细胞代谢组学在中药作用机制中的研究概况
Research Overview of Cell Metabonomics in the Mechanism of Traditional Chinese Medicine
DOI: 10.12677/PI.2022.113027, PDF, HTML, XML, 下载: 42  浏览: 77 
作者: 袁 静, 夏玉凤*:中国药科大学中药学院生药学系,江苏 南京
关键词: 细胞代谢组学中药作用机制Cell Metabolomics Traditional Chinese Medicine Mechanism
摘要: 近年来,中药因其低毒高效的特性受到诸多学者的广泛关注。然而,成分复杂,作用靶点多,作用机制不明确使中药的发展极大受限。细胞代谢组学作为代谢组学一个新的研究方向,将研究系统从生物整体水平缩小到细胞整体水平,可直观反映药物刺激对某种细胞代谢的直接影响,从而更直接地体现中药作用及更有针对性地解释中药作用机制。本文对借助细胞代谢组学探索中药作用机制的概况进行综述,以期为中药作用机制探索提供参考。
Abstract: In recent years, traditional Chinese medicine has been widely concerned by many scholars due to its low toxicity and high efficiency. However, the development of traditional Chinese medicine is greatly restricted owing to its complex composition, numerous targets, and unclear mechanism. As a new research direction of metabolomics, cell metabolomics narrows the research system from the overall level of biology to the overall level of cells, which can intuitively reflect the direct impact of drug stimulation on the metabolism of a certain cell, so as to more directly reflect the effects and explain the mechanism of Chinese medicines in a more targeted manner. This paper reviews the general situation of exploring the mechanism of traditional Chinese medicine by means of cell metabolomics, in order to provide reference for the exploration of the mechanism of traditional Chinese medicine.
文章引用:袁静, 夏玉凤. 细胞代谢组学在中药作用机制中的研究概况[J]. 药物资讯, 2022, 11(3): 216-223. https://doi.org/10.12677/PI.2022.113027

1. 引言

中药是我国传统医学的瑰宝,与时俱进的发展是对其最好的传承,中药的发展离不开传统与现代的结合,从现代科学角度解读传统中药作用原因,阐明中药起效机制是传承与发展中药并将其推向国际至关重要的一环。

代谢组学已成为一种对不同生物基质中代谢物进行定性和定量的尖端技术 [1],可根据代谢水平变化实时揭示机体生理病理状态。将其引入中药研究,既能从整体观角度考察中药的疗效,又能从生物标志物及代谢通路层面考察中药的作用机制,与传统研究手段相比较,代谢组学技术更适合中药作用机制研究 [2]。细胞代谢组学作为代谢组学的一个重要分支,从细胞单元出发,对于阐明中药在细胞水平上的作用机制具有重要意义,目前已广泛应用于中药作用机制的研究。细胞代谢组学除了整体性和系统性的特点外,还更具有针对性。因此,对于特定状态下的作用机制研究,细胞代谢组学具有独特的优势。

2. 细胞代谢组学概述

2.1. 起源与发展

代谢组学是一门通过现代分析技术研究生物体系受到刺激前后代谢图谱及其动态变化的一门学科,这一概念于1999年首次提出 [3],起初主要研究对象为人、实验动物的血浆、尿液及组织等。随着研究的深入,代谢组学研究逐渐深入到细胞水平,Fiehn等 [4] 于2000年提出细胞代谢组学概念,即分析单个细胞或单一细胞类型中内源性小分子代谢物。根据分析对象不同,可分为胞内代谢组学(细胞代谢指纹分析)与胞外代谢组学(细胞代谢足迹分析)。胞内代谢组是细胞内复杂生化活动状态的综合表现,同时与胞外代谢物的分泌存在密切联系,而胞外代谢组能够直接反映细胞与环境间的能量物质交换 [5]。因此,细胞代谢物浓度的改变能够反映细胞基因组的改变和细胞对于外部刺激的响应,这对于系统地理解细胞的性质、功能、寻找生物标志物及筛选活性药物、探索作用机制至关重要 [6]。细胞代谢组学技术,目前已广泛应用于医学众多领域,如疾病的诊断、药物的作用机制及药物研发、毒性评价等。

2.2. 样品前处理方法

细胞内酶系统复杂、代谢活动较快,代谢物轮廓的改变将直接影响代谢组学分析结果,因此细胞样品的前处理是细胞代谢组学分析的关键步骤 [7],前处理主要包括细胞淬灭和代谢物提取。细胞淬灭的目的是快速终止细胞内的酶促反应,使代谢物轮廓“凝固”。理想的淬灭方法是在保证细胞完整的基础上,快速灭活细胞内酶的活性。常见的淬灭方法包括冷甲醇法、液氮淬灭法、低温离心法等。细胞淬灭后,需对代谢产物进行提取,提取除对细胞中代谢物进行提取(胞内代谢组)外,还包括培养上清代谢物质提取(胞外代谢组),溶剂提取为代谢物提取的主要选择,常根据代谢物的种类及极性选择合适提取溶剂 [8]。根据细胞代谢组学研究目的不同,提取方法也不同。如在靶向代谢组学分析中,需要采用特定的提取溶剂和提取方法来提取出特定的组分,而在非靶向代谢组学分析代谢轮廓时,则需要尽可能多地提取出代谢物 [6]。因此,根据细胞种类、代谢物类别、研究目的等的不同,对样品的前处理方法也不尽相同。

2.3. 分析技术

细胞样本在预处理之后,需要运用现代分析手段检测样本中所有代谢物的种类及含量,从而得到大量反映生物样品信息的原始实验数据 [9]。目前使用最广泛的代谢组学研究技术包括核磁共振(NMR)、气相色谱–质谱联用(GC-MS)与液相色谱–质谱联用(LC-MS)。NMR价格较便宜,重复性高,样本可回收用于其他分析,但其灵敏度较低,不能对脂质等物质进行定性。GC具有高色谱分辨率,但需要对样本进行化学衍生化处理。LC则不需要进行衍生化处理,比GC拥有更高的灵敏度与测量范围。GC和LC两种色谱方式与MS联用都能提高灵敏度并且优化目标分析条件,随着质谱仪接口技术的不断更新,LC已能够与四极杆–飞行时间、离子阱–飞行时间等高分辨率质谱仪串联使用进行代谢组学分析,联用分析技术的发展,使破译代谢物信息得到极大便利。但是,目前对细胞代谢物的研究仍然需要克服样本中代谢物过多带来的检测分析困难,代谢组学进一步的发展仍需提高检测的灵敏度、降低背景以及减少样品的准备工作等 [10]。

2.4. 分析方法与数据处理方法

细胞代谢组学可分为靶向细胞代谢组学和非靶向细胞代谢组学,前者是通过标准品对代谢物进行定性定量分析,研究特定的代谢通路、生物活性等,后者则系统全面地检测样品中的代谢物,需对大量数据进行处理分析 [10]。一般情况下,代谢组学产生的原始数据庞大而复杂,需要采用化学计量学和生物信息学技术进行预处理和建模分析 [11]。大致流程如下:预处理(包括归一化、数据转化)原始数据,以标准化实验数据,消除分析过程中带来的误差;将标准化的实验数据通过主成分分析法(PCA)分析降维,归纳和总结研究对象的代谢谱,同时利用(正交)偏最小二乘回归分析法放大组间差异,将不同组进行区分;评价以上所建立的数学模型质量,包括模型的建模能力、预测能力,以及稳定性;最后筛选差异性代谢物,多维统计一般使用变量重要性投影(VIP) > 1作为筛选标准,单维分析则常用相关系数P < 0.05进行筛选。只有建立可靠的数据模型,才有可能从大量的原始代谢数据中筛选出具有统计学意义的差异代谢物,指导相关疾病的机制研究 [8]。

3. 细胞代谢组学在中药作用机制研究中的应用

3.1. 抗炎机制研究

炎症是许多疾病发生的病理基础,其作用在众多疾病的发展和预后过程中不容忽视。短暂的炎症反应是机体的自动防御反应,但长时间持续的炎症则可能会发展为各种疾病,甚至造成机体损伤 [12]。尽管中药抗炎效果显著,但作用机制的不明确,使其在抗炎过程中的高效使用大为受限。

为探索醉茄内酯治疗银屑病时抗炎的潜在机制,Li等 [13] 使用基于LC-MS的非靶向代谢组学对人永生化表皮细胞(HaCaT)内代谢物进行分析,发现醉茄内酯处理显著影响多条代谢途径,尤其是能量代谢、氨基酸代谢、脂质代谢和核酸代谢。采用同样的非靶向细胞内代谢组学。Yan等 [14] 借助1H NMR对黄芩素的抗炎机制进行探索,结果显示其抗炎通路涉及丙氨酸、天冬氨酸和谷氨酸代谢、谷胱甘肽代谢、精氨酸和脯氨酸代谢、D-谷氨酰胺和D-谷氨酸代谢。除采用单一分析技术外,还可使用多分析技术平台对分析结果进行丰富,Noelia等 [15] 使用基于多平台(LC-MS与GC-MS)的非靶向代谢组学方法揭示二氢查耳酮(根皮苷、根皮素)对脂多糖(LPS)刺激的THP-1巨噬细胞代谢物的影响及其抗炎机制。结果显示,二氢查尔酮处理使LPS刺激的巨噬细胞状态趋于正常,代谢物轮廓分析结果表明二氢查尔酮的抗炎机制除促进氨基酸生物合成外,主要表现为甘油磷脂和鞘脂代谢。

中药单体化合物由于其成分单一,所受干扰较小,在研究其抗炎机制时多采用胞内细胞代谢组学,仅对细胞内部代谢物进行分析。相较于单体化合物,中药提取物或复方成分较为复杂。受多种成分影响,细胞的生长坏境也可能会有所差异,因此除常规采用胞内代谢组学外,也有学者将胞内代谢组学与胞外代谢组学相结合进行机制探索。仅采用非靶向胞内代谢组学,Chen等 [16] 借助GC-MS分析用或不用樟树精油处理的BV2小胶质细胞(LPS诱导)的代谢特征。发现LPS刺激使细胞中葡萄糖水平降低,乳酸水平升高,而樟树精油处理能逆转这种情况,研究表明,樟树精油可能通过减弱小胶质细胞的糖酵解能力来介导其抗炎作用。Wang等 [17] 通过LC-MS技术分析在LPS诱导的猪肠上皮细胞(IPEC-J2)中研究假蒟正丁醇提取物的抗炎作用,结果表明,假蒟正丁醇提取物可调节LPS处理的细胞中同型半胱氨酸降解、蛋氨酸代谢、嘧啶代谢、β-丙氨酸代谢和嘌呤代谢等代谢途径。采用非靶向的胞内代谢组学与胞外代谢组学相结合,Lau等 [18] 对芹菜茎与叶提取物处理后细胞内和细胞外代谢物变化进行研究,发现芹菜茎叶提取物处理均能够显著逆转LPS诱导的细胞内(瓜氨酸、脯氨酸、肌酸)和细胞外(瓜氨酸、赖氨酸)代谢产物水平,此外,芹菜叶提取物能逆转细胞内天冬氨酸和细胞外脯氨酸水平;芹菜茎提取物能逆转细胞内组氨酸和细胞外葡萄糖水平。从这些代谢物的改变,推断出芹菜提取物的抗炎机制与精氨酸代谢、三羧酸循环、尿素循环、糖酵解和戊糖磷酸途径有关。因中药提取物是多成分对应多靶点,为减少干扰,分析某一特定的代谢途径或缩小分析范围,靶向细胞代谢组学也常被使用。如Zhang等 [19] 采用基于UPLC-MS的靶向脂质代谢组学揭示雷公藤多苷在人类单核细胞衍生的巨噬细胞中对脂质介质通路具有显著调节作用。

3.2. 抗肿瘤机制研究

癌症是一种慢性健康状况,其发病率和死亡率在世界范围内都在迅速增加 [20]。到目前为止,还没有用于癌症的最佳治疗方案。中药经传统中医实践数千年,目前已被广泛接受为癌症的替代疗法。与化学疗法相比,中药具有毒性低、特异性强、疗效高等优势 [21] [22]。有证据表明,中药联合放化疗能够提高放化疗的疗效,减少放化疗的局限性和弊端 [21]。为阐明中药在癌症治疗中作用机制,已有研究者通过多种分析手段进行了大量研究,细胞代谢组学是其中大有可观的手段之一。

肝癌、结直肠癌、胰腺癌、前列腺癌等的癌症的发病率多年来在全球范围内居高不下,已有研究证明,中药在许多癌症的治疗及预防方面的发挥了不容小觑的作用。譬如,紫草素、薯蓣皂苷、表没食子儿茶素没食子酸酯等均可在结直肠癌中发挥较好的抗肿瘤作用 [23] [24] [25];小檗碱在胰腺癌中能发挥有效作用 [26];复方苦参注射液被(CKI)广泛用于治疗癌性疼痛和包括肝细胞癌(HCC)在内的消化系统肿瘤 [27]。然而绝大部分中药仅被证实有效,却不知如何起效。因此,进行作用机制研究大有必要。

为系统研究紫草素可能涉及的抗肿瘤机制,Chen等 [23] 对紫草素诱导的人结肠癌细胞(SW480)代谢改变进行分析,在代谢组学水平上发现多条显著改变的途径,与转录组学数据相结合,发现紫草素干预SW480后嘌呤代谢、精氨酸生物合成、谷胱甘肽代谢和氨基酸代谢等代谢途径受到显著影响。Wang等 [24] 为揭示薯蓣皂苷对人直肠癌细胞(SW480)的潜在药理作用及机制,使用靶向代谢组学分析发现薯蓣皂苷对SW480直肠癌细胞的代谢干预机制分别与D-谷氨酰胺和D-谷氨酸代谢、丙酮酸代谢、花生四烯酸代谢有关。为从代谢的角度阐明小檗碱抑制胰腺癌细胞(PC)增殖和转移并触发胰腺癌细胞凋亡的生物机制,Liu等 [26] 采用靶向细胞代谢组学方法结合RNA测序和表型成像技术来研究小檗碱对LPS处理的胰腺癌细胞的整体作用。根据结果推测细胞线粒体是小檗碱作用的主要部位,线粒体相关的TCA循环是实现小檗碱抗肿瘤功效的关键代谢途径,并将柠檬酸盐代谢确定为治疗PC的新靶点。Wang等 [27] 利用细胞代谢组学结合系统药理学方法对CKI治疗HCC的潜在机制进行研究,最终EGFR被确定为CKI抗HCC作用的核心靶点。华蟾素(HCS)注射液在中国临床治疗肝细胞癌(HCC)患者已有三十多年的历史,为探索HCS注射抑制HCC的潜在机制,Wu等 [28] 采用基于LC-MS的细胞代谢组学和脂质组学方法对细胞代谢物指纹图谱进行表征,识别与HCS注射对HCC抑制作用相关的潜在生物标志物,代谢途径分析表明,HCS注射液的抗HCC作用主要涉及谷胱甘肽、半胱氨酸和蛋氨酸、鞘脂、脂肪酸生物合成、氨基糖和核苷酸糖的代谢。前列腺癌是临床常见的恶性肿瘤,许多研究表明黄柏对前列腺癌具有重要的治疗意义,木兰素是黄柏的主要成分,研究木兰素对前列腺癌的作用机制具有重要意义,因此,Sun等 [29] 建立基于细胞代谢分析的综合分析策略对人前列腺癌细胞(22RV1)的代谢足迹进行分析,推测木兰素作用于22RV1细胞后,细胞的营养代谢和能量代谢受到干扰,为快速增殖提供物质基础,最终产生抑制作用。

3.3. 神经保护机制研究

神经系统对各种信息的处理均通过神经元完成,神经元的损伤是造成神经功能损害的基础 [30]。保护神经元免受损伤是治疗脑疾病过程中的关键。已有研究表明,很多单味中药、复方等都具有神经保护作用。

为探讨黄芩素对神经毒性的保护机制,Li等 [31] 采用LC-MS法分析黄芩素处理PC12神经细胞(Aβ25-35诱导损伤)后的代谢物改变。结果显示黄芩素可以调节5种代谢物,主要涉及精氨酸和脯氨酸代谢、烟酸盐和烟酰胺代谢,表明黄芩素对Aβ25-35诱导的PC12细胞神经毒性的保护机制可能与代谢紊乱相关。中药方剂栀子豉汤(ZZCD)已被报道对心理亚健康疾病具有潜在的保护作用,然而,仍然缺乏分子机制解释。Zhang等 [32] 基于细胞代谢组学和药理学相结合的策略,研究ZZCD的神经保护作用,40种代谢物被鉴定为神经毒性模型细胞的潜在生物标志物,ZZCD提取物可以调节这些潜在的生物标志物,逆转被干扰的代谢途径,从而发挥神经保护作用。脂质稳态对于神经发育、细胞信号传导和神经传递具有中药意义,脂质代谢的改变已在许多神经系统疾病和神经退行性疾病中得到证实。缝籽蓁甲醚(GM)是中药钩藤中的一种活性生物碱成分,已证实GM在神经保护活性方面具有很强的作用,其可通过调节葡萄糖代谢减少活性氧的产生,从而保护神经元免受氧化应激诱导的细胞死亡。然而,GM是否可以在氧化应激期间调节神经元脂质代谢尚不清楚。为探索GM是否调节氧化应激损伤神经元的脂质代谢,并确定这种神经保护作用的潜在机制,Sun等 [33] 在小鼠海马神经元细胞(HT-22)中使用谷氨酸诱导氧化毒性模型,并进行脂质代谢组学分析。脂质组学分析表明,谷氨酸处理导致多种脂质类别发生不同程度的变化,例如脂肪酸、三酰基甘油、鞘磷脂、心磷脂、溶血磷脂酰胆碱,而GM治疗可以将谷氨酸诱导的脂质紊乱显著回调至稳态水平。这些发现证明GM通过调节脂质代谢来抗谷氨酸诱导的神经元氧化毒性。

3.4. 其他作用机制研究

此外,在中药的其他作用机制方面细胞代谢组学也多被使用,如中药发挥抗氧化、抗缺氧、抗菌等药理作用的机制 [14] [34] [35]。譬如,心可舒(XKS)对改善心肌缺血/再灌注(I/R)损伤具有保护作用,为深入了解XKS抗缺氧/复氧(H/R)损伤的机制,Sun等 [34] 应用H9c2心肌细胞建立H/R模型模拟体内观察到的I/R损伤,采用基于LC-MS的细胞代谢组学方法研究XKS抗H9c2心肌细胞H/R损伤的机制。为探索香菇多糖的抗菌机制,Lan等 [35] 采用基于LC-MS的代谢组学分析,发现香菇多糖能破坏金黄色葡萄球菌中糖酵解和三羧酸循环途径的代谢。

4. 小结与展望

细胞代谢组学作为代谢组学研究的一个新方向,在中药作用机制研究中的应用越来越广泛。但基于细胞代谢组学的中药作用机制研究尚存在一些不足:疾病的发生发展涉及的细胞类型较多,细胞类型的合理选择是确保研究结果可靠真实的关键,单一类型细胞的代谢组学结果是否能反正机体的真实状态是细胞代谢组学的一个难题;对于非单体成分的作用机制研究,尤其是中药、复方提取物,因其未知成分太多,未经筛选的直接作用于细胞,是否能反应中药或复方在体内的真实状态,是另一个值得思考的问题。目前,细胞代谢组学研究的新趋势是与其他组学技术或生化技术相结合,多方面多层次的分析能更准确地研究中药机制。相信随着生命科学技术的不断发展,细胞代谢组学必将推动中医药研究取得更多成果。

NOTES

*通讯作者。

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