Fibroblast growth factor 1 alleviates acetaminophen-induced acute liver injury in mice via improving mitochondrial oxidative stress
PENG Ziying1, NAN Yan2, XIE Xianhai3, LI Xiaoxiao1, GAO Ling1, ZHANG Lei1, YAO Ruina1, YING Lei1, WANG Yang1.
1.Department of Pathophysiology, Wenzhou Medical University,Wenzhou 325035, China; 2.Department of Neonatology, the Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China; 3.Department of Trauma and Emergency, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China
Abstract：Objective: To explore the function and mechanism of fibroblast growth factor 1 (FGF1) on alleviating acute liver injury induced by acetaminophen (APAP) in mice. Methods: ①C57BL/6J mice were randomly divided as the control group, APAP 3 h group, APAP 6 h group, APAP 12 h group, and APAP 24 h group. After intraperitoneal injection of 500 mg/kg APAP, the mice were killed at the corresponding time points.The most significant time point of acute liver injury in APAP mice was determined by observing the content of reactive oxygen species and the number of inflammatory cells using liver DHE staining and F4/80 staining.②C57BL/6J mice were randomly divided as the control group, APAP group, APAP+FGF1 1 mg/kg group,APAP+FGF1 2 mg/kg group, and APAP+FGF1 5 mg/kg group. After 1 h of modeling with a 500 mg/kg dose of APAP, the corresponding dose of FGF1 was intraperitoneally injected. The mice were euthanized 6 h after modeling. The damage area was observed by HE staining of the liver. Liver function level was detected by ALT and AST kits. Oxidative stress levels of the liver were detected by DHE straining, MDA and SOD kits, and gene expression of CAT, SOD2 and NRF2. Inflammation level of the liver was detected by F4/80 staining, and gene expression of CCL5, TNFα and IL-6. Based on the above results, the optimal FGF1 dose for the treatment of acute liver injury induced by APAP was determined; ③C57BL/6J mice were randomly divided as the control group, 3 h APAP group, 3 h APAP+FGF1 group, 6 h APAP group, 6 h APAP+FGF1 group. After modeling with a 500 mg/kg dose of APAP for 1 h, a dose of 2 mg/kg FGF1 was administered intraperitoneally. The mice were euthanized at the corresponding time points after modeling. Then the reagent kit was used to detect ATP content,and Western blot was used to detect the relative expression level of Total Oxphos (CV-ATP5A, CIII-UQCRC2,CIV-MTCO1, CII-SDHB, and CII-NDUFB8) and 3NT proteins. Based on the above results, the mechanism of FGF1 alleviating acute liver injury induced by APAP was investigated; ④HepG2 cells were treated with different concentrations of APAP (0, 1, 5, 10, 15, 20, 40 and 60 mmol/L) for 24 h, and the cell activity was measured by cytotoxicity assay (CCK-8); ⑤In MEM basic culture medium containing 20 mmol/L APAP, different concentrations of FGF1 were added, resulting in FGF1 final concentrations of 0, 0.5, 1, 5 and 10 mg/mL. After 24 h of treatment, the cell activity was detected by CCK-8. Results: ①In acute liver injury induced by APAP in mice,the level of reactive oxygen species significantly increased at 3 h after modeling and reached its peak at 6 h; The number of inflammatory cells significantly increased and reached its maximum at 6 h, and began to decrease at 12 and 24 h; ②In acute liver injury induced by APAP in mice, the expression level of FGF1 protein was significantly reduced (P<0.01); ③After treatment with different doses of FGF1, HE staining of the liver showed a reduction in the area of damage, and significant downregulation of serum ALT and AST; DHE staining of the liver revealed a significant decrease in reactive oxygen species level, downregulation of MDA, upregulation of SOD, and upregulation of gene expression of CAT, SOD2 and NRF2 (P<0.01); F4/80 staining of the liver revealed a significant decrease in the number of inflammatory cells, and downregulation of gene expression of CCL5, TNF α and IL-6 (P<0.01). The overall results showed that the best treatment effect was achieved at a dose of 2 mg/kg; ④APAP at concentrations of 15, 20, 40 and 60 mmol/L can significantly reduce HepG2 cell activity (CCK-8 experiment), and different doses of FGF1 can upregulate the cell activity in HepG2 cell damage caused by 20 mmol/L APAP (P<0.01); ⑤FGF1 can significantly upregulate the ATP content of the liver in mice at 3 h and 6 h liver injury induced by APAP (P<0.05), upregulate the expression of 6 h CIV-MTCO1, CII-SDHB and CINDUFB8 proteins in the Total Oxphos complex, and downregulate the expression of 3 h and 6 h 3-NT proteins (P<0.05). Conclusion: FGF1 can alleviate acute APAP-induced liver injury in mice via inhibiting mitochondrial
oxidative stress.
