Genistein-induced fluid accumulation in ovariectomised rats' uteri is associated with increased cystic fibrosis transmembrane regulator expression

OBJECTIVE: High genistein doses have been reported to induce fluid accumulation in the uteri of ovariectomised rats, although the mechanism underlying this effect remains unknown. Because genistein binds to the oestrogen receptor and the cystic fibrosis transmembrane regulator mediates uterine fluid secretion, we hypothesised that this genistein effect involves both the oestrogen receptor and cystic fibrosis transmembrane regulator. METHODS: Ovariectomised adult female Sprague-Dawley rats were treated with 25, 50, or 100 mg/kg/day genistein for three consecutive days with and without the ER antagonist ICI 182780. One day after the final drug injection, the animals were humanely sacrificed, and the uteri were removed for histology and cystic fibrosis transmembrane regulator mRNA and protein expression analysis using real-time polymerase chain reaction and Western blotting, respectively. The cystic fibrosis transmembrane regulator protein distribution was analysed visually by immunohistochemistry. RESULTS: The histological analysis revealed an increase in the circumference of the uterine lumen with increasing doses of genistein, which was suggestive of fluid accumulation. Moreover, genistein stimulated a dose-dependent increase in the expression of cystic fibrosis transmembrane regulator protein and mRNA, and high-intensity cystic fibrosis transmembrane regulator immunostaining was observed at the apical membrane of the luminal epithelium following 50 and 100 mg/kg/day genistein treatment. The genistein-induced increase in uterine luminal circumference and cystic fibrosis transmembrane regulator expression was antagonised by treatment with ICI 182780. CONCLUSION: Genistein-induced luminal fluid accumulation in ovariectomised rats' uteri involves the oestrogen receptor and up-regulation of cystic fibrosis transmembrane regulator expression, and these findings reveal the mechanism underlying the effect of this compound on changes in fluid volume in the uterus after menopause

Isolation and cloning of human NQO1 promoter in Pgl3 basic vector

Malaria is a major public health problem caused by Plasmodium falciparum, a parasite that infects red blood cells. Recently, several polyphenolic compounds have been reported capable of preventing the progression of malaria parasite. This observation may be related to NAD (P) H: quinon oxidoreductase: a flavoprotein responsible for catalyzing two-electron reduction and detoxification of quinones and their derivatives. In this study the 2123 bp of the 5` upstream of the first transcription start site was successfully isolated. Based on the bioinformatic program, several regulatory regions such as Antioxidant Response Element (ARE) may be responsible for the direct regulation of polyphenols on this enzyme. It was predicted at -477 from the first transcription start site. Upon isolation, this fragment was used in a cloning process into the pGL3 Basic vector and transformed to E.coli DH5a competent cell

Isolation and cloning of human VEGF promoter region in PGL3 basic vector

Worldwide mortality and morbidity from infectious diseases is being replaced by non infectious chronic diseases, such as cancer, obesity, type II diabetes, cardiovascular diseases, neurodegenerative diseases and aging which may involve inflammation. Vascular endothelial growth factor (VEGF) as a potent pro-inflammatory cytokine is elevated in many human diseases, or animal models of human disease, which are mentioned above. Compounds derived from botanic sources, such as polyphenolic compounds express anti-inflammatory activity by modulation of pro-inflammatory gene expression. We hypothesized this effect may related to control regulation of VEGF gene promoter. In this study, the VEGF promoter was isolated using nested PCR to define the transcription factors binding sites. The human VEGF promoter region was cloned in DH5 alpha Ecoli for future investigation

Genistein Induces Increase in Fluid pH, Na+ and HCO3− Concentration, SLC26A6 and SLC4A4 (NBCe1)-B Expression in the Uteri of Ovariectomized Rats

Genistein has been reported to stimulate luminal HCO3− secretion. We hypothesized that genistein mediates this effect via SLC26A6 and SLC4A4 (NBCe1) transporters. Our study aimed to: investigate changes in uterine fluid pH, Na+ and HCO3− concentration and expression of uterine SLC26A6 and NBCe1 under genistein effect. Ovariectomized adult female rats received 25, 50 and 100 mg/kg/day genistein for a week with and without ICI 182780. A day after the last injection, in vivo uterine perfusion was performed to collect uterine fluid for Na+, HCO3− and pH determination. The animals were then sacrificed and uteri were removed for mRNA and protein expression analyses. SLC26A6 and NBCe1-A and NBCe1-B distribution were visualized by immunohistochemistry (IHC). Genistein at 50 and 100 mg/kg/day stimulates uterine fluid pH, Na+ and HCO3− concentration increase. Genistein at 100 mg/kg/day up-regulates the expression of SLC26A6 and SLC4A4 mRNA, which were reduced following concomitant ICI 182780 administration. In parallel, SLC26A6 and NBCe1-B protein expression were also increased following high dose genistein treatment and were localized mainly at the apical membrane of the luminal epithelia. SLC26A6 and NBCe1-B up-regulation by genistein could be responsible for the observed increase in the uterine fluid pH, Na+ and HCO3− concentration under this condition