乳酸介导的SOD2乳酸化修饰在小鼠脑缺血-再灌注损伤中的作用

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周昕祎, 齐雪, 李亚男, 王伟, 赵博, 宋文沁. 乳酸介导的SOD2乳酸化修饰在小鼠脑缺血-再灌注损伤中的作用[J]. 中华急诊医学杂志, 2025, 34(4): 562-566. 复制到剪切板

Zhou Xinyi, Qi Xue, Li Yanan, Wang Wei, Zhao Bo, Song Wenqin. The role of lactate-mediated SOD2 lactylation in cerebral ischemia-reperfusion injury in mice[J]. Chin J Emerg Med, 2025, 34(4): 562-566. 复制到剪切板

乳酸介导的SOD2乳酸化修饰在小鼠脑缺血-再灌注损伤中的作用

周昕祎¹ , 齐雪² , 李亚男² , 王伟² , 赵博² , 宋文沁²

1. 华中科技大学同济医学院附属梨园医院麻醉科，武汉 430077;
2. 武汉大学人民医院麻醉科，武汉 430060

收稿日期: 2024-12-17

基金项目: 国家自然科学基金项目(82401400)；湖北省自然科学基金项目(2024AFB780)

通信作者: 宋文沁，Email：wenqin-1987@163.com.

摘要: 目的探讨乳酸介导的超氧化物歧化酶2(superoxide dismutase 2，SOD2)乳酸化修饰在小鼠脑缺血-再灌注损伤中的作用。方法清洁级雄性C57BL/6小鼠采用随机数字表法分为4组：假手术组(Sham组)、大脑中动脉闭塞/再灌注(middle cerebral artery occlusion/reperfusion, MCAO/R)组(MCAO/R组)、大脑中动脉闭塞/再灌注+2-脱氧-D-葡萄糖(2-Deoxy-D-glucose, 2-DG)组(MCAO/R+2-DG组)、大脑中动脉闭塞/再灌注+乳酸钠组(MCAO/R+Nala组)。MCAO/R组采用线栓法制备脑缺血-再灌注损伤模型；MCAO/R+2-DG组于缺血前90 min腹腔注射250 mg/kg糖酵解抑制剂2-DG；MCAO/R+Nala组于缺血前24 h脑室注射2 μL 100 mmol/L的Nala。乳酸检测试剂盒测定乳酸含量，苏木精-伊红染色(hematoxylin & eosin staining, HE)观察细胞形态，末端脱氧核糖核酸转移酶dUTP断裂末端标记(terminal deoxynucleotidyl transferase dutp nick end labeling, TUNEL)检测细胞凋亡，免疫荧光检测活性氧簇(reactive oxygen species, ROS)，Western blot检测SOD2、超氧化物歧化酶2第114位赖氨酸乳酸化(superoxide dismutase 2 lysine 114 lactylation, SOD2-K114la)、铁调节蛋白2(iron regulatory protein 2, IRP2)、转铁蛋白受体1(transferrin receptor protein 1，TFR1)，对上述指标采用单因素方差进行分析比较。结果与Sham组相比，MCAO/R组中乳酸、SOD2-K114la、TUNEL阳性率、ROS、IRP2、TFR1增加[乳酸：(0.608±0.064) vs. (0.376±0.030)，P < 0.005；SOD2-K114la：(2.311±0.146) vs. (1.009±0.073)，P < 0.0005；TUNEL阳性率：(35.420±2.832) vs. (0.294±0.147)，P < 0.0001；ROS：(3.415±0.229) vs. (1.166±0.155)，P < 0.0001；IRP2：(1.735±0.125) vs. (1.000±0.000)，P < 0.0001；TFR1：(1.611±0.058) vs. (1.000±0.000)，P < 0.0001]，SOD2减少[(0.545±0.062) vs. (1.082±0.088)，P < 0.0001]，HE提示脑损伤；与MCAO/R组相比，MCAO/R+2-DG组中乳酸、SOD2-K114la、TUNEL阳性率、ROS、IRP2、TFR1减少[乳酸：(0.453±0.047) vs. (0.608±0.064)，P < 0.05；SOD2-K114la：(1.764±0.188) vs. (2.311±0.146)，P < 0.05；TUNEL阳性率：(23.800±3.168) vs. (35.420±2.832)，P < 0.005；ROS：(2.640±0.213) vs. (3.415±0.229)，P < 0.005；IRP2：(1.463±0.055) vs. (1.735±0.125)，P < 0.05；TFR1：(1.252±0.081) vs. (1.611±0.058)，P < 0.005]，SOD2增加[(0.727±0.026) vs. (0.545±0.062)，P < 0.05]，HE提示损伤减轻；与MCAO/R组相比，MCAO/R+Nala组中乳酸、SOD2-K114la、TUNEL阳性率、ROS、IRP2、TFR1增加[乳酸：(1.021±0.051) vs. (0.608±0.064)，P < 0.0001；SOD2-K114la：(3.479±0.275) vs. (2.311±0.146)，P < 0.0005；TUNEL阳性率：(53.430±3.551) vs. (35.420±2.832)，P < 0.0001；ROS：(4.687±0.253) vs. (3.415±0.229)，P < 0.0005；IRP2：(2.463±0.117) vs. (1.735±0.125)，P < 0.0001；TFR1：(2.209±0.094) vs. (1.611±0.058)，P < 0.0001]，SOD2减少[(0.286±0.040) vs. (0.545±0.062)，P < 0.005]，HE提示损伤加重。结论脑缺血-再灌注损伤(Cerebral Ischemia Reperfusion Injury, CIRI)后乳酸增多进而SOD2乳酸化修饰增强损伤加重，抑制乳酸生成可减轻脑损伤，其机制与调控铁代谢有关。

关键词: 乳酸 SOD2 乳酸化修饰脑缺血-再灌注损伤糖酵解铁代谢 IRP2 TFR1 缺血-再灌注

The role of lactate-mediated SOD2 lactylation in cerebral ischemia-reperfusion injury in mice

Zhou Xinyi¹ , Qi Xue² , Li Yanan² , Wang Wei² , Zhao Bo² , Song Wenqin²

1. Department of Anesthesiology, Liyuan hospital, Tongji Medical College, Huazhong University of Science and technology, Wuhan 430077, China;
2. Department of Anesthesiology, Renmin Hospital of Wuhan University, Wuhan 430060, China

Fund program: National Natural Science Foundation of China (82401400); National Natural Science Foundation of Hubei (2024AFB780)

Corresponding author: Song Wenqin, Email: wenqin-1987@163.com.

Abstract: Objective To explore the role of lactate in Superoxide dismutase 2 (SOD2) lactylation in cerebral ischemia-reperfusion injury in mice. Methods Male C57BL/6 mice were randomLy (random number) divided into 4 groups: sham operation group (Sham group), Middle Cerebral Artery Occlusion/Reperfusion group (MCAO/R group), Middle Cerebral Artery Occlusion/Reperfusion+2-Deoxy-D-glucose group (MCAO/R+2-DG group), Middle Cerebral Artery Occlusion/Reperfusion+sodium lactate group (MCAO/R+Nala group). Cerebral ischemia reperfusion injury model was established in the mice of MCAO/R group using the thread occlusion. In the MCAO/R+2-DG group, mice received an intraperitoneal injection of 250 mg/kg of 2-DG 90 min before ischemia. Mice in the MCAO/R+ Nala group was given an intraventricular injection of 2 μL of 100 mmol/L Nala 24 h before ischemia. Commercial kits was used to detect lactate levels, Hematoxylin & Eosin Staining (HE) was employed to observe cell morphology, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed to assess cell apoptosis, and immunofluorescence was utilized to detect reactive oxygen species (ROS). Western blot was conducted to measure SOD2, Superoxide Dismutase 2 Lysine 114 Lactylation(SOD2-K114la), Iron regulatory protein 2(IRP2) and transferrin receptor protein 1(TFR1) levels. The above indicators were analyzed and compared by one-way variance. Results Compared with the Sham group, the MCAO/R group showed increased levels of lactate, SOD2-K114la, TUNEL positive rate, ROS, IRP2 and TFR1[lactate: (0.608±0.064) vs. (0.376±0.030), P < 0.005; SOD2-K114la: (2.311±0.146) vs. (1.009±0.073), P < 0.0005; TUNEL positive rate: (35.420±2.832) vs. (0.294±0.147), P < 0.0001; ROS: (3.415±0.229) vs. (1.166±0.155), P < 0.0001; IRP2: (1.735±0.125) vs. (1.000±0.000), P < 0.0001; TFR1: (1.611±0.058) vs. (1.000±0.000), P < 0.0001], while SOD2 decreased[(0.545±0.062) vs. (1.082±0.088), P < 0.0001]. HE staining indicated brain damage. Compared with the MCAO/R group, the MCAO/R+2-DG group showed reduced levels of lactate, SOD2-K114la, TUNEL positive rate, ROS, IRP2, and TFR1[lactate: (0.453±0.047) vs. (0.608±0.064), P < 0.05; SOD2-K114la: (1.764±0.188) vs. (2.311±0.146), P < 0.05; TUNEL positive rate: (23.800±3.168) vs. (35.420±2.832), P < 0.005; ROS: (2.640±0.213) vs. (3.415±0.229), P < 0.005; IRP2: (1.463±0.055) vs. (1.735±0.125), P < 0.05; TFR1: (1.252±0.081) vs. (1.611±0.058), P < 0.005], with higher level of SOD2 [(0.727±0.026) vs. (0.545±0.062), P < 0.05]. Meanwhile, HE staining indicated reduced damage. Compared with the MCAO/R group, the MCAO/R+Nala group showed increased levels of lactate, SOD2-K114la, TUNEL positive rate, ROS, IRP2 and TFR1[lactate: (1.021±0.051) vs. (0.608±0.064), P < 0.0001; SOD2-K114la: (3.479±0.275) vs. (2.311±0.146), P < 0.0005; TUNEL positive rate: (53.430±3.551) vs. (35.420±2.832), P < 0.0001; ROS: (4.687±0.253) vs. (3.415±0.229), P < 0.0001; IRP2: (2.463±0.117) vs. (1.735±0.125), P < 0.0001; TFR1: (2.209±0.094) vs. (1.611±0.058), P < 0.0001], with decreased levels of SOD2 [(0.286±0.040) vs. (0.545±0.062), P < 0.0001]. And HE staining revealed worsened braindamage. Conclusions Increased lactate levels can enhance the lactylation of SOD2, exacerbating brain damage after Cerebral ischemia reperfusion injury(CIRI). Inhibiting lactate production may alleviate brain injury by regulating iron Metabolism.

Key words: Lactate SOD2 Lactylation Cerebral ischemia-reperfusion injury Glycolysis Iro Metabolism IRP2 TFR1 Ischemia-reperfusion

研究发现缺血期间机体氧供不足可迫使细胞进行无氧代谢生成乳酸以提供维持机体基本生存所需的能量^[1]，但在恢复灌注后氧化磷酸化酶的活性仍被抑制而糖酵解依旧处于活跃状态致使乳酸持续升高^[2]。正常情况下适量乳酸能增强神经元的兴奋性和可塑性并参与长期记忆的形成，但在病理状态下过量乳酸可酸化细胞微环境，诱导炎症因子浸润、促使氧自由基堆积并激活通透性转换通道的开放进而导致损伤进一步加重^[3-4]。近期发现乳酸不仅是细胞能量代谢的产物，其还可通过乳酸化修饰影响信号传导和蛋白表达^[5]。然而乳酸在CIRI中是否能通过乳酸化修饰发挥效应？其具体调控机制又是如何?本研究旨在探讨乳酸化修饰在脑缺血-再灌注损伤中的作用及其可能机制。

1 材料与方法 1.1 材料与模型

清洁级雄性C57BL/6小鼠36只(8~10周龄)，采用随机数字表法分为4组：假手术组(Sham组)、大脑中动脉闭塞/再灌注组(MCAO/R组)、大脑中动脉闭塞/再灌注+2-脱氧-D-葡萄糖组(MCAO/R+2-DG组)、大脑中动脉闭塞/再灌注+乳酸钠组(MCAO/R+Nala组)。MCAO/R组采用线栓法缺血1 h再灌注24 h制备小鼠大脑中动脉栓塞模型，MCAO/R+2-DG组于造模前90 min腹腔注射250 mg/kg 2-DG(Sigma, USA)^[6]；MCAO/R+Nala组造模前24 h行侧脑室定位注射100 mmol/L Nala(Sigma, USA) 2 μL^[6]。

线栓法制备MCAO/R模型：小鼠麻醉后仰卧位固定，消毒颈部皮肤延正中线切开。显微镜下暴露左颈总动脉并在近心端结扎，分离结扎颈外动脉，动脉夹夹闭颈总动脉远端，在靠近结扎处剪一小口插入线栓，轻推线栓过颈总动脉分叉处直至稍感阻力随即打结固定。1 h后取出线栓进行再灌注。

1.2 检查方法

乳酸试剂盒测定乳酸含量：将脑组织冰水浴下匀浆，2 500 r/min离心后取上清液为待测样本。严格按说明书配置工作液，利用酶标仪测量并计算乳酸浓度(南京建成)。

HE染色观察细胞形态：再灌注24 h处死小鼠，多聚甲醛固定后取出脑组织，脱水包埋，切片脱脂，苏木精染液浸泡，蒸馏水冲洗，伊红染液复染，显微镜下观察并拍照。

TUNEL检测细胞凋亡：4%多聚甲醛固定脑组织，石蜡包埋切片，透化，dUTP荧光染色，荧光显微镜下观察采集图像。

免疫荧光检测ROS：再灌注24 h处死小鼠，快速取脑置于液氮，制备冰冻切片，DCFH-DA孵育，DAPI染核，荧光倒置显微镜下观察并拍照。

Western blot检测：蛋白上样后电泳仪恒压80 V至溴酚蓝通过上层胶后恒压120 V继续电泳45 min，电转仪恒流200 mA使蛋白转至PVDF膜，封闭后摇床一抗孵育过夜[SOD2 (1:10 000, 武汉三鹰)、SOD2-K114la(1:500, HuaBio)、IRP2(1:1 000, 武汉三鹰)、TRF1(1:1 000, ABclonal)]，含Tween-20的Tris缓冲溶液洗涤，孵育辣根过氧化物酶标记的山羊抗兔二抗，Tris缓冲溶液洗涤后ECL显影液显影。

1.3 统计学方法

应用GraphPad Prism 8.0软件进行统计分析，计量资料以均数±标准差(x±s)表示，设置检验水准α=0.05，组间比较采用单因素方差分析，两两比较采用Turkey检验。以P < 0.05为差异有统计学意义。

2 结果 2.1 小鼠大脑皮质神经元形态、细胞凋亡及ROS的比较

与Sham组相比，MCAO/R组小鼠大脑皮质HE染色可见细胞排列疏松紊乱、数量减少、胞核固缩、形态异常、间质可见明显水肿、空泡化改变，TUNEL染色可见大量凋亡细胞同时ROS荧光强度增强；与MCAO/R组相比，MCAO/R+2-DG组细胞损伤减轻，凋亡减少，ROS荧光强度减弱；与MCAO/R组相比，MCAO/R+Nala组细胞损伤加重，凋亡增多，ROS荧光强度增强。见表 1。

表 1 小鼠TUNEL阳性细胞及ROS荧光强度的比较(x±s, n=3) Table 1 Comparison of TUNEL-positive cells and ROS fluorescence intensity in mice (x±s, n = 3)

组别	TUNEL positive rate (%)	ROS (fold to Sham)
Sham组	0.294±0.147	1.166±0.155
MCAO/R组	35.420±2.832^a	3.415±0.229^a
MCAO/R+2-DG组	23.800±3.168^ab	2.640±0.213^ab
MCAO/R+Nala组	53.430±3.551^ab	4.687±0.253^ab
F值	193.8	140.0
P值	＜0.0001	＜0.0001
注：与Sham组相比，^aP＜0.05；与MCAO/R组相比，^bP＜0.05

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2.2 各组小鼠乳酸、SOD2及其乳酸化修饰的表达变化

与Sham组相比，MCAO/R组小鼠乳酸、SOD2-K114la升高，SOD2降低(P < 0.05)；与MCAO/R组相比，MCAO/R+2-DG组乳酸、SOD2-K114la降低，SOD2升高(P < 0.05)；与MCAO/R组相比，MCAO/R+Nala组乳酸、SOD2-K114la升高，SOD2降低(P < 0.05)。见表 2。

表 2 各组小鼠乳酸、SOD2及其乳酸化水平的表达变化(x±s, n=3) Table 2 Expression changes of lactic acid, SOD2 and SOD2-K114la levels in each goup of mice (x±s, n=3)

组别	乳酸(mmol/gprot)	SOD2	SOD2-K114la
Sham组	0.376±0.030	1.082±0.088	1.009±0.073
MCAO/R组	0.608±0.064^a	0.545±0.062^a	2.311±0.146^a
MCAO/R+2-DG组	0.453±0.047^ab	0.727±0.026^ab	1.764±0.188^ab
MCAO/R+Nala组	1.021±0.051^ab	0.286±0.040^ab	3.479±0.275^ab
F值	101.1	97.3	94.4
P值	＜0.0001	＜0.0001	＜0.0001
注：与Sham组相比，^aP＜0.05；与MCAO/R组相比，^bP＜0.05

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2.3 各组小鼠铁代谢蛋白的表达变化

与Sham组相比，MCAO/R组IRP2、TFR1增多(P < 0.05)；与MCAO/R组相比，MCAO/R+2-DG组IRP2、TFR1减少(P < 0.05)；与MCAO/R组相比，MCAO/R+Nala组IRP2、TFR1增多(P < 0.05)。见表 3。

表 3 各组小鼠铁代谢相关蛋白的表达变化(x±s, n=3) Table 3 Expression changes of iron metabolism-related proteins in each group of mice (x±s, n = 3)

组别	IRP2	TFR1
Sham组	1.000±0.000	1.000±0.000
MCAO/R组	1.735±0.125^a	1.611±0.058^a
MCAO/R+2-DG组	1.463±0.055^ab	1.252±0.081^ab
MCAO/R+Nala组	2.463±0.117^ab	2.209±0.094^ab
F值	138.3	159.4
P值	＜0.0001	＜0.0001
注：与Sham组相比，^aP＜0.05；与MCAO/R组相比，^bP＜0.05

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3 讨论

缺血性脑卒中作为全球第二大致死、致残性疾病，严重威胁了我国居民的身体健康^[7]。早期恢复脑血流是目前治疗缺血性脑卒中的主要方法^[8]，但在恢复血供后常伴随着乳酸等大量酸性代谢产物的释放、进而引起氧化应激增强，血脑屏障破坏等一系列病理现象^[3]，导致神经损伤不但未能减轻反而会加速诱导细胞结构破坏甚至死亡即脑缺血-再灌注损伤。

既往研究认为乳酸作为能量载体可自由穿透血脑屏障，从血液进入脑组织以确保其在脑内的输送和利用^[9]。但近期研究却发现缺血期间脑血流中断，神经元无法获得足够的氧气和营养物质，从而迫使其能量代谢转变为无氧糖酵解以维持基本生存所需的能量。在这一过程中细胞内乳酸浓度持续升高且再灌注后乳酸大量释放又能进一步酸化细胞内环境，促使pH值下降、导致线粒体通透性转换孔(mPTP)持续开放，加剧细胞损害^[10-11]。以上论述表明乳酸在机体内可能发挥了截然不同的作用。

SOD2作为机体内最为重要的抗氧化酶，其可通过催化超氧自由基转化为氧气和过氧化氢以保护细胞免受损伤进而在多种疾病模型中发挥抗氧化作用^[12-13]。本研究结果证实再灌注后SOD2降低，ROS荧光强度增加，同时细胞凋亡增多，抑制乳酸生成后SOD2升高，ROS荧光强度降低，细胞凋亡减少。提示CIRI期间SOD2降低可促使ROS增多导致细胞损伤。那么SOD2又是如何减少的呢？其是否和乳酸有关？课题组前期通过乳酸化组学联合生信分析发现再灌注损伤与细胞能量代谢和氧化应激密切相关，并筛选出SOD2-K114为其乳酸化修饰位点。进一步发现再灌注后MCAO/R组小鼠乳酸、SOD2-K114la升高，脑组织损伤加重，当给予2-DG抑制乳酸生成后乳酸、SOD2-K114la降低，细胞损伤减轻。证实抑制乳酸生成可降低SOD2-K114的乳酸化水平减轻神经损伤。

既往研究显示SOD2降低可促进蛋白复合物中铁离子的释放，催化羟自由基形成进而造成细胞损害^[14-15]，同时另有报道证实SOD2升高能降低心脏、肝脏、肾脏和骨骼肌等组织中活性铁的水平^[16]。同时游离铁和羟自由基的过度积累又会加剧氧化应激，诱导铁死亡的发生^[17-18]。本研究证实CIRI后SOD2降低，同时铁代谢蛋白IRP2、TFR1增多，抑制乳酸生成后铁代谢相关蛋白下调，脑损伤减轻。表明SOD2的抗氧化效应可能与抑制机体铁代谢有关。

在上述研究中笔者还发现外源性给予乳酸可诱导损伤进一步加重，提示乳酸在CIRI中能加重神经损害，虽然有研究认为乳酸可为机体提供能量以保证机体的存活，但在临床工作中乳酸却是反应机体微循环是否良好、血液氧供与组织氧需是否达到平衡的重要指标，异常升高的乳酸是患者不良预后的先兆。本研究结果提示早期对乳酸进行干预可能有利于重症患者的恢复。

综上，本研究表明CIRI后乳酸增多进而SOD2乳酸化修饰增强导致损伤加重，抑制乳酸生成可减轻脑损伤，其机制可能与调控铁代谢有关。

利益冲突 所有作者声明无利益冲突

作者贡献声明 周昕祎：实验操作、文章撰写；齐雪、李亚男：数据统计分析、作图、数据整理；王伟、赵博：研究指导；宋文沁：实验设计、论文审阅

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