Therapeutic effects of melatonin in female mice with central precocious puberty by regulating the hypothalamic Kiss-1/Kiss1R system.

In recent years, central precocious puberty (CPP) in children is becoming more common, which seriously affects their physical and psychological health and requires finding a safe and effective treatment method. The aim of this study was to investigate the therapeutic effect of melatonin on CPP. A CPP model was established by subcutaneous injection of 300 micrograms of danazol into 5-day-old female mice, followed by treatment with melatonin and leuprolide. The vaginal opening was checked daily. Mice were weighed, gonads were weighed, gonadal index was calculated, and gonadal development was observed by hematoxylin and eosin (HE) staining. Serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) levels were measured by ELISA. By using RT-PCR and Western blotting, the mRNA and protein expression of the hypothalamus Kiss-1, Kiss-1 receptor (Kiss1R), gonadotropin-releasing hormone (GnRH), and pituitary GnRH receptor (GnRHR) were identified. The results showed that melatonin delayed vaginal opening time and reduced body weight, gonadal weight and indices in female CPP mice. Melatonin treatment prevents uterine wall thickening and ovarian luteinization in female CPP mice. Melatonin treatment reduces serum concentrations of FSH, LH, and E2 in female CPP mice. Melatonin suppressed the expressions of Kiss-1, Kiss1R and GnRH in the hypothalamus, and the expression of GnRHR in the pituitary of the female CPP mice. Our results suggest that melatonin can inhibit the hypothalamic-pituitary-gonadal (HPG) axis by down-regulating the Kiss-1/Kiss1R system, thereby treating CPP in female mice.

Moringa oleifera leaf extracts protect BMSC osteogenic induction following peroxidative damage by activating the PI3K/Akt/Foxo1 pathway.

OBJECTIVE: We aimed to investigate the therapeutic effects of Moringa oleifera leaf extracts on osteogenic induction of rat bone marrow mesenchymal stem cells (BMSCs) following peroxidative damage and to explore the underlying mechanisms. METHODS: Conditioned medium was used to induce osteogenic differentiation of BMSCs, which were treated with H2O2, Moringa oleifera leaf extracts-containing serum, or the phosphatidyl inositol-3 kinase (PI3K) inhibitor wortmannin, alone or in combination. Cell viability was measured using the MTT assay. Cell cycle was assayed using flow cytometry. Expression levels of Akt, phosphorylated (p)Akt, Foxo1, and cleaved caspase-3 were analyzed using western blot analysis. The mRNA levels of osteogenesis-associated genes, including alkaline phosphatase (ALP), collagen І, osteopontin (OPN), and Runx2, were detected using qRT-PCR. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and ALP activity were detected using commercially available kits. Osteogenic differentiation capability was determined using alizarin red staining. RESULTS: During osteogenic induction of rat BMSCs, H2O2 reduced cell viability and proliferation, inhibited osteogenesis, increased ROS and MDA levels, and decreased SOD and GSH-PX activity. H2O2 significantly reduced pAkt and Foxo1 expression, and increased cleaved caspase-3 levels in BMSCs. Additional treatments with Moringa oleifera leaf extracts partially reversed the H2O2-induced changes. Wortmannin partially attenuated the effects of Moringa oleifera leaf extracts on protein expression of Foxo1, pAkt, and cleaved caspase-3, as well as mRNA levels of osteogenesis-associated genes. CONCLUSION: Moringa oleifera leaf extracts ameliorate peroxidative damage and enhance osteogenic induction of rat BMSCs by activating the PI3K/Akt/Foxo1 pathway.

Thioredoxin mitigates H2 O2 -induced inhibition of myogenic differentiation of rat bone marrow mesenchymal stem cells by enhancing AKT activation.

Thioredoxin (Trx) is a hydrogen acceptor of ribonucleotide reductase and a regulator of some enzymes and receptors. It has been previously shown that significantly elevated levels of Trx expression are associated with the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), but it is not clear how Trx regulates the effects of hydrogen peroxide (H2 O2 ) on myogenic differentiation of BMSCs. Here, we report that rat BMSCs treated with a high dose (150 µm) of H2 O2 exhibited a significant reduction in viability, cell cycling, and superoxide dismutase and glutathione peroxidase levels, and an increase in reactive oxygen species and malondialdehyde levels, which was accompanied by reductions in protein kinase B activation and forkhead Box O1, myogenic differentiation 1 and myogenin expression during myogenic differentiation. Furthermore, treatment with recombinant human Trx significantly mitigated the effects of H2 O2 on the myogenic differentiation of BMSCs, and this was abrogated by cotreatment with wortmannin [a specific phosphatidylinositol 3-kinase inhibitor]. In summary, our results suggest that treatment with recombinant human Trx mitigates H2 O2 -induced oxidative stress and may promote myogenic differentiation of rat BMSCs by enhancing phosphatidylinositol 3-kinase/protein kinase B/forkhead Box O1 signaling.

Antitumour activity of 2-dihydroailanthone from the bark of Ailanthus altissima against U251.

Context The bark of Ailanthus altissima (Mill.) Swingle (Simaroubaceae) is traditionally used to treat ascariasis, diarrhoea, spermatorrhoea, bleeding and gastrointestinal diseases. Objective The objective of this study is to investigate the antitumour activity and mechanism of 2-dihydroailanthone isolated from A. altissima. Materials and methods The U251 cells were treated with 1.00, 4.00 and 8.00 µg/mL of 2-dihydroailanthone for 48 h and the normal cells treated with 20.00 µg/mL of 2-dihydroailanthone were tested as well. Proliferation inhibition of 2-dihydroailanthone on the cells was tested by MTT. Apoptosis and cell-cycle distribution in U251 cells with 1.00, 3.00 and 5.80 µg/mL of 2-dihydroailanthone for 48 h were determined by flow cytometry, respectively. The expression of the apoptosis-related genes and proteins was analysed by RT-PCR and Western blot method, respectively. Results MTT assay revealed that 2-dihydroailanthone inhibited U251 cells proliferation. The cell viability of U251 cells was 62.82, 31.34 and 25.58%, and that of three normal cells was 72.75, 82.74 and 44.92%, respectively. Flow cytometry assay showed that 2-dihydroailanthone induced apoptosis and G0/G1 phase cycle arrest towards U251 cells. The late apoptotic cells were 11.37, 21.73 and 33.83%, and the cells cycle distributed in the G0/G1 accounted for 48.85, 62.77 and 64.40%, respectively. The Western blot and RT-PCR assay showed that up-regulation of pro-apoptotic bax protein and down-regulation of anti-apoptotic bcl-2 protein as well as their mRNA on U251 cells might be related to the apoptosis induction and proliferation inhibition. Conclusion An important bioactive component, 2-dihydroailanthone, has antitumour effects, enlightening a novel source of phytomedicines in tumour therapy.