Alleviation Effect And Mechanism Of α-lipoic Acid On Iron Overload-induced Liver Damage In Rats

Iron is one of the essential trace elements for growth and development in animals.In livestock production,exogenous iron supplementation promote the growth and development of animals.However,the high amount of iron supplementation could induce iron overload in animals.Excessive cellular iron deposition increases the production of reactive oxygen species(ROS),resulting in cellular damage.The iron homeostasis of the animal body is mainly finely regulated by proteins related to iron uptake,iron storage and iron release.It is known that iron metabolism-related gene mutations induce iron overload in the body and cause related damage,but there are few reports on the effect of excess iron on the body’s iron homeostasis.α-lipoic acid(ALA)is the only known water and lipid soluble antioxidant,known as "universal antioxidant".In addition to scavenging reactive oxygen species and free radicals directly,ALA can improve the body’s antioxidant capacity by chelating metal ions and regenerating endogenous antioxidants.Previous studies have shown that ALA can reduce iron deposition in cerebral cortex of rats,liver and duodenum of zebrafish,and alleviates the oxidative damage of fibroblasts,astrocytes and rat kidneys induced by iron overload.Liver is an important "iron reservoir"in the animal body and is the main target organ of iron overload-induced damage.In diseases related to iron metabolism dysregulation,excessive hepatic iron deposition can aggravate hepatic pathogenic damage through oxidative mediators.Whether ALA can alleviate iron overload-induced hepatic iron deposition and liver damage in rats has not been reported,and its regulatory mechanism remains to be further studied.Therefore,in this study,male Sprague Dawley rats were used to investigate the effect of excess iron on iron metabolism and liver health in rats.The effects and mechanism of ALA on hepatic iron deposition and liver damage in iron overload rats were investigated in vivo and in vitro using male Sprague Dawley rats and BRL-3 A cells,respectively.1 Effects of excess iron on iron metabolism and liver health in ratsIron,as an essential trace element for animals,participates in many physiological and biochemical processes.However,excess iron is toxic to animals.The aim of this study was to explore the effects of excess iron on iron metabolism and liver health in rats.Male Sprague Dawley rats aged 6-7 weeks were randomly divided into 2 groups(n=8):control group(Control,Con)and iron-treated group(Iron,Fe).Con group:physiologic saline solution with the same volume as iron dextran was administered intraperitoneally(i.p.)every 3 days;Fe group:iron dextran was administered in a dose of 150 mg/kg i.p.every 3 days.Continuous treatment for 4 weeks,body weight and feed intake were recorded every 3 days.The results showed that,compared with the Con group,Fe significantly reduced body weight,average daily feed intake and average daily iron intake of the rats(P<0.01).Fe led to a significant increase in hemoglobin(HGB),platelet distribution width(PDW),neutrophil(Neu)levels,hematocrit(HCT),Neu(P<0.05)and monocytes(Mon)percentage(P=0.05),but significantly decreased lymphocyte(Lym)percentage(P<0.01)in the blood of rats;significantly elevated plasma iron levels and transferrin saturation(Ts)(P<0.01),but reduced plasma unsaturated iron binding capacity(UIBC)levels(P<0.01).In addition,compared with the Con group,Fe treatment significantly reduced the length of duodenal villus in rats;significantly increased the expression of duodenal iron metabolism-related proteins duodenal cytochrome b(DCYTB)(P<0.01),divalent metal transporter 1(DMT1)(P<0.05),ferritin light chain(FTL)(P<0.01)and FTH(P<0.05)in rats,but did not have a significant effect on ferroportin(FPN)protein expression.Prussian blue staining results indicated that Fe leads to iron deposition in the duodenum of rats.Compared with the Con group,Fe significantly increased rat liver weight,live/body ratio,and hepatic iron content(P<0.01);HE staining results indicated that Fe leads to expansion of the central venous sinus of the rat liver and vacuolarization in hepatocytes,indicating that excess iron induces liver damage.The results of real-time fluorescence quantitative RT-PCR and western blot showed that compared with the Con group,the hepatic iron uptake-related proteins transferrin receptors 1(TFR1),DMT1,ZRT/IRT-like protein 8(ZIP8)and ZRT/IRT-like protein 14(ZIP14)(P<0.01)were significantly down-regulated,and the iron storage protein FTL(P<0.01)and FTH(P<0.05)were significantly up-regulated,but had no significant effect on the expression of iron-releasing protein FPN and hepcidin(P>0.05)in Fe group.In addition,Fe significantly increased the plasma levels of aspartate aminotransferase(AST)/alanine aminotransferase(ALT),alkaline phosphatase(ALP),total bilirubin(Total bilirubin)(P<0.05),as well as hepatic ROS(P<0.05)and malondialdehyde(MDA)levels(P<0.01).The results of transmission electron microscopy showed that Fe induced swelling of mitochondria and increased the number of autophagosomes in liver of rats,which further indicated that excessive iron treatment leads to liver damage.The above results indicate that excessive iron treatment leads to growth arrest in rats,increases circulating iron levels,up-regulates the expression of key proteins in duodenal and hepatic iron metabolism,resulting in iron deposition in the duodenum and liver,inducing oxidative stress and cause liver damage in rats.2 Effects of α-lipoic acid on hepatic iron deposition and liver damage in iron-overloaded ratsALA is both water and lipid soluble antioxidant,which reduces iron deposition in cerebral cortex of rats,liver and duodenum of zebrafish,and improve the oxidative damage of fibroblasts,astrocytes and rat kidneys induced by iron overload.However,it is unclear whether it can alleviate hepatic iron deposition and iron-induced liver damage in ironoverloaded rats.The aim of this study was to explore the effects of ALA on hepatic iron deposition and liver damage in iron-overload rats.Male Sprague Dawley rats aged 6-7 weeks were randomly divided into 4 groups(n=10):control group(Con),iron overload group(Fe),α-lipoic acid group(ALA),and α-lipoic acid+iron overload group(ALA+Fe).The Con group and ALA group were injected with normal saline(the same volume as iron dextran)every 3 days,the Fe group and ALA+Fe group were injected with iron dextran(150 mg/kg)every 3 days,ALA(50 mg/kg)or edible oil(the same volume as ALA)was administered i.p.1 h before iron dextran and saline injection.Continuous treatment for 4 weeks,body weight and food intake were recorded every 3 days.The results showed that compared with Fe group,ALA significantly alleviated the reduction of body weight,average daily food intake,and average daily iron intake in iron-overloaded rats(P<0.05);markedly alleviated the increase of plasma iron levels(P<0.05)and hepatic iron content(P<0.05);greatly alleviated the increase of AST/ALT,ALP,and TBIL of plasma induced by iron overload(P<0.05);significantly alleviated the increase of hepatic ROS and MDA levels(P<0.05),and significantly increased the hepatic antioxidant-related enzyme activities of glutathione peroxidas(GSH-Px)and catalase(CAT)(P<0.05).The above results suggest that ALA alleviates the hepatic oxidative damage induced by iron overload through activating the hepatic antioxidant pathway in rats.At the same time,the results of transmission electron microscopy showed that ALA significantly alleviated the swelling of mitochondria and the increase of autophagosomes in liver of iron overloaded rats,indicating that the hepatic oxidative damage of rats induced by iron overload may be related to excessive autophagy.The proteomic results showed that compared with the Con group,the expression of hepatic autophagy-related proteins cathepsin B(CTSB),cathepsin L(CTSL),cathepsin D(CTSD),cathepsin D(CTSD),lysosomal-associated membrane protein 1(LAMP1),iron metabolismrelated proteins heme oxygenase 1(HO1),FTL and FTH were significantly up-regulated in Fe group rats,but were significantly up-regulated in the ALA+Fe group,and there was an interaction between these proteins.In addition,ALA significantly increased hepatic NAD(P)H quinone dehydrogenase 1(NQO1)protein expression of iron-overloaded rats(P<0.05),suggesting that NQO1 may be involved in the antioxidative effect of ALA on iron overload-induced hepatic oxidative damage in rats.The above results indicate that ALA significantly alleviates iron overload-induced growth inhibition in rats,decreases the levels of circulating iron in iron-overloaded rats,alleviates hepatic iron deposition.ALA alleviates liver damage may be related to NQO1-mediated antioxidant function and autophagy inhibition in iron-overloaded rats.3 Regulation and mechanism of α-lipoic acid on intestinal and hepatic iron metabolism in iron-overloaded ratsStudies have shown that iron homeostasis is mainly maintained by regulating circulating iron levels,which involves duodenal iron absorption and hepatic iron storage.Our previous studies found that ALA significantly reduces hepatic iron content and circulating iron levels in iron-overloaded rats,but the regulatory mechanism remains unclear.Since there is no known physiological mechanism that controls iron excretion,and macrophage-mediated iron recycling is not sufficient to maintain erythropoiesis over long term,efficient absorption of dietary iron in the duodenum is critical for body iron homeostasis.This study aimed to explore the regulatory effect and the mechanism of ALA on intestinal and hepatic iron metabolism in iron-overloaded rats.The experimental design is the same as the previous section.Prussian blue staining confirmed that ALA could significantly alleviate hepatic iron deposition induced by iron overload.Compared with the control group,Fe greatly reduced the expression of hepatic iron uptake proteins TFR1,DMT1,ZIP8,ZIP 14 and the regulators of iron metabolism key proteins IRP1,IRP2 and HIF-1α in rats(P<0.05),but significantly increased the expression of TFR2,iron storage proteins(FTL and FTH)and ferritin autophagy receptor NCOA4(P<0.05).Compared with Fe group,ALA significantly downregulated the expression of hepatic iron storage proteins(FTL and FTH)in rats(P<0.05),significantly up-regulated the expression of the iron uptake protein ZIP14(P<0.05),and had no effect on hepatic iron release,other iron metabolism proteins and regulatory factors(P>0.05).The duodenum is the main site for the absorption of dietary iron into the blood circulation.Compared with the Fe group,ALA significantly alleviated the developmental block of duodenal villi(P<0.05)and the increase in the expression of iron storage proteins FTL and FTH(P<0.05)in iron-overloaded rats.In addition,compared with the Fe group,ALA had no significant effect on the mRNA expression of Dmt1,a key gene for duodenal iron absorption(P>0.05),but significantly alleviated the increase in duodenal DMT1 protein expression induced by iron overload(P<0.05),suggesting that ALA regulates the protein expression of DMT 1 through a post-transcriptional pathway.Since DMT1 is a key protein that determines intestinal iron absorption,we further studied its post-transcriptional regulation mechanism.Bioinformatics tools were used to predict and screen 8 miRNAs that target and regulate DMT1.RT-PCR detection showed that ALA significantly alleviated the decrease of duodenal miR-15b-5p and miR-195-5p expression in iron-overloaded rats(P<0.05).DMT1 3’UTR wild-type and mutant dual-luciferase reporter plasmids of rats were constructed in vitro,and the target miRNAs mimics or miRNAs inhibitors were cotransfected into 293T cells to verify the targeted regulatory effect of miR-15b-5p and miR195-5p on DMT1.The above results indicate that ALA significantly down-regulates the expression of DMT1,a key protein for iron absorption in the duodenum,inhibites duodenal iron efficient absorption,contributing to decrease circulating iron levels,and reduce liver iron deposition in iron-overloaded rats.The downregulation of duodenal DMT1 protein expression was achieved by post-transcriptional mechanisms mediated by miR-15b-5p and miR-195-5p.4 Mechanism of α-lipoic acid alleviating iron overload-induced hepatocyte damage through NQO1 pathway in ratsPrevious study showed that iron overload-induced liver damage in rats may be related to excessive autophagy,which can be effectively alleviate by ALA,possibly by targeting NQO1.However,whether ALA alleviates liver damage in rats through NQO1 and its potential mechanism is unclear.In this study,BRL-3A cells,a normal hepatocytes of rats,were treated with 2 mM Ferric ammonium citrate(FAC)for 24 h to establish a hepatocyte iron overload model in vitro.The cells were treated with 0.5 mM and 1 mM ALA,and 10μM NQO1 inhibitor Dicoumarol(DIC)or transfected NQO1 siRNA to verify the alleviation effect and mechanism of ALA on liver damage in iron overloaded rats.The results showed that FAC treatment of BRL-3A cells for 24 h significantly decreased cell viability and increased intracellular iron and ROS levels(P<0.01),indicating that iron overload is toxic to hepatocytes in rats.ALA had no significant effect on intracellular iron levels(P>0.05),indicating that ALA was not directly involved in hepatic iron chelation in rats.However,ALA significantly alleviated the decrease in cell viability(P<0.05),the increase in intracellular ROS levles(P<0.05),and the decrease in NQO1 protein expression(P<0.01)induced by FAC,and the alleviation effect of ALA on FAC-treated hepatocytes were significantly reversed after DIC treatment and transfection of NQO1 siRNA(P<0.01),indicating that ALA exerted an antioxidant effect through NQO1,thereby alleviating the hepatic oxidative damage of rats induced by iron overload.ALA significantly alleviated the increases in the protein expressions of intracellular autophagy-related proteins LC3B(P<0.05),BECLIN1(P<0.01),LAMP1(P<0.01)and CTSD(P<0.01)induced by FAC.However,both DIC treatment and transfection of NQO1 siRNA significantly reversed the alleviation effect of ALA on the protein expression of LC3B(P<0.05)and LAMP1(P<0.01).The above results indicate ALA alleviates the damage of hepatocytes by up-regulating the expression of the hepatic key antioxidant gene NQO1,reducing the production of ROS induced by iron overload,and inhibiting excessive autophagy.In conclusion,ALA alleviates growth inhibition,decreases duodenal iron content and plasma iron levels,reduces hepatic iron deposition,and alleviates liver damage in ironoverloaded rats.ALA down-regulates the expression of DMT1,a key protein for iron absorption in the duodenum,through miR-15b-5p and miR-195-5 post-transcriptional pathways,inhibits duodenal iron absorption,reduces circulating iron levels and hepatic iron deposition in rats.ALA alleviates liver damage in rats with iron overload by upregulating the expression of the hepatic key antioxidant protein NQO1,reducing the generation of ROS induced by iron overload,and inhibiting excessive cellular autophagy.