c-Jun氨基末端激酶磷酸化在支气管哮喘大鼠气道重塑中的作用及糖皮质激素的影响

目的 研究c-Jun氨基末端激酶(JNK)磷酸化在支气管哮喘(简称哮喘)气道重塑中的作用,探讨糖皮质激素对白细胞介素(IL)-1β、JNK及哮喘气道重塑的影响.方法 将48只SD大鼠按随机数字表法分为对照组、哮喘组、布地奈德组和地塞米松组,每组12只,实验组以卵清白蛋白致敏和激发复制哮喘气道重塑模型,干预组分别于每次雾化激发前以布地奈德雾化或地塞米松腹腔注射干预,对照组以生理盐水代替卵清白蛋白致敏和激发.采用图像分析技术测定支气管壁厚度(Wat)和平滑肌厚度(Wam),ELISA法测定血清、BALF中IL-1β浓度,免疫组织化学检测肺内磷酸化JNK(P-JNK)及其下游物磷酸化c-Jun蛋白表达,Western blot检测肺匀浆P-JNK表达,直线相关分析Wat、Warn与P-JNK蛋白的平均吸光度(mA)的相关性以及P-JNK蛋白的mA与血清、BALF IL-1β浓度的相关性.结果 哮喘组气道壁厚度较对照组明显增加,其血清和BALF中IL-1β浓度分别为(81±4)ng/L、(331±15)ns/L]高于对照组(53±6)ng/L、(130±9)ns/L](t值分别为-8.62、-24.10,均P<0.01);免疫组织化学显示哮喘组P-JNK和P-c-Jun蛋白表达增高;Western blot检测哮喘组P-JNK蛋白的mA为1.66±0.16高于对照组的1.00±0.00(t=-8.35,P<0.01);布地奈德、地塞米松均可抑制JNK的磷酸化;各组Wat、Waln与P-JNK mA均呈高度正相关(r值分别为0.700、0.769,均P<0.01,rg=48),P-JNK的mA与血清、BALF IL-1β浓度均呈显著正相关(r值分别为0.689、0.805,均P<0.01).结论 JNK磷酸化与哮喘气道重塑密切相关;糖皮质激素能抑制JNK磷酸化,其机制之一可能是下调IL-1β表达.

The role of phosphorylation of c-Jun NH2-terminal kinase in airway remodeling of asthmatic rats and the effect of glucocorticoids

Objective To study the role of phosphorylation of c-Jun NH2-terminal kinase(JNK)in asthmatic airway remodeling and to explore the effect of glucocorticoids on IL-1βJNK and airway remodeling. Methods Forty-eight 4-6 week old male SD rats were randomly divided into 4 groups with 12 rats in each group: the control group, the asthma group, the budesonide(BUD)group, and the dexamethasone(DXM)group. The asthma airway remodeling models were made by intra-peritoneal injection of ovalbumin(OVA) on days 1 and 8 and inhalation of OVA every other day for 12 weeks since day 15. The BUD group underwent inhalation of BUD 30 min before every inhalation;the DXM group received intra-peritoneal injection of DXM 30 min before every inhalation; while the control group received normal saline instead of OVA. The histopathology and ultrastroctural changes of pulmonary tissues were observed by light microscope and transmission electron microscope(TEM).The total bronchial wall thickness(Wat)and the airway smooth muscle thickness(Wam)were measured by image analysis system. The concentrations of IL-1βin serum and BALF were tested by sandwich ELISA.The protein expressions of P-JNK and P-c-Jun were detected by immunohistochemistry technique. Lung tissue extracts were analyzed for phesphorylation of JNK by Western blot.Linear correlation analysis was used to evaluate correlations between Wat and P-JNK protein(mA),Warn and P-JNK protein(mA),P-JNK protein(mA)and levels of IL-1βin serum,P-JNK protein(mA)and levels of IL-1βin BALF.Results In the asthma group, HE-staining showed inflammatory cell infiltration around bronchi and mucous gland hyperplasia.TEM examination showed airway smooth muscle and collagen fiber proliferation,and widening of intercellular distance.The Wat and Wam of the asthma group were significantly higher than those of the control group, while the thickness of airway wall in the glucocorticoid intervention groups became significantly decreased. The concentrations of IL-1βin serum and BALF of the asthma group(81)ng/L,(331)ng/L]were significantly higher than those of the control group(53)ng/L,(130)ng/L](t = - 8.62 and t = - 24.10, both P < 0.01).Mean absorbance values (by immunohistochemistry) of P-JNK and P-c-Jun in the asthma group were significantly higher than those of the control group, and the mean absorbance values of P-JNK and P-c-Jun in the BUD and DXM groups were significantly lower than those of the asthma group, but higher than those of the control group( F = 223.59 and F = 76.53, both P < 0. 01). Absorbance(by Western blot)of P-JNK in the control, asthma, BUD, and DXM groups were (1.00),(1.66),(1.18),and(1.29), respectively; that of the asthma group was significantly higher than that of the control group, while absorbance of P-JNK in the BUD and the DXM groups were significantly lower than that of the asthma group, but higher than that of the control group(F=17.84, P < 0.05).Strong positive correlations were found between War or Wam and P-JNK (mA)(r =0. 700 and r = 0.769,P < 0.01, respectively, n =48). Strong positive correlations were also found between P-JNK(mA) and concentrantion of IL-1?in serum or BALF(r =0. 689 and r =0. 805 , P <0. 01, respectively, n=48).Conclusion Phosphorylation of JNK is closely related to asthma airway remodeling. Glucocorticoids can inhibit phosphorylation of JNK, one mechanism of which may be down-regnlation of IL-βexpression.