The Mechanism Of Sodium Sulfite-induced Hepatocyte Apoptosis,Necroptosis And Pyroptosis

Sodium sulfite is commonly used as a food additive to prevent excessive oxidation of nutrients and inhibit the growth of microorganisms.However,there are concerns that human daily intake of sodium sulfite may exceed the allowable daily intake(ADI).Research has shown that overconsumption of sodium sulfite can lead to liver damage,disorders in liver lipid metabolism,and liver cell death.Different types of cell death,such as apoptosis,necroptosis,and pyroptosis,are closely associated with liver injury.While existing studies have demonstrated the role of sodium sulfite in hepatocyte apoptosis,its impact on necroptosis and pyroptosis remains unclear.Therefore,it is important to investigate the mechanisms of sodium sulfite on these different forms of cell death to enhance our understanding of its hepatotoxic effects.Recent reports have highlighted that various modes of cell death are interconnected,with certain cell death marker proteins acting as regulators that can trigger other types of cell death.For instance,phosphorylated mixed lineage kinase domain-like protein MLKL(p-MLKL),a marker of necroptosis,not only induces cell membrane rupture but also leads to lysosomal membrane leakage and the release of cathepsin B(CTSB)into the cytoplasm.Studies have confirmed that the release of CTSB into the cytoplasm plays a crucial role in activating NOD-like receptor pyrin domain-containing protein 3(NLRP3)inflammasome-dependent pyroptosis.However,p-MLKL as an upstream regulator of cell pyroptosis is still under investigation.This study utilizes normal mouse liver cell line AML-12 and C57BL/6 mice to explore the mechanisms of sodium sulfite-induced hepatocyte apoptosis,necroptosis,pyroptosis,and necrosis both in vitro and in vivo.The research focuses on the molecular mechanism of pyroptosis involving the apoptosis marker protein p-MLKL.The main research contents and results are as follows:(1)Sodium sulfite induced hepatocyte apoptosis,necroptosis and pyroptosisThe toxicity of sodium sulfite to AML-12 cells was assessed using the thiazolyl blue(MTT)method,lactate dehydrogenase(LDH)method,and Calcein-AM/PI staining.Results indicated significant cell toxicity at 2 m M concentration of sodium sulfite.Evaluation of C57BL/6 mouse liver coefficient through hematoxylin and eosin(HE)staining,F4/80 immunohistochemistry,aspartate aminotransferase(AST),and alanine aminotransferase(ALT)content revealed sodium sulfite-induced liver enlargement,inflammatory infiltration,macrophage activation,and elevated AST and ALT levels,suggesting liver damage in C57BL/6 mice.Subsequent staining of AML-12 cells with Annexin V-FITC/PI and TUNEL demonstrated an increase in apoptotic cells and TUNEL-positive cells upon exposure to sodium sulfite.Protein expression levels of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(BAX),cytochrome c(Cyt-c),and cysteine in AML-12 cells and C57BL/6 mouse livers were measured,along with caspase-3.Sodium sulfite was found to up-regulate BAX,Cyt-c,and caspase-3,while down-regulating Bcl-2,ultimately leading to hepatocyte apoptosis.Additionally,RIPK1,RIPK3,p-MLKL,and MLKL protein levels were assessed,showing activation of necroptosis markers with sodium sulfite treatment.Furthermore,NLRP3,caspase-1,GSDMD-N,interleukin-1β(IL-1β),and interleukin-18(IL-18)proteins were examined,revealing activation of pyroptosis markers,indicating induction of hepatocyte pyroptosis by sodium sulfite.(2)Sodium sulfite induced hepatocyte apoptosis,necroptosis and pyroptosis by inhibiting mitophagy(1)Sodium sulfite inhibits mitophagy in hepatocytes.Initially,adenosine triphosphate(ATP),cyclooxygenase I(Cox I),cyclooxygenase I(Cox II),mitochondrial reactive oxygen species(mtROS),and reactive oxygen species(ROS)levels were measured in AML-12 cells.The findings revealed that sodium sulfite led to elevated ATP,ROS,and mtROS levels,as well as decreased Cox I and Cox II levels.Subsequently,transmission electron microscopy was utilized to examine the mitochondrial structure of AML-12 cells.The results indicated that sodium sulfite exposure caused disappearance of inner cristae and rupture of the outer membrane in AML-12 cell mitochondria.Western blot analysis was then performed to assess mitophagy marker proteins,including autophagy microtubule-associated protein light chain protein 3 II(LC3 II),autophagy receptor protein(p62),and PTEN-induced mitophagy in AML-12 cells and C57BL/6 mouse livers.The results demonstrated that sodium sulfite down-regulated LC3 II,PINK1,and Parkin protein expression levels,while up-regulating p62 protein expression.LC3 II immunofluorescence and mitochondrial probe Mitotracker red co-localization staining were utilized to label mitochondria enclosed by autophagosomes.The findings revealed that sodium sulfite decreased the co-localization of LC3 II and Mitotracker red,suggesting that sodium sulfite impairs mitochondrial function and hinders mitophagy.(2)Sodium sulfite triggers hepatocyte apoptosis,necroptosis,and pyroptosis through the inhibition of mitophagy.To investigate this,AML-12 cells and C57BL/6mice were exposed to sodium sulfite and subsequently treated with Mito-TEMPO and NAC,which are scavengers of mtROS and ROS.Western blot analysis was then performed to assess the levels of marker proteins associated with apoptosis,necroptosis,and pyroptosis.The findings revealed that Mito-TEMPO and NAC treatment mitigated the sodium sulfite-induced cell death pathways.Additionally,the use of the mitophagy activator UA further supported these results.Overall,the study suggests that sodium sulfite promotes apoptosis,necroptosis,and pyroptosis by elevating mtROS and ROS levels while impeding mitophagy.(3)Sodium sulfite induced pyroptosis through necroptosis marker protein pMLKL-mediated lysosomal membrane permeabilization(1)The necroptosis marker protein p-MLKL mediates sodium sulfite-induced lysosomal membrane leakage.Initially,Western blot analysis was conducted to assess the protein expression levels of lysosomal membrane-associated protein 1(LAMP1),lysosomal membrane-associated protein 2(LAMP2),and CTSB in AML-12 cells and C57BL/6 mouse livers.The findings revealed that sodium sulfite led to an increase in the protein expression levels of LAMP1,LAMP2,and CTSB.Subsequently,AML-12 cells exposed to sodium sulfite were stained with AO and Lysotracker green,demonstrating a decrease in the red-green fluorescence ratio of AO staining and the green fluorescence intensity of Lysotracker green staining.These results suggest that sodium sulfite triggers lysosomal membrane leakage.Following this,immunofluorescence co-localization staining of p-MLKL and LAMP1 was carried out on AML-12 cells and mouse liver sections,revealing that sodium sulfite induced the co-localization of p-MLKL and LAMP1,indicating the translocation of p-MLKL to lysosomes.The study utilized si RNA interference technology to downregulate MLKL protein expression and assess the protein levels of LAMP1,LAMP2,and CTSB.Colocalization staining of AO,Lysotracker green,CTSB,and Lysotracker red was also conducted.The findings demonstrated that si MLKL notably mitigated the sodium sulfite-induced increase in LAMP1,LAMP2,and CTSB protein expression.Additionally,the red-green fluorescence ratio of AO staining decreased,the green fluorescence intensity of Lysotracker green staining weakened,and the colocalization of CTSB and Lysotracker red diminished.These results suggest that sodium sulfite triggers lysosomal membrane leakage and CTSB release from lysosomes via p-MLKL.(2)Sodium sulfite triggers hepatocyte pyroptosis by causing lysosomal membrane leakage through the necroptosis marker protein p-MLKL.Initially,si RNA technology was employed to reduce MLKL expression in AML-12 cells,or the MLKL inhibitor GW806742 X was administered to C57BL/6 mice.Co-localization staining of NLRP3 and CTSB immunofluorescence was observed in AML-12 cells and C57BL/6 mouse sections.The findings demonstrated that sodium sulfite notably enhanced the colocalization of NLRP3 and CTSB,a phenomenon that was mitigated by si MLKL or GW806742 X.Subsequent treatment with the CTSB inhibitor CA074-ME and the NLRP3 inhibitor MCC950 in sodium sulfite-infected AML-12 cells and C57BL/6 mice revealed a significant reduction in cell pyroptosis-related protein expression levels.Both CA074-ME and MCC950 effectively alleviated sodium sulfite-induced cell pyroptosis.Finally,the role of p-MLKL in sodium sulfite-induced pyroptosis was investigated using si MLKL or the MLKL inhibitor GW806742 X,which demonstrated a considerable reduction in pyroptosis.Collectively,these results suggest that sodium sulfite induces pyroptosis through the p-MLKL/CTSB/NLRP3 pathway.In summary,This research comprehensively investigated the impact of sodium sulfite on hepatocyte apoptosis,necroptosis,and pyroptosis both in vivo and in vitro.The study revealed that sodium sulfite triggers hepatocyte death by promoting mtROS accumulation and suppressing mitophagy,leading to apoptosis,necroptosis,and pyroptosis.Moreover,the necroptosis-associated protein p-MLKL relocates to the lysosomal membrane,resulting in lysosomal membrane permeabilization and the release of CTSB from lysosomes,ultimately inducing NLRP3-dependent cell pyroptosis.These findings contribute to a deeper understanding of the hepatotoxicity mechanism of sodium sulfite and offer valuable insights for the toxicological assessment of this compound.