Preliminary Research On Immune Response Of High Mobility Group Box 1 From Schizothorax Prenanti Under Bacterial Stress

High mobility group box 1(HMGB1)is a highly conserved nuclear protein.Studies have shown that it can participate in the immune response under pathogenic stress on some fish species,but there is lack of the related studies on cold water fish.Schizothorax prenanti is a unique sub-cold water fish in the upper reaches of the Yangtze River.With the expansion of farming scale and deterioration of the environment in the past decade year,health farming of S.prenanti was threatened by various diseases.Therefore,the immune response mechanism of the S.prenanti under pathogenic is urgently needed.In order to research the immune response of SpHMGB1 under bacterial stress,the experiment was consisted by three parts.Part I:The basic bio-information of SpHMGB1 was analyzed.Firstly,the HMGB1 homologous gene achieved from Transcriptome Shotgun Assembly databases of S.prenanti spleen was used to design primers.Then the CDS region of SpHMGB1 was amplified by T-cloning technology and amino acid sequence was analyzed by related biological software.Part II: The response situation of SpHMGB1 was investigated after bacterial stress.Firstly,the relative expression of SpHMGB1 was investigated in different tissue of healthy S.prenanti.Then live and heat-killed Streptococcus agalactiae and Aeromonas hydrophila were used as bacterial stressor in vivo and in vitro,separately.The relative expression of SpHMGB1 was detected in blood,spleen and trunk kidney of S.prenanti after bacterial infection in vivo.Meanwhile,the relative expression of SpHMGB1 was detected in head kidney macrophages of S.prenanti after bacterial stimulation in vitro.Part III: The preliminary research of SpHMGB1 mechanism was studied.Firstly,the correlation between SpHMGB1 and cytokine(interleukin-1β and tumor necrosis factor-α)expression was analyzed.Then,the function of the SpHMGB1-B peptide was detected by the respiratory burst,phagocytic activity,cytokines(IL-1β、TNF-α、IL-10、IRF1、IRF7、CXL11-1 and CXL11-2)and TLR4 relative expression in HK macrophages.The specific results area as follows:1.SpHMGB1 cloning and bioinformatics analysisThe CDS of SpHMGB1 was 615 bp encoding a putative protein of 204 amino acid residues.The predicted molecular mass of SpHMGB1 was 23.5 k Da and the theoretical p I(isoelectric point)was 6.79.Domain analysis showed that SpHMGB1 consisted of box A(7-77 AA residues),box B(94-162 AA residues)and a acidic tail(183-204 AA residues).SpHMGB1 has the identity of 64.7%-90.7% with HMGB1 from vertebrates.Phylogenetic analysis showed that SpHMGB1 and fish HMGB1 were clustered together,and the relationship of SpHMGB1 was most closely to Danio rerio and Carassius auratus.The secondary structure prediction showed that SpHMGB1 mainly consisted of α-helical and random coils;The tertiary structure results showed the highly similarity to HMGB1 of Mus musculus and Danio rerio with mainly forms “helix-turn-helix” spatial structure.These results indicated that SpHMGB1 was a family member of HMGB1,and had a highly conservation in evolution.2.Effect of SpHMGB1 relative expression after bacterial stressSpHMGB1 was universal expressed in blood,muscles,skin,spleen,gill,heart,intestines,hepatopancreas,brain and mid-kidney.In blood,the relative expression of SpHMGB1 was significantly up-regulated at 4h-24 h after S.agalactiae infection(aih),with the highest expression at 24 aih.In spleen,the relative expression of SpHMGB1 showed up-regulated tendency with significant difference at 72 aih by S.agalactiae infection.After A.hydrophila infection,the relative expression of SpHMGB1 was significantly up-regulated at0.5aih-72 aih with the highest expression at 72 aih in blood.Meanwhile,the relative expression of SpHMGB1 was significantly up-regulated at 6 aih-120 aih in spleen and at0.5aih-6aih in mid-kidney,separately.In vitro,the relative expression of SpHMGB1 was significantly up-regulated at 0.5 h-48 h after heat-killed S.agalactiae stimulation in HK macrophages.However,down-regulation response was observed at 0.5 h-24 h after heat-killed A.hydrophila stimulation.These results indicated that SpHMGB1 was able to participate in response of bacterial pathogenic stress.3.Immune-modulatory effect of SpHMGB1-B box peptide on macrophagesThere was a positively correlation between the relative expression of SpHMGB1 and cytokine(IL-1β and TNF-α)with the correlation of 0.827 and 0.693,separately.To research the immune-regulatory effect of SpHMGB1,the HK macrophages were treated with the synthetic SpHMGB1-B box peptide.The results showed that SpHMGB1-B box peptide could activate macrophage respiratory burst,enhance phagocytic activity and up-regulate relative expression of cytokines inluding IL-1β,TNF-α,IL-10,IRF,IRF7,CXCL11-1 and CXCL11-2.At the same time,the relative expression of TLR4 was significantly up-regulated in head-kidney macrophage.These results suggested that SpHMGB1-B box peptide had immune-regulatory effects on HK macrophages.In summary,HMGB1 of S.prenanti had highly conserved in evolution and widely distributed in tissues.It was able to participate in the immune response after live and heat-killed S.agalactiae and A.hydrophila stress in vivo and in vitro,separately.The synthetic SpHMGB1-B box peptide has immunomo-regulatory effects on HK macrophage.This study provided a theoretical reference for the immune response mechanism of SpHMGB1 in bacterial disease.