Study On The Mechanism Of Hepatic Fibroblast Growth Factor 21 In Alleviating Liver And Kidney Injury Induced By 5-Fluorouracil Through Inhibiting Pathways Of Ferroptosis

Objective: 5-Fluorouracil(5-FU)is one of the most commonly used antimetabolic chemotherapeutic drugs in clinic in recent decades,and hepatorenal toxicity is its common side effect.This greatly limits the clinical application of 5-FU.Current studies have shown that 5-FU can cause liver and kidney injury through inducing apoptosis,inflammatory response,oxidative stress and lipid peroxidation,but the mechanism of it remains unclear.Fibroblast growth factor 21(FGF21)is a kind of protein mainly secreted by liver,which plays an important role in maintaining the homeostasis of glucose and lipid metabolism.Current studies have shown that FGF21 can ameliorate liver injury caused by decreased autophagy,endoplasmic reticulum stress,mitochondrial dysfunction and oxidative stress as a stress hormone.On the other hand,hepatic FGF21 is an important modulator in liver participated interorgan crosstalk.Studies have shown that the serum level of FGF21 significantly increased in patients diagnosed with chronic kidney disease with renal dysfunction,patients suffering from long-term dialysis and patients diagnose with acute kidney injury.However,the effects of hepatic FGF21 on renal function and renal diseases remain unclear.In the present study,5-FU was intraperitoneally injected into the mice to build a model of liver and kidney injury,which proved that the liver and kidney injury caused by 5-FU was related to ferroptosis.And then we explored the effect of hepatic FGF21 on 5-FU induced liver and kidney injury and the mechanism of it.At the same time,this study explored whether there was liver-kidney crosstalk in this pathological state mediated by FGF21.Methods: In the first part of this study,5-FU was intraperitoneally injected into the wild-type C57BL/6J mice to build a model of liver and kidney injury.After experiment,the serum samples,liver tissues and kidney tissues of mice were collected,and various indicators associated to ferroptosis were detected to explore the mechanism of liver and kidney injury caused by 5-FU in mice.HK-2 cells were treated with 5-FU to build the model of renal tubular cell injury induced by 5-FU in vitro,so as to further verify the mechanism of 5-FU induced kidney injury.In the second part of this study,whole body FGF21 knockout mice and wild-type mice were compared to explore the effect of FGF21 on 5-FU induced liver ferroptosis and its mechanism.Adenovirus was injected into the tail vein of mice to overexpress FGF21 in liver to further verify the effect of FGF21 on liver ferroptosis caused by 5-FU and its mechanism.In the third part of this study,whole body FGF21 knockout mice and wild-type mice were compared to explore the effect of FGF21 on 5-FU induced kidney ferroptosis and its mechanism.The effects of hepatic FGF21 on 5-FU induced kidney ferroptosis and the existence of liver-kidney crosstalk mediated by FGF21 in this pathological process were verified by injecting adenovirus into the tail vein of mice to overexpress FGF21 in liver and the study on liver specific FGF21 knockout mice.1.The changes of bodyweight,general morphology of liver and kidney and liver and kidney organ indexes of mice were observed.2.Pathological changes of mouse liver and kidney were detected by H&E staining and PAS staining.3.Serum ALT and AST assay kits were used to detect the changes of liver function in mice.Serum creatinine and BUN test kit were used to detect the changes of renal function in mice.4.Iron content in serum was detected by serum iron content detection kit.Iron content in liver and kidney tissues was detected by tissue iron detection kit.5.Quantitative RT-PCR was used to detect m RNA expressions of genes related to iron metabolism and ferroptosis in liver and kidney of mice.Western blot assay was used to detect the changes of iron metabolism and ferroptosis related proteins in liver and kidney of mice.6.ROS levels in liver and kidney tissues were detected by dihydrogen ethidium(DHE)superoxide anion fluorescence probe.7.Immunohistochemistry(IHC)was used to assess changes in the expression of 4-hydroxynonenal(4-HNE)in liver and kidney tissues.8.The contents of lipid peroxides malondialdehyde(MDA)and glutathione(GSH)in liver and kidney tissues were measured by detection kits.9.Iron content in HK-2 cells was detected by iron content detection kit.10.Quantitative RT-PCR was used to detect m RNA expression changes of genes related to iron metabolism and ferroptosis in HK-2 cells.Western blot assay was used to detect the changes of iron metabolism and ferroptosis related proteins in HK-2 cells.11.MDA and GSH contents in HK-2 cells were detected by detection kit.12.The cells were treated with 5-Fluorouracil(100 μM,24 h)after being pretreated with ferrostatin-1(Fer-1),deferoxamine(DFO),GSH and N-acetyl-L-cysteine(NAC).The ROS levels of cells were detected by DCFH-DA(2′,7′-dichlorofluorescin diacetate) probe and the survival rate of HK-2 cells was determined by CCK8.13.After Fer-1 pretreatment,the cells were treated with 5-Fluorouracil(100 μM,24 h).MDA content of the cells was detected by the kit,and iron metabolism and ferroptosis related protein expression were detected by Western blot.14.Iron particle deposition in liver tissues was detected by Prussian Blue Iron Stain Kit.15.The interaction between FGF21 and HO-1 at the protein level was verified by co-immunoprecipitation.16.The number of apoptotic bodies in liver tissues was detected by TUNEL staining.Results: Part I: 1.5-FU treatment caused weight loss and liver function impairment in mice.2.5-FU injection caused iron accumulation in the liver of mice.3.5-FU treatment induced ROS,MDA and 4-HNE(the product of lipid peroxidation)accumulation in the liver while the content of GSH was decreased.4.5-FU treatment caused cystic dilatation of renal tubules,decreased glomerular vascular cluster area and widened cystic space in kidney of mice.5-FU treatment caused kidney function impairment in mice.5.5-FU treatment induced increased non-ferritin binding iron intake and enhanced iron autophagy,leading to increased iron content in kidney of mice.6.5-FU treatment induced ROS accumulation in the kidney while the content of GSH was decreased.5-FU treatment inhibited the expression of NRF2 and downstream antioxidant factors regulated by NRF2.7.5-FU treatment induced MDA and 4-HNE accumulation in the kidney,and increased the expression of Acyl-Co A synthetase longchain family member 4(ACSL4).8.5-FU treatment caused iron accumulation in HK-2 cells.9.5-FU treatment decreased the expression of cystine/glutamate reverse transporter and the content of GSH while increased the content of MDA in HK-2 cells.10.Pretreatments with Fer-1,DFO,GSH and NAC improved ROS accumulation and decrease in cell viability of HK-2 cells induced by 5-FU.11.Fer-1 pretreatment improved MDA accumulation in HK-2 cells induced by 5-FU.12.Fer-1 pretreatment improved the changes of iron metabolism and ferroptosis related protein expression in HK-2 cells caused by 5-FU.Part II: 1.FGF21 knockout exacerbated the decline in the bodyweight and food intake of mice caused by 5-FU.2.FGF21 knockout aggravated the decline in organ index of liver caused by 5-FU.The serum AST and ALT levels were further increased by 5-FU treatment after FGF21 knockout.FGF21 knockout aggravated 5-FU-induced cell apoptosis in the liver of mice.3.FGF21 knockout aggravated the increase of iron intake and iron autophagy in the liver of mice caused by 5-FU treatment.4.FGF21 knockout aggravated the increase of ROS content and decrease of GSH content in liver of mice caused by 5-FU treatment.5.FGF21 knockout aggravated the accumulation of MDA and 4-HNE in liver of mice caused by 5-FU treatment.6.FGF21-overexpression failed to improve the decline in the bodyweight and food intake of mice caused by 5-FU.7.FGF21-overexpression in liver improved the decrease of liver organ index and liver function impairment induced by 5-FU treatment.8.FGF21-overexpression in liver improved the increase of iron intake and iron accumulation in liver mice caused by 5-FU.9.FGF21-overexpression in liver improved the ROS and MDA accumulation in liver of mice induced by 5-FU.10.FGF21 inhibited HO-1 overexpression in liver induced by 5-FU.11.FGF21 interacted with HO-1 at the protein level.Part III: 1.FGF21 knockout aggravated the decrease of glomerular vascular cluster area and the widening of capsule space in kidney of mice caused by 5-FU.The serum creatinine and BUN levels were further increased by 5-FU treatment after FGF21 knockout.2.FGF21 knockout aggravated the accumulation of iron in kidney of mice caused by the increase of non-ferritin binding iron intake and iron autophagy.3.FGF21 knockout aggravated the increase of ROS content and decrease of GSH content in kidney of mice caused by 5-FU treatment.FGF21 knockout robustly inhibited the expression of NRF2 after 5-FU treatment as well as the downstream antioxidant factors regulated by NRF2.4.FGF21 knockout aggravated the accumulation of MDA and 4-HNE and the increase of ACSL4 expression in kidney of mice after 5-FU treatment.5.FGF21-overexpression relieved the decrease of Bowman’s capsule area,glomerular vascular cluster area and the increase of cystic space in kidney tissue caused by 5-FU treatment.6.FGF21-overexpression relieved the accumulation of iron in kidney of mice caused by the increase of non-ferritin binding iron intake and iron autophagy.7.FGF21-overexpression relieved the increase of ROS content and decrease of GSH content in kidney of mice caused by 5-FU treatment.FGF21-overexpression induced the expression of NRF2 and rescued the decrease of NRF2 and the downstream antioxidant factors regulated by NRF2 induced by 5-FU treatment in kidney.8.The pathological changes of kidney in liver specific FGF21 knockout mice induced by 5-FU were more similar to those in FGF21 whole body knockout mice,and more serious than those in wild-type mice.9.After 5-FU treatment,the degree of iron accumulation in kidney of liver-specific FGF21 knockout mice was more similar to that of FGF21 whole body knockout mice,and more serious than that of wild-type mice.10.The expression of NRF2 and its downstream antioxidant factors in kidney of liver specific FGF21 knockout mice and FGF21 whole body knockout mice decreased,and the decrease was more obvious after 5-FU treatment.Conclusion: Ferroptosis may be a therapeutic target for liver and kidney injury caused by 5-FU.FGF21 is a regulator of ferroptosis and has a protective effect on organ injury associated with ferroptosis.Hepatic FGF21 regulates liver-kidney crosstalk attenuated kidney injury induced by 5-FU.