Riboflavin recovery of spermatogenic dysfunction via a dual inhibition of oxidative changes and regulation of the PINK1-mediated pathway in arsenic-injured rat model.

Arsenic trioxide (As2O3) poisoning and associated potential lesions are of a global concern. Inversely, riboflavin (vitamin B2, VB2) as a component of flavoproteins could play a vital role in the spermatogenic enzymatic reactions. Thus, this research aimed to explore potential beneficial roles of VB2 during As2O3-injured-toxicity. Rats were randomly allocated into 4 groups (n=8/group) and challenged as follows (for 30 days continuously): Group 1 received normal saline; Group 2 was treated with 3 mg As2O3/L; Group 3 received 40 mg VB2/L; Group 4 received 3 mg As2O3/L + 40 mg VB2/L. Both As2O3 and VB2 were dissolved in deionized water. Malondialdehyde (MDA), Glutathione Peroxidase (GSH-Px), Superoxide dismutase (SOD), and Catalase (CAT) were assessed for the oxidative profile, while TAS (Total Antioxidative Status) levels were evaluated for the antioxidant system, in both serum and testicular tissue. P<0.05 was considered statistically significant. The results show that As2O3 significantly decreased the body weight, testicular weight and testis volume, semen quality and testicular cell count (p<0.05). Furthermore, MDA content in the testicular tissue of the As2O3 group rats was significantly higher in comparison to the vehicle group (p<0.05). Likewise, TAS and the activities of GSH-Px, CAT and SOD were reduced (p<0.05) when compared to the control. As(2)O(3) induced testicular damage and seminiferous tubular atrophy. Monodansylcadaverine assays mirrored the histopathology observations. Meanwhile, As2O3 upregulated the expression of mitophagy-related genes including PINK1, Parkin, USP8, LC3-I, Fis1 and Mfn2. The p38 gene, responsible to stress stimuli, was also upregulated by As2O3 administration. Meanwhile, exposure to VB2 led to a significant decrease of the expression levels of mitophagy related genes. Our study revealed that VB2 supplementation protected testicular structures against As2O3-induced injury via a dual inhibition of oxidative changes and a regulation of the PINK1-mediated pathway.

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.

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.

Investigation of the Properties and Effects of Salvia Officinalis L. on the Viability, Steroidogenesis and Reactive Oxygen Species (ROS) Production in TM3 Leydig Cells in Vitro.

The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.