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Journal of Central South University Medical Sciences logoLink to Journal of Central South University Medical Sciences
. 2025 Oct 28;50(10):1800–1810. [Article in Chinese] doi: 10.11817/j.issn.1672-7347.2025.250460

硝化应激通过糖酵解途径促进乳腺癌的恶性生物学行为

Nitrosative stress promotes the malignant biological behavior of breast cancer through the glycolytic pathway

WANG Yalu 1,1, GONG Mingyan 1, ZHU Yunhao 1, XU Yuxiang 1, HU Jianming 1,2,✉, LUO Chenghua 1,2,✉
Editor: 彭 敏宁
PMCID: PMC12949866  PMID: 41656811

Abstract

Objective

Breast cancer is one of the most common malignant tumors in women, and some patients face poor therapeutic efficacy and recurrence. Tumor cells are extremely dependent on the energy produced by the glycolytic pathway, which not only provides adenosine triphosphate (ATP) for rapid tumor cell proliferation, but also creates favorable conditions for tumor cell growth by regulating the acidity of the tumor microenvironment. Nitrosative stress refers to a pathophysiological state characterized by intracellular accumulation of reactive nitrogen species (RNS). Studies have found that nitrosative stress is closely associated with the occurrence and progression of cancer. This study aims to investigate the effects of nitrosative stress and tumor cell glycolysis, to provide new ideas for breast cancer treatment.

Methods

Postoperative tumor tissues from breast cancer patients at the First Affiliated Hospital of Shihezi University were collected. Immunohistochemistry (IHC) staining was used to analyze the expression levels of inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (3-NT) in breast cancer tissues and adjacent normal breast tissues. Fluorescent probes were used to detect nitric oxide (NO) and reactive oxygen species (ROS) levels in normal breast cells (MCF-10A) and breast cancer cells (MDA-MB-231 and MCF-7). Western blotting was used to compare the expression levels of iNOS and 3-NT between normal and breast cancer cells. The optimal concentrations of the peroxynitrite (ONOO⁻) donor (SIN-1) and the scavenger (FeTMPyP) were determined using cell counting kit-8 (CCK-8). Glycolysis levels were measured using a glycolysis assay kit. CCK-8, Transwell, and wound-healing assays were further used to detect the proliferation, migration, and invasion abilities of MDA-MB-231 and MCF-7 cells under the effects of the two drugs. Western blotting was used to detect the expression levels of E-cadherin, N-cadherin, and vimentin, as well as glycolysis-related proteins hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), and enolase 1 (ENO1).

Results

The expression levels of iNOS and 3-NT in breast cancer tissues were significantly higher than those in normal breast tissues (P<0.001), NO (P<0.01), ROS (P<0.001), iNOS (P<0.05), and 3-NT (P<0.01) levels in breast cancer cell lines (MDA-MB-231 and MCF-7) were all significantly higher than those in normal breast cells (MCF-10A). Breast cancer cells exhibited higher glycolysis levels (P<0.001). Treatment with the nitrosative-stress down-regulating drug FeTMPyP significantly inhibited glycolysis levels (P<0.001), and the expression of glycolytic enzymes HK2, PKM2, and ENO1 was significantly decreased (all P<0.05). The proliferation, migration, and invasion abilities of the two breast cancer cell lines were weakened, and the expression of EMT-related protein E-cadherin was increased, while the expressions of N-cadherin and vimentin were significantly decreased (all P<0.05). After treatment with the nitrosative-stress up-regulating drug SIN-1, glycolysis levels were significantly enhanced (P<0.001), and the expression of glycolytic enzymes HK2, PKM2, and ENO1 was significantly increased (all P<0.05). The proliferation, migration, and invasion abilities of the 2 breast cancer cell lines were enhanced, and the expression of EMT-related protein E-cadherin was reduced, while the expressions of N-cadherin and vimentin were significantly increased (all P<0.05).

Conclusion

Nitrosative stress can promote the malignant biological behavior of breast cancer cells through the glycolytic pathway.

Keywords: breast cancer, reactive oxygen species, NO, nitrosative stress, glycolysis, malignant biological behavior

乳腺癌是女性最常见的恶性肿瘤之一[1]，主要亚型包括三阴性乳腺癌、人类表皮生长因子受体2(human epidermal growth factor receptor 2，HER2)阳性乳腺癌、雌激素受体(estrogen receptor，ER)+/HER2-型乳腺癌[2]。尽管目前已有内分泌治疗、放化疗、免疫治疗、手术治疗等多种治疗手段[3]，但部分患者仍面临疗效不佳和复发的问题。因此，深入探究乳腺癌进展的机制，并探寻乳腺癌新的治疗靶点至关重要。

诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)是产生一氧化氮(nitric oxide，NO)的关键酶[4]，NO与活性氧(reactive oxygen species，ROS)反应可以产生大量的过氧亚硝基离子(peroxynitrite，ONOO-)[5]，ONOO-作用于蛋白质肽链中的酪氨酸残基，使其形成3-硝基酪氨酸(3-nitrotyrosines，3-NT)而发生硝基化修饰(nitration modification)，从而影响疾病的发展[6]。ONOO-引起的硝化应激作为一种重要的氮代谢紊乱在人体心血管系统疾病[7]，如心力衰竭、心肌缺血、主动脉瘤、动脉粥样硬化中发挥重要作用，影响机体的病理生理过程[8-9]。不仅如此，硝化应激还可以通过干扰细胞中的信号转导[10]、线粒体能量代谢、信使核糖核酸转录[11]、蛋白质翻译后修饰和离子通道功能等方式影响疾病的发生、发展[12]。已有研究[13]发现ONOO-与乳腺癌密切相关，然而，其在乳腺癌进展中的作用机制尚不明确。

有氧糖酵解作为癌症进展的中心因素[14-15]，以葡萄糖摄取和乳酸产生速率增高为主要特征[16]，其过程为葡萄糖经糖酵解转化为丙酮酸，并最终生成乳酸[17-18]。研究[19-20]表明，糖酵解过程产生的三磷酸腺苷(adenosine triphosphate，ATP)足以促进癌症的生长，并赋予其选择性生长优势。

本研究旨在通过荧光探针检测细胞内NO和活性氧水平，通过蛋白质印迹法检测iNOS及细胞硝化应激水平；通过药物干预调控细胞硝化应激；通过细胞计数试剂盒-8(cell counting kit-8，CCK-8)实验、划痕试验、Transwell实验、蛋白质印迹实验检测硝化应激对乳腺癌恶性生物学行为的影响；并通过糖酵解相关试剂盒分析乳腺癌细胞糖酵解代谢的变化，以探讨硝化应激通过介导糖酵解促进乳腺癌细胞恶性生物学行为的作用机制，为乳腺癌的治疗提供新策略。

1. 材料与方法

1.1. 伦理声明

本研究获得石河子大学第一附属医院医学伦理委员会批准(批准号：KJ2023-433-01)，且所有患者均签署知情同意书。

1.2. 细胞系和细胞培养

乳腺癌细胞系(三阴型MDA-MB-231、ER+型MCF-7)购自武汉赛维尔生物科技有限公司，MCF-10A细胞购自上海葵赛生物细胞库，均已通过短串联重复序列检测(short tandem repeats，STR)认证，无支原体污染，MDA-MB-231和MCF-7细胞培养于含有10%胎牛血清(fetal bovine serum，FBS)的GibicoTM杜尔贝科改良伊格尔培养基(GibicoTM Dulbecco’s modified Eagle medium，Gibico-DMEM)中，MCF-10A培养于上海葵赛生物细胞库所产的专用培养基，均置于37 ℃、5% CO2的湿润环境中培养。

1.3. 主要试剂

SIN-1购自美国Sigma公司；青、链霉素(双抗)和高糖型DMEM均购自美国Gibico公司；FBS购自中国VivaCell公司；3-NT一抗购自英国Abcam公司；iNOS一抗、E-cadherin一抗、N-cadherin一抗、Vimentin一抗、己糖激酶2(hexokinase 2，HK2)一抗、丙酮酸激酶M2型(pyruvate kinase M2，PKM2)一抗、烯醇化酶1(enolase 1，ENO1)一抗均购自武汉三鹰生物技术有限公司；FeTMPyP和NO探针购自美国MCE公司；ROS荧光检测探针、葡萄糖检测试剂盒、ATP检测试剂盒均购自上海碧云天生物技术有限公司；乳酸检测试剂盒购自南京建成生物有限公司；CCK-8试剂购自中国Biosharp生物有限公司。

1.4. 方法

1.4.1. 获取乳腺癌标本

2023年12月至2024年6月收集石河子大学第一附属医院乳腺癌患者的术后肿瘤组织，获得患者正常乳腺组织和乳腺癌组织共计6对，进行包埋获取病理切片。

1.4.2. 蛋白质印迹法

在正常乳腺细胞MCF-10A和乳腺癌细胞MDA-MB-231，MCF-7中加入200 μL蛋白质裂解液(蛋白质裂解液꞉蛋白酶抑制剂为1꞉100)，采用细胞刮板法收集蛋白质裂解液，以12 000 r/min离心30 min并收集上清。加入50 μL上样缓冲液，99 ℃煮10 min，获得蛋白质印迹样品。将10 μL蛋白质印迹样品通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE)分离，转印至聚偏氟乙烯(polyvinylidene fluoride，PVDF)膜，室温封闭2 h。于4 ℃过夜孵育相应一抗14~16 h，室温孵育种属一致的二抗2 h。使用ECL发光试剂进行曝光。使用ImageJ分析灰度值，并进行归一化处理。

1.4.3. 糖酵解相关指标测定

将正常乳腺细胞分为对照组、SIN-1药物处理组、SIN-1+FeTMPyP药物处理组，乳腺癌细胞分为对照组、FeTMPyP药物处理组、FeTMPyP+SIN-1药物处理组。葡萄糖、乳酸测定需收集药物处理后的各组细胞进行计数，并取各组细胞上清至EP管中，12 000 r/min离心5 min收集上清液，使用葡萄糖/乳酸检测试剂盒对不同组别样品进行处理，使用全纳米波长酶标仪检测OD值以确定葡萄糖(630 nm)和乳酸(530 nm)的含量，所测数值均与各组细胞数量进行归一化处理。ATP测定需将药物处理后的不同组别的6孔板细胞进行计数，使用ATP检测试剂盒提取ATP后，用化学发光仪测定化学荧光信号强度，所测数值均与各组细胞数量进行归一化处理。

1.4.4. CCK-8法检测细胞增殖水平及细胞活力

将上述各组细胞按每孔3 000个细胞铺于96孔板中，待细胞6 h贴壁后，给不同组别细胞更换药物培养基，待药物作用48 h后，每孔加入10 μL CCK-8试剂，置于37 ℃培养箱中，培养2 h后，使用酶标仪(450 nm)检测OD值。细胞增殖活性=(实验组-空白组)/(对照组-空白组)×100%。空白组(即纯培养基组)用于调零。

1.4.5. 细胞划痕试验检测细胞迁移能力

将乳腺癌细胞MDA-MB-231和MCF-7铺于6孔板中，待每孔细胞密度长至70%~80%，更换药物培养基并进行划痕，拍摄药物作用0、24、48 h的细胞划痕。使用ImageJ对各组细胞迁移情况进行测量，计算24 h迁移能力=(0 h划痕宽度-24 h划痕宽度)/0 h划痕宽度×100%；48 h迁移能力=(0 h划痕宽度-48 h划痕宽度)/0 h划痕宽度×100%。

1.4.6. Transwell试验检测细胞侵袭能力

对小室进行基底胶铺胶处理，水化，药物处理后的不同组别细胞进行消化计数，向上室中接种 200 μL无血清的细胞悬液，下室中加入600 μL含20%血清的培养基，置于37 ℃细胞培养箱中培养。36 h后将小室取出，用PBS清洗3遍后，用4%多聚甲醛溶液固定30 min，结晶紫染色20 min，用棉签轻轻擦拭小室内部细胞，干燥后，于倒置显微镜下拍照，并使用ImageJ软件进行侵袭细胞数量计数。上述实验均独立进行，重复3次。

1.4.7. 免疫组织化学染色

乳腺癌组织及癌旁正常乳腺组织切片放入60 ℃烘箱中烤30 min，经过二甲苯和乙醇脱水、枸橼酸高温修复、过氧化氢孵育10 min后，加入iNOS(稀释比1꞉1 600)、3-NT(稀释比1꞉100)一抗，放置在4 ℃冰箱中孵育过夜后，使用PBS清洗3遍，再加入兔源二抗孵育30 min，加入二氨基联苯胺(diaminobenzidine，DAB)显色5 min，苏木精染色 3 min、盐酸-乙醇分色5 s，自来水返蓝5 min后使用乙醇脱水，封片。染色强度评分：无着色为0分，弱阳性为1分，中等阳性为2分，强阳性为3分。阳性细胞比例评分：无阳性肿瘤细胞为0分，1%~25%为1分，26%~50%为2分，51%~75%为3分，76%~100%为4分。最终分值=染色强度评分×阳性细胞比例评分，其中<4分为阴性，≥4分为阳性。

1.5. 统计学处理

采用GraphPad prism 9.5.1软件分析所有实验结果。实验数据用均值±标准差表示。使用t检验对正常乳腺组织与乳腺癌组织之间iNOS和3-NT表达水平差异进行分析，使用单因素方差分析对正常乳腺细胞与乳腺癌细胞硝化应激水平、糖酵解水平表达差异，以及对2种乳腺癌细胞MDA-MB-231和MCF-7中药物处理后细胞恶性生物学行为及糖酵解水平变化进行分析。P<0.05为差异有统计学意义。

2. 结 果

2.1. 乳腺癌组织硝化应激水平显著高于正常组织

免疫组织化学染色发现，相较于正常乳腺组织，乳腺癌组织中iNOS的表达水平显著上调(P<0.001，图1A)。为进一步验证该结果，在细胞水平检测了人正常乳腺细胞MCF-10A和乳腺癌细胞MDA-MB-231、MCF-7中iNOS的表达情况。蛋白质印迹结果显示，2种乳腺癌细胞中iNOS表达均明显高于正常乳腺细胞MCF-10A(均P<0.05，图1B)。本研究采用特异性荧光探针DAF-FM DA及DHE探针，对3种细胞的NO及ROS水平进行测定，发现在乳腺癌细胞中NO及ROS的含量均明显高于正常乳腺细胞(均P<0.05，图1C)。通过检测3种细胞的硝化反应的特异性标志物3-NT水平，本研究发现乳腺癌细胞中的3-NT水平显著升高(P<0.01，图1D)，这一结果在组织水平得到进一步验证(P<0.001，图1E)。

图1.

图1

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乳腺癌组织硝化应激水平显著高于正常组织

Figure 1 Nitric oxide synthase and nitrative stress levels are significantly higher in breast cancer tissues than in normal tissues

A: Difference in the expression levels of iNOS in breast cancer tissues and adjacent normal tissues; B: Differences in iNOS expression levels between normal mammary cells and breast cancer cells; C: NO and ROS levels between normal mammary cells and breast cancer cells; D: Differences in 3-NT expression levels between normal mammary cells and breast cancer cells; E: Difference in the expression levels of 3-NT in breast cancer tissues and adjacent normal tissues. *P<0.05, **P<0.01, ***P<0.001. iNOS: Inducible nitric oxide synthase; NO: Nitric oxide; ROS: Reactive oxygen species; 3-NT: 3-nitrotyrosine; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase. IHC: Immunohistochemistry.

2.2. 硝化应激促进乳腺癌细胞增殖

使用过氧亚硝基离子清除剂FeTMPyP和供体SIN-1处理人正常乳腺细胞MCF-10A及乳腺癌细胞MDA-MB-231、MCF-7，通过CCK-8实验确定2种药物的最适药物浓度[MDA-MB-231细胞：SIN-1 (100 μmol/L)，FeTMPyP(10 μmol/L)。MCF-7细胞：SIN-1(200 μmol/L)，FeTMPyP(10 μmol/L)。MCF-10A细胞：SIN-1(200 μmol/L)，FeTMPyP(20 μmol/L)](均P<0.01，图2A~2B、2D~2E)，蛋白质印迹检测发现药物在最适浓度时刚好可以对正常乳腺细胞MCF-10A，乳腺癌细胞MDA-MB-231、MCF-7的硝化应激水平产生明显变化(均P<0.05，图2C、2F)。使用2种药物的最适浓度处理乳腺癌细胞MDA-MB-231和MCF-7，CCK-8实验显示，降低ONOO-可显著抑制2种乳腺癌细胞的增殖能力，而补充ONOO-则可部分恢复其增殖能力(均P<0.001，图2G~2H)。

图2.

图2

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硝化应激增强乳腺癌细胞的增殖能力

Figure 2 Nitrative stress enhances the proliferation ability of breast cancer cells

A and B: Normal breast cell MCF-10A cell viability levels under the effect of different concentrations of ONOO- providing agent SIN-1 and ONOO- scavenging agent FeTMPyP; C: Changes in the level of 3-NT in MCF-10A after administration of SIN-1 and FeTMPyP; D and E: Breast cancer cell viability levels in the presence of different concentrations of the ONOO- providing agent SIN-1 and the ONOO- scavenging agent FeTMPyP; F: Changes in the level of 3-NT in MDA-MB-231 and MCF-7 after administration of SIN-1 and FeTMPyP; G and H Changes in proliferation level of breast cancer cells after addition of SIN-1 and FeTMPyP. *P<0.05, **P<0.01, ***P<0.001. Drug concentration in MCF-10A: SIN-1 (200 μmol/L), FeTMPyP (20 μmol/L); Drug concentration in MDA-MB-231 cells: SIN-1 (100 μmol/L), FeTMPyP (10 μmol/L); Drug concentration in MCF-7 cells: SIN-1 (200 μmol/L), FeTMPyP (10 μmol/L). ONOO-: Peroxynitrite; SIN-1: 3-Morpholinosydnonimine hydrochloride.

2.3. 硝化应激促进乳腺癌细胞迁移、侵袭能力 及EMT进程

使用上述最适浓度的过氧亚硝基离子清除剂FeTMPyP和供体SIN-1处理乳腺癌细胞MDA-MB-231及MCF-7，发现降低ONOO-可显著抑制2种乳腺癌细胞的迁移、侵袭能力(均P<0.05，图3A~3D)。同样，降低ONOO-可显著抑制2种乳腺癌细胞的上皮-间充质转化(epithelial-mesenchymal transition，EMT)进程，即：当ONOO-减少时，E-cadherin蛋白表达上调，而N-cadherin和vimentin蛋白表达均下调(均P<0.05，图3E~3F)。

图3.

图3

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硝化应激促进乳腺癌细胞迁移、侵袭及EMT进程

Figure 3 Nitrative stress promotes the migration, invasion, and EMT process of breast cancer cells

A and B: Changes in migration level of breast cancers after addition of SIN-1 and FeTMPyP; C and D: Changes in invasion level of breast cancers after addition of SIN-1 and FeTMPyP; E and F: E-cadherin, N-cadherin, and vimentin protein expression levels of breast cancer cells after addition of SIN-1 and FeTMPyP. *P<0.05, **P<0.01, ***P<0.001. EMT: Epithelial-mesenchymal transition.

2.4. 硝化应激增强乳腺癌细胞的糖酵解能力

使用糖酵解检测试剂盒检测正常乳腺细胞MCF-10A和2种乳腺癌细胞MDA-MB-231、MCF-7中的糖酵解水平，发现2种乳腺癌细胞中的糖酵解能力明显高于正常乳腺细胞(均P<0.001，图4A)。同时，当硝化应激水平降低时，正常乳腺细胞MCF-10A和乳腺癌细胞MDA-MB-231、MCF-7的葡萄糖水平均显著上调，乳酸、ATP水平显著下调，而硝化应激水平升高可显著增强正常乳腺细胞和2种乳腺癌细胞的糖酵解活性(均P<0.001，图4B~4E)。为进一步明确硝化应激水平与乳腺癌糖酵解之间的关联，本研究检测了糖酵解途径的关键酶HK2、PKM2、ENO1在2种药物作用下的变化情况。结果表明，当硝化应激水平降低时，乳腺癌细胞MDA-MB-231、MCF-7的糖酵解相关激酶均明显降低(P<0.05，图4F~4G)。

图4.

图4

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硝化应激促进乳腺癌细胞的糖酵解进程

Figure 4 Nitrative stress promotes the glycolytic process in breast cancer cells

A: Differences in glycolysis levels between normal mammary cells and breast cancer cells; B: Changes in glycolysis levels of MCF-10A in breast cells after administration of SIN-1 and FeTMPyP; C-E: Changes in glycolysis levels of MDA-MB-231 and MCF-7 in breast cancer cells after administration of SIN-1 and FeTMPyP; F and G: HK2, PKM2, and ENO1 protein expression levels of breast cancer cells after addition of SIN-1 and FeTMPyP. *P<0.05, **P<0.01,***P<0.001. HK2: Hexokinase 2; PKM2: Pyruvate kinase M2; ENO1: Enolase 1.

3. 讨 论

恶性肿瘤细胞与正常细胞之间的代谢具有明显差异，代谢重编程是恶性肿瘤的重要特征之一，其中Warburg效应(即有氧糖酵解增强)尤为突出[20]；而肿瘤细胞对糖酵解途径产生的能量极其依赖[21-22]。基于这一认知，靶向肿瘤糖酵解已成为极具潜力的治疗策略[23]。本研究通过检测糖酵解相关指标如葡萄糖消耗、乳酸及ATP产生水平，发现乳腺癌细胞MDA-MB-231、MCF-7的糖酵解水平显著高于正常乳腺细胞MCF-10A，这一发现与既往研究[42]结果高度一致。

ONOO-的产生作为硝化应激的关键指标[25]，在细胞氧化还原稳态中起重要作用，过量ONOO-可导致蛋白质硝基化修饰，进而干扰细胞的正常生理功能[26]。值得注意的是，虽然已有研究[27]揭示ONOO-在心血管疾病和细胞凋亡调控中起重要作用，但其在肿瘤代谢重编程中的功能仍知之甚少。本研究发现，乳腺癌细胞MDA-MB-231、MCF-7中iNOS水平明显高于正常乳腺细胞，这与既往研究[28]一致，而NO与ROS结合产生的ONOO-水平明显高于正常乳腺细胞MCF-10A，即乳腺癌细胞的硝化应激水平高于正常乳腺细胞。同时，检测硝化应激对乳腺癌细胞恶性生物学行为的影响，发现降低乳腺癌细胞的硝化应激水平后，其恶性生物学行为也得到明显抑制，即硝化应激与乳腺癌发展呈正相关。进一步研究硝化应激对乳腺癌糖酵解能力的影响，发现降低乳腺癌细胞的硝化应激水平后，乳腺癌细胞的糖酵解水平受到抑制，提示硝化应激可能通过影响能量代谢通路促进乳腺癌细胞的发展。

目前对于肿瘤糖代谢的研究集中于DNA损伤、相关信号通路的激活、某些分子的直接或间接作用，而本研究发现硝化应激在乳腺癌中普遍升高，并将硝化应激与肿瘤糖代谢联系起来，对未来乳腺癌的早期诊断和药物治疗至关重要。目前对乳腺癌的诊断主要依赖影像学检查(如乳腺X线、超声、磁共振)及病理活检，或许可以通过检测组织中的iNOS、ONOO-的表达辅助影像学检查提高检出率，使其作为乳腺癌早期诊断的关键突破点。现如今对乳腺癌的治疗已进入个体化精准治疗时代，化疗仍是主要的治疗手段，但存在预后较差的问题，本研究证实硝化应激通过调控糖酵解途径促进乳腺癌恶性进展，这一机制为乳腺癌的靶向治疗提供了新的靶点。iNOS作为ONOO-生成的关键酶，其高表达是乳腺癌硝化应激激活的核心驱动因素，因此，靶向抑制iNOS活性可能成为乳腺癌治疗的有效策略。

本研究也存在局限性：仅采用了2种乳腺癌细胞系(MDA-MB-231、MCF-7)及1种正常乳腺细胞系(MCF-10A)进行体外实验，虽然这2种乳腺癌细胞系是乳腺癌研究中常用的经典模型，但无法完全模拟临床乳腺癌的异质性。而体外组织只收集到6对正常乳腺组织及乳腺癌组织，例数较少，不同患者之间存在个体差异，因此本研究结果直接进行临床转化价值有限。另外，本研究缺乏动物模型验证。体内肿瘤微环境复杂，涉及多种细胞的相互作用，而糖酵解环节复杂，有多种蛋白质分子及相关酶的参与[29]，如糖酵解的相关激酶HK2、PKM2、ENO1等，而硝化应激可以使蛋白质发生硝基化修饰，但本研究尚未能明确硝化应激作用的具体靶点及其下游效应网络，或许硝化应激可能引起糖酵解相关激酶发生硝基化修饰，或通过激活某些信号通路，如缺氧诱导因子α(hypoxia-inducible factor 1α，HIF-1α)、核因子κB(nuclear factor kappa B，NF-κB)等作用共同影响乳腺癌的恶性进展。此外，肿瘤代谢是一个复杂的调控网络，除糖酵解途径外还有脂肪酸合成、谷氨酰胺代谢等，本研究仅聚焦硝化应激与糖酵解的相互作用，未探讨硝化应激是否参与其他代谢途径，这将是未来研究的重要方向。后续可进一步通过体内实验、大量临床样本验证、分子靶点筛选等深入探讨乳腺癌的发生、发展机制，并推动研究成果向临床转化，为改善乳腺癌患者的疗效及预后提供新的策略。

基金资助

新疆生产建设兵团科技计划项目(2024DA041)；国家自然科学基金(82103290)；新疆生产建设兵团科技研究指导性项目(2023ZD029)；新疆维吾尔自治区人力资源和社会保障厅“天池英才”青年博士项目(CZ003103)；石河子大学第一附属医院临床与基础研究项目(LC2024003)；石河子大学国家大学生创新创业训练计划项目(202510759025)；石河子大学青年创新拔尖人才项目(CXBJ202207)；新疆生产建设兵团重点领域科技攻关项目(2023AB0S8)。

This work was supported by the Xinjiang Production and Construction Corps Science and Technology Plan Project (2024DA041), the National Natural Science Foundation (82103290), the Xinjiang Production and Construction Corps Science Research Guidance Project (2023ZD029), the Xinjiang Uygur Autonomous Region Human Resources and Social Security Department “Tianchi Talent” Program Project (CZ003103), the Clinical and Basic Research Program of the First Affiliated Hospital of Shihezi University (LC2024003), the Shihezi University National Project for College Students’ Innovation and Entrepreneurship Training (202510759025), the Shihezi University Young Innovative Talents Project (CXBJ202207), and the Xinjiang Production and Construction Corps Key Technology Research Project (2023ABOS8), China.

利益冲突声明

作者声称无任何利益冲突。

作者贡献

王雅璐 实验操作，数据整理，论文撰写；龚明妍、朱运豪、许宇翔 实验操作；胡建明、罗成华 论文指导与修改。所有作者阅读并同意最终的文本。

Footnotes

http://dx.chinadoi.cn/10.11817/j.issn.1672-7347.2025.250460

原文网址

http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/2025101800.pdf

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