Effect of Polygonum cuspidatum Aqueous Extract on Porcine epidemic diarrhoea virus Replication in IPEC-J2 Cells

doi:10.7668/hbnxb.20195708

Abstract

Abstract:

The aim of this study was to explore the reproductive adaptability of PEDV in porcine intestinal epithelial cells(IPEC-J2)and to evaluate the inhibitory effect of Polygonum cuspidatum water extract on it in vitro.The effects of different trypsin concentrations and different time on the activity of IPEC-J2 cells and the expression of PEDV N protein in cells were evaluated by cytotoxicity assay and Western Blot assay.The virus titer,PEDV N gene transcription level and N protein expression were detected by TCID₅₀,Real-time Fluorescence Quantitative PCR and Western Blot.The effect of water extract of P.cuspidatum on the proliferation activity of IPEC-J2 cells was detected by CCK-8 method.Further,the inhibitory effect of P.cuspidatum water extract on the expression of PEDV N gene and N protein was detected by Real-time Fluorescence Quantitative PCR and Western Blot experiments,and the effect of P.cuspidatum water extract on each stage of PEDV life cycle was explored.The results showed that when the concentration of trypsin was 2 μg/mL,trypsin had no effect on the cells.At this time,PEDV showed the best reproductive adaptability and reached the highest virulence value after 36 h.When the concentration of P.cuspidatum water extract was lower than 100 μg/mL,there was no cytotoxicity at 12,24 h.The water extract of P.cuspidatum significantly inhibited the expression of N gene under Co-treatment and Post-treatment,and significantly inhibited the expression of PEDV N gene mRNA and N protein in a dose-dependent manner.The water extract of P.cuspidatum had a significant inhibitory effect on PEDV N gene mainly in the replication and proliferation stage of the virus life cycle,and had no significant inhibitory effect in other stages.In summary,2 μg/mL trypsin had the best adaptability to PEDV reproduction,and the water extract of P.cuspidatum had a significant inhibitory effect on PEDV in vitro,which mainly acted on the replication and value-added stage of PEDV life cycle.

Key words: Polygonum cuspidatum water extract, PEDV, IPEC-J2, Adaptability, Inhibition in vitro

CLC Number:

S858.28

WANG Yuxuan, YANG Zhi, LIU Haoyang, WANG Xinghai, YANG Fulin, LU Hongde, HE Zhiyuan, DONG Hong. Effect of Polygonum cuspidatum Aqueous Extract on Porcine epidemic diarrhoea virus Replication in IPEC-J2 Cells[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 215-224. doi: 10.7668/hbnxb.20195708.

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Figures/Tables 9

Tab.1 RT-qPCR primer information

引物名称 Primer names	序列(5'—3') Sequences(5'—3')
PEDV N	Forward:ACCAGTCCAAGAACAGAAACCAGTC
	Reverse:CATCCTTGACAGCAGCCACCAG
GAPDH	Forward:AAGGAGTAAGAGCCCCTGGA
	Reverse:TCTGGGATGGAAACTGGAA

Fig.1 The effects of different concentrations of trypsin on the expression level of PEDV N protein at different time Different letters indicated significant difference(P<0.05).The same as Fig.2,4.

Fig.2 The effects of different concentrations of trypsin on the cell activity at different time

Fig.3 Cytopathic observation chart A,C,E,G.Control group;B,D,F,H.PEDV infected cells.

Fig.4 Growth characteristics of PEDV in IPEC-J2 cells A.The growth curve of PEDV LW/L strain in IPEC-J2 cells;B.Changes in mRNA expression of PEDV N gene; C.The PEDV N protein expression was detected by Western Blot;D.Gray value analysis.

Fig.5 The effect after aclding P.cuspidatum water extract on cytotoxicity of IPEC-J2 cells

Fig.6 The effect of adding P.cuspidatum water extract in IPEC-J2 cells in different ways on the expression of PEDV N gene A.All-treatment group;B.Pre-treatment group;C.Co-treatment group;D.Post-treatment group;E.Direct-treatment group.

Fig.7 Effects of different concentrations of P.cuspidatum water extract on the expression of PEDV N gene and N protein levels A.PEDV N gene expression level;B.The PEDV N protein expression was detected by Western Blot;C.Gray value analysis.

Fig.8 The expression of N gene in PEDV life cycle in IPEC-J2 cells affected by P.cuspidatum water extract A.Adsorption stage(PEDV and Pol.c 4 ℃ 2 h);B.Penetration stage(PEDV 4 ℃ 2 h,Pol.c 37 ℃ 3 h);C.Replicative stage(PEDV 37 ℃ 1 h,Pol.c 37 ℃ 12 h);D.Release stage(PEDV 37 ℃ 1 h,Pol.c 37 ℃ 2 h).

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