PLIN5 deficiency ameliorates metabolic dysfunction‑associated fatty liver disease by inhibiting ferroptosis

Figure 1.. PLIN5 deficiency inhibits ferroptosis in lipotoxic hepatocytes in vitro and in vivo. (A) Fe2+ fluorescence intensity in AML12 cells observed using a fluorescence microscope (magnification, ×10), with red indicating Fe2+ fluorescence and green indicating nuclei. (B) Western blot analysis of GPX4 protein expression in BSA- or PAOA-treated cells was performed, with GAPDH used as an internal control. (C) Fe2+ content in the livers of mice in the WT HFD group and PLIN5−/− HFD group was measured (n=5 mice/group). (D) Liver GSH content of mice in each group was detected (n=5 mice/group). (E) Liver MDA content of mice in each group was detected (n=5 mice/group). (F) mRNA levels of GPX4 in the liver tissues of mice in the WT HFD and PLIN5−/− HFD groups were analyzed by quantitative polymerase chain reaction (n=5 mice/group). (G) Western blot analysis of GPX4 protein expression in the liver tissues of mice from the WT HFD and PLIN5−/− HFD groups was performed, with tubulin used as an internal control (n=3 mice/group). All experiments were repeated at least three times (n=3). *P<0.05, **P<0.01, ***P<0.001, n.s., not significant. Fe2+, ferrous ion; GSH, glutathione; GPX4, GSH peroxidase 4;…

Figure 2.. Overexpression of PLIN5 promotes lipid accumulation and ferroptosis in hepatocytes. (A) mRNA levels of PLIN5 levels were detected via quantitative polymerase chain reaction. (B) PLIN5 protein expression was detected via western blotting. (C) LD changes in cells were observed using a microscope (magnification, ×10), with nuclei stained blue and LDs stained red. Quantitative analysis was performed using ImageJ. (D) Fe2+ fluorescence intensity in cells was observed using a fluorescence microscope (magnification, ×10), with nuclei stained green and Fe2+ fluorescence stained red, followed by quantitative analysis. (E) MDA levels were detected in the three cell groups. (F) GSH levels were detected in the three groups of cells. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s., not significant. Fe2+, ferrous ion; GSH, glutathione; LD, lipid droplet; MDA, malondialdehyde; PLIN5, perilipin 5; FER-1, ferrostatin-1.

Figure 3.. PLIN5 affects ferroptosis through ATF3/CHOP/CHAC1 signaling. (A) DEGs in mouse liver tissues from the PLIN5−/− HFD group are shown compared to those in the WT HFD group (red dots represent upregulated genes, whereas blue dots represent downregulated genes). (B) DEGs associated with ferroptosis in the PLIN5−/− HFD and WT HFD groups were analyzed (log2 FC>1, P<0.05). (C) KEGG pathway and GO term enrichment analyses of different genes were performed. (D) mRNA levels of PRC1, LTF, GDPD5, FSCN1, MYC, ATF3 and PLIN2 in the liver tissues of WT HFD and PLIN5−/− HFD mice were measured (n=5 mice/group). (E) mRNA levels of ATF3, CHOP and CHAC1 in the liver tissues of mice in each group were detected (n=5 mice/group). (F) Western blot analysis of ATF3, CHOP and CHAC1 protein expression in the liver tissues of mice from the WT HFD and PLIN5−/− HFD groups was performed, with tubulin as the internal reference. Semi-quantitative analysis performed using ImageJ (n=3 mice/group). All experiments were repeated at least three times (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s., not significant. ATF3, activating transcription factor 3; CHAC1, cation transport regulator-like protein 1;…

Figure 4.. PLIN5 affects ferroptosis via ATF3/CHOP/CHAC1 signaling. The mRNA expression levels of (A) GPX4, (B) ATF3, (C) CHOP and (D) CHAC1 in cells from each group were measured. (E) Western blot analysis of GPX4, ATF3, CHOP and CHAC1 protein expression in cells from different groups was performed, normalized to tubulin as an internal control. Semi-quantitative analysis was performed using ImageJ. All experiments were repeated at least three times. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant. ATF3, activating transcription factor 3; CHAC1, cation transport regulator-like protein 1; CHOP, C/EBP homologous protein; FER-1, ferrostatin-1; GPX4, glutathione peroxidase 4; PAOA, palmitic acid and oleic acid; PLIN5, perilipin 5.

Figure 5.. Knockdown of ATF3 alleviates lipid accumulation and ferroptosis induced by overexpression of PLIN5. (A) ATF3 mRNA expression in AML12 cells was analyzed by quantitative polymerase chain reaction 48 h after transfection with shATF3 plasmid. (B) Semi-quantitative analysis of ATF3 protein expression in AML12 cells 48 h after transfection with shATF3 plasmid was performed, normalized to tubulin as an internal control, using ImageJ. (C) LDs were observed in cells from different intervention groups using a microscope (magnification, ×10), with nuclei stained blue and LDs stained red and quantitative analysis was performed using ImageJ. (D) Fe2+ fluorescence intensity in cells from different intervention groups was observed using a fluorescence microscope, with nuclei stained green and Fe2+ fluorescence stained red, followed by quantitative analysis. (E) MDA content was measured in cells from different intervention groups. (F) GSH content was measured in cells from different intervention groups. All experiments were repeated at least three times. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant. ATF3, activating transcription factor 3; Fe2+, ferrous ion; GSH, glutathione; LD, lipid…