aPKC及ERK在慢性阻塞性肺疾病大鼠中对NRF2-γ-GCS的调控作用

aPKC and ERK regulate NRF2-γ-GCS in rat lung with chronic obstructive pulmonary disease

目的:探讨慢性阻塞性肺疾病(COPD)大鼠模型中非典型蛋白激酶C(aPKC)、细胞外信号调节激酶(ERK)对红系衍生的核因子2相关因子2(NRF2)-γ-谷氨酰半胱氨酸合成酶(γ-GCS)的调控。方法:雄性Wistar大鼠16只,随机分COPD模型组和对照组。检测γ-GCS活性,γ-GCS的基因表达和NRF2、磷酸化的aP-KC(p-aPKCι/ζ)、磷酸化的ERK(p-ERK)的蛋白质表达。结果:(1)γ-GCS活性在COPD组中明显高于对照组。(2)γ-GCS mRNA广泛高表达于COPD组支气管平滑肌细胞,而对照组在相应部位呈弱表达。对照组肺匀浆中有一定量γ-GCS mRNA表达,但相对于COPD组仍较低。(3)p-aPKC、p-ERK、NRF2、γ-GCS在COPD组支气管上皮细胞胞浆中高表达,而对照组相应部位呈弱表达。COPD组γ-GCS和p-aPKC、p-ERK、NRF2蛋白表达皆有明显上调。(4)NRF2蛋白表达与γ-GCS蛋白、mRNA表达呈正相关,p-aPKCι/ζ、p-ERK与NRF2蛋白表达也呈正相关。结论:aPKC、ERK可能通过使NRF2转录活性增加的方式上调NRF2,进而上调γ-GCS的表达,在COPD的氧化应激机制中可能起重要作用。

AIM： To investigate atypical protein kinase C （aPKC）, extracellular signal regulated kinnase （ERK） regulating NF - E2 - related factor 2 （NRF2） -γ -gutamylcysteine synthetase （γ-GCS） and the effect on lung of rats with chronic obstructive pulmonary disease （COPD）. METHODS： The rat COPD model was established by intra-tracheal instillation of lipopolysaccharide twice and exposure to cigarette smoke daily. The γ-GCS activity was measured. The expression of γ-GCS mRNA in lung tissue was examined by in situ hybridization （ISH） and reverse transcription - polymerase chain reaction （ RT - PCR）. The protein expressions of p-aPKCЛ, p - ERK, NRF2 and γ-GCS in lung tissue were detected by immunohistochemistry （IH） and Western blotting, respectively. RESULTS ： （1） The γ-GCS activity was higher in COPD group than that in control group. （2） The expression of γ-GCS mRNA in the COPD group was stronger than that in control group. ISH showed that the γ-GCS mRNA was expressed in alveolar epithelium and bronchio- lar smooth muscle cell in the COPD group. （3） The protein expressions of p - aPKC, p - ERK, NRF2, γ-GCS were significantly higher than those in control group. IH showed that p - aPKC, p - ERK, NRF2, γ-GCS proteins were expressed in alveolar and bronchiolar epithelium in the COPD group. （4） There was a positive correlation between NRF2 and γ-GCS. γ-GCS mRNA, p- aPKCЛ, p- ERK were also positively correlated with NRF2. CONCLUSION： By upregulat- ing the signal transduction of NRF2 - γ- GCS, the ERK and aPKCЛ may play an important role in the mechanism of COPD formation.