L-Arginine protects mouse Leydig cells against T-2 toxin-induced apoptosis in vitro.

To explore the protective mechanism of L-arginine against T-2 toxin-induced apoptosis in mouse Leydig cells, we investigated whether L-arginine can prevent T-2 toxin-induced apoptosis in mouse Leydig cells and explored the underlying mechanisms. Leydig cells were isolated and cultured with control, T-2 toxin (10 nM), L-arginine (0.25, 0.5, and 1.0 mM), and T-2 toxin (10 nM T-2 toxin) + L-arginine (0.25, 0.5, or 1.0 mM) for 24 h. Cells and supernatants were harvested to examine proliferation of the cells, the apoptosis rate, activity of caspase-3 and mitochondria, and the gene expression levels of Bcl-2, Bax, PARP, and caspase-3. Results showed that proliferation and mitochondrial activity of Leydig cells were inhibited by administration of T-2 toxin. Bcl-2 gene expression levels was decreased, while the gene expression levels of Bax and PARP were increased, which could trigger mitochondria-mediated apoptosis, activate downstream caspase-3, and then increased caspase-3 at both activity and gene expression levels. The expression of the Bcl-2 gene was upregulated and the expression of Bax, caspase-3, and PARP gene were downregulated when L-arginine was added to the cultured cells. The results of this study showed that L-arginine could block T-2 toxin-induced apoptosis in mouse Leydig cells by regulating specific intracellular death-related pathways.

Protective effects of l-arginine against testosterone synthesis decreased by T-2 toxin in mouse Leydig cells.

The testosterone levels decreased by T-2 toxin in mouse Leydig cells were reported previously. It is not known, however, whether l-arginine improves the situation and what's the mechanism. Leydig cells were isolated and cultured with control, 10 nM T-2 toxin, 0.25, 0.5 or 1.0 mM l-arginine, and 10 nM T-2 toxin supplemented with 0.25, 0.5 or 1.0 mM l-arginine for 24 h. Cells and supernatants were collected to detect the mRNA expression and activities of P450scc (cholesterol side-chain cleavage enzyme), 3ß-HSD-1 (3ß-hydroxysteroid dehydrogenase/isomerase-1) and StAR (steroidogenic acute regulatory protein). Results revealed that l-arginine increased the testosterone levels declined by T-2 toxin and up-regulated the activities and mRNA expression of P450scc, 3ß-HSD-1 and StAR down-regulated by T-2 toxin in Leydig cells. Therefore, we concluded that l-arginine ameliorated the testosterone levels decreased by T-2 Toxin via regulating the mRNA expression and activities of P450scc, 3ß-HSD-1 and StAR in mouse Leydig cells.

Delayed effects of autophagy on T-2 toxin-induced apoptosis in mouse primary Leydig cells.

T-2 toxin is a type-A trichothecene produced by Fusarium found in several food commodities worldwide. T-2 toxin causes reproductive disorders, genotoxicity, and testicular toxicity in animals. Our previous research has reported that T-2 toxin can induce apoptosis via the Bax-dependent caspase-3 activation in mouse primary Leydig cells. However, little is known about the functions of autophagy and the cross talk between autophagy and apoptosis after exposure to T-2 toxin in Leydig cells. This study investigated these problems in mouse primary Leydig cells. Results showed that T-2 toxin treatment upregulated LC3-II and Beclin-1 expression, suggesting that T-2 toxin induced a high level of autophagy. Pretreatment with chloroquine (an autophagy inhibitor) and rapamycin (an autophagy inducer) increased and decreased the rate of apoptosis, respectively, in contrast to T-2 toxin-treated group. Autophagy delayed apoptosis in the T-2 toxin-treated Leydig cells. Therefore, autophagy may prevent cells from undergoing apoptosis by reducing T-2 toxin-induced cytotoxicity.

l-arginine protects against T-2 toxin-induced male reproductive impairments in mice.

l-arginine is beneficial for reproductive health; however, whether l-arginine may confer protection against T-2 toxin-induced reproductive impairment is not known. To address this, we used a mice model treated with T-2 toxin to investigate protective effects of l-arginine. Experimentally, we pre-treated mice with designed diet of l-arginine supplementation prior to the T-2 toxin-injected intraperitoneally exposure and then assessed semen quality, fertility and serum testosterone concentration. The results showed that l-arginine improved semen quality (e.g., live spermatozoa, abnormal spermatozoa, and acrosomal integrity of spermatozoa), testicular and cauda epididymal sperm counts, efficiency of sperm production and serum testosterone concentration in mice treated with T-2 toxin. In addition, l-arginine could increase pregnancy rate and decrease fetal resorption rate in females mated with T-2 toxin exposed males. Collectively, these findings suggest that dietary l-arginine supplementation may protect male reproductive impairments in mice treated with T-2 toxin through improving semen quality and serum testosterone levels.

l-arginine protects against oxidative damage induced by T-2 toxin in mouse Leydig cells.

To explore the protective mechanism of l-arginine against T-2 toxin-induced oxidative damage in mouse Leydig cells, Leydig cells were isolated and cultured with control, T-2 toxin (10 nM), l-arginine (0.25, 0.5, and 1.0 mM), and T-2 toxin (10 nM T-2 toxin) with l-arginine (0.25, 0.5, or 1.0 mM) for 24 hours. Cells and supernatants were harvested to examine cell viability, activities, and messenger RNA (mRNA) expression of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT), malondialdehyde (MDA) content, and DNA damage. Results showed that T-2 toxin significantly reduced cell viability, improved MDA content and DNA damage, and decreased activities and mRNA expression of GSH-Px, SOD, and CAT. However, l-arginine reduced T-2 toxin-induced oxidative damage and tended to maintain normal levels. Furthermore, l-arginine upregulated mRNA expressions of GSH-Px, SOD, and CAT. Collectively, l-arginine, due to its antioxidative ability, could ameliorate T-2 toxin-induced cytotoxicities in mouse Leydig cells by regulating oxidative stress.

T-2 toxin induces apoptosis via the Bax-dependent caspase-3 activation in mouse primary Leydig cells.

To explore the toxic effect of T-2 toxin on mouse Leydig cells and its underlying molecular mechanisms, we isolated Leydig cells from mature mice, set-up Leydig cells culture, treated cells with T-2 toxin, evaluated cell proliferation, detected the caspase-3 activity, mitochondrial activity and apoptosis rate, and measured the mRNA levels of Bcl-2, Bax, PARP and caspase-3. T-2 toxin inhibited cell proliferation at concentrations higher than 10-9 M or time more than 12 h, T-2 toxin also decreased Bcl-2 expression at the mRNA levels and mitochondrial activity at concentrations higher than 10-9 M. While, T-2 toxin increased the mRNA expressions of Bax and PARP at concentrations higher than 10-8 M and 10-9 M, respectively, triggered mitochondria-mediated apoptosis, activated downstream caspase-3, and then increased caspase-3 at the activity and mRNA levels at concentrations higher than 10-9 M. These data showed that T-2 toxin appears to activate specific intracellular death-related pathways leading to Bax-dependent caspase-3 activation and the induction of apoptosis in Leydig cells.

T-2 toxin inhibits gene expression and activity of key steroidogenesis enzymes in mouse Leydig cells.

T-2 toxin is one of the mycotoxins, a group of type A trichothecenes produced by several fungal genera including Fusarium species, which may lead to the decrease of the testosterone secretion in the primary Leydig cells derived from the mouse testis. The previous study demonstrated the effects of T-2 toxin through direct decrease of the testosterone biosynthesis in the primary Leydig cells derived from the mouse testis. In this study, we further examined the direct biological effects of T-2 toxin on steroidogenesis production, primarily in Leydig cells of mice. Mature mouse Leydig cells were purified by Percoll gradient centrifugation and the cell purity was determined by 3ß-hydroxysteroid dehydrogenase (3ß-HSD) staining. To examine T-2 toxin-induced testosterone secretion decrease, we measured the transcription levels of 3 key steroidogenic enzymes and 5 enzyme activities including 3ß-HSD-1, P450scc, StAR, CYP17A1, and 17ß-HSD in T-2 toxin/human chorionicgonadotropin (hCG) co-treated cells. Our previous study showed that T-2 toxin (10(-7) M, 10(-8) M and 10(-9) M) significantly suppressed hCG (10 ng/ml)-induced testosterone secretion. The studies demonstrated that the suppressive effect is correlated with the decreases in the levels of transcription of 3ß-HSD-1, P450scc, and StAR (P<0.05) and also in enzyme activities of 3ß-HSD-1, P450scc, StAR, CYP17A1, and 17ß-HSD (P<0.05).

[Protective effect of mouse 2.5s nerve growth factor on PC12 cells from injury induced by 2, 5-hexanedione].

OBJECTIVE: To explore whether the nerve growth factor has protective effects on PC12 cells from injury induced by 2, 5-hexanedione. METHODS: With PC12 cells as the model of neurons, different concentrations of NGF were added into the culture of PC12 cells. Then cell viability was tested with MTT. The DNA fragment was observed with agarose gel electrophoresis. The apoptosis ratio was tested with flow cytometry (FACS). The p53 protein was detected with western blot. The differences among the groups were compared. RESULTS: Cell viabilities were increased with the increase of the concentrations of NGF (P < 0.05). The DNA fragment, the apoptosis ratio and the expression of p53 were all decreased with the increase of the concentrations of NGF (P < 0.05). CONCLUSION: The NGF might have direct nutritional effects on PC12 cells, and protect them from injury induced by 2, 5 HD. Moreover, it might also have anti-apoptosis effect to some extent.