Abstract:

Objective To explore the role of the Toll-like receptor 4/ nuclear factor κB（TLR4/NF-κB）pathway in intestinal mucosal damage induced by Cryptosporidium parvum infection in mice. Methods Thirty 4-week-old BALB/c male mice were randomly divided into 1-week infection group and 2-week infection group and the non-infection group (n = 10 in each group). The mouse model of Cryptosporidium parvum infection was established intragastrically with 1×10⁵ oocysts. Uninfected mice have normal diet and drinking water. Ten infected mice were sacrificed at 7 days after infection (1-week infection group) and 14 days after infection (2-week infection group), respectively. The normal control mice were sacrificed at 14 days after infection. The intestinal tissue was collected for observing pathological alterations of intestinal mucosa. The relative expression of TLR4 mRNA and NF-κB p65 mRNA in intestinal mucosa was detected by qRT-PCR. The relative expression of TLR4 and NF-κB p65 in intestinal mucosa was detected by Western blotting. The expression of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in intestinal mucosa was detected by ELISA. Result HE staining showed atrophy and shedding of intestinal villi in the infection group, with submucosal edema and a significant gap between muscle layers, while the intestinal villi were intact in the non-infection group. qRT-PCR showed that the relative expression of TLR4 mRNA in the intestinal mucosa in infection group at week 1 and week 2 was 2.3 ± 0.4 and 3.5 ± 0.1, respectively, higher than the non-infection group (1.0 ± 0.0) (P < 0.05, P < 0.01), and the relative expression of NF-κB p65 mRNA in the intestinal mucosa in infection group at week 1 and week 2 was 2.6 ± 0.3 and 3.6 ± 0.2, higher than the non-infection group (1.1 ± 0.1) (P < 0.05, P < 0.01). Western blotting showed that the protein level of TLR4 in the intestinal mucosa in infection group at week 1 and week 2 was 0.4 ± 0.0 and 0.6 ± 0.0, higher than the non-infection group (0.2 ± 0.0) (P < 0.05, P < 0.01), and that of NF-κB p65 in infection group at week 1 and week 2 was and 0.6 ± 0.0 and 0.8 ± 0.1, higher than the non-infection group (0.4 ± 0.0) (P < 0.05, P < 0.01). ELISA results showed that the expression level of IL-1β in the intestinal mucosa in infection group at week 1 and week 2 was 33.4 ± 2.2 and 46.1 ± 2.5, higher than the non-infection group (22.3 ± 5.0) (P < 0.01), and that of TNF-α in infection group at week 1 and week 2 was 45.7 ± 2.0 and 55.4 ± 3.6, higher than the non-infection group (25.7 ± 9.3) (P < 0.01). Conclusion Cryptosporidium parvum infection may activate the TLR4/NF-κB signaling pathway, up-regulate the expression of TLR4 and NF-κB p65, and promote the release of inflammatory factors TNF-α and IL-1β, thus inducing intestinal mucosal inflammation.

Key words: Cryptosiporidium parvum, Toll-like receptor, TLR4/NF-κB pathway;, Interleukin 1β, Tumor necrosis factor α