ANGPTL8 accelerates liver fibrosis mediated by HFD-induced inflammatory activity via LILRB2/ERK signaling pathways

Figures

• 

  Fig. 1. Clinical significance of ANGPTL8 in patients with cirrhosis. (A) ANGPTL8 concentration in the serum of healthy volunteers (control), patients with hepatic-fibrosis (hepatocirrhosis), and patients with hepatocellular carcinoma. ***p < 0.001 (one-way ANOVA). (B-L) Cirrhotic patients were divided into three different categories according to their LSM (liver stiffness measurement) as assessed by vibration-controlled transient elastography (FibroScan). Patient characteristics, such as sex, weight, age andserum concentrations of Tbil, ALT, AST, Alb, WBC, PLT, and ANGPTL8 in different groups of patients. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA).

• 

  Fig. 2. ANGPTL8 deficiency suppresses HFD-induced hepatic steatosis. (A) The mRNA expression of ANGPTL8 in the liver of mice induced by NCD (normal chow diet) and HFD (high fatty diet) (left is fasting, right is feeding, n = 3 per group). ***p < 0.001 (Student's t-test). (B-D) The mRNA expression of ANGPTL8 in HepG2 (B), H22 (C) and primary mouse liver hepatocytes (D) treated with the fatty acid oleic acid (OA), **p < 0.01, ****p < 0.0001(Student's t-test). (E&F) Hepatic steatosis was confirmed by macroscopic examination in ANGPTL8 KO or control mice (WT) given with NCD or HFD for 28 (E) or 36 weeks (F), n = 8 per group. (G&H) Hepatic steatosis was confirmed by H&E staining in ANGPTL8 KO or control mice (WT) given NCD or HFD for 28 or 36 weeks, (n = 8 per group), Scale bars, 100 μm and 50 μm for H&E staining. (I) Quantitative analysis of Hepatic TG in ANGPTL8 KO and WT mice treated with NCD or HFD 36 weeks (upper is NCD, lower is HFD, n = 3 per group). *p < 0.05(Student's t-test).

• 

  Fig. 3. ANGPTL8 deficiency attenuates HFD-induced liver fibrosis. (A) Liver fibrosis was evaluated by H&E staining, Masson, Sirius red staining, and IHC for the fibrosis markers (α-SMA, Collagen I) and macrophage activation markers (F4/80). Scale bars, 50 μm and 20 μm for H&E staining, Sirius red staining and IHC in ANGPTL8 KO or control mice (WT) given HFD for 28 weeks. Masson and Sirius red staining, and IHC for α-SMA, Collagen I, and F4/80 (above). Five images of each liver and five livers from different mice were quantified for each group (below), n = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001(Student's t-test). (B) Liver fibrosis was evaluated by H&E staining, Masson, Sirius red staining and IHC for α-SMA, Collagen I, and F4/80. Scale bars, 50 μm and 20 μm for H&E staining, Sirius red staining and IHC in ANGPTL8 KO or control mice (WT) given HFD for 36 weeks. The Masson, Sirius red staining and IHC for α-SMA, Collagen-I, and F4/80 positive liver fibrosis were measured (above). Five images of each liver and five livers from different mice were quantified for each group (below), n = 5 per group. *p < 0.05, ***p < 0.001(Student's t-test). (C) The protein levels of α-SMA, Collagen…

• 

  Fig. 4. ANGPTL8 knockout inhibits HFD-stimulated inflammatory activity in mice. (A) Volcano plot analysis of the RNA sequencing results for the indicated samples. Stringent set (p < 0.05) of hepatic genes in HFD-fed ANGPTL8 KO mice compared with HFD-fed WT mice (n = 3, per group). (B) GO analysis of the RNA sequencing results between the two groups. (C&D) The protein levels of F4/80, IL-6, IL-1β, and Collagen I were determined by western blot of protein extracts from the livers of ANGPTL8 KO or control mice (WT) after fasting and treated for 12 h with NCD (n = 3). (C) β-Tubulin was used as an internal control. Protein expression was normalized to that of β-Tubulin (D). *p < 0.05, **p < 0.01(Student's t-test). (E&F) The protein levels of F4/80, IL-6, IL-1β, and Collagen I in the livers of ANGPTL8 KO or control (WT) mice after 2 h feeding with NCD were determined by western blot. (E). β-Tubulin was used as an internal control. Protein expression was normalized to that of β-Tubulin (F). n = 3 per group. (G&H) Mouse inflammatory cytokine antibody array (protein chip, GSM-INF-1, Ray Biotech Inc.) detection of inflammatory factors in the serum of ANGPTL8 KO or control (WT) mice fed with HFD…

• 

  Fig. 5. ANGPTL8 directly activates HSCs by interacting with the receptor LILRB2 on hepatic stellate cells. (A) Conceptual model for ANGPTL8-mediated activation of hepatic stellate cells. (B) Representative images of oil red O staining (a) and intracellular TG levels (b) in ANGPTL8 OE and control LX2 cells. scale bar: 50 μm and 20 μm. (C-E) Western blot analysis of rANGPTL8- (C&D) and ANGPTL8 OE- (E) induced protein changes of Collagen I, α-SMA, and TGFβ1 in LX2 cells, the ratio to GAPDH is shown in the middle as numbers, and the numbers represent the average of 3 independent experiments. (F) Immunofluorescence detection of Desmin, α-SMA, Collagen I and TGFβ1 in mouse primary hepatic stellate cells induced by ANGPTL8 recombinant protein. The mouse primary hepatic stellate cells were fixed and stained with antibodies Desmin, α-SMA, Collagen I, TGFβ 1 and visualized with secondary antibody. Microphotographs were taken with a fluorescence microscope. scale bar:50 μm. (G) Immunoprecipitation examination of the interaction between ANGPTL8 and LILRB2. The Co-IP experiment was performed using anti-ANGPTL8 antibody in LX2 cell lysates and then analyzed by western blot with anti-ANGPTL8 and…

• 

  Fig. 6. ANGPTL8-induced HSC activation by LILRB2/ERK signaling modulates autophagy. (A) The protein levels of ERK1/2, p-ERK1/2, NF-κB, p-NF-κB, and PirB were determined by western blot of extracts from the livers of ANGPTL8 KO and control mice (WT) mice given HFD for 36 weeks. The ratio to β-tubulin is shown in the middle as numbers, and the numbers represent the average of 3 independent experiments. *p < 0.05(Student's t-test). (B&C) The protein levels of ERK1/2, p-ERK1/2, NF-κB, and p-NF-κB were determined by western blot in LX2 cells treated with rANGPTL8 (B) and ANGPTL8 OE (C). The ratio to GAPDH or β-tubulin is shown in the middle as numbers, and the numbers represent the average of 3 independent experiments. (D&E) ANGPTL8 activates the autophagy process by interacting with LILRB2. The protein levels of LC3-II/LC3-I and Beclin-1 were determined by western blot in LX2 cells treated with exogenous ANGPTL8 protein. The ratio to β-tubulin or LC3-II/LC3-I is shown in the middle as numbers, and the numbers represent the average of 3 independent experiments (D). mRFP-GFP-LC3 expression lentivirus was used to infect LX2 cells for 24 h, then the cells were treated with ANGPTL8 protein for…

• 

  Fig. 7. Effect of metformin on HFD-induced liver fibrosis. (A) Analysis of the effect of metformin (Met) on ANGPTL8 expression in mouse liver cells by RT-PCR and qPCR. (B) Schematic diagram of Met treatment of HFD-induced liver fibrosis in mice. (C) Macroscopic examination of livers to analyze the potential therapeutic effects of metformin on HFD-induced liver fibrosis in WT mice (n = 6 per group). (D) Level in Met and PBS treated mice (*p < 0.05, n = 6 per group). Liver fibrosis was evaluated by H&E staining, Masson, Sirius red staining and IHC for α-SMA, Collagen I, and F4/80. Scale bars, 50 μm and 20 μm for H&E staining, Sirius red staining and IHC in ANGPTL8KO or WT mice given HFD for 24 weeks and treated with Met for 4 weeks. Five images of each liver, and five livers from different mice, were quantified for each group (*p < 0.05, n = 6 per group). (E) The mRNA expression of α-SMA and TGFβ1 was determined by qPCR in LX2 cells treated with metformin and rANGPTL8 for 1 h. (F) The protein levels of α-SMA and TGFβ1, were determined by western blot in LX2 cells treated with metformin and rANGPTL8 for 1 h. (For interpretation of the references to color in this figure legend, the reader is…

• 

  Fig. 8. Schematic representation of the proposed mechanism of ANGPTL8 as an inflammatory factor that promotes HFD-induced NAFLD-associated liver fibrosis by activating LILRB2/ERK signaling in hepatic stellate cells. The elevated fatty acid levels upon long-term exposure to HFD up-regulate liver ANGPTL8. ANGPTL8, in turn, functions as a proinflammatory factor promoting the expression and secretion of the inflammatory factor TGFβ1. Secreted TGFβ1 in turn promotes the expression and secretion of ANGPTL8. Secreted ANGPTL8 interacts with the receptor LILRB2 on the membrane of HSCs thereby activating downstream ERK signaling and increasing the expression of known liver fibrosis genes. Metformin can inhibit the expression of ANGPTL8 and attenuate HFD-induced hepatic fibrosis in mouse livers.