The Role Of FGF21 In High Level Iron Induced Hepatocyte Ferroptosis And The Mechanism Study

Iron is an essential trace element to maintain the health of the body,and the liver as an iron storage organ maintains the body’s iron balance.Disturbance of its iron metabolism can lead to the occurrence of diseases(such as liver injury,liver fibrosis and liver cancer).Iron overload can lead to cell ferroptosis,which is mainly characterized by accumulation of reactive oxygen radicals(ROS)and lipid peroxidation produced by polyunsaturated fatty acids.Many diseases are accompanied by ferroptosis,so it can be used as a new direction for disease treatment.FGF21 has the effect of reducing ROS and anti-stress,but whether FGF21 can inhibit iron overload-induced hepatocyte ferroptosis and liver injury has not been reported.The purpose of this research was to study the effect of high level iron on iron metabolism,ferroptosis and FGF21 expression in hepatocytes and liver tissues,and to further study the effect and possible mechanism of FGF21 on cellular ferroptosis.The details are as follows:Experiment 1.Effect of high concentration iron on ferroptosis and FGF21 expression in primary hepatocytesTreat primary hepatocytes with ferric citrate(FAC)for 24 h.The results showed that:1000 μM FAC treatment for 24 h significantly reduced the gene expression of iron transporter receptor TFR1(P < 0.05);increased the gene expression of iron efflux FPN and iron storage regulators FTH and FTL(P < 0.05);increase the free iron content of cells(P <0.05).Subsequent studies found that iron overload can significantly increase cellular oxidative stress and lipid peroxidation levels(P < 0.01).The expressions of GPX4 and PTGS2,the key factors of ferroptosis,were determined.Compared with the Con group,the gene and protein expressions of GPX4 in the high iron group were significantly reduced(P< 0.05);while the expression of PTGS2 gene was significantly up-regulated(P < 0.05);The relative expression levels of NRF2 gene and protein were significantly reduced(P < 0.05),CAT gene expression was significantly reduced(P < 0.05),and HO-1 expression was significantly increased(P < 0.05).In addition,in the high iron group,the content of MDA in the cell culture fluid was significantly increased(P < 0.05),and the content of lipid peroxidation product GSH was significantly reduced(P < 0.05).Further research showed that compared with the control group,iron overload significantly increased the gene and protein expression levels of FGF21(P < 0.05),and the content of FGF21 in the cell culture medium of the iron-treated group was also significantly higher(P < 0.05).The above results show that:(1)iron treatment with high concentration will lead to iron overload in hepatocytes and up-regulate the expression of FGF21.(2)iron overload will induce ferroptosis in primary hepatocytes.Experiment 2.Effect of high-dose iron on liver ferroptosis and FGF21 expression14-week-old male C57BL/6J rats were randomly divided into two groups,and iron dextran(ID group,0.1 mg/g BW,n = 8)or PBS(Con group,control,n = 8)were injected intraperitoneally once a day.For 13 consecutive days.The mice were starved overnight on the 13 th night,and the next morning the blood glucose concentration was measured.Then samples of mouse heart blood,liver,spleen and adipose tissue were collected.The results showed that high-dose iron injection did not affect the intake of mice,but there was a tendency to reduce body weight,and significantly reduce the average daily weight gain and fasting blood glucose of mice(P < 0.05),while increasing liver and spleen weight(P < 0.05),and reduce the weight of adipose tissue compared with the control group(P < 0.05).Further research shows that high-dose iron treatment does not change the serum triglyceride content of mice,but significantly reduces the HDL-C content(P < 0.01),and significantly increases the LDL-C content(P < 0.01),and has the down trend of serum total cholesterol(P = 0.08).In addition,the serum ALT and AST contents of the ID group were significantly higher than those of the control group(P < 0.01),and the H&E staining results of liver tissue also showed that high-dose iron injection caused severe liver damage in mice.The tissue iron,serum iron,TIBC and TF% of mice injected with high-dose iron were significantly higher than that of the control group(P < 0.01),and the TF% was greater than 60%,and the serum UIBC was significant decrease(P < 0.01).Compared with the control group,ID group was significantly reduced the expression of TFR1 gene and protein(P < 0.01),while the expression of FPN gene and protein responsible for iron excretion was significantly increased(P < 0.01),The expression of iron storage protein complex FTL and FTH was up-regulated(P < 0.01),and the m RNA expression of HAMP,an indicator of iron homeostasis,increased significantly(P < 0.01).In addition,compared with the control group,the expression levels of ferroptosisrelated genes SLC7 ALL,PTGS2 and GPX4,and the oxidative stress activation genes NRF2 and HO-1 in the ID group were significantly increased(P < 0.05);the expression level of the antioxidant gene CAT and Enzyme activity was significantly reduced(P < 0.05);MDA and hydroxyl radical content were significantly increased(P < 0.05),indicating that the body’s iron overload led to increased levels of oxidative stress,lipid peroxidation and ferroptosis in liver.Further research found that,compared with the control group,the expression level of liver FGF21 was significantly higher in the ID group(P < 0.05).The above results indicate that high-dose iron treatment will increase the body’s oxidative stress and lipid peroxidation levels,and cause ferroptosis in liver and liver damage.At the same time,high-dose iron treatment increased the expression of FGF21 in the liver,suggesting that it may be involved in the regulation of ferroptosis.Experiment 3.The role and possible mechanism of FGF21 in hepatocytes ferroptosis induced by high concentration of ironPrimary hepatocytes were treated with FAC(1000 μM)and 100 ng/m L FGF21 recombinant protein to study the role and possible mechanism of FGF21 in high iron-induced ferroptosis and possible mechanism.The results showed that compared with the highconcentration iron treatment group,the addition of FGF21 recombinant protein can significantly reduce FAC-induced free iron content in cells(P < 0.05),but increased the expression of iron efflux gene FPN and iron storage gene FTH(P < 0.05).In addition,the addition of recombinant protein FGF21 restored the high-dose iron-reduced GPX4,NRF2,HO-1 and CAT gene expression(P < 0.05);and inhibited the high-dose iron up-regulated PTGS2 gene expression(P < 0.05),and the high-dose iron inhibiting NRF2 protein phosphorylation was also reversed by FGF21(P < 0.05).In addition,compared with the FAC group,the FGF21 recombinant protein and FAC group,the cell MDA content was significantly reduced(P < 0.05),GSH content was significantly increased(P < 0.05),cell lipid peroxidation and oxidative stress levels decreased significantly(P < 0.01).In summary,high levels of iron will stimulate increased expression and secretion of FGF21 in the liver,and supplementation of FGF21 in vitro can inhibit the occurrence of oxidative stress and lipid peroxidation,thereby protecting the liver from ferroptosis induced by high levels of iron.