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This study investigated how Guilou compound affects migration and invasion of human adenomyosis-derived cells (AMDC), and whether these effects involve the Rho/ROCK signaling pathway. Using CCK-8 to identify an optimal non–cell-viability-affecting concentration (100 mg/L), AMDC were treated with Guilou compound (50 or 100 mg/L) and assessed by scratch and Transwell assays for migration and invasion, alongside Western blotting/RT-qPCR for RhoA, RhoB, RhoC, ROCK1, and ROCK2; a rescue experiment used the ROCK-pathway agonist U-46619 (1 μmol/L) with Guilou compound (100 mg/L). Guilou compound significantly inhibited AMDC migration and invasion and down-regulated RhoA, RhoC, ROCK1, and ROCK2 at both protein and mRNA levels, while U-46619 increased these protein levels and Guilou compound reversed that up-regulation; RhoB showed no significant changes. The paper links its findings to adenomyosis mechanisms and is centrally about adenomyosis—specifically Guilou compound’s inhibition of AMDC migration and invasion via Rho/ROCK signaling.
Abstract
Background Adenomyosis (AM) is a common and difficult gynecological disease, but its specific mechanism has not been fully elucidated. Traditional Chinese Medicine (TCM) has certain advantages in the treatment of AM, preliminary studies have shown that Guilou compound-releasing intrauterine system can significantly reduce the proliferation and invasion ability of endometrial cells in rats with AM model. Objective To investigate the effect of Guilou compound on the migration and invasion of human adenomyosis derived cells (AMDC), and study its effect on Rho/ROCK signaling pathway. Methods The experiment was completed from October 2021 to April 2023 in Central Laboratory of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine. The CCK-8 assay was used to detect AMDC cell viability and screen the optimal drug concentration. AMDC were treated with Guilou compound (50 and 100 mg/L), and the cell migration viability was detected by scratch assay, and cell invasion ability was detected by Transwell assay. The expression of RhoA, RhoB, RhoC, ROCK1, and ROCK2 at protein and mRNA levels was detected by Western Blotting/RT-qPCR assay. In the Rescue experiment, AMDC were treated with agonist U-46619 (1 μmol/L) and Guilou compound (100 mg/L), and the protein expression of RhoA, RhoB, RhoC, ROCK1, and ROCK2 was detected by Western Blotting. Results Compared with the control group, the survival rate of AMDC treated with 100 mg/L mass concentration of Guilou compound was not significantly decreased (P>0.05), which was the optimal drug concentration without affecting the cell viability, 50 and 100 mg/L mass concentrations of Guilou compound were selected for subsequent experiments. Compared with the control group, the migration and invasion ability of AMDC in the Guilou compound (50 mg/L, 100 mg/L) group was significantly inhibited (P<0.001), the protein and mRNA expression levels of RhoA, RhoC, ROCK1, and ROCK2 were significantly down-regulated (P<0.001), while the protein expression of RhoA, RhoC, ROCK1 and ROCK2 in the U-46619 1 μmol/L group were significantly up-regulated (P<0.001). Compared with the U-46619 1 μmol/L group, the protein expression of RhoA, RhoC, ROCK1, and ROCK2 in the U-46619 (1 μmol/L) combined with Guilou compound (100 mg/L) group was significantly down-regulated (P0.05) . Conclusion Guilou compound can effectively inhibit the migration and invasion ability of AMDC with the optimal drug concentration of 100 mg/L that does not affect the cell viability, Guilou compound can reverse the up-regulation of U-46619 on the protein expression of RhoA, RhoC, ROCK1 and ROCK2, thus inhibiting the the continuous progression of ectopic lesions of AM, which may be closely related to the regulation of Rho/ROCK pathway.
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Zhongguo quanke yixue (Aug 2024)
Effect of Guilou Compound on Migration and Invasion of Adenomyosis Derived Cells
Abstract
Background Adenomyosis (AM) is a common and difficult gynecological disease, but its specific mechanism has not been fully elucidated. Traditional Chinese Medicine (TCM) has certain advantages in the treatment of AM, preliminary studies have shown that Guilou compound-releasing intrauterine system can significantly reduce the proliferation and invasion ability of endometrial cells in rats with AM model. Objective To investigate the effect of Guilou compound on the migration and invasion of human adenomyosis derived cells (AMDC), and study its effect on Rho/ROCK signaling pathway. Methods The experiment was completed from October 2021 to April 2023 in Central Laboratory of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine. The CCK-8 assay was used to detect AMDC cell viability and screen the optimal drug concentration. AMDC were treated with Guilou compound (50 and 100 mg/L), and the cell migration viability was detected by scratch assay, and cell invasion ability was detected by Transwell assay. The expression of RhoA, RhoB, RhoC, ROCK1, and ROCK2 at protein and mRNA levels was detected by Western Blotting/RT-qPCR assay. In the Rescue experiment, AMDC were treated with agonist U-46619 (1 μmol/L) and Guilou compound (100 mg/L), and the protein expression of RhoA, RhoB, RhoC, ROCK1, and ROCK2 was detected by Western Blotting. Results Compared with the control group, the survival rate of AMDC treated with 100 mg/L mass concentration of Guilou compound was not significantly decreased (P>0.05), which was the optimal drug concentration without affecting the cell viability, 50 and 100 mg/L mass concentrations of Guilou compound were selected for subsequent experiments. Compared with the control group, the migration and invasion ability of AMDC in the Guilou compound (50 mg/L, 100 mg/L) group was significantly inhibited (P<0.001), the protein and mRNA expression levels of RhoA, RhoC, ROCK1, and ROCK2 were significantly down-regulated (P<0.001), while the protein expression of RhoA, RhoC, ROCK1 and ROCK2 in the U-46619 1 μmol/L group were significantly up-regulated (P<0.001). Compared with the U-46619 1 μmol/L group, the protein expression of RhoA, RhoC, ROCK1, and ROCK2 in the U-46619 (1 μmol/L) combined with Guilou compound (100 mg/L) group was significantly down-regulated (P0.05) . Conclusion Guilou compound can effectively inhibit the migration and invasion ability of AMDC with the optimal drug concentration of 100 mg/L that does not affect the cell viability, Guilou compound can reverse the up-regulation of U-46619 on the protein expression of RhoA, RhoC, ROCK1 and ROCK2, thus inhibiting the the continuous progression of ectopic lesions of AM, which may be closely related to the regulation of Rho/ROCK pathway.
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