山豆根多糖对猪圆环病毒Ⅱ型感染猪肺泡巨噬细胞炎性因子水平的影响

Effect of Sophora subprosrate Polysaccharide on the Levels of Inflammatory Factors in 3D4/2 Cells Infected with Porcine Circovirus VirusⅡType

试验旨在研究山豆根多糖(SSP)对猪肺泡巨噬细胞3D4/2感染猪圆环病毒Ⅱ型(Porcine circovirus virusⅡtype,PCV2)后炎性因子水平的影响。本试验设空白对照组、PCV2感染组、脂多糖(LPS)对照组和3种浓度(100、200和400μg/mL)的山豆根多糖组,采用PCV2体外感染猪肺泡巨噬细胞,在培养液中分别加入100、200和400μg/mL山豆根多糖培养24 h后收集样品,用酶联免疫吸附法(ELISA)测定细胞分泌单核细胞趋化蛋白-1(MCP-1)、白细胞介素-8(IL-8)水平和胞内环氧合酶-1(COX-1)、环氧合酶-2(COX-2)的酶活性,实时荧光定量PCR和Western blotting法分别检测诱导型一氧化氮合酶(iNOS)、COX-2基因及其蛋白表达。结果显示,PCV2感染猪肺泡巨噬细胞后极显著提高了MCP-1、IL-8分泌水平和细胞内COX-1、COX-2酶活性(P<0.01),iNOS、COX-2 mRNA和蛋白表达水平均极显著提升(P<0.01);100、200、400μg/mL山豆根多糖处理后,3D4/2细胞MCP-1、IL-8分泌水平和细胞内COX-1、COX-2酶活性均显著或极显著降低(P<0.05;P<0.01),其中400μg/mL山豆根多糖处理后效果最显著,细胞内iNOS、COX-2基因mRNA表达水平同样显著或极显著降低(P<0.05;P<0.01);100、200、400μg/mL山豆根多糖处理后,显著或极显著抑制了由PCV2诱导的iNOS蛋白表达量的增加(P<0.05;P<0.01),经200、400μg/mL山豆根多糖处理后显著抑制COX-2蛋白表达量的增加,400μg/mL山豆根多糖处理效果最佳。综上所述,山豆根多糖可在一定程度上抑制PCV2感染所引起的促炎症因子mRNA表达的升高及其蛋白的表达,起到一定的缓解炎症的作用。

To investigate the effect of Sophora subprosrate polysaccharide(SSP)on the levels of inflammatory factors in 3D4/2 cells infected by Porcine circovirus virusⅡtype(PCV2),in this study,blank control group,PCV2 group,lipopolysaccharide(LPS)group and three concentrations of SSP group(100,200 and 400μg/mL)were established,respectively.3D4/2 cells was infected by PCV2 in vitro,and then the cells were treated with different concentrations of SSP for 24 h.The secretions of monocyte chemotactic protein 1(MCP-1)and interleukin-8(IL-8)and the enzyme activities of cyclooxygenase-1(COX-1)and cyclooxygenase-2(COX-2)were detected by ELISA assays.The mRNA expression of inducible nitric oxide synthase(iNOS)and COX-2 genes were detected by Real-time quantitative PCR,and the protein expression of iNOS and COX-2 were detected by Western blotting.The results showed that the secretions of MCP-1,IL-8 and the activities of intracellular COX-1 and COX-2 were extremely significantly higher after PCV2 infection than those in the blank control group,the mRNA expression levels and protein expression of intracellular iNOS and COX-2 were also extremely significantly increased(P<0.01);After treatment with SSP at 100,200 and 400μg/mL,the secretions of MCP-1,IL-8 and the activities of COX-1 and COX-2 in the cells were significantly or extremely significantly decreased(P<0.05,P<0.01),the effect was most significant in 400μg/mL SSP group,and the mRNA expression of intracellular iNOS and COX-2 gene was also significantly or extremely significantly decreased(P<0.05,P<0.01).The increase of iNOS protein expression induced by PCV2 was significantly or extremely significantly inhibited by treatment with SSP at 100,200 and 400μg/mL(P<0.05,P<0.01).The increase of COX-2 protein expression was significantly inhibited by treatment with SSP at 200 and 400μg/mL,and the best effect was obtained at 400μg/mL.The results showed that SSP could inhibit the increase of mRNA expression and protein expression of pro-inflammatory factors caused by PCV2 infection and played a certain role in alleviating inflammation.