关键词: 白术多糖; 猪繁殖与呼吸综合征病毒(PRRSV); Marc-145细胞; 氧化应激

Abstract: 【Objective】 This study used African green monkey embryonic kidney cells (Marc-145) as a model to investigate the protective effects and mechanisms of Atractylodes macrocephala polysaccharides (AP) on Porcine reproductive and respiratory syndrome virus(PRRSV)-induced damage in Marc-145 cells. 【Method】 Marc-145 cells that had grown to 80%-90% fusion were infected with PRRSV at different dilutions (10^-1 to 10^-10),and a blank control group was set up.After continued culture,the number of wells with cytopectic effect (CPE) at each dilution was recorded daily.After the lesion was stable,the half tissue culture infection dose (TCID₅₀) of the virus was calculated by Reed-Muench method.Marc-145 cells were seeded in 96-well cell culture plates at a density of no less than 1×10⁶/mL per well.The cells were infected with 100 TICD₅₀ PRRSV for 0,12,24,48 and 96 h,respectively.Random photographs were taken under a microscope at each time point to observe the cell morphology.Marc-145 cells were divided into control group (Mock) and groups treated with different concentrations of AP (200,400,800,1 600,3 200 and 6 400 μg/mL).The optimal concentration range was determined using the cytotoxicity assay.Marc-145 cells were divided into control,model,treatment (AP-treated (200,400 and 800 μg/mL) and 1 μmol/mL ribavirin) groups.The following three administration methods were used to evaluate the effect of AP on PRRSV infection:①Pre-infection administration:Cells were incubated with AP and ribavirin for 24 h,followed by simultaneous challenged with PRRSV for another 24 h in treatment and model groups.②Co-infection administration:Cells were incubated with AP or ribavirin together with PRRSV for 24 h,while model group was simultaneously challenged with PRRSV alone for 24 h;③Post-infection administration:After simultaneous challenged with PRRSV for 24 h in treatment and model groups,cells in treatment groups were incubated with different concentrations of AP or ribavirin for 24 h,while cells in model group was incubated with 2% FBS DMEM for 24 h.In all treatment groups,cells in control group were incubated with 2% FBS DMEM for 24 h as a control.After the respective treatments,all groups were replaced with 2% FBS DMEM and further cultured for 48 h before detection.The virus challenge titer used in this experiment was 100 TCID₅₀.The 50% tissue culture infective dose (TCID₅₀) assay was used to evaluate the effects of AP on PRRSV titer at different stages of infection,while Real-time quantitative PCR was used to measure the effects on relative expression of PRRSV ORF7 gene.The effects of AP on cellular superoxide dismutase (SOD) activity,malondialdehyde (MDA) and glutathione (GSH) contents were measured using colorimetric assays,and the expression of PRRSV N protein and the Nrf2/HO-1 pathway proteins was analyzed using Western blotting. 【Result】 The titer of PRRSV was calculated to be 10^-6.6 TCID₅₀/mL based on the number of CPE wells at 24 h post-infection using the Reed-Muench method.Compared with 0 h post-PRRSV infection,cells exhibited aggregation and contraction at 24 h post-infection,indicating that 24 h was the optimal infection time for this strain.After incubating Marc-145 cells with AP for 24 h,cell viability was significantly increased in the 200-800 μg/mL AP groups compared with control group (P＜0.05).Therefore,200-800 μg/mL AP was selected as the optimal concentration range for subsequent experiments.Compared with model group,AP significantly reduced the TCID₅₀ value,relative expression of PRRSV ORF7 gene,MDA content,and expression of PRRSV N protein during the endocytosis and adsorption stages (P＜0.05).It also increased SOD activity and GSH content and activated the Nrf2/HO-1 pathway proteins (P＜0.05).However,no significant effects were observed during the intracellular replication stage (P＞0.05). 【Conclusion】 The results of this experiment indicated that,200-800 μg/mL AP protectd cells from oxidative stress damage during the endocytosis and adsorption stages of PRRSV infection by activating the Nrf2/HO-1 pathway.

Key words: Atractylodes macrocephala polysaccharides; Porcine reproductive and respiratory syndrome virus (PRRSV); Marc-145 cells; oxidative stress