七氟烷诱导神经元HO-1 mRNA表达信号转导通路的研究

目的探讨七氟烷诱导神经元血红素氧合酶1(HO-1)基因表达的信号转导通路。方法将培养7d的新生大鼠海马神经元随机分为4组:正常培养组(C组)、2%七氟烷组(S1组)、4%七氟烷组(S2组)和4%七氟烷+εV1-2组(V组)。C组神经元按正常培养方法培养。S1组和S2组神经元分别给予2%或4%七氟烷处理60min后继续培养24h。V组在神经元给予4%七氟烷处理同时在培养液中分别加入εV1-2使其终浓度为50μmol/L后同S2组处理。收集神经元进行HO-1mRNA和PKCε、Nrf2和HO-1蛋白表达的检测。结果与C组比较,S1组神经元HO-1mRNA(F=1194.09,P=0.000)和HO-1(F=967.63,P=0.000)蛋白表达增加,PKCε(F=876.13,P=0.000)和Nrf2(F=940.31,P=0.000)蛋白表达增加。与S1组比较,S2组神经元HO-1mRNA(F=1194.09,P=0.000)和HO-1(F=967.63,P=0.000)蛋白表达增加,PKCε(F=876.13,P=0.000)和Nrf2(F=940.31,р=0.000)蛋白表达增加。与S2组比较,V组神经元HO-1mRNA(F=1194.09,P=0.000)和HO-1(F=967.63,P=0.000)蛋白表达减少,PKCε(F=876.13,P=0.000)和Nrf2(F=940.31,P=0.000)蛋白表达减少。结论七氟烷通过PKCε/Nrf2信号转导通路诱导神经元HO-1mRNA的表达。

The Kinase Signal Pathway of Sevoflurane-induced Neuron HO-1 Gene Expression

Objective To investigate signal pathway associated with neuron hemeoxygenase-1（HO-1） expression in sevoflurane-induced. Methods Newborn （24-48h） Wistar rats were decapitated and hippocampus tissue was dissected and cut into 1mm × 1mm ×1mm pieces. Then digestion with 0.125% trypsin, centrifuged at 800 rpm/min for 5 min at 4℃, and suspended in a medium containing DMEM supplemented to 25 mmol/L glucose, 10% fetal bovine serum, 10% horse serum, and 2mmol/L glutamine. Cells were plated at 1.0 105/mL on poly-Dlysine-treated 6-well （2mL/well） plates and were treated with 10μmol/L cytosine arabinoside on day 4 to minimize glial growth. One-half of the medium was replaced twice a week with medium consisting of DMEM （4.5g/L glucose）/F12（1：1）, 5% fetal bovine serum and 5% horse serum. Cells were used after growing for 7 days. Cells were randomLy divided into 4 groups： control group （C group）, 2% sevoflurane group（S1 group）, 4% sevoflurane group（S2 group）and 4% sevoflurane ＋ εV1-2 group（V group）. Control cells were cultured normally. Group S 1 and S2 cells preconditioned with 2% or 4% sevoflurane, then cultured normally 24 hours. Group V cells culture medium was added 50μmol/LεV1-2 and preconditioned with 4% sevoflurane,then cultured normally 24 hours. The expression of HO-1-mRNA, PKCε, Nrf2 and HO-1 protein were detected. Results The expression of HO-1-mRNA （P〈 0.05）, HO-1 （P〈 0.05）, PKCε（P〈 0.05） and Nrf2 （P 〈 0.05） increased in group S1 comparing with group C. The expression of HO-1-mRNA （P〈 0.05）, HO-1（P〈0.05）, PKCε（P〈0.05） and Nrf2 （P〈0.05） increased in group S2 comparing with group S1. The expression of HO-1-mRNA （P〈0.05）, HO-1（P〈0.05）, PKCε（P〈 0.05） and Nrf2 （P〈0.05） decreased in group V comparing with group S2. Conclusion Sevoflurane induces neuron HO-1 gene expression through PKCε/Nrf2 kinase cell signal transduetion pathways.