| Mineral elements are indispensable nutrients for animals to maintain life and production.They participate in a series of physiological and biochemical processes of animals,and play an important role in growth,reproduction and immune defence of animals.There are synergistic and antagonistic interactions among mineral elements,and the phenomenon of abuse of mineral additives is widespread in production practice,the metabolic levels and physiological function of ruminants have great changes under different feeding conditions or during special growth periods,the imbalance of mineral element homeostasis and immune dysfunction often occur,the resulting low production performance and high incidence of diseases cause huge economic losses to the livestock industry.Therefore,it is of great scientific significance and practical value to study and maintain mineral element homeostasis for healthy breeding.In order to explore the effects of mineral element homeostasis imbalance on ruminants,sheep were used as experimental animals in this study to establish the model of mineral element homeostasis imbalance by intravenous injecting metal ion chelator EDTA(4%,60 m L/h,8 h/d,continuous injection for 3 days).Peripheral blood of sheep was collected before and after intravenous injection and during recovery period,then the serum and neutrophils were separated,and elements in each group were detected by ICP-MS.Serum results showed that Zn,P and other11 elements were significantly or extremely significantly lower after intravenous injection of EDTA than before(P<0.05),among them,the levels of P,Zn,K,Rb and As returned to normal levels 7 days after the completion of injection.The levels of Ca,Cu and Sr increased after intravenous injection,and Cu did not return to the control level 7 days after the completion of injection.The results of neutrophils detection showed that the levels of Hg and Cs in neutrophils were significantly lower after intravenous injection than before(P<0.05),but had no significant effects on other elements.In order to explore the effects of mineral element homeostatic imbalance on metabolism of neutrophils,peripheral blood was collected before and after intravenous injection of EDTA and neutrophils were isolated.LC-MS/MS was used for non-targeted metabolomics analysis of neutrophils.The results showed that in the positive ion mode,a total of 182 metabolites were significantly different before and after intravenous injection of EDTA(P<0.05),of which 72 were up-regulated and 110 were down-regulated.A total of 95 metabolites with significant differences were identified in negative ion mode(P<0.05),49 were up-regulated and 46 were down-regulated.KEGG pathway annotation and enrichment analysis showed that,these differential metabolites were mainly involved in the categories of amino acid metabolism,lipid metabolism,nucleotide metabolism and other pathways,and biological amine metabolism changed a lot,monoamine metabolism was weakened,polyamine metabolism was enhanced.Correlation analysis was performed between top 20 differential metabolites and common mineral elements by spearman algorithm in the mode of positive and negative ions,the results showed that,P,K,Ca,Mn,Cu,Zn had the highest correlation with differential metabolites of neutrophils,followed by Se,and the correlation between each mineral element and each differential metabolite were also different.For exploring the effects of mineral element homeostatic imbalance on gene transcription of neutrophils in peripheral blood,unique molecular identifier absolute quantitative transcriptome analysis was performed on neutrophils in peripheral blood before and after intravenous injection of EDTA in this study.The results showed that a total of 1561 genes were differentially expressed in peripheral blood neutrophils of sheep before and after intravenous injection of EDTA,among which 1390 genes were up-regulated and 171 genes were down-regulated.S100A8,S100A9 and S100A12 of the S100 family were extremely significantly increased after intravenous injection(P<0.01),and they were at the forefront of genes which were significantly up-regulated.The GO enrichment analysis showed that the functions of up-regulated differentially expressed genes were mainly concentrated in the process of inflammatory and immune-related,stress response and cellular activation.KEGG enrichment analysis showed that the up-regulated genes were mainly concentrated in NF-κB,Toll-like receptor,glycolysis/gluconeogenic,NOD-like receptor and other signaling pathways.In order to further explore the effects of mineral element homeostasis imbalance on neutrophils function and serum related indicators,the respiratory burst function of neutrophils and serum anti-oxidative,inflammatory factors,metalloproteinases were detected in this study.The results showed that the number of neutrophils activated in peripheral blood after intravenous injection of EDTA was extremely significantly higher than that in control group(P<0.01),the number of neutrophils in resting state was significantly lower than that in control group(P<0.05).The mineral element homeostasis imbalance of serum induced by intravenous injection of EDTA significantly increased the level of IL-6 in serum(P<0.05)and inhibited the level of MMP7(P<0.05),one week after the end of intravenous injection,the levels of IL-6 and MMP7 in serum returned to those before intravenous injection.The level of TIMP1 continued to increase after intravenous injection and was significantly higher than that in control group(P<0.05).The activities of CuZn-SOD and TNOS were significantly increased after intravenous injection of EDTA(P<0.05),and they recovered to the normal group one week later.In addition,the levels of creatinine and urea nitrogen in serum increased after intravenous injection of EDTA(P<0.05)and returned to normal levels one week after the end of intravenous injection.In conclusion,the imbalance model of serum mineral element homeostasis was successfully established through intravenous injection of EDTA in sheep.Mineral element homeostasis imbalance affects the metabolic process of neutrophils through the signaling pathways such as amino acid metabolism,lipid metabolism,nucleotide metabolism,and influences the gene transcription levels of neutrophils.These changes enhance the respiratory burst capacity of neutrophils and alter the levels of serum related indexes such as IL-6 and CuZn-SOD,finally affect the normal function of neutrophils in the inflammatory response,and is not conducive to the immune defense of the body and kidney health.Metabolites such as N1-Acetylspermine,N8-Acetylspermidine,PS(18:0/20:3),3-Methylcrotonylglycine and 5-Hydroxytryptophan are expected to serve as diagnostic markers for mineral element homeostasis or metabolic imbalance in ruminants.Meanwhile,genes S100A8,S100A9 and S100A12 may serve as biomarkers for inflammatory response caused by mineral element homeostasis imbalance in ruminants.These results can provide reference for the study on the mineral element homeostasis imbalance in ruminants. |
PDF Full Text Request