N-乙酰半胱氨酸下调NOX4表达抑制博莱霉素诱导纤维化小鼠的氧化损伤
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摘要
目的观察活性氧清除剂N-乙酰半胱氨酸(NAC)对博莱霉素诱导的肺纤维化小鼠的影响,探讨NAC抑制肺纤维化的可能机制。方法将45只SPF级雌性昆明小鼠随机分为3组:对照组(气管内雾化生理盐水)、纤维化组(气管内博莱霉素3 mg/kg溶于100μL生理盐水内雾化)、NAC处理组(气管内雾化博莱霉素后,NAC 400 mg.kg-1.d-1溶于100μL生理盐水内灌胃)。处理后第28 d收集样本。小鼠左肺置于10%中性甲醛固定,石蜡包埋切片后行HE与Masson染色;右肺碱水解法检测肺组织羟脯氨酸的含量;比色法检测血清丙二醛(MDA)的浓度和总抗氧化能力(T-AOC);提取肺组织总蛋白和总RNA,分别采用Western blot和RT-PCR法检测NADPH氧化酶1(NOX1)、NOX2及NOX4的基因和蛋白表达水平。结果 NAC处理组小鼠肺组织病理损伤较纤维化组减轻,气道上皮下胶原沉积及肺组织羟脯氨酸含量明显减少(P<0.05),血清MDA浓度较纤维化组显著降低(P>0.05),血清T-AOC较纤维化组升高(P=0.029)。纤维化组NOX1/2/4基因及蛋白表达较对照组显著增强(P<0.05),NAC干预后NOX1/2/4基因及蛋白表达水平均有所下调,NOX4蛋白表达明显低于纤维化组(P=0.001)。与对照组比较差异无统计学意义(P>0.05)。结论 NAC能显著改善博莱霉素诱导的小鼠肺纤维化,其机制与抑制NOX4蛋白表达、下调氧化应激损伤有关。
Objective To evaluate the effects of N-acetylcysteine(NAC) on bleomycin-induced lung fibrosis in mice and to investigate the therapeutic mechanisms of NAC on lung fibrosis.Methods Forty-five KM female mice were randomly divided into 3 groups.The mice in the control group were administered with saline aerosol intratracheally.The mice in the fibrosis group were administered with bleomycin(3 mg/kg) dissolved in normal saline aerosol intratracheally.The mice in the NAC group were gastric perfused with NAC at a dose of 400 mg·kg-1·d-1 after administering bleomycin aerosol intratracheally.All animals were sacrificed 28 days after the treatments.The left lung was fixed in 10% neutral formalin,then stained with hematoxylin eosin and Masson's trichrome respectively for the pathological examination.The right lung was sampled and the content of hydroxyproline(HYP) was assayed by alkaline hydrolysis method.The serum was collected and the concentrations of malondialdehyde(MDA) and total antioxidant capacity(T-AOC) were measured by colorimetric method.The RNA and total tissue protein were extracted for the examination of NOX1/2/4 by RT-PCR and Western blot respectively.Results NAC prevented lung fibrosis induced by bleomycin with significantly reducing lung collagen accumulation and the level of HYP in the NAC group(P<0.05).The serum concentration of MDA were reduced and serum T-AOC raised by treating NAC after intratracheal administration of bleomycin(P<0.05).NOX1/2/4 gene and protein expression were increased in the fibrosis group compared with the control group.NAC had no effect on the gene expression of NOX1/2/4(P>0.05),but inhibitted the NOX4 protein expression in lung tissue significantly(P<0.05).Conclusion NAC inhibits the expression of NOX4 and prevents bleomycin-induced lung fibrosis in mice.
Objective To evaluate the effects of N-acetylcysteine(NAC) on bleomycin-induced lung fibrosis in mice and to investigate the therapeutic mechanisms of NAC on lung fibrosis.Methods Forty-five KM female mice were randomly divided into 3 groups.The mice in the control group were administered with saline aerosol intratracheally.The mice in the fibrosis group were administered with bleomycin(3 mg/kg) dissolved in normal saline aerosol intratracheally.The mice in the NAC group were gastric perfused with NAC at a dose of 400 mg·kg-1·d-1 after administering bleomycin aerosol intratracheally.All animals were sacrificed 28 days after the treatments.The left lung was fixed in 10% neutral formalin,then stained with hematoxylin eosin and Masson's trichrome respectively for the pathological examination.The right lung was sampled and the content of hydroxyproline(HYP) was assayed by alkaline hydrolysis method.The serum was collected and the concentrations of malondialdehyde(MDA) and total antioxidant capacity(T-AOC) were measured by colorimetric method.The RNA and total tissue protein were extracted for the examination of NOX1/2/4 by RT-PCR and Western blot respectively.Results NAC prevented lung fibrosis induced by bleomycin with significantly reducing lung collagen accumulation and the level of HYP in the NAC group(P<0.05).The serum concentration of MDA were reduced and serum T-AOC raised by treating NAC after intratracheal administration of bleomycin(P<0.05).NOX1/2/4 gene and protein expression were increased in the fibrosis group compared with the control group.NAC had no effect on the gene expression of NOX1/2/4(P>0.05),but inhibitted the NOX4 protein expression in lung tissue significantly(P<0.05).Conclusion NAC inhibits the expression of NOX4 and prevents bleomycin-induced lung fibrosis in mice.
引文
1 Hunninghake GW.Antioxidant therapy for idiopathic pulmonaryfibrosis.N Engl J Med,2005,353:2285-2287.
2 Behr J,Maier K,Degenkolb B,et al.Antioxidative and clinicaleffects of high dose N-acetylcysteine in fibrosing alveolitis.Adjunctive therapy to maintenance immunosuppression.Am JRespir Crit Care Med,1997,156:1897-1901.
3 Meyer A,Buhl R,Magnussen H.The effect of oral N-acetylcysteineon lung glutathione levels in idiopathic pulmonary fibrosis.EurRespir J,1994,7:431-436.
4 Kinnula VL,Fattman CL,Tan RJ,et al.Oxidative stress inpulmonary fibrosis:a possible role for redox modulatory therapy.AmJ Respir Crit Care Med,2005,172:417-422.
5 Akasaki T,Ohya Y,Kuroda J,et al.Increased expression ofgp91phox homologues of NAD(P)H oxidase in the aortic mediaduring chronic hypertension:involvement of the renin-angiotensinsystem.Hypertens Res,2006,29:813-820.
6 Looi YH,Grieve DJ,Siva A,et al.Involvement of Nox2 NADPHoxidase in adverse cardiac remodeling after myocardial infarction.Hypertension,2008,51:319-325.
7 Waghray M,Cui Z,Horowitz JC,et al.Hydrogen peroxide is adiffusible paracrine signal for the induction of epithelial cell deathby activated myofibroblasts.FASEB J,2005,19:854-856.
8 Larios JM,Budhiraja R,Fanburg BL,et al.Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts.J Biol Chem,2001,276:17437-17441.
9李伟峰,胡玉洁,袁伟锋,等.气管内滴入与雾化博莱霉素致小鼠肺纤维化模型的比较研究.南方医科大学学报,2012,32:221-225.
10 Mikawa K,Nishina K,Takao Y,et al.ONO-1714,a nitric oxidesynthase inhibitor,attenuates endotoxin-induced acute lung injury inRabbits.Anesth Analg,2003,97:1751-1755.
11 Ashcroft T,Simpson JM,Timbrell V.Simple method of estimatingseverity of pulmonary fibrosis on a numerical scale.J Clin Pathol,1988,41:467-470.
12 Rahman I,MacNee W.Oxidative stress and regulation of glutathionein lung inflammation.Eur Respir J,2000,16:534-554.
13 Bando M,Hosono T,Mato N,et al.Long-term efficacy of inhaledN-acetylcysteine in patients with idiopathic pulmonary fibrosis.Intern Med,2010,49:2289-2296.
14 Tomioka H,Kuwata Y,Imanaka K,et al.A pilot study ofaerosolized N-acetylcysteine for idiopathic pulmonary fibrosis.Respirology,2005,10:449-455.
15 Waghray M,Cui Z,Horowitz JC,et al.Hydrogen peroxide is adiffusible paracrine signal for the induction of epithelial cell deathby activated myo broblasts.FASEB J,2005,19:854-856.
16 Martyn KD,Frederick LM,von Loehneysen K,et al.Functionalanalysis of Nox4 reveals unique characteristics compared to otherNADPH oxidases.Cell Signal,2006,18:69-82.
2 Behr J,Maier K,Degenkolb B,et al.Antioxidative and clinicaleffects of high dose N-acetylcysteine in fibrosing alveolitis.Adjunctive therapy to maintenance immunosuppression.Am JRespir Crit Care Med,1997,156:1897-1901.
3 Meyer A,Buhl R,Magnussen H.The effect of oral N-acetylcysteineon lung glutathione levels in idiopathic pulmonary fibrosis.EurRespir J,1994,7:431-436.
4 Kinnula VL,Fattman CL,Tan RJ,et al.Oxidative stress inpulmonary fibrosis:a possible role for redox modulatory therapy.AmJ Respir Crit Care Med,2005,172:417-422.
5 Akasaki T,Ohya Y,Kuroda J,et al.Increased expression ofgp91phox homologues of NAD(P)H oxidase in the aortic mediaduring chronic hypertension:involvement of the renin-angiotensinsystem.Hypertens Res,2006,29:813-820.
6 Looi YH,Grieve DJ,Siva A,et al.Involvement of Nox2 NADPHoxidase in adverse cardiac remodeling after myocardial infarction.Hypertension,2008,51:319-325.
7 Waghray M,Cui Z,Horowitz JC,et al.Hydrogen peroxide is adiffusible paracrine signal for the induction of epithelial cell deathby activated myofibroblasts.FASEB J,2005,19:854-856.
8 Larios JM,Budhiraja R,Fanburg BL,et al.Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts.J Biol Chem,2001,276:17437-17441.
9李伟峰,胡玉洁,袁伟锋,等.气管内滴入与雾化博莱霉素致小鼠肺纤维化模型的比较研究.南方医科大学学报,2012,32:221-225.
10 Mikawa K,Nishina K,Takao Y,et al.ONO-1714,a nitric oxidesynthase inhibitor,attenuates endotoxin-induced acute lung injury inRabbits.Anesth Analg,2003,97:1751-1755.
11 Ashcroft T,Simpson JM,Timbrell V.Simple method of estimatingseverity of pulmonary fibrosis on a numerical scale.J Clin Pathol,1988,41:467-470.
12 Rahman I,MacNee W.Oxidative stress and regulation of glutathionein lung inflammation.Eur Respir J,2000,16:534-554.
13 Bando M,Hosono T,Mato N,et al.Long-term efficacy of inhaledN-acetylcysteine in patients with idiopathic pulmonary fibrosis.Intern Med,2010,49:2289-2296.
14 Tomioka H,Kuwata Y,Imanaka K,et al.A pilot study ofaerosolized N-acetylcysteine for idiopathic pulmonary fibrosis.Respirology,2005,10:449-455.
15 Waghray M,Cui Z,Horowitz JC,et al.Hydrogen peroxide is adiffusible paracrine signal for the induction of epithelial cell deathby activated myo broblasts.FASEB J,2005,19:854-856.
16 Martyn KD,Frederick LM,von Loehneysen K,et al.Functionalanalysis of Nox4 reveals unique characteristics compared to otherNADPH oxidases.Cell Signal,2006,18:69-82.