Lactobacillus Plantarum LP-1 Supernatant Modulates The Anti-PEDV Effect Of Intestinal Calcium Ions

Porcine Epidemic Diarrhea（PED）is an acute,highly contagious intestinal disease of pigs caused by Porcine epidemic diarrhea virus（PEDV）.Clinical signs are vomiting,severe diarrhea,dehydration and eventually death of the piglets.The disease has a very high infection rate in lactating piglets,with the mortality rate of more than 95%after infection,causing great harm to the pig industry.Since 2010,the immunization effect of PEDV vaccine is getting worse and worse,the immunized pigs also have outbreaks of PEDV and the mortality rate of newborn piglets after immunization is more than90%.In such an environment,it is urgent to find new ways to control PED.Probiotics can maintain the microbial homeostasis of the intestinal environment by regulating the immune system of the host intestinal mucosa,so that it can promote nutrient absorption,maintain the balance of the intestinal flora and protect intestinal health and so on.It has been shown that several probiotic metabolites have anti-viral effects and can regulate calcium ion concentration in rat small intestinal epithelial cells.In the early stage of our laboratory,a strain of Lactobacillus plantatus was successfully isolated,named as LP-1,and the preliminary test showed that the supernatant of this strain had inhibitive effect on PEDV infection,but the specific mechanism was still unknown.Ca²⁺,as an important second messenger in intracellular signal transduction,is an essential element for maintaining and regulating the physiological functions of the body.When Ca²⁺homeostasis is disrupted,it will cause diseases and metabolic disorders.Studies have shown that some viral infections could modulate intracellular calcium levels or calcium channels to promote self-infection and replication.It is unclear whether PEDV infection can cause an increase in calcium ion concentration and to promote self-replication.Based on this,it is important to investigate the role of Ca²⁺in the process of PEDV infection and whether Lactobacillus plantarum LP-1 supernatant is antiviral by regulating Ca²⁺,both of which provide a theoretical basis for a new approach to PEDV control.The contents and results of this thesis are as follows.Study on the anti-PEDV-LJX strain of Lactobacillus plantarum LP-1 supernatant:The growth curve of Lactobacillus plantarum LP-1 was measured and 12h was determined as the optimal time to obtain the supernatant.MTT assay detected LP-1S diluted 1/4 times as the maximum non-toxic dose for IPEC-J2 cells,and Western-blot method measured 1/4 times dilution as the optimal concentration of anti-PEDV and has a concentration dependence.LP-1S treatment of IPEC-J2 cells significantly reduced PEDV N protein expression at 8h,12h and 24h,and TCID50 results showed that LP-1S pretreatment group significantly reduced virus titer at 12h,24h and 48h.It showed that Lactobacillus plantarum LP-1 supernatant had good anti-PEDV effect.Study on the change of extracellular Ca²⁺concentration on PEDV replication:The standard curve of Ca²⁺concentration was established by flame atomic absorption method,and the extracellular Ca²⁺concentration was detected by flame atomic absorption method,and the extracellular Ca²⁺concentration after PEDV infection was lower than that of the blank control group.The intracellular Ca²⁺concentration was detected by calcium fluorescence probe Fluo-3AM,and the intracellular Ca²⁺fluorescence intensity in PEDV-infected groups was extremely significantly higher than that in the blank control group.MTT method was used to detect EGTA cytotoxicity,and EGTA was used to chelate extracellular Ca²⁺,and absolute fluorescence quantitative PCR and Western-blot were used to detect the PEDV M gene copy number and N protein expression in IPEC-J2 cells,the PEDV M gene copy number and N protein expression were significantly lower in the EGTA pretreatment group than in the PEDV group.To determine the effect of EGTA on PEDV proliferation,Ca Cl₂ was added to supplement extracellular calcium ions after treating the cells with EGTA,and absolute fluorescence quantitative PCR and Western-blot assays revealed that the addition of Ca Cl₂ restored PEDV M gene copy number and N protein expression.Treatment of cells with the calcium channel inhibitor bepridil hydrochloride revealed that the pretreatment group with bepridil hydrochloride reduced PEDV M gene copy number and N protein expression.Pretreatment of cells with the intracellular calcium chelator BAPTA-AM revealed that BAPTA-AM pretreatment also reduced PEDV M gene copy number and N protein expression.These findings suggest that intra-and extracellular Ca²⁺plays an important role in PEDV infection.Study on the anti-PEDV effect of calcium ions regulated by Lactobacillus plantarum LP-1 supernatant:extracellular Ca²⁺concentration was detected by flame atomic absorption,and LP-1S restored the decrease in extracellular Ca²⁺concentration due to PEDV infection at 2h,8h,24h and 48h.The intracellular Ca²⁺concentration was detected with the calcium fluorescent probe Fluo-3AM,and the LP-1S-treated group was able to reduce the abnormal increase in calcium concentration caused by PEDV infection.The expression of TRPV6 was quantified by relative fluorescence,and the expression of TRPV6 was elevated in the PEDV group and reduced in the LP-1S pretreatment group in the presence of PEDV infection.The expression of TRPV6 was detected by Western-blot,and LP-1S was able to reduce the expression of TRPV6 in the presence of PEDV infection at 6h and 24h.The expression of PMCA1b was quantified by relative fluorescence,and LP-1S was able to elevate the expression of PMCA1b in the presence of PEDV infection.The expression of PMCA1b was measured by Western-blot at 6h and 12h,and LP-1S was able to elevate the protein expression of PMCA1b under PEDV infection.The results suggest that Lactobacillus plantarum LP-1 supernatant can regulate TRPV6 and PMCA1b to reduce the amount of Ca²⁺entering the cells to achieve anti-PEDV effect.Preliminary studies on the effect of interfering with TRPV6 and PMCA1b on PEDV replication:the transfected plasmid reached a stable transfection state at 24h,and the interference efficiency was about 40%using Western-blot assay.The intracellular Ca²⁺concentration after interfering with TRPV6 and PMCA1b was detected using the calcium probe Fura-2AM.Interfering with PMCA1b could reduce the permeability of the cell membrane to calcium ions,and the translocation of calcium ions was decreased;substantially increased the intracellular calcium ion concentration,and the intracellular calcium ion after interfering with TRPV6 was slightly increased.The PEDV N protein,PEDV M gene and viral titer of the TRPV6-interfered group detected by using absolute fluorescence quantitative PCR,Western-blot,and TCID50were significantly higher than those of the PEDV group.Interference with TRPV6 led to a significant increase in PEDV that was inconsistent with expectations may due to the changes in the expression of other calcium ion signaling pathways caused by compensatory mechanisms.The role of TRPV6 in the specific compensatory mechanism during the process of PEDV infection needs to be further investigated.The PEDV N protein,PEDV M gene and virus titer in the PMCA1b group were significantly higher than in the PEDV group,suggesting that the increase in intracellular Ca²⁺concentration after interference with PMCA1b was the main cause of increased PEDV replication.In conclusion:intra-and extracellular Ca²⁺play an important role in PEDV infection,and Lactobacillus plantarum LP-1 supernatant can modulate intracellular Ca²⁺concentration to achieve anti-PEDV effect by regulating the expression of TRPV6and PMCA1b.TRPV6 and PMCA1b play a crucial role in PEDV infection.