The Effect Of Cold Exposure On Tight Junction Injury Of Mouse Colonic Epithelium

In the cold regions of northern China,low temperature in winter and spring can easily lead to high incidence of digestive tract diseases in young animals,and the resulting diarrhea,poor digestion,slow development and even death have seriously endangered the healthy development of animal husbandry economy.Although modern intensive farming model has become mature,cold stress induced by force majeure factors such as transportation,power outage and semi-open farming is still one of the serious challenges faced by animal husbandry.As the organ with the largest contact area with the external environment,the intestine is more vulnerable to environmental low temperature.Intestinal health is closely related to the integrity of the intestinal barrier.In each segment of the intestinal structure,the colon flora is the most abundant.Once the barrier is damaged,it will cause leakage of toxins and bacteria in the intestinal cavity,resulting in intestinal inflammation and even systemic inflammatory response syndrome.At present,the effect of cold exposure on colonic epithelial mechanical barrier is not clear,but many studies have shown that intestinal inflammation and microorganisms are closely related to intestinal mechanical barrier.Therefore,it is of great significance to explore the effect of cold exposure on intestinal microecology and intestinal barrier and related mechanisms.In order to preliminarily explore the effect of cold exposure on the mechanical barrier of colon epithelial tissue,male C57BL/6 mice of 4-week-old were selected and placed in an artificial climate chamber at 4°C for three weeks each day.The intestinal permeability and morphological structure of colon were detected after cold exposure.The results showed that cold exposure increased the intestinal permeability of mice,shortened the colon and destroyed the morphological structure of colon tissue.To further explore the damage effect of cold exposure on colon tissue,RNA sequencing was performed in mice colon tissue after cold exposure.The results showed that there were 261 differentially expressed genes after cold exposure,which were mainly enriched in growth,signal transduction mechanism,post-translational modification and other functions.The most enriched genes were MAPK,Erb B signaling pathway and cytokine receptor interaction.The transcript GSEA found that the differently expressed genes were related to calcium binding and protein folding,suggesting that cold exposure may affect the function of endoplasmic reticulum.In addition,the analysis of GSEA results showed that the differently expressed genes after cold exposure were correlated with the binding process of histone acetyltransferase.Combined with the research hotspots in recent years,this study locked the deacetylase SIRT1,and found that cold exposure significantly inhibited the expression of SIRT1 in colon tissue.Therefore,SIRT1 was selected as the research target in subsequent experiments.Combined with the above results,we detected the organelle function,intercellular tight junction,redox level and microbial diversity of colonic epithelial tissue.It was found that cold exposure could induce endoplasmic reticulum stress and endogenous apoptosis,damage the tight junction of colon in mice,and lead to mitochondrial fusion,division disorder and redox imbalance.The results of microbial diversity showed that cold exposure up-regulated the abundance of Bacteroidetes and Bacteroidetes S24-7,and down-regulated the abundance of Conchaetes,and the difference between cold exposure group and control group was significant.In order to confirm the effect of cold exposure-induced tight junction damage and flora structure disorder on the susceptibility of acute colitis in mice,DSS was used to establish a mouse model of acute colitis after cold exposure.The fecal occult blood kit and HE staining were used to detect the colon length,DAI and tissue damage score of mice in each group.The m RNA expression level of pro-inflammatory cytokines in mice colon was detected by q RT-PCR.The results showed that the DAI score of mice in the cold exposure + DSS group was significantly higher than that in the DSS group after the fifth day of modeling,and there was no significant difference in tissue damage score.The colon length was significantly lower than that in the DSS group,and the m RNA expression levels of pro-inflammatory cytokines(TNFα,IL-6,IL-1β)were significantly higher than those in the DSS group.The results suggested that cold exposure accelerated the development of DSS-induced acute colitis.In order to further explore the mechanism of colon epithelial tissue injury induced by cold exposure,the endoplasmic reticulum stress model was constructed by Tg on caco-2 colon tool cells.The expression level of SIRT1 was knocked down by RNA interference.Hochst staining,Western Blot and immunofluorescence were used to detect apoptosis and endoplasmic reticulum stress,tight junction and apoptosis-related indicators in each group.The results of cell experiments showed that SIRT1 knock-down increased the protein expression of endoplasmic reticulum stress pathway-related effectors(GRP78,CHOP,p-e If2α/e If2α),decreased the expression of tight junction protein ZO-1,and aggravated Tg-induced apoptosis,promoting the expression of apoptosis-related proteins(Bax/Bcl-2,caspase3,cytochrome c).The results in vitro showed that the knock-down of SIRT1 aggravated endoplasmic reticulum stress-induced apoptosis and intercellular tight junction damage.The above results show that cold exposure can destroy the tight junction of colon epithelium,aggravate endoplasmic reticulum stress and apoptosis,lead to mitochondrial fusion and division disorders,and also destroy the structure of colon flora,and accelerate the development of DSS-induced acute colitis.In vitro cold exposure experiments showed that inhibition of SIRT1 expression aggravated endoplasmic reticulum stress in mice,and then induced apoptosis and epithelial cell tight junction damage.