Effect Of LPS And Bacillus Subtilis On The Expression Of Immune Related Genes In Ctenopharyngodon Idellus

This study was to investigate the toxicity of lipopolysaccharide (LPS) to grass carp fry and the influence of Bacillus subtilis on the expression of immune-related gene in grass carp (Ctenopharyngodon idellus).1. In this study, E-LPS was injected intraperitoneally to adult healthy grass carp by different doses (0mg/kg,5mg/kg,15mg/kg,30mg/kg,45mg/kg,65mg/kg,80mg/kg) to detect the toxicity of LPS. And grass carp try of2days post fertilization were treated with1000μg/mL,800μg/mL,500μg/mL,200μg/mL,100μg/mL and0μg/mL E-LPS solution. In the following14days after been injected, there was no death or other abnormal conditions on adult grass carp, that indicat E-LPS is almost non-toxic to adult grass carp; but to the grass carp fry, which was lower mortality rate at50-500ug/mL of E-LPS and only the highest concentration (1000ug/mL) of E-LPS was able to induce grass carp fry100%mortality rate for72h. From the phenomenons we could obtained that the LD50for48h of E-LPS was489.24μg/mL. The strongger resistance to the Aeromonas hydrophila was observed when group grass carp fry bathed with E-LPS, and the immune protection rate were more than70%.2. A. hydrophila crude lipopolysaccharide (C-LPS) and pure E. coli LPS (E-LPS) were injected intraperitoneally to grass carp with4mg/kg respectively and injection of PBS as a control. After48h, total RNA were taken to detect the expression of TNF-a, IL-1β, IL-10and TLR4genes by the RT-PCR in all collected tissues. The results showed, compared to control group, the expression of TNF-a and IL-1β genes in head kidney had significantly up-regulated in E-LPS group and the expression of IL-1β gene was significantly lower in C-LPS group. The TLR4had significantly decreased in the two experimental groups. In spleen the gene IL-1β in C-LPS group was significantly up-regulated compared to control group. Regard to the TLR4, it had significantly decreased in the two experimental groups. In kidney, TNF-a and IL-1β, IL-10levels were significantly increased respectively in E-LPS group and C-LPS group compared to control group, but the expression of TLR4had no significant difference in the two experimental groups. In liver, TNF-a, IL-10and TLR4levels were significantly higher in the two experimental groups, while the expression of IL-1β was significantly up-regulated in E—LPS group compared to the control group and the C-LPS group. In intestine, the level of TNF-a was significantly down-regulated in the two experimental groups compared to the control group; as regard to the genes of IL-1β and TLR4, which had no significant difference compared to the control group. These indicate that C-LPS and E-LPS can regulate the expression of cytokine to adjust the immune reaction of the host, and the LPS could not make the TLR4up-regulation, which is different from the results in the mammal.3. The grass carp were randomly divided into two groups. The fish were fed the basel diet in the control group and the basel diet supplemented with B. subtilis (B. s)(1×109cell/kg) for28d in the probiotic groups. The two groups were then randomly divided into three treaments which challenged with PBS, A. hydrophila and E-LPS respectively (Con+PBS, Con+A. h, Con+LPS, B.s+PBS, B. s+A. h, B.s+LPS). The expression level of the selected genes (TNF-α, IL-1β, IL-10and TLR4) in head kidney, spleen, kidney, liver of the fish were determined by qRT-PCR (quantitative real-time RT-PCR) after challenge. The results showed that the expression level of TNF-a, IL-1β and TLR4genes were significantly up-regulated by B. subtilis in all selected tissues while the expression of IL-10had significantly up-regulated in head kidney, spleen and liver but down-regulated in intestine after28d. B. s supplementation reduced fish mortality by A. hydrophila infection in the probiotic groups comparing with the control group. And the expression of IL-10were also significantly higher in B. s+A. h comparied with Con+A. h in different time point. Besides, the expression of TNF-α, IL-1β in B. s+A. h were significantly down-regulated in other tissues with exception of intestine. It was suggested that B. subtilis can regulate the immune response of grass carp to inhibition or reduce inflammation caused by pathogenic bacteria. After E-LPS injection, the expression of TNF-α and IL-1β were significantly higher in B.s+LPS group than Con+LPS group at1hpi and6hpi, and there was no significantly difference at24hpi, while the expression of TLR4relative stable in Con+LPS group and had significantly down-regulation than the B. s+LPS group. In liver, TNF-a mRNA level was only significantly increased at6hpi, and the expression of IL-10was significantly increased in all phase in B.s+LPS group than Con+LPS group. In intestine, after1hpi, the expression of TNF-α was highest, and the mRNA level of IL-10was significantly higher in B.s+LPS group than Con+LPS group. It was suggested that B. subtilis was able to induce the rapid adjustment the level of cytokines induced by the E-LPS to promote the immune response of fish fed B. subtilis. About TLR4, that illustrated the LPS could not a direct influence the mRNA level TLR4, but it may affect the expression indirectly through other pathways. B. s feeding can induce the expression of TLR4, but the specific mechanism is not well understood, which need to be further researched.