Knockdown of glucocorticoid receptor (GR) using a combined RNAi and Cre-LoxP system and promotion of GR expression by Youguiyin

X.-Y. Li, J.-F. Sun, X.-Y. Xu

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, ChengDu, China. xxy0618@sina.com

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• Pharmacology

BACKGROUND AND OBJECTIVES: It is well documented that the decrease of glucocorticoid receptor (GR) can lead to Yin-yang Deficiency Syndrome in the Traditional Chinese Medicine theory. Youguiyin a famous and traditional Chinese medicine is often used to treat Yang Deficiency Syndrome, but its mechanism of action and target remains unknown. We aimed to establish one cell model whose the GR gene had been decreased and observe the effect of the traditional Chinese medicine Youguiyin on GR gene expression at mRNA level.

MATERIALS AND METHODS: Established recombinant plasmids of GR gene by combination use RNAi and the Cre-LoxP system, stably transfected the recombinant plasmids and Cre-ERT2 plasmid into the murine macrophage RAW264.7 cells and selected with G418 and hygromycin B respectively, then used 4-TH to induce Cre-ERT2 plasmid to express. RT-PCR and Western blot methods to validate the change of GR gene at mRNA and protein level were employed. Feed orally SD male rats with Youguiyin and got their blood serum, used these blood serum to culture RAW264.7–Cre-GR (-,F) cells, detected the change of GR gene at mRNA level by RT-PCR.

RESULTS: We successfully constructed two recombinant plasmids of GR gene which can make GR gene’s expression to decrease significantly in RAW264.7 cells. The blood serum which contained Youguiyin can enhance the expression of GR mRNA.

CONCLUSIONS: Combination use RNAi and the Cre-LoxP system can decrease GR gene’s expression in the murine macrophage RAW264.7 cells. Youguiyin can enhance the expression of GR mRNA.

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