白花前胡素类化合物抗炎活性以及分子作用机制的研究
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摘要
炎症本质是机体应对各种损伤性刺激实现自我保护的复杂防御反应,众多种类的细胞以及活性成分参与了这个过程。在炎症初期,多种炎症因子经炎性细胞释放后,对损伤因子进行稀释、杀伤以及清除,同时,炎症因子也参与了机体受损部位的组织再生,促进伤口的修复和愈合。然而,炎症反应一旦过于剧烈或持久,则会因为炎症因子本身所具有的机体破坏性而对正常组织和细胞造成损伤,诱发各种炎症疾病。因此,炎症就像机体的一把双刃剑,适当的炎症反应会帮助机体清除损伤,抵御外来感染,促进伤口愈合；而过度的炎症反应则会反过来促使多种炎症疾病的发生和发展,影响人们的健康。
急性肺损伤(acute lung injury, ALI)是一种典型的过度性炎症参与的呼吸系统危重症,其定义是：心源性以外的各种肺内外致病因素引起的急性,进行性缺氧性呼吸衰竭,是肺部炎症和通透性增加的综合征。多种临床疾病均可诱发急性肺损伤,如肺炎、脓毒血症、以及胰腺炎等。研究认为,肺内过度性、失控性炎症反应是各种病因所致ALI的根本原因。
目前,临床上初步使用的ALI治疗药物主要有血管舒张剂(一氧化氮、前列环素的吸入治疗)、糖皮质激素、抗凝剂人类重组体活化蛋白C (activated proteinC,APC)、酮康唑(ketoconazole)、抗氧化剂N-乙酰半胱氨酸(NAC)等。然而,这些药物的疗效在各临床试验中还未完全得到证实,其用法用量还有着较大争议,并且有着许多严重的不良反应。因此,迄今为止,治疗ALI药物的总体研究进展是令人较为失望的,临床期待更为安全有效的治疗药物。
中药前胡为伞形科植物白花前胡(Peucedanum praeruptorum Dunn)的干燥根,具有散风清热、降气化痰等功效,临床常用于风热咳嗽痰多、痰热喘满、咯痰黄稠等。前期的研究显示白花前胡总提取物能够明显抑制二甲苯所致的小鼠耳肿胀和蛋清所致的大鼠足肿胀。同时,白花前胡提取物对卵蛋白(OVA)诱导的小鼠气道变态炎症反应也有明显的保护作用,这表明白花前胡良好的体内抗炎活性。然而,这些工作的主要研究对象是白花前胡粗提取物,并没有涉及白花前胡素单体化合物,同时,其抗炎作用机制也没有进行探讨。研究显示,白花前胡的主要有效成分为白花前胡素(Praeruptorin)等香豆素类化合物,如白花前胡甲素(Praeruptorin A, PA)、白花前胡乙素(Praeruptorin B, PB)、白花前胡丙素(Praeruptorin C, PC)、白花前胡丁素(Praeruptorin D,PD)以及白花前胡E素(Praeruptorin E, PE)。因此,在本研究中,我们研究了白花前胡素单体化合物(包括白花前胡甲素、丙素、丁素以及E素)的抗炎活性。我们利用脂多糖(LPS)建立体外巨噬细胞炎症模型,在细胞和分子水平上,从炎症相关因子NO、iNOS、TNF-α、IL-6的表达以及炎症信号通路NF-kB、MAPK和STAT的活化来研究白花前胡素类化合物的抗炎活性以及相关作用机制。由于白花前胡在临床上的应用主要是针对肺部疾病的治疗,因此,我们建立了LPS或者HC1诱导的急性肺损伤小鼠模型,检测白花前胡素类化合物对模型小鼠肺泡灌洗液中炎性细胞浸润、蛋白渗出以及炎症细胞因子释放的影响,同时,我们对小鼠肺组织中MPO的活力以及NF-κB通路的活化进行探讨,旨在研究和阐明白花前胡素类化合物对急性肺损伤的保护作用以及相关的分子机制。
研究方法和实验结果分为两大部分：
一、白花前胡丙素(PC)、白花前胡丁素(PD)以及白花前胡E素(PE)抑制
脂多糖诱导的巨噬细胞炎症反应
1、白花前胡丙素(PC)、丁素(PD)以及E素(PE)剂量的选择：小鼠巨噬细胞株RAW264.7细胞以1×104/孔的密度铺于96孔板后过夜贴壁,待各白花前胡素以2、4、8、16、32μg/ml剂量刺激细胞24h后,采用MTT法对细胞活力进行检测。结果显示,PC、PD以及PE在2-16μg/ml剂量范围内对细胞活力没有显著影响(差异无统计学意义)。因此本研究选取2-16μg/ml剂量范围应用于后续实验。
2、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制巨噬细胞中炎症介质NO的产生：待各白花前胡素以2、4、8、16μg/ml剂量与1μg/ml的LPS共同刺激细胞18h后,用Griess法检测细胞培养液上清中NO的含量。结果显示,PC、PD以及PE均可剂量依赖性地抑制NO的释放,其IC50值分别为7.6、5.6和5.8μg/ml。
3、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制巨噬细胞中iNOS蛋白的表达：提取细胞总蛋白后,采用Western blot法检测巨噬细胞中iNOS的含量。结果显示,在正常情况下,巨噬细胞中基本无iNOS的表达,而在LPS的刺激下,iNOS的表达显著增高。PD以及PE在16μg/ml剂量下均可显著抑制iNOS蛋白的高表达。
4、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制巨噬细胞中炎症细胞因子的产生：小鼠巨噬细胞株RAW264.7细胞以2×105/孔的密度铺于24孔板后过夜贴壁,PC、PD和PE以2、4、8、16μg/ml剂量与1μg/ml的LPS共同刺激细胞18h。用ELISA法检测发现各白花前胡素可显著抑制细胞培养液上清中TNF-a和IL-6的含量。PC、PD以及PE抑制TNF-a的IC50值为40.7、17.3和18.5μg/ml而对于IL-6,PC、PD以及PE的ICso值分别为11.9、4.9和5.1μg/ml。
5、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制iNOS、TNF-α和IL-6mRNA的表达：待各白花前胡素以4、8、16μg/ml剂量与1μg/ml的LPS共同刺激细胞6h后,提取细胞总RNA并逆转录,用Real-time PCR法检测细胞中iNOS、TNF-α和IL-6mRNA的表达。结果显示,在正常情况下,巨噬细胞中iNOS、 TNF-α和IL-6mRNA的表达均很少,而经过LPS刺激之后,其表达量显著增高。PC、PD以及PE均能剂量依赖性地抑制上述基因的表达。其中,在16μg/ml的剂量时,PC对iNOS、TNF-a和IL-6mRNA表达的抑制率分别为53%、35%和55%；PD的抑制率为75%、45%和72%；而PE的抑制率分别为78%、47%和70%。
6、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制巨噬细胞中NF-kB通路的激活
a：PC、PD和PE抑制LPS诱导的NF-κB亚基p65的核转运：提取细胞核蛋白,用Western blot法检测细胞核蛋白中p65的含量。结果发现,与对照细胞相比,1μg/ml剂量的LPS明显增加了巨噬细胞核蛋白中p65的含量；而各白花前胡素以16μg/ml剂量预处理细胞1h后,显著抑制了LPS引起的胞核p65含量的增加,但并不影响核内参蛋白lamin B的表达,说明白花前胡丙素、丁素以及E素可抑制NF-kB亚基p65由胞浆向胞核的转运。这一结果在细胞免疫荧光实验中也得到了证实。
b:PC、PD和PE抑制LPS诱导的IκB蛋白降解：与对照组细胞相比,1μg/ml剂量的LPS刺激30min后明显减少了巨噬细胞中1κB蛋白的含量；而白花前胡丙素、丁素以及E素以16μg/ml预处理细胞可显著抑制Iκ3蛋白含量的减少。
7、白花前胡丙素(PC)、丁素(PD)以及E素(PE)不能抑制巨噬细胞中MAPK通路的激活：MAPK是一类在各种组织细胞中广泛存在的蛋白激酶,由p38、细胞外信号调节激酶(ERK1/2)和C-Jun氨基末端激酶(JNK)三条信号通路组成。我们采用Western blot方法研究PC、PD和PE对MAPK通路的影响。结果显示：与对照组相比,1μg/ml剂量的LPS显著诱导了巨噬细胞中磷酸化p38(p-p38)、磷酸化ERK1/2(p-ERK1/2)以及磷酸化JNK (p-JNK)蛋白的增加。白花前胡丙素、丁素和E素(16μg/ml)对p-p38和p-JNK蛋白无明显影响,但可诱导p-ERK1/2蛋白的增加。
8、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制脂多糖诱导的巨噬细胞中磷酸化STAT-3(p-STAT-3)蛋白的表达,但对磷酸化STAT-1(p-STAT-1)蛋白表达无影响：首先,我们用Western blot方法检测LPS诱导的STAT-1和STAT-3蛋白磷酸化的时间依赖性。结果发现,STAT-1和STAT-3蛋白的磷酸化在LPS刺激2h后开始出现,并可至少持续至LPS刺激后6h。因此,我们选取中间时间4h作为后续实验的时间点。与对照细胞相比,单独给药的PC、PD和PE对p-STAT-1和p-STAT-3蛋白均无影响；而与LPS组细胞相比,上述白花前胡素类化合物(16μg/ml)可显著抑制LPS诱导的p-STAT-3蛋白的增加,但对p-STAT-1蛋白无影响；阳性对照药物,JAK-STAT通路抑制剂AG490,对LPS诱导的p-STAT-1和p-STAT-3蛋白均可抑制。说明PC、PD和PE对JAK-STAT通路中的STAT-3蛋白具有选择性。
二、白花前胡甲素(PA)、白花前胡丙素(PC)、白花前胡丁素(PD)以及白花前胡E素(PE)对急性肺损伤小鼠模型的影响
1、白花前胡甲素(PA)、丙素(PC)、丁素(PD)以及E素(PE)对LPS诱导的急性肺损伤小鼠模型的影响：PA、PC、PD和PE以80mg/kg剂量灌胃给药,1h后,小鼠腹腔注射10%戊巴比妥钠(60mg/kg),待麻醉后滴鼻给予50μlLPS (40μg/ml).小鼠在给予LPS四小时后处死,行肺部灌洗。以灌洗液为样品,检测其中的炎性细胞数和细胞因子的水平。结果显示,PD和PE对急性肺损伤小鼠的细胞浸润以及炎症细胞因子释放均有明显的改善作用,而PA和PC几乎无影响。
a)白花前胡丁素(PD)和白花前胡E素(PE)剂量依赖性地减少肺损伤中炎性细胞的浸润：对照组小鼠肺泡灌洗液中的中性粒细胞数浓度为(15.0±3.90)×103/ml,而在LPS模型组,中性粒细胞浓度达到(648±45.8)×103/ml。80mg/kg的PD和PE明显抑制了中性粒细胞的浸润,其浓度分别减少至(285±48.6)×103/ml和(381±56.4)×103/ml;
b)白花前胡丁素(PD)和白花前胡E素(PE)剂量依赖性地抑制炎症细胞因子的释放：对照组小鼠肺泡灌洗液中的TNF-α和IL-6含量均很少,而LPS模型组中的细胞因子释放显著增多,TNF-a和IL-6的含量分别达到(6.645±0.70)和(1.096±0.149) ng/ml。80mg/kg的PD和PE可显著抑制LPS诱导的细胞因子释放。对于TNF-α,PD和PE两组含量分别减少至(3.379±0.586)和(3.962±0.646)ng/ml;而对于IL-6,PD和PE两组的含量分别降至(0.488±0.084)和(0.523±0.081) ng/ml。
c)白花前胡丁素(PD)和白花前胡E素(PE)剂量依赖性地抑制肺泡灌洗液中总蛋白含量：用Bradford法检测小鼠肺泡灌洗液中总蛋白的浓度,结果显示PD和PE均可抑制灌洗液中蛋白含量的增加,说明PD和PE对LPS诱导的肺部蛋白渗出具有改善作用。
2、白花前胡丁素(PD)和白花前胡E素(PE)抑制肺组织中MPO的活力：MPO活力反应了中性粒细胞在组织的分布多少。我们分别检测了PD和PE对肺组织中MPO活力的影响。结果显示,PD可将LPS诱导的MPO活力从(0.802±0.057)U/g减少至(0.631±0.038)U/g；而PE则将模型组的(0.830±0.074)U/g抑制至(0.581±0.058)U/g。
3、白花前胡丁素(PD)和白花前胡E素(PE)抑制LPS诱导的肺部病理变化：小鼠处死后,取肺叶固定、切片后进行HE染色。光学显微镜观察发现,对照组小鼠肺组织肺泡结构完整,肺泡壁无水肿,肺实质无明显炎症细胞浸润；LPS模型组小鼠肺泡壁则明显水肿增厚,可见大量的肺间质中性粒细胞浸润,有明显的出血、水肿,显示出严重的肺部炎症和肺水肿现象。而PD和PE显著减轻了LPS诱导的肺部损伤。
4、白花前胡丁素(PD)和白花前胡E素(PE)抑制LPS诱导的肺组织中NF-kB通路的活化：小鼠处死后,取全肺用液氮研磨,分别提取肺组织中的胞浆和胞核蛋白,Western blot方法检测胞浆中IκB蛋白和胞核中p65蛋白的含量。结果显示,PD和PE均可抑制肺组织蛋白中IκB蛋白的降解,以及p65的核转运。
5、白花前胡丁素(PD)和白花前胡E素(PE)对HCl诱导的急性肺损伤小鼠具有保护作用：PD和PE以80mg/kg剂量灌胃给药,1h后,小鼠腹腔注射10%戊巴比妥钠(60mg/kg),待麻醉后滴鼻给予5Oμ1HC1(0.1mol/L)。小鼠在给予LPS四小时后处死,行肺部灌洗。以灌洗液为样品,检测其中的炎性细胞数和细胞因子的水平。结果显示：
a)白花前胡丁素(PD)和白花前胡E素(PE)显著减少HCl诱导的肺部炎性细胞浸润：对照组小鼠肺泡灌洗液中的中性粒细胞数浓度很低,而在HCl模型组,中性粒细胞显著增加。80mg/kg的PD和PE明显抑制了中性粒细胞的浸润,其抑制率分别为49.7%和40.0%。
b)白花前胡丁素(PD)和白花前胡E素(PE)显著减少HCl诱导的炎症因子IL-6的释放：对照组小鼠肺泡灌洗液中有少量IL-6表达,而HCl可显著诱导IL-6的释放。80mg/kg的PD和PE明显抑制了肺泡灌洗液中的IL-6含量,其抑制率分别为44.1%和39.6%。
c)白花前胡丁素(PD)和白花前胡E素(PE)显著减少HCl诱导的蛋白渗出：Bradford法检测发现对照组小鼠肺泡灌洗液中有一定的蛋白含量,HCl可明显诱导灌洗液中总蛋白的增加,其含量约为对照组的3倍。80mg/kg的PD和PE可明显抑制蛋白渗出,其抑制率分别为29.2%和36.0%。
d)白花前胡丁素(PD)和白花前胡E素(PE)抑制HCl诱导的肺部病理变化：同LPS诱导的肺损伤模型一样,PD和PE也可明显改善HCl诱导的肺部炎症和水肿现象。
基于上述实验结果和文献分析,本文的结论如下：
本研究建立LPS诱导的巨噬细胞炎症模型以及LPS/HC1诱导的小鼠急性肺损伤模型,在细胞和动物水平上对白花前胡素类化合物的抗炎活性进行了研究。研究结果显示,白花前胡素类化合物,特别是白花前胡丁素(PD)以及白花前胡E素(PE)在体外以及体内均具有良好的抗炎活性。这为白花前胡的临床应用及其现代药理学研究提供了更多的理论和实验依据。
1.白花前胡素类化合物通过抑制iNOS蛋白和mRNA的表达,从而抑制LPS诱导巨噬细胞中炎症介质NO的产生。同时,白花前胡素类化合物还可显著减少炎症细胞因子TNF-α和IL-6的mRNA表达。这些结果显示白花前胡素类化合物良好的体外抗炎活性。
2.本论文研究了白花前胡素类化合物对三条主要炎症信号通路的影响,发现该类化合物为NF-kB以及STAT-3的双通路抑制剂,这合理解释了白花前胡素类化合物的抗炎活性。另一方面,NF-kB以及STAT-3这两条信号通路本身以及它们之间的cross-talk在众多肿瘤疾病,特别是肝细胞癌的发生和发展中起到极为重要的作用,曾有不少研究报道了白花前胡素类化合物抗肿瘤活性以及抗肿瘤耐药的作用,本研究对白花前胡素类化合物的抗肿瘤作用的分子机制起到了一定的启示作用。
3.本研究的一大创新点在于首次报道了白花前胡丁素(PD)以及白花前胡E(PE)素对急性肺损伤小鼠的保护作用。急性肺损伤是一种具有很高病死率的呼吸系统危重综合症,临床尚无特别安全有效的治疗药物。本研究通过建立LPS和HCl诱导的两种肺损伤模型,发现PD和PE可明显改善肺损伤小鼠的中性粒细胞浸润、细胞因子释放以及蛋白渗出三大病理特征。同时,PD和PE还可抑制肺组织中增加的MPO活力以及NF-κB的激活。这些结果为PD和PE作为急性肺损伤治疗药物的开发提供了动物实验基础。
Inflammation is a highly complex process to defend against foreign challenge or tissue injury, which involves various cell types (such as lymphocyte, macrophage, granulocyte, and endothelial cell, etc.) and multi-components (cytokines, vascular active substances, chemokines, adhesion molecules, inflammation-related enzymes). Inflammation seems like a double-edged sword:proper inflammatory response will help our body clearing the damage, fending off infection and promoting wound healing. However, excessive inflammatory response will in turn promote the occurrence and development of lots of inflammatory diseases, therefore affecting people's health.
Acute lung injury (ALI) is a life-threatening syndrome characterized by severe lung inflammation and increased microvascular permeability, and acute respiratory distress syndrome (ARDS) is the most severe form of lung injury. A variety of clinical disorders, such as pneumonia, aspiration of gastric contents, sepsis, major trauma, acute pancreatitis, can induce the occurrence of ALI. Studies suggest that the excessive, uncontrolled inflammatory response in the lung is the root cause of ALI. Clinical uses of corticosteroids, prostaglandins, prostacyclin, surfactant, lisofylline, ketoconazole and N-ace-tylcysteine have shown unsatisfactory significant improvements in patients' mortality, regardless of the unwanted side effects of these drugs. Thus, a drug with better therapeutic effects and fewer side effects is urgently needed.
The dry root of Peucedanum praeruptorum Dunn has been long used in traditional Chinese medicine for its remarkable effects on respiratory diseases. Praeruptorin A, B, C, D and E(PA, PB, PC, PD and PE), a class of pyranocoumarin, have been confirmed to be the main constituents in this herb. Previous studies showed the protective effects of total coumarins extracted from Peucedanum praeruptorum Dunn (TCP) on dimethylbenzene-induced ear swelling in mice and egg white-induced paw swelling in rats. In addition, TCP has been demonstrated to inhibit the airway hyper-responsiveness and inflammation in a mouse model of allergic airway disease. These data showed the anti-inflammatory activities of TCP in vivo. However, the pharmacological effect and the underlying mechanism of each Praeruptorin have not been investigated. In the present study, the anti-inflammatory activities of PC, PD and PE were investigated in LPS-stimulated murine macrophages. The effects of these Praerutorins on the expression of inflammatory mediators and cytokines, as well as activation of NF-kB, MAPK and STAT pathways were examined. In addition, the acute lung injury mouse models induced by LPS or HC1were established, and the effects of Praerutorins on inflammatory cells influx, cytokine release, protein leakage, MPO activity, as well as NF-kB activation were detected to investigate the protective effects and underlying mechanism of these compounds in ALI mice.
The major research methods and results are divided into two parts:
part I:Praeruptorin C, D and E (PC, PD and PE) inhibited inflammatory responses in LPS-activated macrophages
1. Praeruptorin C, D and E (PC, PD and PE) inhibited NO production in LPS-activated macrophage
RAW264.7cells were co-cultured with2,4,8and16μg/ml praeruptorins and1 μg/ml LPS for18h. NO production in the cell culture medium were detected by Griess reagent. Our results showed that stimulation with LPS resulted in a9-fold increase in NO production compared with control, and then treatment with praeruptorins inhibited NO production in a dose-dependent manner with the IC50values of7.6μg/ml (PC),5.6μg/ml(PD), and5.8μg/ml (PE).
2. Cytotoxicity study of Praeruptorin C, D and E (PC, PD and PE) in macrophages
To investigate whether the inhibitory effect of Praeruptorins on NO production is due to a cytotoxic effect in macrophages, the cytotoxicity of Praeruptorins was assessed. Cells were treated with Praeruptorins at a dose of2,4,8,16and32μg/ml for24h. The results showed that treatment with Praeruptorins did not affect the RAW264.7cell viability at doses ranging from2to16μg/ml.
3. Praeruptorin C, D and E (PC, PD and PE) inhibited iNOS expression in LPS-activated macrophage
RAW264.7cells were co-cultured with4,8and16μg/ml praeruptorins and1μg/ml LPS for18h. The total protein of RAW264.7cells was extracted, and the iNOS expression was then investigated by Western blot. The results showed that stimulation with LPS resulted in marked increase of iNOS expression compared with control cells. Treatment with praeruptorins inhibited iNOS expression in a dose-dependent manner.
4. Praeruptorin C, D and E (PC, PD and PE) inhibited TNF-a and IL-6release in LPS-activated macrophage
RAW264.7cells were co-cultured with2,4,8and16μg/ml praeruptorins and1μg/ml LPS for18h. TNF-a and IL-6production in the cell culture medium were detected by ELISA method. The results showed that stimulation with LPS resulted in marked increase of TNF-a and IL-6production compared with control cells. Treatment with praeruptorins inhibited cytokines release in a dose-dependent manner. The IC50values of PC, PD and PE for inhibition of TNF-a production were40.7,17.3, and18.5μg/ml, respectively. For inhibition of IL-6production, the IC50values of PC, PD and PE were11.9,4.9, and5.1μg/ml, respectively.
5. Praeruptorin C, D and E (PC, PD and PE) inhibited TNF-a, IL-6and iNOS mRNA expression in LPS-activated macrophage
RAW264.7cells were co-cultured with2,4,8and16μg/ml praeruptorins and1μg/ml LPS for6h. Total cellular RNA from the treated cells was extracted by using Trizol reagent. Then, the mRNA expressions of TNF-a, IL-6and iNOS were quantitated by Real-time-PCR methods. The results showed that praeruptorins inhibited TNF-a, IL-6and iNOS mRNA expression in a dose-dependent manner. Treatment with16μg/ml PC, PD or PE lead to35%,45%, and47%inhibition of TNF-a mRNA expression, respectively (Fig.3c). For inhibition of IL-6mRNA expression, same concentration of PC, PD, or PE caused55%,72%, and70%inhibition, respectively. For inhibition of iNOS mRNA expression, Treatment with16μg/ml PC, PD or PE lead to53%,75%, and78%inhibition.
6. Praeruptorin C, D and E (PC, PD and PE) inhibited NF-kB activation in LPS-activated macrophage
Western blot and immunofluorescence analysis were performed to examine whether praeruptorins depresses NF-kB activation. The results showed that the p65subunit of NF-kB was translocated into the nucleus after LPS challenge for1h. However, treatment with praeruptorins markedly inhibited the translocation of p65. In the immunofluorescence assay, a low level of p65activity was observed in control macrophages. When stimulated by LPS for1h, p65was activated and translocated into nuclei, whereas treatment with praeruptorins inhibited the p65nuclear translocation.
We then investigated the effect of praeruptorins on LPS-induced IκB-a degradation to examine the molecular mechanisms by which praeruptorins inhibit NF-kB transcriptional activity. Our results showed that LPS treatment induced a marked degradation of the proteins. In contrast, loss of cytoplasmic IkB-α was inhibited by praeruptorins.
7. Praeruptorin C, D and E (PC, PD and PE) inhibited STAT-3activation in LPS-activated macrophage
The results showed that LPS-induced tyrosine phosphorylation of STAT1and STAT3started at2-h post-LPS stimulation, and the significant phosphorylation levels lasted to6h. Thus, we investigated the effect of praeruptorins on STAT pathway at4h after LPS stimulation. Pretreatment of16μg/ml praeruptorins did not affect LPS-stimulated STAT1phosphorylation, but they markedly suppressed STAT3tyrosine phosphorylation without impairing total STAT3proteins. AG490, a positive control, markedly inhibited phosphorylation on both STAT1and STAT3.
8. Praeruptorin C, D and E (PC, PD and PE) did not inhibited MAPKs activation in LPS-activated macrophage
To investigate the effects of pyranocoumarins on LPS-induced MAPK pathway, the phosphorylation levels of p38, ERK1/2, and JNK were examined. Our results showed that PC, PD, and PE did not inhibit LPS-induced phosphorylation of p38and JNK. On the contrary, these compounds significantly augmented the LPS-induced ERK1/2phosphorylation.
part Ⅱ:Praeruptorin D and E attenuate lipopolysaccharide/hydrochloric acid induced acute lung injury in mice
1. Effects of Praeruptorin A, C, D and E (PA, PC, PD and PE) on inflammatory cell counts and cytokine levels in Bronchoalveolar lavage fluid (BALF) of LPS-induced ALI mice
To examine the effects of Praeruptorins on LPS-induced pulmonary inflammation, PA, PC, PD or PE at a dose of80mg/kg were gavaged1h before intranasal administration of LPS. BALF was collected4h after LPS challenge. LPS markedly induced the increase of total cell and PMNs counts, as well as the release of TNF-a and IL-6in BALF. Pretreatment with PD and PE, but not PA and PC, significantly reduced these changes. There was no significant difference between PD and PE group.
2. Dose response relationship of Praeruptorin D and E (PD and PE)
A set of experiments were performed to study the dose response relationship of PD and PE. Our results showed that mice treated with80mg/kg PD or PE alone showed no significant difference in total cell and PMNs counts, when compared with control group. In LPS challenged mice, oral administration of both PD and PE led to a dose-dependent lowering of the total cell and PMNs counts in BALF. PD at doses of20,40and80mg/kg led to10%,33%and58%decrease of PMNs counts, respectively, whilst same doses of PE caused11%,43%and53%inhibition, respectively.
Because vascular leakage is another hallmark of ALI, we also examined total protein concentrations in BALF. The results showed that PD and PE depressed LPS-induced protein contents in a dose-dependent manner.
Both PD and PE dose-dependently reduced the LPS-induced increase of TNF-a and IL-6levels. PD at doses of80mg/kg led to51%and59%inhibitions of TNF-a and IL-6production, respectively. Similarly,80mg/kg of PE caused56%decrease of TNF-a level and51%inhibition of IL-6release, respectively.
3. Pretreatment with Praeruptorin D or E (PD or PE) decreased MPO activity in LPS-induced ALI mice
MPO activity in tissues is an indicator of PMNs infiltration. The results showed that instillation of LPS resulted in a significant increase in MPO activity in mouse lung tissues. In parallel with the number of PMNs, the level of MPO activity was partially inhibited by both PD and PE.
4. Pretreatment with Praeruptorin D or E (PD or PE) attenuated lung histopathology changes in LPS-induced ALI mice.
In the present study, no evident histological alteration was observed in lung specimens of normal mice. However, the instillation of LPS resulted in significant lung injury, evidenced by a large amount of neutrophil infiltration around the pulmonary vessel and interstitial spaces, a marked swelling of the alveolar walls, and severe hemorrhage as well as alveolar structural damage. These pathological changes were improved by pretreatment with80mg/kg of PD or PE.
5. Pretreatment with Praeruptorin D or E (PD or PE) inhibited NF-kB activation in LPS-induced ALI mice
We evaluated IkB-α degradation and nuclear NF-kB p65expression by Western blot analysis to investigate the molecular mechanism whereby treatment with PD and PE attenuated the development of acute lung injury. In our study, mice treated with LPS showed significant decrease of IκB-α protein, when compared with control group. In contrast, loss of cytoplasmic IκB-a was markedly inhibited by pretreatment with PD or PE.
On the other hand, mice challenged with LPS showed significant increase of NF-κB-p65subunits in the nucleus, as compared with control group. However, treatment with80mg/kg of PD or PE markedly inhibited the NF-icB-p65translocation.
6. Pretreatment with Praeruptorin D or E (PD or PE) protected mice from HCl-inducedALI
To evaluate the protective effects of PD and PE in the HCl-induced ALI mouse model, mice were gavaged with80mg/kg of PD or PE1h before HC1challenge, BALF and lung tissue samples were collected4h after HC1challenge. HC1instillation significantly induced increases of inflammatory cell counts, IL-6level and total protein concentration in BALF. However, HC1did not increase the release of TNF-a (data not shown). The HCl-induced PMNs influx, IL-6production and protein extravasation were dramatically reduced by oral administration of both PD and PE.
The effects of PD and PE on lung histopathology in HCl-treated mice were also examined. The results showed that the pathological changes induced by HC1 instillation, such as severe hemorrhage, neutrophil infiltration and alveolar structural damage, were attenuated by both PD and PE.
Based on the above results, the conclusions of this paper are as follows:
1. Praeruptorin C, D and E (PC, PD and PE) inhibited LPS-induced macrophage inflammatory response
A:PC, PD and PE inhibited NO production and iNOS expression in LPS-activated macrophage.
B:PC, PD and PE inhibited TNF-a and IL-6releases and mRNA expressions in LPS-activated macrophage.
C:PC, PD and PE inhibited NF-kB and STAT-3activation in LPS-activated macrophage.
2. Praeruptorin D and E (PD and PE) attenuate lipopolysaccharide/hydrochloric acid induced acute lung injury in mice.
A:PD and PE protected mice from LPS-induced acute lung injury.
a):PD and PE inhibited the inflammatory cells influx, cytokines release and protein leakage in BALF of LPS-challenged mice.
b):PD and PE inhibited the MPO activity in the lungs in of LPS-challenged mice.
c):PD and PE attenuated the pathological changes in the lung of LPS-challenged mice.
B:PD and PE protected mice from HCl-induced acute lung injury.
a):PD and PE inhibited the inflammatory cells influx, IL-6release and protein leakage in BALF of HCl-challenged mice.
b):PD and PE attenuated the pathological changes in the lung of HCl-challenged mice.
急性肺损伤(acute lung injury, ALI)是一种典型的过度性炎症参与的呼吸系统危重症,其定义是：心源性以外的各种肺内外致病因素引起的急性,进行性缺氧性呼吸衰竭,是肺部炎症和通透性增加的综合征。多种临床疾病均可诱发急性肺损伤,如肺炎、脓毒血症、以及胰腺炎等。研究认为,肺内过度性、失控性炎症反应是各种病因所致ALI的根本原因。
目前,临床上初步使用的ALI治疗药物主要有血管舒张剂(一氧化氮、前列环素的吸入治疗)、糖皮质激素、抗凝剂人类重组体活化蛋白C (activated proteinC,APC)、酮康唑(ketoconazole)、抗氧化剂N-乙酰半胱氨酸(NAC)等。然而,这些药物的疗效在各临床试验中还未完全得到证实,其用法用量还有着较大争议,并且有着许多严重的不良反应。因此,迄今为止,治疗ALI药物的总体研究进展是令人较为失望的,临床期待更为安全有效的治疗药物。
中药前胡为伞形科植物白花前胡(Peucedanum praeruptorum Dunn)的干燥根,具有散风清热、降气化痰等功效,临床常用于风热咳嗽痰多、痰热喘满、咯痰黄稠等。前期的研究显示白花前胡总提取物能够明显抑制二甲苯所致的小鼠耳肿胀和蛋清所致的大鼠足肿胀。同时,白花前胡提取物对卵蛋白(OVA)诱导的小鼠气道变态炎症反应也有明显的保护作用,这表明白花前胡良好的体内抗炎活性。然而,这些工作的主要研究对象是白花前胡粗提取物,并没有涉及白花前胡素单体化合物,同时,其抗炎作用机制也没有进行探讨。研究显示,白花前胡的主要有效成分为白花前胡素(Praeruptorin)等香豆素类化合物,如白花前胡甲素(Praeruptorin A, PA)、白花前胡乙素(Praeruptorin B, PB)、白花前胡丙素(Praeruptorin C, PC)、白花前胡丁素(Praeruptorin D,PD)以及白花前胡E素(Praeruptorin E, PE)。因此,在本研究中,我们研究了白花前胡素单体化合物(包括白花前胡甲素、丙素、丁素以及E素)的抗炎活性。我们利用脂多糖(LPS)建立体外巨噬细胞炎症模型,在细胞和分子水平上,从炎症相关因子NO、iNOS、TNF-α、IL-6的表达以及炎症信号通路NF-kB、MAPK和STAT的活化来研究白花前胡素类化合物的抗炎活性以及相关作用机制。由于白花前胡在临床上的应用主要是针对肺部疾病的治疗,因此,我们建立了LPS或者HC1诱导的急性肺损伤小鼠模型,检测白花前胡素类化合物对模型小鼠肺泡灌洗液中炎性细胞浸润、蛋白渗出以及炎症细胞因子释放的影响,同时,我们对小鼠肺组织中MPO的活力以及NF-κB通路的活化进行探讨,旨在研究和阐明白花前胡素类化合物对急性肺损伤的保护作用以及相关的分子机制。
研究方法和实验结果分为两大部分：
一、白花前胡丙素(PC)、白花前胡丁素(PD)以及白花前胡E素(PE)抑制
脂多糖诱导的巨噬细胞炎症反应
1、白花前胡丙素(PC)、丁素(PD)以及E素(PE)剂量的选择：小鼠巨噬细胞株RAW264.7细胞以1×104/孔的密度铺于96孔板后过夜贴壁,待各白花前胡素以2、4、8、16、32μg/ml剂量刺激细胞24h后,采用MTT法对细胞活力进行检测。结果显示,PC、PD以及PE在2-16μg/ml剂量范围内对细胞活力没有显著影响(差异无统计学意义)。因此本研究选取2-16μg/ml剂量范围应用于后续实验。
2、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制巨噬细胞中炎症介质NO的产生：待各白花前胡素以2、4、8、16μg/ml剂量与1μg/ml的LPS共同刺激细胞18h后,用Griess法检测细胞培养液上清中NO的含量。结果显示,PC、PD以及PE均可剂量依赖性地抑制NO的释放,其IC50值分别为7.6、5.6和5.8μg/ml。
3、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制巨噬细胞中iNOS蛋白的表达：提取细胞总蛋白后,采用Western blot法检测巨噬细胞中iNOS的含量。结果显示,在正常情况下,巨噬细胞中基本无iNOS的表达,而在LPS的刺激下,iNOS的表达显著增高。PD以及PE在16μg/ml剂量下均可显著抑制iNOS蛋白的高表达。
4、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制巨噬细胞中炎症细胞因子的产生：小鼠巨噬细胞株RAW264.7细胞以2×105/孔的密度铺于24孔板后过夜贴壁,PC、PD和PE以2、4、8、16μg/ml剂量与1μg/ml的LPS共同刺激细胞18h。用ELISA法检测发现各白花前胡素可显著抑制细胞培养液上清中TNF-a和IL-6的含量。PC、PD以及PE抑制TNF-a的IC50值为40.7、17.3和18.5μg/ml而对于IL-6,PC、PD以及PE的ICso值分别为11.9、4.9和5.1μg/ml。
5、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制iNOS、TNF-α和IL-6mRNA的表达：待各白花前胡素以4、8、16μg/ml剂量与1μg/ml的LPS共同刺激细胞6h后,提取细胞总RNA并逆转录,用Real-time PCR法检测细胞中iNOS、TNF-α和IL-6mRNA的表达。结果显示,在正常情况下,巨噬细胞中iNOS、 TNF-α和IL-6mRNA的表达均很少,而经过LPS刺激之后,其表达量显著增高。PC、PD以及PE均能剂量依赖性地抑制上述基因的表达。其中,在16μg/ml的剂量时,PC对iNOS、TNF-a和IL-6mRNA表达的抑制率分别为53%、35%和55%；PD的抑制率为75%、45%和72%；而PE的抑制率分别为78%、47%和70%。
6、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制巨噬细胞中NF-kB通路的激活
a：PC、PD和PE抑制LPS诱导的NF-κB亚基p65的核转运：提取细胞核蛋白,用Western blot法检测细胞核蛋白中p65的含量。结果发现,与对照细胞相比,1μg/ml剂量的LPS明显增加了巨噬细胞核蛋白中p65的含量；而各白花前胡素以16μg/ml剂量预处理细胞1h后,显著抑制了LPS引起的胞核p65含量的增加,但并不影响核内参蛋白lamin B的表达,说明白花前胡丙素、丁素以及E素可抑制NF-kB亚基p65由胞浆向胞核的转运。这一结果在细胞免疫荧光实验中也得到了证实。
b:PC、PD和PE抑制LPS诱导的IκB蛋白降解：与对照组细胞相比,1μg/ml剂量的LPS刺激30min后明显减少了巨噬细胞中1κB蛋白的含量；而白花前胡丙素、丁素以及E素以16μg/ml预处理细胞可显著抑制Iκ3蛋白含量的减少。
7、白花前胡丙素(PC)、丁素(PD)以及E素(PE)不能抑制巨噬细胞中MAPK通路的激活：MAPK是一类在各种组织细胞中广泛存在的蛋白激酶,由p38、细胞外信号调节激酶(ERK1/2)和C-Jun氨基末端激酶(JNK)三条信号通路组成。我们采用Western blot方法研究PC、PD和PE对MAPK通路的影响。结果显示：与对照组相比,1μg/ml剂量的LPS显著诱导了巨噬细胞中磷酸化p38(p-p38)、磷酸化ERK1/2(p-ERK1/2)以及磷酸化JNK (p-JNK)蛋白的增加。白花前胡丙素、丁素和E素(16μg/ml)对p-p38和p-JNK蛋白无明显影响,但可诱导p-ERK1/2蛋白的增加。
8、白花前胡丙素(PC)、丁素(PD)以及E素(PE)抑制脂多糖诱导的巨噬细胞中磷酸化STAT-3(p-STAT-3)蛋白的表达,但对磷酸化STAT-1(p-STAT-1)蛋白表达无影响：首先,我们用Western blot方法检测LPS诱导的STAT-1和STAT-3蛋白磷酸化的时间依赖性。结果发现,STAT-1和STAT-3蛋白的磷酸化在LPS刺激2h后开始出现,并可至少持续至LPS刺激后6h。因此,我们选取中间时间4h作为后续实验的时间点。与对照细胞相比,单独给药的PC、PD和PE对p-STAT-1和p-STAT-3蛋白均无影响；而与LPS组细胞相比,上述白花前胡素类化合物(16μg/ml)可显著抑制LPS诱导的p-STAT-3蛋白的增加,但对p-STAT-1蛋白无影响；阳性对照药物,JAK-STAT通路抑制剂AG490,对LPS诱导的p-STAT-1和p-STAT-3蛋白均可抑制。说明PC、PD和PE对JAK-STAT通路中的STAT-3蛋白具有选择性。
二、白花前胡甲素(PA)、白花前胡丙素(PC)、白花前胡丁素(PD)以及白花前胡E素(PE)对急性肺损伤小鼠模型的影响
1、白花前胡甲素(PA)、丙素(PC)、丁素(PD)以及E素(PE)对LPS诱导的急性肺损伤小鼠模型的影响：PA、PC、PD和PE以80mg/kg剂量灌胃给药,1h后,小鼠腹腔注射10%戊巴比妥钠(60mg/kg),待麻醉后滴鼻给予50μlLPS (40μg/ml).小鼠在给予LPS四小时后处死,行肺部灌洗。以灌洗液为样品,检测其中的炎性细胞数和细胞因子的水平。结果显示,PD和PE对急性肺损伤小鼠的细胞浸润以及炎症细胞因子释放均有明显的改善作用,而PA和PC几乎无影响。
a)白花前胡丁素(PD)和白花前胡E素(PE)剂量依赖性地减少肺损伤中炎性细胞的浸润：对照组小鼠肺泡灌洗液中的中性粒细胞数浓度为(15.0±3.90)×103/ml,而在LPS模型组,中性粒细胞浓度达到(648±45.8)×103/ml。80mg/kg的PD和PE明显抑制了中性粒细胞的浸润,其浓度分别减少至(285±48.6)×103/ml和(381±56.4)×103/ml;
b)白花前胡丁素(PD)和白花前胡E素(PE)剂量依赖性地抑制炎症细胞因子的释放：对照组小鼠肺泡灌洗液中的TNF-α和IL-6含量均很少,而LPS模型组中的细胞因子释放显著增多,TNF-a和IL-6的含量分别达到(6.645±0.70)和(1.096±0.149) ng/ml。80mg/kg的PD和PE可显著抑制LPS诱导的细胞因子释放。对于TNF-α,PD和PE两组含量分别减少至(3.379±0.586)和(3.962±0.646)ng/ml;而对于IL-6,PD和PE两组的含量分别降至(0.488±0.084)和(0.523±0.081) ng/ml。
c)白花前胡丁素(PD)和白花前胡E素(PE)剂量依赖性地抑制肺泡灌洗液中总蛋白含量：用Bradford法检测小鼠肺泡灌洗液中总蛋白的浓度,结果显示PD和PE均可抑制灌洗液中蛋白含量的增加,说明PD和PE对LPS诱导的肺部蛋白渗出具有改善作用。
2、白花前胡丁素(PD)和白花前胡E素(PE)抑制肺组织中MPO的活力：MPO活力反应了中性粒细胞在组织的分布多少。我们分别检测了PD和PE对肺组织中MPO活力的影响。结果显示,PD可将LPS诱导的MPO活力从(0.802±0.057)U/g减少至(0.631±0.038)U/g；而PE则将模型组的(0.830±0.074)U/g抑制至(0.581±0.058)U/g。
3、白花前胡丁素(PD)和白花前胡E素(PE)抑制LPS诱导的肺部病理变化：小鼠处死后,取肺叶固定、切片后进行HE染色。光学显微镜观察发现,对照组小鼠肺组织肺泡结构完整,肺泡壁无水肿,肺实质无明显炎症细胞浸润；LPS模型组小鼠肺泡壁则明显水肿增厚,可见大量的肺间质中性粒细胞浸润,有明显的出血、水肿,显示出严重的肺部炎症和肺水肿现象。而PD和PE显著减轻了LPS诱导的肺部损伤。
4、白花前胡丁素(PD)和白花前胡E素(PE)抑制LPS诱导的肺组织中NF-kB通路的活化：小鼠处死后,取全肺用液氮研磨,分别提取肺组织中的胞浆和胞核蛋白,Western blot方法检测胞浆中IκB蛋白和胞核中p65蛋白的含量。结果显示,PD和PE均可抑制肺组织蛋白中IκB蛋白的降解,以及p65的核转运。
5、白花前胡丁素(PD)和白花前胡E素(PE)对HCl诱导的急性肺损伤小鼠具有保护作用：PD和PE以80mg/kg剂量灌胃给药,1h后,小鼠腹腔注射10%戊巴比妥钠(60mg/kg),待麻醉后滴鼻给予5Oμ1HC1(0.1mol/L)。小鼠在给予LPS四小时后处死,行肺部灌洗。以灌洗液为样品,检测其中的炎性细胞数和细胞因子的水平。结果显示：
a)白花前胡丁素(PD)和白花前胡E素(PE)显著减少HCl诱导的肺部炎性细胞浸润：对照组小鼠肺泡灌洗液中的中性粒细胞数浓度很低,而在HCl模型组,中性粒细胞显著增加。80mg/kg的PD和PE明显抑制了中性粒细胞的浸润,其抑制率分别为49.7%和40.0%。
b)白花前胡丁素(PD)和白花前胡E素(PE)显著减少HCl诱导的炎症因子IL-6的释放：对照组小鼠肺泡灌洗液中有少量IL-6表达,而HCl可显著诱导IL-6的释放。80mg/kg的PD和PE明显抑制了肺泡灌洗液中的IL-6含量,其抑制率分别为44.1%和39.6%。
c)白花前胡丁素(PD)和白花前胡E素(PE)显著减少HCl诱导的蛋白渗出：Bradford法检测发现对照组小鼠肺泡灌洗液中有一定的蛋白含量,HCl可明显诱导灌洗液中总蛋白的增加,其含量约为对照组的3倍。80mg/kg的PD和PE可明显抑制蛋白渗出,其抑制率分别为29.2%和36.0%。
d)白花前胡丁素(PD)和白花前胡E素(PE)抑制HCl诱导的肺部病理变化：同LPS诱导的肺损伤模型一样,PD和PE也可明显改善HCl诱导的肺部炎症和水肿现象。
基于上述实验结果和文献分析,本文的结论如下：
本研究建立LPS诱导的巨噬细胞炎症模型以及LPS/HC1诱导的小鼠急性肺损伤模型,在细胞和动物水平上对白花前胡素类化合物的抗炎活性进行了研究。研究结果显示,白花前胡素类化合物,特别是白花前胡丁素(PD)以及白花前胡E素(PE)在体外以及体内均具有良好的抗炎活性。这为白花前胡的临床应用及其现代药理学研究提供了更多的理论和实验依据。
1.白花前胡素类化合物通过抑制iNOS蛋白和mRNA的表达,从而抑制LPS诱导巨噬细胞中炎症介质NO的产生。同时,白花前胡素类化合物还可显著减少炎症细胞因子TNF-α和IL-6的mRNA表达。这些结果显示白花前胡素类化合物良好的体外抗炎活性。
2.本论文研究了白花前胡素类化合物对三条主要炎症信号通路的影响,发现该类化合物为NF-kB以及STAT-3的双通路抑制剂,这合理解释了白花前胡素类化合物的抗炎活性。另一方面,NF-kB以及STAT-3这两条信号通路本身以及它们之间的cross-talk在众多肿瘤疾病,特别是肝细胞癌的发生和发展中起到极为重要的作用,曾有不少研究报道了白花前胡素类化合物抗肿瘤活性以及抗肿瘤耐药的作用,本研究对白花前胡素类化合物的抗肿瘤作用的分子机制起到了一定的启示作用。
3.本研究的一大创新点在于首次报道了白花前胡丁素(PD)以及白花前胡E(PE)素对急性肺损伤小鼠的保护作用。急性肺损伤是一种具有很高病死率的呼吸系统危重综合症,临床尚无特别安全有效的治疗药物。本研究通过建立LPS和HCl诱导的两种肺损伤模型,发现PD和PE可明显改善肺损伤小鼠的中性粒细胞浸润、细胞因子释放以及蛋白渗出三大病理特征。同时,PD和PE还可抑制肺组织中增加的MPO活力以及NF-κB的激活。这些结果为PD和PE作为急性肺损伤治疗药物的开发提供了动物实验基础。
Inflammation is a highly complex process to defend against foreign challenge or tissue injury, which involves various cell types (such as lymphocyte, macrophage, granulocyte, and endothelial cell, etc.) and multi-components (cytokines, vascular active substances, chemokines, adhesion molecules, inflammation-related enzymes). Inflammation seems like a double-edged sword:proper inflammatory response will help our body clearing the damage, fending off infection and promoting wound healing. However, excessive inflammatory response will in turn promote the occurrence and development of lots of inflammatory diseases, therefore affecting people's health.
Acute lung injury (ALI) is a life-threatening syndrome characterized by severe lung inflammation and increased microvascular permeability, and acute respiratory distress syndrome (ARDS) is the most severe form of lung injury. A variety of clinical disorders, such as pneumonia, aspiration of gastric contents, sepsis, major trauma, acute pancreatitis, can induce the occurrence of ALI. Studies suggest that the excessive, uncontrolled inflammatory response in the lung is the root cause of ALI. Clinical uses of corticosteroids, prostaglandins, prostacyclin, surfactant, lisofylline, ketoconazole and N-ace-tylcysteine have shown unsatisfactory significant improvements in patients' mortality, regardless of the unwanted side effects of these drugs. Thus, a drug with better therapeutic effects and fewer side effects is urgently needed.
The dry root of Peucedanum praeruptorum Dunn has been long used in traditional Chinese medicine for its remarkable effects on respiratory diseases. Praeruptorin A, B, C, D and E(PA, PB, PC, PD and PE), a class of pyranocoumarin, have been confirmed to be the main constituents in this herb. Previous studies showed the protective effects of total coumarins extracted from Peucedanum praeruptorum Dunn (TCP) on dimethylbenzene-induced ear swelling in mice and egg white-induced paw swelling in rats. In addition, TCP has been demonstrated to inhibit the airway hyper-responsiveness and inflammation in a mouse model of allergic airway disease. These data showed the anti-inflammatory activities of TCP in vivo. However, the pharmacological effect and the underlying mechanism of each Praeruptorin have not been investigated. In the present study, the anti-inflammatory activities of PC, PD and PE were investigated in LPS-stimulated murine macrophages. The effects of these Praerutorins on the expression of inflammatory mediators and cytokines, as well as activation of NF-kB, MAPK and STAT pathways were examined. In addition, the acute lung injury mouse models induced by LPS or HC1were established, and the effects of Praerutorins on inflammatory cells influx, cytokine release, protein leakage, MPO activity, as well as NF-kB activation were detected to investigate the protective effects and underlying mechanism of these compounds in ALI mice.
The major research methods and results are divided into two parts:
part I:Praeruptorin C, D and E (PC, PD and PE) inhibited inflammatory responses in LPS-activated macrophages
1. Praeruptorin C, D and E (PC, PD and PE) inhibited NO production in LPS-activated macrophage
RAW264.7cells were co-cultured with2,4,8and16μg/ml praeruptorins and1 μg/ml LPS for18h. NO production in the cell culture medium were detected by Griess reagent. Our results showed that stimulation with LPS resulted in a9-fold increase in NO production compared with control, and then treatment with praeruptorins inhibited NO production in a dose-dependent manner with the IC50values of7.6μg/ml (PC),5.6μg/ml(PD), and5.8μg/ml (PE).
2. Cytotoxicity study of Praeruptorin C, D and E (PC, PD and PE) in macrophages
To investigate whether the inhibitory effect of Praeruptorins on NO production is due to a cytotoxic effect in macrophages, the cytotoxicity of Praeruptorins was assessed. Cells were treated with Praeruptorins at a dose of2,4,8,16and32μg/ml for24h. The results showed that treatment with Praeruptorins did not affect the RAW264.7cell viability at doses ranging from2to16μg/ml.
3. Praeruptorin C, D and E (PC, PD and PE) inhibited iNOS expression in LPS-activated macrophage
RAW264.7cells were co-cultured with4,8and16μg/ml praeruptorins and1μg/ml LPS for18h. The total protein of RAW264.7cells was extracted, and the iNOS expression was then investigated by Western blot. The results showed that stimulation with LPS resulted in marked increase of iNOS expression compared with control cells. Treatment with praeruptorins inhibited iNOS expression in a dose-dependent manner.
4. Praeruptorin C, D and E (PC, PD and PE) inhibited TNF-a and IL-6release in LPS-activated macrophage
RAW264.7cells were co-cultured with2,4,8and16μg/ml praeruptorins and1μg/ml LPS for18h. TNF-a and IL-6production in the cell culture medium were detected by ELISA method. The results showed that stimulation with LPS resulted in marked increase of TNF-a and IL-6production compared with control cells. Treatment with praeruptorins inhibited cytokines release in a dose-dependent manner. The IC50values of PC, PD and PE for inhibition of TNF-a production were40.7,17.3, and18.5μg/ml, respectively. For inhibition of IL-6production, the IC50values of PC, PD and PE were11.9,4.9, and5.1μg/ml, respectively.
5. Praeruptorin C, D and E (PC, PD and PE) inhibited TNF-a, IL-6and iNOS mRNA expression in LPS-activated macrophage
RAW264.7cells were co-cultured with2,4,8and16μg/ml praeruptorins and1μg/ml LPS for6h. Total cellular RNA from the treated cells was extracted by using Trizol reagent. Then, the mRNA expressions of TNF-a, IL-6and iNOS were quantitated by Real-time-PCR methods. The results showed that praeruptorins inhibited TNF-a, IL-6and iNOS mRNA expression in a dose-dependent manner. Treatment with16μg/ml PC, PD or PE lead to35%,45%, and47%inhibition of TNF-a mRNA expression, respectively (Fig.3c). For inhibition of IL-6mRNA expression, same concentration of PC, PD, or PE caused55%,72%, and70%inhibition, respectively. For inhibition of iNOS mRNA expression, Treatment with16μg/ml PC, PD or PE lead to53%,75%, and78%inhibition.
6. Praeruptorin C, D and E (PC, PD and PE) inhibited NF-kB activation in LPS-activated macrophage
Western blot and immunofluorescence analysis were performed to examine whether praeruptorins depresses NF-kB activation. The results showed that the p65subunit of NF-kB was translocated into the nucleus after LPS challenge for1h. However, treatment with praeruptorins markedly inhibited the translocation of p65. In the immunofluorescence assay, a low level of p65activity was observed in control macrophages. When stimulated by LPS for1h, p65was activated and translocated into nuclei, whereas treatment with praeruptorins inhibited the p65nuclear translocation.
We then investigated the effect of praeruptorins on LPS-induced IκB-a degradation to examine the molecular mechanisms by which praeruptorins inhibit NF-kB transcriptional activity. Our results showed that LPS treatment induced a marked degradation of the proteins. In contrast, loss of cytoplasmic IkB-α was inhibited by praeruptorins.
7. Praeruptorin C, D and E (PC, PD and PE) inhibited STAT-3activation in LPS-activated macrophage
The results showed that LPS-induced tyrosine phosphorylation of STAT1and STAT3started at2-h post-LPS stimulation, and the significant phosphorylation levels lasted to6h. Thus, we investigated the effect of praeruptorins on STAT pathway at4h after LPS stimulation. Pretreatment of16μg/ml praeruptorins did not affect LPS-stimulated STAT1phosphorylation, but they markedly suppressed STAT3tyrosine phosphorylation without impairing total STAT3proteins. AG490, a positive control, markedly inhibited phosphorylation on both STAT1and STAT3.
8. Praeruptorin C, D and E (PC, PD and PE) did not inhibited MAPKs activation in LPS-activated macrophage
To investigate the effects of pyranocoumarins on LPS-induced MAPK pathway, the phosphorylation levels of p38, ERK1/2, and JNK were examined. Our results showed that PC, PD, and PE did not inhibit LPS-induced phosphorylation of p38and JNK. On the contrary, these compounds significantly augmented the LPS-induced ERK1/2phosphorylation.
part Ⅱ:Praeruptorin D and E attenuate lipopolysaccharide/hydrochloric acid induced acute lung injury in mice
1. Effects of Praeruptorin A, C, D and E (PA, PC, PD and PE) on inflammatory cell counts and cytokine levels in Bronchoalveolar lavage fluid (BALF) of LPS-induced ALI mice
To examine the effects of Praeruptorins on LPS-induced pulmonary inflammation, PA, PC, PD or PE at a dose of80mg/kg were gavaged1h before intranasal administration of LPS. BALF was collected4h after LPS challenge. LPS markedly induced the increase of total cell and PMNs counts, as well as the release of TNF-a and IL-6in BALF. Pretreatment with PD and PE, but not PA and PC, significantly reduced these changes. There was no significant difference between PD and PE group.
2. Dose response relationship of Praeruptorin D and E (PD and PE)
A set of experiments were performed to study the dose response relationship of PD and PE. Our results showed that mice treated with80mg/kg PD or PE alone showed no significant difference in total cell and PMNs counts, when compared with control group. In LPS challenged mice, oral administration of both PD and PE led to a dose-dependent lowering of the total cell and PMNs counts in BALF. PD at doses of20,40and80mg/kg led to10%,33%and58%decrease of PMNs counts, respectively, whilst same doses of PE caused11%,43%and53%inhibition, respectively.
Because vascular leakage is another hallmark of ALI, we also examined total protein concentrations in BALF. The results showed that PD and PE depressed LPS-induced protein contents in a dose-dependent manner.
Both PD and PE dose-dependently reduced the LPS-induced increase of TNF-a and IL-6levels. PD at doses of80mg/kg led to51%and59%inhibitions of TNF-a and IL-6production, respectively. Similarly,80mg/kg of PE caused56%decrease of TNF-a level and51%inhibition of IL-6release, respectively.
3. Pretreatment with Praeruptorin D or E (PD or PE) decreased MPO activity in LPS-induced ALI mice
MPO activity in tissues is an indicator of PMNs infiltration. The results showed that instillation of LPS resulted in a significant increase in MPO activity in mouse lung tissues. In parallel with the number of PMNs, the level of MPO activity was partially inhibited by both PD and PE.
4. Pretreatment with Praeruptorin D or E (PD or PE) attenuated lung histopathology changes in LPS-induced ALI mice.
In the present study, no evident histological alteration was observed in lung specimens of normal mice. However, the instillation of LPS resulted in significant lung injury, evidenced by a large amount of neutrophil infiltration around the pulmonary vessel and interstitial spaces, a marked swelling of the alveolar walls, and severe hemorrhage as well as alveolar structural damage. These pathological changes were improved by pretreatment with80mg/kg of PD or PE.
5. Pretreatment with Praeruptorin D or E (PD or PE) inhibited NF-kB activation in LPS-induced ALI mice
We evaluated IkB-α degradation and nuclear NF-kB p65expression by Western blot analysis to investigate the molecular mechanism whereby treatment with PD and PE attenuated the development of acute lung injury. In our study, mice treated with LPS showed significant decrease of IκB-α protein, when compared with control group. In contrast, loss of cytoplasmic IκB-a was markedly inhibited by pretreatment with PD or PE.
On the other hand, mice challenged with LPS showed significant increase of NF-κB-p65subunits in the nucleus, as compared with control group. However, treatment with80mg/kg of PD or PE markedly inhibited the NF-icB-p65translocation.
6. Pretreatment with Praeruptorin D or E (PD or PE) protected mice from HCl-inducedALI
To evaluate the protective effects of PD and PE in the HCl-induced ALI mouse model, mice were gavaged with80mg/kg of PD or PE1h before HC1challenge, BALF and lung tissue samples were collected4h after HC1challenge. HC1instillation significantly induced increases of inflammatory cell counts, IL-6level and total protein concentration in BALF. However, HC1did not increase the release of TNF-a (data not shown). The HCl-induced PMNs influx, IL-6production and protein extravasation were dramatically reduced by oral administration of both PD and PE.
The effects of PD and PE on lung histopathology in HCl-treated mice were also examined. The results showed that the pathological changes induced by HC1 instillation, such as severe hemorrhage, neutrophil infiltration and alveolar structural damage, were attenuated by both PD and PE.
Based on the above results, the conclusions of this paper are as follows:
1. Praeruptorin C, D and E (PC, PD and PE) inhibited LPS-induced macrophage inflammatory response
A:PC, PD and PE inhibited NO production and iNOS expression in LPS-activated macrophage.
B:PC, PD and PE inhibited TNF-a and IL-6releases and mRNA expressions in LPS-activated macrophage.
C:PC, PD and PE inhibited NF-kB and STAT-3activation in LPS-activated macrophage.
2. Praeruptorin D and E (PD and PE) attenuate lipopolysaccharide/hydrochloric acid induced acute lung injury in mice.
A:PD and PE protected mice from LPS-induced acute lung injury.
a):PD and PE inhibited the inflammatory cells influx, cytokines release and protein leakage in BALF of LPS-challenged mice.
b):PD and PE inhibited the MPO activity in the lungs in of LPS-challenged mice.
c):PD and PE attenuated the pathological changes in the lung of LPS-challenged mice.
B:PD and PE protected mice from HCl-induced acute lung injury.
a):PD and PE inhibited the inflammatory cells influx, IL-6release and protein leakage in BALF of HCl-challenged mice.
b):PD and PE attenuated the pathological changes in the lung of HCl-challenged mice.
引文
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