Expression of Genes Related to Toll-Like Receptor 4 (TLR4) Signaling Pathway-Related Molecules and Intestinal Barrier Function

The primers of the related genes were obtained from the NCBI website, and all of the primer sequences are summarized in Supplementary Table 1. The expression of the genes related to TLR4 signaling pathway-related molecules, such as the myeloid differentiation factor 88(MyD88), TLR4, TNF receptor-associated factor-6 (TRAF6), interferon-beta (IFN-β), Interleukin-1 beta (IL-1β), TUMOR necrosis factor-α (TNF-α), IL-6, Leucine Rich Repeat And Pyrin Domain Containing 3 (NLRP3), Interferon regulatory factor 3 (IRF3), TANK-binding kinase-1 (TBK1), and Nuclear Factor kappa-Light-Chain-Enhancer of Activated B-p65 (NF-κB p65), were analyzed by reverse transcription quantitative real-time PCR (RT-PCR).

For RT-PCR, the total RNA extraction protocol of various gut regions used in the current study was similar to what was described above. The extracted eligible RNA was reversely transcribed using PrimeScript™ RT reagent kit (RR047A, TaKaRa, Dalian, China) according to the manufacturer's instructions. Immediately after the total RNA were obtained, cDNA was produced by PrimeScript™ RT reagent kit (RR047A, TaKaRa, Dalian, China) according to the manufacturer's instructions. The reaction mixture in RT-PCR contained 0.5 μg cDNA, 5 μL SYBR Premix Ex Taq II (2×), 0.2 μL of 10 μM forward or reverse primer (Bioline, Luckenwalde, Germany) and 3.6 μL dH2O in a total volume of 10 μl. The relative expression of a gene was calculated relative to the mean expression of ileum in control group using the ΔΔCt method (23), with GAPDH as the internal control.

Similarly, the expressions of genes related to the intestinal barrier function, such as genes encoding tight junction (TJ) proteins, including the claudin family, such as Claudin 1 (CLDN1), Claudin 4 (CLDN4), occludin, the zonula occluden family, such as zona occludens 2 (ZO2), and the genes encoding mucin, such as Mucin12 (MUC12), Mucin13 (MUC13), and Mucin20 (MUC20), were analyzed by RT-PCR as above.

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