The Role And Mechanism Of MtROS-mediated Autophagy And Ferroptosis In Liver Injury Induced By Fluoride

Objective:The liver is an important target organ for fluoride exposure-induced damage,but the detailed mechanism of fluoride-induced liver damage still needs to be studied.This project investigates the role of mitochondrial reactive oxygen species（mtROS）,autophagy,and ferroptosis in liver injury caused by sodium fluoride（NaF）through in vivo and in vitro experiments,especially the mechanism of mtROS-mediated autophagy and ferroptosis in this process,so as to provide scientific basis for the prevention and control of liver injury caused by endemic fluorosis.Methods:In vivo experiments:48 adult Sprague-Dawley（SD）rats（two months old,weighing 180-250 g）were randomly divided into four groups:a control group（water fluoride concentration below 1 mg/L）,and three NaF-treated groups（25 mg/L,50 mg/L,and 100 mg/L NaF）,with 12 rats in each group ingested NaF by ad libitum drinking.After 2 months of NaF treatment,all rats were sacrificed,and the livers were removed.The expression levels of ferroptosis key proteins glutathione peroxidase 4（GPX4）,acyl-Co A synthetase long chain family member 4（ACSL4）,ferritin heavy chain 1（FTH1）and transferrin receptor（TFRC）,and autophagy key proteins microtubule-associated protein 1 light chain 3（LC3）and sequestosome 1（SQSTM1/p62）in rat livers were detected by western blot.In vitro experiments:a NaF-treated rat hepatocytes（BRL3A）model was constructed at different doses（0,20,40,and 60 mg/L NaF）,in addition,BRL3A cells were treated with ferroptosis inhibitor ferrostatin-1（Fer-1）,autophagy agonist rapamycin（RAPA）,and mtROS inhibitor（Mito-TEMPO）in advance for 1 h,followed by NaF staining for 24 h.The expression levels of GPX4,ACSL4,FTH1,TFRC,LC3-II,p62,and mitochondrial import receptor subunit TOM20 homolog（TOMM20）proteins in BRL3A cells were detected by western blot.The m RFP-GFP-LC3 dual fluorescent labeling method detects autophagic flux.JC-1 fluorescent probe,Mito SOX Red mitochondrial superoxide fluorescent probe,and Ferro Orange fluorescent probes were used to detect mitochondrial membrane potential（MMP）,mtROS,and Fe²⁺,respectively.The levels of glutathione（GSH）,superoxide dismutase（SOD）,and malondialdehyde（MDA）were detected by spectrophotometry.Results:In vivo experiments:1.The effect of NaF on ferroptosis in rats:Compared with the control group,the expression levels of GPX4 and FTH1 were decreased in the 100 mg/L NaF group（P<0.05）,and the expression levels of ACSL4 and TFRC were increased（P<0.05）.2.The effect of NaF on autophagic flux in rats:The expression of LC3-II and p62 was also significantly increased in the 100 mg/L NaF group compared with the control group（P<0.05）.In vitro experiments:1.The effect of NaF on ferroptosis in BRL3A cells:The expression of GPX4 and FTH1 decreased and that of ACSL4 and TFRC increased in the 60 mg/L NaF group compared with the control group（P<0.05）;the content of Fe²⁺in the 60 mg/L NaF group also increased by Ferro Orange fluorescence probe compared with the control group（P<0.05）;meanwhile,the contents of GSH and SOD decreased while MDA increased（P<0.05）.2.The effect of NaF on autophagic flux in BRL3A cells:Western blot showed that the expression of LC3-II and p62 increased in the 60 mg/L NaF group compared with the control group（P<0.05）.m RFP-GFP-LC3 dual fluorescence showed that the number of red puncta representing autolysosomes decreased and the number of yellow puncta representing autophagosomes increased in the 60 mg/L NaF group compared with the control group（P<0.05）.3.The effect of NaF on the mitochondria of BRL3A cells:JC-1 fluorescent probe detection results that NaF exposure induced a decrease in MMP in BRL3A cells（P<0.05）;in addition,the localization of mtROS by the Mito SOX Red fluorescent probe revealed that NaF caused a significant increase in mtROS in BRL3A cells（P<0.05）;meanwhile,60 mg/L NaF upregulated the expression of TOMM20 compared with the control group in BRL3A cells（P<0.05）.4.The effect of RAPA on the autophagic flux of BRL3A cells:Western blot showed that,compared with the NaF group,the protein expression of LC3-II was increased and that of p62 was decreased in the combined intervention group of RAPA and NaF（P<0.05）;in addition,the m RFP-GFP-LC3 dual fluorescence found that an increased number of red puncta and a decreased number of yellow puncta（P<0.05）.5.The effect of RAPA on ferroptosis in BRL3A cells:The protein expression of GPX4 was increased and that of ACSL4 was decreased in the combined intervention group of RAPA and NaF compared with the NaF group（P<0.05）;the Fe²⁺content was significantly decreased;the content of GSH and SOD was increased;and the content of MDA was decreased（P<0.05）.6.The effect of Fer-1 on ferroptosis in BRL3A cells:Western blot results showed that,compared with the NaF group,the protein expression of GPX4 was increased and that of ACSL4 was decreased in the combined intervention group of Fer-1 and NaF（P<0.05）;the accumulation of intracellular Fe²⁺decreased;meanwhile,the contents of GSH and SOD increased,and the contents of MDA decreased（P<0.05）.7.The effect of Fer-1 on autophagic flux in BRL3A cells:Compared with the NaF group,the protein expression of LC3-II and p62 was decreased in the combined intervention group of Fer-1 and NaF（P<0.05）;meanwhile,the number of red puncta increased and the number of yellow puncta decreased（P<0.05）.8.The effect of Mito-TEMPO on ferroptosis in BRL3A cells:Western blot results showed that,compared with the NaF group,GPX4 expression was increased and ACSL4 expression was decreased in the combined intervention group of Mito-TEMPO and NaF（P<0.05）;the content of intracellular Fe²⁺decreased;meanwhile,the contents of GSH and SOD increased,and that of MDA decreased.9.The effect of Mito-TEMPO on autophagic flux in BRL3A cells:Compared with the NaF group,the expression levels of LC3-II and p62 were decreased in the combined intervention group of Mito-TEMPO and NaF（P<0.05）;the number of red puncta was increased,and the number of yellow puncta was decreased（P<0.05）.Conclusion:There is an interaction between NaF-induced autophagic flux blockage and ferroptosis in rat liver and BRL3A cells,and the process is regulated by mtROS,ultimately leading to liver injury.