Zinc-α2-glycoprotein 1 attenuates non-alcoholic fatty liver disease by negatively regulating tumour necrosis factor-α

Figure 1 Expression of AZGP1 in liver tissues of chronic hepatitis B and non-alcoholic fatty liver disease patients. IHC-stained AZGP1 is showed in human liver tissues (A) of two chronic hepatitis B (CHB) and non-alcoholic fatty liver disease (NAFLD) patients. AZGP1 mRNA (B) and protein (C) expression is showed in liver tissues of another group of CHB and NAFLD patients (n = 3 each). ^aP < 0.05 vs CHB group. CHB: Chronic hepatitis B; NAFLD: Non-alcoholic fatty liver disease.

Figure 2 Inhibition of inflammation by AZGP1 in LO2 cells. TNF-α, IL-1β, IL-6, NF-κB, and MCP-1 mRNA and protein expression was detected in LO2 cells, pEX-3 ± PA/OV-AZGP1 ± PA groups (A-B), and sh-CTRL ± PA/sh-AZGP1 ± PA groups (D-E). IF staining of LO2 cells shows nuclei (blue), AZGP1 protein (green), and TNF-α protein (red) in these groups (C/F). ^aP < 0.05 vs pEX-3 or sh-CTRL cells; ^aP < 0.05 vs pEX-3 + PA or sh-CTRL + PA cells.

Figure 3 Regulation of lipid metabolism by AZGP1 in LO2 cells. ORO staining of lipids in LO2 cells is shown in pEX-3 ± PA/OV-AZGP1 ± PA groups (A) and sh-CTRL ± PA/sh-AZGP1 ± PA groups (B). SREBP-1c, LXR, FAS, ACC, SCD-1, FXR, PPAR-α, FATP, CPT-1A, and adiponectin mRNA and protein expression was detected in the pEX-3 ± PA/OV-AZGP1 ± PA groups (C/E) and sh-CTRL ± PA/sh-AZGP1 ± PA groups (D/F). ^aP < 0.05 vs pEX-3 or sh-CTRL cells; ^cP < 0.05 vs pEX-3 + PA or sh-CTRL + PA cells.

Figure 4 Proliferation and apoptosis regulation by AZGP1 in LO2 cells. PCNA and cyclin D1 mRNA (A/E) and protein (B/F) expression was detected in LO2 cells. Cell viability was determined using CCK-8 assays (C/G). Cell cycle and proliferation analyses were conducted by flow cytometry (D/H). Bcl2 and caspase 3 mRNA (I/L) and protein (J/M) expression was detected in LO2 cells. The rate of apoptosis was tested using flow cytometry (K/N). ^aP < 0.05 vs pEX-3 or sh-CTRL cells; ^cP < 0.05 vs pEX-3 + PA or sh-CTRL + PA cells.

Figure 5 AZGP1 exerts multiple functions by blocking TNF-α in LO2 cells. All experiments were performed in LO2 cells, which were divided into six groups (pEX-3, OV-AZGP1, OV-AZGP1 + OV-TNF-α, sh-CTRL, sh-AZGP1, and sh-AZGP1 + sh-TNF-α). AZGP1, TNF-α, NF-κB, IL-6, PCNA, cyclin D1, Bcl2, and caspase 3 mRNA (A, F) and protein (B, G) expression was detected. ORO staining (C, H) of LO2 cells is shown. Cell viability (D, I) was determined using CCK-8 assays. The rate of apoptotic LO2 cells was tested using flow cytometry (E, J). ^aP < 0.05 vs pEX-3 or sh-CTRL cells; ^cP < 0.05 vs OV-AZGP1 or sh-AZGP1 cells.

Figure 6 Expression of AZGP1/TNF-α, inflammation, and lipid metabolism regulation by AZGP1 in the non-alcoholic fatty liver disease mouse model. Liver tissue images after HE (A) and Sirius red (B) staining are shown in four mouse groups [control (con), HFD, HFD + CCl₄, and HFD + CCl₄ + AZGP1]. Levels of AZGP1 and TNF-α mRNA (C) and protein (D) were detected in the four mouse groups. IF staining (E) with DAPI (blue) and for AZGP1 (green) and TNF-α (red) was also examined in mice. Serum ALT levels (F) were examined using an ELISA kit in the four mouse groups. IL-1β, IL-6, NF-κB, and MCP-1 mRNA (G) and protein (H) expression levels were detected in the four groups. Representative images of ORO staining (I) are shown for the four groups. SREBP-1c, LXR, FAS, ACC, SCD-1, FXR, PPARα, FATP, CPT-1A, and adiponectin mRNA (J) and protein (K) expression was detected in the four groups. ^aP < 0.05 vs control mice; ^cP < 0.05 vs HFD + CCl₄ mice.