| 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.
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