| Neurotransmitters and their related metabolites play important roles in immune regulation.Pinctada fucata martensii is the most important species for marine pearl cultured in China.In this study,we screened the key neurotransmitters and related metabolites in transplantation immunity by analyzing the metabolome and transcriptome of P.f.martensii after transplantation.The immunomodulatory functio ns and mechanisms of these substances in P.f.martensii were analyzed by using transcriptomic,metabolomic,and molecular cell biology techniques.The main results are as follows:1.Screening of key neurotransmitters and related metabolites in transplanta t io n immunity in P.f.martensii:Metabolome analysis showed that a total of 38neurotransmitters and related metabolites were detected in the serum before and after transplantation,and 35 substances were increased after transplantation.Transcripto me analysis showed that acetylcholine receptors,histamine receptors and ionotropic glutamate receptors were significantly up-regulated after transplantation.The results of combined metabolomic and transcriptomic analysis showed that some pathways,such as Neuroactive ligand-receptor interaction pathway were significantly enriched.Based on these results,3 neurotransmitters（Acetylcholine（ACh）,histamine（HA）,gluta mic acid（Glu））and 2 metabolites（putrescine（Put）and spermine（Spm））were selected as the targets for the follow-up studies.2.The function and mechanism of ACh in the immune regulation of P.f.martensii:（1）ACh was injected before LPS stimulation,and serum immune indexes and hemocytes transcriptome were detected at 24 h after LPS stimulation.Compared with the control group,ACh pretreatment could significantly reduce the content of IL-17 in serum.Transcriptome analysis showed that ACh pretreatment inhibited NF-κB and other immune-related signaling pathways as well as cell proliferation and apoptosis signaling pathways and could induce the expression level of calmodulin（CaM）genes.These results indicated that ACh pretreatment can inhibit LPS-induced inflammator y response,apoptosis,and proliferation through calcium signaling and NF-κB and other signaling pathways.（2）ACh or nAChR blocker was injected before transplantation to activate or block the cholinergic system.Serum immune indexes and hemocyte transcriptome were detected at 6 and 24 h after transplantation.Compared with the control group,the activity of antioxidant enzymes in serum was significantly up-regulated and the content of IL-17 was significantly down-regulated after ACh pretreatment,while the opposite results were obtained in the nAChR blocker pretreatment group.Transcriptome analysis showed that NF-κB and other immune-related signaling pathways were inhibited after ACh pretreatment,the expression of CaM gene was induced,whereas the opposite results were observed in nAChR blocker pretreatment group.The intracellular Ca2+concentration of hemocytes was significantly increased after incubation with ACh in vitro,but decreased significa nt ly after incubation with ACh＿nAChR blocker.Apoptosis and autophagy detection results showed that ACh treatment inhibited the apoptosis and autophagy of hemocytes.After ACh treatment,the survival rate of pearl oysters after transplantation showed no significant change,whereas the nucleus retention rate significantly increased.These results suggested that ACh may inhibit the transplantation-induced inflammator y response,apoptosis,and autophagy while increasing the antioxidant capacity and improving the retention rate through calcium,NF-κB,and other signaling pathways after activating the cholinergic system.Metabolome analysis showed that the contents of 24 phosphatidylcholines（PC）in serum were up-regulated after ACh pretreatment.PCs were synthesized from ACh through ACh E and CDP-choline pathways which were proven by experiments.After PC pretreatment,the activities of SOD and CAT in serum were significantly increased,and the content of IL-17 was significantly decreased.Transcriptome analysis showed that some pathways such as“Toll and Imd signaling pathway”were inhibited,and"apoptosis"as well as other pathways were activated after PC pretreatment.These results suggested that ACh enhanced antioxidant capacity and suppressed inflammatory responses of P.f.martensii by synthesizing PC.3.The function and mechanism of HA in the immune regulation of P.f.martensii:（1）HA was injected before LPS stimulation,and serum immune indexes and hemocytes transcriptome were detected at 24 h after LPS stimulation.Compared with the control group,the IL-17 content and antibacterial ability in serum were significantly decreased after HA pretreatment,and the activity of GSH-Px was significantly increased.Transcriptome analysis showed that celular“NOD-like receptor signaling pathway”and other immunity-related pathways were inhibited after HA pretreatment,and the expression level of CaM genes was significantly down-regulated.These results suggested that HA may inhibit LPS-induced inflammatory response,humoral immunit y,and apoptosis,and enhance antioxidant capacity through calcium signaling pathway.（2）HA was injected before transplantation,and serum immune indexes and hemocytes transcriptome were detected at 24 h after transplantation.Compared with the control group,the activities of SOD and CAT in serum were significantly increased after HA pretreatment,and the antibacterial ability of serum was significantly decreased.Transcriptome analysis showed that celular immune pathways such as“NOD-like receptor signaling pathway”and“apoptosis”pathway were inhibited,and the expression of CaM genes was significantly down-regulated after HA pretreatment.However,signaling pathways such as“Jak-STAT signaling pathway”and“mTOR signaling pathway”were induced.Intracel ular Ca2+concentration of hemocytes was significantly decreased at 3 and 6 h after incubation with HA in vitro.Apoptosis and autophagy detection results showed that HA inhibited the apoptosis and autophagy levels of hemocytes after transplantation.Pearl culture experiments showed that HA treatment had no significant effect on the survival and retention rate of pearl oysters.These results indicated that HA may inhibit the level of humoral immunity,apoptosis,and autophagy induced by transplantation,and improve the antioxidant capacity through calcium signaling,Jak-STAT,and other signaling pathways.4.The regulatory function and mechanism of Glu in transplantation immunity:Glu was injected before transplantation,and serum immune indexes and hemocytes transcriptome were detected at 24 h after transplantation.Compared with the control group,the activities of SOD and CAT in serum were significantly increased after Glu pretreatment,while IL-17 content and antibacterial activity showed no significa nt change.Transcriptome analysis showed that“Toll and Imd signaling pathway”and other immune signaling pathways were inhibited after Glu pretreatment.The expressions of CaM and GluR genes were significantly down-regulated,while the expressions of“apoptosis”and“mTOR signaling pathway”related genes were significantly up-regulated.The intracellular Ca2+concentration of hemocytes was significantly increased after incubation with Glu in vitro.Apoptosis and autophagy detection results showed that Glu inhibited the apoptosis and autophagy levels of hemocytes after transplantation.Pearl culture experiments showed that the survival rate showed no significant change after Glu treatment,but the retention rate was increased.These results indicated that Glu inhibited transplantation-induced apoptosis and autophagy and increased antioxidant capacity and retention rate through calcium and mTOR and other signaling pathways.5.The function and mechanism of polyamines in the immune regulation of P.f.martensii:（1）Put or Spm was injected before LPS stimulation,and serum immune indexes and hemocytes transcriptome were detected at 24 h after LPS stimulat io n.Compared with the control group,the activity of GSH-Px in serum was significa nt ly increased after Put pretreatment,and the antibacterial ability was decreased,but the content of IL-17 showed no significant change.Compared with the control group,the activity of GSH-Px in serum was significantly increased after Spm pretreatment,and the IL-17 content was decreased,but the antibacterial ability showed no significa nt change.Transcriptome analysis showed that after Put pretreatment,the expression of CaM genes and the“Glutamatergic synapse“pathway-related genes were up-regulated and the expression of the“Apoptosis”pathway-related genes was down-regulated,while the opposite results were obtained after Spm pretreatment.These results suggested that Put and Spm participated in the regulation of intracellular calcium signals and enhanced the antioxidant capacity of pearl oysters after LPS stimulat io n.Compared with Spm,Put treatment can better inhibit LPS-induced humoral immunit y and apoptosis.（2）Put was injected before transplantation,serum immune indexes and hemocytes transcriptome were detected at 24 h after transplantation.Compared with the control group,the activities of SOD and CAT in serum were significantly increased after Put pretreatment,and the content of IL-17 was significantly decreased.Transcriptome analysis showed that the“Toll and Imd signaling pathway”,“apoptosis”and other pathways were inhibited after Put pretreatment,and the expression of CaM genes was significantly down-regulated.However,the expression of mTOR and other signaling pathways-related genes were significantly up-regulated after Put pretreatment.The intracellular Ca2+concentration of hemocytes was significantly down-regulated after Put incubation in vitro.Apoptosis and autophagy detection results showed that Put inhibited the apoptosis and autophagy levels of hemocytes after transplantation.These results indicated that Put inhibited transplantation-induced inflammatory response,apoptosis,and autophagy and enhanced antioxidant capacity through calcium signaling,mTOR,and other signaling pathways. |
