曹迷霞 陈奇 杨剑 刘梦倩 贾妮娜 韦英益 胡庭俊

摘要：【目的】探究山豆根多糖（SSP）對猪圆环病毒Ⅱ型（PCV2）感染RAW264.7细胞增殖活性及炎症相关因子的影响，揭示SSP对PCV2感染免疫细胞炎症相关因子的调控作用。【方法】PCV2体外感染RAW264.7细胞建立炎症模型，以不同浓度（25、50、100、200、400、800和1600 μg/mL）SSP进行培养处理，然后采用CCK-8和ELISA分别测定SSP对PCV2体外感染RAW264.7细胞增殖活性及炎症相关因子（IL-1β、IL-8和MCP-1）分泌水平和胞内环氧合酶-1（COX-1）活性的影响。【结果】与细胞对照组相比，SSP浓度≤400 μg/mL对RAW264.7细胞增殖活性无显著影响（P>0.05），但SSP浓度达800和1600 μg/mL时RAW264.7细胞增殖活性极显著降低（P<0.01，下同），且随培养时间的延长，细胞增殖活性呈先降低后升高的变化趋势，于培养48 h时达最低值。PCV2感染RAW264.7细胞后其增殖活性极显著降低，炎症相关因子IL-1β、IL-8和MCP-1分泌水平及胞内COX-1活性极显著升高;100～400 μg/mL SSP能极显著提高PCV2感染RAW264.7细胞增殖活性，且能有效降低RAW264.7细胞的IL-1β、IL-8和MCP-1分泌水平及胞内COX-1活性。具体表现为：与PCV2模型组相比，100 ?g/mL SSP能显著降低PCV2感染RAW264.7细胞的IL-1β和MCP-1分泌水平（P<0.05，下同）;200 ?g/mL SSP能极显著降低PCV2感染RAW264.7细胞的MCP-1分泌水平，同时显著降低细胞IL-1β和IL-8的分泌水平及胞内COX-1活性;400 ?g/mL SSP能极显著降低PCV2感染RAW264.7细胞的IL-1β、IL-8和MCP-1分泌水平及胞内COX-1活性。【结论】SSP对RAW264.7细胞增殖活性无显著影响，也未表现出细胞毒性作用，且100～400 μg/mL SSP能极显著提高PCV2感染RAW264.7细胞增殖活性，并通过调节PCV2感染免疫细胞的炎症相关因子水平而发挥抗炎作用。

关键词： 猪圆环病毒Ⅱ型;山豆根多糖;RAW264.7细胞;增殖活性;炎症相关因子

中图分类号： S853.74                     文献标志码： A 文章编号：2095-1191（2021）02-0439-09

Abstract：【Objective】This study aimed to investigate the proliferation activity and inflammation-related factors of Sophora subprostrate polysaccharide（SSP） on porcine circovirus Ⅱ（PCV2） infected RAW264.7 cells， and to reveal its regulation on PCV2 infected immune cells inflammation-related factors. 【Method】 Established the inflammatory model of PCV2 infected RAW264.7 cells in vitro， and then cultured with different concentrations（25， 50， 100， 200， 400， 800 and 1600 μg/mL） of SSP. CCK-8 method was used to determine the proliferation activity of RAW264.7 cells and PCV2 infec-ted RAW264.7 cells with SSP at different concentrations. The effect of SSP at different concentrations on inflammation-related factors（IL-1β， IL-8， MCP-1 and COX-1） in PCV2 infected RAW264.7 cells were determined by enzyme linked immunosorbent assay（ELISA）. 【Result】Compared with the cell control group， the results showed that the concentration of SSP≤400 μg/mL had no significant effect on RAW264.7 cell viability（P>0.05）， but SSP concentrations at 800 μg/mL and 1600 μg/mL  extremely significantly decreased the viability of RAW264.7 cells（P<0.01，the same below）. After PCV2 infection， the proliferation activity of RAW264.7 cells was extremely decreased， the secretion levels of inflammatory cytokines IL-1β， IL-8 and MCP-1 and the activity of intracellular COX-1 were extremely increased. 100-400 μg/mL of SSP exttemely increased the viability of PCV2 infected RAW264.7 cells， and the secretion levels of IL-1β， IL-8， MCP-1 and the activity of COX-1 in RAW264.7 cells were decreased compared to PCV2 group. Compared with PCV2 model group， 100 μg/mL of SSP significantly decreased IL-1β and MCP-1 secretion levels in PCV2 infected RAW264.7 cells（P<0.05， the same below）; 200 μg/mL of SSP extremely decreased the secretion level of MCP-1， significantly decreased the secretion levels of  IL-1β and IL-8， and the activity of COX-1 in PCV2 infected RAW264.7 cells; SSP at 400 μg/mL of could extremely reduce the levels of IL-1β， IL-8 and MCP-1 and the activity of COX-1 in PCV2 infected RAW264.7 cells. 【Conclusion】 SSP has no significant effect on the proliferation activity of RAW264.7 cells， and shows no cytotoxicity. 100-400 μg/mL of SSP extremely increases the proliferation activity of PCV2 infected RAW264.7 cells， and play an anti-inflammatory effect via regulating the secretion levels of inflammation-related factors.

Key words： porcine circovirus Ⅱ;  Sophora subprostrate polysaccharide; RAW264.7 cell; proliferation activity; inflammation-related factors

Foundation item： National Natural Science Foundation of China（31960715）; Project of Guangxi Graduate Education Innovation（YCBZ2020004）

0 引言

【研究意义】猪圆环病毒（Porcine circovirus，PCV）是圆环病毒科（Circoviridae）圆环病毒属（Circovirus）的成员之一，无囊膜，含有共价闭合的环状单链DNA基因组，主要包括PCV I型（PCV1）、PCV II型（PCV2）和PCV III型（PCV3）等基因型（刘国阳等，2019;张柱青等，2019;苏芮等，2020）。其中，PCV1对猪为非致病性，而PCV2和PCV3均对猪表现出致病性（贺会利等，2017）。PCV2能引发一大类免疫抑制性的多系统传染病，主要侵害机体的免疫系统（邓文芳等，2020）。单核细胞和巨噬细胞是PCV2的靶细胞，PCV2感染可引起仔猪断奶后多系统衰竭综合症、皮炎和肾病综合征、增生性坏死性间质性肺炎和繁殖障碍等，是猪圆环病毒相关疾病（Porcine circovirus associated disease，PCVD）的主要病原（方博，2019;沈顺新和和玉丹，2020）。山豆根多糖（Sophora subprostrate polysaccharide，SSP）是一种从越南槐（Sophora tonkinensis Gapnep.）根和根茎中经干燥后提取获得的多糖，具有多种生物活性，如抗炎、抗氧化、抗病毒及抗肿瘤等（彭湘君等，2012）。山豆根多糖可通过调节一氧化氮（NO）分子水平而影响免疫细胞内的cAMP/cGMP和6-keto-PGF1信号体系，进而调节免疫功能（帅学宏等，2010）。已有……