Genipin inhibits rotavirus-induced diarrhea by suppressing viral replication and regulating inflammatory responses

Abstract

Rotavirus is the leading cause of acute gastroenteritis among young children worldwide. However, agents specifically designed to treat rotavirus infection have not been developed yet. In this study, the anti-rotavirus and anti-inflammatory effects of genipin, a chemical compound found in the fruit of Gardenia jasminoides, were evaluated. Genipin had an antiviral effect against the human rotavirus Wa and SA-11 strains in vitro, and it inhibited two distinct stages of the viral replication cycle: attachment and penetration (early stage) in pre-treatment and assembly and release (late stage) in post-treatment. Additionally, genipin downregulated nitric oxide synthase and pro-inflammatory cytokines in lipopolysaccharide-stimulated RAW264.7 cells and rotavirus-infected Caco-2 cells. Oral administration of genipin before and after viral infection with the murine rotavirus epidemic diarrhea of infant mice strain led to a reduced duration of diarrhea and faecal viral shedding and to decreased destruction of the enteric epithelium. Genipin could have potential as a natural compound with preventive and therapeutic effects against infection and colitis caused by rotavirus.

Figure 1

Inhibition effects of genipin on NO, PGE₂, and pro-inflammatory cytokines in Caco-2 cells. Genipin suppressed rotavirus-infected (A) NO and (B) PGE₂ in Caco-2 cells. Effect of genipin on suppression of rotavirus-infected production of pro-inflammatory cytokines, (C) IL-6, (D) IL-10, (E) IL-1β, and (F) TNF-α in Caco-2 cells. Caco-2 cells were pre- and post-treated with genipin at different concentrations of 10, 50, 100, and 150 µM/mL and incubated for 24 h. Untreated control cells were inoculated with fresh media only. Data are presented as mean ± SEM; **p < 0.005, ***p < 0.001, ****p < 0.0001.

Figure 2

Inhibitory activity of genipin against rotavirus using real-time PCR as a viral titre assay. Genipin at 10, 50, 100, and 150 µM/mL inhibited rotavirus in (A) pre-treatment and (B) post-treatment on MA104 cells. Caco-2 cells were (C) pre- and (D) post-treated with genipin at different concentrations. Data are presented as mean ± SEM; *p < 0.01, **p < 0.005, ***p < 0.001, ****p < 0.0001.

Figure 3

Inhibitory activity of genipin against rotavirus in MOI-dependent manners. Genipin at 100 µM/mL was pre- and post-treated with different MOIs of 0.1, 0.5, and 1. Control was infected rotavirus without genipin. Pre; pre-treatment, Post; post-treatment. Data are presented as mean ± SEM; *p < 0.01, **p < 0.005, ***p < 0.001, ****p < 0.0001.

Figure 4

Effects on the viricidal activity of genipin. Viricidal activity of genipin against rotavirus in MA104 cells. Viral infectivity was determined by qPCR. Data are presented as mean ± SEM; *p < 0.01, **p < 0.005.

Figure 5

The effect of the genipin kinetics of rotavirus release. Genipin pre- and post-treatment affects the kinetics of rotavirus release. Rotavirus (Wa strain)-infected MA104 cell supernatants were harvested at selected times during the rotavirus replication cycle (0 to 48 h). Data are presented as mean ± SEM; *p < 0.01, **p < 0.005, ****p < 0.0001.

Figure 6

Effects of genipin treatment in vivo. (A) Percentage of total diarrhea; (B) diarrhea score alterations in EDIM-infected neonatal mice. The percentage of total diarrhea in EDIM-infected neonatal mice was calculated according to the frequency of diarrhea, and the diarrhea score was determined according to the colour and shape of stool samples collected each day. Black, PBS group; blue, EDIM-infected group; red, pre-treated with genipin group; green, post-treated with genipin group. Percentage and score data are expressed as mean ± SEM. *p < 0.01, **p < 0.005, ***p < 0.001, ****p < 0.0001, compared with the EDIM-infected group.

Figure 7

Anti-rotavirus effect of genipin on viral RNA shedding titre. The experiments were evaluated for 8 days, and viral RNA copies were determined by qPCR. Black, PBS group; blue, EDIM-infected group; red, pre-treated with genipin group; green, post-treated with genipin group. Percentage and score data are expressed as mean ± SEM. ****p < 0.0001, compared with EDIM-infected group.

Figure 8

Schematic diagram of genipin experiments. (A) Pre-treatment with genipin before rotaviral infection and (B) post-treatment with genipin after infection suppressed viral replication cycle in intestinal epithelial cells, respectively, and both decreased diarrhea frequency and score as well as inflammatory cytokine levels.