In this study, the exopolysaccharides of Chlorella sp. (CEP) were isolated to obtain the purified fraction CEP4. Characterization results showed that CEP4 was a sulfated heteropolysaccharide. The main monosaccharide components of CEP4 are glucosamine hydrochloride (40.8%) and glucuronic acid (21.0%). The impact of CEP4 on the immune activity of RAW264.7 macrophage cytokines was detected, and the results showed that CEP4 induced the production of nitric oxide (NO), TNF-α, and IL-6 in a dose-dependent pattern within a range of 6 μg/mL. A total of 4824 differentially expressed genes (DEGs) were obtained from the results of RNA-seq. Gene enrichment analysis showed that immune-related genes such as NFKB1, IL-6, and IL-1β were significantly upregulated, while the genes RIPK1 and TLR4 were significantly downregulated. KEGG pathway enrichment analysis showed that DEGs were significantly enriched in immune-related biological processes, including toll-like receptor (TLR) signaling pathway, cytosolic DNA-sensing pathway, and C-type lectin receptor signaling pathway. Protein–protein interaction (PPI) network analysis showed that HSP90AB1, Rbx1, ISG15, Psmb6, Psmb3, Psmb8, PSMA7, Polr2f, Rpsa, and NEDD8 were the hub genes with an essential role in the immune activity of CEP4. The preliminary results of the present study revealed the potential mechanism of CEP4 in the immune regulation of RAW264.7 macrophages, suggesting that CEP4 is a promising immunoregulatory agent.

Chlamydomonas reinhardtii polysaccharides (CRPs) are bioactive compounds derived from C. reinhardtii, yet their potential in cancer therapy remains largely unexplored. This study optimized the ultrasound-assisted extraction conditions using response surface methodology and proceeded with the isolation and purification of these polysaccharides. The optimal extraction conditions were identified as a sodium hydroxide concentration of 1.5%, ultrasonic power of 200 W, a solid-to-liquid ratio of 1:25 g/mL, an ultrasonic treatment time of 10 min, and a water bath duration of 2.5 h, yielding an actual extraction rate of 5.71 ± 0.001%, which closely aligns with the predicted value of 5.639%. Infrared analysis revealed that CRP-1 and CRP-2 are α-pyranose structures containing furoic acid, while CRP-3 and CRP-4 are β-pyranose structures containing furoic acid. Experimental results demonstrated that all four purified polysaccharides inhibited the proliferation of cervical (HeLa) hepatoma (HepG-2) and colon (HCT-116) cancer cells, with CRP-4 showing the most significant inhibitory effect on colon cancer and cervical cancer, achieving inhibition rates of 60.58 ± 0.88% and 40.44 ± 1.44%, respectively, and significantly reducing the migration of HeLa cells. DAPI staining confirmed that the four purified polysaccharides inhibit cell proliferation and migration by inducing apoptosis in HeLa cells. CRP-1 has the most significant inhibitory effect on the proliferation of liver cancer cells. This study not only elucidates the potential application of C. reinhardtii polysaccharides in cancer therapy but also provides a scientific basis for their further development and utilization.