Effect of NMDA receptor agonist and antagonist on spermatogonial stem cells proliferation in 2- and 3- dimensional culture systems.

BACKGROUND: The main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801) on the proliferation of spermatogonial stem cells (SSCs) in two-dimensional (2D) and three-dimensional (3D) culture systems. METHODS AND RESULTS: The SSCs of male NMRI mice were isolated by enzymatic digestion and cultured for two weeks. Then, the identity of SSCs was validated by anti-Plzf and anti-GFR-α1 antibodies via immunocytochemistry (ICC). The proliferation capacity of SSCs was evaluated by their culture on a layer of the decellularized testicular matrix (DTM) prepared from mouse testis, as well as two-dimensional (2D) with different mediums. After two weeks of the initiation of proliferation culture on 3D and 2D medium, the pre-meiotic at the mRNA and protein levels were evaluated via qRT-PCR and flow cytometry methods, respectively. The results showed that the proliferation rate of SSCs in 3D culture with 50 mM glutamic acid and 20 mM D-serine was significantly different from other groups after 14 days treatment. mRNA expression levels of promyelocytic leukemia zinc finger (Plzf) in 3D cultures supplemented by 20 mM D-serine and 50 mM glutamic acid were considerably higher than the 3D control group (p < 0.001). The flow cytometry analysis revealed that the amount of Plzf in the 2D-culture groups of SSCs with 20 mM MK-801 was considerably lower compared to the 2D-culture control group (p < 0.001). CONCLUSIONS: This study indicated that decellularized testicular matrix supplemented with D-serine and glutamic acid could be considered a promising vehicle to support cells and provide an appropriate niche for the proliferation of SSCs.

Potentilla reptans L. postconditioning protects reperfusion injury via the RISK/SAFE pathways in an isolated rat heart.

BACKGROUND: Our previous study indicated that Potentilla reptans root has a preconditioning effect by its antioxidant and anti-apoptotic effects in an isolated rat heart ischemia/reperfusion (IR) model. In the present study, we investigated the post-conditioning cardio-protective effects of Potentilla reptans and its active substances. METHODS: The ethyl acetate fraction of P. reptans root (Et) was subjected to an IR model under 30 min of ischemia and 100 min of reperfusion. To investigate the postconditioning effect, Et was perfused for 15 min at the early phase of reperfusion. RISK/SAFE pathway inhibitors, 5HD and L-NAME, were applied individually 10 min before the ischemia, either alone or in combination with Et during the early reperfusion phase. The hemodynamic factors and ventricular arrhythmia were calculated during the reperfusion. Oxidative stress, apoptosis markers, GSK-3ß and SGK1 proteins were assessed at the end of experiments. RESULTS: Et postconditioning (Etpost) significantly reduced the infarct size, arrhythmia score, ventricular fibrillation incidence, and enhanced the hemodynamic parameters by decreasing the MDA level and increasing expression of Nrf2, SOD and CAT activities. Meanwhile, Etpost increased the BCl-2/BAX ratio and decreased Caspase-3 expression. The cardioprotective effect of Etpost was abrogated by L-NAME, Wortmannin (a PI3K/Akt inhibitor), and AG490 (a JAK/STAT3 inhibitor). Finally, Etpost reduced the expression of GSK-3ß and SGK1 proteins pertaining to the IR group. CONCLUSION: P. reptans reveals the post-conditioning effects via the Nrf2 pathway, NO release, and the RISK/SAFE pathway. Also, Etpost decreased apoptotic indexes by inhibiting GSK-3ß and SGK1 expressions. Hence, our data suggest that Etpost can be a suitable natural candidate to protect cardiomyocytes during reperfusion injury.

Probiotic supplementation attenuates hippocampus injury and spatial learning and memory impairments in a cerebral hypoperfusion mouse model.

Probiotics are referred to species of living microscopic organisms may help conserve the normal balance of the digestive system and/or manage diseases. A number of autoimmune, psychiatric, cardiovascular and cerebrovascular disorders may be associated with the imbalance of gut microbiota. This study examines the effect of 21 days consumption of multistrain probiotics on hippocampus injury, spatial and learning memory and some potential molecular mechanisms in a mouse model with cerebral hypoperfusion. Cerebral hypoperfusion was established in the mouse model by bilateral common carotid artery occlusion (BCCAO) for 20 min and 24 h reperfusion. Mixtures of several probiotic bacteria at concentrations of 107, 108 and 109 CFU/day were orally administrated for 3 weeks before the BCCAO. Spatial and learning memory, histological damage and apoptosis were assessed in the CA1, CA3 and dentate gyrus (DG) of the hippocampus 24 h after ischemia. The malondialdehyde (MDA) content and brain-derived neurotrophic factor (BDNF) level were measured by ELISA technique. Prophylactic of probiotic considerably reduced the number of apoptotic cells and neuronal death in the CA1, CA3 and DG of the hippocampus at all three concentrations (P < 0.001). In addition, probiotics reduced spatial memory impairment and neurological dysfunction only at the 109-CFU/day (P < 0.01). Nonetheless, probiotics did not change the levels of BDNF and MDA in the hippocampus (P > 0.05). According to the findings, the daily prophylactic ingestion of probiotics reduced hippocampus damage and prevented the spatial learning and memory deficit by suppressing apoptosis in the mouse model with cerebral hypoperfusion. Probiotic supplementation may be suggested as a useful preventive dietary strategy for groups susceptible to cerebrovascular diseases.

Cardioprotective and anti-apoptotic effects of Potentilla reptans L. root via Nrf2 pathway in an isolated rat heart ischemia/reperfusion model.

BACKGROUND: Previous studies have shown that proanthocyanidins have cardioprotective effects which are mediated via the release of nitric oxide (NO) ultimately resulting in increasing the antioxidant activity. We have investigated to show whether 1) the total extract and ethyl acetate fraction (Et) of Potentilla reptans root have an ischemic preconditioning (IPC) effect, 2) P. reptans has antioxidant and cardioprotective effects mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and scavenging of reactive oxygen species (ROS), 3) NO, caspase-3 and Bcl-2/Bax are involved in the IPC effect of P. reptans. METHODS: Male Wistar rats were divided into 10 groups. The isolated hearts were subjected to 30 min of ischemia and 100 min of reperfusion. The P. reptans was applied before the main ischemia. The infarct size was estimated by triphenyl-tetrazolium chloride staining. The hemodynamic parameters and ventricular arrhythmias were calculated during the reperfusion. Antioxidant markers and immunohistochemistry assays were determined at the end of the protocol. RESULTS: The Et significantly decreased the infarct size, arrhythmia scores, ventricular fibrillation incidence, and enhanced the hemodynamic parameters in a concentration-dependent manner against the ischemia/reperfusion group. SOD and CAT activity were increased and MDA level was decreased in response to the Et. Meanwhile, Et attenuated the suppression of Nrf2 expression and reduced the apoptotic indexes. The cardioprotective effect of P. reptans was abrogated by L-NAME. CONCLUSIONS: P. reptans demonstrated that the cardioprotective preconditioning effects via NO release, Nrf2 pathway, and antioxidant activity lead to a decrease in the apoptotic index.