2.5 Isolation of RNA，cDNA synthesis and qRT-PCR
Total RNA was isolated using the TRIzol reagent(CoWin Bioscience，Beijing，China).Its quality was assessed by electrophoresis on 1%agarose gels based on the integrity of 28S，18Sand 5S bands after ethidium bromide staining.The total RNA concentration was quantified by measuring absorbance at 260nm and 280nm.cDNA was synthesized from RNA by PrimeScript TM RT reagent Kit with gDNA Eraser(TaKa-Ra，Japan)according to the instruction.qRT-PCR was performed on the CFX 96TM Real-Time System(C1000TM Thermal Cycler，BIO-RAD，USA).Primer sequences(Table 1)of GAPDH、Caspase-3、i NOS、Bcl-2、Bax and Fas were referred to our previous study［38］while the others(ATF-3、JNK、p38、Akt)were designed by Primer Premier 5.0 software(Applied Biosystems)with the cDNA sequences obtained from National Center of Biotechnology Information(NCBI)［GenBank：NM_012912.2，NM_053829.1，NM_031020.2，BF563829.1，respectively］.All of these primers were synthesized by Beijing Genome Institute(BGI-Shenzhen，China).For qRT-PCR，each reaction was run in triplicate and contained the following：10.0μL Premix Ex Taq TM II(Tli RNaseH Plus，2×Conc.，TaKaRa，Japan)，0.8μL Forward primer and Reverse primer(2.5μM)，0.4μL ROX Refrence DyeⅡ(50×Conc.，TaKaRa，Japan)and 100 ng cDNA template in a final reaction volume of 20μL.
Table 1 Primer sequences of target genes for qRT-PCR
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Continued table
After a pre-incubation step at 95℃for 30s to activate DNA polymerase，PCR amplification was 40 cycles of denaturation at 95℃for 5s and annealing at 60℃(except 64℃for Akt，Fas，i NOS)for 30 s.After amplification，the melting curves were generated in the range 65～95℃with increments of 0.5℃every 5 seconds.The emission data were quantified using the cycle threshold value.Data were normalized to GAPDH and presented as the mean fold change by comparing with the control group.