西番莲多糖提取物对猪圆环病毒2型感染RAW264.7细胞氧化应激的影响

为研究西番莲多糖提取物（Passiflora edulis polysaccharide extract，PEPE）对猪圆环病毒2型（Porcine circovirus type 2，PCV2）感染RAW264.7细胞氧化应激相关因子的影响，本试验探讨了PEPE对氧化应激的调节作用。在96孔细胞培养板中每孔加入100 μL浓度为1×10⁶个/mL的RAW264.7细胞，分别设细胞对照组、PEPE组（25、50、100、200、400、800和1 600 μg/mL），分别于培养箱培养24和48 h，用MTT法检测细胞活性，筛选PEPE的药物安全浓度范围。试验分为以下6个组：细胞对照组、病毒组、PEPE高（400 μg/mL）、中（200 μg/mL）、低（100 μg/mL）剂量组及维生素C组，其中细胞对照组加入含10% FBS的DMEM培养液，其余组加入PCV2病毒液，孵育2 h后在PEPE组加入对应浓度的PEPE溶液，VC组加入配制好的VC溶液，细胞对照组和病毒组加入含10% FBS的DMEM培养液，培养48 h，同样用MTT法检测细胞活性，以研究PEPE对PCV2感染RAW264.7细胞活性的影响。按照上述6个试验组分组，处理同上，将细胞培养12 h，收集样品用于检测氧化应激相关因子水平。用Griess法和DCFH-DA荧光探针分别检测RAW264.7细胞上清中NO含量和细胞内活性氧（ROS）水平、OPT荧光法检测细胞内还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）含量，化学发光法检测细胞内黄嘌呤氧化酶（XOD）、髓过氧化物酶（MPO）和诱导型一氧化氮合酶（iNOS）活力。结果显示，PEPE对RAW264.7细胞的安全浓度范围为25~400 μg/mL。PCV2感染RAW264.7细胞后，细胞活性显著降低（P<0.05），而不同浓度PEPE处理组均能提高细胞活性。RAW264.7细胞被PCV2感染后，细胞分泌NO、ROS水平，GSSG含量及XOD、MPO和iNOS活性显著升高（P<0.05），感染细胞GSH含量显著降低（P<0.05）；PEPE作用于PCV2感染的RAW264.7细胞，显著降低感染细胞NO、ROS水平、GSSG含量及XOD、MPO和iNOS活性（P<0.05），100 μg/mL PEPE处理组细胞GSH水平显著升高（P<0.05）。本试验结果表明，PEPE能提高PCV2感染RAW264.7细胞的抗氧化能力，有利于缓解病毒感染所致氧化应激。

Effect of Passiflora edulis Polysaccharide Extract on Oxidative Stress of Porcine Circovirus Type 2 Infected RAW264.7 Cells

The study was aimed to investigate the effect of Passiflora edulis polysaccharide extract (PEPE) on oxidative stress-related factors of RAW264.7 cells infected with Porcine circovirus type 2 (PCV2) and explore the role of PEPE on oxidative stress.First,100 μL RAW264.7 cells of 1×10⁶ cells/mL were added into 96-well cell culture plate in each well.Cell control group and PEPE groups (25,50,100,200,400,800 and 1 600 μg/mL) were respectively set up and cultured in the incubator for 24 and 48 h,respectively.MTT assay was used to detect cell activity and screen the safe concentration range of PEPE.The experiment was then divided into the following six groups:Cell control group,virus group,PEPE high (400 μg/mL),medium (200 μg/mL) and low (100 μg/mL) dose groups and vitamin C group,among them,the cell control group was added with 10% FBS-containing DMEM culture medium,and the other groups were added with PCV2 virus liquid.After incubating for 2 h,the corresponding concentration of PEPE solution were added to the PEPE groups,the prepared VC solution was added to the VC group,and DMEM culture medium containing 10% FBS was added to cell control group and virus group,and cultured for 48 h.MTT assay was also used to detect cell activity to study the effect of PEPE on the activity of RAW264.7 cells infected with PCV2.Finally,in accordance with the above 6 test component groups,the treatment was the same as above,the cells were cultured for 12 h,and samples were collected to detect the levels of oxidative stress related factors.Griess method and DCFH-DA fluorescent probe label method were used to analyze the levels of nitric oxide (NO) and reactive oxygen species (ROS) in RAW264.7 cells supernatant.The levels of glutathione (GSH) and oxidized glutathione (GSSG) were analyzed by OPT fluorescence assay.The activities of xanthine oxidase (XOD),myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) were analyzed by chemiluminescence technique.The results of this study showed that the safe concentration range of PEPE on RAW264.7 cells was 25-400 μg/mL.After PCV2 infected RAW264.7 cells,the cell viability was significantly reduced (P<0.05),and the PEPE group with different concentrations could improve the cell viability.The secreting levels of NO and ROS,GSSG content and activities of XOD,MPO and iNOS were significantly increased (P<0.05),while GSH content was significantly decreased (P<0.05) after PCV2 infected RAW264.7 cells.When PCV2-infected cells were treated with PEPE,the production of NO,ROS,GSSG,as well as the activities of XOD,MPO and iNOS were significantly decreased (P<0.05),while the level of GSH in 100 μg/mL PEPE treatment group was significantly increased (P<0.05).The results showed that PEPE enhanced antioxidant capacity of PCV2-infected RAW264.7 cells,which was conducive to easing the oxidative stress caused by virus infection.