The effect of Apium Graveolens L., Levisticum Officinale and Calendula Officinalis L. on cell viability, membrane integrity, steroidogenesis, and intercellular communication in mice Leydig cells in vitro

Abstract

Several plants have the potential to protect essential reproductive processes such as spermatogenesis or steroidogenesis, however, effective concentrations and main mechanisms of action are still unknown. This in vitro study was aimed to assess the effects of Apium graveolens L., Levisticum officinale, and Calendula officinalis L. extracts on the structural integrity, functional activity and gap junctional intercellular communication (GJIC) in mice Leydig cells. TM3 cells were grown in the presence of experimental extracts (37.5, 75, 150 and 300 µg/ml) for 24 h. For the present study, high-performance liquid chromatography analysis was used to quantify flavonoids or phenolic acids. Subsequently, Leydig cell viability was assessed by alamarBlue assay, while the cell membrane integrity was detected by 5 carboxyfluorescein diacetate-acetoxymethyl ester. The level of steroid hormones production was determined by enzyme-linked immunosorbent assay. Additionally, GJIC was assessed by scalpel loading/dye transfer assay. According to our results, Apium graveolens L. significantly increased the viability and cell membrane integrity at 75 µg/ml (109.0±4.3 %) followed by a decline at 300 µg/ml (89.4±2.3 %). In case of Levisticum officinale and Calendula officinalis L. was observed significant decrease at 150 µg/ml (88.8±11.66 %, 87.4±6.0 %) and 300 µg/ml (86.2±9.3 %, 84.1±4.6 %). Furthermore, Apium graveolens L. significantly increased the progesterone and testosterone production (75 and 150 µg/ml) however, Levisticum officinale and Calendula officinalis L. significantly reduced steroid hormones synthesis at 150 and 300 µg/ml. Finally, the disturbance of GJIC was significantly affected at 300 µg/ml of Levisticum officinale (82.5±7.7 %) and Calendula officinalis L. (79.8±7.0 %). The balanced concentration ratio may support the Leydig cell function, steroidogenesis as well as all essential parameters that may significantly improve reproductive functions.