Exogenous glucose reduces hypoxic injury of C8-D1A astrocytes by regulating metabolism
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摘要:目的 探讨缺氧对星形胶质细胞能量代谢和细胞活性的影响，并验证补充葡萄糖的潜在保护作用。方法 将小鼠小脑星形胶质细胞系C8-D1A分为对照组、缺氧组、缺氧联合葡萄糖（10 mmol/L）组、缺氧联合高剂量葡萄糖（15 mmol/L）组。通过CCK-8和ATP法检测细胞活力，采用Seahorse XF细胞能量分析仪检测细胞的线粒体呼吸功能与糖酵解水平，通过qPCR和蛋白质印迹法检测细胞糖代谢、A1/A2表型及凋亡相关基因和蛋白的表达水平，并检测细胞培养上清中葡萄糖、乳酸和脑源性神经营养因子的水平，采用活性氧荧光探针评估氧化应激水平。结果 C8-D1A细胞在缺氧6 h后出现增殖抑制，缺氧12 h细胞凋亡的线粒体途径启动，缺氧24 h呈现信号通路p-Akt/Akt抑制；缺氧条件下C8-D1A的能量代谢模式由氧化磷酸化向糖酵解转换，这种代谢转换过程伴随着葡萄糖摄取增加、乳酸生成增多及糖代谢关键酶基因表达的上调。补充10或15 mmol/L葡萄糖能够增强C8-D1A细胞糖酵解通量，表现为葡萄糖摄取基因（葡萄糖转运蛋白1）的表达升高，糖酵解相关酶基因（己糖激酶、丙酮酸激酶、丙酮酸脱氢酶激酶）的转录增强；其中，10 mmol/L葡萄糖能够调控凋亡相关信号通路，抑制细胞凋亡的线粒体途径，并减轻氧化应激效应；同时，补充葡萄糖还促进C8-D1A细胞向神经保护性A2表型极化，激活环磷酸腺苷反应元件结合蛋白-脑源性神经营养因子信号通路，增加脑源性神经营养因子的分泌。结论 缺氧导致星形胶质细胞C8-D1A发生增殖抑制、凋亡增加及代谢重编程等功能紊乱，补充葡萄糖可改善上述异常，其神经保护效应可能通过代谢支持、凋亡抑制及表型重塑共同介导。Abstract:Objective To investigate the effects of hypoxia on energy metabolism and cell viability in astrocytes and to verify the potential protective effect of glucose supplementation.Methods Mouse cerebellar astrocytes, C8-D1A, were assigned to control group, hypoxia group, hypoxia plus glucose (10 mmol/L) group, or hypoxia plus high-dose glucose (15 mmol/L) group. Cell viability was detected by cell counting kit 8 (CCK-8) assay and adenosine triphosphate (ATP) measurement. Mitochondrial respiratory function and glycolysis level were determined using a Seahorse XF Cell Energy Analyzer. Quantitative polymerase chain reaction and Western blotting were performed to detect the expression levels of genes and proteins related to glucose metabolism, A1/A2 phenotype, and apoptosis. In addition, the levels of glucose, lactate, and brain-derived neurotrophic factor (BDNF) in the culture supernatant were measured, and reactive oxygen species (ROS) fluorescent probes were used to assess oxidative stress levels.Results The proliferation of C8-D1A cells was inhibited after 6 h of hypoxia, the mitochondrial apoptotic pathway was activated after 12 h, and the p-Akt/Akt signaling pathway was suppressed after 24 h. Under hypoxia, the cellular energy metabolism shifted from oxidative phosphorylation to glycolysis, accompanied by increased glucose uptake, elevated lactate production, and upregulation of key glycolytic enzyme genes. Supplementation with 10 and 15 mmol/L glucose enhanced glycolytic flux of the C8-D1A astrocytes, as evidenced by elevated expression of the glucose uptake gene (glucose transporter 1) and enhanced transcription of glycolysis-related enzyme genes (hexokinase, pyruvate kinase, pyruvate dehydrogenase kinase). Among them, 10 mmol/L glucose regulated apoptosis-related signaling pathways, inhibited the mitochondrial pathway of cell apoptosis, and alleviated oxidative stress. Meanwhile, glucose supplementation promoted the polarization of C8-D1A astrocytes toward the neuroprotective A2 phenotype, activated the cAMP-response element-binding protein-BDNF signaling pathway, and increased the secretion of BDNF.Conclusion Hypoxia induces functional disorders such as inhibited proliferation, increased apoptosis and metabolic reprogramming in C8-D1A astrocytes. Exogenous glucose supplementation can ameliorate these abnormalities. The neuroprotective effects of glucose are likely achieved through the concerted actions of metabolic support, apoptosis attenuation, and phenotypic remodeling.
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Keywords:
- astrocytes /
- energy metabolism /
- apoptosis /
- hypoxia /
- glucose /
- A2 astrocytes /
- neuroprotection
星形胶质细胞作为脑能量代谢的核心参与者，在维持中枢神经系统稳态中发挥关键作用[1-2]。在生理条件下，星形胶质细胞通过葡萄糖转运蛋白（glucose transporter，GLUT）1从血液中摄取葡萄糖，并表现出糖酵解代谢优势，其糖酵解途径中的关键限速酶[己糖激酶（hexokinase，HK）、丙酮酸激酶（pyruvate kinase M，PKM）、6-磷酸果糖激酶（6-phosphofructokinase，PFK）]呈高表达，促使葡萄糖高效转化为丙酮酸[3]。随后，在丙酮酸脱氢酶激酶（pyruvate dehydrogenase kinase，PDK）的调控下，星形胶质细胞限制丙酮酸进入三羧酸循环，转而通过乳酸脱氢酶（lactate dehydrogenase，LDH）催化将其转化为乳酸。乳酸经单羧酸转运体（monocarboxylate transporter，MCT）1/4释放至胞外，被神经元摄取并作为主要能量底物[4]。在缺氧条件下，星形胶质细胞通过缺氧诱导因子1α（hypoxia-inducible factor 1α，HIF-1α）介导的代谢重编程机制调节葡萄糖-乳酸代谢轴，维持能量稳态[5]，表现为：促进GLUT1/4的表达，增加葡萄糖摄取；激活糖酵解关键酶基因的转录，增强糖酵解通量；增加乳酸生成，通过MCT1/4介导的乳酸穿梭为神经元提供替代能源，优化能量底物分配；协同激活多重保护机制，包括分泌神经营养因子支持突触可塑性、增强糖原分解代谢、重构谷氨酸循环、维持谷胱甘肽的氧化还原稳态等[6-7]。
葡萄糖作为大脑最主要的能量底物，在缺氧环境下的作用具有双向性：一方面，它通过糖酵解途径维持细胞基本ATP水平；另一方面，其无氧代谢产物乳酸的过量积累可能引发细胞内酸中毒[8]。但外源性补充葡萄糖对缺氧的星形胶质细胞究竟是发挥保护作用还是产生损伤效应尚无定论，特别是葡萄糖的保护作用是否超越单纯的能量供应，而涉及氧化应激、细胞表型转化与凋亡调控等多重机制，仍有待深入探索。本研究利用小鼠星形胶质细胞系C8-D1A探讨缺氧对星形胶质细胞的存活和糖代谢的影响，以及补充葡萄糖在调控星形胶质细胞糖代谢和表型极化中的作用及其潜在机制。
1 材料和方法
1.1 细胞与主要试剂
C8-D1A细胞购于中国典型培养物保藏中心；DMEM、葡萄糖溶液购自美国Gibco公司；线粒体压力测试试剂盒、糖酵解压力测试试剂盒购于美国Agilent公司；CCK-8购于日本同仁化学研究所；葡萄糖测定试剂盒、乳酸测定试剂盒购于南京建成生物工程研究所；脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）检测试剂盒购于上海酶联生物科技有限公司；ATP测定试剂盒、活性氧检测试剂盒购于上海碧云天生物技术公司；引物由生工生物工程（上海）股份有限公司合成；HiScriptⅢ RT SuperMix（货号R323-01）、ChamQ SYBR qPCRMaster Mix（货号Q321-02）购自南京诺唯赞生物科技有限公司；HIF-1α抗体（货号36169S）、HK1抗体（货号2024T）、HK2抗体（货号2867T）、PKM2抗体（货号4053T）、PKM1/2抗体（货号3190T）、乳酸脱氢酶同工酶A（lactate dehydrogenase A，LDHA）抗体（货号2012S）、顺乌头酸酶2（aconitase 2，ACO2）抗体（货号6517S）、Bcl-2抗体（货号3498S）、Bax抗体（货号2772S）、Akt抗体（货号9272S）、磷酸化Akt（phosphorylated Akt，p-Akt）抗体（货号9271S）、环磷酸腺苷反应元件结合蛋白（cAMP response element-binding protein，CREB）抗体（货号9197S）、磷酸化CREB（phosporylated CREB，p-CREB）抗体（货号9198S）、caspase 8抗体（货号4927S）、caspase 3抗体（货号9665S）、cleaved caspase 3抗体（货号9661S）、HRP偶联的羊抗鼠IgG（货号7076S）、HRP交联的羊抗兔IgG（货号7074S）购于美国Cell Signaling Technology公司，GLUT1抗体（货号ab115730）、磷酸甘油酸激酶1（phosphoglycerate kinase 1，PGK1）抗体（货号ab199438）、MCT4抗体（货号ab308528）、S100钙结合蛋白A10（S100 calcium binding protein A10，S100A10）抗体（货号ab76472）购自英国Abcam公司，β-肌动蛋白抗体（货号A2228）购于美国Sigma公司。
1.2 细胞培养及分组处理
将C8-D1A细胞培养于含10% FBS、1%青霉素-链霉素混合液的DMEM中，在5% CO2、37 ℃、饱和湿度条件下培养，每2~3 d传代1次。实验分为2个部分：（1）将C8-D1A细胞分为对照组和缺氧组。生理状态下，成年动物脑组织中的氧浓度普遍维持在2%~5%[9-10]。为观察缺氧是否对C8-D1A细胞生长造成损伤效应，选择0.3%氧浓度的缺氧模型，观察缺氧对细胞存活和代谢的影响。（2）将C8-D1A细胞分为对照组、缺氧组、缺氧联合葡萄糖组、缺氧联合高剂量葡萄糖组。采用0.3%氧浓度进行缺氧处理，缺氧处理24 h后进行葡萄糖干预。健康成人的空腹血糖水平约4.9~6.4 mmol/L，脑脊液葡萄糖浓度通常为血糖浓度的30%~60%[11]。健康小鼠的空腹血糖水平约3.9~6.7 mmol/L，餐后血糖可短暂升高至8.3~11.1 mmol/L[12]。体外细胞系的培养基常用DMEM高糖和DMEM低糖2种培养基，DMEM低糖培养基更接近生理浓度，用于模拟正常代谢，其葡萄糖浓度为5.5 mmol/L。参照人体、小鼠、细胞的生理基准、病理阈值建立体外葡萄糖干预模型，将5 mmol/L葡萄糖作为正常对照浓度（对照组、缺氧组采用此浓度）；结合补糖后脑脊液浓度变化和糖尿病血糖阈值，以10 mmol/L葡萄糖模拟补糖干预的脑脊液浓度（缺氧联合葡萄糖组采用此浓度），15 mmol/L葡萄糖模拟糖尿病性脑病特征浓度（缺氧联合高剂量葡萄糖组采用此浓度）[13-14]。
1.3 检测指标与方法
1.3.1 CCK-8法检测细胞存活情况
将处于对数增殖期的C8-D1A细胞用胰蛋白酶消化并计数，以5×103个/孔接种于96孔板中，各组处理完毕后，每孔加入10 μL CCK-8溶液，轻轻晃动摇匀，将培养板放入培养箱中继续孵育1 h，然后用酶标仪测定450 nm波长处的光密度值。以对照组的细胞存活率为100%，计算各实验组的相对细胞存活率。
1.3.2 ATP法测定细胞代谢活力
将细胞以5×103个/孔的密度接种到96孔板，各组处理完毕后，加入50 μL裂解液，离心后的上清液即为含有ATP的细胞裂解液，吸取20 μL转移至不透明的黑色96孔检测板，按说明书加入底物溶液后，将检测板放入化学发光检测仪中，测量并记录每个孔的光信号值，根据标准曲线计算样品的ATP浓度。
1.3.3 Seahorse XF糖酵解压力测试和线粒体压力测试
采用Seahorse XF细胞能量分析仪（美国Agilent公司）检测细胞的线粒体呼吸功能与糖酵解水平。将C8-D1A细胞以8×103个/孔的密度接种于Seahorse XF细胞培养板。探针板每孔加入200 μL校准液，放在无CO2的37 ℃培养箱中。各组处理完毕后进行检测。测定氧气消耗速率（oxygen consumption rate，OCR）时，用补充有10 mmol/L D-葡萄糖、1 mmol/L丙酮酸盐和2 mmol/L L-谷氨酰胺的Seahorse XF DMEM替换完全培养基。测定细胞外酸化速率（extra cellular acidification rate，ECAR）时，将完全培养基替换为含4 mmol/L L-谷氨酰胺的Seahorse XF DMEM。培养体系总体积为180 μL，使细胞在无CO2培养箱中于37 ℃平衡1 h。分别在探针板载药孔加入葡萄糖、寡霉素和2-脱氧葡萄糖，保证终浓度分别为10 mmol/L、2 μmol/L和50 mmol/L；设置程序，上机检测。最后进行BCA蛋白定量，将OCR和ECAR进行标准化处理。
1.3.4 qPCR检测相关基因的表达
收集各组细胞，采用TRIzol法提取总RNA，应用紫外可见分光光度计进行定量，取1 μg总RNA进用反转录，然后进行qPCR扩增。采用Primer3（version 4.1.0）在线设计各目的基因的上、下游引物（表 1）。PCR程序：95 ℃预变性30 s，95 ℃ 5 s、60 ℃ 30 s共40个循环。以β-肌动蛋白作为内参基因，用2－ΔΔCt法计算各组目的基因的表达量，将对照组的表达量设定为1，各实验组的表达量为相对于对照组的变化倍数。
表 1 qPCR引物序列Table 1 Sequences of the primers for qPCRGene Primer-forward (5'-3') Primer-reverse (5'-3') β-actin GTGGGAATGGGTCAGAAGGA CTTCTCCATGTCGTCCCAGT Bcl-2 AGCCCAATGCCCTCCAGAGC TCTCAAGCCTTCACGCAAGTTCAG Bax TTGCCCTCTTCTACTTTGCTAG CCATGATGGTTCTGATCAGCTC GLUT1 GACACTTGCCTTCTTTGCCA AAGAGACAGGAATGGGCGAA MCT1 TGGCTGTCATGTATGCTGGA TTGAAAGCAAGCCCAAGACC MCT4 CCCTGGCTGCTATCATCTGT GCTGTAGAAAGGCCCAATCG S100A10 GTGCTCATGGAACGGGAGT AAAGCTCTGGAAGCCCACTT CD109 CATTGAGCAACATTGCCTGAGA GTCACCTACTACACTCCTTTGT H2-T23 TCAGAGTAACGACGAATCTCAC GTTCAGGGAGATGTAATCCTGG BDNF GAAGAGCTGCTGGATGAGGA GTTTGCGGCATCCAGGTAAT HIF-1α CCAGCAGACCCAGTTACAGA TGAGTGCCACTGTATGCTGA HK1 CTGGGTGAGATCGTCCGTAA CACGTGCTGTTTAGACCCAG HK2 CTCTCTCTCAACCCTGGCAA GGCAGTCACTCTCGATCTGA PKM CTTCATTCAGACCCAGCAGC CCGAGCCACATTCATTCCAG PGK1 GACTTTGGACAAGCTGGACG CCAGGTGGCTCATAAGGACA PDK1 CCGATTCAGGTTCACGTCAC CTCCCTGGAAGTACTGTGCA LDHA AGCTGCTGATCGTCTCCAAT GCAACATTCACACCACTCCA qPCR: Quantitative polymerase chain reaction; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2 associated X protein; GLUT1: Glucose transporter 1; MCT1/4: Monocarboxylate transporter 1/4; S100A10: S100 calcium binding protein A10; H2-T23: Histocompatibility 2, T region locus 23; BDNF: Brain-derived neurotrophic factor; HIF-1α: Hypoxia-inducible factor 1α; HK1/2: Hexokinase 1/2; PKM: Pyruvate kinase M; PGK1: Phosphoglycerate kinase 1; PDK1: Pyruvate dehydrogenase kinase 1; LDHA: Lactate dehydrogenase A. 1.3.5 蛋白质印迹法检测相关蛋白的表达
收集各组细胞，使用RIPA裂解液裂解并提取细胞总蛋白，然后在4 ℃下以9 200×g离心10 min，取上清。采用BCA法进行蛋白定量。取10 μg蛋白进行SDS-PAGE。转印至PVDF膜后，将膜放在5%脱脂奶粉溶液中室温封闭1 h；用TBST洗涤3次后，加入适量稀释于5% BSA中的一抗（稀释比例1∶1 000），4 ℃孵育过夜。用TBST洗涤3次，然后加入HRP偶联的二抗（稀释比例1∶5 000）室温孵育1 h，再用TBST洗涤3次。滴加ECL化学发光液，采用化学发光成像分析仪在暗室中曝光、显影、拍照。
1.3.6 葡萄糖消耗量和乳酸产生量检测
用葡萄糖测定试剂盒和乳酸测定试剂盒测量葡萄糖消耗量和乳酸产生量。将细胞以5×103个/孔的密度接种到96孔板，处理完毕后，收集上清液，依据细胞内总蛋白的浓度校准，根据说明书检测葡萄糖消耗和乳酸积累。用酶标仪分别测定505 nm和530 nm波长处的光密度值，根据标准曲线计算测试样品中葡萄糖消耗量和乳酸产生量。
1.3.7 活性氧检测
将C8-D1A细胞以3×104/mL的密度接种到共聚焦小皿，处理完毕后，加入用探针稀释液按1∶5 000稀释的2’, 7’-二氯二氢荧光素二乙酸酯（2’, 7’-dichlorodihydrofluorescein diacetate，DCFH-DA）并孵育20 min。然后，使用EVOSTM M7000智能成像系统（美国ThermoFisher公司）在激发/发射波长488/525 nm条件下拍照，分析荧光密度。
1.3.8 BDNF检测
按试剂盒说明书收集处理完毕的细胞上清液，依据细胞内总蛋白的浓度校准后依次加入样品、标准品各50 μL，除空白孔外，每孔加入HRP标记的检测抗体100 μL，37 ℃孵育60 min。洗涤5次后，加入底物A、B各50 μL，37 ℃避光孵育15 min。每孔加入终止液50 μL。用酶标仪检测450 nm波长处的光密度值，根据标准曲线计算样品中BDNF的水平。
1.4 统计学处理
采用GraphPad Prism 8.0软件进行统计学分析。所有实验独立重复3次。计量资料以x±s表示，两组之间的比较采用独立样本t检验。检验水准（α）为0.05。
2 结果
2.1 缺氧对C8-D1A细胞存活和糖代谢的影响
2.1.1 缺氧导致C8-D1A细胞损伤
将C8-D1A细胞缺氧处理不同时间后进行检测。结果显示，与对照组相比，缺氧6 h组的细胞活力开始下降（P＜0.01，图 1A）；缺氧12 h、24 h组凋亡相关基因Bcl-2/Bax的蛋白和mRNA比值降低，缺氧24 h组细胞内p-Akt/Akt蛋白比值降低（均P＜0.05，图 1B~1E）。上述结果表明C8-D1A细胞在缺氧6 h后细胞增殖被抑制，在缺氧12 h开始启动细胞凋亡的线粒体途径，在缺氧24 h信号通路p-Akt/Akt被抑制。
图 1 缺氧导致C8-D1A细胞损伤Fig. 1 Hypoxia induces C8-D1A cell injuryA: Hypoxia-induced viability reduction of C8-D1A cells (CCK-8); B-D: Hypoxia decreases p-Akt/Akt and Bcl-2/Bax protein (Western blotting); E: Hypoxia decreases Bcl-2/Bax mRNA (qPCR). *P<0.05, **P<0.01. n＝3, x±s. CCK-8: Cell counting kit 8; p-: Phosphorylated; Akt: Protein kinase B; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2 associated X protein; qPCR: Quantitative polymerase chain reaction.
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2.1.2 缺氧条件下C8-D1A细胞的能量代谢由氧化磷酸化向糖酵解转换
与对照组相比，缺氧组的基础OCR、备用呼吸容量、ATP关联OCR、最大OCR被抑制（均P＜0.01，图 2A~2E），而基础糖酵解水平、糖酵解容量和糖酵解储备明显提升（均P＜0.01，图 2F~2I）。这提示在缺氧情况下，C8-D1A细胞的胞外酸化能力明显强于有氧呼吸水平，糖酵解成为主要供能途径。
图 2 缺氧条件下C8-D1A细胞的能量代谢由氧化磷酸化向糖酵解转换Fig. 2 C8-D1A energy metabolism shifts from oxidative phosphorylation to glycolysis under hypoxiaThese are the results of the Seahorse XF glycolysis stress test and mitochondrial stress test. A: Dynamic changes of OCR; B-E: Quantification of basal OCR, ATP-linked OCR, maximal OCR, and spare respiratory capacity OCR, respectively; F: Dynamic changes of ECAR; G-I: Quantification of glycolytic, glycolytic capacity and glycolytic reserve ECAR, respectively. **P<0.01. n＝3, x±s. OCR: Oxygen consumption rate; ECAR: Extra cellular acidification rate; ATP: Adenosine triphosphate; Con: Control; Hy: Hypoxic exposure; FCCP: Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; Rot: Rotenone; AA: Antimycin A; 2-DG: 2-deoxy-D-glucose.下载:
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2.1.3 缺氧条件下C8-D1A细胞通过增强葡萄糖摄取和糖代谢关键酶的表达维持能量稳态
由图 3可见，在缺氧3、6、12和24 h，葡萄糖消耗和乳酸产生呈现时间依赖性增加；C8-D1A细胞HIF-1α的mRNA和蛋白表达水平呈现时间依赖性增加；GLUT1的mRNA表达水平明显升高但呈先升后降趋势，而HK1、PKM、PGK1、PDK1、MCT4、LDHA的mRNA表达水平呈持续性升高趋势，这些糖代谢相关分子的蛋白表达均表现为持续性升高趋势。这提示在急性缺氧条件下，C8-D1A细胞通过激活HIF-1α信号通路上调GLUT1蛋白表达，增强葡萄糖摄取能力；同时促进糖酵解关键酶的表达，增加糖酵解通量，并加速乳酸的生成和转运，从而实现以糖酵解为主的快速能量供应模式转变。
图 3 缺氧通过增强葡萄糖摄取及糖代谢关键酶基因的表达维持C8-D1A细胞的能量稳态Fig. 3 Hypoxia sustains cellular energetics through enhancing glucose uptake and glycolytic enzyme expression in C8-D1A cellsA: Hypoxia enhances glucose consumption (glucose kit); B: Hypoxia enhances lactate production (lactate kit); C: Hypoxia upregulates HIF-1α mRNA expression (qPCR); D: Hypoxia upregulates GLUT1 mRNA expression (qPCR); E-H: Hypoxia enhances the mRNA expression of essential glycolytic enzymes (qPCR); I, J: Hypoxia enhances the mRNA expression of lactate production and transport genes (qPCR); K-Q: Hypoxia enhances protein expression of glycolytic enzymes (Western blotting). *P<0.05, **P<0.01. n＝3, x±s. HIF-1α: Hypoxia-inducible factor 1α; qPCR: Quantitative polymerase chain reaction; GLUT1: Glucose transporter 1; HK1/2: Hexokinase 1/2; PKM: Pyruvate kinase M; PGK1: Phosphoglycerate kinase 1; PDK1: Pyruvate dehydrogenase kinase 1; MCT4: Monocarboxylate transporter 4; LDHA: Lactate dehydrogenase A.下载:
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2.2 补充葡萄糖对缺氧条件下C8-D1A细胞存活和糖代谢的影响
2.2.1 补充葡萄糖上调缺氧C8-D1A细胞糖代谢相关酶基因的表达
由图 4可见，补充葡萄糖后，缺氧C8-D1A细胞的葡萄糖消耗量明显增加（P＜0.01），糖代谢相关分子的mRNA表达水平均升高（均P＜0.05），且HK1和PKM2的蛋白表达水平也增高（均P＜0.05），提示糖酵解代谢增强；此外，补充葡萄糖后，缺氧C8-D1A细胞的ACO2蛋白表达明显升高（P＜0.05），提示ACO2介导的三羧酸循环代谢活性增强。以上结果提示，补充葡萄糖能够上调缺氧C8-D1A细胞的糖代谢水平，这种改变不仅能够满足细胞在缺氧状态下的能量需求，还可能通过维持细胞能量稳态而发挥潜在的神经保护作用。
图 4 补充葡萄糖促进缺氧C8-D1A细胞糖代谢、上调糖代谢相关酶基因的表达Fig. 4 Exogenous glucose supplementation enhances glucose metabolism and upregulates glycolytic metabolism-related genes in C8-D1A cells under hypoxiaA: Exogenous glucose enhances glucose consumption (glucose kit); B: Exogenous glucose upregulates GLUT1 mRNA expression (qPCR); C-G: Exogenous glucose enhances the mRNA expression of essential glycolytic enzymes (qPCR); H, I: Exogenous glucose enhances the mRNA expression of lactate transport genes (qPCR); J-O: Exogenous glucose enhances protein expression of glycolytic enzymes (Western blotting). *P<0.05, **P<0.01. n＝3, x±s. GLUT1: Glucose transporter 1; qPCR: Quantitative polymerase chain reaction; Con: Control; Hy: Hypoxic exposure; HG: Hypoxia+glucose (10 mmol/L); HGG: Hypoxia+high dose glucose (15 mmol/L); HK1/2: Hexokinase 1/2; PKM: Pyruvate kinase M; PGK1: Phosphoglycerate kinase 1; PDK1: Pyruvate dehydrogenase kinase 1; MCT1/4: Monocarboxylate transporter 1/4; ACO2: Aconitase 2.下载:
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2.2.2 补充葡萄糖上调缺氧C8-D1A细胞抗凋亡基因的表达
CCK-8检测结果显示，补充葡萄糖可增强缺氧C8-D1A细胞的存活率（P＜0.01，图 5A）；ATP活力检测亦证实，补充葡萄糖后缺氧C8-D1A细胞代谢活力增强（P＜0.05，图 5B）。为排除高浓度葡萄糖在缺氧条件下可能引发的氧化应激效应，采用DCFH-DA荧光探针法对C8-D1A细胞内活性氧水平进行了检测，结果显示，补充葡萄糖后缺氧C8-D1A细胞的数量增多、活性氧荧光密度降低（P＜0.05，图 5C~5E），表明研究中所观察到的高糖效应并非由氧化应激介导。凋亡相关分子检测结果显示，补充葡萄糖后缺氧C8-D1A细胞的Bcl-2/Bax的mRNA比值上调（P＜0.01，图 5F），凋亡起始信号分子caspase 8及凋亡执行分子cleaved caspase 3/caspase 3的蛋白比值降低（P＜0.05，图 5G~5I）。这些结果提示，缺氧条件下，补充葡萄糖能够抑制细胞内源性凋亡途径和阻断下游caspase级联反应，增强C8-D1A细胞的缺氧耐受性。
图 5 补充葡萄糖提高缺氧C8-D1A细胞活力、上调抗凋亡基因的表达Fig. 5 Exogenous glucose enhances viability and upregulates anti-apoptotic genes in C8-D1A cells under hypoxiaA: Exogenous glucose enhances C8-D1A cell viability (CCK-8); B: Exogenous glucose enhances ATP production (ATP kit); C-E: Exogenous glucose reduces reactive oxygen species generation (DCFH-DA method); F: Exogenous glucose upregulates Bcl-2/Bax mRNA (qPCR); G-I: Effect of exogenous glucose on cleaved caspase 3/caspase 3 and caspase 8 protein (Western blotting). *P<0.05, **P<0.01. n＝3, x±s. CCK-8: Cell counting kit 8; ATP: Adenosine triphosphate; DCFH-DA: 2', 7'-dichlorodihydrofluorescein diacetate; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2 associated X protein; qPCR: Quantitative polymerase chain reaction; Con: Control; Hy: Hypoxic exposure; HG: Hypoxia+glucose (10 mmol/L); HGG: Hypoxia+high dose glucose (15 mmol/L); caspase: Cysteine aspartic acid specific protease.下载:
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2.2.3 补充葡萄糖诱导缺氧C8-D1A细胞向A2表型极化并增加BDNF分泌
由图 6可见，补充葡萄糖对缺氧C8-D1A细胞A1型星形胶质细胞标志物H2-T23的表达无明显影响（P＞0.05），而A2型星形胶质细胞标志物S100A10和CD109的mRNA表达水平及S100A10蛋白表达水平上调（P＜0.01），表明缺氧条件下补充葡萄糖可促进星形胶质细胞向A2表型极化。有趣的是，补充葡萄糖后缺氧C8-D1A细胞的乳酸生成量降低（P＜0.01），这与LDHA mRNA表达水平下调一致，考虑可能是细胞通过负反馈调节机制避免因糖酵解过度激活导致的乳酸蓄积和细胞内酸中毒。尽管补充葡萄糖后缺氧C8-D1A细胞乳酸生成减少，但神经保护因子BDNF的mRNA表达水平和蛋白分泌量均增加，并且BDNF的关键上游调控因子CREB磷酸化水平增高，提示葡萄糖可能通过激活CREB-BDNF信号通路发挥神经保护作用。
图 6 补充葡萄糖导致缺氧C8-D1A细胞向A2表型极化并增加BDNF分泌Fig. 6 Exogenous glucose induces A2 polarization and enhances BDNF secretion in C8-D1A cells under hypoxiaA: The effect of exogenous glucose supplementation on A1-type astrocyte gene mRNA expression (qPCR); B, C: Exogenous glucose enhances A2-type astrocyte gene mRNA expression (qPCR); D: The effect of exogenous glucose on lactate production (lactate kit); E: Effect of glucose supplementation on LDHA mRNA expression (qPCR); F: Exogenous glucose enhances BDNF mRNA expression (qPCR); G: Exogenous glucose enhances BDNF secretion (ELISA kit); H-J: Exogenous glucose upregulates p-CREB/CREB and S100A10 (Western blotting). *P<0.05, **P<0.01. n＝3, x±s. qPCR: Quantitative polymerase chain reaction; LDHA: Lactate dehydrogenase A; BDNF: Brain-derived neurotrophic factor; ELISA: Enzyme-linked immunosorbent assay; p-: Phosphorylated; CREB: cAMP response element-binding protein; S100A10: S100 calcium binding protein A10; H2-T23: Histocompatibility 2, T region locus 23; Con: Control; Hy: Hypoxic exposure; HG: Hypoxia+glucose (10 mmol/L); HGG: Hypoxia+high dose glucose (15 mmol/L).下载:
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3 讨论
稳定且持续的血氧血糖供给是脑功能正常运转的必要条件。急性缺氧暴露可导致认知功能下降，以注意力分散、反应速度减慢、记忆编码和检索效率降低为主要表现[15]，其发生和发展伴随能量供应不足、葡萄糖代谢紊乱甚至继发性脑损伤等不良后果[16]。星形胶质细胞作为中枢神经系统中含量最丰富、分布最广泛的细胞，在维持神经元能量稳态中发挥核心作用。因此，本研究观察了急性缺氧对星形胶质细胞功能的影响，并探讨了补充葡萄糖在调控细胞代谢重编程与表型极化中的作用及潜在机制。研究结果发现，缺氧条件下细胞葡萄糖消耗量与乳酸生成量随缺氧时间延长呈时间依赖性上升，表现为更高的糖酵解水平、糖酵解容量及糖酵解储备，而线粒体功能相关参数包括基础OCR、备用呼吸容量、ATP关联OCR、最大OCR及ATP产量均明显降低，提示在缺氧环境中C8-D1A细胞能量代谢更倾向于依赖糖酵解途径。在分子机制层面，缺氧24 h可显著上调C8-D1A细胞GLUT1、糖酵解关键酶（HK1、HK2、PGK1、PKM）以及乳酸代谢相关酶（LDHA、MCT1、MCT4）在mRNA和蛋白水平的表达。以上结果表明，急性缺氧可诱导C8-D1A细胞发生显著的代谢重编程，表现为糖酵解活性增强而氧化代谢减弱，该现象与既往研究中缺氧调控糖代谢的机制[17]一致。随着缺氧时间（3、6、12、24 h）的延长，C8-D1A细胞的存活发生改变。C8-D1A细胞在缺氧6 h即出现显著的增殖抑制，而促/抗凋亡平衡因子（Bcl-2/Bax）比值在12 h降低；生存信号通路的抑制（p-Akt/Akt比值降低）迟至24 h，这也为后续在24 h这一关键节点进行葡萄糖干预提供了实验依据。
补充葡萄糖在缺氧下对C8-D1A细胞展现出多维度保护效应。在细胞存活层面，葡萄糖提升了细胞能量状态和增殖活力（ATP水平），这种能量支持从上游延缓了细胞凋亡线粒体途径启动（Bcl-2/Bax）并阻断下游caspase级联反应。在功能层面，葡萄糖还促进C8-D1A细胞向神经保护性A2表型极化并激活CREB-BDNF信号通路，增加BDNF分泌，这与A2型星形胶质细胞通过代谢支持促进神经修复的理论相吻合[18-19]。在代谢层面，葡萄糖展现出代谢整合能力：通过上调ACO2蛋白表达促进三羧酸循环运行，可能提供更多还原当量与代谢中间物；进一步上调糖酵解通路关键分子（GLUT1、HK1/2、PGK1、PKM、PDK1、MCT1/4）的表达，增强糖酵解通量。我们推测增加的糖酵解可能使中间产物如葡萄糖-6-磷酸更多流入磷酸戊糖途径，从而满足细胞的抗氧化需求——这一假设在缺氧C8-D1A细胞经葡萄糖处理后活性氧水平降低的结果中得到印证。有趣的是，本研究观察到外源性葡萄糖反而引起乳酸水平下降。结合课题组前期发现——5 mmol/L乳酸可缓解而20 mmol/L乳酸加剧神经炎症[8]，我们推测葡萄糖可能通过优化乳酸代谢，避免因糖酵解过度激活导致乳酸蓄积和炎症反应，进而发挥保护作用。这说明补充葡萄糖的保护效应并非单一靶点，是细胞应对缺氧应激、重塑生存平衡的综合体现。
本研究证明，缺氧导致星形胶质细胞C8-D1A发生增殖抑制、凋亡增加及代谢重编程等功能紊乱，补充外源性葡萄糖可改善上述异常，其神经保护作用可能通过代谢支持（整合与增强细胞代谢能力）、凋亡抑制（减轻线粒体途径凋亡与阻断caspase级联反应）以及表型重塑（激活A2型神经保护表型）实现。这提示星形胶质细胞在缺氧条件下对葡萄糖的利用可能超越了传统的能量供给角色，还存在尚未被充分认识的细胞保护机制，为靶向星形胶质细胞代谢以改善缺氧相关神经损伤提供了新的实验依据和潜在干预策略。
图 1 缺氧导致C8-D1A细胞损伤
Fig. 1 Hypoxia induces C8-D1A cell injury
A: Hypoxia-induced viability reduction of C8-D1A cells (CCK-8); B-D: Hypoxia decreases p-Akt/Akt and Bcl-2/Bax protein (Western blotting); E: Hypoxia decreases Bcl-2/Bax mRNA (qPCR). *P<0.05, **P<0.01. n＝3, x±s. CCK-8: Cell counting kit 8; p-: Phosphorylated; Akt: Protein kinase B; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2 associated X protein; qPCR: Quantitative polymerase chain reaction.
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图 2 缺氧条件下C8-D1A细胞的能量代谢由氧化磷酸化向糖酵解转换
Fig. 2 C8-D1A energy metabolism shifts from oxidative phosphorylation to glycolysis under hypoxia
These are the results of the Seahorse XF glycolysis stress test and mitochondrial stress test. A: Dynamic changes of OCR; B-E: Quantification of basal OCR, ATP-linked OCR, maximal OCR, and spare respiratory capacity OCR, respectively; F: Dynamic changes of ECAR; G-I: Quantification of glycolytic, glycolytic capacity and glycolytic reserve ECAR, respectively. **P<0.01. n＝3, x±s. OCR: Oxygen consumption rate; ECAR: Extra cellular acidification rate; ATP: Adenosine triphosphate; Con: Control; Hy: Hypoxic exposure; FCCP: Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; Rot: Rotenone; AA: Antimycin A; 2-DG: 2-deoxy-D-glucose.
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图 3 缺氧通过增强葡萄糖摄取及糖代谢关键酶基因的表达维持C8-D1A细胞的能量稳态
Fig. 3 Hypoxia sustains cellular energetics through enhancing glucose uptake and glycolytic enzyme expression in C8-D1A cells
A: Hypoxia enhances glucose consumption (glucose kit); B: Hypoxia enhances lactate production (lactate kit); C: Hypoxia upregulates HIF-1α mRNA expression (qPCR); D: Hypoxia upregulates GLUT1 mRNA expression (qPCR); E-H: Hypoxia enhances the mRNA expression of essential glycolytic enzymes (qPCR); I, J: Hypoxia enhances the mRNA expression of lactate production and transport genes (qPCR); K-Q: Hypoxia enhances protein expression of glycolytic enzymes (Western blotting). *P<0.05, **P<0.01. n＝3, x±s. HIF-1α: Hypoxia-inducible factor 1α; qPCR: Quantitative polymerase chain reaction; GLUT1: Glucose transporter 1; HK1/2: Hexokinase 1/2; PKM: Pyruvate kinase M; PGK1: Phosphoglycerate kinase 1; PDK1: Pyruvate dehydrogenase kinase 1; MCT4: Monocarboxylate transporter 4; LDHA: Lactate dehydrogenase A.
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图 4 补充葡萄糖促进缺氧C8-D1A细胞糖代谢、上调糖代谢相关酶基因的表达
Fig. 4 Exogenous glucose supplementation enhances glucose metabolism and upregulates glycolytic metabolism-related genes in C8-D1A cells under hypoxia
A: Exogenous glucose enhances glucose consumption (glucose kit); B: Exogenous glucose upregulates GLUT1 mRNA expression (qPCR); C-G: Exogenous glucose enhances the mRNA expression of essential glycolytic enzymes (qPCR); H, I: Exogenous glucose enhances the mRNA expression of lactate transport genes (qPCR); J-O: Exogenous glucose enhances protein expression of glycolytic enzymes (Western blotting). *P<0.05, **P<0.01. n＝3, x±s. GLUT1: Glucose transporter 1; qPCR: Quantitative polymerase chain reaction; Con: Control; Hy: Hypoxic exposure; HG: Hypoxia+glucose (10 mmol/L); HGG: Hypoxia+high dose glucose (15 mmol/L); HK1/2: Hexokinase 1/2; PKM: Pyruvate kinase M; PGK1: Phosphoglycerate kinase 1; PDK1: Pyruvate dehydrogenase kinase 1; MCT1/4: Monocarboxylate transporter 1/4; ACO2: Aconitase 2.
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图 5 补充葡萄糖提高缺氧C8-D1A细胞活力、上调抗凋亡基因的表达
Fig. 5 Exogenous glucose enhances viability and upregulates anti-apoptotic genes in C8-D1A cells under hypoxia
A: Exogenous glucose enhances C8-D1A cell viability (CCK-8); B: Exogenous glucose enhances ATP production (ATP kit); C-E: Exogenous glucose reduces reactive oxygen species generation (DCFH-DA method); F: Exogenous glucose upregulates Bcl-2/Bax mRNA (qPCR); G-I: Effect of exogenous glucose on cleaved caspase 3/caspase 3 and caspase 8 protein (Western blotting). *P<0.05, **P<0.01. n＝3, x±s. CCK-8: Cell counting kit 8; ATP: Adenosine triphosphate; DCFH-DA: 2', 7'-dichlorodihydrofluorescein diacetate; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2 associated X protein; qPCR: Quantitative polymerase chain reaction; Con: Control; Hy: Hypoxic exposure; HG: Hypoxia+glucose (10 mmol/L); HGG: Hypoxia+high dose glucose (15 mmol/L); caspase: Cysteine aspartic acid specific protease.
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图 6 补充葡萄糖导致缺氧C8-D1A细胞向A2表型极化并增加BDNF分泌
Fig. 6 Exogenous glucose induces A2 polarization and enhances BDNF secretion in C8-D1A cells under hypoxia
A: The effect of exogenous glucose supplementation on A1-type astrocyte gene mRNA expression (qPCR); B, C: Exogenous glucose enhances A2-type astrocyte gene mRNA expression (qPCR); D: The effect of exogenous glucose on lactate production (lactate kit); E: Effect of glucose supplementation on LDHA mRNA expression (qPCR); F: Exogenous glucose enhances BDNF mRNA expression (qPCR); G: Exogenous glucose enhances BDNF secretion (ELISA kit); H-J: Exogenous glucose upregulates p-CREB/CREB and S100A10 (Western blotting). *P<0.05, **P<0.01. n＝3, x±s. qPCR: Quantitative polymerase chain reaction; LDHA: Lactate dehydrogenase A; BDNF: Brain-derived neurotrophic factor; ELISA: Enzyme-linked immunosorbent assay; p-: Phosphorylated; CREB: cAMP response element-binding protein; S100A10: S100 calcium binding protein A10; H2-T23: Histocompatibility 2, T region locus 23; Con: Control; Hy: Hypoxic exposure; HG: Hypoxia+glucose (10 mmol/L); HGG: Hypoxia+high dose glucose (15 mmol/L).
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表 1 qPCR引物序列
Table 1 Sequences of the primers for qPCR
Gene Primer-forward (5'-3') Primer-reverse (5'-3') β-actin GTGGGAATGGGTCAGAAGGA CTTCTCCATGTCGTCCCAGT Bcl-2 AGCCCAATGCCCTCCAGAGC TCTCAAGCCTTCACGCAAGTTCAG Bax TTGCCCTCTTCTACTTTGCTAG CCATGATGGTTCTGATCAGCTC GLUT1 GACACTTGCCTTCTTTGCCA AAGAGACAGGAATGGGCGAA MCT1 TGGCTGTCATGTATGCTGGA TTGAAAGCAAGCCCAAGACC MCT4 CCCTGGCTGCTATCATCTGT GCTGTAGAAAGGCCCAATCG S100A10 GTGCTCATGGAACGGGAGT AAAGCTCTGGAAGCCCACTT CD109 CATTGAGCAACATTGCCTGAGA GTCACCTACTACACTCCTTTGT H2-T23 TCAGAGTAACGACGAATCTCAC GTTCAGGGAGATGTAATCCTGG BDNF GAAGAGCTGCTGGATGAGGA GTTTGCGGCATCCAGGTAAT HIF-1α CCAGCAGACCCAGTTACAGA TGAGTGCCACTGTATGCTGA HK1 CTGGGTGAGATCGTCCGTAA CACGTGCTGTTTAGACCCAG HK2 CTCTCTCTCAACCCTGGCAA GGCAGTCACTCTCGATCTGA PKM CTTCATTCAGACCCAGCAGC CCGAGCCACATTCATTCCAG PGK1 GACTTTGGACAAGCTGGACG CCAGGTGGCTCATAAGGACA PDK1 CCGATTCAGGTTCACGTCAC CTCCCTGGAAGTACTGTGCA LDHA AGCTGCTGATCGTCTCCAAT GCAACATTCACACCACTCCA qPCR: Quantitative polymerase chain reaction; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2 associated X protein; GLUT1: Glucose transporter 1; MCT1/4: Monocarboxylate transporter 1/4; S100A10: S100 calcium binding protein A10; H2-T23: Histocompatibility 2, T region locus 23; BDNF: Brain-derived neurotrophic factor; HIF-1α: Hypoxia-inducible factor 1α; HK1/2: Hexokinase 1/2; PKM: Pyruvate kinase M; PGK1: Phosphoglycerate kinase 1; PDK1: Pyruvate dehydrogenase kinase 1; LDHA: Lactate dehydrogenase A. [1] 范杏飞,赵云鹏,王多祥,等.缺血性脑卒中后小鼠脑组织中星形胶质细胞的异质性分析[J].海军军医大学学报,2023,44(7):778-784. DOI: 10.16781/j.CN31-2187/R.20230192. [2] BORBOR M, YIN D, BROCKMEIER U, et al. Neurotoxicity of ischemic astrocytes involves STAT3-mediated metabolic switching and depends on glycogen usage[J]. Glia, 2023, 71(6): 1553-1569. DOI: 10.1002/glia.24357. [3] GOYAL M S, HAWRYLYCZ M, MILLER J A, et al. Aerobic glycolysis in the human brain is associated with development and neotenous gene expression[J]. Cell Metab, 2014, 19(1): 49-57. DOI: 10.1016/j.cmet.2013.11.020. [4] XIONG X Y, TANG Y, YANG Q W. Metabolic changes favor the activity and heterogeneity of reactive astrocytes[J]. Trends Endocrinol Metab, 2022, 33(6): 390-400. DOI: 10.1016/j.tem.2022.03.001. [5] BEARD E, LENGACHER S, DIAS S, et al. Astrocytes as key regulators of brain energy metabolism: new therapeutic perspectives[J]. Front Physiol, 2021, 12: 825816. DOI: 10.3389/fphys.2021.825816. [6] LINNERBAUER M, WHEELER M A, QUINTANA F J. Astrocyte crosstalk in CNS inflammation[J]. Neuron, 2020, 108(4): 608-622. DOI: 10.1016/j.neuron.2020.08.012. [7] CICCONE C, DÖTTERER S E, VOLD JENSEN S, et al. Potential for flexible lactate shuttling between astrocytes and neurons to mitigate against diving-induced hypoxia[J]. Front Neuroanat, 2025, 19: 1607396. DOI: 10.3389/fnana.2025.1607396. [8] FEI X, CHEN L, GAO J, et al. p53 lysine-lactylated modification contributes to lipopolysaccharide-induced proinflammatory activation in BV2 cell under hypoxic conditions[J]. Neurochem Int, 2024, 178: 105794. DOI: 10.1016/j.neuint.2024.105794. [9] ZHANG K, ZHOU Y, ZHAO T, et al. Reduced cerebral oxygen content in the DG and SVZ in situ promotes neurogenesis in the adult rat brain in vivo[J]. PLoS One, 2015, 10(10): e0140035. DOI: 10.1371/journal.pone.0140035. [10] CHRISTIE I N. Astrocytes: orchestrators of brain gas exchange and oxygen homeostasis[J]. J Physiol, 2026, 604(4): 1682-1694. DOI: 10.1113/JP288934. [11] RAZA M U, CHHABRA K H. Osmotic minipump implantation for increasing glucose concentration in mouse cerebrospinal fluid[J]. J Vis Exp, 2023(194): 10.3791/65169. DOI: 10.3791/65169. [12] VISKAITIS P, TESMER A L, LIU Z, et al. Orexin neurons track temporal features of blood glucose in behaving mice[J]. Nat Neurosci, 2024, 27(7): 1299-1308. DOI: 10.1038/s41593-024-01648-w. [13] 《持续葡萄糖监测临床应用专家共识》专家组. 持续葡萄糖监测临床应用专家共识2024[J]. 国际内分泌代谢杂志, 2024, 44(6): 430-445. DOI: 10.3760/cma.j.cn121383-20240911-00124. [14] American Diabetes Association Professional Practice Committee. 2. Diagnosis and classification of diabetes: standards of care in diabetes-2025[J]. Diabetes Care, 2025, 48(1 Suppl 1): S27-S49. DOI: 10.2337/dc25-S002. [15] 米娜, 成祥, 朱玲玲. 高原环境下认知能力评估技术与军事应用展望[J]. 军事医学, 2024, 48(12): 926-930. DOI: 10.7644/j.issn.1674-9960.2024.12.007. [16] SHI D, CHEN J, LI M, et al. Closing the loop: autonomous intelligent control for hypoxia pre-acclimatization and high-altitude health management[J]. Natl Sci Rev, 2025, 12(5): nwaf071. DOI: 10.1093/nsr/nwaf071. [17] WANG Q, WANG P, QIN Z, et al. Altered glucose metabolism and cell function in keloid fibroblasts under hypoxia[J]. Redox Biol, 2021, 38: 101815. DOI: 10.1016/j.redox.2020.101815. [18] LU W, WEN J. Metabolic reprogramming and astrocytes polarization following ischemic stroke[J]. Free Radic Biol Med, 2025, 228: 197-206. DOI: 10.1016/j.freeradbiomed.2025.01.002. [19] GUO H, FAN Z, WANG S, et al. Astrocytic A1/A2 paradigm participates in glycogen mobilization mediated neuroprotection on reperfusion injury after ischemic stroke[J]. J Neuroinflammation, 2021, 18(1): 230. DOI: 10.1186/s12974-021-02284-y.
