Mechanistic insights into restoration of gut barrier and microbiota homeostasis by allicin and Qingchang Huashi formula in Cryptosporidium parvum-infected immunosuppressed murine model.

Nitazoxanide (NTZ), the only FDA-approved anti-Cryptosporidium drug, shows inconsistent efficacy in immunocompromised patients. This study employed HCT-8 cell-based cytotoxicity/parasite inhibition assays and an immunosuppressed murine model to explore the anti-C. parvum efficacy and mechanisms of combined allicin and Qingchang Huashi Formula (QHF). In vitro, WST-1-based cytotoxicity screening preceded inoculation of 4 × 104 C. parvum oocysts to assess parasitic inhibition. In vivo, sixty immunosuppressed ICR mice were divided into control (C), model-infected (M), paromomycin-treated (P), allicin-treated (A), QHF-treated (F), and allicin + QHF (AF)-treated. On day 1, groups M, P, A, F, and A + F were orally infected with 3 × 104 C. parvum oocysts each; on day 2, the P, A, F, and A + F groups received daily intragastric administration of the respective drugs for 8 days (1-8 dpi), before euthanasia and 16S rRNA sequencing of rectal feces. In vitro results showed a significant parasitic inhibition at 750 μg/mL (allicin): 1500 μg/mL (QHF) (p < 0.05). In vivo A + F combined treatment significantly reduced fecal C. parvum burden, attenuated inflammation (IL-6, IL-1β, and TNF-α), upregulated IL-10, strengthened antioxidant defense (SOD, GSH-Px, and T-AOC), and reduced MDA levels (p < 0.05). Combined treatment significantly ameliorated intestinal damage, upregulated tight junction proteins (occludin, claudin-1, and ZO-1) and NLRP6 expression, while inhibiting caspase-1 (p < 0.05). 16S rRNA sequencing revealed treatment significantly restored the abundance of beneficial flora, including Firmicutes_A, Bacteroidota, Actinobacteriota (phyla), and Ligilactobacillus, Bacteroides_H, Dwaynesavagella, and UBA3282 (genera), along with regulation of energy metabolic pathways and amino acid synthesis (p < 0.05). Conclusively, allicin and QHF ameliorated the C. parvum-induced damage through gut barrier restoration and microbiota modulation, holding great significance in both public health and livestock sectors.