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Article Open Access

Intestinal CD4+ T cells treated with Pseudostellaria heterophylla polysaccharide improve insulin resistance in BNL CL.2 cells by modulating PI3K/AKT signaling and energy metabolism

  • Authors:
    • Yongjun Kan
    • Yingying Liu
    • Yating Huang
    • Li Zhao
    • Jiang Chang
    • Wensheng Pang
    • Wenxiong Lin
    • Juan Hu
  • View Affiliations / Copyright

    Affiliations: Institute of Materia Medica, Fujian Academy of Chinese Medical Sciences, Fuzhou, Fujian 350003, P.R. China, Clinical Research Institute, The Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350003, P.R. China, School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, P.R. China, Fujian Key Laboratory for Agroecological Processes and Safety Monitoring, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, P.R. China
    Copyright: © Kan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 317
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    Published online on: September 11, 2025
       https://doi.org/10.3892/mmr.2025.13682
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Abstract

Pseudostellaria heterophylla polysaccharide PF40 has shown potential in alleviating insulin resistance by modulating CD4+ T cells in the intestinal tissue of rats with type 2 diabetes mellitus. To further elucidate the underlying mechanism, CD4+ T cells were isolated from the intestinal tissue of rats treated with PF40 (P‑T) and co‑cultured with insulin‑resistant (IR)‑BNL CL.2 cells. Oxidative stress was assessed by measuring reactive oxygen species, malondialdehyde and superoxide dismutase activity, while apoptosis was evaluated by flow cytometry. Insulin sensitivity was examined by glucose uptake and consumption assays. Protein expression related to the PI3K/AKT pathway was determined by western blotting, and targeted energy metabolomics was performed to analyze glycolysis and the tricarboxylic acid cycle. P‑T treatment reduced oxidative stress in IR‑BNL CL.2 cells by reducing reactive oxygen species and malondialdehyde levels, while increasing superoxide dismutase activity. Additionally, P‑T inhibited apoptosis and improved insulin sensitivity, as evidenced by the increased glucose uptake and consumption. Mechanistically, P‑T decreased phosphorylated‑insulin receptor substrate‑1 expression, leading to activation of the PI3K/AKT signaling pathway, which enhanced glucose metabolism. Targeted energy metabolomics analysis further revealed that P‑T regulated glycolysis and the tricarboxylic acid cycle, ameliorating energy metabolism dysfunction. Notably, the combined treatment of PF40 and metformin indicated potential synergistic effects. These findings highlight the critical role of intestinal CD4+ T cells in PF40‑mediated metabolic regulation, suggesting that targeted modulation of intestinal immune cell homeostasis may offer a promising strategy for the prevention and treatment of insulin resistance.
View Figures

Figure 1

Effects of different insulin
concentrations and intervention durations on glucose consumption in
IR-BNL CL.2 cells. (A) Effects of various insulin concentrations
(0-1×10−9 mol/l) on glucose consumption in IR-BNL CL.2
cells. **P<0.01 vs. untreated control group. (B) Effects of
different intervention durations of insulin (1×10−7
mol/l) on glucose consumption in IR-BNL CL.2 cells. Data are
presented as the mean ± SD, n=6. **P<0.01 vs. control group.
IR-BNL CL.2, insulin-resistant BNL CL.2.

Figure 2

Stability of the IR-BNL CL.2 cell
model and characteristics of co-culture with CD4+ T
cells. (A) Analysis of cell viability in IR-BNL CL.2 and
CD4+ T cells co-cultured at different ratios (1-40:1).
(B) Changes in glucose consumption in IR-BNL CL.2 cells at various
time points (24-60 h) following insulin intervention. **P<0.01
vs. control. Data are presented as the mean ± SD, n=6. *P<0.05,
**P<0.01 vs. untreated IR-BNL CL.2 cells. IR-BNL CL.2,
insulin-resistant BNL CL.2.

Figure 3

Effects of treated CD4+ T
cells on glucose uptake of BNL CL-2 cells. (A) Glucose consumption
in the different treatment groups. (B) Glucose degradation ratio in
the different treatment groups. Data are presented as the mean ±
SD, n=6. ##P<0.01 vs. BNL CL.2 control group;
**P<0.01 vs. Mod-T group. IR-BNL CL.2, insulin-resistant BNL
CL.2; Con-T, control; Mod-T, model; P-T, PF40; M-T, metformin;
PM-T, combined PF40 and metformin.

Figure 4

Effects of differently treated
CD4+ T cells on (A) MDA and (B) SOD levels in IR-BNL
CL.2 cells. Data are presented as the mean ± SD, n=5.
##P<0.01 vs. Con-T group; *P<0.05, **P<0.01 vs.
Mod-T group. MDA, malondialdehyde; SOD, superoxide dismutase;
IR-BNL CL.2, insulin-resistant BNL CL.2; Con-T, control; Mod-T,
model; P-T, PF40; M-T, metformin; PM-T, combined PF40 and
metformin.

Figure 5

Effects of differently treated
CD4+ T cells on ROS levels in IR-BNL CL.2 cells. (A)
Representative ROS images for each treatment group. (B) Statistical
analysis of ROS levels across treatment groups. Data are presented
as the mean ± SD, n=6. ##P<0.01 vs. Con-T group;
*P<0.05, **P<0.01 vs. Mod-T group. ROS, reactive oxygen
species; IR-BNL CL.2, insulin-resistant BNL CL.2; Con-T, control;
Mod-T, model; P-T, PF40; M-T, metformin; PM-T, combined PF40 and
metformin.

Figure 6

Effects of differently treated
CD4+ T cells on the apoptosis of IR-BNL CL.2 cells. (A)
Representative plots of apoptosis in each treatment group. (B)
Quantification of apoptosis levels across treatment groups, based
on the percentages of cells in the Q1-2 quadrants as determined by
flow cytometry. Data are presented as the mean ± SD, n=6.
##P<0.01 vs. Con-T group; **P<0.01 vs. Mod-T
group. IR-BNL CL.2, insulin-resistant BNL CL.2; Con-T, control;
Mod-T, model; P-T, PF40; M-T, metformin; PM-T, combined PF40 and
metformin.

Figure 7

Effects of differently treated
CD4+ T cells on the IRS-1/PI3K/AKT signaling pathway in
IR-BNL CL.2 cells. (A) Representative western blot images showing
protein and p-protein expression levels of IRS-1, PI3K and AKT
across the different treatment groups. Relative protein expression
levels of (B) p-IRS-1/IRS-1, (C) p-PI3K/PI3K, and (D) p-AKT/AKT.
Data are presented as the mean ± SD, n=3. ##P<0.01
vs. Con-T group; *P<0.05, **P<0.01 vs. Mod-T group. Con-T,
control; Mod-T, model; P-T, PF40; M-T, metformin; PM-T, combined
PF40 and metformin; IRS-1, insulin receptor substrate-1; p-,
phosphorylated.

Figure 8

Targeted energy metabolomics analysis
of the different treatment groups. (A) PCA of metabolites in the
different treatment groups. OPLS-DA was performed between the (B)
Con-T and Mod-T groups, and the (C) Mod-T and PM-T groups. (D) Heat
maps showing the relative content of key glycolysis products across
different treatment groups. (E) Redundancy analysis. Data are
presented as the mean ± SD, n=4 or 5. (F) Heat map showing the
relative content of key TCA cycle products across different
treatment groups. Mod-T. Con-T, control; Mod-T, model; P-T, PF40;
M-T, metformin; PM-T, combined PF40 and metformin; PCA, principal
component analysis; OPLS-DA, orthogonal partial least squares
discriminant analysis; TCA, tricarboxylic acid.
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Copy and paste a formatted citation
Spandidos Publications style
Kan Y, Liu Y, Huang Y, Zhao L, Chang J, Pang W, Lin W and Hu J: Intestinal CD4<sup>+</sup> T cells treated with <em>Pseudostellaria heterophylla</em> polysaccharide improve insulin resistance in BNL CL.2 cells by modulating PI3K/AKT signaling and energy metabolism. Mol Med Rep 32: 317, 2025.
APA
Kan, Y., Liu, Y., Huang, Y., Zhao, L., Chang, J., Pang, W. ... Hu, J. (2025). Intestinal CD4<sup>+</sup> T cells treated with <em>Pseudostellaria heterophylla</em> polysaccharide improve insulin resistance in BNL CL.2 cells by modulating PI3K/AKT signaling and energy metabolism. Molecular Medicine Reports, 32, 317. https://doi.org/10.3892/mmr.2025.13682
MLA
Kan, Y., Liu, Y., Huang, Y., Zhao, L., Chang, J., Pang, W., Lin, W., Hu, J."Intestinal CD4<sup>+</sup> T cells treated with <em>Pseudostellaria heterophylla</em> polysaccharide improve insulin resistance in BNL CL.2 cells by modulating PI3K/AKT signaling and energy metabolism". Molecular Medicine Reports 32.6 (2025): 317.
Chicago
Kan, Y., Liu, Y., Huang, Y., Zhao, L., Chang, J., Pang, W., Lin, W., Hu, J."Intestinal CD4<sup>+</sup> T cells treated with <em>Pseudostellaria heterophylla</em> polysaccharide improve insulin resistance in BNL CL.2 cells by modulating PI3K/AKT signaling and energy metabolism". Molecular Medicine Reports 32, no. 6 (2025): 317. https://doi.org/10.3892/mmr.2025.13682
Copy and paste a formatted citation
x
Spandidos Publications style
Kan Y, Liu Y, Huang Y, Zhao L, Chang J, Pang W, Lin W and Hu J: Intestinal CD4<sup>+</sup> T cells treated with <em>Pseudostellaria heterophylla</em> polysaccharide improve insulin resistance in BNL CL.2 cells by modulating PI3K/AKT signaling and energy metabolism. Mol Med Rep 32: 317, 2025.
APA
Kan, Y., Liu, Y., Huang, Y., Zhao, L., Chang, J., Pang, W. ... Hu, J. (2025). Intestinal CD4<sup>+</sup> T cells treated with <em>Pseudostellaria heterophylla</em> polysaccharide improve insulin resistance in BNL CL.2 cells by modulating PI3K/AKT signaling and energy metabolism. Molecular Medicine Reports, 32, 317. https://doi.org/10.3892/mmr.2025.13682
MLA
Kan, Y., Liu, Y., Huang, Y., Zhao, L., Chang, J., Pang, W., Lin, W., Hu, J."Intestinal CD4<sup>+</sup> T cells treated with <em>Pseudostellaria heterophylla</em> polysaccharide improve insulin resistance in BNL CL.2 cells by modulating PI3K/AKT signaling and energy metabolism". Molecular Medicine Reports 32.6 (2025): 317.
Chicago
Kan, Y., Liu, Y., Huang, Y., Zhao, L., Chang, J., Pang, W., Lin, W., Hu, J."Intestinal CD4<sup>+</sup> T cells treated with <em>Pseudostellaria heterophylla</em> polysaccharide improve insulin resistance in BNL CL.2 cells by modulating PI3K/AKT signaling and energy metabolism". Molecular Medicine Reports 32, no. 6 (2025): 317. https://doi.org/10.3892/mmr.2025.13682
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