Total RNA of hypothalamus samples was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The ratio of optical density at 260 nm and 280 nm (OD 260/OD 280) was limited in the range of 1.8–2.1 to ensure the purity and quality of RNA. Then 2 μg of total RNA was reverse transcribed into cDNA using the TUREscript 1st Stand cDNA Synthesis Kit (Aidlab Biotechnologies, China). After adding the SYBR Green PCR Master Mix (TaKaRa Biotechnology, China), RT-qPCR was performed using the ABI StepOnePlus System (Applied Biosystems, USA) following the conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 60 s, and 72°C for 60 s. The target mRNA primers were designed by Premier 5.0 and synthesized by Sangon Biotech (Shanghai, China). The primer sequences for GRfwd were as follows: 5ʹ-CACATCTCACCCGCACCGATTG-3ʹ, rev: 5ʹ-TTGGACAAACACGGATGCCTGAC-3ʹ; for CRHfwd: 5ʹ-TGCCAAGGGAGGA GAAGAGA GC-3ʹ, rev: 5ʹ-GACAGAGCCACCAGCAGCATG-3ʹ; for GAPDHfwd: 5ʹ-CTGGAGAAACCTGCCAAGTATG-3ʹ, rev: 5ʹ-GGTGGAAGAATGGGAGTTG CT-3ʹ; and GAPDH was used as an endogenous control. The gene expression levels of CRH and GR were calculated with the method of 2−ΔΔCT.

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