没食子酸通过负向调控APOC3减轻铁死亡并改善创伤性脑损伤引起的神经功能缺损-动脉网

Gallic acid alleviates ferroptosis by negatively regulating APOC3 and improves nerve function deficit caused by traumatic brain injury

Nature 等信源发布 2025-03-06 19:00

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可切换为仅中文

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Abstract

摘要

Traumatic brain injury (TBI) is more common than ever and is becoming a global public health issue. A variety of secondary brain injuries occur after TBI, including ferroptosis characterized by iron-dependent lipid peroxidation. Gallic acid is a kind of traditional Chinese medicine, which has many biological effects such as anti-inflammatory and antioxidant.

创伤性脑损伤（TBI）比以往任何时候都更加普遍，正在成为一个全球性的公共卫生问题。TBI后会发生多种继发性脑损伤，其中包括以铁依赖性脂质过氧化为特征的铁死亡。没食子酸是一种中药，具有抗炎和抗氧化等多种生物学效应。

We further investigated whether Gallic acid can improve the neurological impairment caused by ferroptosis after TBI by targeting APOC3. Weighted gene coexpression network analyses (WGCNA) and 3 kinds of machine-learning algorithms were used to find the potential biomarkers. Then the HERB database was used to select the Chinese herb that acted on the target gene APOC3.

我们进一步研究了没食子酸是否可以通过靶向APOC3来改善TBI后由铁死亡引起的神经功能损伤。使用加权基因共表达网络分析（WGCNA）和三种机器学习算法来寻找潜在的生物标志物。然后利用HERB数据库筛选作用于目标基因APOC3的中草药。

Finally, we selected Gallic acid as a drug targeting APOC3 and verified by Western blotting. The effect of Gallic acid on the improvement of neurological function was studied by Nissl staining and FJB staining. Finally, the effect of Gallic acid on the cognitive ability of TBI mice was explored through behavioral experiments.

最后，我们选择了没食子酸作为靶向APOC3的药物，并通过Western blotting进行了验证。通过尼氏染色和FJB染色研究了没食子酸对神经功能改善的作用。最后，通过行为实验探讨了没食子酸对TBI小鼠认知能力的影响。

Gallic acid can inhibit the expression level of APOC3 and thus inhibit the level of ferroptosis after TBI. It can also reduce the degeneration of nerve tissue by inhibiting ferroptosis and improve the neurological function deficit. The behavioral experiment proved that Gallic acid can alleviate the behavioral cognitive impairment caused by TBI.

没食子酸可以抑制APOC3的表达水平，从而抑制TBI后铁死亡的水平。它还可以通过抑制铁死亡减少神经组织的退化，并改善神经功能缺损。行为学实验表明，没食子酸能够减轻TBI引起的行为认知障碍。

Gallic acid can reduce ferroptosis by inhibiting APOC3, and then alleviate neurological impairment after TBI. .

没食子酸可通过抑制APOC3减少铁死亡，进而减轻TBI后神经功能损伤。

Introduction

简介

TBI is a major global health problem and a leading cause of death and disability

创伤性脑损伤是全球主要的健康问题之一，也是导致死亡和残疾的主要原因。

. It occurs as a result of direct impact or impact to the head from factors such as motor vehicles, crush and assault

它是由直接撞击或因机动车、挤压和攻击等因素对头部的撞击造成的。

. Even non-fatal injuries can lead to severe lifelong disability, which has significant implications for the injured and their families, as well as for medical costs

即使是非致命伤也可能导致严重终身残疾，这对伤者及其家人以及医疗费用都有重大影响。

，

. The damage caused by TBI to brain tissue can be divided into primary damage and secondary damage. There are many forms of secondary brain injury, including brain tissue edema, blood brain barrier (BBB) destruction, reactive oxygen species (ROS) production and inflammation

创伤性脑损伤对脑组织的损害可以分为原发性损害和继发性损害。继发性脑损伤有多种形式，包括脑组织水肿、血脑屏障（BBB）破坏、活性氧（ROS）产生和炎症。

，

. Cell death can be divided into apoptosis, necrosis, autophagy and ferroptosis

细胞死亡可以分为凋亡、坏死、自噬和铁死亡。

，

. Active intervention to the secondary injury of TBI can improve the neurological impairment and improve the prognosis of patients

对TBI的继发性损伤进行积极干预，可以减轻神经功能损害，改善患者预后。

。

Ferroptosis is a new type of cell death discovered in recent years

铁死亡是近年来发现的一种新型细胞死亡方式

. It is a kind of oxidative cell death induced by small molecules, which is iron ion dependent

它是一种由小分子诱导的、铁离子依赖性的氧化性细胞死亡。

. Its occurrence is caused by the imbalance between the generation and degradation of intracellular lipid reactive oxygen species

其发生是由细胞内脂质活性氧的生成与降解之间的不平衡引起的。

. Ferroptosis inducers directly or indirectly act on glutathione peroxidase (GPXs) through different pathways, leading to the reduction of cellular antioxidant capacity, ROS accumulation, and finally oxidative cell death

铁死亡诱导剂通过不同途径直接或间接作用于谷胱甘肽过氧化物酶（GPXs），导致细胞抗氧化能力下降、活性氧（ROS）积累，最终引发氧化性细胞死亡。

，

. Ferroptosis is not only related to the occurrence and development of TBI, but the key proteins in the related signaling pathway can also become targets for drug action

铁死亡不仅与TBI的发生和发展有关，其相关信号通路中的关键蛋白也可成为药物作用的靶点。

，

。

In this study, we performed gene expression level analysis on the downloaded dataset to obtain differentially expressed genes between TBI patients and normal subjects. Combined the downloaded data with WGCNA and machine-learning algorithm including Support Vector Machines (SVM), least absolute shrinkage and selection operator (LASSO) regression, and Random Forest (RF).

在本研究中，我们对下载的数据集进行了基因表达水平分析，以获取TBI患者与正常受试者之间的差异表达基因。结合下载的数据与WGCNA及包括支持向量机（SVM）、最小绝对收缩和选择算子（LASSO）回归以及随机森林（RF）在内的机器学习算法进行了分析。

，

. Ultimately, we screened out the differentially expressed genes between normal brain tissue and TBI brain tissue for further study. Using the HERB database, a specialized Chinese medicine high-throughput experimental and reference database, the Chinese herb (fruit of Axillary choerospondias) that acts on the target gene was selected.

最终，我们筛选出正常脑组织与TBI脑组织之间的差异表达基因以进行进一步研究。利用专门的中药高通量实验和参考数据库HERB数据库，选取作用于靶基因的中药（腋果香树果实）。

. Fruit of Axillary choerospondias is the dried and mature fruit of Southern Jujube, a plant in the Urticaceae family

漆树科植物南酸枣的干燥成熟果实。

. Its main component contains Gallic acid, chemical name is 3, 4, 5-trihydroxybenzoic acid, molecular formula C

其主要成分含没食子酸，化学名为3,4,5-三羟基苯甲酸，分子式为C

, which is a polyphenolic organic compound

，这是一种多酚类有机化合物

. Gallic acid has anti-inflammatory, anti-mutation, anti-oxidation, anti-free radical and other biological activities, and these biological activities are closely related to the secondary damage after TBI, which is considered to be the potential mechanism of its treatment of TBI

没食子酸具有抗炎、抗突变、抗氧化、抗自由基等多种生物活性，这些生物活性与TBI后的继发性损伤密切相关，被认为是其治疗TBI的潜在机制。

. Previous studies have shown that Gallic acid can improve the neurological deficit after TBI through anti-inflammatory and antioxidant, but whether it can treat TBI by anti-ferroptosis has not been reported

以往的研究表明，没食子酸可以通过抗炎和抗氧化改善TBI后的神经功能缺损，但其是否能通过抑制铁死亡治疗TBI尚未见报道。

。

In order to clarify the effect of Gallic acid on ferroptosis in brain tissue caused by TBI, we first explored the effect of Gallic acid on the expression level of key protein of ferroptosis. Then, the characteristic changes of ferroptosis in mitochondria were observed under electron microscope, and iron deposition levels in tissues were detected by Perl’s staining to further verify the inhibitory effect of Gallic acid on ferroptosis.

为了进一步明确没食子酸对TBI引起的脑组织铁死亡的影响，我们首先探讨了没食子酸对铁死亡关键蛋白表达水平的影响；随后通过电镜观察线粒体铁死亡特征性改变，并用普鲁士蓝染色检测组织中铁沉积水平，以进一步验证没食子酸对铁死亡的抑制作用。

Finally, the protective effect of Gallic acid on neural function and behavioral cognition after TBI was confirmed through the neurodegeneration level and animal behavior experiments..

最后，通过神经变性水平和动物行为实验，证实了没食子酸对TBI后神经功能和行为认知的保护作用。

Materials and methods

材料与方法

Data collection and potential drugs screening

数据收集与潜在药物筛选

The gene expression profile (GSE2871) was obtained from the GEO database (

基因表达谱（GSE2871）从GEO数据库获得（

https://www.ncbi.nlm.nih.gov/geo/

), which was sequenced using the GPL85 platform. At early post-injury timepoint, animals will be sacrificed, brain regions (parietal cortex and hippocampus, ipsilateral and contralateral to injury) will be dissected and RNA isolated. RNA will be used to synthesize cRNA probes for microarray hybridization.

），该平台使用GPL85进行测序。在损伤后的早期时间点，将对动物实施安乐死，解剖脑区（顶叶皮层和海马体，损伤同侧和对侧），并提取RNA。RNA将用于合成cRNA探针以进行微阵列杂交。

We identified a number of potential drugs that may act on some of the TBI-associated genes identified in this study in the HERB database (.

我们在HERB数据库中识别出一些可能作用于本研究中发现的与TBI相关基因的潜在药物。

http://herb.ac.cn/

) and other public databases (

）以及其他公共数据库（

https://drugcentral.org/

）。

Identifification of DEGs and GSEA

DEGs和GSEA的鉴定

The “Limma” R package was used to screen differentially expressed genes (DEGs) between TBI and normal samples, and genes with

使用“Limma”R包筛选TBI和正常样本之间的差异表达基因（DEGs），并筛选出符合标准的基因，

< 0.05 and |log2FC| >1 were regarded as DEGs. GSEA-4.1.0 was used to input the expression data and phenotypic data, and the five most significantly up-regulated pathways and the most significantly down-regulated pathways were plotted, respectively.

<0.05 且 |log2FC| >1 被视为差异表达基因（DEGs）。使用 GSEA-4.1.0 输入表达数据和表型数据，并分别绘制五个最显著上调的通路和最显著下调的通路。

Screening of the critical genes

筛选关键基因

To find out the core genes that were altered after TBI, the downloaded dataset was used to construct a weighted gene co-expression network using the “WGCNA” R package. Then, we performed a cluster analysis of the samples and calculated the pearson correlation coefficient between each pair of genes to evaluate the expression similarity of genes and acquire a correlation matrix.

为了找出脑外伤后改变的核心基因，使用下载的数据集通过“WGCNA”R包构建了一个加权基因共表达网络。然后，我们对样本进行了聚类分析，并计算了每对基因之间的皮尔逊相关系数，以评估基因的表达相似性并获得相关矩阵。

We further used a soft threshold function to transform the correlation matrix into a weighted neighborhood matrix, and a soft join algorithm was used to select the optimal soft threshold to ensure that gene correlations fit the scale-free distribution to the greatest extent possible. Subsequently, the neighborhood matrix was transformed into a topological overlap matrix.

我们进一步使用软阈值函数将相关矩阵转换为加权邻域矩阵，并使用软连接算法选择最佳软阈值，以确保基因相关性尽可能符合无标度分布。随后，将邻域矩阵转换为拓扑重叠矩阵。

After obtaining the co-expression modules, the key modules were screened out by correlation analysis, and the genes of the key modules were regarded as the key genes. Based on WGCNA screening, DEGs and key genes were intersected to obtain the target genes..

获得共表达模块后，通过相关性分析筛选出关键模块，并将关键模块中的基因视为关键基因。基于WGCNA筛选，将差异表达基因（DEGs）与关键基因取交集，得到目标基因。

Identification of TBI hub genes based on machine-learning algorithms

基于机器学习算法的TBI关键基因识别

Least absolute shrinkage and selection operator (LASSO) is a regression analysis method that performs both gene selection and classification

最小绝对收缩和选择算子（LASSO）是一种回归分析方法，可同时进行基因选择和分类。

. First, the R package glmnet (Version4.1.2) was used to fit the logistic LASSO regression model. Next, the SVM-RFE algorithm was used to screen potential genes using the “e1071” R package. In addition, the random forest (RF) algorithm was also conducted to screen potential genes using the “randomForest” R package.

首先，使用R包glmnet（版本4.1.2）拟合逻辑LASSO回归模型。接下来，使用“e1071”R包通过SVM-RFE算法筛选潜在基因。此外，还利用“randomForest”R包进行了随机森林（RF）算法以筛选潜在基因。

Finally, the intersection of the genes obtained by LASSO, SVM-RFE and RF machine-learning algorithms was taken by Veen graph as the hub genes of TBI..

最后，通过Veen图取LASSO、SVM-RFE和RF机器学习算法获得的基因交集作为TBI的关键基因。

Experimental animals and grouping

实验动物与分组

The male ICR mice (6–8 weeks, 20–30 g) used in this experiment were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., LTD., and fed in the mouse house for 7 days for the experiment. The temperature of the mouse house is controlled at 22 ~ 25

本实验所用的雄性ICR小鼠（6-8周龄，20-30克）购自杭州梓源实验动物技术有限公司，并在鼠房中饲养7天以进行实验。鼠房温度控制在22至25℃之间。

◦

C, the humidity is 70% ~ 75%, and the water and food are adequately supplied. All animal experiments were conducted in accordance with the animal welfare policy of The Affiliated Huai’an Hospital of Xuzhou Medical University and were approved by The Affiliated Huai’an Hospital of Xuzhou Medical University.

C组的湿度为70%～75%，水和食物供应充足。所有动物实验均按照徐州医科大学附属淮安医院的动物福利政策进行，并经徐州医科大学附属淮安医院批准。

The experimental animals were randomly divided into Sham group, TBI group and TBI + Gal group (Gallic acid, 40 mg/kg, ip). In Sham group, the bone window was opened but not traumatized. The TBI group was traumatized but not treated. Gallic acid (MedChemExpress, USA) is administered once 30 min after TBI and then once daily.

实验动物被随机分为假手术组（Sham组）、TBI组和TBI+Gal组（没食子酸，40 mg/kg，腹腔注射）。在假手术组中，打开骨窗但不造成创伤。TBI组则施加创伤但不进行治疗。没食子酸（MedChemExpress，美国）在TBI后30分钟给药一次，随后每天一次。

The experimental section of this study fully complies with the ARRIVE guidelines..

本研究的实验部分完全符合ARRIVE指南。

The construction of TBI model

TBI模型的构建

The Feeney free-fall impact method was used to construct the TBI model

Feeney自由落体撞击法被用于构建TBI模型。

. First, the mice were anesthetized with isoflurane by an inhalation anesthesia machine (M5209, Changsha Maiyue Biotechnology Co., Ltd.) and fixed in a stereoscope, then the local hair was removed and disinfected with iodophor. Then the scalp was cut about 1.5 cm along the median line of the mouse skull, exposing the skull and stripping the periosteum.

首先，使用吸入式麻醉机（M5209，长沙迈跃生物科技有限公司）用异氟烷对小鼠进行麻醉，并固定在立体显微镜下，然后去除局部毛发并用碘伏消毒。随后沿小鼠颅骨中线切开头皮约1.5厘米，暴露颅骨并剥离骨膜。

Drilling was then done with a 2 mm diameter cranial rotatory to create a bone window of about 5 mm diameter, exposing the parietal lobe with as little damage to the dura as possible. Parameters for setting up the craniocerebral injury percussion apparatus are as follows: weight mass 20 g, drop height 15 cm, impact depth 1.5 mm.

然后用直径为 2 毫米的颅骨旋转钻头进行钻孔，以创建一个直径约为 5 毫米的骨窗，尽量减少对硬脑膜的损伤并暴露顶叶。设置颅脑损伤撞击装置的参数如下：重量质量 20 克、下降高度 15 厘米、冲击深度 1.5 毫米。

After the attack, the mice were hemostatic and the scalp was tightly sutured, and then returned to the original cage for feeding. All operations should pay attention to the principle of asepsis, and the instruments should be autoclaved and disinfected in advance during the operation..

攻击完成后，对小鼠进行止血，并将头皮紧密缝合，然后放回原来的笼子中饲养。所有操作均应注意无菌原则，术中所用器械应提前进行高温高压灭菌消毒。

Quantitative real-time PCR

定量实时PCR

Total RNA extraction and cDNA reverse transcription of mouse brain tissue were performed by the Vazyme (Vazyme Biotech, Nanjing, China) kit. Using GAPDH as internal parameter, 2

小鼠脑组织的总RNA提取和cDNA反转录使用了Vazyme（Vazyme Biotech，南京，中国）试剂盒。以GAPDH为内参，2

−ΔΔCt

method was used to calculate the expression levels. Primer sequences for target genes:

该方法用于计算表达水平。目标基因的引物序列：

STK39 (Forward)5’ - CAAACCCAGGCAAGAACGC − 3’ ;

STK39（正向）5’ - CAAACCCAGGCAAGAACGC − 3’ ;

STK39 (Reverse)5’ - GCCACAGCTCATCTTTGACCAC − 3’ ;

STK39（反向）5’ - GCCACAGCTCATCTTTGACCAC − 3’ ;

Kcnd3 (Forward)5’ - CACCAGTCGCTCCAGCCTTAA − 3’ ;

Kcnd3（正向）5’ - CACCAGTCGCTCCAGCCTTAA − 3’ ;

Kcnd3 (Reverse)5’ - GACGACATTGCTGGTTATGGAAG − 3’ ;

Kcnd3（反向）5’ - GACGACATTGCTGGTTATGGAAG − 3’ ;

Apoc3 (Forward)5’ - GAGTCCGATATAGCTGTGGTGG − 3’ ;

Apoc3（正向）5’ - GAGTCCGATATAGCTGTGGTGG − 3’ ;

Apoc3 (Reverse)5’ - GTTGGTTGGTCCTCAGGGTTAG − 3’ ;

Apoc3（反向）5’ - GTTGGTTGGTCCTCAGGGTTAG − 3’ ;

FOXE3 (Forward)5’ - CGACTGTTTCGTCAAGGTGC − 3’ ;

FOXE3（正向）5’ - CGACTGTTTCGTCAAGGTGC − 3’ ;

FOXE3 (Reverse)5’ - CGTTGTCGAACATGTCAGCG − 3’ ;

FOXE3（反向）5’ - CGTTGTCGAACATGTCAGCG − 3’ ;

CHRNB1 (Forward)5’ - CCGTTATCCTTAGTGTTGTGGTC − 3’ ;

CHRNB1（正向）5’ - CCGTTATCCTTAGTGTTGTGGTC − 3’ ;

CHRNB1 (Reverse)5’ - AGTGATGTGGTTCAGGGAGTTG − 3’ ;

CHRNB1（反向）5’ - AGTGATGTGGTTCAGGGAGTTG − 3’ ;

NPW (Forward)5’ - CTGCTAGAGCCTTCGGAGAGAC − 3’ ;

NPW（正向）5’ - CTGCTAGAGCCTTCGGAGAGAC − 3’ ;

NPW (Reverse)5’ - ATCGGTTCTTGGGCCTGACA − 3’ ;

NPW（反向）5’ - ATCGGTTCTTGGGCCTGACA − 3’ ;

GAPDH (Forward)5’ - CCTCGTCCCGTAGACAAAATG − 3’ ;

GAPDH（正向）5’ - CCTCGTCCCGTAGACAAAATG − 3’ ;

GAPDH (Reverse)5’ - TGAGGTCAATGAAGGGGTCGT − 3’ .

GAPDH（反向）5’ - TGAGGTCAATGAAGGGGTCGT − 3’。

Western blotting

蛋白质印迹法

The tissue blocks were washed with pre-cooled PBS for 2–3 times to remove the blood stain, cut into small pieces and placed in a homogenizing tube. 2 homogenizing beads of 4 mm were added, and the lysate of 10 times the tissue volume was added (protease inhibitor was added before use), and homogenizing procedure was set for homogenizing.

组织块用预冷的PBS洗涤2-3次以去除血渍，切成小块并放入匀浆管中。加入2颗4毫米的匀浆珠，并加入10倍于组织体积的裂解液（使用前添加蛋白酶抑制剂），设置匀浆程序进行匀浆。

Take out the homogenated tube, place the ice lysate for 30 min, and shake every 5 min to ensure complete tissue cracking; The supernatant was collected by centrifugation at 12,000 rpm at 4℃ for 10 min. Protein concentration was quantified using the protein quantification kit (Servicebio, China). Add a certain amount of loading buffer and boil at 95℃ for 10 min to complete sample preparation.

取出匀浆管，将冰裂解液放置30分钟，并每隔5分钟摇匀以确保组织完全裂解；在4℃下以12,000 rpm离心10分钟收集上清液。使用蛋白质定量试剂盒（Servicebio，中国）对蛋白浓度进行定量。加入一定量的上样缓冲液并在95℃煮沸10分钟，完成样品制备。

Then the sample is applied and electrophoresis is performed successively until the sample reaches the lower edge of the gel. Then the gel was transferred and closed for 1 ~ 2 h. After closure, the primary antibody was incubated overnight (4℃). The next day, the secondary antibody was washed three times with PBS buffer for 5 min each time, and then added to the incubator and incubated at room temperature for 30 min; And then clean it again with PBS buffer for three times, 10 min each time.

然后加入样品并连续进行电泳，直至样品到达凝胶的下边缘。之后转移凝胶并封闭1~2小时。封闭结束后，加入一抗并在4℃下孵育过夜。第二天，用PBS缓冲液洗涤三次二抗，每次5分钟，然后放入孵育箱中在室温下孵育30分钟；接着再用PBS缓冲液清洗三次，每次10分钟。

Luminescent solution (Servicebio, China) was configured and protein band detection and gray scale analysis were performed by imaging system (CLINX, China). The main reagents include primary antibody TfR1 (1:1000, Proteintech, USA), NOX2 (1:1000, Proteintech, USA) and GPX4 (1:1000, Proteintech, USA), secondary antibody Polyclonal Goat Anti-Mouse IgG labeled by HRP (1:2000, Servicebio, China), HRP labeled goat anti-Rabbit secondary antibody (1:2000, Servicebio, China)..

发光液（Servicebio，中国）配置，使用成像系统（CLINX，中国）进行蛋白条带检测及灰度分析。主要试剂包括一抗TfR1（1:1000，Proteintech，美国）、NOX2（1:1000，Proteintech，美国）和GPX4（1:1000，Proteintech，美国），二抗为HRP标记的多克隆山羊抗小鼠IgG（1:2000，Servicebio，中国）、HRP标记的山羊抗兔二抗（1:2000，Servicebio，中国）。

Transmission electron microscopy

透射电子显微镜

2 mm brain tissue around the site of brain injury and normal brain tissue at the same site were cut into 1mm

在脑损伤部位周围2毫米的脑组织和同一部位的正常脑组织被切成1毫米。

and immersed in electron microscope fixative (G1102-100ML, Servicebio, China). The tissue blocks were fixed at room temperature for 2 h away from light, and then stored in a refrigerator at 4℃. After the tissue blocks were removed, the surface fixing solution was washed with PBS (PH = 7.4) for 3 times, and the washing time was 15 min each time.

并浸入电子显微镜固定液（G1102-100ML，Servicebio，中国）。将组织块在室温下避光固定 2 小时，然后存放在 4℃ 的冰箱中。取出组织块后，用 PBS（PH = 7.4）洗涤表面固定液 3 次，每次洗涤时间为 15 分钟。

The rinsed tissue blocks were fixed with 0.1 mol/L PBS (PH = 7.4) prepared with 1% osmic acid and then placed in a dark room at room temperature for 2 h. After that, the tissue blocks were rinsed with PBS (PH = 7.4) for 3 times, and each rinsing time was 15 min. Finally, different concentrations of ethanol and 100% acetone were dehydrated.

冲洗后的组织块用含1%锇酸配制的0.1 mol/L PBS（PH = 7.4）固定，然后置于室温暗室中2小时。之后，用PBS（PH = 7.4）冲洗组织块3次，每次冲洗时间为15分钟。最后，使用不同浓度的乙醇和100%丙酮进行脱水。

After completing the above operations, the tissue was sliced with a thickness of 60–80 nm. The tissue sections were double-stained with uranium lead and then left to dry overnight at room temperature. Finally, the sections were analyzed under transmission electron microscopy..

完成上述操作后，将组织切成60-80纳米的薄片。组织切片用铀铅进行双重染色，然后在室温下干燥过夜。最后，在透射电子显微镜下对切片进行分析。

Immunohistochemistry

免疫组织化学

We first dewaxed the tissue sections according to the previous experimental procedures

我们首先按照之前的实验步骤对组织切片进行脱蜡处理

. After rinsing with PBS for three times, it was incubated in hydrogen peroxide solution in the dark, and then rinsed with PBS solution for three times. The tissue was uniformly covered with 3% BSA in the tissue chemical circle and closed at room temperature for 30 min. The main reagents include primary antibody TfR1 (1:1000, Proteintech, USA), NOX2 (1:1000, Proteintech, USA) and GPX4 (1:1000, Proteintech, USA).

用PBS冲洗三次后，将其置于过氧化氢溶液中暗孵，然后用PBS溶液再次冲洗三次。组织在组织化学圈内均匀覆盖3% BSA，并在室温下封闭30分钟。主要试剂包括一抗TfR1（1:1000，Proteintech，美国），NOX2（1:1000，Proteintech，美国）和GPX4（1:1000，Proteintech，美国）。

After adding the primary antibody, place it in a refrigerator at 4 °C overnight and rinse it the next day. After rinsing the primary antibody and adding the second antibody, incubate in room temperature for 50 min. Secondary antibody Polyclonal Goat Anti-Mouse IgG labeled by HRP (1:2000, Servicebio, China), HRP labeled goat anti-Rabbit secondary antibody (1:2000, Servicebio, China).

加入一抗后，放置于4°C冰箱过夜，次日进行冲洗。冲洗完一抗并加入二抗后，在室温下孵育50分钟。二抗为HRP标记的多克隆山羊抗小鼠IgG（1:2000，Servicebio，中国），HRP标记的山羊抗兔二抗（1:2000，Servicebio，中国）。

After completing the above steps, retain the core by DAB color development method for 3 min and then rinse. Then wash with hematoxylin differentiation solution and then rinse with water, and finally, hematoxylin is rinsed after returning to blue. In the last step, the sections were dehydrated in anhydrous ethanol and closed under a microscope..

完成上述步骤后，用DAB显色方法保留核心3分钟，然后冲洗。接着用苏木精分化液洗涤，再用水冲洗，最后苏木精返蓝后冲洗。在最后一步中，将切片置于无水乙醇中脱水，并在显微镜下封片。

Perl’s staining

Perl染色

First, the paraffin sections of the brain tissue were dewaxed to water. Prepare the Prussian blue dye, add it to the section and dye for 1 h. Then use distilled water to wash the excess dye on the surface of the section. The sections were stained with nuclear fast red staining solution for 3 min and then the excess dye solution was rinsed.

首先，将脑组织的石蜡切片脱蜡至水。准备普鲁士蓝染液，将其加入切片中染色1小时。然后用蒸馏水冲洗切片表面多余的染料。切片用核快红染色液染色3分钟，然后冲洗掉多余的染液。

Finally, the slices are dehydrated and sealed with neutral gum. Image J 1.53 software was used for image processing..

最后，将切片脱水并用中性胶密封。使用Image J 1.53软件进行图像处理。

Nissl staining

尼氏染色

The paraffin sections were dewaxed to water and then washed 3 times with PBS. The slices were then incubated by Nissl staining solution (Beyotime Biotechnology, China) for 10 min. The analysis results were photographed with an optical microscope (Nikon 80 i, Japan).

石蜡切片脱蜡至水，然后用PBS洗涤3次。随后，将切片用尼氏染色液（碧云天生物技术，中国）孵育10分钟。分析结果使用光学显微镜（Nikon 80i，日本）拍照记录。

Fluoro-Jade B staining

氟代翡翠B染色

First, the paraffin sections are dewaxed to water, and then the FJB working liquid is added. The specific process was as follows: the pen circle was organized, 50% ice acetic acid was used as solvent, FJB working liquid (Merck, Germany) was configured at 1: 400, the diluted FJB green fluorescent probe was added, and the nucleus was re-dyed with DAPI after 4.

首先，将石蜡切片脱蜡至水，然后加入FJB工作液。具体过程如下：用笔圈组织，使用50%冰醋酸作为溶剂，按1:400配置FJB工作液（Merck，德国），加入稀释的FJB绿色荧光探针，4分钟后用DAPI复染细胞核。

◦

C overnight. Images were collected after sealing.

C过夜。密封后收集图像。

Lesion degree assessment

病变程度评估

We first stained the tissue with HE. The simple process is as follows: (1) Dewaxing paraffin sections to water; (2) Hematoxylin staining; (3) eosin staining; (4) dehydration seal. We started the section from the defect edge to the normal tissue edge with a thickness of 30 μm. Then the brain tissue defect volume was calculated using NIH Image J software (Bethesda, MD, USA)..

我们首先用HE对组织进行染色。简单过程如下：（1）石蜡切片脱蜡至水；（2）苏木精染色；（3）伊红染色；（4）脱水封片。我们从缺损边缘到正常组织边缘开始切片，厚度为30μm。然后使用NIH Image J软件（美国马里兰州贝塞斯达）计算脑组织缺损体积。

Evans blue extravasation assay

伊文思蓝外渗试验

We measured the amount of EB dye in mouse brain tissue 3 days after TBI to assess the extent of BBB destruction. In simple terms, after injecting EB dye (2%, 2 µL/g) through the tail vein, the mice were anesthetized and then injected with PBS through the left ventricle of the heart to eliminate the localized dye from the sinus bleeding.

我们在TBI后3天测量了小鼠脑组织中EB染料的含量，以评估BBB破坏的程度。简而言之，通过尾静脉注射EB染料（2%，2 µL/g）后，将小鼠麻醉，并通过心脏左心室注入PBS，以消除因窦出血而残留的局部染料。

The brain tissue was then weighed, then the sample was soaked in formamide solution and homogenized at a concentration of 200 mg of tissue per milliliter. 37.

然后称重脑组织，随后将样本浸泡在甲酰胺溶液中，并以每毫升200毫克组织的浓度进行均质化。

◦

C warm bath for 48 h, centrifuge at 6000 rpm for 20 min and remove the supernatant. The absorbance of the mixture at 632 nm was measured using a spectrophotometer (BioTek, Winooski, VT, USA).

C 在48小时温浴后，以6000转/分钟离心20分钟并去除上清液。使用分光光度计（BioTek，威努斯基，佛蒙特州，美国）测量混合物在632纳米处的吸光度。

Behavioral experiments

行为实验

The perception and memory abilities of mice were investigated using the Novel Object Recognition (NOR) experiment

使用新颖物体识别（NOR）实验研究了小鼠的感知和记忆能力。

. Within 3 days of the training stage, an identical object (yellow cube) was placed in a box (100 cm in diameter and 50 cm in height) at a position 20 cm away from the wall, and then the mouse was placed in the middle of the two objects for it to explore by itself. In the experiment phase, a familiar object (yellow cube) was replaced with a new object (white cylinder), and then the mouse was placed in the middle of the two objects and allowed to explore freely for 5 min.

在训练阶段的3天内，将一个相同的物体（黄色立方体）放置在一个盒子里（直径100厘米，高50厘米），位置距离墙壁20厘米，然后将小鼠放在两个物体中间，让其自行探索。在实验阶段，将熟悉的物体（黄色立方体）替换为一个新物体（白色圆柱体），然后将小鼠放在两个物体中间，自由探索5分钟。

New object recognition rate (NORI): the proportion of old object recognition time to all object recognition time..

新物体识别率（NORI）：旧物体识别时间占所有物体识别时间的比例。

The Morris water maze experiment is divided into training and experimental stages

Morris水迷宫实验分为训练和实验阶段。

. The whole process was recorded and evaluated using a video tracking system (Anhui Zhenghua Biological Instrument Equipment Co., LTD. : Huaibei, China). Specific experiments are as follows: During the training phase (the first 7 days), 4 training sessions are performed daily. The mice were randomly placed into the water from a quadrant facing the wall of the pool.

整个过程使用视频跟踪系统（安徽正华生物仪器设备有限公司：淮北，中国）进行记录和评估。具体实验如下：在训练阶段（前7天），每天进行4次训练。小鼠被随机从面向池壁的象限放入水中。

If the mouse successfully found the platform within 90 s, it was allowed to stay on the platform for 10 s, and if the mouse failed to find the platform within the specified time, it was guided to the platform for 10 s with a guide stick. The water temperature was kept at 24.

如果小鼠在90秒内成功找到平台，则允许其在平台上停留10秒；如果小鼠在规定时间内未能找到平台，则用引导棒将其引导至平台并停留10秒。水温保持在24摄氏度。

◦

C during the experiment. In the experimental stage (day 8 of the water maze test), the platform was removed from the water, and the mice were put into the pool from the opposite quadrant of the platform for a single test. Each mouse swam for 90 s. The movement track of the mice, the number of times they crossed the platform and the time they stayed in the quadrant where the platform was located were recorded..

实验期间的C。在实验阶段（水迷宫测试的第8天），将平台从水中移除，并将小鼠从平台相对的象限放入池中进行单次测试。每只小鼠游泳90秒。记录小鼠的运动轨迹、穿越平台的次数以及它们停留在平台所在象限的时间。

Statistic analysis

统计分析

Statistical analyses were performed and plotted using GraphPad Prism 8.4.2 software (San Diego, CA, USA) and SPSS 20.0. Data were presented as means ± SD. (*

使用GraphPad Prism 8.4.2软件（美国加利福尼亚州圣地亚哥）和SPSS 20.0进行统计分析并绘图。数据以均值±标准差表示。（*

< 0.05, **

< 0.01, ***

< 0.001).

< 0.001)。

Results

结果

Screening of key modules and genes based on WGCNA and machine-learning algorithms

基于WGCNA和机器学习算法的关键模块和基因筛选

To explore biomarkers that are altered after TBI, this study retrospectively analyzed data on gene expression from TBI and normal samples in GSE2871 by setting the cut-off value as

为了探索TBI后发生改变的生物标志物，本研究通过设定截断值，回顾性分析了GSE2871中TBI和正常样本的基因表达数据。

< 0.05 and |log2FC| >1. 107 DEGs were identified, including 47 up regulated genes and 60 down regulated genes (Fig.

<0.05 且 |log2FC| >1。共鉴定出107个差异表达基因（DEGs），其中包括47个上调基因和60个下调基因（图。

A, B). Analysis was performed to identify differentially expressed genes between TBI patients and normal controls. First of all, the soft threshold was selected for subsequent co-expression network construction (Fig.

A、B）。进行分析以鉴定TBI患者与正常对照之间的差异表达基因。首先，选择软阈值用于后续的共表达网络构建（图。

C, D). The essence was to make the constructed network more consistent with the characteristics of scale-free networks. WGCNA was used to construct a co-expression network module and visually display the gene correlation of the modules. Co-expression modules were shown in a hierarchical cluster plot (Fig. .

C、D）。其本质是为了使构建的网络更符合无标度网络的特性。使用WGCNA构建共表达网络模块，并以可视化方式展示模块中的基因相关性。共表达模块展示在层次聚类图中（图。

E). Multiple modules were shown to be associated with TBI through the moduletrait correlation studies. Each cell contains the corresponding correlation and

E). 通过模块特征相关性研究，显示多个模块与TBI相关。每个单元格包含相应的相关性及

-value (Fig.

-值（图。

F). We show the association between module membership and gene importance using scatter plots (Fig.

F). 我们使用散点图展示了模块隶属度与基因重要性之间的关联（图。

G). The module “MEturquoise” had high association with TBI and was selected as TBI related module. By WGCNA screening, DEGs were crossed with key genes to obtain target genes (Fig.

G). 模块“MEturquoise”与TBI高度相关，并被选为TBI相关模块。通过WGCNA筛选，将差异表达基因（DEGs）与关键基因交叉，获得目标基因（图。

H). To further identify the hub genes of TBI, we selected three machine-learning algorithms to screen the target genes. The LASSO regression approach was used to narrow down the nine overlapping features, and nine variables were were further used in subsequent analyses (Fig.

H). 为了进一步确定TBI的关键基因，我们选择了三种机器学习算法来筛选目标基因。使用LASSO回归方法将九个重叠特征进行缩小，并在后续分析中进一步使用了九个变量（图。

I). The SVM-RFE analysis showed that a total of 86 potential genes were identified when the accuracy of SVM model was the best (Fig.

I). SVM-RFE分析显示，当SVM模型的准确性最佳时，共鉴定出86个潜在基因（图。

J). Meanwhile, the RF algorithm identified 32 genes at the lowest error rate (Fig.

同时，RF算法在最低错误率下识别出了32个基因（图。

K). Finally, seven hub genes changed after TBI were obtained according to the above three machine-learning algorithms, which were STK39, KCND3, APOC3, FOXE3, CHRNB1, LOC103691092 and NPW (Fig.

最后，根据上述三种机器学习算法，获得了七个在TBI后发生变化的关键基因，分别是STK39、KCND3、APOC3、FOXE3、CHRNB1、LOC103691092和NPW（图。

L).

L）。

Fig. 1

图1

Screening of key modules and genes based on WGCNA and machine-learning algorithms. (

基于WGCNA和机器学习算法的关键模块和基因筛选。

) Volcano plot of DEGs. (

火山图展示了差异表达基因（DEGs）。

) Heat map of DEGs. (

) 差异表达基因的热图。(

，

) The soft threshold was selected for subsequent co-expression network construction. (

软阈值被选用于后续的共表达网络构建。

) The cluster dendrogram of co-expression genes in TBI. (

创伤性脑损伤中共表达基因的聚类树状图。

) The module-trait relationship heat map. (

模块-特征关系热图。

) Associations between module membership and gene importance is depicted in a scatter plot. (

模块成员与基因重要性之间的关联在散点图中描绘。

) A Venn diagram was made to obtain the intersection of the target genes screened by the two methods. (

通过绘制维恩图，获取两种方法筛选出的目标基因的交集。

我

) The Log (Lambda) value of the genes in the LASSO model and the most proper log (Lambda) value in the LASSO model. (

LASSO 模型中基因的 Log(Lambda) 值和 LASSO 模型中最合适的 Log(Lambda) 值。

) The optimum accuracy rate of the SVM model based on the characteristic genes. (

基于特征基因的SVM模型的最佳准确率。

) The RF module based on the characteristic genes. (

基于特征基因的RF模块。

) The Venn diagram showing the overlapping genes in LASSO, SVM, and RF modules.

) 显示LASSO、SVM和RF模块中重叠基因的维恩图。

Full size image

全尺寸图像

Analysis of core genes and screening of potential drugs

核心基因分析与潜在药物筛选

In view of the relatively small sample size and low confidence of the screened gene set, we further verified the meaningful genes by conducting q-PCR analysis on TBI mice. The results showed that only APOC3 and KCND3 had significant differences as well as the screened gFig. (Fig.

鉴于筛选出的基因集样本量相对较小且置信度较低，我们进一步通过在TBI小鼠上进行q-PCR分析来验证有意义的基因。结果显示，只有APOC3和KCND3存在显著差异，与筛选结果一致（图. (Fig.）。

A). The pathophysiological changes after TBI included ROS production, edema, inflammation, angiogenesis and metabolic related changes. We performed ssGSEA on the seven genes, and the results showed that these genes were closely related to these pathophysiology ofFig. (Fig.

A). TBI后病理生理变化包括ROS产生、水肿、炎症、血管生成和代谢相关变化。我们对这七个基因进行了ssGSEA，结果显示这些基因与这些病理生理学密切相关。

B). We looked at the previous literature and found, the formation of ROS is closely related to FOXE3, CHENB1 and APOC3. Otherwise, APOC3 is closely related to inflammation caused by TBI. We identified a number of potential drugs that may act on some of the TBI-associated genes identified in this study in HERB database and the other public dataFig. (Fig. .

B). 我们查阅了以往的文献，发现活性氧（ROS）的形成与FOXE3、CHENB1和APOC3密切相关。此外，APOC3与TBI引起的炎症反应密切相关。我们在HERB数据库及其他公共数据中识别出一些可能作用于本研究发现的部分TBI相关基因的潜在药物。图（Fig.）。

C). In view of the consistency of the results of the previous bioinformatics analysis and q-PCR experiment, as well as its rich role in various pathological processes, APOC3 was selected for subsequent research.We found Gallic acid acting on APOC3 in the HERB database. Western blotting results showed that Gallic acid could inhibit the increase of APOC3 afterFig. (Fig. .

C). 鉴于先前的生物信息学分析和q-PCR实验结果的一致性，以及其在多种病理过程中的丰富作用，选择APOC3进行后续研究。我们在HERB数据库中发现没食子酸作用于APOC3。蛋白质印迹结果表明，没食子酸能够抑制图中所示的APOC3增加。

D, E).

D, E)。

Fig. 2

图2

Screening of core genes and potential drugs. (

核心基因和潜在药物的筛选。

) q-PCR results of core genes. (

) q-PCR核心基因结果。(

) Differential expression of core gene-enriched pathways. (

核心基因富集通路的差异表达。

) Potential drugs and regulated pathological processes. (

潜在药物和受调控的病理过程。

) The expression levels of APOC3 in different groups. (*

) 不同组中APOC3的表达水平。(*

< 0.05, **

< 0.01, and ***

小于0.01，且***

< 0.001).

< 0.001)。

Full size image

全尺寸图像

Gallic acid alleviates ferroptosis in brain tissue of TBI mice by inhibiting APOC3

没食子酸通过抑制APOC3减轻TBI小鼠脑组织中的铁死亡

Analysis of the correlation between APOC3 and key protein of iron death from STRING (

APOC3与铁死亡关键蛋白的相关性分析来自STRING (

https://cn.string-db.org

) : Protein-Protein Interaction (PPI network) database found that it was closely related to GPX4, NOX2 and Fig. (Fig.

）：蛋白质-蛋白质相互作用（PPI网络）数据库发现它与GPX4、NOX2和图密切相关。（图

A). The characteristic changes of mitochondrial ferroptosis were observed under electron microscopy, and it was found that the mitochondrial membrane shrank and mitochondrial ridge disappeared after TBI, and the number of mitochondria with characteristic damage of ferroptosis in the brain tissue of TBI mice after Gallic acid treatment was improved compared with that in the TBI gFig. (Fig. .

A). 在电子显微镜下观察到线粒体铁死亡的特征性变化，发现TBI后线粒体膜收缩、线粒体嵴消失，而经没食子酸处理后，TBI小鼠脑组织中具有铁死亡特征损伤的线粒体数量较TBI组有所改善（图. 。

B). Further Western blotting results showed that the expression level of GPX4 in brain tissue after TBI was decreased, and the expression level of GPX4 in brain tissue after Gallic acid treatment was significantly increased. The expression levels of NOX2 and TfR1 in the TBI group were higher than those in the normal brain tissue, and the expression levels of NOX2 and TfR1 in the Gallic acid treatment group were significantly lower than those in the TBI gFig. (Fig. .

B). 进一步的蛋白质印迹结果显示，TBI后脑组织中GPX4的表达水平降低，而经没食子酸处理后脑组织中GPX4的表达水平显著升高。TBI组中NOX2和TfR1的表达水平高于正常脑组织，而没食子酸治疗组中NOX2和TfR1的表达水平显著低于TBI组（图. 。

C-F). The immunohistochemical results were consistent with those of Western blotting, confirming that Gallic acid could inhibit the level of iron death afterFig. (Fig.

C-F)。免疫组化结果与西方印迹结果一致，证实没食子酸可抑制铁死亡水平。

G-J). Perl’s staining showed increased iron deposits in TBI tissues and decreased iron deposits in the brain tissues of mice treated with Gallic Fig. (Fig.

G-J). Perl染色显示，TBI组织中铁沉积增加，而用没食子酸处理的小鼠脑组织中铁沉积减少。（图。

K, L).

K，L）。

Fig. 3

图 3

Gallic acid alleviates ferroptosis caused by TBI. (

没食子酸减轻了TBI引起的铁死亡。

) PPI analysis of APOC3 and key protein of ferroptosis. (

) APOC3与铁死亡关键蛋白的PPI分析。 (

) Changes of mitochondria under electron microscope. (

电子显微镜下线粒体的变化。

C语言

) The expression levels of GPX4, NOX2 and TfR1 were detected by Western blotting. (

通过蛋白质印迹法检测了GPX4、NOX2和TfR1的表达水平。

) Quantitative analysis of Western blotting results of GPX4, NOX2 and TfR1. (

GPX4、NOX2 和 TfR1 的蛋白质印迹结果的定量分析。

) Representative images of immunohistochemical staining of GPX4, NOX2 and TfR1 in the injured cortex. (

受伤皮层中GPX4、NOX2和TfR1免疫组织化学染色的代表性图像。

) Quantitative analysis of immunohistochemical staining. (

免疫组织化学染色的定量分析。

) Representative images of Perl’s staining in each group. (

`) 各组Perl染色的代表性图像。(`

) Quantitative analysis of iron deposition level in tissues. (*

) 组织中铁沉积水平的定量分析。 (*

< 0.05, **

< 0.01, and ***

< 0.01，且 ***

< 0.001).

< 0.001)。

Full size image

全尺寸图像

Gallic acid alleviates the neurodegeneration caused by TBI

没食子酸减轻了TBI引起的神经退行性病变

To determine the degree of neuronal necrosis and degenerative changes after TBI, we analyzed the level of neuronal necrosis in brain tissues through Nissl staining. Results showed that the number of normal neurons decreased after TBI, and the degree of neuronal necrosis in the Gallic acid treatment group was lower than that in the TBI gFig. (Fig. .

为了确定TBI后神经元坏死和退行性变化的程度，我们通过尼氏染色分析了脑组织中神经元坏死的水平。结果显示，TBI后正常神经元数量减少，没食子酸治疗组的神经元坏死程度低于TBI组（图. 。

A, C). The deformed neurons were labeled by FJB staining, and the number of denatured neurons increased after TBI, while the number of denatured neurons decreased after Gallic acid treatFig. (Fig.

A、C）。变形的神经元通过FJB染色标记，TBI后变性神经元数量增加，而经没食子酸处理后变性神经元数量减少。图（Fig.

B, D). Then the degree of tissue defect was analyzed by HE staining, and the results showed that the degree of brain tissue defect in the Gallic acid treatment group was lighter than that in the TBI gFig. (Fig.

B、D）。然后通过HE染色分析组织缺损的程度，结果显示，没食子酸治疗组的脑组织缺损程度比TBI组轻。

E, G). We further studied the effect of Gallic acid on BBB after TBI. Evans blue extravasation assay showed that Gallic acid could improve BBB damage caused by TBI. Reduced Evans blue exudation in brain tiFig. (Fig.

E, G）。我们进一步研究了棓酸对TBI后BBB的影响。Evans blue外渗实验表明，棓酸可以改善TBI引起的BBB损伤。脑组织中Evans blue渗出减少。图（Fig.）。

F, H).

F，H）。

Fig. 4

图4

Gallic acid protects neurons and brain tissue after TBI. (

没食子酸在TBI后保护神经元和脑组织。

，

) Representative images of each group with Nissl staining and their quantitative analysis. (

尼氏染色的各组代表性图像及其定量分析。

，

) The representative images of FJB staining in each group and their quantitative analysis. (

各组FJB染色的代表性图像及其定量分析。

，

) HE staining and quantitative analysis of tissue defect degree (

) HE染色及组织缺损程度的定量分析 (

= 5). (

，

) Representative pictures of EB extravasation and quantitative analysis of EB leakage (

）EB渗出的代表性图片和EB泄漏的定量分析（

= 5). (*

< 0.05, **

< 0.01, and ***

小于0.01，且***

< 0.001).

< 0.001)。

Full size image

全尺寸图像

Gallic acid alleviates neurologic deficits and behavioral cognitive impairment caused by TBI

没食子酸减轻了TBI引起的神经功能缺损和行为认知障碍。

The effect of Gallic acid on the neurological impairment and behavioral cognitive impairment caused by TBI was investigated through behavioral experiments. mNSS scores were performed on day 1, day 3, day 7 and day 14 after TBI, and the differences among all groups were compared on day 14. The Morris water maze experiment was carried out on mice at days 14–21.

通过行为学实验，研究了没食子酸对TBI引起的神经损伤和行为认知障碍的影响。在TBI后第1天、第3天、第7天和第14天进行mNSS评分，并在第14天比较各组间的差异。莫里斯水迷宫实验于第14-21天在小鼠上进行。

NOR experiment was performed on the 28th day after TBI. The timeline and schematic of behavioral experiments are shown bFig. (Fig. .

NOR实验在TBI后第28天进行。行为实验的时间线和示意图见图bFig.（图。

A). The mNSS score results showed that the score of the Gallic acid treatment group was lower than that of the TBI group, suggesting that Gallic acid treatment can reduce the degree of brain inFig. (Fig.

A). mNSS评分结果显示，没食子酸治疗组的评分低于TBI组，表明没食子酸治疗可以减轻脑部损伤程度。

B). NORI in mice after TBI was significantly lower than that in normal mice. However, NORI in the Gallic acid treatment group was significantly higher than that in the TBI group, suggesting that Gallic acid can significantly improve the memory and learning ability of mice afterFig. (Fig.

B). TBI后小鼠的NORI显著低于正常小鼠。然而，没食子酸治疗组的NORI显著高于TBI组，表明没食子酸可以显著改善小鼠在图（Fig.）之后的记忆和学习能力。

C). In the Morris water maze experiment, the number of times that mice passed through the hidden platform area in water and the proportion of time spent in the quadrant where the platform was located after TBI were significantly lower than that of normal mice, indicating that the learning ability and memory of mice after TBI were significantly decreased.

C). 在Morris水迷宫实验中，TBI后小鼠在水中通过隐藏平台区域的次数以及在平台所在象限停留的时间比例均显著低于正常小鼠，表明TBI后小鼠的学习能力和记忆能力显著下降。

After Gallic acid treatment, the number of mice crossing the hidden platform region was increased compared with the TBI group, and the proportion of time spent in the quadrant where the platform was located was significantly increased compared with the TBI gFig. (Fig. .

经没食子酸处理后，小鼠穿越隐藏平台区域的次数较TBI组增加，并且在平台所在象限停留的时间比例较TBI组显著增加（图X）。

D-F).

D-F）。

Fig. 5

图5

Gallic acid alleviates behavioral cognitive impairment caused by TBI. (

没食子酸减轻了TBI引起的行为认知障碍。

) Behavioral experiment timeline and schematic diagrams. (

行为实验时间线与示意图。

) mNSS score of each group (

) 各组的mNSS评分 (

= 5). (

C语言

) NORI of each group (

) 每组的NORI (

= 5). (

) Representative swimming tracks of mice in each group (

) 每组小鼠的代表性游泳轨迹 (

= 5). (

) Quantitative analysis of time proportion in target quadrant (

目标象限时间比例的定量分析

= 5). (

) Quantitative analysis of the number of crossing platform area (

) 对跨越平台区域数量的定量分析 (

= 5). (*

< 0.05, **

< 0.01, and ***

小于0.01，且***

< 0.001).

< 0.001)。

Full size image

全尺寸图像

Discussion

讨论

With the rapid development of social economy, the number of motor vehicles has soared, infrastructure has blossomed everywhere, and brain injuries caused by traffic accidents and falling injuries from high places have also followed

随着社会经济的迅速发展，机动车数量激增，基础设施遍地开花，由交通事故和高处坠落导致的脑损伤也随之而来。

. TBI is a disabling injury with a high fatality rate, and most of the patients need surgical treatment for primary brain injury

TBI是一种致残性损伤，死亡率高，大多数患者需要手术治疗原发性脑损伤。

，

. However, in addition to the primary brain injury, the subsequent secondary brain injury, such as inflammation, edema, BBB destruction, and ROS production will continue to cause damage to nerve cells

然而，除了原发性脑损伤外，随后的继发性脑损伤，如炎症、水肿、血脑屏障破坏和活性氧产生等，会继续对神经细胞造成损害。

，

. If the secondary injury is not controlled in time, the patient’s prognosis will also be worse

如果不及时控制继发性损伤，患者的预后也会更差。

. Ferroptosis is a newly defined form of cell death in recent years, which plays an important role in the occurrence and development of many diseases, including TBI

铁死亡是近年来新定义的一种细胞死亡形式，在包括创伤性脑损伤在内的多种疾病的发生发展中发挥重要作用。

. Our results on the changes of GPX4, NOX2 and TfR1 expression levels, characteristic changes of mitochondria under electron microscopy and iron deposition levels in brain tissue further prove that ferroptosis is involved in the secondary pathophysiological changes of traumatic brain injury.

我们关于GPX4、NOX2和TfR1表达水平的变化、电镜下线粒体的特征性改变以及脑组织中铁沉积水平的结果进一步证明，铁死亡参与了创伤性脑损伤的继发性病理生理变化。

Ferroptosis is a new type of iron-dependent programmed cell death, which is different from apoptosis, cell necrosis and autophagy

铁死亡是一种新型的铁依赖性程序性细胞死亡，与细胞凋亡、细胞坏死和自噬不同。

. The main mechanism of ferroptosis is that under the action of ferrobivalent or ester oxygenase, the highly expressed unsaturated fatty acids on the cell membrane are catalyzed to undergo lipid peroxidation, thus inducing cell death

铁死亡的主要机制是在亚铁或酯氧酶的作用下，细胞膜上高表达的不饱和脂肪酸被催化发生脂质过氧化，从而诱导细胞死亡。

. In addition, it also showed a decrease in GPX4, the regulatory core enzyme of the antioxidant system (glutathione system)

此外，它还显示出抗氧化系统（谷胱甘肽系统）的调节核心酶GPX4的减少。

. There are also characteristic changes in morphology, which are mainly manifested as the shrinkage of the mitochondrial double membrane structure and the disappearance of mitochondrial ridge

。形态学上也有特征性的变化，主要表现为线粒体双层膜结构的收缩和线粒体嵴的消失。

四十三

. Our experimental results also confirm this point.

我们的实验结果也证实了这一点。

Both WGCNA and machine-learning algorithms are advanced algorithms for finding key genes in genomics that can quickly help us find core genes in diseases through computers

WGCNA和机器学习算法都是用于在基因组学中寻找关键基因的高级算法，能够通过计算机快速帮助我们找到疾病中的核心基因。

. In this study, we used WGCNA and three machine-learning methods (LASSO, SVM-RFE, RF) to identify the core genes after TBI. We focused on exploring the genes that express proteins and verified them by q-PCR. The bioinformatics analysis was consistent with the experimental results of two genes: APOC3 and KCND3.

本研究使用WGCNA和三种机器学习方法（LASSO、SVM-RFE、RF）来鉴定TBI后的核心基因。我们重点关注表达蛋白质的基因，并通过q-PCR进行验证。生物信息学分析与两个基因（APOC3和KCND3）的实验结果一致。

Then, through functional enrichment of genes and pharmacological prediction through online networks, we found that Gallic acid can act on APOC3, and APOC3 is closely related to ROS, inflammation, lipid metabolism and other physiological and pathological processes. Further experiments are conducted to study the effect of Gallic acid on TBI, mainly to determine whether it can reduce nerve function deficits caused by TBI by inhibiting ferroptosis..

然后，通过对基因的功能富集和在线网络的药理预测，我们发现没食子酸可作用于APOC3，而APOC3与活性氧、炎症、脂质代谢等生理病理过程密切相关。进一步实验研究没食子酸对TBI的影响，主要是确定其是否能够通过抑制铁死亡来减轻TBI引起的神经功能缺损。

The results showed that Gallic acid inhibited the expression of APOC3 after TBI. PPI analysis showed that APOC3 was closely related to ferroptosis key proteins. In addition, the characteristic ferroptosis manifestations of mitochondria in the brain tissues of mice treated with Gallic acid were reduced under electron microscopy, and the changes in the expression levels of ferroptosis key proteins were also correspondingly reduced, which was also confirmed by immunohistochemistry.

结果显示，没食子酸抑制了TBI后APOC3的表达。PPI分析显示APOC3与铁死亡关键蛋白密切相关。此外，透射电镜下观察到经没食子酸处理的小鼠脑组织中线粒体的铁死亡特征性表现减轻，铁死亡关键蛋白的表达水平变化也相应减少，免疫组化亦证实了这一点。

Of course, the iron content in the tissues of mice in the Gallic acid group also decreased correspondingly, which jointly confirmed that Gallic acid could reduce the ferroptosis level. We also found that Gallic acid can reduce brain tissue defects and protect BBB. The results of the behavioral experiments show that Gallic acid can alleviate the neurological impairment caused by TBI.

当然，没食子酸组小鼠组织中的铁含量也相应下降，共同证实了没食子酸能够降低铁死亡水平。我们还发现没食子酸能够减轻脑组织损伤，保护血脑屏障（BBB）。行为学实验结果显示，没食子酸能够缓解TBI引起的神经功能损伤。

By analyzing the chemical structure formula of Gallic acid, we also found that Gallic acid and curcumin have the same chemical group: Phenol. Phenol can be used in pharmaceutical industry as raw material and additive in pharmaceutical synthesis.

通过分析没食子酸的化学结构式，我们还发现没食子酸和姜黄素拥有相同的化学基团：酚。酚类物质在医药工业中可作为医药合成的原料和添加剂使用。

. For example, parabens have analgesic, anti-inflammatory, antibacterial and other biological activities, and are often used in the preparation of painkillers, anti-inflammatory drugs, anti-inflammatory drugs, etc

例如，对羟基苯甲酸酯具有镇痛、抗炎、抗菌等生物活性，常用于制备镇痛药、抗炎药、抗菌药等。

. It is likely that this is the main group in which Gallic acid exerts its therapeutic effects. We will analyze drugs with similar structures in subsequent studies, focusing on the structures that play the main pharmacological role.

它很可能是没食子酸发挥其治疗作用的主要群体。我们将在后续研究中分析具有类似结构的药物，重点关注发挥主要药理作用的结构。

As shoFig. Fig.

如图所示。图。

, Gallic acid reduces the accumulation of free iron caused by TBI and the excessive consumption of GPX4 by inhibiting the expression level of APOC3, thus reducing ferroptosis caused by lipid peroxidation. To sum up, Gallic acid can inhibit ferroptosis caused by TBI. Secondly, after Gallic acid treatment, brain tissue defects and BBB damage degree were reduced.

，没食子酸通过抑制APOC3的表达水平，减少TBI引起的游离铁积累和GPX4的过度消耗，从而减轻脂质过氧化引起的铁死亡。综上所述，没食子酸能够抑制由TBI引起的铁死亡。其次，经没食子酸处理后，脑组织缺损和血脑屏障损伤程度均有所减轻。

Gallic acid treatment reduced nerve necrosis and degenerative changes in mice..

没食子酸治疗减少了小鼠的神经坏死和退行性变化。

However, it is important to acknowledge that the exact molecular mechanisms linking APOC3 inhibition by Gallic acid to the modulation of GPX4, NOX2, and TfR1 remain incompletely resolved. While our findings demonstrate correlative associations, causality between APOC3 suppression and downstream ferroptosis-related pathways requires further validation.

然而，需要承认的是，将没食子酸抑制APOC3与GPX4、NOX2和TfR1的调控联系起来的确切分子机制仍未完全阐明。虽然我们的研究结果表明了相关性关联，但APOC3抑制与下游铁死亡相关通路之间的因果关系仍需进一步验证。

Future studies utilizing APOC3-specific genetic models or in vitro mechanistic assays are necessary to delineate the precise regulatory interactions and confirm Gallic acid’s direct role in this network..

未来的研究需要利用APOC3特异性遗传模型或体外机制测定来详细描述精确的调控相互作用，并确认没食子酸在此网络中的直接作用。

Fig. 6

图6

Schematic diagram of Gallic acid treatment inhibiting TBI-induced ferroptosis.

没食子酸治疗抑制TBI诱导的铁死亡的示意图。

Full size image

全尺寸图像

Data availability

数据可用性

The raw data supporting the conclusions of this article will be made available by the corresponding author on request.

本文支持结论的原始数据将由通讯作者应要求提供。

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Acknowledgements

致谢

This work was financially supported by Postgraduate Research&Practice Innovation Program of Jiangsu Province (SJCX23-1413). This work was supported by the Huai’an Science and Technology Plan Project (No. HAB202321).

本工作得到了江苏省研究生科研与实践创新计划（SJCX23-1413）的资助。本工作得到了淮安市科技计划项目（编号：HAB202321）的支持。

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Author notes

作者笔记

Yu Liu, Xiaojia Fu and Jing Li contributed equally.

刘宇、傅晓佳和李静贡献相同。

Authors and Affiliations

作者与所属机构

Department of Neurosurgery, The Affiliated Huai’an Hospital of Xuzhou Medical University, Huai’an, 223022, China

徐州医科大学附属淮安医院神经外科，淮安，223022，中国

Yu Liu, Xiaojia Fu, Jing Li, Jianqiang Guo, Zongren Zhao & Jinyu Zheng

刘宇，傅晓佳，李静，郭建强，赵宗仁，郑金玉

Xuzhou Medical University, Xuzhou, 221000, China

徐州医科大学，徐州，221000，中国

Xiaojia Fu & Jianqiang Guo

傅晓佳和郭建强

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Contributions

贡献

Yu Liu and Xiaojia Fu wrote the main manuscript text. Jing Li and Jianqiang Guo visualized the figures. Zongren Zhao and Jinyu Zheng reviewed and edited the final manuscript. All authors reviewed the manuscript.

刘宇和傅晓佳撰写了主要的手稿文本。李静和郭建强对图表进行了可视化。赵宗仁和郑金玉审阅并编辑了最终手稿。所有作者都审阅了手稿。

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通讯作者

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联系方式：

Zongren Zhao

赵总人

或

Jinyu Zheng

郑金玉

。

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The authors declare no competing interests.

作者声明不存在竞争性利益。

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以下是电子补充材料的链接。

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Liu, Y., Fu, X., Li, J.

刘, Y., 傅, X., 李, J.