Study On The Effects And Mechanism Of Selenomethionine Against Porcine Deltacoronavirus Infection

Selenium,a micronutrient essential for humans and animals,has many important biological functions,including enhancing the antioxidant function,anti-viral infections and improving immune functions.Selenium methionine(Se-Met)is the organic selenide with the highest bioavailability.Studies have shown that supplementation of appropriate amount of Se-Met can promote growth performance,nutrient availability of animals and improve antioxidant capacity.Porcine deltacoronavirus(PDCoV)is a newly discovered porcine intestinal coronavirus in recent years,which could cause severe watery diarrhea in pigs and lead to death in newborn piglets.It mainly invades small intestine and causes acute necrosis of intestinal epithelial cells of piglets and small intestinal villi atrophy and fall off,which in turn decreases feed digestion and absorption,which seriously affects the healthy development of pig industry.At present,studies have shown that the toxicity of porcine enteric coronavirus is associated with the production of reactive oxygen species(ROS),however the mechanism is not clearly demonstrated.In this study,we measured that growth performance,nutrient apparent digestibility,disaccharidase activity,antioxidant capacity and intestinal barrier damage in piglets artificially infected with PDCoV,and studied the antioxidative and barrier protecting effects of Se-Met against PDCoV infection.The effects of Se-Met on PDCoV infection and its potential regulatory mechanism were studied with cells infected with PDCoV,which provides a theoretical basis for the development of nutritional antiviral feed additives.1.Research of the Se-Met on growth performance and antioxidant capacity in piglets infected with PDCoVTwelve eight-day-old healthy piglets(Duroc×Landrace×Yorkshire)with similar weights(2.47±0.15 kg)were randomly assigned into four groups(n=3):the control group(basal diet),the Se-Met group(basal diet+0.3 mg/Kg Se),the PDCoV group(basal diet),the Se-Met+PDCoV group(basal diet+0.3 mg/Kg Se).The trial lasted for 21 days after 3 d adaptation.On day 14 after the experiment started,the piglets in the PDCoV group and the Se-Met+PDCoV group were orally administrated with PDCoV solution(109 TCID50/mL)per piglet,the piglets in the control group and the Se-Met group were orally administrated with cell culture medium.The clinical signs in all piglets were observed daily,the feed intake,body weight and diarrhea of piglets were recorded,the intestinal disaccharidase activities and antioxidant enzyme and the content of virus in feces and tissues of piglets were measured.The results showed the average daily gain(ADG)of piglets,apparent digestibility of crude protein and calcium in piglets were significantly increased(P<0.05),the activities of maltase,lactase in jejunum and sucrase in ileum and the GSH-PX levels in jejunum,ileum and serum of piglets were significantly increased in the Se-Met group when compared to the control group(P<0.05).Compared with the PDCoV group,the ADG and ADFI were significant increased and F/G of piglets was markedly reduced in the Se-Met+PDCoV group(0-21d in the experiment)(P<0.05),the nutrient apparent digestibility(crude protein,crude fat,calcium and phosphorus),the activities of disaccharidases in jejunum and ileum and the levels of SOD,GSH-PX in jejunum and ileum of piglets were improved(P<0,05).In addition,the supplementation of Se-Met could relieve diarrhea,shorten the time of the fecal viral shedding and decrease viral load in the tissues of piglets infected with PDCoV.These results indicated that the supplementation of Se-Met could increase growth performance,nutrient apparent digestibility,intestinal disaccharidases activities and antioxidant capacity in piglets infected with PDCoV,and further improve the effect of anti-PDCoV infection in piglets.2.Effects of Se-Met on PDCoV-induced intestinal barrier injurySe-Met could increase antioxidant capacity in piglets infected with PDCoV.In order to study the effects of Se-Met on PDCoV-induced intestinal barrier injury,the experimental design was also the same as experiment 1,the mucus and tissue samples of jejunum and ileum and serum was collected after piglets were slaughtered.The mucus rheological properties,pathological changes and morphology were measured.Serum was collected to measure intestinal permeability.Tight junction protein,inflammatory factors and apoptosis-associated indexes in jejunum and ileum of piglets were detected.The results showed that the supplementation of Se-Met could increase mucus viscosity and mucus elastic modulus(G')and viscous modulus(G")of the jejunum and ileum in infected or non-infected piglets,which indicated that the supplementation of Se-Met improved intestinal mucus properties in piglets infected with PDCoV.Compared with the PDCoV group,intestinal histopathological injury and intestinal mucosal morphology were significantly improved in Se-Met+PDCoV group.(P<0.05).Also,in the Se-Met+PDCoV group,the levels of D-lactate and diamine oxidase(DAO)in serum of piglets were significantly decreased,the gene expression of ZO-1 and Occludin were significantly increased compared with the PDCoV group(P<0.05).The results showed that the supplementation of Se-Met could improve intestinal barrier function in piglets infected with PDCoV.The Se-Met+PDCoV group significantly down-regulated the levels of inflammatory factors(IL-1 ? IL-6,TNF-? and IL-10)in jejunum and ileum of piglets compared with the PDCoV group(P<0.05),which suggested that the supplementation of Se-Met reduced the inflammatory damage caused by PDCoV infection.In terms of intestinal cell apoptosis,the activities of Caspase3 and Caspase9 and the expression of apoptosis factors(Bax,Cascase3 and Cascase9)in the jejunum and ileum of piglets were observed to decline notably,and the expression level of anti-apoptotic factor(Bcl2)was observed to increase significantly in the Se-Met+PDCoV group compared with the PDCoV group(P<0.05).The results indicated that the supplementation of Se-Met decreased intestinal cell apoptosis in piglets infected with PDCoV.In addition,compared with the control group,the expression level of Bc12 factor in jejunum and ileum of piglets in Se-Met group was significantly increased(P<0.05).These results suggest that Se-Met can improve intestinal mucus properties and intestinal barrier function,reduce intestinal inflammatory response,inhibit intestinal cell apoptosis and protect the integrity of intestinal mucosal barrier in piglets infected with PDCoV.3.Research of Se-Met on gut microbiota and short-chain fatty acids in piglets infected with PDCoVIn order to study the effects of Se-Met on gut microbiota and short-chain fatty acids in piglets infected with PDCoV,the colonic contents of piglets were collected at the end of the experiment.Gut microbiota profiles in the colon of piglets were investigated using 16S rRNA sequencing.The short-chain fatty acids(SCFAs),including acetic acid,propionic acid and butyric acid,were detected using high performance liquid chromatography.The results showed there was no difference in the intestinal microbiota of piglets between the control group and the Se-Met group(P>0.05).Compared with the PDCoV group,the relative abundance of Coriobacteriaceae(Family),Subdoligranulum(Genus)and Prevotellaceae_UCG-003(Genus)in piglets were significantly increased(P<0.05),and the relative abundance of Prevotellaceae_NK3B31_group and Prevotella₂ in piglets were significantly decreased in the Se-Met+PDCoV group(P<0.05).The contents of acetic acid,propionic acid and butyric acid in colon of piglets,in Se-Met+PDCoV group were significantly increased compared with PDCoV group(P<0.05).In addition,there appeared some correlation between bacterial genera and SCFAs in colon of the piglets.The result showed that Se-Met could regulate intestinal homeostasis and energy metabolism of piglets infected with PDCoV.4.Effects and mechanism of Se-Met against PDCoV infectionSe-Met could inhibit intestinal barrier damage induced by PDCoV infection in piglets,among them,cell apoptosis is closely related to the pathogenesis of PDCoV.The protective effects of Se-Met against PDCoV-induced cell apoptosis were investigated using the ST cells as a model,which can effectively support the growth of PDCoV in cell cultures.First,the maximum safe concentration of Se-Met on ST cells was determined by MTT method.Then,the different concentration of Se-Met was incubated with ST cells for 24 h,inoculated with PDCoV(MOI=1),incubated with Se-Met for 24 h,the effect of Se-Met on PDCoV replication was analyzed.The results showed Se-Met inhibited PDCoV replication in ST cells,the inhibition was dose-dependent with Se-Met concentration.Next,the different concentration of Se-Met was incubated with ST cells for 24 h,inoculated with PDCoV(MOI=0.1),incubated with Se-Met for 24 h,and the cell apoptosis indexes(caspase3 and caspase9 activity,apoptosis rate),markers of oxidative stress(ROS,GSH-PX,SOD and MDA levels),and Nrf2,HO-1 expression were measured to study the effects and possible mechanism of Se-Met on cell apoptosis induced by PDCoV.The results showed that Se-Met could reduce Caspase3 and Caspase9 activities and apoptosis levels induced by PDCoV,the effection was dose-dependent with Se-Met concentration,which suggested that Se-Met had a significant protective effect on PDCoV-induced apoptosis.In the NAC-pretreated cells,the NAC+PDCoV group significantly decreased Caspase3 and Caspase9 activities and apoptosis levels compared with the PDCoV group(P<0.01),which indicated that the apoptosis induced by PDCoV was related to oxidative stress.Besides,Se-Met enhanced the activities of SOD,GSH-PX and reduced the content of MDA and ROS,which described that the protective effect of Se-Met on apoptosis induced by PDCoV was related to its antioxidant capacity.Furthermore,the Se-Met+PDCoV group increased mRNA levels and protein expression of Nrf2 and downstream HO-1 compared with the PDCoV group(P<0.05),which suggested that Se-Met might exert antioxidant effects through activation of Nrf2 signaling pathway.After pretreating with Nrf2 inhibitor(ML385)on ST cells,the expressions of Nrf2 and HO-lgene and protein were significantly decreased on ST cells infected or non-infected with PDCoV(P<0.05),and Nrf2 inhibitor eliminated the protective effects of Se-Met on PDCoV-infected ST cells,which suggested that Se-Met alleviates PDCoV infection by improving Nrf2 expression in ST cells.These results indicated that Se-Met could inhibit the replication of PDCoV on ST cells and cell apoptosis induced by PDCoV via activating Nrf2 pathway,in which Nrf2 plays a crucial role.In conclusion,Se-Met could improve growth performance,alleviate the diarrhea,inhibit PDCoV replication in piglets infected with PDCoV.The protective effect of Se-Met might be to improve the activity of intestinal antioxidant enzymes via activating Nrf2 pathway,inhibit intestinal cell apoptosis,strengthen intestinal barrier and finally reduce the occurrence of diarrhea in piglets infected with PDCoV.The study provides a scientific basis for dietary supplementation of Se-Met to protect piglets from PDCoV-infected diarrhea.