Activation of the CXCL16/CXCR6 pathway promotes lipid deposition in fatty livers of apolipoprotein E knockout mice and HepG2 cells

Abstract

Non-alcoholic fatty liver disease (NAFLD), characterised by early lipid accumulation and subsequent inflammation in the liver, is becoming a worldwide challenge due to its increasing prevalence in developing and developed countries. This study aimed to investigate the role of CXC chemokine ligand 16 (CXCL16) and its receptor CXC chemokine receptor 6 (CXCR6) in NAFLD under inflammation. We used IL-1β stimulation in human hepatoblastoma cell line (HepG2) for in vitro studies and casein injection in apolipoprotein E knockout mice in vivo to induce inflammatory stress. The effects of inflammation on cholesterol accumulation were examined by histochemical staining and a quantitative intracellular cholesterol assay. The gene and protein expression of molecules involved in CXCL16/CXCR6 pathway and extracellular matrix (ECM) were examined by real-time polymerase chain reaction (PCR) and Western blotting. The fluorescence intensity of reactive oxygen species (ROS) was assessed by flow cytometry. Results showed that significantly elevated levels of serum amyloid protein A in casein-injected mice confirmed the successful induction of inflamed NAFLD model. Inflammation significantly increased lipid accumulation in livers compared with the high-fat diet group and the controls. Furthermore, inflammation increased the expression of CXCL16, CXCR6, and adisintegrin and metalloproteinase domain-containing protein 10 (ADAM10) in livers, accompanied with increased ECM expression and ROS production. These effects were further confirmed by in vitro studies. Interestingly, CXCL16 gene knockdown in HepG2 cells induced by CXCL16 siRNA resulted in decreased lipid accumulation, ECM excretion, and ROS production. These findings demonstrated that inflammation-mediated activation of CXCL16/CXCR6 is involved in the progression of NAFLD.

Keywords: CXCL16/CXCR6 pathway; Non-alcoholic fatty liver disease; inflammatory stress.

Figure 1

Establishment of inflamed NAFLD model. ApoE KO mice were fed with a normal diet containing 4% fat (Control), a high-fat diet containing 21% fat and 0.15% cholesterol (HF group), or a HF diet with 10% casein injection (HF+casein group) for 8 weeks (n=8). The levels of SAA in the serum of three groups were measured by enzyme linked immunosorbent assay (A). The results are expressed as the means ± SD (n=8). **P<0.01 vs. Control. The protein expression of CD68, TNF-α, and MCP-1 in the livers of the mice was measured by immunohistochemical staining (B, brown colour, original magnification ×400). The protein expression of TNF-α and MCP-1 in the livers of the mice was further checked by Western blotting. The identical total protein extracted from liver tissues was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-mouse polyclonal antibodies against TNF-α, MCP-1, or β-actin which was used as an internal control. The histogram represents the means ± SD of the densitometric scans of the protein bands from the mice in each group, normalised by comparison with β-actin (C and D). *P<0.05 vs. Control, **P<0.01 vs. Control. HepG2 cells were treated without (Control) or with 30 µg/ml of cholesterol (CHO group), 5 ng/ml of IL-1β (IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β (CHO+IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA (CHO+IL-1β + siCXCL16 group), or 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA negative control (CHO+IL-1β + sicontrol group) for 24 hours. Total RNA was extracted from the HepG2 cells and cDNA was aquired by reverse transcription. The mRNA expression of TNF-α and MCP-1 in HepG2 cells was determined by real-time PCR. β-actin served as the housekeeping gene (E). Results represent the means ± SD.**P<0.01 vs. Control, ##P<0.01 vs. CHO+IL-1β. The protein expression of TNF-α and MCP-1 in HepG2 cells was checked by Western blotting. The identical total protein extracted from the HepG2 cells was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-human polyclonal antibodies against MCP-1, TNF-α, or anti-human monoclonal antibody against β-actin which was used as an internal control. The histogram represents the means ± SD of the densitometric scans for TNF-α and MCP-1, normalised by comparison with β-actin (F and G). *P<0.05 vs. Control, **P<0.01 vs. Control, ##P<0.01 vs. CHO+IL-1β.

Figure 2

Inflammation induced lipid accumulation in hepatic cells in vivo and in vitro. ApoE KO mice were fed with a normal diet containing 4% fat (Control), a high fat diet containing 21% fat and 0.15% cholesterol (HF group), or with 10% casein injection (HF+casein group) for 8 weeks (n=8). The lipid accumulation in livers was checked by HE staining (A, I-III, ×400), Filipin staining (B, IV-VI, ×200). Quantitative assay of intracellular free cholesterol and cholesterol ester was used to further evaluate lipid accumulation in liver tissues (C). The results are expressed as the means ± SD (n=8). **P<0.01 vs. Control. HepG2 cells were treated without (Control) or with 30 µg/ml of cholesterol (CHO group), 5 ng/ml of IL-1β (IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β (CHO+IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA (CHO+IL-1β + siCXCL16 group), or 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA negative control (CHO+IL-1β + sicontrol group) for 24 hours. The lipid accumulation in HepG2 cells was checked by Filipin staining (D, I-VI, ×200). Quantitative assay of intracellular free cholesterol and cholesterol ester was used to check lipid accumulation as described in the section of materials and methods (E). Values are expressed as the means ± SD of triplicate wells from four experiments. **P<0.01 vs. Control, ##P<0.01 vs. CHO+IL-1β.

Figure 3

Inflammation induced the activation of the CXCL16/CXCR6 pathway in hepatic cells. ApoE KO mice were fed with a normal diet containing 4% fat (Control) a high fat diet containing 21% fat and 0.15% cholesterol (HF group), or a high fat diet with a 10% casein injection (HF+casein group) for 8 weeks (n=8). The protein expression of CXCL16/CXCR6 pathway components in the three groups of mice was checked by immunohistochemistry (A I-IX, ×400) and Western blotting. The identical total protein extracted from liver tissues was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-mouse polyclonal antibodies against CXCL16, CXCR6, ADAM10, or β-actin which was used as an internal control. The histogram represents the means ± SD of the densitometric scans for the protein bands of CXCL16/CXCR6 pathway components, normalised by comparison with β-actin (B and C). **P<0.01 vs. Control. HepG2 cells were treated without (Control) or with 30 µg/ml of cholesterol (CHO group), 5 ng/ml of IL-1β (IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β (CHO+IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA (CHO+IL-1β + siCXCL16 group), or 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA negative control (CHO+IL-1β + sicontrol group) for 24 hours. Total RNA was extracted from the HepG2 cells and cDNA was acquired by reverse transcription. The mRNA expression of CXCL16, CXCR6, and ADAM10 in HepG2 cells was determined by real-time PCR. β-actin served as the housekeeping gene (D). Results represent the means ± SD. *P<0.05 vs. Control, **P<0.01 vs. Control, #P<0.05 vs. CHO+IL-1β, ##P<0.01 vs. CHO+IL-1β. The protein expression of CXCL16, CXCR6, and ADAM10 in HepG2 cells was checked by Western blotting. The identical total protein extracted from the HepG2 cells was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-human polyclonal antibodies against CXCL16, CXCR6, ADAM10, or anti-human monoclonal antibody against β-actin which was used as an internal control. The histogram represents means ± SD of the densitometric scans for CXCL16, CXCR6 and ADAM10, normalised by comparison with β-actin (E and F). *P<0.05 vs. Control, **P<0.01 vs. Control, #P<0.05 vs. CHO+IL-1β, ##P<0.01 vs. CHO+IL-1β. Immunofluorescent staining of CXCL16 and ADAM10 in HepG2 cells (G). The cells were stained with DAPI to visualise nuclei (blue), and with Alexa Fluor 488 and Alexa Fluor 594 to visualise the distribution of ADAM10 (green) and CXCL16 (red) proteins.

Figure 4

Effects of CXCL16/CXCR6 pathway activation on the expression of ECM components in the livers of ApoE KO mice and in HepG2 cells. ApoE KO mice were fed with a normal diet containing 4% fat (Control), a high fat diet containing 21% fat and 0.15% cholesterol (HF group), or with 10% casein injection (HF+casein group) for 8 weeks (n=8). Immunohistochemical staining shows the expression of collagen I and α-SMA in the livers of ApoE mice (A I-VI, ×400). The protein expression of collagen I and α-SMA in livers was checked by Western blotting. The identical total protein extracted from liver tissues was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-mouse polyclonal antibodies against collagen I, α-SMA, or β-actin which was used as an internal control. The histogram represents the means ± SD of the densitometric scans for the protein bands of collagen I and α-SMA, normalised by comparison with β-actin (B and C). **P<0.01 vs. Control. HepG2 cells were treated without (Control) or with 30 µg/ml of cholesterol (CHO group), 5 ng/ml of IL-1β (IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β (CHO+IL-1β group), 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA (CHO+IL-1β + siCXCL16 group), or 30 µg/ml of cholesterol + 5 ng/ml of IL-1β + CXCL16 siRNA negative control (CHO+IL-1β + sicontrol group) for 24 hours. Total RNA was extracted from the HepG2 cells and cDNA was acquired by reverse transcription. The mRNA expression of collagen I and α-SMA was determined by real-time PCR. β-actin served as the housekeeping gene (D). Results represent the means ± SD. *P<0.05 vs. Control, **P<0.01 vs. Control, #P<0.05 vs. CHO+IL-1β, ##P<0.01 vs. CHO+IL-1β. The protein expression of collagen I and α-SMA in HepG2 cells was checked by Western blotting. The identical total protein extracted from the HepG2 cells was isolated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were subjected to Western blotting using anti-human polyclonal antibodies against collagen I, α-SMA, or anti-human monoclonal antibody against β-actin which was used as an internal control. The histogram represents the means ± SD of the densitometric scans for the protein bands of collagen I and α-SMA, normalised by comparison with β-actin (E and F). **P<0.01 vs. Control, #P<0.05 vs. CHO+IL-1β, ##P<0.01 vs. CHO+IL-1β. HepG2 cells were washed with Dulbecco’s phosphate buffered saline and incubated in the dark for 6 hours with 50 μmol/l 5-(and-6)-chloromethyl-2’7’-dichlorodihydrofluorescein diacetate (green fluorescence) (G). The fluorescence intensities of ROS production was quantified by Cell Quest software. Data represent the means ± SD (H). **P<0.01 vs. Control, ##P<0.01 vs. CHO+IL-1β.