrf2,LC3,P62,SLC7A11,GPX-4 were detected by Western blot.The co-localization of SLC7A11 and Lamp2 was detected by Laser Confocal Microscope.7.After H₂O₂,CQ,SFN,Compound-C administration treatment,the expression of AMPK,P-AMPK,BECN1 and P-BECN1 were detected,and P-BECN1 and SLC7A11 binding was detected by immunoprecipitation.Results:1.After the treatment of CCl₄the liver of rats showed obvious bubble-like degeneration,mitochondrial crinkling,significant increase of ALT and AST in serum,accumulation of ROS,Fe²⁺,Caspase-3 and decrease of GSH/GSSG ratio in liver tissue.Compared with the model group,SFN could alleviate the bubble-like degeneration of liver tissue and mitochondrial damage,decrease ALT and AST in rat serum,decrease ROS,Fe²⁺,Caspase-3 and increase the GSH/GSSG ratio in rat liver tissue.The protein expression in rat liver tissues was detected by Western blot,and it was found that SFN activated Nrf2 to promote autophagy and upregulated the expression of SLC7A11 and GPX-4.Immunohistochemical staining showed that compared with the model group SFN can upregulate the expression of SLC7A11 and GPX-4.2.Nrf2 knockdown attenuated the decrease of ALT and AST in serum,decrease of ROS,Fe²⁺,Caspase-3 and increase of GSH/GSSG ratios in liver tissues caused by SFN.Western blot showed that Nrf2 knockdown attenuated SFN-activated autophagy and SLC7A11,GPX-4 upregulation.Immunohistochemical staining showed that the upregulation of SLC7A11 and GPX-4 caused by SFN was diminished after Nrf2knockdown.3.Inhibition of autophagy with CQ attenuated the decrease of ALT,AST in serum,decrease of ROS,Fe²⁺,Caspase-3 and increase of GSH/GSSG ratio in rat liver tissues caused by SFN.Surprisingly,CQ did not inhibit the activation of Nrf2 and the upregulation of SLC7A11 and GPX-4 caused by SFN.Immunohistochemical staining indicated that CQ treatment did not inhibit the upregulation of SLC7A11 and GPX-4by SFN.Detection of SLC7A11 expression in the cell membrane and cytoplasm in liver tissues revealed that CQ inhibited SLC7A11 expression in the cell membrane and increased SLC7A11 accumulation in the cytoplasm.Immunofluorescence revealed that CQ promoted co-localization of SLC7A11 with Lamp2.4.H₂O₂ induced L02 and BRL cell injury and showed accumulation of ROS,Fe²⁺,Caspase-3 and decreased GSH/GSSG ratio.Compared with the model group,SFN can increase the Cell viability,decrease ROS,Fe²⁺,Caspase-3,Lip-ROS,and increase the GSH/GSSG ratio of L02 and BRL cells.Transmission electron microscopy revealed that the model group showed significant mitochondrial damage in L02 cell,which was alleviated by SFN treatment.In order to determine whether the cell injury was related to ferroptosis,we selected ferroptosis inhibitor,apoptosis inhibitor,necrosis inhibitor,and autophagy inhibitor to detect the protective effect on hepatocytes.The results showed that ferroptosis inhibitors could effectively alleviate the decrease of cell viability.The protein expression in L02 and BRL cells revealed that SFN activated Nrf2 to promote autophagy and upregulated the expression of SLC7A11 and GPX-4.Autophagy double-labeled adenovirus assay showed that SFN activates autophagy.In order to demonstrate that autophagy activation is associated with increased Nrf2expression,we constructed an Nrf2 overexpression plasmid.After overexpression of Nrf2,the transformation from LC3I to LC3II increased and the expression of P62decreased,indicating that overexpression of Nrf2 activates autophagy.Chromatin immunoprecipitation showed that Nrf2 binds to DNA of Atg5 and that Nrf2 binding to DNA of Atg5 increased after SFN treatment.5.Nrf2 inhibitor ML385 and si Nrf2 were used to verify the protective effect of Nrf2 on cells.Inhibition of Nrf2 reversed the decrease of ROS,Fe²⁺,Caspase-3 and increase of GSH/GSSG ratio in L02 and BRL cells caused by SFN.Inhibition of Nrf2impaired SFN-activated autophagy and upregulation of SLC7A11 and GPX-4.6.Inhibition of autophagy by CQ or si LC3 attenuated the decrease of ROS,Fe²⁺,Caspase-3 and increase of GSH/GSSG ratio in L02,BRL cells caused by SFN.CQ did not inhibit the activation of Nrf2 and the upregulation of SLC7A11 and GPX-4 by SFN in L02 and BRL cells.CQ inhibited the uptake of cystine by SLC7A11,inhibited SLC7A11 expression in the cell membrane and increased SLC7A11 accumulation in the cytoplasm and increased the co-localization of SLC7A11 and Lamp2 in L02 cells.7.L02 cell was used to explore why CQ inhibits SLC7A11 membrane localization.CQ can promote phosphorylation at the 172 site of AMPK,resulting in an increase in phosphorylation levels at the 93 site of BECN1.The binding of SLC7A11 and P-BECN1 increased significantly after CQ treatment.To verify that the effect of CQ was related to the phosphorylation of AMPK and BECN1.We chose Compound-C,an AMPK inhibitor,to detect the phosphorylation levels of AMPK and BECN1,and the results showed that Compound-C significantly inhibited the phosphorylation of AMPK and BECN1,and Compound-C could reduce the formation of SLC7A11-P-BECN1complexes.Conclusion:1.The process of acute liver injury is related to ferroptosis,and acute liver injury induced by CCl₄can be alleviated by inhibiting ferroptosis.2.SFN can counteract ferroptosis and alleviate acute liver injury through the co-regulation of Nrf2 and autophagy.3.Nrf2 activation can induce the expression of SLC7A11,GPX-4 and autophagy-related proteins.4.Nrf2-dependent autophagy is a key step in promoting SLC7A11 membrane localization to alleviate acute liver injury.