The Influences Of Probiotic Bacillus On Intestinal Epithelial Apoptosis Of Grass Carp Ctenopnaryngodon Idella

In intensive aquacultural rearing,Aeromonas hydrophila regarded as an opportunistic pathogen is one of the major pathogenic bacteria that causes intestinal inflammation in fish and remains a significant problem with farming grass carp.It is very regarded that probiotics instead of antibiotics to prevent diseases.Strains of Bacillus subtilis Ch9 was isolated from the intestinal tract of grass carp Ctenopharyngodon idella.In our previous studies show that B.subtilis Ch9 effect on the growth and enhance the immune response of grass carp.In this study,influence of B.subtilis Ch9 on intestinal apoptosis of grass carp challenged with Aeromonas hydrophila was studied.Groups of grass carp were orally intubated with B.subtilis or phosphate buffer saline(PBS)for 3 days and subjected to oral A.hydrophila challenge.We divided the experiment fish into three groups,1)Control: oral PBS at 1,3,5 and 6 d;2)A.hydrophila group: oral PBS at 1,3,5 d and challenged with A.hydrophila at 6 d;3)B.subtllis + A.hydrophila: oral B.subtllis Ch9 at 1,3,5 d and challenged with A.hydrophila at 6d.Intestinal tissues were collected and prepared for TUNEL assay,real-time quantitative PCR analysis of caspase-3and-8 and bcl-2,histopathological analyses and caspase-3 and-8 activities at 0,12,24,48,72 and 96 h post-challenge.TUNEL analyses demonstrated that B.subtilis induced intestinal cell apoptosis,and caspase-3 and-8 activities increased significantly compared to the control and A.hydrophila groups.The intestinal damage due to A.hydrophila was also reduced.However,mRNA transcript levels of caspase-3 and-8 and bcl-2 did not markedly elevate.These results suggested that B.subtilis Ch9 increased intestinal apoptosis in fish challenged with A.hydrophila.B.subtilis Ch9-induced caspase-dependent apoptosis may play an important role in reducing intestinal damage following A.hydrophila challenge.In order to further studied the mechanism of B.subtilis Ch9,we divided the experiment fish into two groups,1)A.hydrophila group: challenged with A.hydrophila at 1d,and oral PBS at 2 and 4d;2)A.hydrophila + B.subtllis: challenged with A.hydrophila at 1 d,and oral B.subtllis at 2 and 4d.The samples were collected for the TUNEL assay and detection of caspase-3 and caspase-8 activities.Results showed that B.subtilis Ch9 induced apoptosis and reduced the damage on intestinal of grass carp.The effects of B.subtilis Ch9 and A.hydrophila on Caco-2 cell lines apoptosis were studied,including the survival rate,toxic by bacteria and activities of caspase-3and-8.The results showed that B.subtilis Ch9 could induce apoptosis of Caco-2 cell line.The activity of caspase-3 and caspase-8 was tested in four groups,control:cultured with fresh cell culture medium;Ah group(A.hydrophila): cultured with A.hydrophila in fresh cell culture medium;Bs group(B.Subtllis): cultured with B.Subtllis Ch9 in fresh cell culture medium;Ah+Bs group(A.hydrophila + B.Subtllis):co-cultured with A.hydrophila and B.Subtllis Ch9 in fresh cell culture medium.Samples were collected at 0.5 h,1 h,1.5 h,3 h,and 6 h after inoculation.The results showed that B.Subtllis Ch9 could induce apoptosis of Caco-2 cells challenge with A.hydrophila.These results indicated that B.subtilis Ch9 has a protective effect on intestinal epithelial cells.B.subtilis Ch9 reduces the intestinal damage of A.hydrophila infection via increasing apoptosis.