The Pathway Of PI3K/Akt, APKC_ι/ζ-Nrf2 Regulating Cigarette Smoking Extractive Inducing The Expression Of γ-glutamylcysteine Synthetase In The Bronchial Epithelial Cells Of Rats

【Objective】To study the effects of cigarette smoking extractive in different periods on the regulating expression of PI3K(phosphoinositol -3-kinase, PI3K)/Akt, aPKC_ι/ζ,Nrf2 (Nuclear factor–E2 related factor),γ-glutamylcysteine synthetase(γ-GCS)in bronchial epithelial cells of the rats,elucidate the role of cigarette smoking in regulating the expression of PI3K/Akt,aPKC_ι/ζ,Nrf2 nuclear translocation andγ-GCS in the oxidation/antioxidation mechanism.To investigate the effects of PI3K specific inhibitor LY294002 and aPKC_ι/ζ specific inhibitor RO-31-8220 in Nrf2 nuclear translocation and the expression ofγ-GCS,elucidate the correlation of the pathway of PI3K/Akt and aPKC_ι/ζ and the effects of the pathway of PI3K/Akt and aPKC_ι/ζ on activating Nrf2 nuclear translocation and regulating the expression ofγ-GCS in the oxidation/antioxidation mechanism.【Methods】The study consisted of two parts. In vitro the bronchial epithelial cells which were cultured with DMEM/ F12 medium for 24 hours were devided into two parts randomly,a part of the cells were devided into 5 groups:0 hour control group, exposure to cigarette smoking extractive for 1 hour,3 hours,6 hours,12 hours groups(CSE0h,CSE1h,CSE3h, CSE6h, CSE12h), another part of the cells were devided into 5 groups too:0 hour negative control group, CSE3h positive group, LY294002 group,RO-31-8220 group and and both inhibitors group,the inhibitor groups were prepared by the method that the cells were pretreated with 10mM PI3K specific inhibitor LY294002 and/or 3uM aPKC_ι/ζ specific inhibitor RO-31-8220 for 0.5 hour and then cultured with DMEM/ F12 medium containing 10%CSE for 3 hours.To investigate the effects of cigarette smoking extractive in different periods,PI3K specific inhibitor and aPKC_ι/ζ specific inhibitor on Nrf2 nuclear translocation and the expression ofγ-GCS in the bronchial epithelial cells.The nuclear translocation of Nrf2 and expression of Nrf2 protein was detected by immunofluorescence and Western blot. The expression ofγ-GCS protein was detected by immunocytochemistry and western blot.The expressions ofγ-GCS mRNA were detected by reverse transcription- polymerase chain reaction(RT-PCR).The rates of positive cells which expressed p-Akt and the expression of p-Akt protein were examised by flow cytometry and western blot.The expression of p-aPKC_ι/ζ protein were were examised by western blot. The content of reduced glutathione(GSH)and the level of the activity ofγ-GCS was examined.【Results】A part of examination:1.The bronchial epithelial cells of rats exposed to 10% CSE in vitro, GSH content decreased in 1 hour(P<0.05,compared with control group),increased in 3 hours, attained the peaking in 6 hours,went back the normal level in 12 hours(P>0.05,compared with control group).2. Nrf2 was expressed in the cellular cytoplasma in normal condition and CSE 12h group,but in the nucleus in CSE1h,CSE3h,CSE6h,CSE12h groups.Nrf2 protein expressed in cellular nucleus in CSE1h,CSE3h and CSE6h groups ,and there was more significant changes than control group.However,the expression of Nrf2 protein existed in cellular cytoplasma in control group and CSE12h, simultaneously,there was no significant difference each other (P<0.05,compared with control group).3.The expression of p-Akt protein was weak in control group,increased in 1 hour (P<0.05,compared with control group), attained its peaking at 3h(P<0.05,compared with control group),remained the low level. The rate of positive cells expressed p-AKT gradually increased, attained its peaking at 3h, and then remained unchanged at 6h.4. The expression of p-aPKC_ι/ζ protein slightly higher at 1h , evident at 3h which was approximately 3-fold as compared with that of control group, gradually decreased, and then went back the normal level at 12h.5.γ-GCSmRNA,γ-GCS protein,γ-GCS activity increased gradually in time- dependent manner, attained its peaking at 6h,then decreased gradually, refected the normal level at 12h. The expressions ofγ-GCS protein were weakly positive in the cytoplasm of bronchial epithelial cells in control group and CS12h, positive in CS1h group and CS3h group,strongly positive in CS6h group.6. By linear correlation analysis, we found that there were positive correlation between Nrf2 andγ-GCS,γ-GCS activity,p-Akt,p-aPKC_ι/ζ, betweenγ-GCS and GSH,γ-GCS activity,p-aPKC_ι/ζ,Nrf2, between p-Akt and p-aPKC_ι/ζ,Nrf2, between p-aPKC_ι/ζ and GSH,γ-GCS activity,p-Akt ,γ-GCS, Nrf2 , respectively.Another part of eximination:1.GSH contents of three inhibitor groups were more decreasing than CS3h group when the cells were pre-treated with PI3K inhibitor LY294002 and aPKC_ι/ζ inhibitor RO-31-8220 (P<0.05,compared with control group).Compared with control group, GSH contents of three inhibitor groups were increasing,and there was significant difference (P<0.05,compared with control group). There was no significant difference among three inhibitor groups (P>0.05,compared with control group).2. Nrf2 was expressed in the cellular cytoplasma in control group, but in the nucleus in LY294002 group, RO-31-8220 group and both inhibitors group,as showed that PI3K specific inhibitor LY294002 and/or 3uM aPKC_ι/ζ specific inhibitor RO-31-8220 had no effect on Nrf2 nuclear translocation.Nrf2 nuclear protein weakly expressed in control group,but highly expressed in LY294002 group, RO-31-8220 group and both inhibitors group ,and there was significant difference than control group (P<0.05,compared with control group).and there was no significant change than CS3h group, there was no significant difference each other (P>0.05,compared with control group).3. The expression ofγ-GCSmRNA ,γ-GCS protein andγ-GCS activities were weak in control group,enhanced in LY294002 group, RO-31-8220 group and both inhibitors group(P<0.05, compared with contron group).Compared with CS3h, there was significant difference, and there was no significant difference among three inhibitor groups. The expressions ofγ-GCS protein were weakly positive in the cytoplasm of bronchial epithelial cells in control group, positive in LY294002 group, RO-31-8220 group and inhibitors groups (P<0.05, compared with CSE1h group, positive in CSE3h group).4. Both p-Akt and p-aPKC_ι/ζ protein declined in LY294002 group and both inhibitors group, p-Akt protein increased in RO-31-8220 group,however, p- aPKC_ι/ζ protein decreased in RO-31-8220 group.The rate of positive cells expressed p-AKT decreased in LY294002 group and both inhibitors group,increased in RO-31-8220 group.【Conclusions】1. cigarette smoking extractive upregulated the expression ofγ-GCS and the synthesis of GSH.1. Nrf2 nuclear translocation took place during a certain period when the bronchial epithelial cells of the rats were exposed to cigarette smoking extrative,showed that Nrf2 might be the critical transcription factor which regulated the expression ofγ-GCS.2. PI3K specific inhibitor and aPKC_ι/ζ specific inhibitor had an effect on the expression ofγ-GCS protein,γ-GCSmRNA,γ-GCS activity and GSH content, demonstrated that the pathway of PI3k/Akt-Nrf2 and aPKC_ι/ζ-Nrf2 might regulate the expression ofγ-GCS.3. PI3K specific inhibitor inhibited the expression of p-aPKC_ι/ζ,, aPKC_ι/ζ specific inhibitor had no effect on the expression of p-Akt,showed that located the upstream of aPKC_ι/ζ.4. cigarette smoking brought about oxidative stress,and might regulat the expression level ofγ-GCSmRNA and protein through the pathway of PI3K/Akt- aPKC_ι/ζ- Nrf2.