Rnaios

Trizol reagent, RNAios and Real-time PCR kit were obtained from TaKaRa Company (Dalian, China). Anti- MyD88, anti-TRAF6, anti-TRIF and anti-TLR4 antibodies were offered from Abcam Biotechnology (MA, USA). LPS was taken from Sigma-Aldrich (MO, USA). The immunohistochemistry kits and HRP-labeled secondary antibodies were bought from Boster Biotechnology (Wuhan, China). The IL-1β and IL-6 ELISA kit were purchased from Dkewei Biotechnology (Beijing, China). Corilagin was received from China National institutes for food and drug control.

RNAios (TaKaRa, Dalian, China) was used to extract the total RNA. After the concentration and purity of the total RNA were detected, it was reverse transcribed into cDNA by reverse transcription kit (TaKaRa, Dalian, China). Real-time PCR (RT-qPCR) was performed with cDNA as template. The PCR reaction mixture contained 5 μL of TB GreenTM Premix Ex Taq TM II (TaKaRa, Dalian, China), 0.25 μL of forward and reverse primers, respectively, 1 μg of cDNA, and 2 μL of RNase Free H₂O in a 10 μL reaction system. The reaction procedure is as follows: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, extension at 60 °C for 30 s, 45 cycles, extension at 65 °C for 5 s, extension at 95 °C for another 5 min. The primers used in this study were shown in Table S2 and synthesized in Sangon Biotech (Shanghai) Co., Ltd., China. All data were normalized to the 18S rRNA transcript. The mRNA abundance of each gene was calculated by 2^−ΔΔCT method.