核因子κB对肿瘤坏死因子-α刺激的肺泡Ⅱ型上皮细胞NADPH oxidase1基因调控机制的探讨
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摘要
研究背景和目的
急性肺损伤(acute lung injury, ALI)是由各种严重的非心源性致病因素如严重感染、休克、创伤、误吸等引起,以急性进行性加重的呼吸困难和难治性低氧血症为主要表现的一种临床综合征,严重阶段可发展为急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)。ALI/ARDS发病率高,病死率高,缺乏特效治疗,因而其机制研究仍受各国科学家重视。
炎症反应及其诱发的氧化应激是ALI/ARDS重要的发病机制。一方面,以肿瘤坏死因子-α (tumor necrosis factor-a, TNF-a)所代表的细胞因子大量释放为特征的全身过度炎症反应是ALI的主要发病基础。另一方面,TNF-α能诱导内皮细胞、中性粒细胞等产生大量活性氧(Reactive Oxidative Species, ROS),造成过度的氧化应激,与炎症反应协同加重病情。此外,过度的炎症反应可诱导结构细胞内源性ROS生成增多,加重氧化-抗氧化系统失衡,直接损伤结构细胞。炎症及氧化失衡所导致的肺泡上皮细胞凋亡进一步造成肺泡塌陷、肺泡-毛细血管屏障通透性增加,肺泡腔内渗出物增多,诱发或加重肺水肿、肺不张,引起难以纠正的低氧血症。因此,针对ALI患者的肺组织保护策略,除了进行有效的抗感染治疗,同时还需对机体免疫系统合理干预,将过度的炎症反应和氧化应激维持在适当水平,保持肺结构细胞完整性,维持肺泡-毛细血管的屏障功能。
急性肺损伤的炎症反应失控表现为促炎细胞因子和抑炎细胞因子的过度激活与失衡。TNF-α是启动和放大炎症反应的主要炎症介质。ALI/ARDS患者、动物模型的支气管肺泡灌洗液或血清中TNF-α白介素-1β、白介素-8、白介素-10等细胞因子明显增加,其浓度与疾病进展、临床预后密切相关[7]。因此,测定促炎细胞因子和抑炎细胞因子浓度可直观地评估ALI模型的炎症反应水平。
氧化应激的发生,一方面是由于ROS和/或RNS的产生过多；另一方面是由于抗氧化物质的过度消耗。烟酰胺腺嘌呤二核苷酸磷酸氧化酶(nicotinamide adenine dinucleotide phosphate oxidase, NADPH oxidase, NOX)包括7种同源蛋白,共同组成NOX/DUOX家族,是产生ROS的重要来源。NOX/DUOX家族各亚型的分布具有组织细胞特异性,其衍生的ROS的功能也存在差异。吞噬细胞型ROS主要参与宿主防御,非吞噬细胞型ROS则可作为第二信使直接或间接作用于信号传导通路。NOX/DUOX家族各亚型与慢性阻塞性肺疾病、支气管哮喘、急性肺损伤等疾病密切相关,但目前尚缺乏各亚型在肺泡Ⅱ型上皮细胞分布情况的相关研究。筛选出与ALI相关的重要亚型,研究其调控机制,进而调节其ROS的生成,是下调ALI氧化应激损伤的重要途径之一。
本实验室的前期研究表明,NF-κB/p65基因可能通过下调凋亡相关基因Bax/Bcl-2表达比例减少TNF-α诱导的A549细胞凋亡。NF-κB是TNF-α促发、放大炎症反应的信号转导核心,可调控包括NOX/DUOX家族基因在内的众多炎症疾病相关因素的表达。通过干预NF-κB通路下调其下游靶基因的表达水平,可能比单一补充细胞因子拮抗剂或抗氧化剂效果更好。
启动子(promoter)是RNA聚合酶辨认、结合和启动转录的一段DNA序列。真核生物RNA聚合酶本身不能直接识别、结合相应的启动子序列,必须经转录因子的介导辅助才能与之结合。启动子与转录因子的相互作用影响基因转录、翻译等过程,是研究疾病机制的重要切入点。生物信息学软件分析初步结果显示,NOX1启动子近端存在2个NF-κB结合元件,NF-κB活化进入细胞核后可能通过与该元件结合,调控NOX1的转录过程。因此,明确NF-κB对NOX1基因的调控机制,通过下调NF-κB的活性调控NOX1生成ROS,理论上可以减轻TNF-α促发的炎症反应和氧化应激程度,为调控ALI的肺保护策略研究提供更为精准的靶基因。
综上所述,本课题的研究目的如下：
1采用TNF-α刺激肺泡II型上皮细胞(A549),评估其炎症反应、氧化应激、细胞凋亡水平,建立ALI体外细胞炎症模型；
2探讨NF-KB/p65在TNF-α诱导的炎症及氧化应激损伤病理过程中的作用；
3明确参与TNF-α诱导A549细胞产生炎症——氧化应激病理过程的NOX/DUOX家族亚型,分析其与NF-κB的相关性；
4探讨NF-κB对NOX1的调控机制及NOX1启动子区完整性对NF-κB调控作用的影响。
方法
肺泡Ⅱ型上皮细胞来源的A549细胞由广州军区广州总医院医学实验科细胞库提供。A549细胞在含10%灭活小牛血清、100U/mL青霉素和100U/mL链霉素的RPMI-1640培养基中,置于37℃、5%CO2、饱和湿度条件下的培养箱中培养。
1沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮细胞炎症反应和细胞凋亡水平的影响
A549细胞分为5组：空白对照组(不加干预因素)、TNF-α组(仅用TNF-α刺激)p65siRNA组(预转染p65siRNA且用TNF-α刺激)、siRNA阴性对照组(预转染阴性对照siRNA且用TNF-α刺激)、siRNA阳性对照组(预转染阳性对照siRNA且用TNF-α刺激)。进行TNF-α(10ng/mL)刺激前6小时预转染siRNA,倾去转染液后开始以TNF-α刺激24小时。
以RT-PCR法检测NF-κB/p65mRNA的表达水平；免疫印迹法分别检测胞浆和胞核中NF-κB/p65蛋白的表达水平；ELISA法检测细胞培养上清液中IL-1β、IL-4、IL-6、IL-8、IL-10的浓度；MTT法检测细胞存活率；Annexin V/PI法检测细胞凋亡率。
2沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮细胞氧化应激水平的影响
细胞分组同上。以DCFH-DA探针法检测细胞内活性氧水平；比色法检测细胞的丙二醛浓度、总抗氧化能力、总超氧化物歧化酶活力、总谷胱甘肽的浓度。
3沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮NOX/DUOX家族的影响细胞分组同上。Real-time RT-PCR检测NOX/DUOX家族各基因mRNA表达水平；免疫印迹法检测NOX1、NOX2、NOX4蛋白的表达水平。
4转录因子NF-κB/p65对肺泡Ⅱ型上皮细胞NOX1基因调控机制的探讨
Alibaba2.1软件预测NOX1近端启动子区的NF-κB结合元件,据此设计并合成DNA探针；EMSA法检测上述探针与核蛋白中NF-κB是否结合；在此基础上,分别构建含目的NOX1启动子片段(1415bp)的重组pGL3萤光素酶报告基因载体pGL3-NOX1-1415、缺失上述NF-κB阳性结合位点的启动子片段(1327bp)所对应的重组pGL3萤光素酶报告基因载体pGL3-NOX1-1327(缺失载体)；将pGL3-NOX1-1415、pGL3-NOX1-1327分别瞬时转染入A549细胞,同时转染质粒pRL-TK作为内对照,TNF-α刺激24小时,双荧光素酶报告基因系统检测转染细胞的萤光素酶活性。
5统计学分析：应用SPSS13.0软件包进行统计学分析。计量资料以均数±标准差(X±S)表示,所有数据均进行方差齐性检验。采用单因素方差分析进行组间均数比较,满足方差齐性的多重比较采用LSD法,不满足方差齐性的多重比较采用Dunnett's T3法。显著性水准α取0.05,双侧。
结果
1沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮细胞炎症反应和细胞凋亡水平的影响
1.1各组A549细胞NF-KB/p65mRNA表达水平的差异有统计学意义(F=771.003,P<0.001)。 TNF-α组的A549细胞NF-KB/p65mRNA表达水平较空白对照组上调(P<0.05); NF-κB/p65siRNA组的A549细胞NF-κB/p65mRNA表达水平较空白对照组、TNF-α组、阴性对照组下调(P<0.05)。
1.2各组A549细胞胞浆中NF-KB/p65蛋白表达水平的差异有统计学意义(F=231.811,JP<0.001)。NF-κB/p65siRNA组的A549细胞胞浆中NF-KB/p65蛋白表达水平较空白对照组、TNF-α组、阴性对照组下调(P<0.05)。
1.3各组A549细胞胞核中NF-κB/p65蛋白表达水平的差异有统计学意义(F=1206.566, P<0.001), TNF-α组的A549细胞胞核中NF-KB/p65蛋白表达水平较空白对照组上调(P<0.05)。 NF-κB/p65siRNA组的A549细胞胞核中NF-KB/p65蛋白表达水平较TNF-α组、阴性对照组、阳性对照组下调(P<0.05)。
1.4各组A549细胞培养上清液中IL-1β、IL-4、IL-6、IL-8、IL-10的浓度差异有统计学意义(P<0.001)。TNF-α组的A549细胞培养上清液中上述细胞因子浓度较空白对照组上调(P<0.05)；NF-κB/p65siRNA组的A549细胞培养上清液中上述细胞因子浓度较TNF-α组、阴性对照组、阳性对照组下调(P<0.05)。
1.5各组A549细胞存活率的差异有统计学意义(F=125.145,P<0.001)。TNF-α组的A549细胞存活率较空白对照组下调(P<0.05); NF-κB/p65siRNA组的A549细胞存活率较TNF-α组、阴性对照组、阳性对照组上调(P<0.05)；较空白对照组下调(P<0.05)。
1.6各组A549细胞凋亡率的差异有统计学意义(F=155.592, P<0.001)。 TNF-α组的A549细胞凋亡率较空白对照组上调(P<0.05); NF-KB/p65siRNA组的A549细胞凋亡率较TNF-α组、阴性对照组、阳性对照组下调(P<0.05)；较空白对照组上调(P<0.05)。
2沉默NF-KB/p65对TNF-α诱导的肺泡Ⅱ型上皮细胞氧化应激水平的影响
2.1各组A549细胞活性氧、丙二醛的浓度差异有统计学意义(P<0.001)。TNF-α组的A549细胞活性氧、丙二醛的浓度较空白对照组上调(P<0.05)；NF-κB/p65siRNA组的A549细胞活性氧、丙二醛的浓度较TNF-α组、阴性对照组、阳性对照组下调(P<0.05)。
2.2各组A549细胞总抗氧化能力、超氧化物歧化酶、总谷胱甘肽的浓度差异有统计学意义(P<0.001)。TNF-α组的A549细胞的上述指标较空白对照组下调(P<0.05)；NF-κB/p65siRNA组的A549细胞的上述指标较TNF-α组、阴性对照组、阳性对照组上调(P<0.05)。
3沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮NOX/DUOX家族的影响
3.1各组A549细胞NOX1、NOX2、NOX4mRNA表达水平的差异有统计学意义(P<0.001)；TNF-α组的A549细胞上述基因mRNA表达水平较空白对照组上调(P<0.05)。 NF-κB/p65siRNA组的A549细胞上述基因mRNA表达水平较TNF-α组、阴性对照组下调(P<0.05)。
3.2各组A549细胞NOX3、NOX5、DUOX1、DUOX2mRNA的表达水平无统计学差异(P>0.05)。
3.3各组A549细胞NOX1、NOX2、NOX4蛋白表达水平的差异有统计学意义(P<0.001)；TNF-α组的A549细胞上述蛋白的表达水平较空白对照组上调(P<0.05); NF-κB/p65siRNA组的A549细胞上述蛋白表达水平较TNF-α组、阴性对照组下调(P<0.05)。
4转录因子NF-κB/p65对肺泡Ⅱ型上皮细胞NOX1基因调控机制的探讨
4.1Alibaba2.1软件分析结果提示,NOX1近端启动子区约1500bp范围内可预测到2个NF-κB结合元件。元件1称为NOX1/kB1,位于-1095-1086,序列为5'-CAGGAAAAC-3';元件2称为NOX1/κB2:位于-261-252,序列为5'-TAAAATCCCC-3'。
4.2经EMSA检测,元件2可与核蛋白中NF-KB/p65结合。
4.3载体pGL3-NO1-1415、载体pGL3-NOX1-1327经双酶切电泳及测序鉴定,结构正确。
4.4转染载体pGL3-NOX1-1415的各组A549细胞萤光素酶活性的差异有统计学意义(F=142.105,P<0.001)。TNF-α组的A549细胞萤光素酶活性较空白对照组上调(P<0.05); NF-κB/p65siRNA组的A549细胞萤光素酶活性较TNF-α组、阴性对照组下调(P<0.05)；较空白对照组上调(P<0.05)。4.5分别转染pGL3空质粒、载体pGL3-NOX1-1415、载体pGL3-NOX1-1327后,经TNF-α刺激的各组A549细胞萤光素酶活性的差异有统计学意义(F=646.047,P<0.001)。转染载体pGL3-NOX1-1415、pGL3-NOX1-1327的A549细胞萤光素酶活性较转染pGL3者上调(P<0.05)；转染pGL3-NOX1-1415较pGL3-NOX1一1327,A549细胞萤光素酶活性升高(P<0.05)。
结论
1.TNF-α诱导肺泡Ⅱ型上皮细胞A549能够在体外模拟ALI肺结构细胞的炎症——氧化损伤及细胞凋亡；
2.NF-κB/p65参与TNF-α诱导的细胞炎症-氧化损伤的病理生理过程,RNA干扰技术可有效沉默NF-κB/p65并下调炎症反应和氧化应激程度,减少细胞凋亡,达到保护结构细胞的目的。
3.NOX/DUOX家族中NOX1、NOX2、NOX4等亚型参与了TNF-α诱导的细胞炎症-氧化损伤的病理生理过程；
4.NF-κB/p65通过与NOXl启动子区κB结合元件(-261--252bp)结合,调控其转录过程；NOX1启动子区结构的完整性影响NF-κB/p65的结合及调控作用。
综上所述,在TNF-α诱导肺泡Ⅱ型上皮细胞A549产生的炎症—氧化应激环路中,NF-κB/p65可通过与NOX1启动子区KB结合元件(-261--252bp)结合实现对其转录调控作用。
Background and Objection:
Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), have been documented clinically following several pathological states such as serious infection, shock, trauma, aspiration and characterized by progressive respiratory distress and acute severe arterial hypoxemia. As a result of the lack of effective treatment, the incidence and mortality rates of ALI/ARDS are not greatly improved. Therefore, many groups from different countries still focus on the utilizing multiple approaches (biological, genomic and genetic) to clarify the underlying pathophysiological mechanisms by which a wide variety of insults can lead to this common clinical and pathophysiological syndrome.
Massive release of various pro-and anti-inflammatory cytokines including TNF-alpha lead to excessive systemic inflammatory response, which have important effects on the pathogenesis of ALI/ARDS. On the other hand, many studies indicate that TNF-a stimulates the generation of reactive oxygen species (ROS) by human endothelial cells and neutrophils. Reactive oxygen species is one of the most prominent manifestations of oxidative stress and associated with the apoptosis of type Ⅱ alveolar epithel cells. Therefore, besides of the anti-infective therapy, keeping the level of both inflammatory response and oxidative stress in an appropriate level should make contribution to the treatment of ALI.
On the basis of several experimental studies, TNF-a, along with other cytokines, such as IL-β, IL-8, IL-10, is suggested as an important early mediator of ALI. High generation levels of these cytokines had been detected in the plasma, bronchoalveolar lavage fluid of ALI/ARDS patients and animal models, the concentration of which is closely related to the progression and prognosis of ALI.
Excessive generation of ROS/RNS and consumption of the antioxidants break the balance between them, leading to the oxidative stress. The NOX/DUOX oxidase family is considered as a major source of reactive oxygen species. Tissue-specific distribution of NOX/DUOX isoforms affects the function of ROS generated by them. Their distribution in type Ⅱ alveolar epithel cells is still an unsettled subject, so as their contributions to ALL Down-regulating the ROS generation of NOX/DUOX oxidase family can be an effective method to reduce oxidative stress.
NF-κB is of central importance in TNF-a induced-inflammation. It regulates the expression of various inflammation-associated genes, including the NOX/DUOX family. More than700inhibitors of the NF-κB activation pathway, including antioxidants, peptides, small RNA/DNA, have been described. Downregulation of NF-κB-regulated genes by NF-κB silencing can be a better way to ALI treatment than cytokine antagonists or the antioxidants.
A promoter is a region of DNA that facilitates the transcription of a particular gene. Transcription factors regulate the gene transcription by binding to the specific site of the promoter. Several groups reported the mechanisms of transcriptional regulation of NOX1,mentioning NF-κB, in the colon epithelium, macrophage, gastric mucosal cell, vascular smooth muscle cells. There are two typical NF-κB binding elements predicted by Alibaba2.1software in the proximal promoters (-1500bp) of the human NOX1. Therefore, it is necessary for us to investigate wheather the NF-κB signal pathway is involved in the regulations of NOX1-derived ROS, which may facilitate the research on the ALI/ARDS treatment.
The goals of the present study are as followed:
1To simulate an inflammatory environment during ALI/ARDS in vitro, Type Ⅱ alveolar epithelial cells (A549) were cultured and stimulated by TNF-α (10ng/mL), then the levels of inflammatory response, oxidative stress and apoptosis were investigated;
2To investigate the effects of NF-κB p65silencing by RNAi on the level of inflammatory response, oxidative stress and apoptosis in TNF-a-induced A549;
3To investigate the expression of NOX/DUOX oxidase family of A549in the absence or presence of TNF-α, and discuss the relationship between NOX/DUOX oxidase family and NF-κB;
4To obtain one of the NOX/DUOX oxidase isoforms which is closely related to NF-κB, then investigate whether the NF-κB signal pathway was involved in the regulations of it.
5Statistical analysis:Data were expressed as means±standard error (S.E.). The statistical significance of differences among groups was assessed by one-way analysis of variance (ANOVA), followed by least significant difference (LSD) for multiple comparison, as a. post hoc test. All statistical analyses were performed using SPSS13.0. For these comparisons, P<0.05was considered to be statistically significant.
Methods and materials
Type Ⅱ alveolar epithelial cells (A549) were obtained from experimental department of Guangzhou General Hospital of Guangzhou Military command and routinely grown in RPMI-1640supplemented with10%fetal bovine serum (FBS), penicillin (100U/mL), and streptomycin (100U/mL) in a humidified chamber supplemented with5%CO2at37℃.
1The effects of NF-κB p65silencing by RNAi on the level of inflammatory response and apoptosis in TNF-a-induced type Ⅱ alveolar epithel cell
The cells were divided into five groups:control group (cells without any interference factors); TNF-α group (10μg/L TNF-α); NF-KB/p65siRNA group (10μg/L TNF-α50nM NF-κB/p65siRNA); negative siRNA group (10μg/L TNF-α,50nM negative siRNA); positive siRNA group (10μg/L TNF-a,50nM positive siRNA). The RNA interference lasted for6hours; the TNF-a stimulation lasted for24hours.
RT-PCR and Western blotting were performed to analyze the silence efficiency of RNAi targeting NF-kB p65. The consentrations of IL-1β, IL-4, IL-6, IL-8and IL-10in the culture supernatant were measured by ELISA. The survival rate of cell was assessed by the methyl thiazolyl tetrazolium (MTT) assay. The apoptosis rate of cell was detected by Annexin V/PI assay.
2The effects of NF-κB p65silencing by RNAi on the level of oxidative stress in TNF-α-induced type Ⅱ alveolar epithel cell
The cells were divided into five groups as indicated above. The peroxide-sensitive fluorescent probe2',7'-dichlorofluorescein diacetate (DCFH-DA) was used to assess the intracellular ROS production. The concentration of MDA, TAOC, SOD and TGSH was detected by kits using colorimetry.
3The effects of NF-⒘B p65silencing by RNAi on the expression of NOX/DUOX oxidase family of A549
The mRNA expression of NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2gene were detected by real-time RT-PCR. The expression of NOX1, NOX2, NOX4protein were detected by western bloting.
4NF-κB/p65regulates the expression of NOX1of A549
NF-κB binding elements in the proximal promoters (-1500bp) of the human NOX1were predicted by Alibaba2.1software. The binding of the elements with NF-κB was detected by electrophoretic mobility shift assay. pGL3basic vector inserted with the NOX1proximal promoter was constructed, named "pGL3-NOX1-1415"; pGL3basic vector inserted with the NOX1proximal promoter which was absence of the positive element as constructed, named "pGL3-NOX1-1327". They were respectively transfected into A549, then the cells were stimulated with TNF-a. The luciferase level which stands for the expression of reporter gene was monitored on MD SpectraMax M5enzyme-labeled instrument.
Results:
1The effects of NF-κB p65silencing by RNAi on the level of inflammatory response and apoptosis in TNF-a-induced type Ⅱ alveolar epithel cell
RT-PCR and Western blotting were performed to analyze the silence efficiency of RNAi targeting NF-κB p65. The consentrations of IL-1β、IL-8and IL-10in the culture supernatant were measured by ELIS A. The survival rate of cell was assessed by the methyl thiazolyl tetrazolium (MTT) assay. The apoptosis rate of cell was detected by Annexin V/PI assay.
1.1Compared with control group, the expression of NF-KB/p65mRNA was significantly higher in TNF-a group. The expression of NF-κB/p65mRNA in NF-KB/p65siRNA group was significantly lower than that in TNF-α group, negative siRNA group and positive siRNA group.
1.2The expression of NF-κB/p65protein in cytoplasm of NF-κB/p65siRNA group was significantly lower than that in control group, TNF-α group, negative siRNA group and positive siRNA group.
1.3Compared with control group, the expression of NF-KB/p65protein in nucelus was significantly higher in TNF-α group. The expression of NF-KB/p65protein in nucelus in NF-κB/p65siRNA group was significantly lower than that in TNF-α group, negative siRNA group and positive siRNA group.
1.4Compared with control group, the concentrations of IL-1β, IL-4, IL-6, IL-8and IL-10in the culture supernatant were significantly higher in TNF-a group. They are significantly lower in NF-κB/p65siRNA group than that in TNF-α group, negative siRNA group and positive siRNA group.
1.5Compared with control group, the survival rate was significantly lower in TNF-α group. It was significantly higher in NF-κB/p65siRNA group than that in TNF-α group, negative siRNA group and positive siRNA group.
1.6Compared with control group, the apoptosis rate was significantly higher in TNF-α group. It was significantly lower in NF-κB/p65siRNA group than that in TNF-α group, negative siRNA group and positive siRNA group.2The effects of NF-κB p65silencing by RNAi on the level of oxidative stress in TNF-α-induced type Ⅱ alveolar epithel cell
Compared with control group, the concentrations of ROS and MDA were significantly higher in TNF-α group. It was significantly lower in NF-κB/p65siRNA group than that in TNF-α group, negative siRNA group and positive siRNA group. Compared with control group, the concentrations of TAOC SOD and TGSH were significantly lower in TNF-a group. It was significantly higher in NF-KB/p65siRNA group than that in TNF-a group, negative siRNA group and positive siRNA group.
3The effects of NF-κB p65silencing by RNAi on the expression of NOX/DUOX oxidase family of A549
Compared with control group, the expression of NOX1, NOX2and NOX4mRNA were significantly higher in TNF-a group. It was significantly lower in NF-KB/p65siRNA group than that in TNF-a group and negative siRNA group.
Compared with control group, the expression of NOX1, NOX2and NOX4protein were significantly higher in TNF-a group. It was significantly lower in NF-KB/p65siRNA group than that in TNF-a group and negative siRNA group.
The mRNA expression of NOX3, NOX5, DUOXland DUOX2had no statistical significance.
4NF-KB/p65regulates the expression of NOX1of A549
NF-κB binding elements in the proximal promoters (-1500bp) of the human NOX1were predicted by Alibaba2.1software. The binding of the elements with NF-κB was detected by electrophoretic mobility shift assay. pGL3basic vector inserted with the NOX1proximal promoter was constructed, named "pGL3-NOX1-1415"; pGL3basic vector inserted with the NOX1proximal promoter which was absence of the positive element as constructed, named "pGL3-NOX1-1327". They were respectively transfected into A549, then the cells were stimulated with TNF-a. The luciferase level which stands for the expression of reporter gene was monitored on MD SpectraMax M5enzyme-labeled instrument.
4.1Two potential NF-κB responsive cis-acting elements in the proximal promoters of human Nox1genes (1439) were assessed using Alibaba2.1software
4.2the results of EMS A showed a binding of the probe2designed based on Nox1/kB2with NF-κB/p65.
4.3the sequence of the vector "pGL3-NOXl-1415" and "pGL3-NOX1-1327" were identified to be corret.
4.4Compared with control group, the luciferase level were significantly higher in TNF-a group transfected with the vector "pGL3-NOX1-1415". It was significantly lower in NF-κB/p65siRNA group than that in TNF-a group and negative siRNA group.
4.5Compared with control group, the luciferase level were significantly higher in the group transfected with the vector "pGL3-NOXl-1327", but it was significantly lower than that in the group transfected with the vector "pGL3-NOX1-1415".
Conclusion
TNF-a induced excessive inflammatory response and oxidative stress, leading to higher apoptosis rate of A549. NF-κB/p65silencing could down-regulate the over inflammation and oxidative stress induced by TNF-a, and down-regulate the apoptosis rate of A549. TNF-a increased the expression of NOX1, NOX2and NOX4of A549while NF-KB/p65silencing decreased their expression. Activated NF-κB binds to specific elements in NOX1promoter regions to control the transcription. Integrity of the NOX1proximal promoters affected the NF-κB binding. On the whole, NF-κB is an essential role for transcriptional regulation of NOX1in TNF-a induced A549, then affects the NOX1-derived ROS generation.
急性肺损伤(acute lung injury, ALI)是由各种严重的非心源性致病因素如严重感染、休克、创伤、误吸等引起,以急性进行性加重的呼吸困难和难治性低氧血症为主要表现的一种临床综合征,严重阶段可发展为急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)。ALI/ARDS发病率高,病死率高,缺乏特效治疗,因而其机制研究仍受各国科学家重视。
炎症反应及其诱发的氧化应激是ALI/ARDS重要的发病机制。一方面,以肿瘤坏死因子-α (tumor necrosis factor-a, TNF-a)所代表的细胞因子大量释放为特征的全身过度炎症反应是ALI的主要发病基础。另一方面,TNF-α能诱导内皮细胞、中性粒细胞等产生大量活性氧(Reactive Oxidative Species, ROS),造成过度的氧化应激,与炎症反应协同加重病情。此外,过度的炎症反应可诱导结构细胞内源性ROS生成增多,加重氧化-抗氧化系统失衡,直接损伤结构细胞。炎症及氧化失衡所导致的肺泡上皮细胞凋亡进一步造成肺泡塌陷、肺泡-毛细血管屏障通透性增加,肺泡腔内渗出物增多,诱发或加重肺水肿、肺不张,引起难以纠正的低氧血症。因此,针对ALI患者的肺组织保护策略,除了进行有效的抗感染治疗,同时还需对机体免疫系统合理干预,将过度的炎症反应和氧化应激维持在适当水平,保持肺结构细胞完整性,维持肺泡-毛细血管的屏障功能。
急性肺损伤的炎症反应失控表现为促炎细胞因子和抑炎细胞因子的过度激活与失衡。TNF-α是启动和放大炎症反应的主要炎症介质。ALI/ARDS患者、动物模型的支气管肺泡灌洗液或血清中TNF-α白介素-1β、白介素-8、白介素-10等细胞因子明显增加,其浓度与疾病进展、临床预后密切相关[7]。因此,测定促炎细胞因子和抑炎细胞因子浓度可直观地评估ALI模型的炎症反应水平。
氧化应激的发生,一方面是由于ROS和/或RNS的产生过多；另一方面是由于抗氧化物质的过度消耗。烟酰胺腺嘌呤二核苷酸磷酸氧化酶(nicotinamide adenine dinucleotide phosphate oxidase, NADPH oxidase, NOX)包括7种同源蛋白,共同组成NOX/DUOX家族,是产生ROS的重要来源。NOX/DUOX家族各亚型的分布具有组织细胞特异性,其衍生的ROS的功能也存在差异。吞噬细胞型ROS主要参与宿主防御,非吞噬细胞型ROS则可作为第二信使直接或间接作用于信号传导通路。NOX/DUOX家族各亚型与慢性阻塞性肺疾病、支气管哮喘、急性肺损伤等疾病密切相关,但目前尚缺乏各亚型在肺泡Ⅱ型上皮细胞分布情况的相关研究。筛选出与ALI相关的重要亚型,研究其调控机制,进而调节其ROS的生成,是下调ALI氧化应激损伤的重要途径之一。
本实验室的前期研究表明,NF-κB/p65基因可能通过下调凋亡相关基因Bax/Bcl-2表达比例减少TNF-α诱导的A549细胞凋亡。NF-κB是TNF-α促发、放大炎症反应的信号转导核心,可调控包括NOX/DUOX家族基因在内的众多炎症疾病相关因素的表达。通过干预NF-κB通路下调其下游靶基因的表达水平,可能比单一补充细胞因子拮抗剂或抗氧化剂效果更好。
启动子(promoter)是RNA聚合酶辨认、结合和启动转录的一段DNA序列。真核生物RNA聚合酶本身不能直接识别、结合相应的启动子序列,必须经转录因子的介导辅助才能与之结合。启动子与转录因子的相互作用影响基因转录、翻译等过程,是研究疾病机制的重要切入点。生物信息学软件分析初步结果显示,NOX1启动子近端存在2个NF-κB结合元件,NF-κB活化进入细胞核后可能通过与该元件结合,调控NOX1的转录过程。因此,明确NF-κB对NOX1基因的调控机制,通过下调NF-κB的活性调控NOX1生成ROS,理论上可以减轻TNF-α促发的炎症反应和氧化应激程度,为调控ALI的肺保护策略研究提供更为精准的靶基因。
综上所述,本课题的研究目的如下：
1采用TNF-α刺激肺泡II型上皮细胞(A549),评估其炎症反应、氧化应激、细胞凋亡水平,建立ALI体外细胞炎症模型；
2探讨NF-KB/p65在TNF-α诱导的炎症及氧化应激损伤病理过程中的作用；
3明确参与TNF-α诱导A549细胞产生炎症——氧化应激病理过程的NOX/DUOX家族亚型,分析其与NF-κB的相关性；
4探讨NF-κB对NOX1的调控机制及NOX1启动子区完整性对NF-κB调控作用的影响。
方法
肺泡Ⅱ型上皮细胞来源的A549细胞由广州军区广州总医院医学实验科细胞库提供。A549细胞在含10%灭活小牛血清、100U/mL青霉素和100U/mL链霉素的RPMI-1640培养基中,置于37℃、5%CO2、饱和湿度条件下的培养箱中培养。
1沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮细胞炎症反应和细胞凋亡水平的影响
A549细胞分为5组：空白对照组(不加干预因素)、TNF-α组(仅用TNF-α刺激)p65siRNA组(预转染p65siRNA且用TNF-α刺激)、siRNA阴性对照组(预转染阴性对照siRNA且用TNF-α刺激)、siRNA阳性对照组(预转染阳性对照siRNA且用TNF-α刺激)。进行TNF-α(10ng/mL)刺激前6小时预转染siRNA,倾去转染液后开始以TNF-α刺激24小时。
以RT-PCR法检测NF-κB/p65mRNA的表达水平；免疫印迹法分别检测胞浆和胞核中NF-κB/p65蛋白的表达水平；ELISA法检测细胞培养上清液中IL-1β、IL-4、IL-6、IL-8、IL-10的浓度；MTT法检测细胞存活率；Annexin V/PI法检测细胞凋亡率。
2沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮细胞氧化应激水平的影响
细胞分组同上。以DCFH-DA探针法检测细胞内活性氧水平；比色法检测细胞的丙二醛浓度、总抗氧化能力、总超氧化物歧化酶活力、总谷胱甘肽的浓度。
3沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮NOX/DUOX家族的影响细胞分组同上。Real-time RT-PCR检测NOX/DUOX家族各基因mRNA表达水平；免疫印迹法检测NOX1、NOX2、NOX4蛋白的表达水平。
4转录因子NF-κB/p65对肺泡Ⅱ型上皮细胞NOX1基因调控机制的探讨
Alibaba2.1软件预测NOX1近端启动子区的NF-κB结合元件,据此设计并合成DNA探针；EMSA法检测上述探针与核蛋白中NF-κB是否结合；在此基础上,分别构建含目的NOX1启动子片段(1415bp)的重组pGL3萤光素酶报告基因载体pGL3-NOX1-1415、缺失上述NF-κB阳性结合位点的启动子片段(1327bp)所对应的重组pGL3萤光素酶报告基因载体pGL3-NOX1-1327(缺失载体)；将pGL3-NOX1-1415、pGL3-NOX1-1327分别瞬时转染入A549细胞,同时转染质粒pRL-TK作为内对照,TNF-α刺激24小时,双荧光素酶报告基因系统检测转染细胞的萤光素酶活性。
5统计学分析：应用SPSS13.0软件包进行统计学分析。计量资料以均数±标准差(X±S)表示,所有数据均进行方差齐性检验。采用单因素方差分析进行组间均数比较,满足方差齐性的多重比较采用LSD法,不满足方差齐性的多重比较采用Dunnett's T3法。显著性水准α取0.05,双侧。
结果
1沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮细胞炎症反应和细胞凋亡水平的影响
1.1各组A549细胞NF-KB/p65mRNA表达水平的差异有统计学意义(F=771.003,P<0.001)。 TNF-α组的A549细胞NF-KB/p65mRNA表达水平较空白对照组上调(P<0.05); NF-κB/p65siRNA组的A549细胞NF-κB/p65mRNA表达水平较空白对照组、TNF-α组、阴性对照组下调(P<0.05)。
1.2各组A549细胞胞浆中NF-KB/p65蛋白表达水平的差异有统计学意义(F=231.811,JP<0.001)。NF-κB/p65siRNA组的A549细胞胞浆中NF-KB/p65蛋白表达水平较空白对照组、TNF-α组、阴性对照组下调(P<0.05)。
1.3各组A549细胞胞核中NF-κB/p65蛋白表达水平的差异有统计学意义(F=1206.566, P<0.001), TNF-α组的A549细胞胞核中NF-KB/p65蛋白表达水平较空白对照组上调(P<0.05)。 NF-κB/p65siRNA组的A549细胞胞核中NF-KB/p65蛋白表达水平较TNF-α组、阴性对照组、阳性对照组下调(P<0.05)。
1.4各组A549细胞培养上清液中IL-1β、IL-4、IL-6、IL-8、IL-10的浓度差异有统计学意义(P<0.001)。TNF-α组的A549细胞培养上清液中上述细胞因子浓度较空白对照组上调(P<0.05)；NF-κB/p65siRNA组的A549细胞培养上清液中上述细胞因子浓度较TNF-α组、阴性对照组、阳性对照组下调(P<0.05)。
1.5各组A549细胞存活率的差异有统计学意义(F=125.145,P<0.001)。TNF-α组的A549细胞存活率较空白对照组下调(P<0.05); NF-κB/p65siRNA组的A549细胞存活率较TNF-α组、阴性对照组、阳性对照组上调(P<0.05)；较空白对照组下调(P<0.05)。
1.6各组A549细胞凋亡率的差异有统计学意义(F=155.592, P<0.001)。 TNF-α组的A549细胞凋亡率较空白对照组上调(P<0.05); NF-KB/p65siRNA组的A549细胞凋亡率较TNF-α组、阴性对照组、阳性对照组下调(P<0.05)；较空白对照组上调(P<0.05)。
2沉默NF-KB/p65对TNF-α诱导的肺泡Ⅱ型上皮细胞氧化应激水平的影响
2.1各组A549细胞活性氧、丙二醛的浓度差异有统计学意义(P<0.001)。TNF-α组的A549细胞活性氧、丙二醛的浓度较空白对照组上调(P<0.05)；NF-κB/p65siRNA组的A549细胞活性氧、丙二醛的浓度较TNF-α组、阴性对照组、阳性对照组下调(P<0.05)。
2.2各组A549细胞总抗氧化能力、超氧化物歧化酶、总谷胱甘肽的浓度差异有统计学意义(P<0.001)。TNF-α组的A549细胞的上述指标较空白对照组下调(P<0.05)；NF-κB/p65siRNA组的A549细胞的上述指标较TNF-α组、阴性对照组、阳性对照组上调(P<0.05)。
3沉默NF-κB/p65对TNF-α诱导的肺泡Ⅱ型上皮NOX/DUOX家族的影响
3.1各组A549细胞NOX1、NOX2、NOX4mRNA表达水平的差异有统计学意义(P<0.001)；TNF-α组的A549细胞上述基因mRNA表达水平较空白对照组上调(P<0.05)。 NF-κB/p65siRNA组的A549细胞上述基因mRNA表达水平较TNF-α组、阴性对照组下调(P<0.05)。
3.2各组A549细胞NOX3、NOX5、DUOX1、DUOX2mRNA的表达水平无统计学差异(P>0.05)。
3.3各组A549细胞NOX1、NOX2、NOX4蛋白表达水平的差异有统计学意义(P<0.001)；TNF-α组的A549细胞上述蛋白的表达水平较空白对照组上调(P<0.05); NF-κB/p65siRNA组的A549细胞上述蛋白表达水平较TNF-α组、阴性对照组下调(P<0.05)。
4转录因子NF-κB/p65对肺泡Ⅱ型上皮细胞NOX1基因调控机制的探讨
4.1Alibaba2.1软件分析结果提示,NOX1近端启动子区约1500bp范围内可预测到2个NF-κB结合元件。元件1称为NOX1/kB1,位于-1095-1086,序列为5'-CAGGAAAAC-3';元件2称为NOX1/κB2:位于-261-252,序列为5'-TAAAATCCCC-3'。
4.2经EMSA检测,元件2可与核蛋白中NF-KB/p65结合。
4.3载体pGL3-NO1-1415、载体pGL3-NOX1-1327经双酶切电泳及测序鉴定,结构正确。
4.4转染载体pGL3-NOX1-1415的各组A549细胞萤光素酶活性的差异有统计学意义(F=142.105,P<0.001)。TNF-α组的A549细胞萤光素酶活性较空白对照组上调(P<0.05); NF-κB/p65siRNA组的A549细胞萤光素酶活性较TNF-α组、阴性对照组下调(P<0.05)；较空白对照组上调(P<0.05)。4.5分别转染pGL3空质粒、载体pGL3-NOX1-1415、载体pGL3-NOX1-1327后,经TNF-α刺激的各组A549细胞萤光素酶活性的差异有统计学意义(F=646.047,P<0.001)。转染载体pGL3-NOX1-1415、pGL3-NOX1-1327的A549细胞萤光素酶活性较转染pGL3者上调(P<0.05)；转染pGL3-NOX1-1415较pGL3-NOX1一1327,A549细胞萤光素酶活性升高(P<0.05)。
结论
1.TNF-α诱导肺泡Ⅱ型上皮细胞A549能够在体外模拟ALI肺结构细胞的炎症——氧化损伤及细胞凋亡；
2.NF-κB/p65参与TNF-α诱导的细胞炎症-氧化损伤的病理生理过程,RNA干扰技术可有效沉默NF-κB/p65并下调炎症反应和氧化应激程度,减少细胞凋亡,达到保护结构细胞的目的。
3.NOX/DUOX家族中NOX1、NOX2、NOX4等亚型参与了TNF-α诱导的细胞炎症-氧化损伤的病理生理过程；
4.NF-κB/p65通过与NOXl启动子区κB结合元件(-261--252bp)结合,调控其转录过程；NOX1启动子区结构的完整性影响NF-κB/p65的结合及调控作用。
综上所述,在TNF-α诱导肺泡Ⅱ型上皮细胞A549产生的炎症—氧化应激环路中,NF-κB/p65可通过与NOX1启动子区KB结合元件(-261--252bp)结合实现对其转录调控作用。
Background and Objection:
Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), have been documented clinically following several pathological states such as serious infection, shock, trauma, aspiration and characterized by progressive respiratory distress and acute severe arterial hypoxemia. As a result of the lack of effective treatment, the incidence and mortality rates of ALI/ARDS are not greatly improved. Therefore, many groups from different countries still focus on the utilizing multiple approaches (biological, genomic and genetic) to clarify the underlying pathophysiological mechanisms by which a wide variety of insults can lead to this common clinical and pathophysiological syndrome.
Massive release of various pro-and anti-inflammatory cytokines including TNF-alpha lead to excessive systemic inflammatory response, which have important effects on the pathogenesis of ALI/ARDS. On the other hand, many studies indicate that TNF-a stimulates the generation of reactive oxygen species (ROS) by human endothelial cells and neutrophils. Reactive oxygen species is one of the most prominent manifestations of oxidative stress and associated with the apoptosis of type Ⅱ alveolar epithel cells. Therefore, besides of the anti-infective therapy, keeping the level of both inflammatory response and oxidative stress in an appropriate level should make contribution to the treatment of ALI.
On the basis of several experimental studies, TNF-a, along with other cytokines, such as IL-β, IL-8, IL-10, is suggested as an important early mediator of ALI. High generation levels of these cytokines had been detected in the plasma, bronchoalveolar lavage fluid of ALI/ARDS patients and animal models, the concentration of which is closely related to the progression and prognosis of ALI.
Excessive generation of ROS/RNS and consumption of the antioxidants break the balance between them, leading to the oxidative stress. The NOX/DUOX oxidase family is considered as a major source of reactive oxygen species. Tissue-specific distribution of NOX/DUOX isoforms affects the function of ROS generated by them. Their distribution in type Ⅱ alveolar epithel cells is still an unsettled subject, so as their contributions to ALL Down-regulating the ROS generation of NOX/DUOX oxidase family can be an effective method to reduce oxidative stress.
NF-κB is of central importance in TNF-a induced-inflammation. It regulates the expression of various inflammation-associated genes, including the NOX/DUOX family. More than700inhibitors of the NF-κB activation pathway, including antioxidants, peptides, small RNA/DNA, have been described. Downregulation of NF-κB-regulated genes by NF-κB silencing can be a better way to ALI treatment than cytokine antagonists or the antioxidants.
A promoter is a region of DNA that facilitates the transcription of a particular gene. Transcription factors regulate the gene transcription by binding to the specific site of the promoter. Several groups reported the mechanisms of transcriptional regulation of NOX1,mentioning NF-κB, in the colon epithelium, macrophage, gastric mucosal cell, vascular smooth muscle cells. There are two typical NF-κB binding elements predicted by Alibaba2.1software in the proximal promoters (-1500bp) of the human NOX1. Therefore, it is necessary for us to investigate wheather the NF-κB signal pathway is involved in the regulations of NOX1-derived ROS, which may facilitate the research on the ALI/ARDS treatment.
The goals of the present study are as followed:
1To simulate an inflammatory environment during ALI/ARDS in vitro, Type Ⅱ alveolar epithelial cells (A549) were cultured and stimulated by TNF-α (10ng/mL), then the levels of inflammatory response, oxidative stress and apoptosis were investigated;
2To investigate the effects of NF-κB p65silencing by RNAi on the level of inflammatory response, oxidative stress and apoptosis in TNF-a-induced A549;
3To investigate the expression of NOX/DUOX oxidase family of A549in the absence or presence of TNF-α, and discuss the relationship between NOX/DUOX oxidase family and NF-κB;
4To obtain one of the NOX/DUOX oxidase isoforms which is closely related to NF-κB, then investigate whether the NF-κB signal pathway was involved in the regulations of it.
5Statistical analysis:Data were expressed as means±standard error (S.E.). The statistical significance of differences among groups was assessed by one-way analysis of variance (ANOVA), followed by least significant difference (LSD) for multiple comparison, as a. post hoc test. All statistical analyses were performed using SPSS13.0. For these comparisons, P<0.05was considered to be statistically significant.
Methods and materials
Type Ⅱ alveolar epithelial cells (A549) were obtained from experimental department of Guangzhou General Hospital of Guangzhou Military command and routinely grown in RPMI-1640supplemented with10%fetal bovine serum (FBS), penicillin (100U/mL), and streptomycin (100U/mL) in a humidified chamber supplemented with5%CO2at37℃.
1The effects of NF-κB p65silencing by RNAi on the level of inflammatory response and apoptosis in TNF-a-induced type Ⅱ alveolar epithel cell
The cells were divided into five groups:control group (cells without any interference factors); TNF-α group (10μg/L TNF-α); NF-KB/p65siRNA group (10μg/L TNF-α50nM NF-κB/p65siRNA); negative siRNA group (10μg/L TNF-α,50nM negative siRNA); positive siRNA group (10μg/L TNF-a,50nM positive siRNA). The RNA interference lasted for6hours; the TNF-a stimulation lasted for24hours.
RT-PCR and Western blotting were performed to analyze the silence efficiency of RNAi targeting NF-kB p65. The consentrations of IL-1β, IL-4, IL-6, IL-8and IL-10in the culture supernatant were measured by ELISA. The survival rate of cell was assessed by the methyl thiazolyl tetrazolium (MTT) assay. The apoptosis rate of cell was detected by Annexin V/PI assay.
2The effects of NF-κB p65silencing by RNAi on the level of oxidative stress in TNF-α-induced type Ⅱ alveolar epithel cell
The cells were divided into five groups as indicated above. The peroxide-sensitive fluorescent probe2',7'-dichlorofluorescein diacetate (DCFH-DA) was used to assess the intracellular ROS production. The concentration of MDA, TAOC, SOD and TGSH was detected by kits using colorimetry.
3The effects of NF-⒘B p65silencing by RNAi on the expression of NOX/DUOX oxidase family of A549
The mRNA expression of NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2gene were detected by real-time RT-PCR. The expression of NOX1, NOX2, NOX4protein were detected by western bloting.
4NF-κB/p65regulates the expression of NOX1of A549
NF-κB binding elements in the proximal promoters (-1500bp) of the human NOX1were predicted by Alibaba2.1software. The binding of the elements with NF-κB was detected by electrophoretic mobility shift assay. pGL3basic vector inserted with the NOX1proximal promoter was constructed, named "pGL3-NOX1-1415"; pGL3basic vector inserted with the NOX1proximal promoter which was absence of the positive element as constructed, named "pGL3-NOX1-1327". They were respectively transfected into A549, then the cells were stimulated with TNF-a. The luciferase level which stands for the expression of reporter gene was monitored on MD SpectraMax M5enzyme-labeled instrument.
Results:
1The effects of NF-κB p65silencing by RNAi on the level of inflammatory response and apoptosis in TNF-a-induced type Ⅱ alveolar epithel cell
RT-PCR and Western blotting were performed to analyze the silence efficiency of RNAi targeting NF-κB p65. The consentrations of IL-1β、IL-8and IL-10in the culture supernatant were measured by ELIS A. The survival rate of cell was assessed by the methyl thiazolyl tetrazolium (MTT) assay. The apoptosis rate of cell was detected by Annexin V/PI assay.
1.1Compared with control group, the expression of NF-KB/p65mRNA was significantly higher in TNF-a group. The expression of NF-κB/p65mRNA in NF-KB/p65siRNA group was significantly lower than that in TNF-α group, negative siRNA group and positive siRNA group.
1.2The expression of NF-κB/p65protein in cytoplasm of NF-κB/p65siRNA group was significantly lower than that in control group, TNF-α group, negative siRNA group and positive siRNA group.
1.3Compared with control group, the expression of NF-KB/p65protein in nucelus was significantly higher in TNF-α group. The expression of NF-KB/p65protein in nucelus in NF-κB/p65siRNA group was significantly lower than that in TNF-α group, negative siRNA group and positive siRNA group.
1.4Compared with control group, the concentrations of IL-1β, IL-4, IL-6, IL-8and IL-10in the culture supernatant were significantly higher in TNF-a group. They are significantly lower in NF-κB/p65siRNA group than that in TNF-α group, negative siRNA group and positive siRNA group.
1.5Compared with control group, the survival rate was significantly lower in TNF-α group. It was significantly higher in NF-κB/p65siRNA group than that in TNF-α group, negative siRNA group and positive siRNA group.
1.6Compared with control group, the apoptosis rate was significantly higher in TNF-α group. It was significantly lower in NF-κB/p65siRNA group than that in TNF-α group, negative siRNA group and positive siRNA group.2The effects of NF-κB p65silencing by RNAi on the level of oxidative stress in TNF-α-induced type Ⅱ alveolar epithel cell
Compared with control group, the concentrations of ROS and MDA were significantly higher in TNF-α group. It was significantly lower in NF-κB/p65siRNA group than that in TNF-α group, negative siRNA group and positive siRNA group. Compared with control group, the concentrations of TAOC SOD and TGSH were significantly lower in TNF-a group. It was significantly higher in NF-KB/p65siRNA group than that in TNF-a group, negative siRNA group and positive siRNA group.
3The effects of NF-κB p65silencing by RNAi on the expression of NOX/DUOX oxidase family of A549
Compared with control group, the expression of NOX1, NOX2and NOX4mRNA were significantly higher in TNF-a group. It was significantly lower in NF-KB/p65siRNA group than that in TNF-a group and negative siRNA group.
Compared with control group, the expression of NOX1, NOX2and NOX4protein were significantly higher in TNF-a group. It was significantly lower in NF-KB/p65siRNA group than that in TNF-a group and negative siRNA group.
The mRNA expression of NOX3, NOX5, DUOXland DUOX2had no statistical significance.
4NF-KB/p65regulates the expression of NOX1of A549
NF-κB binding elements in the proximal promoters (-1500bp) of the human NOX1were predicted by Alibaba2.1software. The binding of the elements with NF-κB was detected by electrophoretic mobility shift assay. pGL3basic vector inserted with the NOX1proximal promoter was constructed, named "pGL3-NOX1-1415"; pGL3basic vector inserted with the NOX1proximal promoter which was absence of the positive element as constructed, named "pGL3-NOX1-1327". They were respectively transfected into A549, then the cells were stimulated with TNF-a. The luciferase level which stands for the expression of reporter gene was monitored on MD SpectraMax M5enzyme-labeled instrument.
4.1Two potential NF-κB responsive cis-acting elements in the proximal promoters of human Nox1genes (1439) were assessed using Alibaba2.1software
4.2the results of EMS A showed a binding of the probe2designed based on Nox1/kB2with NF-κB/p65.
4.3the sequence of the vector "pGL3-NOXl-1415" and "pGL3-NOX1-1327" were identified to be corret.
4.4Compared with control group, the luciferase level were significantly higher in TNF-a group transfected with the vector "pGL3-NOX1-1415". It was significantly lower in NF-κB/p65siRNA group than that in TNF-a group and negative siRNA group.
4.5Compared with control group, the luciferase level were significantly higher in the group transfected with the vector "pGL3-NOXl-1327", but it was significantly lower than that in the group transfected with the vector "pGL3-NOX1-1415".
Conclusion
TNF-a induced excessive inflammatory response and oxidative stress, leading to higher apoptosis rate of A549. NF-κB/p65silencing could down-regulate the over inflammation and oxidative stress induced by TNF-a, and down-regulate the apoptosis rate of A549. TNF-a increased the expression of NOX1, NOX2and NOX4of A549while NF-KB/p65silencing decreased their expression. Activated NF-κB binds to specific elements in NOX1promoter regions to control the transcription. Integrity of the NOX1proximal promoters affected the NF-κB binding. On the whole, NF-κB is an essential role for transcriptional regulation of NOX1in TNF-a induced A549, then affects the NOX1-derived ROS generation.
引文
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