The Role And Mechanism Of TFE3-mediated Abnormal Lysosomal Function In Fluoride-induced Hepatocyte Toxicity

Part I.Role of lysosomal biogenesis disorders in fluoride-induced hepatotoxicity in SD ratsObjective:Excessive fluoride exposure can cause liver injury,but the exact mechanism needs to be further investigated.In addition,transcription factor E3(TFE3)is a major regulator of lysosomal biogenesis,and nuclear translocation is a prerequisite for its activity.Therefore,the present study was designed to investigate the role of TFE3 nuclear translocation and impaired lysosomal biogenesis in sodium fluoride(Na F)-induced hepatotoxicity.Methods:Forty-eight SD rats(2 months old,weighing 180-250 g)were divided into four groups: a control group(Na F < 1 mg/L)and three treatment groups(25,50 and 100 mg/L Na F)of 12 rats each,and all groups were contaminated by ad libitum drinking,and the feeding and drinking conditions of each group were recorded.After 2 months of poisoning,the liver alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activities were measured by enzyme-linked immunosorbent assay.The expression of autophagy-associated proteins LC3-II and p62,lysosomal biogenesis and lysosomal degradation function-associated proteins CTSD and LAMP2,apoptosis-associated protein cleaved-PARP,and lysosomal biogenesis regulator TFE3 protein were detected by western blot.Results:1.The results of enzyme-linked immunosorbent assay showed that both ALT and AST activities were significantly increased in the 100 mg/L Na F group compared with the control group(P < 0.05),suggesting that fluorine exposure may impair liver function in rats.2.Western blot results showed that the protein expression of CTSD and LAMP2,proteins related to lysosomal biogenesis and lysosomal degradation function,was decreased(P < 0.05),and the protein expression of autophagy-related proteins LC3-II,p62 and apoptosis-related protein cleaved-PARP was increased(P < 0.05)in the livers of rats in the Na F group compared with the control group.Lysosomal biogenesis regulator total TFE3 and nuclear TFE3 protein expression were decreased(P < 0.05).Conclusion:Na F exposure can impair liver function and lead to liver damage in rats.In addition,Na F exposure caused impaired autophagic degradation,impaired lysosomal biogenesis,impaired lysosomal degradation function and increased apoptosis in liver tissues.Meanwhile,Na F exposure can lead to reduced TFE3 nuclear translocation in liver tissues.In conclusion,the hepatotoxicity caused by Na F exposure may be related to impaired lysosomal biogenesis.Part II The role and mechanism of TFE3-mediated lysosomal biogenesis disorder in fluoride-induced BRL3 A cytotoxicityObjective:The purpose of this study was to investigate the role and mechanism of TFE3-mediated lysosomal biogenesis disorder in Na F-induced BRL3 A cytotoxicity and to provide a scientific basis for elucidating the mechanism of Na F-induced hepatotoxicity.Methods:Different doses of Na F-stained cell models(dose groups 0,20,40,60 mg/L Na F),TFE3 overexpression intervention models(control group,60 mg/L Na F group,Ad-null group,Ad-null+60 mg/L Na F group,Ad-TFF3 group,Ad-TFE3+60 mg/L Na F group)and rapamycin(rapamycin,RAPA)intervention models(control,60 mg/L Na F,RAPA,RAPA+60 mg/L Na F),cell survival was measured by CCK-8 kit 24 h after staining,ALT and AST enzyme activities of BRL3 A cells were measured by enzyme-linked immunosorbent assay,and autophagy related proteins LC3-II and p62,lysosomal biogenesis and lysosomal degradation function related proteins CTSD and LAMP2,apoptosis related protein cleaved-PARP,lysosomal biogenesis regulator TFE3 protein expression by western blot,and TFE3 nuclear translocation,lysosomal degradation ability and cellular autophagic flow by immunofluorescence assay.Results:1.CCK-8 results showed that after 24 h of Na F staining of BRL3 A cells,the cell survival rate of each Na F group was significantly reduced compared with the control group,and a dose-effect relationship was observed(P < 0.05).2.The results of enzyme-linked immunosorbent assay showed that both ALT and AST activities were significantly increased in the 60 mg/L Na F group compared with the control group(P < 0.05),suggesting that fluorine exposure may impair liver function in rats.3.Western blot results showed that after staining BRL3 A cells with different doses of Na F,the expression of lysosomal biogenesis and lysosomal degradation function-related proteins CTSD and LAMP2 protein was decreased in the 60 mg/L Na F stained group compared with the control group(P <0.05),and the expression of autophagy-related proteins LC3-II,p62 and apoptosis-related protein cleavedPARP protein expression was increased(P < 0.05),and both total TFE3 and nuclear TFE3 protein expression of lysosomal biogenesis regulators were decreased(P < 0.05).4.Immunofluorescence results showed that the mean fluorescence intensity of nuclear TFE3 decreased continuously with increasing Na F concentration compared with the control group(P < 0.05);in addition,to further confirm the effect of Na F on autophagic degradation in rat hepatocytes,BRL3 A cells were treated with m RFP-GFP-LC3 adenovirus so that autophagosomes and autophagic lysosomes could be distinguished.The results showed that Na F exposure led to an increase in the number of yellow dots and adecrease in the number of red dots compared to the control group.The results of DQ-BSA Green assay showed a progressive decrease in lysosomal degradation in the 20,40 and 60 mg/L Na F groups compared to the control group(P < 0.05).5.To further reveal the regulatory role of TFE3 on lysosome biogenesis in Na F-induced hepatotoxicity,BRL3 A cells were treated with TFE3 overexpressing adenovirus and RAPA.After TFE3 overexpression adenovirus and RAPA intervention,western blot experiments showed that the protein levels of LC3-II,LAMP2 and CTSD were significantly increased in the RAPA+Na F group and Ad-TFE3+Na F group,and the protein levels of p62 and cleaved-PARP were significantly decreased in BRL3 A cells compared with the Na F group.In addition nuclear TFE3 protein levels were significantly increased in the RAPA+Na F group(P < 0.05)6.After TFE3 overexpression adenovirus and RAPA intervention,immunofluorescence results showed that the lysosomal degradation ability and the mean intensity of nuclear TFE3 fluorescence were increased in the RAPA+Na F group and Ad-TFE3+Na F group compared with Na F.Conclusion:Na F induces impaired lysosomal biogenesis by inhibiting nuclear translocation of TFE3,leading to decreased lysosomal degradation,which results in defective autophagy and apoptosis.Ad-TFE3 and RAPA can enhance lysosomal biogenesis and improve autophagic flow by promoting nuclear translocation of TFE3,which in turn alleviates Na F-induced hepatotoxicity.