The Qing Chang Hua Shi Formula combined with allicin alleviates Cryptosporidium parvum infection in immunosuppressed mice and is associated with restoration of NLRP6, IL-18, IFN-γ, and intestinal microbiota

Cryptosporidium parvum is a highly pathogenic zoonotic parasite with limited therapies. Current study explored the anti- C . parvum effect of Qing Chang Hua Shi Formula (QHF) and allicin in vitro in immunosuppressed mice. In vitro , QHF and allicin inhibited over 90% of C. parvum . In vivo , the 8-day treatment reduced fecal oocyst shedding to 12.88% of the model group. Infection disrupted intestinal homeostasis, reduced tight-junction protein expression, and downregulated NLRP6, while inducing systemic inflammation and elevating IL-18/IFN-γ levels, which were further increased by treatment. Treated mice also showed increased abundance of Archangium and Corallococcus and decreased abundance of Clostridioides and Butyricimonas . Predicted microbial functional pathways revealed enrichment of glycolysis-related genes (L-lactate dehydrogenase [LDH], glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) in infected mice, with treatment-associated shifts in amino acid synthesis pathways. Molecular docking suggested potential binding of benzoylpaeoniflorin, baicalin, and wogonoside to C. parvum LDH, ATP/ADP translocase, and gp40/15. Together, these findings indicate that QHF combined with allicin alleviates C. parvum infection in immunosuppressed mice and is associated with restoration of intestinal barrier markers, immune responses, and gut microbiota composition, suggesting a potential therapeutic approach for cryptosporidiosis. IMPORTANCE Current study demonstrates that Qing Chang Hua Shi Formula (QHF) combined with allicin effectively inhibits Cryptosporidium parvum proliferation and concurrently mitigates intestinal damage and dysbiosis induced by C. parvum infection. QHF treatment was associated with upregulation of NLRP6, improved gut microbiota profiles, and elevated IFN-γ levels. These changes correlated with reduced parasite burden and alleviated intestinal injury, suggesting a potential immunomodulatory role that warrants further mechanistic investigation. Metagenomics disclosed that QHF and allicin were associated with changes in predicted microbial functional pathways, including those involved in glycolysis and amino acid metabolism, and with enrichment of certain bacterial taxa, such as Corallococcus and Archangium . Molecular dynamics simulations suggested that benzoylpaeoniflorin and baicalin may bind stably to C. parvum L-lactate dehydrogenase (LDH) and ATP/ADP translocase, raising the hypothesis that they could interfere with parasite energy metabolism. However, enzymatic validation is required to confirm this possibility. These findings suggest a potential therapeutic approach for cryptosporidiosis and highlight alterations associated with C. parvum infection.