Vitamin D Alleviates Porcine Epidemic Diarrhea Virus Infection And The Underlying Mechanisms

In recent years,porcine epidemic diarrhea virus（PEDV）infection is one of the main causes of piglet diarrhea and death in our country,causing serious economic losses.Vaccination used to be an effective method to prevent swine epidemic diarrhea,but due to the rapid mutation of the virus,vaccines are easily invalidated.Therefore,it is urgent to look for possible nutritional measures to inhibit virus replication and alleviate infection symptoms.VD is a fat-soluble vitamin with immune regulation and resistance to pathogenic microorganism.Whether VD can inhibit PEDV replication and relieve the symptoms of virus infection in piglets have not been reported.Therefore,firstly,this study was conducted to investigate whether VD can alleviate the symptoms of PEDV infection in vivo,and then to explore the possible mechanism of VD on inhibiting PEDV replication and alleviating infection symptoms in vitro.Experiment 1 Effects of dietary supplementation with different levels of 25（OH）D₃on growth performance,immune function and antioxidant capacity of weaned pigsIt is generally believed that the biological efficacy of 25（OH）D₃is higher than VD₃,but there are few studies reported the effects of 25（OH）D₃on the growth performance of weaned piglets,and whether 25（OH）D₃can regulate the immune function and antioxidant capacity of weaned piglets.Therefore,the aim of this experiment was to evaluate the effects of different doses of 25（OH）D₃supplementation on growth performance,immune function and anti-oxidative capacity in piglets.Thirty-five weaned pigs were divided into five groups and fed 5.5,43.0,80.5,118.0,155.5μg 25（OH）D₃/kg in a 21-d trial,respectively.No differences were observed in average daily gain（ADG）,average daily feed intake（ADFI）and feed to gain ratio（F/G）.But increasing dietary25（OH）D₃concentration linearly increased serum 25（OH）D₃level（P<0.05）,and decreased CD3+CD4+and CD3+CD8+T cells frequency（P<0.05）and serum complement component 3 level（P=0.05）.In addition,increasing dietary 25（OH）D₃concentration linearly and quadratic increased serum glutathione peroxidase（GSH-Px）activity（P<0.05）,and quadratic increased serum MDA level（P<0.05）.Overall,feeding high-dose 25（OH）D₃to weaned pigs partly improved immune function and anti-oxidative capacity.Experiment 2 Effects of dietary supplementation with 25（OH）D₃on growth performance,intestinal structure and function of PEDV-challenged pigletsThis experiment was conducted to determine whether feeding 25（OH）D₃to weaned pigs would alleviate PEDV infection.Forty-two weaned pigs were allotted to 1 of 6dietary 25（OH）D₃treatments（5.5,5.5,43.0,80.5,118.0,155.5μg 25（OH）D₃/kg diet）for26 d.On d 22 of the trial,all the treatments were orally administrated with PEDV except for one of the 5.5μg 25（OH）D₃/kg treatment,which was challenged with the same volume of sterile saline and served as control.ADG was reduced by PEDV infection（P<0.05）.PEDV administration increased diarrhea index,diarrhea rate,crypt depth and serum diamine oxidase activity（P<0.05）,and reduced villous height and the ratio of villous height to crypt depth.Serum Ig M and complement component 4 levels were also increased by PEDV challenge.However,the diarrhea index（P<0.05）,diarrhea rate（P<0.1）,serum DAO activity（P<0.1）,and crypt depth（P<0.05）were linearly decreased with increasing dietary 25（OH）D₃supplementation.The villous height and the ratio of villous height to crypt depth were increased with increasing dietary 25（OH）D₃supplementation.Furthermore,PEDV challenge significantly increased the gene expression of RIG-Ⅰ,TLR2,My D88,IL-6,IL-8,IFN-λ,LC3Ⅱand ATG5 in the jejunal mucosa（P<0.05）,and increased p-STAT1 and p-STAT3 Protein level;but 155.5μg25（OH）D₃/kg supplementation down-regulated the m RNA abundance of TLR2,TRIF,IL-6,IL-8,IFN-λ1,IFNAR-1,LC3Ⅱand ATG5（P<0.05）,and decreased p-STAT1 and p-STAT3 protein level.These results suggested that dietary supplementation of 155.5μg25（OH）D₃/kg could alleviate intestinal damage and protect against PEDV-induced inflammatory status.Experiment 3 Effects of 1,25（OH）₂D₃on cell cycle and PEDV replication in IPEC-J2 cellsThe aim of this experiment was to investigate whether 1,25（OH）₂D₃could inhibit PEDV replication and alleviate cell damage in IPEC-J2 cells.The results showed that1,25（OH）₂D₃significantly inhibited PEDV-induced cell apoptosis and mitochondrial damage（P<0.05）.PEDV-N gene and protein levels were decreased by 1,25（OH）₂D₃treatment for 48 h（P<0.05）,but not for 24 h.Gene ontology enrichment analysis of transcriptome differential genes found that PEDV affected the cell cycle process.We also found PEDV infection significantly increased G1 phase and decreased S phase of IPEC-J2 cells after 24 h infection（P<0.05）.Blocking the cells in G1 phase significantly increased PEDV-N gene and protein levels（P<0.05）.In addition,1,25（OH）₂D₃significantly decreased the number of cells in G1 phase and increased S phase with PEDV infection（P<0.05）.Compared with PEDV infection group,1,25（OH）₂D₃supplementation significantly increased the gene expression of cyclin D and cyclin E（P<0.05）.Moreover,on the condition of PEDV infection,1,25（OH）₂D₃supplementation increased p-ERK1/2 level,and the regulation of 1,25（OH）₂D₃on cell cycle progression was abrogated by ERK1/2 inhibitor,as well as the m RNA expression of cyclin D.The inhibition of 1,25（OH）₂D₃on PEDV replication was also eliminated by ERK inhibitor.Taken together,these results demonstrated that 1,25（OH）₂D₃supplementation inhibited PEDV replication,and the anti-virus effect of 1,25（OH）₂D₃was mediated in part by regulating cell cycle progression through ERK1/2 signaling pathway.Experiment 4 Effects of 1,25（OH）₂D₃on ROS production and PEDV replication in IPEC-J2 cellsROS plays important roles in intracellular signal transmission and resistance to pathogenic microorganism infection.But whether ROS is involved in inhibition of PEDV replication is still unknown.Therefore,the aim of this experiment was to explore whether 1,25（OH）₂D₃inhibited PEDV replication through ROS production.The results showed that 1,25（OH）₂D₃can significantly increase the level of cellular ROS（P<0.05）.When ROS was reduced by NAC supplementation,the inhibitory effect of 1,25（OH）₂D₃on PEDV replication was also eliminated（P<0.05）.In addition,1,25（OH）₂D₃significantly increased i NOS expression and NO production（P<0.05）,and the effects of1,25（OH）₂D₃on ROS production and inhibition of PEDV replication were eliminated by i NOS inhibitor.Furthermore,both 1,25（OH）₂D₃supplementation and VDR overexpression increased the activity of i NOS promoter in HEK293T cells（P<0.05）.We also found that 1,25（OH）₂D₃can induce ROS production through activating ERK1/2.In summary,1,25（OH）₂D₃could inhibit PEDV replication through inducing ROS production.Experiment 5 Effects of 1,25（OH）₂D₃on cell metabolism and PEDV replication in IPEC-J2 cellsViral infection can disrupt AMPK activity,leading to disorders of cell metabolism.It is unclear whether PEDV infection can affect cell metabolism by regulating AMPK activity,and whether 1,25（OH）₂D₃can regulate cell metabolism and inhibit PEDV replication.In this study,we found PEDV infection increased p-AMPK level,and significantly promoted glucose absorption（P<0.05）,and increased the production of pyruvate and ATP（P<0.05）.In addition,both glycolysis inhibitor and AMPK inhibitor inhibited PEDV replication（P<0.05）,while AMPK activator promoted PEDV replication（P<0.05）.However,on the condition of PEDV infection,1,25（OH）₂D₃supplementation decreased p-AMPK and ATP level（P<0.05）.We also found that1,25（OH）₂D₃supplementation significantly increased LDHA expression and lactic acid level（P<0.05）.Lactate dehydrogenase inhibitor significantly promoted PEDV replication（P<0.05）.Moreover,on the condition of PEDV infection,1,25（OH）₂D₃supplementation increased p-m TOR level,and the inhibition of PEDV replication by1,25（OH）₂D₃was eliminated by rapamycin,an m TOR inhibitor（P<0.05）.The above studies suggested that 1,25（OH）₂D₃can increase p-m TOR and reduce p-AMPK levels,thereby promoting the conversion of pyruvate to lactate and reducing ATP production,and then inhibiting PEDV replication.Experiment 6 Effects of 1,25（OH）₂D₃on PEDV-induced inflammatory response in IPEC-J2 cellsThe aim of this experiment was to investigate whether 1,25（OH）₂D₃could inhibit PEDV-induced inflammatory cytokines expression and its possible mechanism.The experiments showed that PEDV infection for 1 h increased the gene expression of IL-8,IL-19 and CCL20 in IPEC-J2 cells（P<0.05）.PEDV also significantly increased IL-19and CCL20 expression with time increasing（P<0.05）.1,25（OH）₂D₃supplementation not only inhibited IL-8,IL-19 and CCL20 expression induced by PEDV infection for 1 h,but also inhibited IL-19 and CCL20 expression induced by PEDV infection for 24 h（P<0.05）.In addition,we also found that 1,25（OH）₂D₃inhibited the increase of p-NF-κB,p-STAT1 and p-STAT3 levels induced by PEDV infection for 24 h.In addition,1,25（OH）₂D₃supplementation inhibited ISG15 and Mx A expression induced by PEDV（P<0.05）.Furthermore,1,25（OH）₂D₃also inhibited IL-8,IL-19 and ICAM-1 expression induced by poly（I:C）（P<0.05）.Besides,the inhibitory effect of 1,25（OH）₂D₃on IL-19and CCL20 expression was weakened by VDR si RNA,while overexpression of VDR significantly inhibited IL-19 and CCL20 expression induced by PEDV.The above studies indicated that 1,25（OH）₂D₃could inhibit PEDV-induced inflammatory cytokines expression by regulating NF-κB and JAK/STAT signaling pathways.In summary,we found that high doses of 25（OH）D₃（155.5μg/kg）supplementation could alleviate the symptoms of PEDV infection and intestinal injury of PEDV-challenged piglets.In addition,1,25（OH）₂D₃could inhibit PEDV replication in intestinal epithelium cells through regulating cell cycle progression,inducing ROS production and regulating glucose metabolism process,respectively.Moreover,1,25（OH）₂D₃could inhibit inflammatory cytokines expression induced by PEDV through suppressing NF-κB and JAK/STAT signaling pathways,thereby alleviating cell damage.