实验海洋生物学重点实验室知识库

        神经递质作为机体神经内分泌免疫调节网络中的重要“信使”，不仅能够作用于神经细胞传导兴奋，也可以作用于免疫细胞发挥免疫调节作用。近些年，随着神经与免疫系统共同进化起源理论的提出，无脊椎动物神经递质的免疫调控作用逐渐受到人们重视。目前无脊椎动物单胺类、神经肽类以及胆碱类神经递质免疫调控作用的研究已有了部分进展，但氨基酸类神经递质的免疫调控作用却鲜有报道。本研究以长牡蛎为研究对象，采用分子生物学、细胞生物学、免疫学等研究手段，选取两种经典氨基酸类神经递质谷氨酸（glutamic acid，Glu）和γ-氨基丁酸（γ-aminobutyric acid，GABA），检测了二者在免疫应答中的动态变化过程，分析了调节二者间代谢平衡的关键限速酶谷氨酸脱羧酶（glutamic acid decarboxylase，GAD）的分布特征及其表达模式，并进一步探讨了Glu和GABA的免疫调控作用。
        LPS刺激后的长牡蛎血清中Glu和GABA含量分别呈现6 h上升、48 h下降和6 h下降、48 h上升的拮抗变化趋势。通过基因组学分析发现，长牡蛎体内存在一个调节二者间代谢转化的关键限速酶GAD（命名为CgGAD），其cDNA全长1689 bp，能编码一条含有pyridoxal依赖的典型脱羧酶结构域的多肽链。利用转染技术将CgGAD基因转染至HEK293细胞后，CgGAD可以促进HEK293细胞中GABA的合成。CgGAD mRNA及蛋白主要在神经节和血细胞中表达，且其在血细胞中的表达量显著高于神经节，并主要表达在无吞噬能力的颗粒细胞中。CgGAD mRNA表达水平在LPS刺激后呈现先下降后上升的趋势，与GABA浓度变化趋势相一致。
        LPS或GABA刺激能显著影响长牡蛎体液免疫及细胞免疫水平变化。LPS（LPS+PBS）刺激后，长牡蛎血细胞中的促炎因子CgIL-17、CgTNF和免疫效应因子CgSOD、CgBPI的mRNA表达水平显著上升， NOS的蛋白活性显著提高，而在GABA（LPS+GABA）刺激后，此上升趋势被显著抑制。同时，LPS刺激还可以显著诱导长牡蛎血细胞吞噬能力和凋亡水平的增加，而GABA刺激（LPS+GABA）则在免疫应答后期显著抑制了细胞吞噬和凋亡作用的发生。此外，与LPS刺激组相比，GABA+LPS刺激能显著抑制原代培养血细胞内Ca²⁺浓度和ROS水平的升高，而分别加入A型或B型GABA受体抑制剂后，这种抑制作用消失。LPS或G刺激均能引起原代培养长牡蛎血细胞内Ca²⁺浓度和ROS含量显著上升，并促进细胞凋亡的发生。而当Glu与其三种类型受体抑制剂（NMDA型、AMPA型及代谢型）共同刺激时，三种类型受体抑制剂均能在不同时间段显著抑制细胞内Ca²⁺浓度和细胞凋亡率的上升。

    Neurotransmitters, as the vital messenger in neuro-endocrine-immune network, not only have function on signal transmission in nerve cells, but also play immunomodulatory role in immune cells. In recent decades, along with the co-evolution of nervous and immune system speculated, more attention has been paid on the immunomodulatory function of neurotransmitters in invertebrates. At present, the research on the immunomodulatory role of monoamines, neuropeptides and cholines has been reported, however, little has been mentioned as for the fucntion of amino acid neurotransmitters in immune system of invertebrates. In the present study, the dynamic changes of amino acid neurotransmitters, glutamate (Glu) and γ-aminobutyric acid (GABA), were investigated after immune stimulation, the distribution and expression patterns of glutamic acid decarboxylase (GAD) were identified, and the immunomodulatory role of Glu and GABA was further explored in Pacific oyster Crassostrea gigas for better understanding of the immunomodulation of amino acid neurotransmitters in invertebrates.
    After LPS stimulation, the concentration of Glu in serum increased at 6 h and then decreased at 48 h, conversely, the concentration of GABA decreased at 6 h and increased at 48 h, leaving an antagonistic effect of of Glu and GABA. According to the genome information of oyster C. gigas, one rate-limiting enzyme GAD (designed as CgGAD) catalyzing the reaction from Glu to GABA was identified. The full length cDNA of CgGAD was of 1689 bp, encoding a polypeptide of 562 amio acids containing a conserved pyridoxal-dependent decarboxylase domain. After transfected into HEK293 cells, CgGAD could promote the production of GABA. In oyster C. gigas, CgGAD could be detected in ganglion and hemocytes at both mRNA and protein level. Unexpectedly, CgGAD was more abundant in hemocytes than that in ganglion, and was mostly located in the granulocytes without phagocytic capacity. Moreover, CgGAD could also dynamically respond to LPS stimulation, its expression level decreased at first and increased at later stage, which was conformed to the change of GABA after LPS stimulation.

    LPS/GABA stimulation could influence both the humoral and cellular immune response of oysters. After LPS stimulation, the mRNA expressions of pro-inflammatory cytokines (CgIL-17 and CgTNF) and immune effectors (CgSOD and CgBPI), and the NOS activity increased significantly, but these increased trends were remarkably inhibited by GABA stimulation. Similarly, the phagocytosis rate and apoptosis rate of immunocytes also increased obviously after LPS stimulation, whereas the increase was repressed with the addition of GABA. Furthermore, compared with LPS+PBS group, LPS+GABA stimulation could also inhibit the increase of Ca²⁺ concentration and ROS production in the primary cultured hemocytes. However, the inhibitory role of GABA became invalid when the antagonist of GABA A or B receptors was added. Compared with GABA, Glu could increase the Ca²⁺ concentration and the ROS production, and then induce apoptosis of primary cultured hemocytes, however, these effects could be inhibited by three different type receptors (NMDA, AMPA and metabotropic) antagonists of Glu.
    All results collectively suggested that, CgGAD, as GABA synthase, could modulate the dynamic balance between Glu and GABA during the immune response of oysters, and participate in the regulation of neuro-endocrine-immune network. Besides, as the molecular marker of GABAergic system, CgGAD was mainly distributed in the immune tissues of oyster, which also provided a novel insight to the co-evolution between nervous system and immune system. In addition, the antagonistic fluctuation of Glu and GABA in hemolymph during immune response was witnessed, together with their excitory or inhibitory effect on immune response, which benefits for oysters to maintain the immune homeostasis.