| 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 cellThe 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 cellThe 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-'B p65silencing by RNAi on the expression of NOX/DUOX oxidase family of A549The 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 A549NF-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 cellRT-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 cellCompared 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 A549Compared 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 A549NF-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 Alibaba2.1software4.2the 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".ConclusionTNF-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. |
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