Establishment of transgenic zebrafish model regulated by nr1h4 and related studies

doi:10.19803/j.1672-8629.2022.08.12

Abstract

Abstract: Objective A tissue-specific fluorescent labeled transgenic zebrafish model of farnesol X receptor (FXR, gene name nr1h4) was established to provide a visual detection animal model for the screening and safety warning of drugs related to bile acid metabolism. Methods The regulatory sequence of nr1h4 in zebrafish was found on UCSC website and optimized by promoter 2.0 software. Primers were designed and the nr1h4 regulatory sequence of zebrafish was obtained by PCR and constructed at the cloning site of pT2AL200R150G transposable vector. Then pTol2- nr1h4-EGFP plasmid and pCS-TP transposase mRNA were co-injected into zebrafish fertilized egg single cells to screen fluorescent individuals specifically expressed in liver and intestine, and Tg (-1.6nr1h4-EGFP) zebrafish strain was established through genetic screening. Tg (-1.6nr1h4: EGFP) zebrafish larvaes at the fourth day post fertilization were randomly divided into blank control group, solvent control group, glycine-β-muricholic acid group, obeticholic acid group, and low, medium and high concentration (10, 20 and 40 μg·mL^-1) ursodeoxycholic acid group, danning tablet group, rhein group and aloe-emodin group, and were administered continuously for 4 days. And the fluorescence intensity and development of transgenic zebrafish in each group at different times were observed for 4 days, and the fluorescence intensity was analyzed. Results Tg (-1.6nr1h4: EGFP) fluorescent labeled transgenic zebrafish strain was successfully established. Green fluorescence was expressed in the abdomen of zebrafish from the somite stage and mainly concentrated in the liver and intestine at the fourth day post fertilization. Compared with the blank control group, the fluorescence expression level of transgenic zebrafish in glycine-β-muricholic acid group decreased significantly at each administration time (4dpf: P< 0.01; 5~7dpf: P<0.000 1), the fluorescence in obecholic acid group increased significantly at each administration time (P<0.000 1), and there was no significant difference between solvent control group and blank control group, which indicate the effective establishment of the model. Compared with the blank control group, the fluorescence expression level of transgenic zebrafish in low concentration ursodeoxycholic acid group and aloe-emodin group increased gradually from 2 to 4 days after administration (P<0.000 1), and in medium concentration ursodeoxycholic acid group and aloe-emodin group inecreased significantly at each administration time (P<0.000 1), and the fluorescence in high concentration aloe-emodin group increased gradually from 2 to 4 days after administration (5~6dpf: P<0.001; 7dpf: P<0.05), but the fluorescences were almost same between high concentration ursodeoxycholic acid group and blank control group. The fluorescence expression level of transgenic zebrafish in low concentration danning tablet group and rhein group were not different from that of blank control group at each administration time, and the fluorescence in medium concentration danning tablet group increased significantly at each administration time (4 and 7dpf: P<0.000 1; 5 and 6dpf: P<0.001) and in high danning tablet group still increased significantly at each administration time (P<0.000 1), and the fluorescence in medium concentration rhein group increased gradually from 2 to 4 days after administration(5dpf: P< 0.000 1; 6dpf: P<0.001; 7dpf: P<0.01) while in high concentration rhein group began to decrease from 2 to 4 days after administration (P<0.000 1). Conclusion Tg (-1.6nr1h4-EGFP) zebrafish model provides a new animal model for the study of bile acid drug mechanism, drug screening and safety evaluation. Low and medium concentration ursodeoxycholic acid, medium and high concentrationcan danning tablet, medium concentration rhein and all concentration aloe-emodin can promote bile acid transport by up-regulating FXR expression in zebrafish liver and intestine, while high concentration ursodeoxycholic acid have no significant effect on FXR, and high concentration rhein inhibit FXR expression.

Key words: farnesoid X receptor, transgenic zebrafish model, danning tablet, aloe-emodin, rhein, ursodeoxycholic acid, obeticholic acid, glycine-β-muricholic acid

CLC Number:

R994
R979.1

LIU Caiping, CHAO Bao, FENG Yuanzhou, SUN Lan, LUO Yuan, YANG Jingfeng, DONG Wu, WANG Yongan, LI Yueting, ZHAO Baoquan. Establishment of transgenic zebrafish model regulated by nr1h4 and related studies[J]. Chinese Journal of Pharmacovigilance, 2022, 19(8): 873-880.

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