Study Of GABA A Receptor And Safety Evaluation Of Difloxacin(DIF) On Carassais Auratus Gibebiol

The high-speed development of China’s aquaculture industry has motivated all kinds aquatic pathogen, meanwhile, drugs still is the main way for aquatic animal disease prevention and control in China, whose security has attracted the attention of all social sectors. Therefore, it is necessary to pay attention to the fishery drugs safety evaluation to ensure the security of aquatic animals and its breeding ecological environment. γ-aminobutyric acid(GABA) as the main inhibitory neurotransmitter in the central nervous system(CNS), which must bond to GABA receptor to function. GABA receptor can be divided into three types, GABA A receptor, GABA B receptor and GABA C receptor, for they got different pharmacological properties. Among the three receptor types, GABA A receptor is the most important one, beside the GABA and its inhibitors dicentrine binding site, there are other three affinity ligands binging sites, they were benzodiazepine site, barbiturate site, inhibitor picrotoxinin and TBPS site. These affinity ligands could direct or indirect impact the affinity of GABA and GABA A receptor, increase or decrease the open frequency of the chloride ion channel, in turn impact the inhibitory regulation of the CNS. At present, the hot areas of GABA and GABA A receptor research are still focus on the pathological and pharmacological research of mammals disease, as well as the development of new agricultural pesticides, and the related researches in aquaculture are scanty. However, avermectins(AVM), fluoroquinolones and organochlorine drugs also can be used in the aquatic animal disease prevention or treatment. When the drugs binging to the receptor in pathogen they could binging to the receptor in the farmed animals as well, and cause serious damage and these cannot be ignore anymore. Up to now, predecessors’ researches are focus on the influence of drugs on GABA, detect its content change and its affinity to the receptor, or the open frequency of the chloride ion channel, rarely involve in the influence on GABA receptors. However, study the influence of drugs based on the receptors would be much more pertinence and specificity. Therefore, our research treat GABA A receptor as the detection index, clone its γ2 subunit gene, test the expression profile of GABA A receptor in health Carassais auratus gibebiol(C. auratus gibelio) by using realtime PCR, Western blot and immunohistochemistry, then detect the expression changes of GABA A receptor after DIF treatment to evaluate the safety of DIF on C. auratus gibelio. Our research would provide theoretical direction to the construction of Chinese fishery drugs safety assessment system, also it can improve safe usage of fishery drugs, and sustainable and healthy development of Chinese aquaculture industry.The main research of this paper and the results are as follows:(1) In the present study, we cloned and characterized GABA-A receptor γ2 subunit full-length gene of C. auratus gibelio by rapid-amplification of c DNA ends(RACE). The full-length GABA-A receptor γ2 subunit c DNA was 2763 bp in length, contained a 1437 bp open reading frame(ORF), and encoded 477 amino acids which constituting a 55.3 k D protein molecule with an isoelectric point of 9.13. The γ2 subunit protein was hydrophilic. The protein sequence had one signal peptide, consisting of 35 amino acid residue. The sequence of amino acids contained four transmembrane regions, which length of 23、20、23 and 23 aa, involving in electronic transfer catalysis between the internal and external membrane. We found three N-glycosylation sites, two O- glycosylation sites and an extracellular domain which had obvious ligand-gated ion channels’ characteristics. Sequence comparison revealed that the similarity of the γ2 subunit protein all above 89% with other aquatic animals showed that it belongs to GABA A receptor subunits’ family. We conducted a phylogenetic analysis using the neighbor joining(NJ) method. The evolutionary tree showed that the γ2 subunit protein was clustered with the zebra fish which indicated that they are the two most closely related species.(2) Test the expression profile of GABA A receptor in health C. auratus gibelio by using realtime PCR, Western blot and immunohistochemistry. The results of realtime PCR showed that GABA A receptor expressed in all the tested tissues, and the m RNA content got significant difference among all the tissues(P<0.05). The highest expression level of γ2 subunit m RNA was observed in brain followed by gonad, liver, kidney, heart, intestine, muscle and lowest was in the skin. Western blot results showed that the protein of GABA A receptor expressed in all tested tissues, and the highest expression level of γ2 subunit protein was also observed in brain, followed by gonad, liver, kidney, intestine, muscle, heart, and lowest was in the skin. The protein content got significant difference among all the tissues(P<0.05). Immunohistochemistry showed that GABA A receptor distribute mainly in brain followed by kidney and liver. Density analysis showed that the density of cerebellum, mesencephalon, medulla oblongata, telencephalon, kidney and liver successively increasing. Among these tissues, GABA A receptor was abundant in the corpus striatum of telencephalon, plentiful in the optic tectum of mesencephalon, vast in molecular layer and purkinje cells of cerebellum, less in stratum granulosum, beside, the cerebellar lobe that stretch into the mesencephalon got a high distribution. In liver, GABA A receptor uniform distribute on the liver cell membrane, while, concentrated distributed on kidney tubules membrane.(3) Detect the expression changes of GABA A receptor in brain, liver, kidney, muscle and gonad after DIF treatment. The results showed that the expression of GABA A receptor decrease significant in all tested tissues, the m RNA level decreased more faster and more significant than the protein level which decrease lagging behind. Among all tissues, the inhibitory effect of DIF on GABA A receptor in brain was the most significant one, and more enduring which means that it would take a long time to recover to the normal level. Based on these, our research indicated that DIF can significantly inhibit the expression of GABA A receptor, and there would be some neurotoxin to all tissues of aquatic animals with the damage to CNS as the most significant one.In summary, we found GABA A receptor in C. auratus gibelio, and GABA A receptor that consisted by γ2 subunit expressed both in CNS and peripheral tissue, and mainly distributed in brain, while gonad got a higher expression as well. After DIF treatment, the content of GABA A receptor in brain, liver, kidney, muscle and gonad decreased with different levels, and the most significant decrease was in brain. Therefore, DIF was neurotoxin to all tissues of aquatic animals with the damage to CNS as the most significant one. This study would provide some experimental and theoretical basis to the apply of GABA A receptor in aquaculture and fishery drugs safety assessment.