| ADP/ATP Carrier Protein 1(ANT1)is a major molecule in the inner membrane of mitochondria that maintains normal mitochondrial function and plays an important role in regulating autophagy and inflammation.ANT1 can directly or indirectly participate in the regulation of autophagy and mitochondrial autophagy,and can regulate inflammation by promoting or inhibiting the expression of cytokines.In this study,ANT1,High Mobility Group Box1(HMGB1)and Interleukin 17-5(IL17-5)were screened from the genome of Crassostrea gigas.Real-Time Quantitative Polymerase Chain Reaction(RT-q PCR),Western blot,immunofluorescence,and flow cytometry were used to explore the regulation of ANT1 on autophagy and inflammation in C.gigas.The results are as follows:1.Structural characteristics of CgANT1,Cg HMGB1 and Cg IL17-5The open reading frame of CgANT1 is 921 bp,encoding 306 amino acids.CgANT1 contains three Mito＿carr domains,each domain containing two transmembrane regions.The similarity between CgANT1 and other ANT1 s in Homo sapiens,Mus musculus,Danio rerio,Drosophila melanogaster,Mizuhopecten yessoensis,and Caenorhabditis elegans was46.67%-83.01%.Phylogenetic tree analysis showed that CgANT1 clustered with Mizuhopecten yessoensis ANT1(My ANT1)and was relatively conserved in evolution.The open reading frame of Cg HMGB1 was 609 bp,encoding 202 amino acids.Cg HMGB1 consists of two tandem HMG domains(HMG A-box and HMG B-box)and an acidic amino acids C-tail in the C-terminal.By comparing the sequences of Cg HMGB1 and Hs HMGB1,it was found that Cg HMGB1 contained two LPS-binding sites and one antibacterial function site which were similar to Hs HMGB1.The open reading frame of Cg IL17-5 is 399 bp,encoding 132 amino acids.Cg IL17-5 contains a signal peptide and an IL-17 domain.There were two tightly folded alpha helixes and two pairs of antiparallel beta-pleated sheet in the three-dimensional structure analysis of Cg IL17-5,which were similar to Hs IL-17 F.2.The expression characteristics of CgANT1,Cg HMGB1 and Cg IL17-5The tissue distribution and expression pattern of CgANT1,Cg HMGB1 and Cg IL17-5 in haemocytes after V.splendidus stimulation were examined by RT-q PCR.CgANT1 was mainly distributed in adductor muscle and it was also highly distributed in gills and heamocytes.Cg HMGB1 and Cg IL17-5 were highly distributed in haemocytes.After V.splendidus stimulation,the m RNA expression levels of CgANT1 were significantly increased at 12 and 72 h compared to the control group(p < 0.05).The m RNA expression levels of Cg HMGB1 were significantly increased at 3,48 and 72 h compared to the control group(P < 0.05).And the m RNA expression level of Cg IL17-5 was significantly increased at 24 h(p < 0.05).These results suggested that CgANT1,Cg HMGB1 and Cg IL17-5 could response to V.splendidus stimulation.Immunocytochemical analysis showed that CgANT1 was mainly distributed in the cytoplasm of haemocytes and co-located with mitochondria.Cg HMGB1 was located in the nucleus of haemocytes.After V.splendidus stimulation,Cg HMGB1 was able to transfer from nuclear of haemocytes to cytoplasm and release to hemolymph.3.The autophagy of haemocytes in C.gigas induced by V.splendidus stimulationThe methods of flow cytometry,Western blot and immunocytochemistry were used to analyze ROS,mitochondrial membrane potential,autophagy level,autophagy-related gene expression,and Cg LC3 co-localization changes with mitochondria in haemocytes after V.splendidus treated C.gigas.The results showed that ROS of haemocytes was significantly increased after V.splendidus treated C.gigas for 12 h compared with the control group(p <0.01).The mitochondrial membrane potential level and autophagy level of haemocytes were significantly increased after V.splendidus treated C.gigas for 24 h compared with the control group(p < 0.05).Meanwhile,the bands of autophagy related genes Cg LC3 and Cg ATG5 were significantly thicker,and the ratio of Cg LC3Ⅱ/ Cg LC3Ⅰ was increased.The co-location of Cg LC3 and mitochondria was observed in haemocytes after V.splendidus stimulation for 24 h by confocal laser microscopy.The above experiments results proved that V.splendidus could induce autophagy of haemocytes in C.gigas.After V.splendidus treated C.gigas for 24 h,obvious swelling and cilium shedding occurred in gill tissue,and V.splendidus could induce tissue inflammation of C.gigas.4.The regulation of CgANT1 in autophagy of haemocytes induced by V.splendidus stimulationThe regulation of CgANT1 on autophagy of haemocytes in C.gigas was detected after CgANT1 knocked down by RNA interference and V.splendidus stimulation for 24 h.The results showed that the autophagy level of haemocytes in CgANT1-RNAi group was significantly decreased(p < 0.05),compared with the control group.The expression changes of autophagy related genes(Cg Belcin1,Cg LC3,Cg ATG5 and Cg P62)in haemocytes of CgANT1-RNAi group were significantly decreased(p < 0.05)detected by RT-q PCR,and the expression of mitophagy related genes(Cg PINK1 and Cg Parkin)was significantly increased(p < 0.05).In vitro molecular interaction experiments showed that Cg HMGB1 could bind Cg Beclin1.After Cg HMGB1 knocked down by RNA interference and V.splendidus stimulation for 24 h,the autophagy level of haemocytes in Cg HMGB1-RNAi group was significantly decreased compared with the control group(p < 0.05),and the expression of autophagy related genes(Cg Belcin1,Cg LC3,Cg ATG5,and Cg P62)in haemocytes were also significantly decreased(p <0.05).These results suggested that CgANT1 could regulate the autophagy of V.splendidus induced haemocytes in C.gigas by promoting the expression of autophagy related genes.It was noteworthy that CgANT1 could regulate the expression of Cg PINK1 and Cg Parkin,which were the classical mitophagy regulatory pathway molecules.And it could be speculated that CgANT1 regulates the occurrence of mitophagy in haemocytes through PINK1-Parkin signaling pathway.In addition,it could be speculated that Cg HMGB1 regulated autophagy through binding with Cg Beclin1.5.The regulation of CgANT1,Cg HMGB1 and Cg IL17-5 in inflammation induced by V.splendidus stimulationThe regulation of CgANT1 on inflammation in C.gigas was detected after CgANT1 knocked down by RNA interference and V.splendidus stimulation for 24 h.The results showed that the expression levels of cytokines(Cg IL17-5,Cg HMGB1 and Cg TNF1)and antimicrobial peptide(Cgdefh2)related genes in haemocytes of CgANT1-RNAi group were significantly decreased(p < 0.05)detected by RT-q PCR.CgANT1 could regulate the inflammation induced by V.splendidus stimulation through promoting the expression of cytokines(Cg IL17-5 and Cg HMGB1).Cg HMGB1 can recognize and bind multiple pathogens and PAMPs.After r Cg HMGB1 or r Cg IL17-5 stimulated C.gigas for 2 h,Cg MAPKs in haemocytes and gills were phosphorylated and the transcription factor,Cg Rel,was translocated from cytoplasm to nucleus compared with r Trx group.After r Cg HMGB1 or r Cg IL17-5 stimulated C.gigas for 12 h,the m RNA expression levels of Cg IL-17 s and Cgdefh2 were significantly increased in haemocytes compared with r Trx group(p < 0.05).And the gill tissues showed obvious swelling and cilia shedding in r Cg HMGB1 and r Cg IL17-5 stimulation group compared with r Trx group.In conclusion,CgANT1 is involved in the regulation of pathogen-induced autophagy and mitophagy of haemocytes by regulating the expression of autophagy related genes(Cg Belcin1,Cg LC3,Cg ATG5,and Cg P62)and mitophagy related genes(Cg PINK1 and Cg Parkin).CgANT1 can further activate the MAPK signaling pathway by inducing the expression of cytokines(Cg HMGB1 and Cg IL17-5),promote the translocation of Cg Rel from cytoplasm into the nucleus,and then induce the synthesis of cytokines and antimicrobial peptides,participate in the regulation of inflammation and help oysters resist pathogens.This study revealed the regulatory mechanism of CgANT1 on autophagy and inflammation,which provided theoretical guidance for the prevention of shellfish diseases and enriched invertebrate immunology and comparative immunology theories. |
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