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Heterophyllin B enhances the benefits of intermittent fasting in the treatment of metabolic dysfunction‑associated steatotic liver disease via activation of GLP‑1R

  • Authors:
    • Kaimin Li
    • Ligong Deng
    • Lijun Xue
    • Shukun Yao
  • View Affiliations / Copyright

    Affiliations: School of Biological and Medical Engineering, Beijing University of Aeronautics and Astronautics, Beijing 100191, P.R. China, Department of Gastroenterology, Jinan Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250013, P.R. China
    Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 200
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    Published online on: September 19, 2025
       https://doi.org/10.3892/ijmm.2025.5641
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Abstract

Intermittent fasting (IF) has shown particularly promising short‑term effects in improving metabolic dysfunction‑associated steatotic liver disease (MASLD), although its long‑term efficacy remains unclear. Heterophyllin B (HP‑B), a cyclopeptide compound derived from Pseudostellaria heterophylla, is known for its potent anti‑inflammatory and hypoglycemic properties. However, studies investigating the potential role of HP‑B in the management of MASLD are lacking. In vitro, an OA/PA‑induced lipid accumulation model was established using HepG2/Huh‑7 cells. The therapeutic effects of HP‑B and fasting‑mimicking conditions were evaluated through Cell Counting Kit‑8 assay, Oil Red O staining, reverse transcription‑quantitative PCR, and western blot analysis. For in vivo studies, C57BL/6J mice were fed a high‑fat diet and treated with HP‑B, IF, or their combination. Mechanistic validation was performed via adenovirus‑mediated GLP‑1R knockdown. The present study aimed to explore whether HP‑B can serve as an adjunctive supplement to enhance the benefits of IF in the treatment of MASLD. HepG2 and Huh‑7 liver cancer cells treated with oleic acid/palmitic acid (OA/PA) presented significant lipid accumulation, which was attenuated by HP‑B treatment and fasting. The combination treatment markedly reduced lipid levels and oxidative stress, as well as restored the mitochondrial membrane potential, with a synergistic effect over treatment alone. In addition, the combination of HP‑B and fasting upregulated glucagon‑like peptide‑1 receptor (GLP‑1R) and peroxisome proliferator‑activated receptor gamma coactivator 1‑alpha expression, reversing the OA/PA‑induced decline. In high‑fat diet‑fed mice, the combination treatment reduced hepatic lipid accumulation, decreased liver weight, decreased mouse body weight, and improved biochemical indices of liver function. The beneficial effects of HP‑B and fasting were reversed after silencing GLP‑1R with small interfering RNA or Ad‑GLP‑1R, emphasizing the critical role of GLP‑1R in mediating these protective effects. In conclusion, the synergistic effects of HP‑B and fasting on improving lipid metabolism and mitochondrial function are mediated primarily through the regulation of GLP‑1R, making it a promising therapeutic target for the treatment of MASLD and other lipid metabolism‑related disorders.
View Figures

Figure 1

Effects of HP-B treatment and fasting
on OA/PA-induced lipid accumulation and gene expression in HepG2
and Huh-7 liver cancer cells. (A and B) Cell Counting Kit-8
analysis revealed that 10, 25 and 50 μM HP-B did not
significantly alter cell viability, whereas 75, 100 and 200
μM HP-B reduced cell viability in HepG2 and Huh-7 cells. (C
and D) Oil Red O staining of HepG2 and Huh-7 liver cancer cells
treated with OA/PA revealed significant lipid accumulation compared
with the Con group. Both HP-B treatment and fasting reversed
OA/PA-induced lipid accumulation, and the combined HP-B and fasting
treatment further reduced lipid accumulation (Scale bar, 10
μm). (E-H) Quantitative analysis of TC and TG levels in
HepG2 and Huh-7 liver cancer cells. (I and J) Quantitative analysis
of the mRNA levels of lipid synthesis- and uptake-related genes
(SREBP1, FAS and CD36) in HepG2 and Huh-7 liver cancer cells.
**P<0.01 and ***P<0.001 vs. Con;
#P<0.05, ##P<0.01 and
###P<0.001 vs. OA/PA; $P<0.05 and
$$P<0.01 vs. OA/PA + HP-B; &P<0.05
and &&P<0.01 vs. OA/PA + Fasting. HP-B,
heterophyllin B; OA/PA, oleic acid/palmitic acid; TC, total
cholesterol; TG, triglycerides.

Figure 2

Effects of HP-B treatment and fasting
on OA/PA-induced changes in ROS, mitoROS, and the MMP in HepG2 and
Huh-7 liver cancer cells. (A and B) Representative images of the
ROS levels in (A) HepG2 and (B) Huh-7 cells are shown. (C and D)
Quantitative analysis of ROS levels in (C) HepG2 and (D) Huh-7
cells. (E and F) Representative images and quantification of
mitoROS levels in (E) HepG2 and (F) Huh-7 cells are shown. (G and
H) Quantitative analysis of mitoROS levels in (G) HepG2 and (H)
Huh-7 cells. (I and J) Representative images and quantification of
the MMP in (I) HepG2 and (J) Huh-7 cells are shown. Scale bar, 10
μm. *P<0.05, **P<0.01 and
***P<0.001 vs. Con; #P<0.05,
##P<0.01 and ###P<0.001 vs. OA/PA;
$P<0.05 vs. OA/PA + HP-B; &P<0.05
vs. OA/PA + Fasting. HP-B, heterophyllin B; OA/PA, oleic
acid/palmitic acid; ROS, reactive oxygen species; mitoROS,
mitochondrial ROS; MMP, mitochondrial membrane potential.

Figure 3

Effects of HP-B treatment and fasting
on OA/PA-induced changes in GLP-1R and PGC1α expression in HepG2
and Huh-7 liver cancer cells. (A-D) Quantitative analysis of GLP-1R
and PGC1α mRNA levels in HepG2 and Huh-7 liver cancer cells. (E and
F) HP-B treatment or fasting reversed the OA/PA-induced reduction
in protein levels, and the combined treatment had a more pronounced
effect. *P<0.05, **P<0.01 and
***P<0.001 vs. Con; #P<0.05,
##P<0.01 and ###P<0.001 vs. OA/PA;
$P<0.05 and $$P<0.01 vs. OA/PA + HP-B;
&P<0.05 vs. OA/PA + Fasting. HP-B, heterophyllin
B; OA/PA, oleic acid/palmitic acid; GLP-1R, glucagon-like peptide-1
receptor; PGC1α, peroxisome proliferator-activated receptor gamma
coactivator 1-alpha.

Figure 4

Effects of HP-B treatment and fasting
on lipid accumulation and related parameters in the livers of
HFD-fed mice. (A) Glucose tolerance test and AUC quantification.
(B) Insulin tolerance test and AUC quantification. (C and D)
Representative H&E and Oil Red O images showing that HP-B
treatment or fasting treatment reduced liver lipid accumulation to
some extent, whereas the combined HP-B and fasting treatment
further significantly reduced lipid accumulation (Scale bar, 25
μm). (E-G) HP-B treatment or fasting reduced the liver
weight, body weight and liver index. The combined treatment
resulted in a more pronounced reduction in liver weight and body
weight, but the liver index did not obviously change. (H-J) HP-B
treatment and fasting lowered these levels, and the combined
treatment resulted in a more significant reduction in TG and ALT
levels than HP-B treatment alone, as well as a more significant
reduction in TG levels than fasting alone. (K) HP-B treatment or
fasting increased the protein levels of GLP-1R and PGC1α, and the
combined treatment further increased PGC1α protein expression
compared with HP-B treatment alone. *P<0.05,
**P<0.01 and ***P<0.001 vs. Con;
#P<0.05, ##P<0.01 and
###P<0.001 vs. HFD; $P<0.05,
$$P<0.01 and $$$P<0.001 vs. HFD + HP-B;
&P<0.05, &&P<0.01 and
&&&P<0.001 vs. HFD + Fasting. HP-B,
heterophyllin B; HFD, high-fat diet; AUC, area under the curve; TG,
triglycerides; ALT, alanine aminotransferase; AST aspartate
aminotransferase; GLP-1R, glucagon-like peptide-1 receptor; PGC1α,
peroxisome proliferator-activated receptor gamma coactivator
1-alpha.

Figure 5

Effects of GLP-1R silencing on
OA/PA-induced lipid accumulation in HepG2 and Huh-7 liver cancer
cells. (A and C) Reverse transcription-quantitative PCR analysis
showed that the mRNA levels of GLP-1R in HepG2 and Huh-7 cells were
reduced after siGLP-1R transfection. (B and D) Western blot
analysis revealed that siGLP-1R silenced GLP-1R expression in OA/PA
pre-treated HepG2 and Huh-7 cells and reversed the OA/PA-induced
increase in PGC1α protein levels. (E) Oil Red O staining
demonstrated that fasting and HP-B treatment reduced lipid
accumulation induced by OA/PA, but this effect was blocked by
GLP-1R silencing. (F-I) GLP-1R silencing further elevated (F and G)
TG and (H and I) TC levels in HepG2 and Huh-7 cells, counteracting
the reductions achieved by fasting and HP-B treatment.
*P<0.05, **P<0.01 and
***P<0.001 vs. OA/PA; $P<0.05 and
$$$P<0.01 vs. OA/PA + HP-B + Fasting. HP-B,
heterophyllin B; GLP-1R, glucagon-like peptide-1 receptor; OA/PA,
oleic acid/palmitic acid; si-, small interfering; TG,
triglycerides; TC, total cholesterol; NC, negative control.

Figure 6

GLP-1R knockdown impacts fasting and
HP-B treatment in HFD + Ad-NC mice. (A) Western blot analysis
showed that the expression of GLP-1R was decreased in the livers of
HFD mice. (B and C) Glucose tolerance test and insulin tolerance
test. (D and E) H&E staining and Oil Red O staining of liver
sections from HFD-fed mice (Scale bar, 25 μm). (F and G)
Liver weights and body weights of HFD-fed mice. (H) Compared with
the oil/control treatment, fasting and HP-B treatment reduced the
liver index, whereas GLP-1R knockdown reversed this effect. (I)
GLP-1R knockdown reduced the TG content compared with that in
HFD-fed mice, and this effect was counteracted by fasting and HP-B
treatment. (J and K) Fasting and HP-B treatment decreased the ALT
and AST levels, and these reductions were reversed by GLP-1R
knockdown. (L and M) Fasting and HP-B treatment reduced the MDA and
ROS levels, whereas Ad-shGLP-1R injection increased these levels.
*P<0.05, **P<0.01 and
***P<0.001 vs. HFD + Ad-NC; #P<0.05,
##P<0.01, ###P<0.001 vs. HFD +
Ad-sh-GLP-1R; $P<0.05, $$P<0.01 and
$$$P<0.001 vs. HFD + HP-B + IF. GLP-1R, glucagon-like
peptide-1 receptor; HP-B, heterophyllin B; HFD, high-fat diet; NC,
negative control; TG, triglycerides; ALT, alanine aminotransferase;
AST aspartate aminotransferase; MDA, malondialdehyde; ROS, reactive
oxygen species; sh-, short hairpin.
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Li K, Deng L, Xue L and Yao S: Heterophyllin B enhances the benefits of intermittent fasting in the treatment of metabolic dysfunction‑associated steatotic liver disease via activation of GLP‑1R. Int J Mol Med 56: 200, 2025.
APA
Li, K., Deng, L., Xue, L., & Yao, S. (2025). Heterophyllin B enhances the benefits of intermittent fasting in the treatment of metabolic dysfunction‑associated steatotic liver disease via activation of GLP‑1R. International Journal of Molecular Medicine, 56, 200. https://doi.org/10.3892/ijmm.2025.5641
MLA
Li, K., Deng, L., Xue, L., Yao, S."Heterophyllin B enhances the benefits of intermittent fasting in the treatment of metabolic dysfunction‑associated steatotic liver disease via activation of GLP‑1R". International Journal of Molecular Medicine 56.6 (2025): 200.
Chicago
Li, K., Deng, L., Xue, L., Yao, S."Heterophyllin B enhances the benefits of intermittent fasting in the treatment of metabolic dysfunction‑associated steatotic liver disease via activation of GLP‑1R". International Journal of Molecular Medicine 56, no. 6 (2025): 200. https://doi.org/10.3892/ijmm.2025.5641
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Spandidos Publications style
Li K, Deng L, Xue L and Yao S: Heterophyllin B enhances the benefits of intermittent fasting in the treatment of metabolic dysfunction‑associated steatotic liver disease via activation of GLP‑1R. Int J Mol Med 56: 200, 2025.
APA
Li, K., Deng, L., Xue, L., & Yao, S. (2025). Heterophyllin B enhances the benefits of intermittent fasting in the treatment of metabolic dysfunction‑associated steatotic liver disease via activation of GLP‑1R. International Journal of Molecular Medicine, 56, 200. https://doi.org/10.3892/ijmm.2025.5641
MLA
Li, K., Deng, L., Xue, L., Yao, S."Heterophyllin B enhances the benefits of intermittent fasting in the treatment of metabolic dysfunction‑associated steatotic liver disease via activation of GLP‑1R". International Journal of Molecular Medicine 56.6 (2025): 200.
Chicago
Li, K., Deng, L., Xue, L., Yao, S."Heterophyllin B enhances the benefits of intermittent fasting in the treatment of metabolic dysfunction‑associated steatotic liver disease via activation of GLP‑1R". International Journal of Molecular Medicine 56, no. 6 (2025): 200. https://doi.org/10.3892/ijmm.2025.5641
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