Propolis improves intestinal barrier function against Cryptosporidium parvum via NLRP6 inflammasome.

Cryptosporidiosis, a significant global zoonotic disease, poses a serious health threat due to the current lack of effective and specific therapeutic agents, particularly for immunocompromised individuals and young animals. The therapeutic potential and underlying mechanisms of propolis alcohol extract (BP) and water extract (WBP) in Cryptosporidium parvum infection remain incompletely understood. The study assessed the cytotoxicity of BP and WBP, as well as their effects on host cell infection. For the in vivo phase, an immunosuppressed mouse model was employed. Fecal oocyst shedding was quantified via RT-qPCR, and therapeutic efficacy was further evaluated through intestinal histopathological examination. Serum levels of inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-10) and oxidative stress markers (MDA, SOD, GSH-Px) were measured using an enzyme-linked immunosorbent assay to assess immunomodulatory and antioxidant activities. In addition, the expression of intestinal tight junction proteins (ZO-1, occludin, claudin-1) and key components of the NLRP6/caspase-1/IL-18 inflammatory pathway was analyzed by RT-qPCR and Western blot. Molecular docking analysis predicted interactions between major bioactive compounds in propolis, such as hesperidin and quercetin, and the critical adhesion protein gp40/15 of C. parvum. 16S rRNA sequencing revealed alterations in the intestinal microbiota, identifying key genera such as Turicibacter and Ligilactobacillus. Overall, the results of this study provide an important practical basis for the development of new anti-C. parvum drugs. Both BP and WBP significantly reduce C. parvum infection burden, alleviate intestinal inflammation, reduce oxidative stress, and restore intestinal barrier integrity, with BP exhibiting more pronounced antiparasitic and immunomodulatory effects.IMPORTANCECryptosporidium is an important pathogen that causes diarrhea in infants and young children and serious diseases in patients with weakened immune function. Currently, there are no specific drugs for its treatment. This study compared the inhibitory effects of propolis extracted by different methods on Cryptosporidium and revealed its inhibitory mechanism. Propolis can directly target the key virulence factor gp40/15 protein on the surface of insects, interfering with the invasion and colonization of C. parvum. In addition, propolis enhances the anti-C. parvum immune response by activating the host's NLRP6 inflammatome pathway and promoting the production of protective cytokines such as IL-18/IFN-γ. Studies have confirmed that propolis can simultaneously improve intestinal barrier damage and flora imbalance caused by infection. These findings provide a scientific basis for the development of propolis as a natural anti-C. parvum drug. The gp40/15 target and the NLRP6 inflammasome regulatory mechanism also offer new ideas for the research and development of anti-C. parvum drugs.