Flurbiprofen Inhibits Androgen Productions in Rat Immature Leydig Cells

Abstract

Flurbiprofen is one of the nonsteroidal anti-inflammatory drugs. Whether flurbiprofen affects androgen biosynthesis in Leydig cells is still unknown. Immature Leydig cells (ILCs) isolated from 35-day-old male Sprague–Dawley rats were cultured with 0–100 μM flurbiprofen for 24 h and medium androgen levels and Leydig cell mRNA levels were measured. Immature Leydig cells were also incubated with 100 μM flurbiprofen for 3 h in combination with luteinizing hormone (LH), 8bromo-cAMP, 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively, and medium androgen levels were measured. The ROS generation and apoptosis rate were also investigated. The direct effects of flurbiprofen on androgen biosynthetic and metabolizing enzyme activities were measured. Flurbiprofen significantly inhibited basal, LH, and 8bromo-cAMP stimulated androgen production at 10 and 100 μM. Further study demonstrated that flurbiprofen competitively inhibited rat and human testis 3β-hydroxysteroid dehydrogenase (HSD3B) activity with the half maximal inhibitory concentration (IC50) values of 0.95 μM for rat enzyme and 6.31 μM for human enzyme. In addition, flurbiprofen down-regulated the expression of Srd5a1 and Akr1c14 at 1, 10, and 100 μM. Flurbiprofen also down-regulated Lhcgr expression at 100 μM. Flurbiprofen at 10 and 100 μM increased ROS production and apoptosis rate of rat Leydig cells. In conclusion, flurbiprofen directly inhibits HSD3B activity and the expression levels of Srd5a1 and Akr1c14 in rat Leydig cells, thus leading to the reduction of androgen secretion.

Oncostatin M stimulates immature Leydig cell proliferation but inhibits its maturation and function in rats through JAK1/STAT3 signaling and induction of oxidative stress in vitro

“…ROS levels in LC were measured by a flow cytometer using a Reactive Oxygen Species Assay Kit (DCFH‐DA; Beyotime, Shanghai, China) as previously described 19 . This dye is a stable nonpolar compound that readily diffuses into cells and yields dichloro‐dihydrofluorescein diacetate (DCFH).…”

“…Cells were cultured for 24 h, as above. To evaluate early and lately apoptotic activity, an Annexin V‐FITC/PI apoptosis detection kit was used as the manufacturer's instructions (Nanjing Keygen Biotech, Nanjing, China) as previously described 19 . Cells were harvested and washed with cold PBS and then were resuspended in 200 µl the Annexin V‐binding buffer.…”

“…Oxygen Species Assay Kit (DCFH-DA; Beyotime, Shanghai, China) as previously described. 19 This dye is a stable nonpolar compound that readily diffuses into cells and yields dichloro-dihydrofluorescein diac- Relative changes in dichlorofluorescein fluorescence were expressed as fold increase over the control cells and ROS level was calculated.…”

“…ILC was planted into the 12-well plates with a density of 5 × 10 5 cells/well and incubated for 12 h. Then, the medium was changed an Annexin V-FITC/PI apoptosis detection kit was used as the manufacturer's instructions (Nanjing Keygen Biotech, Nanjing, China) as previously described. 19 Cells were harvested and washed with cold PBS and then were resuspended in 200 µl the Annexin V-binding buffer. After cells were stained with 5 µl of FITC-labeled Annexin V and 10 µL of PI, they were instantly measured using FACSCalibur (BD Biosciences).…”