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Article Open Access

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

  • Authors:
    • Ya Li
    • Xiaohan Wang
    • Xuecui Yin
    • Xiaohui Zhu
    • Weiqi Cui
    • Lin Dong
    • Yu Xia
    • Ying Wang
    • Liu Liu
    • Pengyuan Zheng
    • Youcai Tang
  • View Affiliations / Copyright

    Affiliations: Henan Joint International Research Laboratory of Chronic Liver Injury and Henan Provincial Outstanding Overseas Scientists Chronic Liver Injury Workshop, Henan Key Laboratory of Rehabilitation Medicine, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China, Department of Pediatrics, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China, Henan Key Laboratory of Helicobacter pylori, Microbiota and Gastrointestinal Cancer, Marshall Medical Research Center, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China, Department of Pharmacy, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China, Department of Blood Transfusion, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China, Department of Gastroenterology, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450000, P.R. China
    Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 4
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    Published online on: October 14, 2025
       https://doi.org/10.3892/mmr.2025.13714
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Abstract

Metabolic‑associated fatty liver disease (MAFLD) is widely recognized as the most common type of chronic liver disease. As a member of the perilipin (PLIN) family, PLIN5 serves an important role in the regulation of lipid metabolism. Ferroptosis is a form of iron‑dependent non‑apoptotic cell death characterized by lipid peroxidation. Notably, knockout of PLIN5 can attenuate high‑fat diet (HFD)‑induced MAFLD; however, the specific underlying mechanism remains unclear. The present study induced PLIN5 overexpression by transfecting AML12 cells with a pcDNA3.1‑PLIN5 plasmid, and PLIN5 knockdown was achieved using short hairpin RNA‑mediated interference. Subsequently, intracellular ferrous iron (Fe2+) levels were assessed via immunofluorescence staining. Furthermore, a MAFLD model was established in C57BL/6J mice by feeding them a HFD. To establish an in vitro model of hepatic steatosis, AML12 hepatocytes were treated with palmitic acid and oleic acid to induce intracellular lipid accumulation. To further explore the effects of PLIN5 on ferroptosis, liver single‑cell sequencing was conducted and cellular experiments were performed to assess changes in redox and ferroptosis‑related proteins. The current study investigated the effects of PLIN5 on MAFLD in animal and cellular experiments, including the changes in lipid accumulation, redox and ferroptosis‑related markers. The results revealed that genetic knockdown of PLIN5 significantly attenuated lipid accumulation and intracellular Fe2+ levels in AML12 hepatocytes, whereas PLIN5 overexpression markedly exacerbated these parameters. In addition, PLIN5 deficiency substantially reduced malondialdehyde content while enhancing glutathione levels, indicating attenuated oxidative stress. The results of the in vivo studies demonstrated that PLIN5 knockout effectively ameliorated MAFLD progression in mice by suppressing ferroptosis. In conclusion, PLIN5 knockout may delay the progression of MAFLD in mice via ferroptosis inhibition. Therefore, targeting PLIN5 could offer a novel therapeutic strategy to address MAFLD by modulating lipid metabolism and ferroptosis pathways.
View Figures

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; HFD, high-fat diet; MDA, malondialdehyde; PAOA,
palmitic acid and oleic acid; PLIN5, perilipin 5; WT,
wild-type.

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; CHOP, C/EBP homologous protein; DEGs,
differentially expressed genes; FC, fold-change; FSCN1,
fascin actin-bundling protein 1; GDPD5,
glycerophosphodiester phosphodiesterase domain containing 5;
GPX4, glutathione peroxidase 4; HFD, high-fat diet; KEGG,
Kyoto Encyclopedia of Genes and Genomes; ND, normal diet;
LTF, lactotransferrin; PLIN, perilipin; PRC1,
protein regulator of cytokinesis 1; WT, wild-type; BP, biological
process; CC, cellular component; MF, molecular function.

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 droplet; MDA, malondialdehyde; NC, negative
control; PAOA, palmitic acid and oleic acid; PLIN5,
perilipin 5; sh, short hairpin.
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Copy and paste a formatted citation
Spandidos Publications style
Li Y, Wang X, Yin X, Zhu X, Cui W, Dong L, Xia Y, Wang Y, Liu L, Zheng P, Zheng P, et al: <em>PLIN5</em> deficiency ameliorates metabolic dysfunction‑associated fatty liver disease by inhibiting ferroptosis. Mol Med Rep 33: 4, 2026.
APA
Li, Y., Wang, X., Yin, X., Zhu, X., Cui, W., Dong, L. ... Tang, Y. (2026). <em>PLIN5</em> deficiency ameliorates metabolic dysfunction‑associated fatty liver disease by inhibiting ferroptosis. Molecular Medicine Reports, 33, 4. https://doi.org/10.3892/mmr.2025.13714
MLA
Li, Y., Wang, X., Yin, X., Zhu, X., Cui, W., Dong, L., Xia, Y., Wang, Y., Liu, L., Zheng, P., Tang, Y."<em>PLIN5</em> deficiency ameliorates metabolic dysfunction‑associated fatty liver disease by inhibiting ferroptosis". Molecular Medicine Reports 33.1 (2026): 4.
Chicago
Li, Y., Wang, X., Yin, X., Zhu, X., Cui, W., Dong, L., Xia, Y., Wang, Y., Liu, L., Zheng, P., Tang, Y."<em>PLIN5</em> deficiency ameliorates metabolic dysfunction‑associated fatty liver disease by inhibiting ferroptosis". Molecular Medicine Reports 33, no. 1 (2026): 4. https://doi.org/10.3892/mmr.2025.13714
Copy and paste a formatted citation
x
Spandidos Publications style
Li Y, Wang X, Yin X, Zhu X, Cui W, Dong L, Xia Y, Wang Y, Liu L, Zheng P, Zheng P, et al: <em>PLIN5</em> deficiency ameliorates metabolic dysfunction‑associated fatty liver disease by inhibiting ferroptosis. Mol Med Rep 33: 4, 2026.
APA
Li, Y., Wang, X., Yin, X., Zhu, X., Cui, W., Dong, L. ... Tang, Y. (2026). <em>PLIN5</em> deficiency ameliorates metabolic dysfunction‑associated fatty liver disease by inhibiting ferroptosis. Molecular Medicine Reports, 33, 4. https://doi.org/10.3892/mmr.2025.13714
MLA
Li, Y., Wang, X., Yin, X., Zhu, X., Cui, W., Dong, L., Xia, Y., Wang, Y., Liu, L., Zheng, P., Tang, Y."<em>PLIN5</em> deficiency ameliorates metabolic dysfunction‑associated fatty liver disease by inhibiting ferroptosis". Molecular Medicine Reports 33.1 (2026): 4.
Chicago
Li, Y., Wang, X., Yin, X., Zhu, X., Cui, W., Dong, L., Xia, Y., Wang, Y., Liu, L., Zheng, P., Tang, Y."<em>PLIN5</em> deficiency ameliorates metabolic dysfunction‑associated fatty liver disease by inhibiting ferroptosis". Molecular Medicine Reports 33, no. 1 (2026): 4. https://doi.org/10.3892/mmr.2025.13714
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