Vitamin D₃ Regulates The Rotavirus Infection In The Porcine Intestine Via The Endoplasmic Reticulum Stress And Autophagy Pathway

Rotavirus, mainly infection of the mature intestinal epithelial cells, is one of the major pathogenic factors of the diarrhea in piglets. Its prevalence rate is up to 80%, and there is no specific treatment. Previous studies have shown that vitamin D3 has some effect in rotavirus infection. In the present study, we examined whether vitamin D3 regulated rotavirus infection via autophagy and endoplasmic reticulum stress. In experiment 1,24 DLY pigs (26.2±2.02 kg) were used in a 2×2 design, the main factors consisted of rotavirus stress (infection vs. no infection) and dietary levels of vitamin D3 (200 IU vs. 5000 IU). The trial lasted for 7 days. The relative expression of autophagy and endoplasmic reticulum stress-related genes of jejunum and ileum was determined. In experiment 2, the IPEC-J2 cell line infected with rotavirus was used to investigate the effects of vitamin D3 on inhibition of infection and underly autophagy and endoplasmic reticulum stress pathways.1. In vivo, the relative expression of GRP78, XBP1, IRE1, ATF6 and CHOP of jejunum and ileum were significantly up-regulated by the infection of rotavirus(P<0.05), dietary VD3 treatment tended to inhibit the up-regulation. The relative expression of Beclin 1 was significantly increased with VD3 treatment and the stress of rotavirus, while the relative expression of LC3-Ⅱ/Ⅰ and the PR-39 were significantly increased with VD3 treatment (P<0.05).2. Rotavirus infection significantly reduced the IPEC-J2 cell viability. When the multiplicity of infection was 1, the cell viability was significantly decreased (P< 0.05).3. There were no significant improvement on cell viability with vitamin D3 treatment, whereas the concentration of rotavirus antigen and NSP4 decreased with vitamin D3 treatment, when the concentration of vitamin D3 up to 1 μmol/L, the concentration of rotavirus antigen and NSP4 significantly reduced (P< 0.05). The relative expression of GRP78 and CHOP was significantly increased by rotavirus infection (P< 0.05), but the relative expression of GRP78 and CHOP was significantly relieved with vitamin D3 treatment (P< 0.05). There were no significant changes on the relative expression of Beclinland PR-39 by the rotavirus infection. The relative expression of Beclin 1 and PR-39 was significantly increased with the vitamin D3 treatment (P< 0.05).4. The relative expression of GRP78, XBP1, ATF6, CHOP and EDEM was significantly increased by rotavirus infection (P< 0.05), while the relative expression of GRP78, CHOP, XBP1 was significantly decreased with the vitamin D3 treatment (P< 0.05). Under the condition of rotavirus infection, the relative expression of GRP78, PDIA4, calnexin, calreticulin, XBP1, IRE1, ATF6, CHOP and EDEM was significantly increased by TM treatment (P< 0.01), vitamin D3 relieved the relative expression of GRP78, calreticulin, IRE1, ATF6, CHOP and EDEM (P<0.05); The relative expression of GRP78, PDIA4, calnexin, CHOP and EDEM was significantly reduced by 4-PBA treatment(P< 0.01), while there was no significant influence with vitamin D3 treatment. The concentration of capase12 was significantly increased by the stress of rotavirus (P< 0.05), then was reduced with vitamin D3 treatment (P<0.05). Under the condition of rotavirus infection, the concentration of caspase 12 with TM treatment was similar to the concentration with the stress of rotavirus, and the concentration of caspase with 4-PBA treatment was similar to the concentration with vitamin D3 treatment.5. In the autophagy pathway, the concentration of NSP4 were significantly reduced by vitamin D3 treatment (P< 0.05), but there were no effects on the concentration of NSP4 with co-treatment vitmian D3 and the autophagy inhibitor 3-MA or Baf Al. The concentration of NSP4 with co-treatment vitamin D3 and the autophagy inhibitor Rap was similar to by vitamin D3 treatment. The relative expression of LC3-Ⅱwas significantly reduced by the stress of rotavirus and the vitamin D3 treatment (P<0.05). Compared with the group of rotavirus infection and vitamin D3 treatment, the relative expression of LC3-Ⅱwas significantly reduced in the group of rotavirus infection and vitamin D3, and 3-MA treatments. No effect of LC3-II were observed in Baf A1 and Rap treatments. Compared with the group of rotavirus infection, the relative expression of p 62 was significantly reduced (P<0.05), and the relative expression of PMAP23, PG1-5 and PR-39 was significantly increased (P<0.05) by vitamin D3 treatment under the condition of rotavirus infection.In conclusion, these in vivo and in vitro results indicated that ERS and autophagy would be induced by rotavirus. Supplementation with VD3, ERS and autophagy could be relieved through regulating ERS pathways, the mature and degradation of autophagolysosomes.