Effect Of Boron On Immune Regulation Of Splenic Lymphocytes In Ostrich Chicks

African ostrich is the largest two-toed bird in the world.Because of its resistance to roughage,strong resistance and reproductive performance,at the same time,it also has a very high economic value,favored by farmers.The brooding period of African ostriches lasts for 90 days.During this period,the immune system of ostriches have not yet developed and are highly susceptible to diseases,environment,nutrition,stress and other effects,causing certain economic losses to farmers.The spleen is an important place for immune response and contains a large number of immune cells such as lymphocytes and plasma cells.Previous studies have found that the addition of different concentrations of boron in drinking water has an effect on the growth and development of the spleen of African ostrich chicks.So far,little research has been reported on the regulation of immune function of splenic lymphocytes in ostrich chicks.In this experiment,the splenic lymphocytes of fourteen 15-day-old African ostriches were used as the research object.They were treated with boron at final concentrations of 0,0.01,0.1,0.5,1,5,10,25,50 and 100 mmol/L,respectively.Then CCK-8,flow cytometry,ELISA,RNA-Seq and RT-PCR methods were used to study the toxic effect,cell proliferation and apoptosis,cytokine secretion and related signaling pathways of boron on splenic lymphocyte in ostrich chicks,and to elucidate the regulatory mechanism of boron on splenic lymphocyte immune function in ostrich chicks.The research results were as follows:1.Toxic effects of boron on splenic lymphocyte of ostrich chicksWhen the concentration of boron was 0.01,0.1,0.5,1,5 and 10 mmol/L,compared with 0 mmol/L group,the OD value of splenic lymphocyte in boron treatment groups did not change significantly?P > 0.05?,cell viability was not significantly affected.While the concentration of boron was 25,50 and 100 mmol/L,the OD value of each group decreased gradually with the increase of boron concentration?P<0.01?.At this time,boron had significant toxicity to splenic lymphocytes.2.Effect of boron on the proliferation of splenic lymphocytes in ostrich chicksWhen different concentrations of boron and ConA co-stimulated the splenic lymphocytes,compared with the ConA group,while the concentration of boron was 0.01,0.1,0.5,1,5,10 mmol/L,the number of cell accumulation growth was increased,and cell viability showed an increase.When the concentration of boron was 0.1 mmol/L and 10 mmol/L,the cell survival rate was significantly higher than that in ConA group.When the concentration of boron was 25,50 and 100 mmol/L,the number of cell accumulation and growth decreased gradually with the increase of boron concentration,and the cell survival rate decreased with the increase of boron concentration,which was not significantly different from that of ConA group?P>0.05?.3.Effect of boron on apoptosis of splenic lymphocyte in ostrich chicksWhen 0.01?0.1?1?10?50?100 mmol/L boron was applied to the splenic lymphocyte of ostrich chicks,the apoptosis rates ratio were firstly decreased and then increased with the increase of boron dose.When the concentration of boron was 0.01 and 0.1 mmol/L,the apoptotic rate decreased gradually,and reached the lowest value when the concentration of boron was 0.1 mmol/L.Compared with the control group,the difference was significant?P < 0.05?.When boron concentration was 1,10,50 and 100 mmol/L,the apoptotic rate increased gradually with the increase of boron concentration.When boron concentration was 100 mmol/L,the apoptotic rate was higher than that of 0 mmol/L group,but the difference was not significant?P > 0.05?.4.Effect of boron on cytokine secretion by splenic lymphocytes of ostrich chicksWhen boron was applied to the splenic lymphocytes of ostrich chicks,the secretion of IL-6 was higher than that of the 0 mmol/L group when the boron concentration was 0.01 mmol/L,but the difference was not significant?P>0.05?.When the concentration of boron were 0.1,1,10,50,100 mmol/L,with the increase of boron concentration,the secretion of IL-6 gradually decreased,and when the concentration of boron was 100mmol/L,the secretion was significantly lower than that of the 0 mmol/L group?P<0.05?.The secretion of IFN-? was dose-dependent,showing a trend of increasing first and then decreasing,and when the concentration was 1mmol/L,the secretion of IFN-? reached its maximum value,which was extremely significant different from that of the 0 mmol/L group?P < 0.01?.When boron synergized with ConA on spleen lymphocyte of ostrich chicks,the secretion of IL-6 was higher than that of the 0 mmol/L group when the boron concentration was 0.01 mmol/L and 0.1 mmol/L,but the differences were not significant?P>0.05?.Then with the increase of boron concentration,the secretion of IL-6 decreased gradually,and when the concentration of boron was 50 mmol/L and 100 mmol/L,the secretions were significantly lower than that of the 0 mmol/L group?P<0.05?.The secretion of IFN-? in the experimental group was higher than that in the 0 mmol/L group.When the concentration of boron was 0.1 mmol/L,the secretion of IFN-? reached the maximum value,and the difference was extremely significant compared with the 0 mmol/L group?P<0.01?.5.RNA-Seq analysis of splenic lymphocyte response to boron stimulation in ostrich chicks?1?After sequencing by Illumina platform,three ostrich splenic lymphocyte sequencing libraries were obtained,it was 0 mmol/L group?C group?sample library,0.1 mmol/L group?T1 group?sample library and 100 mmol/L group?T2?sample library,which contained 48033271,49772060 and 48738533 Raw Reads,respectively.The Clean Reads of the resulting high quality sequences were 45587992,46909764,46159500,respectively.The ratios of Clean Reads to total Reads were 95%,94% and 95%,respectively.?2?The reads number of the upper ostrich genome in group C,T1 and T2 was 33665674,34095611,30754929,respectively,and the proportion of total reads was 73.84%,72.72%,66.72%,respectively.?3?By analyzing the statistical results of differential expression analysis,there was no differentially expressed genes in T1 group and C group;21 differentially expressed genes in T2 group and C group,of which 18 were up-regulated and 3 were downregulated;there were 43 differentially expressed genes in the T1 group and the T2 group,of which 5 were up-regulated and 38 were down-regulated.?4?In the biological process,metabolic processes,cell proliferation,cell physiological processes and reproductive processes were markedly enriched;in the cellular components,extracellular regions,membranes,macromolecular components,organelles,extracellular domain components,organelles The components,membrane components and cell components were characterized by significant enrichment;in the molecular structure,catalytic activity,binding function,signal transduction activity,molecular transduction activity and molecular function regulation were extremely significant enrichment.?5?Signal pathways for differential gene enrichment mainly include Systemic lupus erythematosus,alcohol dependence,viral carcinogenesis,and Necroptosis6 Real-time fluorescence quantitative verificationGenes that significant alter in Necroptosis signaling pathway were detected,including BIRC2₃,FTH1,and IL-1?.When the concentration of boron was 0.1 mmol/L,the relative expression of BIRC2₃,FTH1,and IL-1? mRNA decreased,compared with 0 mmol/L group,the difference was not significant?P>0.05?.When the boron concentration was 100 mmol/L,the relative expression levels of BIRC2₃,FTH1,and IL-1? mRNA increased,and the difference was extremely significant compared with 0 mmol/L group?P<0.01?.Boron regulated the expression of BIRC2₃,FTH1,and IL-1? genes,affected the activity of Necroptosis signaling pathway,thereby exerting immunoregulatory effects.