Chronic inflammation is a common factor in the pathological processes of multiple human diseases. EPS-LM, an exopolysaccharide (EPS) from the Cordyceps sinensis fungus Cs-HK1, has shown notable anti-inflammatory activities in previous studies. This study aimed to investigate the major signaling events mediating the anti-inflammatory effects of EPS-LM in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cell culture. EPS-LM treatment significantly reduced LPS-induced production of pro-inflammatory mediators, including nitric oxide (NO) and reactive oxygen species (ROS). It also suppressed the expression levels of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88), subsequently delaying the translocation of nuclear factor-kappa B (NF-κB) to the nucleus. Additionally, co-immunoprecipitation (Co-IP) experiments demonstrated that EPS-LM inhibited the binding of TLR4 to MyD88. The ability of EPS-LM to inhibit the TLR4/MyD88/NF-κB pathway, coupled with its capacity to reduce oxidative stress, underscores its multifaceted anti-inflammatory effects. These effects render EPS-LM as a promising candidate for the comprehensive management of various inflammatory and oxidative stress-related conditions, protecting against cell damage.

The exopolysaccharides (EPS) from the mycelial fermentation of Cordyceps sinensis Cs-HK1, especially the low-molecular weight, protein-rich exopolysaccharide fractions (EPS-LM), have previously exhibited significant antioxidant activity. This study further investigated the antioxidant and protective effects of EPS-LM on intestinal epithelial barrier integrity in Caco-2 monolayers challenged with hydrogen peroxide (H₂O₂, 550 μM). EPS-LM contained two major molecular-weight fractions, 25 kDa and 1.7 kDa, with 19.3% total carbohydrate and 28.7% protein content (w/w). Treatment of the cells with EPS-LM (50–200 μg/mL) showed concentration-dependent protective effects against ROS-induced losses of cell viability and epithelial barrier integrity. EPS-LM treatment enhanced the activities of major antioxidant enzymes (SOD, GSH-Px, and CAT) and modulated NRF2 and its downstream target NQO1, consistent with alleviated oxidative stress. It also improved several indicators of intestinal barrier function, including increased transepithelial electrical resistance and upregulation of tight junction proteins (Occludin, ZO-1, and Claudin-1). These results provide new experimental evidence and theoretical basis for the nutraceutical potential of EPS-LM to mitigate oxidative stress and preserve intestinal epithelial barrier integrity.