α- 酮戊二酸的抗氧化作用及其应用

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The Antioxidative Function of Alpha-Ketoglutarate and Its Applications


Abstract

摘要

Alpha-ketoglutarate (AKG) is a crucial intermediate of the Krebs cycle and plays a critical role in multiple metabolic processes in animals and humans. Of note, AKG contributes to the oxidation of nutrients (i.e., amino acids, glucose, fatty acids) and then provides energy for cell processes. As a precursor of glutamate and glutamine, AKG acts as an antioxidant agent as it directly reacts with hydrogen peroxide with formation of succinate, water, and carbon dioxide; meanwhile, it discharges plenty of ATP by oxidative decarboxylation. Recent studies also show that AKG has alleviative effect on oxidative stress as a source of energy and an antioxidant in mammalian cells. In this review, we highlight recent advances in the antioxidative function of AKG and its applications in animals and humans.

阿尔法酮戊二酸(AKG)是克雷布斯循环的关键中间体,在动物和人类的多种代谢过程中起着关键作用。值得注意的是,AKG 有助于营养物质(如氨基酸、葡萄糖、脂肪酸)的氧化,然后为细胞过程提供能量。作为谷氨酸和谷氨酰胺的前体,AKG 作为一种抗氧化剂,直接与过氧化氢反应,形成琥珀酸、水和二氧化碳; 同时,它通过氧化脱羧释放大量的 ATP。最近的研究也表明,AKG 对哺乳动物细胞中作为能量来源和抗氧化剂的氧化应激有缓解作用。本文综述了 AKG 的抗氧化功能及其在动物和人体中的应用。

1. Introduction

1. 引言

Reactive oxygen species (ROS) are oxygen-containing chemical species including superoxide anion, hydrogen peroxide (H2O2), and hydroxyl radicals, and most of which are produced by mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [1]. Of note, excess of ROS could lead to oxidative stress in cells. Oxidative stress is associated with the disorder of proteins, lipid oxidation, and nucleic acid breaks, which may further impair cellular physiological functions. Numerous studies suggested that oxidative stress may result in some pathogenic diseases, such as cancer [2], neurological disorders [3], age-related diseases [4], atherosclerosis [5], inflammation [6], and cardiovascular diseases [7]. Mammals have evolved a series of antioxidant defenses to protect vital biomolecules from oxidative damage. On the one hand, antioxidant agents, such as antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), or nonenzymatic agents, such as glutathione (GSH), vitamin C, and vitamin E, can clean off most of ROS [8]. On the other hand, the excess ROS can also activate many signaling pathways such as mitogen-activated protein kinase (MAPKs), NF-erythroid 2-related factor/antioxidant response element (Nrf2/ARE), and peroxisome proliferator-activated receptor γ (PPARγ), which play a vital role in cellular redox homeostasis and contribute to antioxidative defense [9].

活性氧类(ROS)是含氧的化学物种,包括超氧阴离子、过氧化氢(H2O2)和羟基自由基,其中大部分是由线粒体和烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶产生的。值得注意的是,过量的活性氧会导致细胞中的氧化应激。氧化应激与蛋白质紊乱、脂质氧化和核酸分解有关,这可能进一步损害细胞的生理功能。许多研究表明,氧化应激可能导致一些致病性疾病,如癌症、神经系统疾病、年龄相关疾病、动脉粥样硬化、炎症和心血管疾病。哺乳动物已经进化出一系列的抗氧化防御机制来保护重要的生物分子免受氧化损伤。一方面,抗氧化剂,如抗氧化酶,如超氧化物歧化酶,过氧化氢酶,谷胱甘肽过氧化物酶,或非酶代理,如谷胱甘肽,维生素 c,维生素 e,可以清除大部分的活性氧。另一方面,过量的活性氧还可以激活许多信号通路,如丝裂原活化蛋白激酶(MAPKs)、 nf-throid2相关因子/抗氧化反应元件(Nrf2/ARE)和过氧化物酶体增殖物活化受体 γ (pparγ) ,这些信号通路在细胞氧化还原稳态中起重要作用,有助于抗氧化防御[9]。

Glutamate, as a precursor of GSH, exerts alleviative effects on oxidative stress in medicine and surgery [10]. AKG, as a precursor of glutamine, is cheaper and more stable than glutamine and acts as an antioxidant instead of glutamine in many cellular processes. Many reports demonstrated that AKG can be converted into glutamine by glutamate dehydrogenase (GDH) and glutamine synthetase (GS), which is a sign of antioxidative function. It is evident that AKG could improve antioxidative capacity by promoting glutamine content and antioxidative systems [1112]. Additionally, Chen et al. showed that AKG could significantly improve SOD activity but reduce malondialdehyde (MDA) level, suggesting an improvement of intestinal antioxidative capacity [13]. Recently, more and more studies indicated that AKG could improve antioxidative function against oxidative imbalance in cells, which further contributed to the prevention and treatment of various diseases induced by oxidative stress. Therefore, in this review, we aim to summarize the recent advances of the antioxidative function of AKG and its applications.

谷氨酸作为谷胱甘肽的前体,在药物和手术中对氧化应激有缓解作用[10]。AKG,作为谷氨酰胺的前体,比谷氨酰胺更便宜,更稳定,在许多细胞过程中作为抗氧化剂而不是谷氨酰胺。许多研究表明,谷氨酸脱氢酶和谷氨酰胺合成酶可以将 AKG 转化为谷氨酰胺,这是抗氧化功能的一个标志。显然,AKG 可以通过促进谷氨酰胺含量和抗氧化系统提高抗氧化能力[11,12]。此外,Chen 等人的研究表明,AKG 能显著提高 SOD 活性,但降低丙二醛(MDA)水平,提示了肠道抗氧化能力的提高。近年来,越来越多的研究表明,AKG 可以改善细胞的抗氧化功能失衡,进一步有助于氧化应激诱导的各种疾病的防治。因此,本文就 AKG 的抗氧化功能及其应用的研究进展作一综述。

2. Biochemical Characteristics of AKG

2. AKG 的生化特性

AKG is a weak acid containing two carboxyl groups and a ketone group which is also called 2-ketoglutaric acid or 2-oxoglutaric acid. AKG possesses many physiological functions. On the one hand, AKG could react with ammonia and then be converted into glutamate; subsequently, the glutamate further reacts with ammonia and generates glutamine (Figure 1). On the other hand, AKG reacts with H2O2 as a result of the conversion of succinate, carbon dioxide (CO2), and water (H2O), eventually achieving elimination of H2O2 (Figure 2) [14]. Additionally, AKG could produce plenty of ATP in the TCA cycle and provide energy for intestinal cell processes. Furthermore, AKG performs positive effects on oxidative stress damage in intestinal mucosal cells and contributes to cell redox homeostasis [15]. It has been reported that enteral AKG was oxidized and used by intestinal mucosa, thereby, as an energy donor and antioxidant agent via the TCA cycle. Apart from the above, AKG also exerts antioxidative defense by enzymatic systems and nonenzymatic oxidative decarboxylation.

AKG 是一种含有两个羧基和一个酮基的弱酸,也称为 α-酮戊二酸或 α-酮戊二酸。AKG 具有多种生理功能。一方面,AKG 可以与氨反应,然后转化为谷氨酸; 随后,谷氨酸进一步与氨反应,生成谷氨酰胺(图1)。另一方面,AKG 通过琥珀酸、二氧化碳(CO2)和水(H2O)的转化与 H2O2发生反应,最终消除 H2O2(图2)[14]。此外,AKG 可以在 TCA 循环中产生大量的 ATP,为肠道细胞的生长过程提供能量。此外,AKG 对肠粘膜细胞的氧化应激损伤有积极作用,并有助于细胞氧化还原稳态[15]。据报道,肠内 AKG 被肠黏膜氧化和利用,从而通过 TCA 循环作为能量供应者和抗氧化剂。除此之外,AKG 还通过酶系统和非酶氧化脱羧来发挥抗氧化作用。

Figure 1 图1The conversion of AKG into glutamate and glutamine. AKG 转化为谷氨酸和谷氨酰胺的研究

Figure 2 图2Nonenzymatic oxidative decarboxylation of AKG in hydrogen peroxide decomposition. AKG 的非酶氧化脱羧在过氧化氢分解中的作用

3. Antioxidative Function of AKG

3. AKG 的抗氧化作用

3.1. Antioxidants Activities
3.1. 抗氧化剂活性

The balance between oxidants and antioxidants plays an important role in physiological functions in cells and biomolecules. Antioxidant system comprises enzymatic and nonenzymatic agents. Antioxidative enzymes include SOD, CAT, GSH-Px, and nonenzymatic agents include GSH, vitamin C, vitamin E [10]. AKG is an antioxidant substance which exhibits a vital role in scavenging ROS in organism [16]. Growing studies suggest that AKG serves as a natural antidote of scavenging ammonia by exerting its antioxidative capacity. It has been reported that AKG inhalation showed a protective role in ammonia-induced lung damage in rats [17]. The mechanism may be caused by reducing the levels of lactate dehydrogenase (LDH) and MDA and improving the activities of SOD and CAT and GSH level. Lipid peroxidation is susceptible to ammonia or trauma like burns and eventually produces MDA resulting in membrane injury and even cell apoptosis, while antioxidants such as SOD and GSH-Px are beneficial to prevent the lipid peroxidation and injury [18]. AKG could prevent the lipid peroxidation by increasing SOD, GSH-Px, and CAT activities to facilitate fat metabolism, and then alleviate ethanol-induced hepatotoxicity and hyperammonemia induced by ammonium acetate in rats [1920]. Similarly, AKG also performs chemopreventive role in hepatocarcinogenesis induced by N-nitrosodiethylamine (NDEA) in rats by modulating the levels of antioxidants and lipid peroxide to access normal levels [21]. Furthermore, AKG shows high resistance to ammonia-N stress in hybrid sturgeons as it enhances antioxidant enzymes activity and HSP 70 and HSP 90 gene expression [22]. Besides, cyanide-induced oxidative stress could lead to neurotoxicity, the lipid peroxidation, and dysfunction of membrane especially in brain and kidney of animals like rats [23]. And cyanide is evident to inhibit antioxidative defense such as reducing SOD activity and GSH level [24]. Interestingly, AKG is considered as a natural antagonist of cyanide poisoning because of its chemical structure that is able to bind with cyanide to produce cyanohydrin and further prevent cyanide poisoning or cyanide lethality [2526]. In rat in vitro and vivo models, AKG reduces GSH depletion and DNA damage induced by cyanide [27]. Furthermore, studies demonstrate that AKG alone could prevent brain and liver from cyanide-induced oxidative damage by increasing GSH, SOD, and GSH-Px levels and reducing MDA level in rats, especially when combined with sodium thiosulfate [2829]. Additionally, a recent study indicates that AKG could enhance freeze-thaw tolerance and prevent cell death induced by carbohydrate stress in yeast, and the protective pathway may be involved in the enhanced antioxidant defense [30].

氧化剂和抗氧化剂之间的平衡在细胞和生物分子的生理功能中起着重要作用。抗氧化系统包括酶和非酶两种试剂。抗氧化酶包括超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px) ,非酶促剂包括谷胱甘肽(GSH)、维生素 c、维生素 e [10]。AKG 是一种抗氧化物质,在清除机体活性氧方面发挥重要作用[16]。越来越多的研究表明,AKG 作为一种天然的解毒剂,通过发挥其抗氧化能力清除氨。据报道,AKG 吸入对氨诱导的大鼠肺损伤有保护作用[17]。其作用机制可能与降低乳酸脱氢酶和 MDA 含量,提高 SOD、 CAT 和 GSH 含量有关。脂质过氧化易受氨或烧伤等创伤的影响,最终产生导致膜损伤甚至细胞凋亡的丙二醛,而 SOD 和 GSH-Px 等抗氧化剂则有利于防止脂质过氧化和损伤。AKG 可通过提高 SOD、 GSH-Px 和 CAT 活性,促进脂肪代谢,从而抑制脂质过氧化,进而减轻酒精性肝毒性和乙酸铵所致大鼠高氨血症[19,20]。同样,AKG 也可通过调节抗氧化剂和脂质过氧化物的水平达到正常水平,在 n- 亚硝基二乙胺(NDEA)诱发的大鼠肝癌发生中起预防作用[21]。此外,AKG 显示高抗氨氮胁迫杂交鲟鱼因为它提高了抗氧化酶活性和 hsp70和 hsp90基因表达[22]。此外,氰化物引起的氧化应激可能导致神经毒性、脂质过氧化和膜功能障碍,尤其是在大鼠等动物的脑和肾中。氰化物具有明显的抗氧化防御作用,如降低 SOD 活性和 GSH 水平[24]。有趣的是,AKG 被认为是氰化物中毒的天然拮抗剂,因为它的化学结构能与氰化物结合产生氰醇,进一步防止氰化物中毒或氰化物的致死性[25,26]。在大鼠体内外模型中,AKG 可减少谷胱甘肽损耗和氰化物引起的 DNA 损伤[27]。此外,研究表明,单独使用 AKG 可以通过提高谷胱甘肽、超氧化物歧化酶和谷胱甘肽过氧化物酶的水平,降低大鼠体内丙二醛的水平,特别是与硫代硫酸钠联合使用时,可以预防氰化物引起的大脑和肝脏的氧化损伤。此外,最近的研究表明,AKG 可以增强酵母的抗冻融耐受性,防止碳水化合物胁迫诱导的细胞死亡,其保护途径可能参与了抗氧化防御的增强[30]。

3.2. Nonenzymatic Oxidative Decarboxylation in H2O2 Decomposition
3.2. H2O2分解过程中的非酶氧化脱羧

In regard of antioxidative defense, some studies show that AKG exerts its function by other redox regulatory mechanisms rather than antioxidant activities. A number of studies demonstrate that AKG acts as a source of energy and antioxidant agent on improving physiological metabolism and scavenging ROS to alleviate oxidative stress via nonenzymatic oxidative decarboxylation in H2O2 decomposition. Hydrogen peroxide, one of ROS, is a weak oxidant and cytotoxic and easily causes oxidative stress injury in cells such as cell membrane damage and DNA alterations [31]. Indeed, pyruvate and α-ketoacids exhibit protective effects on H2O2-induced toxicity in vivo and vitro and can cross the blood-brain barrier and scavenge H2O2, which provide a novel therapeutic mode against H2O2-induced brain pathologies. The mechanism may be due to the nonenzymatic oxidative decarboxylation in which ketone group in α-carbon atom is combined with H2O2 to form corresponding carboxylic acid, CO2, and H2O. AKG serves as a key intermediate in the TCA cycle and participates in nonenzymatic oxidative decarboxylation in the H2O2decomposition. It has been demonstrated that AKG significantly elevated antioxidative capacity by decreasing the level of H2O2 in the liver and intestinal mucosa of ducks [32]. Also, AKG performs a protective role in intestinal cells damage induced by H2O2 through mitochondria pathway [33]. Similarly, the protective action of AKG is noticed in alleviating toxic effects of H2O2 inDrosophila melanogaster, other animals, and humans, which provides a strong evidence for the H2O2-scavenging ability of AKG [34]. Thus, AKG can be used as a potent scavenger in nonenzymatic oxidative decarboxylation in H2O2decomposition.

在抗氧化防御方面,一些研究表明,AKG 通过其他氧化还原调节机制发挥作用,而不是通过抗氧化活性发挥作用。大量研究表明,AKG 作为能量和抗氧化剂的来源,改善生理代谢和清除活性氧,以减轻氧化应激,通过非酶氧化脱羧在 H2O2分解。过氧化氢,一种活性氧,是一种弱氧化剂和细胞毒性,很容易导致细胞氧化应激损伤,如细胞膜损伤和 DNA 改变。实际上,丙酮酸和 α- 酮酸对 H2O2所致的体内外毒性具有保护作用,并能通过血脑屏障和清除 H2O2,为治疗 H2O2所致的脑病提供了一种新的治疗模式。其机理可能是由于 α 碳原子中酮基与过氧化氢结合形成相应的羧酸、二氧化碳和过氧化氢的非酶氧化脱羧。AKG 作为 TCA 循环的关键中间体,参与 H2O2分解的非酶氧化脱羧。研究表明,AKG 通过降低鸭肝脏和肠粘膜中 H2O2的水平,显著提高了鸭的抗氧化能力[32]。同时,AKG 在 H2O2通过线粒体途径引起的肠细胞损伤中起保护作用[33]。同样地,AKG 的保护作用可以减轻 H2O2对黑腹果蝇、其他动物和人类的毒性作用,这为 AKG 清除 H2O2的能力提供了有力的证据[34]。因此,AKG 可以作为 H2O2分解过程中非酶氧化脱羧的有效清除剂。

4. The Applications of AKG in Animals and Humans

4. AKG 在动物和人类中的应用

AKG has been widely used in animals and humans as a feed additive and medicine. In animal industry, AKG could effectively improve growth performance, nitrogen utilization, immunity, bone development, intestinal mucosal injury, and oxidative system [3539]. In humans, AKG is extensively used in trauma, aged diseases, postoperative recovery, and other nutritional diseases [40]. In terms of antioxidative function, AKG exhibits a crucial role in multiple diseases involved in aging, cancer, cardiovascular diseases, and neurological diseases. It has been reported that AKG developed its antioxidant capacity to fight against ethanol toxicity and enhance cold tolerance in the model of Drosophila, which provided an effective therapy against ethanol and alcohol poisoning in animals and humans [4142]. Similar protective effect is noticed in lipopolysaccharide-induced liver injury in which AKG provides a new intervention to alleviate liver damage in young pigs [43]. AKG also maintains redox state stabilization for antioxidant defense. Indeed, AKG oxidation plays a beneficial role in maintaining the levels of reductive carboxylation to handle mitochondrial defects in cancer cells [44]. Besides, oral administration of AKG improves blood vessel elasticity by exerting its antioxidant in aging organisms [45]. Additionally, AKG could facilitate the rate of GSH synthesis in human erythrocytes [46]. AKG has been identified to effectively decrease the incidence of cataracts induced by sodium selenite in rat and acted as a scavenger of ROS [47]. Moreover, AKG functions as a neuroprotective agent in ischemic pathology of hippocampus [48]. Furthermore, a novel study demonstrates that AKG could regulate organismal lifespan and prevent age-related diseases by regulating cellular energy metabolism [49]. Interestingly, apart from antioxidative function, AKG is characterized by prooxidative property which can generate active complexes with iron in rat brain homogenates [5051]. Under mild oxidative stress, it results in activating antioxidant system of AKG, thus displaying its protective effects such as strengthening resistance of the yeast cells to oxidative stress [52].

AKG 作为一种饲料添加剂和药物已经在动物和人类中广泛应用。在动物工业中,AKG 能有效地改善生长性能、氮利用、免疫、骨发育、肠粘膜损伤和氧化系统[35-39]。在人类中,AKG 被广泛用于创伤、老年疾病、术后恢复和其他营养性疾病[40]。在抗氧化功能方面,AKG 在衰老、癌症、心血管疾病和神经系统疾病等多种疾病中起着至关重要的作用。在果蝇模型中发现 AKG 具有抗乙醇毒性和增强耐寒性的抗氧化能力,为动物和人类抗乙醇和酒精中毒提供了有效的治疗方法[41,42]。在脂多糖诱导的肝损伤中也发现了类似的保护作用,AKG 为减轻幼猪肝损伤提供了一种新的干预手段[43]。AKG 还维持氧化还原状态稳定抗氧化防御。事实上,AKG 氧化在维持还原羧化水平以处理癌细胞线粒体缺陷方面发挥了有益的作用[44]。此外,AKG 的口服给药通过在衰老生物体中施加抗氧化剂来改善血管弹性[45]。此外,AKG 还能促进人红细胞中谷胱甘肽的合成[46]。已证实 AKG 能有效降低大鼠亚硒酸钠所致白内障的发生率,并能清除 ROS [47]。此外,AKG 在海马缺血病理学中起着神经保护剂的作用[48]。此外,一项新的研究表明,AKG 可以通过调节细胞能量代谢来调节机体寿命和预防与年龄有关的疾病。有趣的是,AKG 除了具有抗氧化功能外,还具有拥有属性促氧化功能,能够在大鼠脑匀浆中产生活性铁配合物[50,51]。在温和的氧化应激下,它能激活 AKG 的抗氧化系统,从而显示其保护作用,例如增强酵母细胞对氧化应激的抗性。

5. Summary and Perspective

5. 总结和观点

AKG serves as a pivotal intermediate and is widely applied in animals and humans. Particularly, AKG primarily exerts its antioxidative function by the following: (1) enhancing antioxidative enzymes activities and nonenzymatic agent levels against oxidative stress and lipid peroxidation, especially in intervention of ammonia and cyanide poisoning; (2) participating in nonenzymatic oxidative decarboxylation in H2O2 decomposition to scavenge ROS and protect organism from various ROS-induced diseases. And AKG provides a promising therapeutic intervention for clinical diseases in animals and humans (Figure 3). Besides the above antioxidative pathways, Nrf2/ARE is an important regulator of antioxidative process that aids to keep redox homeostasis, and it has been proved to perform a vital role in various diseases (i.e., liver injury, traumatic brain injury, and inflammation) induced by oxidative stress [53]. Of particular interest, glutamine has been verified to improve the gene expression of Nrf2 by activating Nrf2/ARE signaling pathway to suppress ROS generation, elevate GSH levels, and prevent apoptosis in intestine [5455]. However, as a precursor of glutamine, whether AKG could directly activate Nrf2/ARE signaling pathway to alleviate oxidative stress or not, relevant research about that is not reported and further study is needed.

AKG 是一种重要的中间体,广泛应用于动物和人类。其中,AKG 主要通过以下途径发挥其抗氧化作用: (1)提高抗氧化酶活性和非酶活性水平,尤其是对氧化应激和脂质过氧化的干预作用; (2)参与 H2O2非酶氧化脱羧的分解,清除 ROS,保护机体免受 ROS 引起的各种疾病的侵袭。AKG 为动物和人类的临床疾病提供了一种有希望的治疗干预(图3)。除了上述的抗氧化途径外,Nrf2/ARE 还是一个重要的抗氧化过程调节因子,有助于维持氧化还原稳态,而且已被证明在氧化应激诱导的各种疾病中起着重要作用。谷氨酰胺通过激活 Nrf2/ARE 信号通路来抑制活性氧产生,提高谷胱甘肽水平,防止肠道细胞凋亡,从而改善 Nrf2的基因表达[54,55]。但作为谷氨酰胺的前体,AKG 能否直接激活 Nrf2/ARE 信号通路以减轻氧化应激抑制作用尚未见报道,有待进一步研究。

Figure 3 图3The antioxidative function of AKG and its applications. ①: antioxidative enzymes activites; ②: nonenzymatic oxidative decarboxylation in hydrogen peroxide decomposition. AKG 的抗氧化功能及其应用: 1: 抗氧化酶活性; 2: 非酶氧化脱羧在过氧化氢分解中的作用

Abbreviations

缩写

AKG:Alpha-ketoglutarate α- 酮戊二酸
ROS: 活性氧:Reactive oxygen species 活性氧类
H2O2:Hydrogen peroxide 过氧化氢
NADPH:Nicotinamide adenine dinucleotide phosphate 烟酰胺腺嘌呤二核苷酸磷酸
SOD: 超氧化物歧化酶:Superoxide dismutase 超氧化物歧化酶
CAT: 猫:Catalase 过氧化氢酶
GSH-Px: 返回文章页面谷歌地图:Glutathione peroxidase 谷胱甘肽过氧化物酶
GSH: 谷胱甘肽:Glutathione 谷胱甘肽
MAPKs:Mitogen-activated protein kinase 有丝分裂原活化蛋白激酶
Nrf2/ARE:NF-Erythroid 2-related factor/antioxidant response element 促红细胞 nf-2相关因子/抗氧化反应元件
PPARγ:Peroxisome proliferator-activated receptor 过氧化物酶体增殖物活化受体γ
GDH: 广东省卫生厅:Glutamate dehydrogenase 谷氨酸脱氢酶
GS: 高级管理人员:Glutamine synthetase 谷氨酰胺合成酶
MDA:Malondialdehyde 丙二醛
CO 一氧化碳2:Carbon dioxide 二氧化碳
H2O:Water 水
LDH: 乳酸脱氢酶:Lactate dehydrogenase 乳酸脱氢酶
NDEA: 返回文章页面 NDEA:N-Nitrosodiethylamine. N- 亚硝基二乙胺

Conflicts of Interest

利益冲突

The authors declare that there are no conflicts of interest regarding the publication of this paper.

作者声明,在发表这篇论文方面没有利益冲突。

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