Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, People's Republic of China.
Key Laboratory of Ecology and Environment Science in Guangdong Higher Education, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, School of Life Sciences, South China Normal University, Guangzhou 510631, People's Republic of China.
Fish Shellfish Immunol. 2018 Feb;73:272-278. doi: 10.1016/j.fsi.2017.12.028. Epub 2017 Dec 22.
Oxidative burst, release of reactive oxygen species/reactive nitrogen species (ROS/RNS) contributed to microorganisms killing, is a vital immune response of crustacean haemocyte. Three morphologic haemocyte types (hyaline cells, HC; semigranular cells, SGC; granular cells, GC) have been defined in crustaceans, and found to play different roles in immune defense. However, oxidative burst activities of different haemocyte subpopulations in crustaceans are currently not documented. In the present study, we investigated the oxidative burst activities of the three haemocyte types in the freshwater prawn Macrobrachium rosenbergii using the common ROS fluorescent probe dichlorofluorescin-diacetate (DCFH-DA). Nitric oxide (NO) donor sodium nitroprusside (SNP) improved the DCF fluorescence in haemocytes, while NO scavenger C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and NO-synthase inhibitor N-monomethyl-l-arginine (L-NMMA) reduced the fluorescence, suggesting that DCF fluorescence intensity could also be modified by intracellular NO level and activity of NO-synthase pathway. ROS/RNS was also produced in the untreated haemocytes. GC contained most non-induced ROS/RNS production, while oxidative activity of HC was rather weak. No significant impact of PMA could be observed on ROS/RNS level in all the three cell types. Both zymosan A (ZA) and lipopolysaccharide (LPS) significantly triggered the production of ROS/RNS in SGC and GC, whereas they had no effect on those of HC, suggesting that SGC and GC were the primary cell types involved in pathogens killing by ROS/RNS pathway. Cytochalasin B (Cyt B) inhibited the ZA-induced ROS/RNS production, but could not change the ROS/RNS level stimulated by LPS. For unstimulated haemocytes, ROS/RNS productions decreased 29.6%, 44.1% and 48.6% in SGC, and decreased 44.5%, 28.4% and 57.3% in GC, in the presence of L-NMMA, Fccp and DPI respectively, whereas apocynin could not modulate DCF fluorescence in both SGC and GC, suggesting that mitochondrial oxidative pathway was relatively more dominant in SGC, and NO-synthase (NOS) pathway appeared more active in GC. For LPS-stimulated haemocytes, oxidative activities decreased 22.9%, 42.9%, 29.6% and 60.0% in SGC, and reduced 40.6%, 25.2%, 26.7% and 70.6% in GC with the presence of L-NMMA, apocynin, Fccp and DPI respectively, suggesting that NADPH-oxidase (NOX) pathway in both SGC and GC was activated by LPS, and it became the predominant oxidative pathway in stimulated SGC, while NOS pathway was the relative main source for ROS/RNS production in stimulated GC.
活性氧爆发（ROS/RNS）是甲壳动物血细胞杀伤微生物的重要免疫反应，其涉及活性氧和活性氮的释放。甲壳动物的血细胞可分为三种形态：透明细胞（HC）、半颗粒细胞（SGC）和颗粒细胞（GC），它们在免疫防御中发挥不同的作用。然而，目前尚未记录甲壳动物不同血细胞亚群的活性氧爆发活性。本研究采用常见的 ROS 荧光探针二氯荧光素二乙酸酯（DCFH-DA），研究了淡水龙虾 Macrobrachium rosenbergii 三种血细胞类型的活性氧爆发活性。一氧化氮（NO）供体硝普酸钠（SNP）增强了血细胞中的 DCF 荧光，而 NO 清除剂 C-PTIO（2-（4-羧基苯基）-4,4,5,5-四甲基咪唑啉-1-氧-3-氧化物）和 NO 合酶抑制剂 N-单甲基-L-精氨酸（L-NMMA）降低了荧光强度，表明细胞内 NO 水平和 NO 合酶途径的活性也可以改变 DCF 荧光强度。未处理的血细胞中也产生了 ROS/RNS。GC 产生的非诱导 ROS/RNS 最多，而 HC 的氧化活性较弱。PMA 对三种细胞类型的 ROS/RNS 水平均无明显影响。几丁质（ZA）和脂多糖（LPS）均显著触发 SGC 和 GC 中 ROS/RNS 的产生，但对 HC 无影响，表明 SGC 和 GC 是通过 ROS/RNS 途径杀伤病原体的主要细胞类型。细胞松弛素 B（Cyt B）抑制 ZA 诱导的 ROS/RNS 产生，但不能改变 LPS 刺激的 ROS/RNS 水平。对于未刺激的血细胞，在存在 L-NMMA、Fccp 和 DPI 的情况下，SGC 中的 ROS/RNS 产生分别降低了 29.6%、44.1%和 48.6%，GC 中的 ROS/RNS 产生分别降低了 44.5%、28.4%和 57.3%，而 apocynin 不能调节 SGC 和 GC 中的 DCF 荧光，表明线粒体氧化途径在 SGC 中更为占主导地位，而 NO 合酶（NOS）途径在 GC 中更为活跃。对于 LPS 刺激的血细胞，在存在 L-NMMA、apocynin、Fccp 和 DPI 的情况下，SGC 中的氧化活性分别降低了 22.9%、42.9%、29.6%和 60.0%，GC 中的氧化活性分别降低了 40.6%、25.2%、26.7%和 70.6%，表明 LPS 激活了 SGC 和 GC 中的 NADPH 氧化酶（NOX）途径，使其成为刺激 SGC 中的主要氧化途径，而 NOS 途径是刺激 GC 中 ROS/RNS 产生的相对主要来源。
