SOCE调控亚临床低钙血症奶牛单核巨噬细胞吞噬和黏附功能的研究

doi:10.16431/j.cnki.1671-7236.2025.08.039

摘要/Abstract

摘要： 【目的】探究钙池操控的钙内流(store operated Ca²⁺ entry,SOCE)对奶牛单核巨噬细胞吞噬和黏附功能的影响。【方法】采集健康奶牛(对照，Con)及亚临床低钙血症奶牛(hypocalcemia,Hyp)外周血液，利用磁珠法分离并体外培养CD14^＋单核巨噬细胞。取亚临床低钙血症奶牛单核巨噬细胞分为3组，分别为Hyp、毒胡萝卜素(thapsigarsin，TG)处理组(Hyp＋TG)和2-氨基乙基联苯基硼酸酯(2-aminoethyl diphenylborinate,2-APB)处理组(Hyp＋2-APB组)。通过流式细胞术检测细胞内Ca^2＋水平,利用实时荧光定量PCR和Western blotting分别检测SOCE相关因子mRNA和蛋白表达量；通过激光共聚焦和免疫荧光检测单核巨噬细胞吞噬和黏附情况，并检测单核巨噬细胞吞噬和黏附因子的表达。【结果】流式细胞术检测结果显示，与Con组相比，Hyp组单核巨噬细胞内Ca^2＋水平极显著降低(P＜0.01)。实时荧光定量PCR结果显示，与Con组相比，Hyp组单核巨噬细胞中钙离子释放激活的钙通道蛋白1(calcium release-activated calcium modulator 1,ORAI1)、基质相互作用分子1(stromal interaction molecule 1,STIM1)、STIM2、选择素L(selectin L,SELL)、ORAI2、ORAI3以及吞噬因子髓过氧化物酶(myeloperoxidase,MPO)基因表达量均极显著或显著降低(P＜0.01；P＜0.05)。Western blotting结果显示，与Con组相比，Hyp组单核巨噬细胞中ORAI2、ORAI3、STIM2、ORAI1和STIM1蛋白表达量极显著或显著降低(P＜0.01；P＜0.05)。同时，与Con组相比，Hyp组单核巨噬细胞吞噬和黏附能力显著或极显著降低(P＜0.05；P＜0.01)。TG处理后，与Hyp组相比，亚临床低钙血症奶牛单核巨噬细胞中ORAI1、ORAI2、MPO、ORAI3、STIM1、STIM2和SELL基因表达量显著或极显著升高(P＜0.05；P＜0.01)，ORAI1、ORAI3、STIM1、STIM2和ORAI2蛋白表达水平显著或极显著上调(P＜0.05；P＜0.01)；细胞吞噬、黏附能力均显著或极显著增强(P＜0.05；P＜0.01)。2-APB处理后，与Hyp组相比，亚临床低钙血症奶牛单核巨噬细胞中ORAI1、ORAI2、MPO、ORAI3、STIM1、STIM2和SELL基因表达量显著或极显著降低(P＜0.05；P＜0.01)，ORAI2、STIM2、ORAI1、ORAI3和STIM1蛋白表达量显著或极显著降低(P＜0.05；P＜0.01)；细胞吞噬、黏附能力极显著或显著减弱(P＜0.01；P＜0.05)。【结论】 TG激活SOCE后，亚临床低钙血症奶牛单核巨噬细胞内Ca²⁺水平升高，细胞吞噬和黏附能力增强；2-APB抑制SOCE后，亚临床低钙血症奶牛单核巨噬细胞内Ca^2＋水平降低，细胞的吞噬和黏附功能减弱，表明SOCE参与调控单核巨噬细胞的吞噬和黏附作用。

关键词: 钙池操控的钙内流(SOCE); 单核巨噬细胞; 吞噬; 黏附

Abstract: 【Objective】 The purpose of this experiment was to explore the effect of store operated Ca^2＋ entry (SOCE) on the phagocytic and adhesion functions of mononuclear macrophages in dairy cows. 【Method】 Peripheral blood of healthy (control，Con)and subclinical hypocalcemia dairy cows (hypocalcemia,Hyp) was collected,and CD14^＋ mononuclear macrophages in the blood were isolated and cultured in vitro using magnetic beads.The mononuclear macrophages of subclinical hypocalcemia dairy cows were divided into three groups: Hyp, thapsigarsin (TG) treatment group (Hyp+TG) and 2-aminoethyl diphenylborinate (2-APB) treatment group (Hyp＋2-APB).The intracellular Ca^2＋ level was detected by flow cytometry.Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of SOCE-related factors.The phagocytosis and adhesion of mononuclear macrophages were detected by laser confocal and immunofluorescence,and the expression of phagocytosis and adhesion factors of mononuclear macrophages was detected. 【Result】 Flow cytometry analysis showed that,compared with Con group,the intracellular Ca^2＋ level of mononuclear macrophages in Hyp group was extremely significantly decreased (P＜0.01).Real-time quantitative PCR results showed that,compared with Con group,the expression of calcium release-activated calcium modulator 1 (ORAI1)，stromal interaction molecule 1 (STIM1),STIM2 and selectin L (SELL) genes of mononuclear macrophages in Hyp group were extremely significantly decreased (P＜0.01)，expression of ORAI2,ORAI3 and myeloperoxidase (MPO) genes were significantly decreased (P＜0.05).Western blotting results showed that,compared with Con group,the expression of ORAI2,ORAI3,STIM2,ORAI1 and STIM1 proteins of mononuclear macrophages in Hyp group were extremely significant or significantly decreased (P＜0.01 or P＜0.05).Meanwhile,compared with Con group,the phagocytic and adhesion abilities of mononuclear macrophages in Hyp group were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).After TG treatment,compared with Hyp group,the expression of ORAI1,ORAI2,MPO,ORAI3,STIM1,STIM2 and SELL genes in mononuclear macrophages of subclinical hypocalcemia dairy cows were significantly or extremely significantly increased (P＜0.05 or P＜0.01)，the expression of ORAI1,ORAI3,STIM1,STIM2 and ORAI2 proteins were significantly or extremely significantly upregulated (P＜0.05 or P＜0.01).The phagocytic and adhesion abilities of cells were significantly or extremely significantly enhanced (P＜0.05 or P＜0.01).After 2-APB treatment,compared with Hyp group,the expression of ORAI1,ORAI2,MPO,ORAI3,STIM1,STIM2 and SELL genes in mononuclear macrophages of subclinical hypocalcemia dairy cows were significantly or extremely significantly decreased (P＜0.05 or P＜0.01)，the expression of ORAI2,STIM2,ORAI1,ORAI3 and STIM1 proteins were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The phagocytic and adhesion abilities of cells were extremely significant or significantly weakened (P＜0.01 or P＜0.05). 【Conclusion】 After SOCE was activated by TG,the Ca^2＋ level in mononuclear macrophages of subclinical hypocalcemia dairy cows increased,and the phagocytic and adhesion abilities enhanced.After SOCE was inhibited by 2-APB,the intracellular Ca^2＋ level in mononuclear macrophages of subclinical hypocalcemia dairy cows decreased,and the phagocytic and adhesion functions weakened，which indicated that SOCE was involved in regulating the phagocytosis and adhesion of mononuclear macrophages.

Key words: store operated Ca^2＋ entry (SOCE); mononuclear macrophage; phagocytic; adhesion

中图分类号:

S856

李思瑶, 季子崴, 李蕾, 张靖淇, 岳柏晨, 尹玉凤, 徐闯, 杨威, 张冰冰. SOCE调控亚临床低钙血症奶牛单核巨噬细胞吞噬和黏附功能的研究[J]. 中国畜牧兽医, 2025, 52(8): 3907-3917.

LI Siyao, JI Ziwei, LI Lei, ZHANG Jingqi, YUE Baichen, YIN Yufeng, XU Chuang, YANG Wei, ZHANG Bingbing. Regulation of Phagocytic and Adhesion Functions of Mononuclear Macrophages by SOCE in Subclinical Hypocalcemia Dairy Cows[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3907-3917.

参考文献

[1] 王鸿宇,宋玉锡,白云龙,等.我国部分规模化牧场奶牛低钙血症的发病情况调查及诊治和防控方案分析[J].黑龙江畜牧兽医,2023,24:57-64. WANG H Y,SONG Y X,BAI Y L,et al.Investigation of the incidence of hypocalcemia in dairy cows in some large-scale pastures in China and analysis on diagnosis and treatment,prevention and control plans[J]. Heilongjiang Animal Science and Veterinary Medicine,2023,24:57-64.(in Chinese)
[2] COUTO S R,DEVRIES T J,DUFFIELD T F,et al.Graduate student literature review:What do we know about the effects of clinical and subclinical hypocalcemia on health and performance of dairy cows[J].Journal of Dairy Science,2021,104(5):6304-6326.
[3] EMAM M H,SHEPLEY E,MAHMOUD M M,et al.The association between prepartum rumination time,activity and dry matter intake and subclinical hypocalcemia and hypomagnesemia in the first 3 days postpartum in Holstein dairy cows[J].Animals (Basel),2023,13(10):1621.
[4] 张伟.胞内Ca^2＋介导糖酵解调控亚临床低钙血症奶牛血液中性粒细胞黏附和吞噬功能[D].大庆:黑龙江八一农垦大学,2023. ZHANG W.Regulation of intracellular Ca^2＋-mediated glycolysis on neutrophil adhesion and phagocytosis function by in subclinical hypocalcemia dairy cows[D].Daqing:Heilongjiang Bayi Agricultural University,2023.(in Chinese)
[5] LENA L,STÉPHANE K,XAVER K,et al.Store-operated calcium entry in skeletal muscle:What makes it different[J].Cells,2021,10(9):2356.
[6] ROSENBERG P,KATZ D,BRYSON V.SOCE and STIM1 signaling in the heart:Timing and location matter[J].Cell Calcium,2019,77:20-28.
[7] 柏佩原,孙爱军.单核巨噬细胞系统在心肌损伤和修复中的作用研究进展[J].中国分子心脏病学杂志,2022,22(4):4845-4852. BAI P Y,SUN A J.Research progress on the role of mononuclear phagocyte system in myocardial injury and repair[J].Molecular Cardiology of China,2021,22(4):4845-4852.(in Chinese)
[8] 张毕红,王先胜,陈东颖,等.围产期奶牛低钙血症及其与其他疾病的关系研究进展[J].中国畜牧杂志,2023,59(4):56-62. ZHANG B H,WANG X S,CHEN D Y,et al.Research progress on cow periparturient hypocalcemia and the relationship with other metabolic disorders[J].Chinese Journal of Animal Science,2023,59(4):56-62.(in Chinese)
[9] MI S,XINYI T,XIN G,et al.Inhibition of calcium signaling prevents exhaustion and enhances anti-leukemia efficacy of CAR-T cells via SOCE-calcineurin-NFAT and glycolysis pathways[J]. Advanced Science,2022,9(9):e2103508.
[10] EMRICH S M,YOAST R E,ZHANG X,et al.ORAI3 and ORAI1 mediate CRAC channel function and metabolic reprogramming in B cells[J].eLife,2023,12:e84708.
[11] CHEN X,LI S,OJCIUS M D,et al.Mononuclear-macrophages but not neutrophils act as major infiltrating anti-leptospiral phagocytes during leptospirosis[J].PLoS One,2017,12(7):e0181014.
[12] PAULA N,NICOLAS D.The role of calcium signaling in phagocytosis[J].Journal of Leukocyte Biology,2010,88(1):57-68.
[13] 张伟,何雨欣,麻芯茹,等.亚临床低钙血症奶牛外周血中性粒细胞的糖异生作用下降[J].中国兽医学报,2023,43(6):1315-1320. ZHANG W,HE Y X,MA X R,et al.Decreased gluconeogenesis pathway of peripheral blood neutrophils in subclinical hypocalcemia dairy cows[J].Chinese Journal of Veterinary Science,2023,43(6):1315-1320.(in Chinese)
[14] YAN J H,QIANG L,ZUN Y F,et al.STIM1 controls calcineurin Akt/mTOR/NFATC2-mediated osteoclastogenesis induced by RANKL/M-CSF[J].Experimental and Therapeutic Medicine,2020,20(2):736-747.
[15] ELENA C,PAOLA I,PAOLA M,et al.Alteration of STIM1/ORAI1-mediated SOCE in skeletal muscle:Impact in genetic muscle diseases and beyond[J].Cells,2021,10(10):2722.
[16] HUANG S A,WU J A,TONG C B,et al.Rectifying attenuated store-operated calcium entry as a therapeutic approach for Alzheimer’s disease[J].Current Alzheimer Research,2020,17(12):1072-1087.
[17] SCREMIN E,AGOSTINI M,LEPARULO A,et al.ORAI2 down-regulation potentiates SOCE and decreases Aβ42 accumulation in human neuroglioma cells[J].International Journal of Molecular Medical Science,2020,21(15):5288.
[18] DONG H,ZHANG Y,SONG R,et al.Toward a model for activation of ORAI channel[J].iScience,2019,16:356-367.
[19] ALEJANDRO E B,GIRISH R,ELENA D,et al.CAPN1 (Calpain1)-dependent cleavage of STIM1 (stromal interaction molecule 1) results in an enhanced SOCE (store-operated calcium entry) in human neonatal platelets[J]. Arteriosclerosis,Thrombosis,and Vascular Biology,2023,43(5):e151-e170.
[20] LIFU S,IRYNA L,VLADIMIR S.Cell adhesion and intracellular calcium signaling in neurons[J].Cell Commun Signal,2013,11(1):94.
[21] ALQINYAH M,ALHAMED A S,ALNEFAIE H O,et al.Targeting store-operated calcium entry regulates the inflammation-induced proliferation and migration of breast cancer cells[J].Biomedicines,2023,11(6):1637.
[22] 何雨欣.ACC1介导SOCE调控肥胖小鼠CD4^＋ Th17细胞IL-17分泌的研究[D].大庆:黑龙江八一农垦大学,2023. HE Y X.Study on the regulation of IL-17 secretion of CD4^＋ Th17 cells of obese mice by SOCE mediated by ACC1[D].Daqing:Heilongjiang Bayi Agricultural University,2023.(in Chinese)
[23] HAMMAD A S,MACHACA K.Store operated calcium entry in cell migration and cancer metastasis[J].Cells,2021,10(5):1246.
[24] CHAUDHARI S,MALLET R T,SHOTORBANI P Y,et al.Store-operated calcium entry:Pivotal roles in renal physiology and pathophysiology[J].Experimental Biology and Medicine,2020,246(3):305-316.
[25] SEUNGEUN S,SUKYUNG S,YONGWOON K,et al.Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity[J].Life Sciences,2023,332(2023):122107.
[26] KUMAR S,DEVI S,SOOD S K,et al.Antibiotic resistance and virulence genes in nisin-resistant Enterococcus faecalis isolated from raw buffalo milk modulate the innate functions of rat macrophages[J].Journal of Applied Microbiology,2019,127(3):897-910.
[27] 王赞嘉,锡林高娃,吴金花,等.牛乳腺炎粪肠球菌cylA基因对牛外周血单核巨噬细胞完全吞噬功能的影响[J].中国动物传染病学报,2023,31(3):178-183. WANG Z J,XI L G W,WU J H,et al.Effect of the bovine Enterococcus faecalis cylA gene on cow mononuclear macrophages phagocytes[J].Chinese Journal of Animal Infectious Diseases,2023,31(3):178-183.(in Chinese)