| The organism clears the pathogens of infection in time through immune response,so as to maintain the immune homeostasis of the organism.Immune cells are the executors of the immune response.In different stages of immune response,immune cells undergo a series of cytological changes,such as migration,proliferation,differentiation,apoptosis,autophagy and so on.Apoptosis and autophagy are the key processes that affect the fate of cells in immune response.In the signal pathway of apoptosis and autophagy,there are many crosstalk molecules and the antiapoptotic protein Bcl-2 is one of them.Bcl-2 regulates apoptosis and autophagy mainly through binding and dissociation with pro-apoptotic protein-Bax and autophagy regulatory protein-Beclin1.In the immune response of invertebrates,the dynamic changes of apoptosis and autophagy of haemocytes and the potential regulatory role of Bcl-2 in it are still unclear.In this study,using the techniques of bioinformatics,molecular biology and cell biology,it was traced the dynamic changes of granulocyte apoptosis and autophagy in immune response of Crassostrea gigas(C.gigas),identified an anti-apoptotic protein Bcl-2(named CgBcl-2)and explored the regulatory mechanism of CgBcl-2 on granulocyte apoptosis and autophagy in immune response.The main results are as follows:1.Dynamic changes of apoptosis and autophagy in granulocytes during immune response after Vibrio.splendidus(V.splendidus)stimulation.To study the dynamic changes of apoptosis and autophagy in granulocytes after V.splendidus stimulated.It was found that at the prime stage of immune response(0-6 h after V.splendidus stimulation)the apoptosis rate of granulocytes was no obvious change,and increased significantly at the middle stage of immune response(12-24 h after V.splendidus stimulation),which was 2.19-fold(p < 0.05)and 1.68-fold(p < 0.05)of those in the seawater group,respectively,and then decreased significantly at the later stage of immune response(72-120 h after V.splendidus stimulation),which was 0.5-fold(p < 0.001),0.6-fold(p < 0.05)and 0.5-fold(p < 0.05)of those in the seawater group,respectively.The proportion of autophagosomes in granulocytes were no obvious change at 0-12 h after V.splendidus stimulation and increased significantly from 24 h to120 h,which was 2.2-fold(p < 0.001),2.14-fold(p < 0.001)and 1.56-fold(p < 0.01),2.46-fold(p< 0.001)and 2.04-fold(p < 0.001)of those in the seawater group,respectively.2.It was identified a CgBcl-2 with relatively conservative molecular structure in C.gigas and the expression of CgBcl-2 reach the highest expression in haemocytes,and could response to the stimulation of V.splendidus.CgBcl-2(CGI＿10006540)is cloned from C.gigas.The c DNA sequence of CgBcl-2 was predicted to contain an open reading frame(ORF)of 684 bp,encoding a protein of 227 amino acid residues.Theoretical isoelectric point of 6.1 was predicted by Expasy and calculated molecular weight of 25.3 k Da.There were three conserved motifs predicted in CgBcl-2,including a BH3motif(residues 91-98),a BH1 motif(residues 130-149)and a BH2 motif(residues 182-195).CgBcl-2 was closely clustered in the invertebrate branch by phylogenetic analysis.The m RNA expression of CgBcl-2 is detected in haemocytes and different tissues(mantle,gills,adductor muscle,hepatopancreas and gonad).It was highest expression in haemocytes,which was 5.82-fold of that in hepatopancreas(p < 0.05).Among the haemocytes,compared with the seawater group,the m RNA expression of CgBcl-2 decreased significantly on the middle stage(12-24 h after V.splendidus stimulation)in immune response and increased significantly in the late stage(48-120 h after V.splendidus stimulation)after stimulation with V.splendidus.Similarly,in granulocytes,the protein expression of CgBcl-2 decreased significantly in the middle stage of response(24 h after V.splendidus stimulation),which was 0.83-fold that of the control group(p < 0.01),and increased significantly in the later stage of response(120 h after V.splendidus stimulation),which was 1.26-fold higher than that of the control group.3.CgBcl-2 can dynamically regulate apoptosis and autophagy of granulocytes in immune response.At the middle stage of immune response(24 h after V.splendidus stimulation),the apoptosisrelated genes CgBax,CgCaspase9 and CgCaspase3 were all significantly upregulated,which were3.34-fold(p < 0.05),3.01-fold(p < 0.01)and 4.03-fold(p < 0.05)of those in the seawater,respectively;autophagy-related genes CgBeclin1,CgATG5 and CgATG10 were all significantly upregulated,which were 5.88-fold(p < 0.001),3.49-fold(p < 0.05)and 2.73-fold(p < 0.05)of those in the swawater group,respectively.After treatment with the Bcl-2 activator thevetiaflavone,the apoptosis-related genes CgBax,CgCaspase9 and CgCaspase3 were all significantly downregulated in granulocytes,which were 0.37-fold(p < 0.05),0.4-fold(p < 0.05)and 0.2-fold(p <0.001)of those in DMSO group,respectively;and the apoptosis rate of granulocytes was significantly down-regulated,which were 0.68-fold(p < 0.05)of that in DMSO group;meanwhile,autophagy-related genes CgBeclin1,CgATG5 and CgATG10 were significantly down-regulated,which were 0.19-fold(p < 0.001),0.44-foid(p < 0.05)and 0.16-fold(p < 0.05)of that in DMSO group,respectively;the proportion of autophagosomes in granulocytes were significantly downregulated,which was 0.39-fold(p < 0.001)of that in the DMSO group.The proportion of granulocytes was significantly up-regulated which was 1.51-fold of that in the DMSO group(p <0.01).At at the later stage of immune response(120 h after V.splendidus stimulation),the apoptosisrelated genes CgBax,CgCaspase9 and CgCaspase3 were all significantly down-regulated,which were 0.66-fold(p < 0.01),0.14-fold(p < 0.01)and 0.19-fold(p < 0.05)of those in the seawater,respectively;autophagy-related genes CgBeclin1,CgATG5 and CgATG10 were all significantly up-regulated,which were 2.16-fold(p < 0.01),9.82-fold(p < 0.001)and 4.39-fold(p < 0.05)of those in the swawater group,respectively.After treatment with the Bcl-2 inhibitor ABT-263,the apoptosis-related genes CgBax,CgCaspase9 and CgCaspase3 were all significantly up-regulated in granulocytes,which were 2.19-fold(p < 0.01),3.55-fold(p < 0.05)and 2.86-fold(p < 0.01)of those in DMSO group,respectively;and the apoptosis rate of granulocytes was significantly upregulated,which were 2.67-fold(p < 0.001)of that in DMSO group;meanwhile,autophagy-related genes CgBeclin1,CgATG5 and CgATG10 were no obvious changes in granulocytes;the proportion of autophagosomes in granulocytes were also no obvious changes.The proportion of granulocytes was significantly down-regulated which was 0.71-fold of that in the DMSO group(p< 0.01).It was further found that the phosphorylation level of CgBcl-2 in granulocytes increased significantly at 120 h after stimulation by V.splendidus.To sum up,after stimulation by V.splendidus,the apoptosis and autophagy of granulocytes also showed dynamic changes.The apoptosis of granulocytes mainly occurred in the middle stage of immune response and autophagy mainly occurred in the middle and later stage of immune response.CgBcl-2 contains typical BH motif,which is expressed in haemocytes and different tissues(mantle,gill,adductor muscle,hepatopancreas and gonad),among which it is the highest expression in haemocytes and respose to the stimulation of V.splendidus.In different stages of immune response,the level of CgBcl-2 involved in regulating apoptosis and autophagy of granulocytes was different:in the middle stage of response(24 h after V.splendidus stimulation),CgBcl-2 regulates both apoptosis and autophagy,while in the late stage of response(120 h after V.splendidus stimulation),CgBcl-2 tend to regulate apoptosis but not autophagy.The results of this study will be helpful to analyze the fate and regulatory mechanism of immune cells in invertebrates in the process of immune response. |
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