Ileum tissue total RNA extraction and qPCR analysis

Total RNA from the ileum tissues was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s procedures. Real-time quantitative PCR was performed using the One Step SYBR PrimeScript plus RT-PCR Kit (TaKaRa Biotechnology Company Limited, Dalian, China) in the 7500 real-time PCR system (Applied Biosystems, Waltham, MA, USA). The expression of Gapdh mRNA served as an internal control, and the results were analyzed using the ΔΔCT method. The qPCR primers were synthesized and purified by GENERAY Biotechnology (Shanghai, China), and the following primers were used: for MuRF-1, 5′-GATGGAAACGCTATGGAGAACC-3′ and 5′-ACGGAAACGACCTCCAGACAT-3′; for krüppel-like factor 4 (klf4), 5′-AGGAACTCTCTCACATGAAGCG-3′ and 5′-GGTCGTTGAACTCCTCGGTC-3′; for resistin-like molecule-beta (Relm-β), 5′-CCATTTCCTGAGCTTTCTGG-3′ and 5′-AGCACATCCAGTGACAACCA-3′; and for trefoil factor 3 (Tff3), 5′-CAGATTACGTTGGCCTGTCTCC-3′ and 5′-ATGCTTGCTACCCTTGGACCAC-3′.