As previously described, total RNA was extracted from lung tissues with TRIzol reagent (Invitrogen, Carlsbad, CA), then reverse transcribed with the Super TaqMan OneStep RT-qPCR Kit (Life Technologies). qRT-PCR was performed on an Applied Biosystems 7500 real-time PCR system with the following cycling program: 40 cycles of incubation at 50 °C for 2 min and 95 °C for 10 min, followed by melting at 95°C for 15 s, then annealing at 60°C for 1 min. Target gene expression levels were normalized to the housekeeping gene, GAPDH, and the fold change was calculated using the 2−ΔΔCt method.

The primers sequences were as follows: CCL2: 5-CCACTCACCTGCTGCTACTCA-TTC-3, 5-CTGCTGCTGGTGATCCTCTTGTAG-3; CXCL2: 5′-CCACTCACCTGC-TGCTACTCATTC-3′ and reverse 5′-CTGCTGCTGGTGATCCTCTTGTAG-3′.

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