Effect Of Acute Cold Stress On Intestinal Mucosal Mechanical Barrier Of Chinese Soft-Shelled Turtle

In order to study the effect of acute cold stress on the intestinal mucosal mechanical barrier of Pelodiscus sinensis,the full-length cDNA of fut2 was cloned.We analyzed fut2 gene structure,similarity to its counterparts in selected animals,putatively coding protein structure and phylogenetic relationship.Tissue expression pattern by using qPCR were also determined.Nest,the morphological structure of intestinal mucosa and the expression of Occludin,diamine oxidase activity,intestinal tissue structure and protein expression levels after acute cold stress were investigated.The effects of acute cold stress on intestinal mucus layer of Chinese soft-shelled turtle were further studied.To investigate the effects of low temperature stress on fut2 and occludin,samples from two temperatures?25 °C and 15 °C?,two bacteria?Gram-negative and positive bacteria?challenge in vivo,and 5 time points?6,12,24,72 h and 7 d?,were used to determine the expression of fut2 and occludin in the intestine.Results:1.Cloning of the fut2 cDNA and analysis of its tissue distribution in Pelodiscus sinensisThe full length of fut2 cDNA of Chinese soft-shelled turtle is 1918 bp,including a 5'untranslated region?5'-UTR?of 380 bp and a 3'-UTR?3'-UTR?of 518 bp,and 1020 bp in open reading frame?ORF?encoding 339 amino acid residues.The Fut2 protein is a lipid-soluble protein,and the protein structure is unstable.The protein has a transmembrane helix structure but no signal peptide,which indicates that the Fut2 protein is a transmembrane protein.By SMARTer it is predicated that it contains a conserved Glyco_transf₁1 domain.Phylogenetic tree shows that fut2 protein in Pelodiscus sinensis is most similar to that in Chrysemys picta bellii and Chelonia mydas.Real-time PCR results showed that fut2 was expressed in all selected tissues,with highest expression in muscle and heart,lowest expression in stomach,kidney and brain.It has moderate expression level in ileum,rectum,lung,spleen and liver.The results of this study provide a theoretical basis for further understanding of fut2 gene function.2.Effect of acute cold stress on intestinal integrity of Chinese soft-shelled turtleThe serum DAO activity of Chinese soft-shelled turtle showed a decreasing trend with the increased duration of acute cold stress.In acute cold stress and rewarming experiment,the activity of DAO in serum of Chinese soft-shelled turtle was significantly increased after 3 days of cold stress.With the recovery of temperature,DAO activity returned to normal level.Histopathological results showed that acute cold stress had no significant effect on the morphology of mucosal epithelium of the intestinal tract?the posterior ileum and large intestine?of Chinese soft-shelled turtle;acute cold stress had no significant effect on intestinal villus length and the ratio of villus length to crypt depth in the posterior ileum.However,the thickness of mucosa in the posterior ileum was significantly reduced in acute cold stress group,but not in the large intestine.3.Effect of acute cold stress on intestinal mucus layer of Chinese soft-shelled turtleAfter acute cold stress,there was no significant change in the number of goblet cells in the posterior ileum of Chinese soft-shelled turtle.The number of goblet cells in the acute cold stress group of the large intestine was significantly lower than that in the control group on day 3.In posterior ileum,the thickness of mucus in the acute cold stress three-day group was significantly higher than that in the control group before acute cold stress and on day 3 after acute cold stress.In large intestine,the thickness of the mucus in the three days acute cold stress group was significantly lower than that in the control group on day 3.The fluorescence intensity of the control group before acute cold stress in the posterior ileum was significantly lower than that of other groups.However,the fluorescence intensity of the acute cold stress three days group had no significant difference compared with that in control group on day 3 after cold stress.4.Effects of bacteria stress and acute cold stress on the expression of key factors fut2 and occludin in intestinal mucous mechanical barrier of Chinese soft-shelled turtleThe expression of fut2 gene in the posterior ileum and large intestine showed a tendency to decrease after infection with Aeromonas hydrophila or Staphylococcus aureus.The expression of occludin gene in the posterior ileum was significantly higher in the acute cold stress group than in the normal temperature control group at the early stage of infection?6 and 12 h?with Aeromonas hydrophila or Staphylococcus aureu.Its expression during the middle and late stages of infection?24,72 h and 7 d?were significantly lowerthan the normothermic control group.The expression of occludin in the large intestine was significantly lower in the acute cold stress group than in the normothermic control group after infection with Aeromonas hydrophila in 6 h and Staphylococcus aureus in the preand post-infection periods?6,12,and 24 h?.The expression of occludin was significantly higher in the 7-day infection group than in the normothermic control group after infection with Staphylococcus aureus.The analysis of different bacterial treatments at the same temperature showed that bacterial stimulation alone did not have a significant effect on the expression of fut2 in the intestine of Chinese soft-shelled turtle,but had a significant effect on the expression of occludin.Conclusions:1.The cDNA sequence of fut2 of Chinese soft-shelled turtle was cloned and bioinformatically analyzed.2.By detecting a series of relevant indicators of intestinal mucosal barrier of Chinese soft-shelled turtle,it can be concluded that under the same cold stress,different segments of the intestines,at least posterior ileum and large intestine,response differently.3.Acute cold stress can reduce the thickness of mucus and the number of goblet cells in the large intestine of Chinese soft-shelled turtle,which suggested that the intestinal mucosa of Chinese soft-shelled turtle was damaged.4.Acute cold stress can affect the intestinal mucosal ability of Chinese soft-shelled turtle against bacterial invasion,and different intestinal segments have different resistance to different bacteria concerning the mechanical barriers.