Analysis Of Portulaca Oleracea L.Extract Inhibiting Porcine Epidemic Diarrhea Virus Replication In Vitro

Porcine epidemic diarrhea(PED)is an acute intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV).It is extremely harmful to piglets and can cause diarrhea,dehydration and even death.Since 2010,PED,which is characterized by high morbidity and high mortality caused by PEDV mutants,has spread throughout the world,resulting in a large number of piglet diseases and deaths,causing huge economic losses to the pig industry.In recent years,despite the progress made in elucidating the molecular epidemiology,diagnosis,prevention and treatment of PED,its epidemic outbreaks continue to increase,posing serious problems for the pig industry and international trade.Portulaca oleracea L.(PO)is an annual herb of the genus Purslane in the family Caryophyllaceae.It is widely distributed in tropical and subtropical regions around the world.It belongs to the dual-use traditional Chinese medicine.Modern pharmacological studies have shown that purslane has various effects such as anti-inflammatory,antibacterial and antiviral,anti-oxidant,lowering blood glucose and lipids,and enhancing immunity.This study firstly evaluated the drug safety range of PO water extract on VH cells and Vero cells,and then VH cells and Vero cells were infected with PEDV to establish an virus infection model in vitro.The inhibitory effect on the virus is determined by the CCK-8 detection method and real-time quantitative PCR method;and determined the effect of drugs during.Analyzing its effect on viral protein replication by western-blot and immunofluorescence.Effects of compounds on the secreted cytokines such as IFN-?,TNF-?,IL-6,IL-12 and IL-22 in PEDV VH cell infection model;western-blot measurement of NF-?B signaling pathway-related proteins in cells of PEDV VH cell infection model expression.The above methods were used to explore the inhibitory effect of purslane extract on porcine epidemic diarrhea virus and its mechanism.The safe concentrations of Portulaca oleracea L.extract on VH cells and Vero cells is ? 25 mg/mL and ? 12.5 mg/mL,respectively.Purslane extract can inhibit the replication of porcine epidemic diarrhea virus on VH cells and Vero cells,and relieve cellular damage caused by virus infection.Western blot analysis showed that the S protein signal was significantly downregulated in PO-treated cells,and the S protein fluorescence signal was decreased in immunofluorescence experiments,indicating that the purslane extract reduced the expression of S protein in a dose-dependent manner.Pretreatment of PO extract at the concentration of 25 mg/mL reduces the overexpression of cytokines TNF-?,IL-22,and IFN-? produced by VH cells stimulated,but it had no significant effect on the expression of IL-6 and IL-12.Purslane extract inhibited the expression of NF-?B and p-NF-?B proteins in VH cells caused by PEHV infection and down-regulated the expression of I?B? protein.At the same time,the expression of MyD88 was inhibited,while the expression of TRIF was not affected,indicating that purslane extract inhibited the activation of NF-?B pathway in VH cells caused by PEHV infection through the MyD88-dependent pathway.Conclusion: Purslane extract can inhibit PEDV replication in VH cells by down-regulating the expression of cytokines(TNF-?,IL-22 and IFN-?)and inhibiting PEDV-activated NF-?B signaling pathway.