FGF21 alleviates acute liver injury by inducing the SIRT1-autophagy signalling pathway

Abstract

Liver injury can lead to different hepatic diseases, which are the mainly causes of high global mortality and morbidity. Autophagy and Sirtuin type 1 (SIRT1) have been shown protective effects in response to liver injury. Previous studies have showed that Fibroblast growth factor 21 (FGF21) could alleviate acute liver injury (ALI), but the mechanism remains unclear. Here, we verified the relationship among FGF21, autophagy and SIRT1 in carbon tetrachloride (CCl₄ )-induced ALI. We established CCl₄ -induced ALI models in C57BL/6 mice and the L02 cell line. The results showed that FGF21 was robustly induced in response to stress during the development of ALI. After exogenous FGF21 treatment in ALI models, liver damage in ALI mice was significantly reduced, as well as serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Consistently, FGF21 also greatly reduced the levels of ALT, AST, pro-inflammatory cytokines interleukin 6 (IL6) and tumour necrosis factor-alpha (TNFα) in ALI cell lines. Mechanistically, exogenous FGF21 treatment efficiently upregulated the expression of autophagy marker microtubule-associated protein light chain-3 beta (LC3 II) and autophagy key molecule coiled-coil myosin-like BCL2-interacting protein (Beclin1), which was accompanied by alleviating hepatotoxicity in CCl₄ -treated wild-type mice. Then, we examined how FGF21 induced autophagy expression and found that SIRT1 was also upregulated by FGF21 treatment. To further verify our results, we constructed an anti-SIRT1 lentit-RNAi to inhibit SIRT1 expression in mice and L02 cells, which reversed the protective effect of FGF21 on ALI. In summary, these results indicate that FGF21 alleviates ALI by enhancing SIRT1-mediated autophagy.

Keywords: Beclin1; CCl4; FGF21; LC3 II; SIRT1; autophagy; liver injury.

FIGURE 1

Upregulation of FGF21 expression during ALI. (A) Changes in FGF21 expression in serum at 6 h and 24 h after CCl₄ injection. (B and C) The expression level of FGF21 in the liver by western blot. (D) Immunohistochemical results of FGF21 in the liver after 6 and 24 h of CCl₄ treatment (scale bar = 50 µm). (E and G) Changes in FGF21 expression in normal hepatocytes L02 treated with different concentrations of CCl₄ for 4 h. (F and H) Changes in FGF21 expression in normal hepatocytes L02 after administration of 10 mmol/L CCl₄ at different time points. *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 2

Exogenous recombinant FGF21 alleviated ALI. (A and B) Serum transaminase expression in mice injected with CCl₄ for 2 h and after that with various doses of exogenous recombinant FGF21. (C) Haematoxylin and eosin staining of the liver after administration of various doses of recombinant exogenous FGF21 (scale bar = 50 µm). (D) The organelles' changes in hepatocytes were detected by transmission electron microscopy after administration of various doses of exogenous recombinant FGF21 (scale bar = 1 µm). (E and F) Transaminase expression in the L02 cells treated with CCl₄ after administering 100 ng/mL exogenous recombinant FGF21 for 2 h. (G–I) The expression of IL6 and TNFα in the L02 cells treated with CCl₄ after administering 100 ng/ml exogenous recombinant FGF21 for 2 h. *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 3

Autophagy was induced during FGF21 alleviation of ALI. (A–C) Mice were injected with CCl₄ for 2 h, followed by various doses of recombinant exogenous FGF21. Then, we measured the expression of Beclin1 and LC3 II in the liver. (D–F) L02 cells treated with CCl₄ were exposed to various doses of exogenous recombinant FGF21, and the expression levels of Beclin1 and LC3 II were measured (The beta‐actin band shown in (D) was the same as the one shown in Figure 4D because Beclin1 and LC3 II in (D) and SIRT1 in Figure 4D were run on the same blot.). *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 4

SIRT1 was upregulated in the process of alleviating ALI. (A and B) Various doses of recombinant exogenous FGF21 protein were given to the CCl₄ injury group, and the expression of FGF21 in the liver was analysed using western blot (The beta‐actin band shown in (D) was the same as the one shown in Figure 3D because SIRT1 in (D) and Beclin1 and LC3 II in Figure 3D were run on the same blot.). (C) Various doses of recombinant exogenous FGF21 were given to the CCl₄ injury group, and the immunohistochemical staining of SIRT1 in the liver was performed (scale bar = 50 µm). (D and F) SIRT1 expression in L02 cells treated with various concentrations of FGF21 for 2 h after CCl₄ treatment. (E and G) L02 cells damaged by CCl₄ were treated with 100 ng/ml FGF21 for various periods, and SIRT1 expression was determined. *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 5

siRNA‐mediated SIRT1 downregulation in liver or hepatocytes significantly suppressed the FGF21‐related upregulation of autophagy. (A–C) Mice were injected with siRNA‐mediated SIRT1 lentivirus, then CCl₄ and/or FGF21 were given to the corresponding groups. Beclin1 and LC3 II in the liver were assayed using western blot. (D and E) The immunohistochemical results of Beclin1 and LC3 II in the liver (scale bar = 50 µm). (F) Immunofluorescence staining of Beclin1 and LC3 II in the liver (scale bar = 50 µm). (G–I) L02 cells were transfected with the siRNA‐mediated SIRT1 lentivirus, then CCl₄ and/or FGF21 were given to the corresponding groups. Next, Beclin1 and LC3 II in the cells were measured. *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 6

The downregulation of SIRT1 mediated by siRNA in liver or hepatocytes significantly reversed the ameliorative effect of FGF21 on ALI. (A and B) Mice were injected with siRNA‐mediated SIRT1 lentivirus, then CCl₄ and FGF21 were given to the corresponding groups. We then measured serum transaminase levels. (C) Haematoxylin and eosin staining of the liver (scale bar = 100 µm). (D and E) After transfecting L02 cells with siRNA‐mediated SIRT1 lentivirus, the corresponding groups were treated with CCl₄ and/or FGF21, and then we measured serum transaminase levels. (F and G) The expression of IL‐6 and TNF‐α in the cells. *p < 0.05, **p < 0.01, ***p < 0.001