Carbenoxolone disodium salt cbx

Dexamethasone (DEX), mifepristone (RU486), and carbenoxolone disodium salt (CBX) were purchased from Sigma, United States. The Erzhi formula was provided by the Department of Pharmacy, Institute of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine. The Erzhi formula was prepared by mixing the alcoholic extract of Ligustrum lucidum Ait (Oleaceae Hoffmanns; Ligustri lucidi fructus) and Eclipta prostrata L (Asteraceae Bercht; Ecliptae herba) in a 1:1 ratio with raw herbs. Ligustri lucidi fructus and Ecliptae herba were purchased from the Beijing Tongrentang Group Tianjin Ping Shan Dao Pharmacy Co. Both Ligustri lucidi fructus and Ecliptae herba were extracted by heating and refluxing with 70% ethanol; the procedure was performed three times, each time for 1 h, and the amount of 70% ethanol was increased by 10 times of raw herbs for each cycle. Then the extracts were combined and concentrated in an electric heating set (98-1-C, Tianjin Taisite Instrument Co., China) at 55°C to form a concentrated solution, and the concentrated solution were luophilized with an electric vacuum drying oven (DZG-6050, Shanghai Pein Experimental Instruments Co., China) at 45°C. Finally, 24.29 g powder was obtained from 100.00 g of Ligustri lucidi fructus and 20.80 g powder was obtained from 100.00 g of Ecliptae herba.

Carbenoxolone disodium salt (CBX; Sigma) was used as a gap junction inhibitor. Human connexin-32 plasmid (Cx32), C-terminally tagged with mEmerald, was obtained from Addgene (plasmid #54054) [48 (link)] while pcDNA 3.1 mock plasmid was purchased from Invitrogen. EVOS fluorescent microscope was used to verify the expression of Cx32 in cells (Additional file 3: Figure S3). Co-cultures of GS-293 and FSHR-293 cells were transiently transfected with Cx32 plasmid to determine the effect of connexin overexpression on cAMP transfer.
For determining cell viability after CBX treatment, co-cultures of GS-293 and FSHR-293 were preincubated with different concentrations of CBX (25, 50, 75 and 100 μM) for 2h in assay medium. Assay medium was then discarded, followed by washing with PBS. Cell viability was then assessed by CellTiter AQueous non-radioactive cell proliferation assay (Promega) following manufacturer’s instructions. Briefly, cells were incubated in DMEM complete medium (without phenol red) containing MTS/PMS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate] solution for 2h (37°C, 5% CO₂). Absorbance was then read at 490 nm, which provides a measure of cellular viability. One-way analysis of variance (ANOVA) was used to determine statistical differences among different samples.