α-mangostin 通过抑制 NAMPT/NAD 控制的炎症反应减轻脂多糖诱导的大鼠急性肺损伤

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α-Mangostin Alleviated Lipopolysaccharide Induced Acute Lung Injury in Rats by Suppressing NAMPT/NAD Controlled Inflammatory Reactions


Abstract

摘要

α-Mangostin (MAN) is a bioactive xanthone isolated from mangosteen. This study was designed to investigate its therapeutic effects on acute lung injury (ALI) and explore the underlying mechanisms of action. Rats from treatment groups were subject to oral administration of MAN for 3 consecutive days beforehand, and then ALI was induced in all the rats except for normal controls via an intraperitoneal injection with lipopolysaccharide. The severity of disease was evaluated by histological examination and hematological analysis. Protein expressions in tissues and cells were examined with immunohistochemical and immunoblotting methods, respectively. The levels of cytokines and nicotinamide adenine dinucleotide (NAD) were determined using ELISA and colorimetric kits, respectively. It was found that MAN treatment significantly improved histological conditions, reduced leucocytes counts, relieved oxidative stress, and declined TNF-α levels in ALI rats. Meanwhile, MAN treatment decreased expressions of nicotinamide phosphoribosyltransferase (NAMPT) and Sirt1 both in vivo and in vitro, which was accompanied with a synchronized decline of NAD and TNF-α. Immunoblotting assay further showed that MAN downregulated HMGB1, TLR4, and p-p65 in RAW 264.7 cells. MAN induced declines of both HMGB1/TLR4/p-p65 and TNF-α were substantially reversed by cotreatment with nicotinamide mononucleotide or NAD. These results suggest that downregulation of NAMPT/NAD by MAN treatments contributes to the alleviation of TLR4/NF-κB-mediated inflammations in macrophage, which is essential for amelioration of ALI in rats.

α-mangostin (MAN)是从山竹中分离得到的一种生物活性黄酮类化合物。本研究旨在探讨其对急性肺损伤(ALI)的治疗作用,并探讨其作用机制。治疗组的大鼠预先连续3天服用口服给药,然后通过腹腔注射脂多糖,在除正常对照组以外的所有大鼠中诱发 ALI。组织学检查和血液学分析评价病情严重程度。免疫组织化学和免疫印迹法检测组织和细胞中蛋白质的表达。分别用酶联免疫吸附试剂盒和比色试剂盒测定细胞因子和烟酰胺腺嘌呤二核苷酸的水平。研究发现,MAN 治疗明显改善了 ALI 大鼠的组织学状况,减少了白细胞计数,减轻了氧化应激,并降低了 tnf-α 水平。同时,MAN 处理降低体内外烟酰胺磷酸核糖基转移酶(NAMPT)和 Sirt1的表达,伴随 NAD 和 tnf-α 的同步下降。免疫印迹实验进一步表明 MAN 下调 RAW 264.7细胞中的 HMGB1、 TLR4和 p-p65。MAN 诱导的 hmgb1/tlr4/p-p65和 tnf-α 的下降在与烟酰胺单核苷酸或 NAD 联合治疗后基本上得到逆转。这些结果提示,MAN 处理下调 NAMPT/NAD 有助于减轻 tlr4/nf-κb 介导的巨噬细胞炎症,这是改善 ALI 所必需的。

1. Introduction

1. 引言

Xanthone is a naturally occurring polyphenol with the hybrid structure of flavone and anthraquinone. Similarly, xanthones also possess various notable bioactivities and have drawn intense attentions worldwide [1]. Increasing researches are conducted to synthesize more effective xanthone derivatives and to explore their possible clinical applications [12]. Among the well investigated bioactivities, the most eye-catching finding is the encouraging potential in treatments of inflammatory diseases [13].

黄酮是一种天然存在的多酚类化合物,具有黄酮和蒽醌的杂化结构。同样,黄酮类化合物也具有多种显著的生物活性,已引起世界范围内的广泛关注[1]。越来越多的研究正在合成更有效的黄酮衍生物,并探索其可能的临床应用[1,2]。在众多的生物活性研究中,最引人注目的发现是在治疗炎症性疾病方面的令人鼓舞的潜力[1-3]。

Although numerous studies have solidly confirmed the anti-inflammatory properties of xanthones both in vivo and in vitro, the underlying mechanisms are still not well understood. It is believed that, as a typical polyphenol, the beneficial effects of xanthone on inflammations result from its antioxidative activities, and downregulation of certain oxidative stress sensitive pathways such as COX-2, NF-κB, and MAPKs is responsible for reduced inflammatory reactions [48]. Indeed, these molecular events are essential for the alleviation of inflammatory manifestations, as all of them are deeply implicated in the development and progression of inflammatory diseases. Consistent with these findings, we also reported that xanthone derivatives substantially inhibited the activation of NF-κB in rodent arthritis models, which subsequently alleviated the arthritis severity [911]. However, the pathways discussed above just serve as part of downstream in the inflammatory cascade, and identification of the molecular targets of xanthones contributing to these critical signaling changes is important for better understanding of their anti-inflammatory mechanisms.

虽然大量的研究已经证实了黄酮类化合物在体内和体外的抗炎作用,但其作用机制尚不清楚。作为一种典型的多酚类化合物,黄酮对炎症的有益作用来源于其抗氧化活性,以及下调某些氧化应激敏感通路,如 COX-2、 NF-κB 和 mapk,从而减少炎症反应[4-8]。事实上,这些分子事件对于减轻炎症表现是必不可少的,因为所有这些都与炎症性疾病的发展和进展有着深刻的联系。与这些发现一致的是,我们还报道了黄酮衍生物在啮齿类关节炎模型中实质上抑制了 NF-κB 的激活,从而减轻了关节炎的严重程度[9-11]。然而,上面讨论的通路只是炎症级联反应下游的一部分,确定促成这些关键信号转导变化的黄酮类分子靶点对于更好地理解其抗炎机制很重要。

Recently, we carried out a metabolomics study to elucidate therapeutic mechanisms of α-mangostin (MAN, a mangosteen derived prenylated xanthone) on experimental arthritis and found that MAN compromised the biosynthesis of nicotinamide adenine dinucleotide (NAD) in fibroblast-like synoviocyte (FLS) via downregulation of nicotinamide phosphoribosyltransferase (NAMPT, a rate-limiting enzyme in the salvage pathway) and subsequently protected the joints from destruction (unpublished). We believe that the decrease in energy metabolism during this process is critical for the clinical outcome. However, the effects of MAN in FLS could have little to do with the alleviated systemic symptoms, because most of the circulating extracellular NAMPT (eNAMPT) and NAD are contributed by leucocytes [1213]. From this point of view, manipulation of NAMPT/NAD in blood cells by xanthone would be much more meaningful in treatments of systemic inflammatory diseases, and exploration of such knowledge will be beneficial to further understand its anti-inflammatory mechanisms. Therefore, in this study, we investigated the therapeutic effects of MAN on lipopolysaccharide induced acute lung injury (ALI) in rats and analyzed its relevance to the regulation of NAMPT/NAD mediated signaling transductions in leucocytes. The results obtained suggest that MAN treatment significantly downregulates NAMPT/NAD and intervenes in energy metabolism, which largely accounts for the alleviation of TLR4/NF-κB mediated inflammations.

最近,我们进行了一项新陈代谢组学研究,以阐明 α- 山竹苷(MAN,一种山竹烯衍生的异冠醚酮)对实验性关节炎的治疗机制,发现 MAN 通过下调烟酰胺磷酸核糖转移酶(NAMPT,一种补救途径中的限速酶)抑制成纤维样滑膜细胞(FLS)中烟酰胺腺嘌呤二核苷酸的生物合成,从而保护关节免受破坏(未发表)。我们相信在这个过程中能量代谢的减少对临床结果是至关重要的。然而,由于大部分循环中的细胞外 NAMPT (eNAMPT)和 NAD 是由白细胞贡献的,所以 MAN 在 FLS 中的作用可能与全身症状的缓解无关。从这个角度来看,黄酮对血细胞 NAMPT/NAD 的调控在全身炎症性疾病的治疗中具有重要意义,探讨这方面的知识将有助于进一步了解其抗炎机制。因此,在这项研究中,我们研究了 MAN 对脂多糖诱导的大鼠急性肺损伤(ALI)的治疗作用,并分析其与 NAMPT/NAD 介导的白细胞信号转导的调节的相关性。结果提示,MAN 治疗可明显降低 NAMPT/NAD 的水平,并干预能量代谢,这在很大程度上解释了 TLR4/NF-κB 介导的炎症反应。

2. Materials and Methods

2. 材料和方法

2.1. Chemicals and Reagents
2.1. 化学品和试剂

MAN with the purity of 98% (based on HPLC-UVD analysis) was brought from SanHerb Bioscience Inc. (Chengdu, Sichuan, China). Lipopolysaccharide (LPS, from the Gram-negative bacteria E. coli 055:B5), phosphate buffered saline (PBS), BCA protein quantitative kit, NAD, nicotinamide mononucleotide (NMN), HRP conjugated streptavidin, and HRP/biotin conjugated secondary antibodies were purchased from KeyGen Biotech (Nanjing, Jiangsu, China). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM) were from TianHang Biotechnology (Hangzhou, Zhejiang, China). Enhanced chemiluminescence (ECL) detection kit, defatted milk powder, and bovine serum albumin (BSA) were from Thermo Scientific (Rockford, IL, USA). Primary antibodies used in both western blot and immunohistochemical assays were obtained from Affinity Biosciences (Cincinnati, OH, USA). Ultrapure water was prepared by using a Milli-Q purification system (Millipore, Bedford, MA, USA).

采用高效液相色谱-紫外分光光度法(HPLC-UVD)分析,纯度为98% 的 MAN 来自四川成都三禾生物科技有限公司。脂多糖(LPS,来自革兰阴性菌 e. coli 055: B5)、磷酸盐缓冲生理盐水(PBS)、 BCA 蛋白定量试剂盒、 NAD、烟酰胺单核苷酸(NMN)、 HRP 结合链霉亲和素及 HRP/生物素结合的二级抗体均购自 KeyGen Biotech (江苏南京)。胎牛血清(FBS)和杜氏改良鹰培养基(DMEM)来自浙江杭州天航生物技术有限公司。增强型化学发光(ECL)检测试剂盒、脱脂奶粉和牛血清白蛋白(BSA)来自 Thermo Scientific (罗克福德,IL,USA)。从 Affinity Biosciences (美国辛辛那提)获得了用于蛋白印迹和免疫组织化学检测的初级抗体。采用 Milli-Q 净化系统(美国马萨诸塞州贝德福德市)制备超纯水。

2.2. LPS Challenge in Rats and Treatments
2.2. LPS 对大鼠的攻击及其治疗

Male SD rats (about 160 g in weight, obtained from Qinglongshan Laboratory Animal Company, Nanjing, Jiangsu, China) were used in this study. All the animal protocols described below were approved by the Ethical Committee of Yijishan Hospital, Wannan Medical College (No. YJS 2018-5-011), and were strictly in accordance with the Guide for the Care and Use of Laboratory Animals (US National Research Council, 2011). Before experimental procedures, all the animals were kept for 7 days to get accommodated. Afterwards, the rats were assigned into 4 groups randomly. Two MAN treated groups received MAN treatments by gavage (suspended in CMC-Na solution) for 3 consecutive days and 15 and 45 mg/kg/day served as the low and high doses, respectively. The other two groups were used as normal healthy and ALI model controls and treated with 0.5% CMC-Na instead. One hour after the last administration, lung injury was induced in all rats except for the normal controls via an intraperitoneal injection of LPS at the dose of 10 mg/kg. The animals were then intensely observed and sacrificed under anaesthesia with chloral hydrate 12 h later. The whole blood was collected through abdominal aorta into anticoagulation/promoting coagulation tubes. Anticoagulated blood was used for complete blood count (CBC) analysis on an automated hematology system (ADVIA 120, Bayer Diagnostics, German), and the coagulated blood was used to separate serum for the serological analyses. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were measured using colorimetric kits (JianCheng Bioengineering Institute, Jiangsu, China), and the concentrations of TNF-α and eNAMPT in serum were determined using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The lung was promptly dissected, washed by cold PBS, and fixed in buffered formalin for further examinations.

本研究选用江苏省南京市青龙山实验动物公司雄性 SD 大鼠(体重约160g)为研究对象。所有下面描述的动物方案都经过了皖南医学院医院伦理委员会的批准。YJS 2018-5-011) ,并严格按照实验室动物护理和使用指南(美国国家研究委员会,2011年)。在实验程序之前,所有的动物被关押7天以获得安置。然后,将大鼠随机分为4组。2个 MAN 治疗组分别给予 MAN 治疗,灌胃(悬浮于 CMC-Na 溶液中) ,连续3天,低剂量和高剂量分别为15和45mg/kg/d。另两组分别作为正常对照组和 ALI 模型对照组,用0.5% CMC-Na 替代治疗。最后一次给药后1小时,除正常对照组外,所有大鼠均出现肺损伤,腹腔注射 LPS 10mg/kg。动物然后强烈观察和处死在水合氯醛麻醉12小时后。全血经腹主动脉进入抗凝/促凝管。在德国拜耳诊断公司 advia120血液分析系统上采用抗凝血进行全血细胞计数(CBC)分析,用凝固血分离血清进行血清学分析。采用比色法测定血清中超氧化物歧化酶(SOD)和丙二醛(MDA)的含量,并按照制造商的说明书采用 ELISA 法测定血清中 tnf-α 和烯胺铂的含量。肺迅速解剖,用冷 PBS 冲洗,用福尔马林缓冲固定,以便进一步检查。

2.3. Histological and Immunohistochemical Examinations
2.3. 组织学及免疫组织化学检查

The fixed lung was embedded in paraffin and then sectioned at 4 μm thickness. After the staining of hematoxylin and eosin, the lung injury was evaluated based on the following histological changes: (a) thickness of the alveolar walls, (b) infiltration or aggregation of inflammatory cells, and (c) alveolar hemorrhage [14]. Each pathological change was scored on the scale of 0-3 based on the severity: 0, no visual changes; 1, slight; 2, obvious; 3, severe. The highest score in sum for each rat is 9 theoretically. Some deparaffinized sections were rehydrated in PBS and treated with 3% hydrogen peroxide at room temperature for 20 min. The heat-mediated epitope retrieval method was adopted to recover antigen reactivity using citric acid treatment by the aid of microwave heating. The nonspecific proteins were blocked with normal goat serum. Then, the blocked slides were incubated with anti-NAMPT or Sirt1 rabbit polyclonal antibodies (dilution ratio 1: 100) at 4°C overnight and subsequently subject to incubation with the goat anti-rabbit IgG biotin-labeled secondary antibody (dilution ratio 1: 2000) for 1 h at room temperature. Signals of tagged proteins were amplified and detected by HRP-conjugated streptavidin incubation and 3,3-diaminobenzidine (DAB) staining. Finally, the slides were counterstained with hematoxylin, and observed using an Olympus BH-2 light microscope coupled with a digital camera (Tokyo, Japan). Specificity of the immune reactions was tested by negative and positive controls. Certain lung specimens treated with PBS instead of primary antibodies were adopted as negative control, and synovium tissue from rats joint was used as positive controls.

固定肺用石蜡包埋后切片,厚度4μm。苏木精-伊红染色后,根据肺泡壁厚度、炎细胞浸润或聚集、肺泡出血等组织学变化评价肺损伤程度。根据病变严重程度,按0-3评分: 0,无视觉改变; 1,轻微; 2,明显; 3,严重。理论上,每只老鼠的总和最高分为9。部分去除的切片在 PBS 中重新水化,并在室温下用3% 的过氧化氢处理20分钟。采用热介导的表位恢复方法,利用柠檬酸处理,借助微波加热恢复抗原反应性。用正常山羊血清阻断非特异性蛋白。然后,将阻断的载玻片与抗 nampt 或 Sirt1兔抗体(稀释比1:100)在4 ° c 条件下孵育过夜,再与山羊抗兔 IgG 生物素标记的二级抗体(稀释比1:2000)孵育1h。用 hrp 标记的链霉亲和素(streptavidin)和3,3- 二氨基联苯胺(DAB)染色,扩增和检测标记蛋白信号。最后,用苏木素对载玻片进行反染,并用奥林巴斯 BH-2型光学显微镜和数码相机(日本东京)进行观察。用阴性和阳性对照检测免疫反应的特异性。以 PBS 替代初级抗体处理的肺组织作为阴性对照,以大鼠关节滑膜组织作为阳性对照。

2.4. Cells Culture and NAD Determination
2.4. 细胞培养和 NAD 测定

Because macrophage is deeply implicated in sepsis and relevant complications, we used RAW 264.7 cells (Jennio Biotech Co., Ltd., Guangzhou, China) in the following experiments to mimic effects in vivo. The cells were grown in DMEM supplemented with 10% FBS and penicillin-streptomycin (100 U/ml) at 37°C under the humid atmosphere containing 5% CO2. Cells were passed every 2 days.

由于巨噬细胞与脓毒症及相关并发症有密切关系,我们在以下实验中使用 RAW 264.7细胞(中国广州詹妮生物技术有限公司)模拟体内效应。细胞在含有5% CO2的潮湿气氛中,在加入10% FBS 和青霉素-链霉素(100u/ml)的 DMEM 培养基中,在37 ° c 条件下生长。细胞每2天传代一次。

To evaluate the effects of MAN on production of NAD, cells were seeded in a 75 cm2 culture flask and treated with MAN with a series of concentrations or in combination with NAD/NMN after an overnight incubation for attachment. After 24 h treatment, the cells were collected and counted. NAD was extracted from cells using the reagent provided in the NAD determination kit with the aid of heating in a boiling water bath (Solarbio biotech, Beijing, China), and supernatant of the extraction was obtained after a high speed centrifugation (10,000 g) at 4°C. The concentrations of NAD in the processed samples were determined according to the manufacturer’s instructions and then normalized to the number of cells to calculate the relative NAD levels in cells.

为了评价 MAN 对 NAD 产生的影响,将细胞种植在75cm2的培养瓶中,用一系列浓度的 MAN 处理细胞,或与 NAD/nmn 联合处理细胞,经过整夜的培养后进行附着。处理24h 后,收集细胞并计数。采用 NAD 测定试剂盒中的试剂,在沸水浴中加热提取细胞中 NAD,在4 ° c 高速离心(10,000 g)获得提取上清液。根据制造商的说明书确定加工样品中 NAD 的浓度,然后将其归一化为细胞数,以计算细胞中相对 NAD 水平。

2.5. Western Blot and ELISA Analysis
2.5. Western Blot 和 ELISA 分析

Cells at exponential growth stage were seeded into 6-well plates at the density of 1×105 cells/well. According to the experimental arrangement, some cells were pretreated with LPS (1 μg/ml) for 1 h after attachment. Afterwards, the cells were treated with MAN or in combination of NAD/NMN for 24 h based on a predetermined schedule. By the end of treatments, supernatants of the culture medium were collected for TNF-α and eNAMPT quantitative analyses using commercial available ELISA kits (R&D Systems, Minneapolis, MN, USA), and the harvested cells were lysed on ice using RIPA buffer supplemented with 1% PMSF. Supernatants from the lysates were obtained after centrifugation at 12,000 RPM under 4°C. Samples containing the equal amount of proteins (10 μg, quantified by BCA method) were subject to SDS-PAGE, and the separated proteins were then transferred onto nitrocellulose filter membranes, which were then blocked with 5% defatted milk for 2 h at room temperature and incubated with primary antibodies at 4°C overnight. The target proteins on the membranes were probed by HRP conjugated secondary antibodies (2 h at room temperature), and specific signals of protein complexes were finally developed and detected using an ECL detection kit on a Tanon 5200 system (Bio-tanon, Shanghai, China) [15].

在细胞密度为1 × 105/well 的条件下,细胞于指数增长期种植于6孔板中。根据实验安排,部分细胞接种 LPS (1μg/ml)预处理1h。然后,根据预定的时间表,用 MAN 或 NAD/NMN 联合处理细胞24小时。在处理结束时,采集培养基上清液,用现有的商品化酶联免疫吸附试剂盒(r & d Systems,Minneapolis,MN,USA)进行 tnf-α 和 anampt 的定量分析,并用 RIPA 缓冲液加1% PMSF 在冰上溶解获得的细胞。在4 ° c 下12,000 RPM 离心后,从裂解液中获得上清液。将含有等量蛋白质(BCA 法定量为10μg)的样品进行 SDS-PAGE,将分离的蛋白质转移到硝化纤维素过滤膜上,然后用5% 脱脂牛奶在室温下封闭2h,并与初级抗体在4 ° c 条件下孵育过夜。用 HRP 标记的二级抗体(室温下2h)检测膜上的目标蛋白,最终制备出蛋白复合物的特异性信号,并在 Tanon 5200系统(上海 bio-Tanon)上用 ECL 检测试剂盒进行检测。

2.6. Molecular Docking Simulation
2.6. 分子对接模拟

The 3D conformation construction and energy minimization of MAN were achieved using the ChemBio3D Ultra 14.0 software (Cambridge, MA, USA). Crystal structures of NAMPT in complex with small molecular ligands were chosen and downloaded from RCSB Protein Data Bank. The docking simulation procedures were mainly performed on the 5LX3 conformation, while 3DKJ and some other cocrystallized structures were used to test results. To prepare the protein for simulation procedures, atoms of water and ligands were removed from the molecule, and polar hydrogens were added by the aid of AutoDockTools 1.5, the graphical interface of AutoDock software. MAN and NAMPT structural data were then converted into the PDBQT format and fed to AutoDock 4.2 for docking simulation. A grid box with the size of 40×40×40 was generated to completely encompass the binding site cleft with the coordinate of PHE 193 as the center reference. Lamarckian genetic algorithm (GA) was adopted. Interactions between MAN and the protein were analyzed and visualized using PyMol graphic system 2.1 (DeLano Scientific, San Carlos, CA) [16].

利用 ChemBio3D Ultra 14.0软件(Cambridge,MA,USA)实现了城域网的三维构型构造和能量最小化。从 RCSB 蛋白质数据库中选择并下载了小分子配体配合物中的 NAMPT 晶体结构。对接模拟主要在5LX3构象上进行,而3DKJ 和其他共晶结构则用于测试结果。为了制备用于模拟过程的蛋白质,利用 AutoDockTools 1.5软件中的图形界面 AutoDock,从分子中去除水原子和配体原子,添加极性氢。然后将 MAN 和 NAMPT 结构数据转换为 PDBQT 格式,并提供给 AutoDock 4.2进行对接仿真。以 PHE 193坐标为中心坐标,生成一个40 × 40 × 40的网格框,完全包围结合部位的裂隙。采用拉马克遗传算法。用 PyMol 图形系统2.1(DeLano Scientific,San Carlos,CA)[16]分析和可视化 MAN 和蛋白质之间的相互作用。

3. Results

3. 结果

3.1. MAN Treatment Alleviated ALI Severity in Rats
3.1. MAN 治疗减轻大鼠 ALI 严重程度

LPS challenge caused severe acute lung injury in rats. The airspace inflammation was characterized by notable alveolar thickening, interstitial edema, and extensive inflammatory cells infiltration. Interstitial patchy hemorrhage was readily observed too. MAN treated rats showed modest lung injury, but the severity was much less than pathological changes in ALI models. MAN at high dose effectively reduced interalveolar septal thickening and alveolar hemorrhage, and cells infiltration was also relieved (Figure 1(a)), which together resulted in a significant reduction in histological scores of lung in rats (Figure 1(b)). CBC analysis revealed that the most significant hematological change under LPS challenge was the enlarged leucocytes population, supporting the inflammatory conditions in vivo. Specifically, neutrophil and monocyte were significantly increased. Individual rat’s responses varied substantially to MAN regimen, especially in the low dose group; however, the tendency of dose-dependent decrease of these indicators was quite obvious (Figure 1(c)). SOD activity was significantly suppressed in ALI rats, and MAN treatments only slightly recovered it. Meanwhile, MDA production was greatly elevated in ALI models, which was completely reversed by MAN treatments. Similar effects of MAN treatments occurred to the levels of TNF-α in serum. The LPS induced overproduction of TNF-α was abrogated by MAN in a dose-dependent manner (Figure 1(d)).

内毒素攻击致大鼠严重急性肺损伤。空气拥有属性的炎症表现为肺泡明显增厚,间质水肿,广泛的炎症细胞浸润。间质出血也很容易观察到。MAN 治疗的大鼠肺损伤较轻,但严重程度远小于 ALI 模型的病理改变。大剂量 MAN 能有效地减少肺泡间隔增厚和肺泡出血,细胞浸润也得到缓解(图1(a)) ,这些都导致了大鼠肺组织学评分的显著降低(图1(b))。CBC 分析显示,在 LPS 刺激下,最显著的血液学变化是白细胞数量增加,支持体内的炎症条件。特别是中性粒细胞和单核细胞明显增多。个体大鼠对 MAN 方案的反应差异很大,特别是在低剂量组,然而,这些指标的剂量依赖性下降趋势是相当明显的(图1(c))。在 ALI 大鼠中,SOD 活性明显受到抑制,而 MAN 处理仅略有恢复。与此同时,ALI 模型中 MDA 含量显著升高,而 MAN 处理完全逆转了这一趋势。MAN 治疗对血清肿瘤坏死因子 -α 水平的影响相似。LPS 诱导的肿瘤坏死因子 α 的过度产生被 MAN 以剂量依赖的方式消除(图1(d))。(a)
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(d)Figure 1 图1Therapeutic effects of MAN on ALI in rats. (a) Histological examination of lung in rats: A-D (100 × magnification), E-H (400 × magnification), representative images selected from normal control, ALI model, MAN treated (low), and MAN treated (high) groups, respectively; (b) quantitative evaluation of pathological changes based on histological examination; (c) main cell types with significant population differences among groups; (d) serological differences among groups. Statistics significance:MAN 对大鼠急性肺损伤的治疗作用。(a)大鼠肺组织学检查: A-D (100倍)、 E-H (400倍)、正常对照组、 ALI 模型组、 MAN 治疗组(低)、 MAN 治疗组(高) ; (b)组织学检查对病理改变进行定量评价; (c)主要细胞类型组间差异显著; (d)组间血清学差异。统计数字的重要性: < 0.01 compared with ALI models. 与 ALI 模型相比

3.2. MAN Treatment Downregulated NAMPT and Sirt1 in ALI Rats
3.2. MAN 治疗下调 ALI 大鼠的 NAMPT 和 Sirt1

The increase in MDA levels in ALI rats suggested that lipid catabolism in vivo could possibly be disruptedThis phenomenon reflects the augmented energy expenditure due to extra demands of inflammatory reactions and suggests that the primary energy resource could have been switched to fatty acid oxidation [17]. As well known, NAMPT-Sirt1 axis plays a central role in the maintenance of energy homeostasis in mammals [17]. Such metabolic alteration will inevitably affect the signaling status. Consistent with our assumption, immunohistochemical examination revealed that expressions of both intracellular NAMPT (iNAMPT) and Sirt1 were remarkably enhanced in ALI rats, and these abnormal changes were restored by MAN treatments (Figures 2(a)and 2(b)). Of note, the suppressive effect of MAN on iNAMPT was especially effective. It brought iNAMPT expression in lung down to the extent even lower than that in normal animals (Figure 2(c)). Similar changes happened to eNAMPT in serum. The levels of circulating eNAMPT seem to remain synchronized with its intracellular counterpart, but its fluctuation was a bit smaller (Figure 2(d)). Simultaneous downregulation of iNAMPT and eNAMPT suggested that MAN substantially intervened in the energy metabolism and would eventually affect NAD consuming signaling transductions, including Sirt1.

ALI 大鼠 MDA 水平升高提示体内脂质分解代谢可能受到干扰。这一现象反映了由于炎症反应的额外需求而增加的能量消耗,并表明主要的能量来源可能已经转化为脂肪酸氧化[17]。众所周知,NAMPT-Sirt1轴在哺乳动物的能量稳态维持中起着中心作用[17]。这种代谢变化不可避免地影响信号转导的状态。与我们的假设一致,免疫组织化学检测显示 ALI 大鼠细胞内 NAMPT (iNAMPT)和 Sirt1的表达均明显增强,而且这些异常变化经 MAN 治疗后得以恢复(图2(a)和图2(b))。值得注意的是,城域网对 iNAMPT 的抑制作用特别有效。它使肺组织中 iNAMPT 的表达下降到比正常动物更低的程度(图2(c))。血清烯胺酶也发生了类似的变化。循环的釉蛋白水平似乎与其细胞内的配对物保持同步,但波动较小(图2(d))。iNAMPT 和 ampt 同时下调表明,MAN 实质上干预了能量代谢,并最终影响 NAD 消耗的信号转导,包括 Sirt1。(a)
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(d)Figure 2 图2Regulation of MAN on NAMPT and Sirt1 城域网对 NAMPT 和 Sirt1的调控 in vivo 在活体内. (a) Expression of iNAMPT in lung; (b) expression of Sirt1 in lung; (c) quantitative results of immunohistochemical assays; (d) levels of eNAMPT in serum. Photographs A-D represent normal control, ALI model, MAN treated (low), and MAN treated (high), respectively. Statistical significance: .(a) iNAMPT 在肺组织中的表达; (b) Sirt1在肺组织中的表达; (c)免疫组化定量检测结果; (d)血清 ampt 水平。照片 A-D 分别代表正常对照、 ALI 模型、 MAN 治疗(低)和 MAN 治疗(高)。统计显著性: < 0.01 compared with ALI models. 与 ALI 模型相比

3.3. MAN Downregulated NAMPT and Sirt1 in RAW 264.7 Cells In Vitro
3.3. MAN 在 RAW 264.7细胞中下调 NAMPT 和 Sirt1

Since leucocytes including macrophage play a critical role in systemic inflammations, we subsequently investigated the effects of MAN on the levels of NAMPT and Sirt1 in RAW 264.7 cells. To mimic the inflammation conditions, the cells were pretreated with LPS, which led to increased productions of iNAMPT, eNAMPT, and Sirt1 (Figures 3(a)3(c)). Overall, the effects of MAN on these LPS induced changes in vitro were similar to those in vivo. MAN stimulus reduced both iNAMPT and eNAMPT in a concentration-dependent manner (Figures 3(a) and 3(b)) and exerted similar but smaller effects on the levels of Sirt1 (Figure 3(c)).

由于包括巨噬细胞在内的白细胞在系统性炎症中起着关键作用,我们随后研究了 MAN 对 RAW 264.7细胞中 NAMPT 和 Sirt1水平的影响。为了模拟炎症状态,细胞被 LPS 预处理,导致 iNAMPT,珐琅质和 Sirt1的产生增加(图3(a)-3(c))。总的来说,MAN 对这些 LPS 诱导的变化的影响与体内相似。MAN 刺激以浓度依赖的方式降低 iNAMPT 和 anampt (图3(a)和3(b) ,对 Sirt1水平产生类似但较小的影响(图3(c))。(a)
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(d)Figure 3 图3Regulation of MAN on NAMPT and Sirt1 in RAW 264.7 cells MAN 对 RAW 264.7细胞中 NAMPT 和 Sirt1的调控 in vitro 在试管中. (a) Expression of iNAMPT in cells; (b) levels of eNAMPT in culture medium; (c) expression of Sirt1 in cells; (d) deacetylation capability of Sirt1 indicated by expression of ace-p65 in cells. Statistical significance: .(a) iNAMPT 在细胞中的表达; (b) ampt 在培养基中的水平; (c) Sirt1在细胞中的表达; (d) Sirt1在细胞中的脱乙酰能力。统计显著性: < 0.05 and 及 < 0.01 compared with LPS treated cells. 与 LPS 处理的细胞进行比较

Both results from in vivo and in vitro assays revealed downregulation of Sirt1 by MAN, which is contradictory to a previous report [8]. MTT assay showed that the cells were sensitive to concentration and treatment duration of MAN. By increased exposure to MAN, the viability of cells was dramatically decreased. In the meantime, downregulation of NAMPT/Sirt1 by MAN was diminished and eventually turned to the opposite. These findings suggested that the stress response of the cells under critical conditions should account for the contrary outcomes.

体内和体外试验的结果都显示了 MAN 对 Sirt1的下调,这与先前的报道[8]相矛盾。MTT 比色法显示,细胞对 MAN 的浓度和处理时间敏感。通过增加男子暴露,细胞的活力急剧下降。同时,城域网对 NAMPT/Sirt1的下调作用减弱,并最终转向相反的方向。这些发现表明,细胞在临界条件下的应激反应应该解释相反的结果。

It also raised a concern about the possible negative effects on inflammations brought by reduced Sirt1, since Sirt1 can inhibit the transcriptional activity of NF-κB via deacetylation of p65 subunit [18]. Indeed, we noticed accumulation of ace-p65 upon MAN treatments, which indicated the crippled deacetylation capability of Sirt1 (Figure 3(d)). At present, it is difficult to conclude if such changes will aggravate the inflammation reactions based on limited experimental evidence of this study; however, at least downregulation of Sirt1 by MAN does not seem to make sense to the eased flame. We therefore mainly focused on the changes of NAMPT/NAD in the subsequent experiments.

Sirt1可能通过 p65亚基的脱乙酰化抑制 NF-κB 的转录活性,从而对 Sirt1的减少可能产生的负面影响也引起了人们的关注。事实上,我们注意到 ace-p65在 MAN 处理中的积累,这表明 Sirt1的残缺脱乙酰能力(图3(d))。目前,基于本研究有限的实验证据,很难得出这种变化是否会加重炎症反应的结论; 然而,至少 MAN 对 Sirt1的下调对缓和的火焰似乎没有意义。因此,在随后的实验中,我们主要关注 NAMPT/NAD 的变化。

3.4. MAN Inhibits Enzymic Activity of NAMPT by Binding to the Catalytic Site
3.4. MAN 通过结合到催化位点抑制 NAMPT 的酶活性

Previously, we found that MAN treatment efficiently reduced production of NADin vivo (unpublished). Given the newly developed conception about feedback between energy metabolism and inflammation [19], manipulation of NAD production by MAN could be crucial for its anti-inflammatory bioactivities. Due to structure similarities with endogenous substrates, we assumed that, apart from its effects on NAMPT expression, MAN could also inhibit the catalytic activity of NAMPT directly. Results generated from simulation docking were clustered and ranked by binding energy, and the one with lowest binding energy was deemed as the most stable conformation and further analyzed. We showed that the energetic gap from the largest conformation population of MAN docking to NAMPT spanned from 7.2 to 8.02 kcal/mol. This docking result provided us a tool to roughly evaluate the stability of constructed complexes and suggested that MAN could bind to NAMPT steadily. As shown in Figure 4, the cleft in active binding site of NAMPT is composed by both A and B subunits of the homodimer (colored by blue and purple, respectively), and the cavity shape was perfectly fit to the structure of MAN. The two residues situated deeply in the cleft (Phe193 from subunit A and Typ18 from subunit B) play critical roles for binding of NAMPT to the ligands [20]. Similar to the interactions with validated ligands, Phe193 and Typ18 formed a strong face-to-face ππ interaction with the aromatic ring of MAN, and the planar structure of MAN was especially favored for the sandwich-like insertion [20]. Additionally, multiple hydrogen bonds were found between MAN and Asp219 (subunit A), Arg196 (subunit A), and Arg392 (subunit B) in NAMPT, which further stabilized the interactions. As the groove constructed by Phe193 and Typ18 is crucial for substrate binding and Asp219 determines catalytic specificity [20], the occupation by MAN in the binding site could result in reduced catalytic activity of NAMPT.

在此之前,我们发现 MAN 治疗有效地降低了体内 NAD 的产生(未发表)。考虑到能量代谢与炎症反馈的新概念,MAN 控制 NAD 的产生可能对其抗炎生物活性至关重要。由于与内源性底物的结构相似,我们认为,除了对 NAMPT 表达的影响外,MAN 还可以直接抑制 NAMPT 的催化活性。模拟对接产生的结果按结合能进行聚类和排序,结合能最低的结构被认为是最稳定的结构,并进行了进一步分析。我们发现,从 MAN 对接的最大构象种群到 NAMPT 的能隙跨度为7.2ー8.02 kcal/mol。这一对接结果为我们粗略评估构建的配合物的稳定性提供了一个工具,并表明城域网可以稳定地与 NAMPT 结合。如图4所示,NAMPT 活动结合部位的裂隙由同二聚体的 a 亚基和 b 亚基组成(分别用蓝色和紫色标记) ,空腔形状与 MAN 结构完全吻合。位于裂隙深处的两个残基(Phe193来自 a 亚基和 Typ18来自 b 亚基)对 NAMPT 与配体的结合起着关键作用[20]。与已验证的配体相似,Phe193和 Typ18与 MAN 的芳环形成了强的面对面 π-π 相互作用,MAN 的平面结构特别有利于夹心结构的插入[20]。此外,在 NAMPT 发现 MAN 与 Asp219(a 亚单位)、 Arg196(a 亚单位)和 Arg392(b 亚单位)之间存在多重氢键,进一步稳定了相互作用。由于 Phe193和 Typ18构筑的凹槽对底物的结合起着至关重要的作用,Asp219测定了催化特异性[20] ,城域网占据结合位点可能导致 NAMPT 的催化活性降低。

Figure 4 图4Direct interaction between MAN and NAMPT revealed by molecular docking simulation analysis. 分子对接模拟分析揭示城域网与 NAMPT 之间的直接相互作用

3.5. MAN Inhibited TLR4/NF-κB Mediated Inflammation by Affecting NAD Production
3.5. MAN 通过影响 NAD 的产生抑制 TLR4/NF-κB 介导的炎症反应

Due to the effects of MAN on expression and catalytic activity of NAMPT, biosynthesis of NAD is expected to be suppressed under MAN treatments. As expected, MAN significantly decreased the NAD production in LPS treated RAW 264.7 cells (Figure 5(a)). With the decrease of NAD production, we found a synchronized decline of TNF-α in culture medium (Figure 5(b)). Because inflammatory cytokine secretion is universally controlled by the NF-κB pathway, this change would be attributable to the downregulation of NF-κB. This assumption was subsequently proved, as LPS induced expression of p-p65 was completely abrogated by MAN (Figure 5(b)). Meanwhile, MAN treatments reduced the expression of TLR4 and HMGB, an endogenous agonist of TLR4, and partner molecule that amplifies LPS induced TLR4 action [21] (Figure 5(b)). These results suggested that the metabolic changes observed in this study have a logical correlation with TLR4/NF-κB mediated inflammation under MAN treatments.

由于 MAN 对 NAMPT 表达和催化活性的影响,NAD 的生物合成有望在 MAN 处理下得到抑制。与预期一样,MAN 显著降低 LPS 处理的 RAW 264.7细胞的 NAD 产生(图5(a))。随着 NAD 产生的减少,我们发现在培养基中肿瘤坏死因子 α 同步下降(图5(b))。由于炎性细胞因子的分泌普遍受 NF-κB 通路的调控,这种变化可能与 NF-κB 的下调有关。当 LPS 诱导的 p-p65表达被 MAN 完全消除后,这一假设得到了证实(图5(b))。同时,MAN 处理降低了内源性 TLR4激动剂 TLR4和 HMGB 的表达,以及增强 LPS 诱导的 TLR4作用的伙伴分子[21](图5(b))。这些结果提示,本研究中观察到的代谢变化与 TLR4/NF-κB 介导的 MAN 治疗下的炎症反应有一定的逻辑关系。(a)
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(c)Figure 5 图5Downregulation of MAN on NAD production contributed to reduce TLR4/NF- 城域网对 NAD 生产的下调有助于降低 TLR4/NF-κB activation in RAW 264.7 cells RAW 264.7细胞中的 b 激活 in vitro 在试管中. (a) Production of NAD in cells; (b) MAN inhibited activation of TLR4/NF- . (a)在细胞内产生 NAD; (b) MAN 抑制 TLR4/NF-的活化κB in a concentration-dependent manner; (c) cotreatments with NAD/NMN reversed MAN induced inhibition on TLR4/NF- (c) NAD/NMN 逆转 MAN 对 TLR4/NF-的抑制作用κB: A, secretion of TNF- 乙: a,肿瘤坏死因子-的分泌α by cells; B, expression of p-p65 in cells; C, expression of HMGB1 and TLR4 in cells. Statistical significance: B、 p-p65在细胞中的表达、 c、 HMGB1和 TLR4在细胞中的表达。统计学意义: < 0.01compared with LPS treated cells; 与 LPS 处理的细胞比较; < 0.01 compared with LPS+MAN treated cells. 与 lps + man 处理的细胞比较

To further validate the connection between NAD production and TLR4/NF-κB activation, we cotreated the cell with NAD/NMN (main precursor of NAD in mammals) to restore the decreased intracellular levels of NAD under MAN treatments. Cotreatment with both NAD and NMN significantly reversed the MAN induced inhibition of NF-κB activation and TNF-α release (Figure 5(c)), which strongly supported our hypothesis. Further, we found that the MAN induced reduction in expressions of TLR4 and HMGB1 was also restored by the supplementary with NAD and NMN (Figure 5(c)). All these evidences suggested that MAN reduced the NAD production via multiple means, which consequently resulted in downregulation of the TLR4/NF-κB pathway and alleviated inflammation.

为了进一步验证 NAD 的产生与 TLR4/NF-κB 活化之间的关系,我们将 NAD/nmn (哺乳动物 NAD 的主要前体)与 TLR4/NF-κB 共同处理,以恢复 MAN 处理下降的细胞内 NAD 水平。联合 NAD 和 NMN 治疗可显著逆转 MAN 诱导的 NF-κB 激活和 tnf-α 释放抑制(图5(c)) ,这有力地支持了我们的假设。此外,我们还发现,通过辅以 NAD 和 NMN,MAN 诱导的 TLR4和 HMGB1表达下降也得到了恢复(图5(c))。这些证据表明,MAN 通过多种途径降低 NAD 的产生,从而导致 TLR4/NF-κB 通路下调,减轻炎症反应。

4. Discussion

4. 讨论

Due to its high abundance in nature and versatile bioactivities, MAN is one of the most investigated naturally occurring xanthones. Its potentials in therapies of immune and inflammatory disorders are especially meaningful, because MAN rich mangosteen pericarp has been used as a traditional medicine to cure infections and inflammations for centuries in Southern Asia [22]. In previous studies, we noticed that MAN treatments were always accompanied with body weight loss in rats [10] and assumed this could be due to disrupted metabolismin vivo. The current study firmly confirmed this hypothesis and further revealed its relevance to the anti-inflammatory properties.

由于其丰富的自然资源和多样的生物活性,人是研究最多的天然黄酮之一。它在免疫和炎症疾病治疗方面的潜力特别有意义,因为富含 MAN 的山竹果皮已经在南亚作为传统药物用于治疗感染和炎症几个世纪了[22]。在以前的研究中,我们注意到 MAN 治疗总是伴随着大鼠体重的减轻,并假设这可能是由于在体内的新陈代谢紊乱。目前的研究坚定地证实了这一假设,并进一步揭示了其相关性的抗炎属性。

Most researches on anti-inflammatory activities of xanthones mainly emphasized their antioxidative capability [57]. We also noticed that the SOD activity could be restored by MAN treatments; however, this effect was not so significant. In comparison, much bigger differences were observed concerning the levels of MDA among groups. Such changes served as an important indicator for relieved oxidative stress and inflammation but could also reflect the altered lipid metabolism profile in vivo, because the lipid peroxidation is usually connected to accumulation of fatty acids in blood [23].

黄原酮类化合物的抗炎活性研究大多侧重于其抗氧化能力[5-7]。我们还注意到,超氧化物歧化酶(SOD)活性可以通过 MAN 处理恢复,但这种效果并不明显。相比之下,各组间丙二醛水平差异较大。这种变化是氧化应激和炎症缓解的重要指标,但也可能反映体内脂质代谢的改变,因为脂质过氧化通常与血液中脂肪酸的积累有关。

Available evidences show that Sirtuins regulate key aspects of lipid metabolism, and Sirt1 promotes lipid mobilization and oxidation of fatty acids [24]. We found MAN downregulated Sirt1 both in vivo and in vitro. Given its well recognized anti-inflammatory role [25], the reduction in Sirt1 expression seems to deteriorate the inflammatory conditions. However, on the other hand, fatty acid has been recognized as the main energy resource to fuel inflammation reactions [17]. From this perspective, downregulation of Sirt1 would be beneficial to inflammation treatments by reducing energy supply. Nevertheless, at present, we can hardly conclude its net effects and further studies should be performed to resolve this paradox.

现有证据表明,Sirtuins 调节脂质代谢的关键方面,Sirt1促进脂肪动员和脂肪酸氧化[24]。我们发现 MAN 在体内和体外都下调了 Sirt1。由于其公认的抗炎作用[25] ,减少 Sirt1表达似乎恶化炎症状况。然而,另一方面,脂肪酸被认为是助长炎症反应的主要能量来源[17]。从这个角度来看,Sirt1的下调通过减少能量供应对炎症治疗是有益的。然而,目前我们很难得出其净效应的结论,需要进一步的研究来解决这一悖论。

Accumulating evidences support that, apart from its decisive role in NAD biosynthesis, extracellular NAMPT also acts as an adipokine cooperating in glucose and lipid metabolism. By exerting an insulin-mimetic effect, NAMPT regulates carbohydrate metabolism and promotes fat deposition. Hence, overexpression of NAMPT is usually linked to development of obesity [13]. Taken the negative effects on body weight into consideration, it is reasonable to assume that MAN would downregulate eNAMPT in serum, and this was confirmed in this study. More importantly, this metabolic change has a profound impact on ALI in rats, as an increasing number of reports suggest that eNAMPT is an emerging proinflammatory cytokine [1213] and is implicated in the pathogenesis of ALI [26]. eNAMPT appears to be produced through a posttranslational modification from iNAMPT, although the underlying mechanisms still remains elusive [13]. Yoon et al. found that Sirt1 rigorously controlled eNAMPT secretion via deacetylation of iNAMPT [27]. This clue shed some light on the elucidation of therapeutic effects of MAN on ALI. On one hand, MAN reduced the expression of iNAMPT and inhibited its catalytic activity, which led to decreased biosynthesis of NAD and supply of precursor for eNAMPT production; on the other hand, MAN compromised posttranslational modification of iNAMPT by reducing NAD production and Sirt1 expression. All these factors cooperatively contribute to the declined eNAMPT levels. Taken together, manipulation of NAD production could be essential for the therapeutic effects of MAN on ALI via inhibiting eNAMPT mediated inflammation reactions.

越来越多的证据表明,细胞外的 NAMPT 除了在 NAD 生物合成中起决定性作用外,还可以作为一种脂肪因子参与葡萄糖和脂质代谢的合作。通过发挥类胰岛素作用,NAMPT 调节糖代谢并促进脂肪沉积。因此,NAMPT 的过度表达通常与肥胖的发展有关[13]。考虑到对体重的负面影响,可以合理地假设 MAN 下调血清中的烯胺基转移酶,本研究证实了这一假设。更重要的是,这种代谢变化对大鼠 ALI 有着深远的影响,因为越来越多的报道表明,烯胺化酶是一种新兴的促炎性细胞因子,与 ALI 的发病机制有关。珐琅铂似乎是通过 iNAMPT 的翻译后修饰生产的,尽管其基本机制仍然难以捉摸。Yoon 等人发现 Sirt1通过 iNAMPT 的脱乙酰基严格控制珐琅 pt 的分泌。这一线索有助于阐明 MAN 对 ALI 的治疗作用。一方面,MAN 降低了 iNAMPT 的表达并抑制了其催化活性,从而导致 NAD 生物合成的减少和生产搪瓷的前体供应的减少; 另一方面,MAN 通过减少 NAD 的生产和 Sirt1的表达而损害了 iNAMPT 的翻译后修饰。所有这些因素共同作用导致釉蛋白水平下降。综上所述,控制 NAD 的产生对 MAN 通过抑制烯胺铂介导的炎症反应治疗 ALI 是必不可少的。

Increased leucocyte counts and neutrophil/macrophage recruitment in lung are important hallmarks of sepsis induced ALI [28], which will contribute to the increase in eNAMPT under pathological conditions [1226]. The dramatic reduction in neutrophil/monocyte counts under MAN treatments would benefit the improvement of inflammation in ALI rats by reducing circulating eNAMPT. Moreover, leucocytes are indispensable components in the innate immune system to defend infections based on the identification of pathogen-associated molecular patterns by TLRs. However, hyperactivation of this defensive mechanism will also perpetuate unfavorable inflammations. Results from this study showed that reduction in energy metabolism caused by MAN substantially inhibited TLR4/NF-κB activation in macrophage. Despite we still do not know why the decrease in NAD leads to reduced expression of TLR4, there are some available clues to clarify this phenomenon from other perspectives. Camp et al. have shown that eNAMPT is a unique endogenous agonist of TLR4. It can potently activate the TLR4/NF-κB pathway alone without the need of partner molecules [29]. Logically, downregulation of eNAMPT could result in reduced TLR4/NF-κB activation under MAN treatments. Considering their roles in lipid mobilization and fat deposition, decreases in Sirt1 and eNAMPT under MAN treatments could cut down free saturated fatty acids in blood, which will further curb the activation of TLR4/NF-κB, because they can bind to TLR4 and directly elicit inflammation reactions [30]. Due to the high energy expenditure under inflammation conditions, oxygen supply cannot meet the demands sufficiently, and a hypoxia circumstance develops [17], which would cause necrosis of cells and subsequent release of HMGB1 [31]. These molecular events ultimately strengthen the activation of TLR4 and aggravate the inflammation. MAN treatments achieved a low metabolism status and greatly alleviated hypoxia mediated HMGB1 release. All these evidences suggested that MAN could restore the innate immune intolerance in leucocytes by controlling energy metabolism.

白细胞计数增加和肺中性粒细胞/巨噬细胞增多是脓毒症引起 ALI 的重要特征,在病理条件下,这将有助于 ampt 的增加[12,26]。MAN 治疗下中性粒细胞/单核细胞数量的显著减少有利于改善 ALI 大鼠的炎症反应,其机制可能是通过减少循环的釉蛋白。此外,根据 TLRs 对病原体相关分子模式的鉴定,白细胞是先天免疫系统中防御感染不可缺少的组成部分。然而,这种防御机制的过度激活也将使不利的炎症永久化。本研究结果表明,MAN 导致的能量代谢下降,实质上抑制了巨噬细胞 TLR4/NF-κB 的活化。尽管我们仍然不知道为什么 NAD 的减少导致 TLR4的表达减少,但是有一些可用的线索可以从其他角度阐明这一现象。坎普等人已经证明,烯胺基转移酶是一个独特的内源性 TLR4激动剂。它可以单独有效地激活 TLR4/NF-κB 通路,而不需要合作分子[29]。从逻辑上讲,在 MAN 处理下,ampt 的下调可能导致 TLR4/NF-κB 活性降低。考虑到它们在脂肪动员和脂肪沉积中的作用,在 MAN 治疗下减少 Sirt1和烯胺铂可以减少血液中游离的饱和脂肪酸,这将进一步抑制 TLR4/NF-κB 的活化,因为它们可以结合 TLR4,直接引起炎症反应[30]。由于炎症状态下的高能量消耗,氧供应不能充分满足需求,缺氧环境发生,导致细胞坏死,随后释放 HMGB1。这些分子事件最终加强了 TLR4的活化,加重了炎症反应。MAN 处理可以达到低代谢状态,大大减轻缺氧介导的 HMGB1释放。这些证据表明,MAN 通过控制能量代谢,可以恢复白细胞的先天性免疫不耐受。

5. Conclusion

5. 总结

As a well known naturally occurring bioactive compound, MAN possesses a notable clinical potential in treatments of many diseases. This study provides further evidences to support its anti-inflammatory properties and partially elucidates the underlying mechanisms from a unique perspective. The results of this study suggested that MAN suppressed TLR4/NF-κB mediated inflammation reactions by manipulation of NAMPT/NAD, and the regulation of fat metabolism could be an effective therapeutic strategy in therapies of inflammation related disorders.

作为一种众所周知的天然生物活性化合物,MAN 在许多疾病的治疗方面具有显著的临床潜力。本研究为其抗炎作用提供了进一步的证据,并从独特的角度部分阐明了其作用机制。本研究结果提示,MAN 通过调控 NAMPT/NAD 抑制 TLR4/NF-κB 介导的炎症反应,调节脂肪代谢可能是治疗炎症相关性疾病的有效策略。

Data Availability

数据可用性

The data used to support the findings of this study are included within the article.

支持这项研究结果的数据包括在本文中。

Conflicts of Interest

利益冲突

The authors report no conflicts of interest.

作者报告没有利益冲突。

Authors’ Contributions

作者贡献

Mengqing Tao and Jia Jiang contribute equally to the manuscript.

陶和蒋家对这部手稿贡献相当。

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