【作者】 李秀英；

【导师】 罗百灵；

【作者基本信息】 中南大学 ， 呼吸内科， 2009， 博士

【摘要】 第一部分动物部分Nrf2在慢性阻塞性肺疾病大鼠模型中的作用及与IKKα/β的关系目的探讨核因子Nrf2在慢性阻塞性肺疾病发病中的作用及与NF-KB和IKKα/β之间的关系。方法36只Wistar大鼠随机分为A,B,C三个组,各12只,A组为对照组,B组为慢性阻塞性肺疾病模型组,C组为15d-PGJ2干预组,采用熏烟+气管内滴注脂多糖的方法建立模型;C组在建立模型的基础上第1天和第14天静脉注射15d-PGJ2(0.3 mg/kg)干预。各组分别进行一般情况的观察,30天后检测大鼠肺功能,经微机处理计算出FEV0.3/FVC、R1及Cdyn。测定肺功能后,进行肺泡灌洗左肺,取其上清液,检测总抗氧化能力和SOD的检测。取左肺组织固定、病理切片,部分HE染色光镜观察病理改变结果,部分切片免疫组化和右肺抽提mRNA,RT-PCR法检测Nrf2,NF-KB p65和IKKα/β的表达。所有结果应用SPSS12.0软件系统进行统计分析。结果(1)慢性阻塞性肺疾病的大鼠模型成功,模型组大鼠FEV0.3/FVC(%)和Cdyn(动态肺顺应性)明显低于正常对照组,差异有显著性(均P＜0.05),R1(气道阻力)明显高于正常对照组,差异有显著性(均P＜0.05),干预组介于了两组之间,气流受限和肺通气功能较模型组有明显改善,差异有统计学意义(P＜0.05)。(2)大鼠肺组织肺泡灌洗液氧化能力的检测示:模型组的SOD和T-AOC的水平均明显低于对照组,干预组介入两者之间,差异均有统计学意义。(3)大鼠肺及支气管组织的病理学观察模型组病理切片示模型组的肺组织破坏、增殖及炎症程度明显大于对照组,干预组介于两者之间。(4)免疫组化测得模型组中Nrf2、NF-KBp65和IKKα/β的阳性表达明显高于对照组,干预组NF-KBp65和IKKα/β介于两者之间,而与Nrf2的表达明显高于模型组,差异有显著性(均P＜0.05)。(5)RT-PCR测得模型组中Nrf2、NF-KB p65和IKKβ的mRNA的表达明显高于对照组,干预组NF-KBp65和IKKβ介于两者之间,而与Nrf2的表达明显高于模型组,差异有显著性(均P＜0.05),IKKα在三组中都无显著异常。结论(1)采用被动香烟吸入以及气管内注入脂多糖,成功复制了COPD大鼠模型。(2) COPD模型组中15d-PGJ2有抗氧化应激和抗炎症的作用,这一作用可能与Nrf2的增加有关。(3) Nrf2可能通过抑制IKKβ的表达,而抑制NF-κBp65的表达水平。第二部分细胞部分Nrf2通过抑制IKKβ途径下调支气管上皮细胞NF-κBp65和IL-8的表达目的探讨香烟烟雾提取物刺激后支气管上皮细胞后Nrf2的表达,及其是否通过IKKβ途径下调气道上皮细胞NF-κBp65和IL-8的表达。方法培养支气管上皮细胞分为7组A:正常对照组;B:烟草提取物(CSE)刺激组;C:15d-PGJ2预处理后CSE刺激组;D:Nrf2siRNA后CSE刺激组;E:阴性干扰后CSE刺激组F组和G组为IKKInhibitor抑制后的C和D组。首先通过烟草提取物的刺激使细胞氧化应激的水平增加,再通过15d-PGJ2预处理和Nrf2siRNA的方法来增加或降低Nrf2的水平,并用RT-PCR法及westblot的方法观察各组细胞NF-κBp65和IL-8的表达与Nrf2的表达的关系,及抑制IKKs后NF-κBp65和IL-8的表达及与Nrf2的表达的关系。所有结果应用SPSS12.0软件系统进行统计分析。结果(1)支气管上皮细胞在CSE的刺激下,Nrf2、IKKα/β、NF-κb p65以及IL-8的表达水平均有明显升高,差异有统计学意义。(2)在支气管上皮细胞中,PGJ2可增加Nrf2的表达,而Nrf2siRNA可成功抑制了Nrf2的表达。Nrf2的表达增加,同时伴有IKKα/β、NF-κBp65和IL-8的明显降低;Nrf2的表达被抑制后,同时伴有IKKα/β、NF-κBp65和IL-8的明显增加,差异均有统计学意义。这些变化在转录水平和翻译水平是一致的。(3)用IKK inhibitor进行干预后发现,PGJ2仍然可以增加Nrf2的表达,而Nrf2siRNA也可成功抑制了Nri2的表达。在转录水平,IKK被抑制后,IKKα/β、NF-κBp65和IL-8的都有明显的降低,差异有统计学意义,但是并不随着Nrf2的增加而降低,同时也不伴随Nrf2的降低而升高。结论(1)Nrf2在支气管上皮细胞中有抗炎症作用。(2)Nrf2抑制炎症的作用点可能位于IKKs。(3)IKKβ在Nrf2抗炎作用中起关键作用。更多还原

【Abstract】 Part 1 The function of Nrf2 in the chronic obstructive pulmonary disease and its relation Nrf2 and IKKα/βin rat model of chronic obstructive pulmonary diseaseObjective To study the effect of Nrf2 in the pathogenesis of chronic obstructive pulmonary disease and the relation between Nrf2 and IKKα/β.Methods 36 male rat are divided into 3 groups in random.A group were the normal control;B group were the rat models of COPD;C group were the drug intervened rat.Building the COPD model by passive smoking and endotracheal injection of LPS,and the drug intervention on the base of building the rat models of COPD,injection of 15d-PGJ2(0.3mg/kg). Watching the general condition,after 30 days,testing the pulmonary function,calculate the FEV0.3/FVC、R1 and Cdyn by computer.After that, alveolus lavaging,measure total antioxidant capacity in alveolus lavage fluid.We tested the pathological change in pulmonary tissue,the expression of Nrf2,NF-Kb and IKKα/βby the paraffin-embedded Immunohistochemistry and RT-PCR.All results were statistically analysed by SPSS 12.0 software.Results(1) The COPD model were succeed.Lung function tests showed that FEV0.3/FVC and Cdyn values were significantly lower in the group with COPD than in the normal control group(P＜0.05);RI values were significantly higher in the group with COPD than in the normal control group(P＜0.05),the intervened between the other two,airflow limitation and pulmonary ventilation were improved significantly(P＜0.05).(2) The antioxidant capacity of Rat lung lavage fluid oxidation test showed: the model group of SOD and T-AOC were significantly lower than the control group,the intervention group between the other two,the differences were statistically significant.(3) Pathological observed the rat pulmonary and brochia tissue:the pathological slices of the model rat had the most destroyed,proliferation and inflammation;the intervened rat were better than the model.the differences were significant.(4) Comparing with the rats of normal,the expression of Nrf2,NF-KB and IKKα/βin lungs increased significantly(P＜0.05)in the rat with COPD by Immunohistochemistry.the expression of NF-KB and IKKα/βin lungs of intervened rat was between the previous two;but the expression of Nrf2 increased more than the model rat.All of them were significantly different(P＜0.05).(5) Comparing with the rats of normal,the expression of Nrf2,NF-κB and IKKβmRNA in lungs increased significantly in the rat with COPD by RT-PCR,the expression of NF-κB and IKKα/βin lungs of intervened rat was between the previous two;but the expression of Nrf2 increased more than the model rat.All of them were significantly different(P＜0.05).and the expression of Nrf2 mRNA in lungs was positively related with the pulmonary function in the model group and the intervened group.but,the expression of IKKαhad no statistical difference in the three groups.All of the Results were statistical analysed by SPSS 12.0.Conclusion(1) The rat model of COPD were reproduced by passive inhalation of cigarette and intratracheal injection of LPS.(2) 15d-PGJ2 have the role of anti-oxidation and anti-inflammation in COPD model,which may be related to the increase of Nrf2.(3) Nrf2 inhibited the expression of NF-KBp65 level,which may be by inhibiting the expression of IKKβ. Part 2 Nrf2 reduced NF-KBp65 and IL-8 expression in bronchial epithelial cells by inhibiting IKKβObjective Exploit the expression of Nrf2 in bronchial epithelial cells after the stimulation of cigarette smoke extract,and whether modulating the NF-KBp65 and IL-8 expression through the IKKβin airway epithelial cells.Methods Cultured the bronchial epithelial cells,divided into 7 group: group A:normal control group;group B:stimulated cigarete smoke extract(CSE);group C:pretreated with 15d-PGJ2 and stimulated CSE; group D:pretreated with Nrf2 siRNA and stimulated CSE;group E: pretreated with negative interference;Group F:pretreated with IKK Inhibitor and after CSE stimulation and 15d-PGJ2 pretreatment;group G: pretreated with IKK Inhibitor and after CSE stimulation and Nrf2 siRNA pretreatment.First of all,the level of oxidative stress of the cells increased by stimulation of CSE,and then through the 15d-PGJ2 pretreatment and Nrf2siRNA methods to increase or decrease the level of Nrf2,RT-PCR and westblot used to observe the expression of NF-KBp65 and IL-8 and their relationship with the expression of Nrf2,and after inhibited IKKs,the relationship between the expression of Nrf2 and NF-KBp65/IL-8 expression.All of the Results were statistical analysed by SPSS12.0. Results(1)CSE stimulated human bronchial epithelial cells,the expression of Nrf2,IKKs,NF-κBp65 and IL-8 were significantly increased.(2)Compared the intervention with 15d-PGJ2 pretreatment and CSE stimulation to the intervention with CSE single,Nrf2 expression was significantly increased,with the significantly decreasing of IKKs, NF-κBp65 and IL-8;Nrf2 siRNA successful inhibited the gene expression of Nrf2,Compared the intervention with Nrf2 siRNA and CSE to the intervention with a single CSE,Nrf2 expression was significantly reduced,with the significantly increasing of IKKs, NF-κBp65 and IL-8.(3) After inhibited the expression of IKKs by the IKK inhibitors,the expression of NF-κBp65 and IL-8 has decreased,and the expression of Nrf2 still be significantly increased by Nrf2 inducer and still be significantly decreased by Nrf2siRNA agent,but cannot changed the exprssion of NF-κBp65 and IL-8.Conclusions(1) Nrf2 in bronchial epithelial cells had the role anti-inflammatory.(2) Nrf2 inhibited the inflammation by IKKs.(3) IKKβplay a key role in the anti-inflammatory effects of Nrf2.更多还原