Effect of matrine in MAC-T cells and their transcriptome analysis: A basic study.

Matrine, an alkaloid derived from herbal medicine, has a wide range of biological activities, including antibacterial. Matrine was toxic to multiple cells at high concentrations. Bovine mammary epithelial cells (MAC-T) could be used as model cells for cow breast. Matrine was a feasible option to replace antibiotics in the prevention or treatment of mastitis against the background of prohibiting antibiotics, but the safe concentration of matrine on MAC-T cells and the mechanism of action for matrine at different concentrations were still unclear. In this study, different concentrations of matrine (0.5, 1, 1.5, 2, 2.5 and 3 mg/mL) were used to treat MAC-T cells for various time periods (4, 8, 12, 16 and 24 h) and measure their lactic dehydrogenase (LDH). And then the optimal doses (2 mg/mL) were chosen to detect the apoptosis at various time periods by flow cytometry and transcriptome analysis was performed between the control and 2 mg/mL matrine-treated MAC-T cells for 8 hours. The results showed that matrine was not cytotoxic at 0.5 mg/mL, but it was cytotoxic at 1~3 mg/mL. In addition, matrine induced apoptosis in MAC-T cells at 2 mg/mL and the proportion of apoptosis cells increases with time by flow cytometry. RNA-seq analysis identified 1645 DEGs, 676 of which were expressed up-regulated and 969 were expressed down-regulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the following pathways were linked to matrine-induced toxicity and apoptosis, including cytokine-cytokine receptor interaction pathway, viral protein interaction with cytokine and cytokine receptor, P53 and PPAR pathway. We found 7 DEGs associated with matrine toxicity and apoptosis. This study would provide a basis for the safety of matrine in the prevention or treatment of mastitis.

Progesterone Induces Apoptosis and Steroidogenesis in Porcine Placental Trophoblasts.

Placentation and placental steroidogenesis are important for pregnancy and maternal−fetal health. As pregnancy progresses, the main site of progesterone (P4) synthesis changes from the corpus luteum to the placenta, in which placental trophoblasts are the main cell type for P4 synthesis. Therefore, this study investigated the effects of P4 on apoptosis and steroidogenesis in porcine placental trophoblasts and the underlying molecular mechanisms. Porcine placental trophoblasts were treated with different concentrations of P4 for 48 h in a serum-free medium in vitro. Cell number, steroidogenesis, and relevant gene and protein expression levels were detected. A high dose of P4 (10.0 µM) significantly increased P4 (p < 0.01), androstenedione (p < 0.05), testosterone (p < 0.05), and estradiol (p < 0.05) production in porcine placental trophoblasts compared with that in control cells, while a low dose of P4 (1 × 10−3 µΜ) had no marked impact on steroid production. The mRNA expression of apoptosis-related genes (CASP3, CASP8, and Bax) (p < 0.05) and steroidogenesis-related genes (CYP11A1, CYP19A1, and StAR) (p < 0.01) was upregulated, and the expression of HSD3B and HSD17B4 was inhibited (p < 0.05) in the porcine placental trophoblasts treated with high doses of P4. Low doses of P4 had a lighter effect on gene expression than high doses. The expression of apoptosis-related proteins CASP3 (p < 0.05), and Bax (p < 0.01) and steroidogenesis-related proteins CYP19A1 (p < 0.05) and StAR (p < 0.01) was raised, but the proliferation-related protein CCND2 (p < 0.01) was downregulated in the pTr cells treated with high dose of P4. In comparison, a low dose of P4 inhibited the expression of Bax, CYP11A1 (all p < 0.01), and CCND2 (p < 0.05), but the expression of CASP3 (p < 0.05) and StAR (p < 0.01) was upregulated. In summary, excessive P4 can induce the apoptosis of porcine placental trophoblasts and lead to abnormal steroidogenesis in the placenta and hormone imbalance.

Supplementation of EGF, IGF-1, and Connexin 37 in IVM Medium Significantly Improved the Maturation of Bovine Oocytes and Vitrification of Their IVF Blastocysts.

The quality and developmental capacity of oocytes derived from in vitro maturation (IVM) remain unsatisfactory, which greatly impairs the efficiency and application of embryo technologies. The present experiment was designed to investigate the effect of the supplementation of EGF, IGF-1, and Cx37 in an IVM medium on the maturation quality and development ability of bovine oocytes. The cytoplasmic maturation events of oocytes and the quality of in vitro fertilization (IVF) blastocysts were examined to investigate the relative mechanisms. Our results showed that the nuclear maturation and blastocyst development after the IVF of oocytes treated with 25 µg/mL Cx37 or the combination of 50 ng/mL EGF and 100 ng/mL IGF-1 were significantly increased compared to those of the control group (p < 0.05). Furthermore, the blastocyst rate, and blastocyst total cell number and survival rate after vitrification of the EGF+IGF-1+Cx37 group, were significantly higher than those of the control group (p < 0.05), but lower than those of the FSH+LH+EGF+IGF-1+Cx37 group (p < 0.05). The transzonal projection (TZP) intensity, glutathione (GSH) level, and mitochondrial function of the EGF+IGF-1+Cx37 group were significantly higher than that of the control group, and lower than those of the FSH+LH+EGF+IGF-1+Cx37 group, in contrast to the results of the reactive oxygen species (ROS) levels. In conclusion, our results showed that the supplementation of 50 ng/mL EGF, 100 ng/mL IGF-1, and 25 µg/mL Cx37 in the IVM of bovine oocytes significantly improved their quality and developmental ability by increasing the TZP, mitochondrial function, and GSH level.

Influence of N-acetylcysteine on steroidogenesis and gene expression in porcine placental trophoblast cells.

N-acetylcysteine (NAC) is a widely used anti-inflammatory agent and antioxidant in vivo and in vitro. As a nutritional supplement, NAC can improve production and reproductive performances in animals through enhancing placental function and regulating hormone production. Trophoblast proliferation and steroid hormone production are two major functions in the placenta. We hypothesized that the effects of NAC on placental function is due to its direct and indirect effects on gene expression in placental trophoblast cells (pTr). To evaluate this hypothesis, we investigated the effects of NAC on steroidogenesis, gene expression, and cell proliferation in porcine pTr in vitro. pTr were treated with NAC in serum-free medium for 24 h with different concentrations (0, 0.1 µM, 1.0 µM, 10.0 µM, 0.1 mM, 1.0 mM, and 10.0 mM). Low-dose NAC (1 µM) stimulated pTr proliferation and decreased progesterone production, while increasing estradiol production (P < 0.05). High-dose NAC (10 mM) suppressed cell proliferation (P < 0.05), but had no effect on steroidogenesis. Low-dose NAC increased CCDN1 and decreased CASP3 and CASP8 mRNA levels (P < 0.05), whereas high-dose NAC decreased CDK4 and CCDN1 and increased CASP3 mRNA levels (P < 0.05). NAC had no effect on the mRNA abundance of StAR and HSD3B. Low-dose NAC upregulated CYP19A1 mRNA expression, and high-dose NAC downregulated CYP11A1 mRNA abundance (P < 0.05). Only low-dose NAC increased NOS3 mRNA abundance and tetrahydrobiopterin reduction (BH4/BH2 ratio). We conclude that NAC may act directly and indirectly on pTr with a dose-dependent manner and may regulate placental function by affecting pTr differentiation via regulating pTr steroid synthesis, cell proliferation, and apoptosis in sows.

Effects of N-carbamylglutamate on steroidogenesis and relative abundances of mRNA transcripts in pig placental trophoblasts.

Supplementation of N-carbamylglutamate (NCG) improves gestation outcomes, with increased piglet within-litter uniformity of birth weight and reduced peripheral steroid concentrations in pregnant sows and ewes. It was hypothesized that the effect of NCG on placental function results from direct effects on the placental trophoblasts. There, therefore, was investigation of the effects of NCG on pig placental trophoblast (pTr) steroidogenesis, mRNA transcript abundance, and cell proliferation in vitro. The pTr were treated with NCG in serum-free medium for 24-48 h. Treatment with NCG inhibited pTr progesterone, androstenedione, testosterone (all P <  0.01), and estradiol (P <  0.05) production, whereas it promoted (P <  0.05) pTr proliferation. Treatment with NCG suppressed (P <  0.05) the relative abundances of CYP11A1, CYP19A1, and CASP3 and increased abundances of CCDN1 (P <  0.01) and CDK4 (P <  0.05) mRNA transcripts in pTr, whereas NCG treatment had no effect (P >  0.10) on relative abundances of StAR, HSD17B4, or HSD3B mRNA transcripts. Treatments with NCG can increase pTr cell numbers of sows through upregulating CCND1 and CDK4 and suppressing CASP3 mRNA transcript abundances, while modulating steroidogenesis through effects on CYP11A1 and CYP19A1 mRNA transcript abundances. It is concluded that NCG may have a direct action on pTr and may regulate placental function by suppressing pTr differentiation as a consequence of lesser steroid synthesis while promoting pTr proliferation and inhibiting apoptosis in sows.