Regulation Of Intracellular Ca²⁺ Mediated Glycolysis On Neutrophil Adhesion And Phagocytosis Function In Subclinical Hypocalcemia Dairy Cows

In recent years,with the continuous optimization and adjustment of dairy production management level,high-quality dairy cattle breeding and nutrition strategy,China’s dairy industry has been booming,and milk production has also increased.However,hypocalcemia in dairy cows is still one of the important factors for the healthy and sustainable development of China’s dairy industry.Hypocalcemia in dairy cows is a disease caused by the imbalance of calcium homeostasis in perinatal dairy cows.According to research,subclinical hypocalcemia can cause secondary diseases,including ketosis,retained placenta,mastitis and other inflammatory diseases,causing immeasurable economic losses to the dairy industry.Neutrophils,as the most abundant white blood cells,play an important role in the innate immune response.They are the first to reach the site of infection and eliminate pathogens through adhesion and phagocytosis.It is generally believed that neutrophils obtain energy through glycolysis.However,it is not clear how glycolysis of peripheral blood neutrophils in subclinical hypocalcemia of dairy cows regulates adhesion and phagocytosis.Our previous study found that there was a causal relationship between the decrease of adhesion and phagocytosis of peripheral blood neutrophils and intracellular Ca²⁺concentration in subclinical hypocalcemia cows,but the specific mechanism was not clear.Therefore,this experiment intends to obtain the differential genes of neutrophils in peripheral blood of dairy cows with subclinical hypocalcemia by transcriptome technology,further analyze the effect of glycolysis on neutrophil adhesion and phagocytosis,and further clarify the effect of calcium pool-operated calcium entry channel（SOCE）related factors on glycolysis ability,and then regulate the mechanism of neutrophil adhesion and phagocytosis in dairy cows with subclinical hypocalcemia.In order to explore the relationship between glycolysis,adhesion and phagocytosis of neutrophils in subclinical hypocalcemia cows,the serum of 3-day-old dairy cows was collected and divided into healthy control group(Ca²⁺>2.10 mmol/L)and subclinical hypocalcemia group(Ca²⁺<2.00 mmol/L)according to the blood calcium Ca²⁺level,with 6 cows in each group.Blood was collected from the cows tail vein,and neutrophils were isolated.RNA sequencing was performed to analyze differential expression,and differentially expressed genes were analyzed by GO and KEGG enrichment.On the other hand,the level of Ca²⁺in the cytoplasm of neutrophils was detected by flow cytometry,the adhesion and phagocytosis were detected by confocal laser scanning microscopy.The m RNA levels of glycolytic pathway rate-limiting enzymes Hexokinase II（HKII）,Pyruvate type 2（PKM2）,Lactate Dehydrogenase A（LDHA）and gluconeogenesis gene fructose 1-6-bisphosphate kinase 1（FBP1）were detected by real-time fluorescence quantitative PCR.The results showed that the differential genes of subclinical hypocalcemia cows were enriched in the glucose metabolism pathway.The m RNA levels of HKII,PKM2 and LDHA decreased,while the m RNA level of FBP1 increased,indicating that the glycolytic ability decreased.At the same time,the intracellular Ca²⁺level and adhesion and phagocytosis of neutrophils were decreased.It indicated that the decrease of adhesion and phagocytosis of neutrophils in peripheral blood of subclinical hypocalcemia cows was related to the decrease of glycolysis ability.In order to explore how glycolytic capacity affects neutrophil adhesion and phagocytosis,we added HKII inhibitor Benserazide-d3 and FBP1 inhibitor MB05032 to change glycolytic capacity.Flow cytometry was used to detect intracellular Ca²⁺levels in neutrophils.Laser confocal was used to detect adhesion and phagocytosis.The m RNA levels of HKII,FBP1 and SOCE pathway-related molecules（ORAI1,ORAI2,ORAI3,STIM1,STIM2）were detected by quantitative real-time PCR.Western Blot was used to detect the transcription of SOCE pathway related molecules.The results showed that when Benserazide-d3 was added,the glycolytic ability of neutrophils decreased significantly,the adhesion and phagocytosis ability decreased significantly,and the Ca²⁺level in neutrophils and the m RNA and protein expression of SOCE pathway-related molecules did not change significantly.When MB05032 was added,the glycolysis ability of central granulocytes increased significantly,the adhesion and phagocytosis ability increased significantly,and the intracellular Ca²⁺level and the m RNA level and protein level of SOCE pathway related molecules did not change significantly.Therefore,this result suggests that the glycolytic capacity of neutrophils regulates adhesion and phagocytosis,but Ca²⁺levels and SOCE pathway-related molecules are not affected.In order to explore the effect of intracellular Ca²⁺level on glycolytic capacity of neutrophils,we added Thapsigargin or 2-Aminoethoxy-diphenyl borate（2-APB）to change intracellular Ca²⁺level.Under the stimulation of Thapsigargin,the m RNA level and protein level of SOCE pathway related molecules in the healthy control group and the subclinical hypocalcemia group increased significantly,the intracellular Ca²⁺level increased significantly,the glycolysis ability increased,and the adhesion and phagocytosis function increased significantly.After 2-APB treatment,the m RNA and protein levels of SOCE pathway-related molecules in neutrophils,whether in the healthy control group or the subclinical hypocalcemia group,decreased significantly,intracellular Ca²⁺levels decreased,glycolytic capacity decreased,adhesion and phagocytosis decreased significantly.This result indicates that intracellular Ca2+levels regulate glycolytic capacity.In summary,SOCE changes intracellular Ca²⁺levels and mediates glycolysis to regulate the adhesion and phagocytosis of neutrophils in peripheral blood of subclinical hypocalcemia cows.