[Inhibitory effect of phloroglucinol on eutopic stromal cells and ectopic stromal cells from endometriosis and its...

Objective: To investigate the inhibitory effects of phloroglucinol on eutopic stromal cells and ectopic stromal cells from endometriosis and its molecular mechanism. Methods: The effect of phloroglucinol on the survival rate of endometriosis-derived eutopic stromal cells hEM15A and ectopic stromal cells ihESCs was detected by cell counting kit 8 method. The effect of phloroglucinol on cell migration were evaluated by scratch test and Transwell migration test. The level of reactive oxygen species (ROS) in cells was detected by the fluorescence probe method. The expression of nuclear factor erythroid 2-related factor 2 (NRF2), kelch like ECH associated protein 1 (KEAP1), glutathione peroxidase 4 (GPX4) proteins in KEAP1/NRF2/GPX4 pathway was detected by western blot. Results: (1) The results of cell counting kit 8 method showed that phloroglucinol significantly reduced the cell viability of hEM15A and ihESCs [(47.89±1.93)% and (81.55±6.16)% after treatment with 5.550 mmol/L for 48 hours, (18.44±6.64)% and (66.09±1.63)% after treatment with 5.550 mmol/L for 72 hours, (23.20±4.95)% and (51.05±7.16)% after treatment with 11.100 mmol/L for 48 hours, (3.62±0.23)% and (40.14±2.63)% after treatment with 11.100 mmol/L for 72 hours; all P<0.05]. (2) Scratch test results showed that the cell migration rates of hEM15A cells and ihESCs cells at 24 hours time points without phloroglucinol (0 mmol/L) were (41.02±2.85)% and (41.60±1.77)%, respectively. Compared with the 0 mmol/L group of the respective cells, the migration rates of hEM15A and ihESCs cells treated with different concentrations (2.775, 5.550, 8.325, 11.100, 13.875 mmol/L) of phloroglucinol at 24 hours were significantly decreased [hEM15A cells were (21.28±2.96)%, (9.97±3.42)%, (3.39±0.79)%, (3.26±0.47)% and (2.38±0.11)%, ihESCs cells were (20.31±2.17)%, (17.17±1.44)%, (3.68±0.94)%, (2.96±1.19)% and (2.57±0.43)%; all P<0.05]. (3) The results of Transwell migration test showed that the numbers of migrated hEM15A and ihESCs cells were 196.00±14.00 and 73.67±5.51, respectively, at 24 hours without phloroglucinol (0 mmol/L) treatment. Compared with the respective 0 mmol/L group of the respective cells, there were no significant changes in the numbers of migration of the two cells in the 2.775 mmol/L group at 24 hours (190.30±42.59 and 68.33±16.56, respectively; all P>0.05). After treatment with the remaining higher concentration of phloroglucinol, the migration ability of hEM15A and ihESCs cells were significantly reduced (hEM15A cells were 131.70±29.96, 117.70±4.51, 83.33±12.42 and 42.33±13.87, ihESCs cells were 42.33±8.51, 41.00±9.54, 18.33±7.37 and 15.67±2.52; all P<0.05). (4) The results of ROS level detected by the fluorescence probe method showed that there were no significant changes in ROS levels in hEM15A cells and ihESCs cells after treatment with 2.775 mmol/L phloroglucinol for 24 hours (1.21±0.12 and 1.16±0.11, respectively; all P>0.05). After treatment for 48 and 72 hours, the levels of ROS in the two kinds of cells were significantly increased (2.75±1.15 and 2.76±0.74 in hEM15A cells, 2.82±0.72 and 2.90±0.82 in ihESCs cells; all P<0.05). (5) Western blot results showed that the expressions of KEAP1 in hEM15A cells and ihESCs cells were significantly decreased after phloroglucinol treatment (0.34±0.16 and 0.33±0.08; all P<0.05), and the expressions of NRF2 and GPX4 were significantly up-regulated (hEM15A cells were 2.88±0.44 and 2.09±0.06, ihESCs cells were 3.43±0.49 and 1.73±0.14; all P<0.05). Conclusion: Phloroglucinol could affect the survival of eutopic stromal cells and ectopic stromal cells from endometriosis by inducing oxidative stress, and its mechanism may be related to the activation of KEAP1/NRF2/GPX4 pathway and the imbalance of redox homeostasis.