Impairment Of Autophagy And Apoptosis In Swine Small Intestine By Selenium Deficiency

Selenium（Se）is one of the indispensable elements in the process of regulating body health.The deficiency of Se can lead to a variety of tissue injuries,such as apoptosis,autophagy.In severe cases even leads to organ failure and disease.The intestine,as one of the important target organs of selenium,whose damage caused by selenium deficiency should be paid more attention.However,there are still insufficient studies and reports on the specific mechani sm of intestinal damage caused by Se deficiency.In this paper,a pilot study was conducted by establishing a porcine small intestinal Se deficiency model in vivo and in vitro,aiming to elucidate the specific mechanism by which Se deficiency causes damage to the porcine small intestine.The experimental white pigs were divided into two groups,The test used large white pigs were divided into two groups,and fed diets with different Se contents（selenium-deficient diet:0.007 mg/kg,normal diet:0.300 mg/kg）to establish a model of Se deficiency in pig.An in vitro Se deficiency model of porcine intestinal epithelial cells was established by culturing IPEC-J2 cells in the normal and Se deficient media respectively.To explore the specific mechanism by which Se deficiency causes injury in the porcine small intestine,we performed ultrastructural observation of small intestinal tissues and cells,combined with fluorescent staining and flow cytometry analysis of IPEC-J2 cells injury.The m RNA and protein expression levels of genes involved in autophagy,apoptosis,endoplasmic reticulum（ER）stress and calcium homeostasis were determined using qRT-PCR and Western blot.The mRNA expression of selenoproteins and the trace element content in the small intestine were detected.The findings are as follows:（1）The expression of selenoproteins was down-regulation in porcine small intestinal tissues and IPEC-J2 cells of the Se deficient group.The results demonstrated the success of constructing in vivo and in vitro test models of intestinal Se deficiency in pigs.Se deficiency resulted in increased Ca and decreased B,Cu,Ni,V,Al levels in the porcine small intestine.（2）Se deficiency resulted in loose villus structure,fragmented hair,shrunken intestinal glands,shrunken blood vessels within the central chylous duct,and fewer goblet cells in the small intestine of pigs.The ultrastructural observation showed that there were obvious autophagic vesicles,autophagosomes,nuclear and apoptotic bodies in the small intesti ne and IPEC-J2 cells of Se deficient pigs.The surface structure of small intestinal tissue is broken,and the fissures are furrowed back.Selenium deficiency disrupts the structural barrier of the porcine small intestine,promotes autophagy of enterocytes,and leads to apoptosis.（3）MDC staining results showed that autophagosomes were increased in IPEC-J2 cells exposed to Se deficiency,suggesting that Se deficiency promotes IPEC-J2 cells.The results of intracytoplasmic calcium staining showed increased intracytoplasmic calcium in IPEC-J2 cells in the Se deficient group,indicating that selenium deficiency causes intracellular calcium overload in porcine intestinal epithelial cells.ROS staining results showed increased ROS in IPEC-J2 cells of the Se-deficient group,which suggested that Se deficiency disrupted the antioxidant balance in the porcine small intestine.The results of cellular AO/EB,fluorescence staining of mitochondrial membrane potential,and flow cytometry analysis together indicated that apoptotic cells were increased in the Se-deficient group,and the above results suggested that Se deficiency led to apoptosis of intestinal epithelial cells.（4）The results showed that the mRNA and protein expression levels of Ca homeostasis-related genes Ca MKK,calpain,and AMPK were significantly increased,whereas the protein expression of SERCA was decreased in the Se-deficient group.The mRNA expression of autophagy-related genes,LC3,ATG5,ATG16,and m TOR,the protein expression of Beclin1,LC3-1,and LC3-2 were increased.The protein expression of mTOR was decreased significantly in the Se-deficient group.The above detection results indicated that Se deficiency could trigger Ca²⁺overload and promote autophagy in the porcine small intestine.（5）The expression of the genes related to apoptosis and ER stress in small intestine and cells of pigs was detected.The m RNA expressions of the ER stress-related genes IRE,XBP1,and CHOP were significantly upregulated in the Se-deficient group.The protein expressions of CHOP,ATF6,PERK,and GRP78 were elevated.The mRNA expressions of apoptosis-related genes Bax,Bcl2,Caspase 3,Caspase 8,Caspase 9,and cytc were increased in the Se-deficient group,while the protein expressions of Bax,Bcl2,bad,Caspase 3,Caspase 9,and cytc were increased.The above results suggested that Se deficiency triggered ER stress and apoptosis in the porcine small intestine.In conclusion,Se deficiency can induce ROS accumulation and intracytoplasmic Ca overload and promote autophagy in the porcine small intestine,and trigger endoplasmic reticulum stress to cause apoptotic injury in the porcine small intestine.The conclusion provides a foundation for further exploring the mechanism of Se deficiency intestinal diseases,prov iding a new target for the treatment and prevention of Se deficiency related diseases,and providing a decision-making basis for animal husbandry and healthy breeding.