Regulation And The Mechanism Of Ileal Tight Junctions By SIRT2 Under Cold Stress

Exposure to a cold environment can lead to the occurrence of cold stress in livestock and poultry,and loss of livestock due to extreme weather occurs in northern China and alpine regions.As one of the tissues connected with the external environment,the endoplasmic reticulum and other organelles in the intestinal epithelium are powerful enough to cope with the complex intestinal environment,but under stress,the endoplasmic reticulum can be overloaded,resulting in dysfunction of the intestinal epithelium and imbalance of the internal environment,which can be harmful to the function and health status of the intestine and lead to the occurrence of intestinal inflammatory diseases.As an NAD⁺-dependent nicotinamide deacetylase that regulates protein acetylation modification,SIRT2 is widely involved in various biological processes such as aging,differentiation,and cell death,and is widely expressed in various tissues and organs,but its role in regulating stress function in livestock is not entirely known.Therefore,this experiment focuses on the hot issue of a high incidence of various intestinal diseases in cold-stressed livestock and intends to investigate the response of mouse ileum tissue to cold exposure,and to reveal the mechanism of SIRT2 regulation of ileal structure and function in cold-stressed mice,thereby providing the scientific basis and therapeutic means for the prevention in cold-stressed livestock.Therefore,in this experiment,C57BL/6J mice were randomly divided into a normothermic control group and a cold exposure group and treated with a temperature of 4°C for 3 h per day for 3 weeks under the condition of cold exposure.The morphological and ultrastructural changes of mouse ileal tissues,the expression of tight junction-related proteins,pro-inflammatory cytokines,endoplasmic reticulum stress,and autophagy-related indicators,as well as the level of SIRT2 expression and acetylation modification of Fox O1 in each group,were examined by detecting changes in mouse intestinal permeability using HE,Masson staining,TEM detection,immunofluorescence and Western blot.The SIRT2^-/-mice were subjected to cold exposure,and the above techniques were used to detect the relevant indexes to investigate the regulatory effect of SIRT2 on cold exposure injury to ileal tight junctions.Subsequently,in vitro experiments were performed using the mouse small intestinal epithelial cell line MODE-K to simulate endoplasmic reticulum stress,and plasmid transfection,Western blot,and immunofluorescence techniques were used to investigate the regulatory mechanism of SIRT2 on tight junction injury under stress conditions.The results showed that chronic cold exposure resulted in increased intestinal permeability and altered ileal morphological structure;Western blot results showed that cold exposure down-regulated the expression of tight junction-related proteins（ZO-1,Claudin1,and Occludin）;increased pro-inflammatory cytokines（TNF-α,IL-1β,and IL-6）and endoplasmic reticulum stress-related indicators（GRP78,CHOP,p-e IF2α,and XBP1s）;meanwhile,the ratio of LC3II/LC3I,the expressions of p62 and Beclin1 increased;chronic cold exposure inhibited the expression of SIRT2,Increase Fox O1 acetylation modification.Knockdown of SIRT2 in the cold exposure state alleviated the downregulation of ileal tight junction-related proteins（ZO-1,Claudin1,and Occludin）,reduced intestinal permeability,and alleviated structural damage caused by cold exposure.Enhanced the expression of autophagy-related proteins（LC3II and Beclin1）and down-regulated the expression of p62.Knockdown of SIRT2 in a cold-exposed state increased endoplasmic reticulum expansion with spherical mitochondrial electron density in ileal epithelial cells,suppressed CHOP expression,promoted XBP1s expression,and had no significant effect on GRP78 and p-e IF2αexpression,accompanied by high expression of pro-inflammatory cytokine IL-6.SIRT2 knockdown significantly increased the expression of AC-Fox O1.Inhibition of SIRT2 in the endoplasmic reticulum stress state was able to upregulate ZO-1 expression and was accompanied by high AC-Fox O1 expression and increased autophagosome numbers.Subsequently,an autophagy agonist model and an autophagy inhibition model were constructed using rapamycin and 3-MA based on the sh-SIRT2+Tg model,respectively,to analyze the effects of autophagy on ZO-1 and Claudin1.The results showed that inhibition of autophagy could significantly decrease the expression of ZO-1 and down-regulate the expression of Claudin1compared with the Tg+sh-SIRT2 treatment group.In conclusion,cold exposure can cause the occurrence of endoplasmic reticulum stress,damage tight junctions,and increase intestinal permeability in mice ileum;SIRT2 deletion can upregulate the acetylation modification of Fox O1 under stress,enhance the occurrence of autophagy,alleviate tight junction damage and reduce intestinal permeability;autophagy may serve as a recovery pathway for the disturbance of ileal environment under stress.