Data Sets of ESR1 Expression in HESC cultures and ESR1 and PR levels in Wistar rat's endometrium when treated with Adansonia digitata root bark fractions.
Description
Adansonia digitata Linn root bark extract alters oestrogen and progesterone activities in the female Wistar rat. We hypothesized that components of the extract may act through the regulation of oestrogen and progesterone transduction. We obtained fractions from Aqueous methanol extract of Adansonia digitata root bark through Column chromatography with different solvents; acetyl acetate, methanol, and distilled water, to yield acetyl acetate-methanol fraction (AAMF); Aqueous methanol fraction (AQMF); and Aqueous fraction (AQF). The fractions were investigated for their effect on the gene expression of oestrogen receptor alpha (ESR1) in Human endometrial stromal cell (HESC) line (Data 1). The effect of the acetyl acetate-methanol fraction of the extract on the location and levels of ESR1 and progesterone receptor alpha-beta (PR) in the mature virgin female Wistar rat's uterus was also investigated (Data 2 - 6). The results revealed that AAMF at 50 – 500 µg /ml downregulates ESR1 gene expression in HESC cultures. In the Wistar rat's uterus, AAMF at 150 - 300 mg/kg bodyweight caused a reduction in membrane-located ESR1 levels in the endometrial glands and epithelium, but increased PR levels in the endometrial stroma.
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[Data 1]. HESC cultures were incubated for 72 hours in plates with Dulbecco’s Modified Eagle’s Medium (DMEM-F12) to which 0 – 500 µg /ml of AAMF, AQMF, and AQF were added. AAMF and AQMF were first dissolved with 0.1% DMSO (5µl DMSO in 5 ml DMEM-F12) while AQF was dissolved in double distilled water and filtered through a 0.20 µm filter. The cells were harvested from each group and the RNA was isolated using Qiagen’s isolation kit as per the manufacturer’s manual. Isolated RNA was subjected to QIAGEN® One-Step RT-PCR Reverse transcription technique using primers for the human βeta actin gene (ACTB) and oestrogen receptor alpha (ESRI). The qPCR amplification was in a conventional qPCR machine. The components of the PCR products were separated through gel electrophoresis (1 X tris acetate buffer, subjected to 90 volts – 60Hz electricity for 45 minutes ) and the resulting bands were viewed under a Gel UV transilluminator (M-15 UVP Transilluminator, with a UVP VisiDoc-It Imaging System California USA. The intensity of the bands in pixels was analyzed using ImageJ. Data generated for the intensity of ESR1 and ACTB bands were expressed in pixels. Data 2 - 6]. The oestrous cycle of mature virgin female Wistar rats was synchronized. The rats were treated with different doses of AAMF per os for 7 days. Subsequently, under anaesthesia, the uterus was harvested and fixed in 10% paraformaldehyde and processed for ESR1 and PR proteins by H-Dab immunostaining technique using IHC-P protocol of DB Biotech, Kosice, Slovakia, for Anti-ESR1 and Anti-PR rabbit clonal antibodies. Photomicrographs of each section of the uterus (myometrium and endometrium- stroma, gland, and epithelium) were made. The intensity of H-Dab staining of ESR1 and PR in Wistar rat’s uterine tissue was measured by imageJ tool analysis. The stain intensity was measured and presented in pixels.