
==== Front
Zhong Nan Da Xue Xue Bao Yi Xue Ban
Zhong Nan Da Xue Xue Bao Yi Xue Ban
zndx
Journal of Central South University Medical Sciences
1672-7347
中南大学出版社 湖南省长沙市湘雅路110号湘雅医学院

39311784
1672-7347(2024)06-0878-12
10.11817/j.issn.1672-7347.2024.240240
240240
Articles
MARK4在甲基苯丙胺诱导急性肾损伤中的作用
Role of MARK4 in methamphetamine-induced acute kidney injuryhttp://orcid.org/0000-0002-2006-7857
汤 进 TANG Jin 1
胡 国铅 HU Guoqian 1
曾 梁 ZENG Liang 1
赵 东升 ZHAO Dongsheng 1
唐 贵疆 TANG Guijiang 1
刘 建业 LIU Jianye 1
http://orcid.org/0009-0007-5086-1895
申 丽君 SHEN Lijun 2
1 中南大学湘雅三医院泌尿外科 长沙 410013
1 Department of Urology, Third Xiangya Hospital, Central South University Changsha 410013
2 中南大学湘雅三医院血液净化中心 长沙 410013
2 Blood Purification Center, Third Xiangya Hospital, Central South University Changsha 410013 China
田 朴 executive-editor
申丽君，Email: 25441325@qq.com, ORCID: 0009-0007-5086-1895
汤进，Email: ajin530@139.com, ORCID: 0000-0002-2006-7857

28 6 2024
49 6 878889
09 4 2024
©Journal of Central South University (Medical Science). All rights reserved.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ 开放获取(Open access)：本文遵循知识共享许可协议，允许第三方用户按照署名-非商业性使用-禁止演绎4.0(CC BY-NC-ND 4.0)的方式，在任何媒介以任何形式复制、传播本作品(https://creativecommons.org/licenses/by-nc-nd/4.0/)。
目的

甲基苯丙胺(methamphetamine，METH)是一种非法的精神活性物质，可以损害身体的各个器官，其中泌尿系统是其毒性作用的重要靶标之一。本研究旨在探讨微管亲和调控激酶4(microtubule affinity regulating kinase 4，MARK4)在METH诱导急性肾损伤(acute kidney injury，AKI)中的作用。

方法

选取10只健康成年雄性C57BL/6小鼠，随机分为对照组和METH组，每组5只。METH组腹腔注射METH(20 mg/kg，每天1次，连续3 d)，对照组腹腔注射同等体积的生理盐水。末次注射24 h后处死小鼠，通过血清肌酐、血尿素氮和肾脏HE染色检测AKI模型是否建立成功；蛋白质组学技术筛选METH诱导AKI的肾脏组织与正常肾脏组织之间差异表达的蛋白质，并进行基因本体(gene ontology，GO)分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)分析和生物信息学分析；蛋白质印迹法验证蛋白质组学数据的准确性，选择目标分子，检测小鼠肾脏中MARK4和cleaved caspase-3表达情况。进一步探讨MARK4在METH诱导AKI中的作用。首先在BUMPT细胞中建立METH毒性模型，筛选出合适的METH处理浓度和时间；使用抑制剂抑制MARK4的表达，检测METH处理后BUMPT细胞的活力以及cleaved caspase-3表达情况。

结果

蛋白质组学分析共筛选出差异表达蛋白质17个，其中上调11个，下调6个(均P<0.05)。METH处理后小鼠肾脏中MARK4和cleaved caspase-3的表达水平升高(均P<0.05)。BUMPT细胞的活性随METH处理浓度升高而逐渐降低(均P<0.05)，其中4 mmol/L的METH处理后BUMPT细胞活力下降至60%。与对照组相比，MARK4和cleaved caspase-3的表达水平随着METH处理浓度的升高和处理时间的延长而升高(均P<0.05)，呈浓度和时间依赖性。抑制MARK4表达可以改善METH引起的BUMPT细胞活性下降情况，降低cleaved caspase-3表达，减少BUMPT细胞的细胞凋亡。

结论

MARK4在METH诱导AKI小鼠模型中高表达，MARK4通过调控细胞凋亡来介导METH中毒所致AKI。

Objective

Methamphetamine (METH) is an illicit psychoactive substance that can damage various organs, with the urinary system being one of its significant targets. This study aims to explore the role of microtubule affinity-regulating kinase 4 (MARK4) in METH-induced acute kidney injury (AKI).

Methods

A total of 10 healthy adult male C57BL/6 mice were randomly divided into a control group and a METH group, 5 mice in each group. The METH group was administered METH (20 mg/kg, intraperitoneally, once daily for 3 consecutive days), while the control group received an equal volume of physiological saline. The mice were executed 24 hours after the final injection, and the success of the AKI model was detected by blood serum creatinine, blood urea nitrogen, and renal HE staining. Proteins differentially expressed between kidney tissues with METH-induced AKI and normal kidney tissues were screened by proteomics techniques and subjected to gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and bioinformatics analysis. The accuracy of proteomic data was validated using Western blotting, and the expression levels of MARK4 and cleaved caspase-3 in mouse kidneys were measured. We further explored the role of MARK4 in METH-induced AKI. Firstly, a METH toxicity model was established in BUMPT cells to screen the appropriate concentration and time of METH treatment; the viability of BUMPT cells after METH treatment and the expression of cleaved caspase-3 were detected by interfering with MARK4 expression through inhibitors.

Results

The proteomic analysis of kidney tissues from METH and control groups screened for a total of 17 differentially expressed proteins, of which 11 were up-regulated and 6 were down-regulated (all P<0.05). The expression levels of MARK4 and cleaved caspase-3 were elevated in the kidneys of METH-treated mice (both P<0.05). The activity of BUMPT cells gradually decreased with increasing METH treatment concentration (all P<0.05), where the viability of BUMPT cells decreased to about 60% after METH treatment at 4 mmol/L. Compared with the control group, expression levels of MARK4 and cleaved caspase-3 were increased with higher METH concentrations and longer exposure times in a concentration- and time-dependent manner (all P<0.05). Inhibition of MARK4 expression improved METH-induced decrease in BUMPT cell activity, down-regulated the expression of cleaved caspase-3, and decreased the apoptosis of BUMPT cells induced by METH.

Conclusion

MARK4 is highly expressed in a mouse model of METH-induced AKI, and MARK4 mediates METH-induced AKI by regulating cell apoptosis.

甲基苯丙胺
急性肾损伤
微管亲和调控激酶4
细胞凋亡
methamphetamine
acute kidney injury
microtubule affinity regulating kinase 4
cell apoptosis
湖南省科技创新领军人才(拔尖)支持计划2023RC1033┫。This work was supported by the Scientific and Technological Innovation Leading Talents (top) Support Program of Hunan Province China ┣2023RC1033
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pmc甲基苯丙胺(methamphetamine，METH)俗称“冰毒”，是一种高度成瘾性的拟交感神经剂，可增强儿茶酚胺的释放并阻止其再吸收[1]。METH是一种被广泛滥用的毒品，全球有超过1 720万使用者[2-3]。长期滥用METH可使滥用者出现体重减轻、营养不良、厌食、恶心、失眠、注意力不集中、不愈性溃疡、心脑血管疾病、急性肾衰等多种症状和临床表现[4-8]，会对全身多个系统造成不可逆性损伤，其中泌尿系统是冰毒毒性作用重要靶标之一[9-10]。在诸多METH引起的健康问题中，急性肾损伤(acute kidney injury，AKI)在滥用者中很常见[11-14]。对苯丙胺中毒患者的肾脏进行活检，组织病理学分析示广泛的肾小管变性和坏死，间质水肿和出血，小血管闭塞，肾髓质中白细胞浸润[13, 15-17]。临床和动物模型研究[11-13, 18]也表明METH可引起肾脏损伤。有研究[19]表明苯丙胺类似物在人肾小管上皮细胞损伤的自噬和凋亡机制中发挥了重要作用。然而，METH诱导肾脏损伤的具体机制仍有待研究。

微管亲和调控激酶4(microtubule affinity regulating kinase 4，MARK4)是一种丝/苏氨酸激酶，能将微管相关蛋白(microtubule-associated protein，MAP)磷酸化，并将其从微管上解离，进而提高微管的动力，对细胞分裂和细胞周期的调控、细胞极性的确定以及细胞形态的变化等起重要的作用[20]。实验研究[21-26]表明MARK4在微管组织和脂肪生成、细胞凋亡、氧化应激、炎症等过程中起重要作用。同时MARK4可以通过多条通路来调控细胞凋亡[21, 24, 27-29]。例如过表达的MARK4可通过JNK通路诱导脂肪细胞凋亡[27]。在不同的模型中，MARK4可以通过不同的通路来调控细胞凋亡，但目前尚无研究验证MARK4在METH诱导AKI模型中的作用。

本研究通过建立METH中毒的小鼠模型，测定METH处理后小鼠肾脏中MARK4和细胞凋亡蛋白分子cleaved caspase-3的表达，并且选择BUMPT细胞系作为体外模型，使用抑制剂降低MARK4表达后，探讨MARK4与METH诱导的细胞凋亡之间的关联性。本研究旨在探讨MARK4在METH诱导的肾脏损伤中的作用，为今后治疗METH诱导的AKI时选择新的治疗靶点并开展新的治疗方案提供前期的实验依据。

1 材料与方法

1.1 主要试剂

血清肌酐(serum creatinine，SCr)检测试剂盒(货号C011-2-1)购自南京建成生物工程研究所；血尿素氮(blood urea nitrogen，BUN)检测试剂盒(货号BC1535)购自北京索莱宝科技有限公司；苏木精-伊红(HE)染色试剂盒(货号G1120)购自北京索莱宝科技有限公司；BCA蛋白质定量试剂盒(货号A55864)购自美国Thermo Fisher Scientific公司；抗体MARK4(货号AF0693)购自美国Affinity公司；DDhD1(货号ab241975)和Saa4(货号ab92540)为美国Abcam公司产品；cleaved caspase-3(货号9661)购自美国Cell Signaling Technology公司；GAPDH(货号60004-1-Ig)、β-actin(货号66009-1-Ig)购自武汉三鹰生物技术有限公司；山羊抗兔二抗(货号925-32211)、山羊抗鼠二抗(货号925-68070)购自美国LI-COR公司；MARK4 inhibitor 1(货号E2818)购自美国Selleck公司；细胞计数试剂盒-8(cell counting kit-8，CCK-8)(货号G4103-5ML)购自武汉赛维尔生物科技有限公司；乳酸脱氢酶(lactate dehydrogenase，LDH)检测试剂盒(货号C0016)和TUNEL检测试剂盒(货号C1086)购自上海碧云天生物科技公司。

1.2 动物及细胞

选取体重大约25 g的无特定病原体级C57BL/6雄性小鼠，8~10周龄，购自湖南斯莱克景达实验动物有限公司。小鼠寄养在中南大学实验动物学部。本研究严格按照中南大学实验伦理道德规范设计所有的实验方案和操作，已通过中南大学实验动物福利伦理委员会批准(审批号：CSU-2024-0083)。BUMPT细胞(小鼠近端肾小管细胞系)从中南大学湘雅二医院获得。

1.3 方法

1.3.1 建立METH致AKI的小鼠模型

C57BL/6小鼠适应环境后随机分为对照组和METH组，每组各5只。METH组腹腔注射METH(20 mg/kg)，每日1次，连续3 d；对照组同方式注射等体积生理盐水。给药后24 h处死小鼠，进行样本采集。

1.3.2 采集和处理实验动物样本

腹腔注射麻醉剂麻醉小鼠后，剪掉小鼠眼睛周围的毛发，用眼钳夹住眼球，迅速拔出眼球，血液流入分离凝胶/前凝血的静脉采血管。收集血液的采血管于室温下静置15 min后于室温离心15 min。将上清液转移到新的离心管中，再次于4 ℃离心15 min。获得的上清液为血清，可立即检测或置于-80 ℃冰箱中保存。取血后，立即处死小鼠，冰上提取肾脏，用于切片染色或蛋白质检测。

1.3.3 SCr和BUN测定

根据检测试剂盒说明书进行SCr和BUN的检测。

1.3.4 HE染色

于4 ℃环境下将小鼠肾脏置于4%多聚甲醛中固定48 h，然后用石蜡包埋并切片4 μm。使用HE染色试剂盒并根据说明书进行染色。

1.3.5 蛋白质组学及生物信息学分析

通过蛋白质组学技术筛选METH诱导AKI的肾脏组织与正常肾脏组织之间差异表达的蛋白质，并进行基因本体(gene ontology，GO)分析、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)分析和生物信息学分析。数据分析采用Proteome Discover 2.4(美国Thermo Fisher Scientific公司)。

1.3.6 蛋白质印迹法

采用蛋白质印迹法验证蛋白质组学结果的准确性，提取肾脏组织蛋白质进行MARK4、DDhD1、Saa4蛋白质的检测。使用组织蛋白质抽提试剂盒从小鼠肾脏提取总蛋白质，并使用BCA试剂盒对蛋白质浓度进行测量。将蛋白质样品经过变性处理并通过SDS-PAGE进行分离，之后将蛋白质转移至硝酸纤维素膜上。使用5%脱脂牛奶封闭膜1 h，随后添加一抗于4 ℃孵化过夜。次日，用TBST洗膜并加入二抗，于室温孵化1 h后进行洗涤和显影处理。采用ImageJ软件分析MARK4、DDhD1、Saa4、cleaved caspase-3的蛋白质相对表达量，并对蛋白质印迹法结果条带进行灰度值测量分析，用目标蛋白与相应内参蛋白GAPDH或者β-Actin的测量结果相比得到相对值。

1.3.7 免疫荧光试验

首先用与HE染色相同方法将石蜡切片进行脱蜡处理，之后将组织用柠檬酸钠置于沸水15 min进行抗原修复后，自然降至室温，用PBS洗3次，5 min/次。用含0.3%Triton的5%BSA封闭2 h，后孵育MARK4抗体(1꞉100)，于4 ℃过夜。PBS清洗一抗3次，5 min/次，加入荧光二抗在室温下孵育2 h后用PBS清洗二抗3次，5 min/次，用DAPI染核10 min，PBS清洗3次，5 min/次，封片并拍照观察。

1.3.8 细胞培养与处理

在37 ℃、5% CO2的细胞培养箱中用高糖DMEM完全培养基培养BUMPT细胞。BUMPT细胞每2~3 d传代1次，并用不同浓度(1.0、2.0、3.0、4.0和 5.0 mmol/L)的METH进行处理。选取4.0 mmol/L METH处理不同时间(0、3、6、12、24 h)后进行相关实验。MARK4抑制剂(MARK4 inhibitor 1)采用的浓度为0.5 μmol/L，在METH暴露前2 h开始给药，并持续于METH处理全程。

1.3.9 细胞活力检测

使用CCK-8进行细胞活力检测。把约2 000个对数生长期BUMPT细胞接种至96孔板，每组设6复孔及空白对照孔。细胞于37 ℃孵育后，加入不同浓度METH继续培养24 h。加入10 μL CCK-8，孵育2 h，注意避免气泡及光照影响。在酶标仪中以450 nm波长测量吸光度(optical density，OD)值，细胞生存率=(实验组OD值-空白对照OD值)/(对照组OD值-空白对照OD值)×100%。

1.3.10 LDH释放检测

使用LDH检测试剂盒检测每组中凋亡细胞释放的LDH。将METH处理后的96孔板细胞离心5 min，收集上清液并将其在室温下与LDH工作试剂混合物孵育30 min。随后在490和650 nm波长下检测每个孔的OD值，并计算凋亡细胞的LDH释放百分比。

1.3.11 TUNEL检测

使用TUNEL检测试剂盒检测BUMPT细胞的凋亡情况。将细胞取出细胞培养皿，去上清后用PBS洗3次，3 min/次；取出已贴好组织的切片并进行脱蜡处理；用4%多聚甲醛固定20 min后用PBS洗3次，3 min/次；破膜用含0.3% Triton X-100的PBS孵育 10 min，后加入TUNEL染色工作液，并于37 ℃避光孵育1 h；PBS洗3次，3 min/次，然后用DAPI染核10 min，再次用PBS洗3次，3 min/次，最后封片并拍照观察。

1.4 统计学处理

使用统计学软件GraphPad Prism9对数据绘图及统计分析，所有数据均以均数±标准差表示，组间比较采取t检验或单因素方差分析，多样本比较采用多因素方差分析。P<0.05为差异具有统计学意义。

2 结 果

2.1 METH可引起小鼠AKI

与对照组相比，METH组的小鼠SCr以及BUN明显升高，差异均具有统计学意义(均P<0.05，图1A和1B)。肾脏组织的HE染色结果表明：与对照组相比，METH组小鼠的肾小球囊壁层增生、肾小管扩张、上皮扁平、刷状缘损伤、上皮细胞肿胀(图1C)，结果与SCr结果一致。TUNEL染色结果(图1D)显示METH处理后肾脏组织出现TUNEL阳性，说明METH处理后肾脏组织细胞发生损伤。

图1 METH可引起小鼠急性肾损伤

Figure 1 METH induces acute kidney injury in mice

A: SCr level; B: BUN level; C: HE staining to detect renal histomorphology (blue arrowheads point to glomerular capsule wall layer hyperplasia, black arrowheads point to tubular dilatation, epithelial flattening, and brush border injury, and red arrowheads point to epithelial cell swelling); D: Results of TUNEL coloring. Data are represented as mean±standard deviation. **P<0.01, ***P<0.001 vs the CTL group. METH: Methamphetamine; CTL: Control; SCr: Serum creatinine; BUN: Blood urea nitrogen.

2.2 差异蛋白质筛选结果

蛋白质组学共发现可定量蛋白质7 172个，以差异倍数(fold change，FC)=1.2倍且P<0.05为差异筛选条件，共筛选出差异蛋白质17个，其中11个上调，6个下调(表1)。火山图见图2。

表1 METH暴露后肾脏组织差异表达基因

Table 1 Differentially expressed genes in kidney tissues after METH exposure

基因名称	差异表达倍数	变化类型	基因名称	差异表达倍数	变化类型	
MARK4	2.393 665 158	上调	EIF2S3Y	1.233 432 614	上调	
DDhD1	1.636 203 866	上调	GATC	1.215 657 312	上调	
TMEM63a	1.280 988 593	上调	CYBA	-0.830 637 778	下调	
HSPBAP1	1.259 036 145	上调	C4B	-0.827 848 873	下调	
SMCO4	1.256 487 401	上调	SSU72	-0.811 349 230	下调	
ASB13	1.256 015 038	上调	CLDN2	-0.752 628 505	下调	
RNF126	1.252 722 493	上调	DDA1	-0.732 601 790	下调	
TPRKB	1.247 191 011	上调	Saa4	-0.694 915 254	下调	
OSBPL10	1.238 343 902	上调				

图2 差异蛋白质火山图

Figure 2 Differential protein volcano map

Horizontal coordinates are protein variance multiplicity, and distance from the 0 point indicates the degree of variance, with the left side of the 0 point being down-regulated and the right side of the 0 point being up-regulated. The vertical coordinate is ⁃log10(P-value), and the distance from the 0 point indicates the degree of difference. The red points in the figure are up-regulated differentially expressed proteins, the blue points are down-regulated differentially expressed proteins, and the gray points represent non-significant differentially expressed proteins. FC: Fold change.

2.3 差异蛋白质的GO和KEGG分析

GO分析显示：在生物学过程方面，差异蛋白质在炎症反应、先天性免疫应答、细胞内信号转导存在富集；在细胞组成方面，差异蛋白质在细胞质、胞液、树突、细胞核、神经元胞体存在富集；在分子功能方面，差异蛋白质在金属离子结合、ATP结合存在富集(图3)。KEGG分析显示：差异蛋白质富集较多的是白细胞跨内皮层迁移(图4)。

图3 差异基因基因本体富集分析结果

Figure 3 Results of gene ontology enrichment analysis of differential genes

In the graph, x-axis enrichment score is the enrichment score, and y-axis is the information of the respective top 5 terms of BP/CC/MF. The larger the bubble, the more proteins the entry contains, the color of the bubble changes from yellow-red, and the smaller the value of its enrichment P value, the greater the degree of difference.

图4 前20位差异表达基因的KEGG信号通路分析气泡图

Figure 4 Bubble plots of the differentially expressed gene top 20 KEGG signaling pathway analysis

The x-axis in the graph represents the enrichment score, and the y-axis is the top 20 pathway information. The size of bubbles in the entries represents the amount of differential proteins included, the larger the bubbles the more differential proteins, the color of bubbles changes from red-green-blue-violet, and the smaller the enrichment P-value, the greater the degree of significance. KEGG: Kyoto Encyclopedia of Genes and Genomes.

2.4 METH导致小鼠肾脏组织中MARK4和cleaved caspase-3表达升高

蛋白质印迹法结果显示：与对照组相比，METH组MARK4、DDhD1表达增加，Saa4表达减少(图5)，趋势与蛋白质组学结果一致。在METH暴露后小鼠肾脏中MARK4表达增多1.5倍左右(P<0.05，图5A和图5B)。METH暴露后小鼠肾脏中凋亡蛋白cleaved caspase-3的表达升高2.5倍左右(P<0.05，图5A和图5E)。进一步对肾脏组织切片进行的免疫荧光染色结果显示：METH处理后肾脏组织中MARK4阳性增多，且主要表达在肾小管(图5F)。METH暴露后，MARK4与cleaved caspase-3同时增高，这说明MARK4可能与METH诱导细胞凋亡有关。

图5 METH暴露后小鼠肾脏中MARK4、DDhD1、Saa4、cleaved caspase-3的表达变化

Figure 5 Change of MARK4, DDhD1, Saa4, and cleaved caspase-3 expression in the kidneys of mice after METH exposure

A: Protein expression of MARK4, DDhD1, Saa4, and cleaved caspase-3 in kidney tissues after METH treatment detected by Western blotting; B-E: Statistical analysis of gray scale value of MARK4 (B), DDhD1 (C), Saa4 (D), and cleaved caspase-3 (E) protein; F: Graph of the results of MARK4 immunofluorescence staining of mouse kidney tissues. Data are represented as mean±standard deviation. *P<0.05, **P<0.01 vs the CTL group. CTL: Control; METH: Methamphetamine; MARK4: Microtubule affinity regulating kinase 4.

2.5 METH处理后BUMPT细胞活力降低，MARK4和cleaved caspase-3的表达水平随着METH浓度升高而升高

CCK-8结果表明：不同浓度的METH处理24 h后，BUMPT细胞的活力随着METH浓度的升高而下降。与对照组相比，1 mmol/L的METH处理后，METH组细胞的活力差异无统计学意义(P>0.05)；2、3、4、5 mmol/L的METH处理后，METH组的细胞活力均显著降低(均P<0.05，图6A)。蛋白质印迹法结果表明：METH处理24 h后BUMPT细胞中cleaved caspase-3的表达水平随着METH浓度的升高而升高，当METH浓度达4 mmol/L时，cleaved caspase-3的表达接近对照组的3.5倍左右(P<0.001，图6B和6D)。

图6 不同浓度的METH处理BUMPT细胞24 h后细胞的活力及MARK4和cleaved caspase-3的表达变化

Figure 6 Changes in cell viability and MARK4 and cleaved caspase-3 expression in BUMPT cells after 24 hours of exposure to different concentrations of METH

A: Statistical analysis of cell viability of BUMPT cells treated with different concentrations of METH for 24 hours as detected by cell counting kit-8 (CCK-8); B: Protein expression of MARK4 and cleaved caspase-3 of BUMPT cells after treatment with different concentrations of METH as detected by Western blotting; C and D: Statistical analysis of gray scale value of MARK4 (C) and cleaved caspase-3 (D) protein. Data are represented as mean±standard deviation. *P<0.05, **P<0.01, ***P<0.001 vs the control group. METH：Methamphetamine; MARK4: Microtubule affinity regulating kinase 4.

METH处理后MARK4的表达明显升高，在BUMPT细胞中MARK4的表达水平随着METH处理的浓度升高而增加，且METH浓度在4 mmol/L时，MARK4的表达量最高，接近对照组的4倍，差异有统计学意义(P<0.001，图6B和6C)。

2.6 BUMPT细胞中MARK4及cleaved caspase-3的d蛋白质的表达量随着METH处理时间延长而增加

随着处理时间的延长，MARK4及cleaved caspase-3蛋白质的表达量显著增加，在24 h时的表达量约为对照组的3倍，差异具有统计学意义(P<0.001，图7)。

图7 不同时间的METH处理后BUMPT细胞MARK4和cleaved caspase-3表达情况

Figure 7 Expression of MARK4 and cleaved caspase-3 in BUMPT cells after METH exposure at different times

A: Western blotting detection of protein expression of MARK4 and cleaved caspase-3 after METH treatment of BUMPT cells for different times; B and C: Statistical analysis of gray scale values of MARK4 (C) and cleaved caspase-3 (D) protein. Data are represented as mean±standard deviation. *P<0.05, **P<0.01, ***P<0.001 vs the control group. METH: Methamphetamine; MARK4: Microtubule affinity regulating kinase 4.

2.7 抑制MARK4表达可减少METH诱导的细胞凋亡

MARK4抑制剂对于BUMPT细胞的活力无明显影响(图8A)。随着抑制剂浓度的增加，METH+MARK4 inhibitor 1组细胞活力较METH组逐步升高，抑制剂浓度于0.5 μmol/L时细胞活力较METH组提升约1倍左右(P<0.001，图8B)。选取MARK4抑制剂浓度为0.5 μmol/L进一步探索MARK4在METH诱导细胞凋亡中的具体作用。LDH释放检测结果显示：加入MARK4 inhibitor 1后可以明显减少(10%左右)METH引起的LDH的释放(P<0.05，图8C)。TUNEL染色检测BUMPT细胞凋亡情况结果表明：加入MARK4 inhibitor 1后TUNEL染色阳性结果较METH组减少(图8D)，MARK4抑制剂能够有效降低MARK4的表达，且凋亡相关分子cleaved caspase-3的表达也随着下降(均P<0.05，图8E~8G)。上述结果提示抑制MARK4的表达可以减少METH所致BUMPT细胞凋亡。

图8 抑制MARK4表达后，METH诱导的细胞凋亡减少

Figure 8 Inhibition of MARK4 expression reduces METH-induced apoptosis of BUMPT cells

A and B: Statistical analysis graph of cell viability of BUMPT cells treated with different concentrations of MARK4 inhibitor 1(A) and METH+different concentrations of MARK4 inhibitor 1 (B) for 24 hours by CCK-8 (the concentration of METH was 4 mmol/L); C: Results of the LDH release assay; D: Graph of TUNEL staining results; E: Western blotting detection of protein expression of MARK4 and cleaved caspase-3 in METH and METH+MARK4 inhibitor 1-treated BUMPT cells for 24 hours; F and G: Statistical analysis of gray value of MARK4 (F) and cleaved caspase-3 (G) protein. Data are represented as mean±standard deviation.*P<0.05, **P<0.01, ***P<0.001 vs the CTL; †P<0.05 vs the METH group. MARK4: Microtubule affinity regulating kinase 4; METH: Methamphetamine; CTL: Control; CCK-8: Cell counting kit-8; LDH: Lactate dehydrogenase.

3 讨 论

本研究通过体内及体外实验初步探究了METH介导AKI的机制。首先本研究建立了METH诱导AKI的小鼠模型，既往METH急性中毒的模型研究[30-31]大部分集中在中枢神经系统，采用的剂量和频次也各不相同，如单次腹腔注射5、10和20 mg/kg，每日注射2~4次，或者15、30、40 mg/kg，每日注射1~2次。考虑药物来源及实验成本等因素，本研究使用C57BL/6小鼠来建立METH诱导AKI的动物模型，使用METH进行腹腔注射，剂量为20 mg/kg，每日1次，连续3 d。通过SCr、BUN的升高及肾脏HE染色的病理改变和TUNEL染色证实METH诱导AKI的动物模型构建成功。

目前尚未有研究报道METH所致AKI的分子机制，为探索潜在机制，本研究利用蛋白质组学的方法对肾脏组织进行检测，共筛选出17个差异表达的蛋白质，其中11个上调，6个下调，其中MARK4变化最明显。MARK属于钙/钙调蛋白依赖性蛋白激酶的1个亚家族。MARK家族在维持细胞极性、微管动力学、细胞周期控制、程序性细胞死亡、信号转导以及神经元迁移和分化等方面发挥重要作用[29]。在哺乳动物中MARK由4种亚型(MARK1~MARK4)组成，其中MARK4基因存在2种可变剪接的亚型，分别为MARK4L和MARK4S，MARK4L在睾丸、大脑、肾、肝和肺中高表达，而MARK4S在睾丸、心脏和大脑中表达较高[32]。MARK4S在正常脑组织中上调而MARK4L在肝癌、神经胶质瘤细胞和神经元祖细胞中上调，表明这2种亚型的功能不同[33]。既往研究[21-26]表明MARK4在微管组织、脂肪生成和细胞凋亡、氧化应激和炎症中起重要作用。MARK4控制下游靶基因表达，参与细胞周期进程、炎症和多种信号级联反应，如哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR）、核转录因子-κappaB(nuclear factor kappa-B，NF-κB)和Hippo。MARK4是mTOR信号通路mTORC1复合物的负调节因子，限制mTOR通路的激活[34]。MARK4通过激活IKKα/NF-κB信号通路促进线粒体氧化应激和炎症[35]。而在乳腺癌细胞中MARK4上调可通过抑制Hippo信号转导来增加增殖[36-37]。MARK4与多种人类疾病密切相关，例如在脑卒中时MARK4在皮质神经元中表达升高并可使tau蛋白的磷酸化和聚集，导致神经退行性改变[38]。研究[24]显示MARK4在代谢性疾病中发挥作用，在脂肪细胞中MARK4通过激活c-Jun氨基末端激酶1和抑制p38 MAPK通路促进脂肪生成，诱导脂肪细胞凋亡。在动脉粥样硬化病变中MARK4可与Nod样受体蛋白3(Nod-like receptor family pyrin domain containing 3，NLRP3)炎症小体相互作用，促进NLRP3炎症小体激活[35, 39]。研究[40]显示：在心肌梗死模型中，MARK4通过促进MAP4的磷酸化调节心肌细胞收缩力，抑制MARK4可以减轻急性心肌梗死损伤。MARK4在神经损伤疾病、神经退行性疾病以及代谢性疾病、心血管疾病以及癌症等多种疾病中发挥重要作用，提示在METH毒性损伤中也可能发挥着一定的作用。有研究[19]探究了合成卡西酮诱导的肾毒性的细胞机制，其中包括募集自噬和激活凋亡细胞死亡，自噬和凋亡之间的相互作用可介导人工合成卡西酮在人肾细胞中引发的毒性。卡西酮是一种天然存在的β-酮苯丙胺类似物，存在于卡西草叶中。合成卡西酮是该化合物的衍生物[41]，考虑到不同苯丙胺之间相似的结构和作用机制，这些化合物具有相似的毒代动力学和毒理学特征[42]，笔者认为METH可能也是通过凋亡途径介导肾脏的损伤。同时，研究[21, 24, 27-29]表明MARK4通过多条通路来调控细胞凋亡，因此笔者猜想MARK4通过调控细胞凋亡来介导METH中毒的肾脏损伤。本研究对肾脏组织进行蛋白质印迹法检测发现MARK4和细胞凋亡蛋白分子cleaved caspase-3表达都明显升高，表明METH诱导细胞凋亡的同时伴有MARK4表达的升高，提示MARK4可能与METH诱导细胞凋亡有关。为验证猜想，本研究在细胞水平上进行进一步的探索。

本研究选取小鼠肾小管上皮细胞系BUMPT在体外建立METH毒性模型，使用不同浓度的METH处理BUMPT细胞24 h，利用CCK-8法检测细胞的活力，蛋白质印迹法检测凋亡分子cleaved caspase-3的表达，结果发现细胞活力呈浓度依赖性下降，凋亡分子cleaved caspase-3的表达呈浓度依赖性上调。本研究中METH引起的BUMPT细胞活力和细胞凋亡变化与合成卡西酮诱导人肾小管上皮细胞系(HK-2)凋亡的变化[19]一致。

在此基础上，本研究进一步检测了MARK4的表达与细胞凋亡之间的关系。使用不同浓度和作用时间的METH处理BUMPT细胞后发现：在正常的情况下，MARK4表达量很低，但是METH处理后，其表达量呈METH浓度依赖性升高。当METH浓度在 4 mmol/L时，MARK4的表达量最高，约对照组的4倍，同时METH诱导的细胞毒性与MARK4的表达趋势一致。用浓度为4 mmol/L的METH对BUMPT细胞设置了不同的时间梯度，发现MARK4的表达随着作用时间的延长而升高，并在处理24 h时最高，大约为对照组的3倍。

为确定MARK4在METH诱导肾小管细胞凋亡中作用，通过使用MARK4抑制剂抑制MARK4的活性，检测MARK4对METH诱导的细胞凋亡的影响。结果表明MARK4抑制剂本身对细胞活力无明显影响，抑制MARK4表达后METH处理仍可诱导BUMPT细胞发生凋亡，但与METH组相比，细胞凋亡减少且差异具有统计学意义。这说明MARK4与METH诱导的BUMPT细胞凋亡有关，抑制MARK4的表达可以减轻METH所致的细胞凋亡。

本研究的创新性体现在：1)建立了METH致AKI的小鼠模型；2)通过蛋白质组学筛选出了METH致AKI的差异表达蛋白质；3)发现METH暴露可引起小鼠肾小管上皮细胞BUMPT的损伤，如细胞活力下降、细胞凋亡增加；4)发现抑制MARK4的表达可以改善METH引起的细胞凋亡作用，说明MARK4在METH引起细胞凋亡中起重要作用。

本研究虽证明了抑制MARK4的表达可以抑制METH诱导的细胞凋亡，但是仍然存在一些局限性：第一，仅采用BUMPT细胞进行体外实验，并未进行体内的相关验证，不能充分模拟METH在体内的生物学效应及作用过程，缺乏细胞与细胞之间或者体内微环境的影响。第二，单独细胞系并不能证明在所有类型的细胞中抑制MARK4均可减轻METH诱发的细胞凋亡。第三，没有进一步探索MARK4在METH诱导的细胞凋亡中的具体调控机制。

综上，本研究表明METH可诱导小鼠肾小管上皮细胞发生凋亡，抑制MARK4可以减少METH诱导的肾小管上皮细胞凋亡，这将为今后MARK4可能成为METH所致AKI的潜在治疗靶点提供前期实验依据。

基金资助

湖南省科技创新领军人才(拔尖)支持计划(2023RC1033)。This work was supported by the Scientific and Technological Innovation Leading Talents (top) Support Program of Hunan Province, China (2023RC1033).

利益冲突声明

作者声称无任何利益冲突。

作者贡献

汤进、刘建业 论文选题与修改，实验操作；胡国铅 协助选题，初稿撰写；曾梁、赵东升、唐贵疆 协助实验，文献检索，数据分析；申丽君 选题指导，数据分析，论文修改。所有作者阅读并同意最终的文本。

原文网址

http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202406878.pdf

http://dx.chinadoi.cn/10.11817/j.issn.1672-7347.2024.240240
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