| Selenium(Se)is an essential trace element in the growth and development stages of animals.In addition,Se deficiency poses a serious threat to the swine industry and further endangers human health through the accumulation in the food chain.Clinically,Se deficiency causes damage to digestive systems and causes diarrhea in animals,but the molecular mechanism by which Se deficiency affects the small intestine is still unclear.Harbin is located in a Se-deficient belt in China.Case studies were conducted on swine farms and free-range households in the surrounding areas of Harbin to elucidate the epidemiological conditions of Se deficiency-related diseases in swine in the region and to determine the molecular mechanism by which the ileum is damage by Se deficiency in swine.The study was conducted from October 2017 to October 2018.The statistics included the number of cases of Se deficiency in swine,the age of the cases,and the time of onset to calculate the incidence,time of occurrence,and age of incidence.Then,in vitro and in vivo models of Se deficiency were established to explore the molecular mechanism by which Se deficiency leads to swine ileum injury.IPEC-J2 cells were cultured in Se-deficient medium for 5 days to establish an in vitro model.Furthermore,24 pure white male castrated swine were divided into two groups and fed with a Se-deficient diet(Se content 0.007 mg/kg)and a normal diet(Se content 0.300 mg/kg)for 16 weeks to establish the in vivo model.Pathological observation and ultrastructure observation of cells and tissues were used to investigate the damage to the swine ileum due to Se deficiency.The m RNA and protein expression levels of genes related to the TNF-? necroptosis pathway,NF-?B inflammatory pathway,and Th1/Th2 balance were detected by q RT-PCR and Western blot to reveal the mechanism of the effects of Se deficiency on the swine ileum.The results showed the following:(1)The results of the epidemiological survey of Se deficiency-related diseases of swine in Harbin showed that from October 2017 to October 2018,the incidence of Se deficiency-related diseases of swine in free-range households in Harbin and the surrounding areas was higher than that of swine from farmers;the incidence of affected suckling piglets of free-range farmers was higher than that of affected weaned piglets and fattening swine.The results showed that in Harbin and the surrounding areas,only a few suckling swine from farmers had Se deficiency-related diseases due to intensive farming,and the free-range households' swine faced a certain risk of Se deficiencyrelated diseases.Hence,we should pay close attention to elemental Se supplementation of feed,treatment of symptomatic animals,and timely prevention.(2)In the in vitro test,after 5 days of Se-deficient culture,IPEC-J2 cells could still not reach 80% of the value-added range,and a large number of cells failed to differentiate,adhere,and adopt a spherical shape.Flow cytometry analysis of necroptosis showed that Se-deficient culture increased the cell necroptosis rate by 4.5%.Se deficiency has a severe impact on the proliferation and differentiation of IPEC-J2 cells and leads to cell necroptosis.(3)In the in vivo test,after 16 weeks of Se-deficient feeding,the clinical temperature of the test swine was normal.However,the test swine showed depression,slow movements,gray skin,and weak muscles.The experimental swine had subcutaneous edema in the abdomen and buttocks.The experimental swine swung back and forth while walking and exhibited hind limb tremors during forced exercise,with mild diarrhea.A postmortem examination showed that the skin of the affected swine was pale yellow with yellowish-white jelly infiltration under the skin of the neck and buttocks.The muscles were gray and had mulberry heart disease.There was more gas in the stomach.Some intestinal belching was purple and red,with a small amount of bleeding from the mucosa.The pathological structure of swine ileum tissue also changed significantly,and the number of goblet cells at the edge of ileum villi was significantly reduced,the ileum villi edge was blurred,and inflammatory cell infiltration occurred in the tissue.In addition,the length of the ileum villi in the Se-deficient group was significantly lower than that in the normal group,and the depth of the ileum crypts in the Se-deficient group was significantly higher than that in the normal group.The ratio of depth was also significantly reduced compared to the normal group.Scanning electron microscopy and transmission electron microscopy experiments were used to observe the ultrastructure of swine ileum.The results showed that the velum on the surface of Se-deficient swine had ruptured,the surface of the velvet was uneven,the ileum microvilli were lost,and the cells in the ileum exhibited many typical features of necroptosis.These factors include cell damage,nuclear membrane rupture,leakage of chromatin,disintegration of nuclear structure,swelling of organelles,mitochondrial ridge breakage,mitochondrial burst,and even contents overflowing in the cytoplasm.The above observation results showed that Se deficiency caused damage to swine ileum tissue,destroyed its digestive function and intestinal mucosal barrier and led to ileum tissue necroptosis and inflammatory reactions.(4)Detection of the genes related to the NF-?B pathway and Th1/Th2 balance in the IPEC-J2 cells and swine ileum tissue revealed that the m RNA and protein levels of IL-1?,i NOS,COX-2,and NF-?B in the Se-deficient group were significantly upregulated and the m RNA levels of IFN-?,IL-10,and IL-4 were significantly downregulated,indicating that Se deficiency could cause Th1/Th2 imbalance and NF-?B pathway activation.(5)Detection of the genes related to the TNF-? necroptosis pathway in the IPEC-J2 cells and swine ileum tissue revealed that the m RNA and protein levels of TNF-?,RIPK3,MLKL,and FAS were significantly upregulated in the Se-deficient group.These results indicate that Se deficiency could activate the TNF-? necroptosis pathway.In summary,this experiment first investigated the epidemiology of Se-deficient swine in Harbin,and the results showed that there were multiple occurrences of Se deficiency-related diseases in suckling piglets and weaned piglets,and the incidence of Se deficiency-related diseases in the swine in free-range households was higher than that in farms.The experiment also elucidated the molecular mechanism of Se deficiency-induced swine ileum injury.Se deficiency caused swine ileum tissue damage,caused Th1/Th2 imbalance,activated the NF-?B inflammation pathway,and activated the TNF-? programmed necroptosis pathway.These findings provided new clues for the prevention and assessment of diseases caused by Se deficiency in the swine industry. |
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