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.

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