Rapamycin inhibits B16 melanoma cell viability invitro and invivo by inducing autophagy and inhibiting the mTOR/p70­S6k pathway.

Rapamycin is an immunosuppressant that has been shown to prevent tumor growth following organ transplantation. However, its exact mode of antitumor action remains unknown. The present study used the B16-F10 (B16) murine melanoma model to explore the antitumor mechanism of rapamycin, and it was revealed that rapamycin reduced B16 cell viability in vitro and in vivo. In addition, in vitro and in vivo, the results of western blotting showed that rapamycin reduced Bcl2 expression, and enhanced the protein expression levels of cleaved caspase 3 and Bax, indicating that it can induce the apoptosis of B16 melanoma cells. Furthermore, the results of cell cycle analysis and western blotting showed that rapamycin induced B16 cell cycle arrest in the G1 phase, based on the reduction in the protein expression levels of CDK1, cyclin D1 and CDK4, as well as the increase in the percentage of cells in G1 phase. Rapamycin also significantly increased the number of autophagosomes in B16 melanoma cells, as determined by transmission electron microscopy. Furthermore, the results of RT-qPCR and western blotting showed that rapamycin upregulated the protein expression levels of microtubule-associated protein light chain 3 (LC3) and Beclin-1, while downregulating the expression of p62 in vitro and in vivo, thus indicating that rapamycin could trigger cellular autophagy. The present study revealed that rapamycin in combination with chloroquine (CQ) further increased LC3 expression compared with that in the CQ group, suggesting that rapamycin induced an increase in autophagy in B16 cells. Furthermore, the results of western blotting showed that rapamycin blocked the phosphorylation of p70 ribosomal S6 kinase (p70-S6k) and mammalian target of rapamycin (mTOR) proteins in vitro and in vivo, thus suggesting that rapamycin may exert its antitumor effect by inhibiting the phosphorylation of the mTOR/p70-S6k pathway. In conclusion, rapamycin may inhibit tumor growth by inducing cellular G1 phase arrest and apoptosis. In addition, rapamycin may exert its antitumor effects by inducing the autophagy of B16 melanoma cells in vitro and in vivo, and the mTOR/p70-S6k signaling pathway may be involved in this process.

Generation of Porcine Angiogenin 4-Expressing Pichia pastoris and Its Protection against Intestinal Inflammatory Injury.

Antimicrobial peptides have been extensively studied as potential alternatives to antibiotics. Porcine angiogenin 4 (pANG4) is a novel antimicrobial peptide in the angiogenin (ANG) family, which may have a regulatory effect on intestinal microflora. The object of present study is obtained pANG4 protein by heterologous expression, so as to explore the biological function of recombinant pANG4 (rpANG4). The pANG4 was expressed in Pichia pastoris (P. pastoris) and anti-inflammatory effects were investigated in intestinal porcine epithelial cell line-J2 (IPEC-J2) and mice. Purified rpANG4 had bacteriostatic activity and did not cause hemolysis or cytotoxicity at concentrations below 128 µg/mL. Purified rpANG4 increased the activity of IPEC-J2 and reduced apoptosis in vitro. rpANG4 reduced the pro-inflammatory gene expression and upregulated tight junction protein gene expression during inflammation. rpANG4 alleviated lipopolysaccharide (LPS)-induced liver and spleen damage, intestinal inflammation, jejunal apoptosis genes' expression, and improved immune function in an in vivo mice model. rpANG4 increased tight junction protein gene expression in jejunum, thereby improving the jejunum intestinal barrier function. In conclusion, rpANG4 had antibacterial activity, inhibited intestinal inflammation, improved intestinal barrier function, and alleviated liver and spleen damage. The current study contributes to the development of antibiotic substitutes and the improvement of animal health.

2-Hydroxy-4-Methylselenobutanoic Acid Promotes Follicle Development by Antioxidant Pathway.

Selenium (Se) is assumed to promote the follicle development by attenuating oxidative stress. The current study was developed to evaluate the effects of dietary 2-hydroxy-4-methylselenobutanoic acid (HMSeBA) supplementation on the follicle development in vivo and on the function of ovarian granulosa cells (GCs) in vitro. Thirty-six gilts were randomly assigned to fed control diet (CON), Na2SeO3 diet (0.3 mg Se/kg) or HMSeBA diet (0.3 mg Se/kg). The results showed that HMSeBA and Na2SeO3 supplementation both increased the total selenium content in liver and serum compared with control, while HMSeBA increased the total selenium content in liver compared with Na2SeO3 group. HMSeBA tended to increase the total selenium content in ovary compared with control. HMSeBA and Na2SeO3 supplementation both increased the weight of uteri in gilts at the third estrus. Moreover, HMSeBA supplementation down-regulated the gene expression of growth differentiation factor-9 (GDF-9) and bone morpho-genetic protein-15 (BMP-15) in cumulus-oocyte complexes (COCs). HMSeBA supplementation decreased malondialdehyde (MDA) content in serum, liver and ovary, increased activity of T-AOC in liver, TXNRD in ovary and GPX in serum, liver and ovary, while up-regulated the liver GPX2, SOD1 and TXNRD1, ovarian GPX1 gene expression. In vitro, HMSeBA treatment promoted GCs' proliferation and secretion of estradiol (E2). HMSeBA treatment increased the activity of T-AOC, T-SOD, GPX, TXNRD and decreased MDA content in GCs in vitro. Meanwhile, HMSeBA treatment up-regulated SOD2 and GPX1 gene expression in GCs in vitro. In conclusion, HMSeBA supplementation is more conducive to promoting follicle development by antioxidant pathway.

Effects of Energy and Dietary Fiber on the Breast Development in Gilt.

To study the effects of energy and dietary fiber on breast development in gilts and its possible mechanisms, 32 gilts (Landrace × Yorkshire) were randomly allocated into a 2 × 2 factorial design to receive a diet with low or high energy [LE: 33.37 MJ/d digestible energy (DE); HE: 41.87 MJ/d DE] and low or high fiber (LF: 0.3 kg/d dietary fiber, HF: 0.6 kg/d dietary fiber). The weight of breast tissue was recorded. The mammary glands were collected for further analyses. The high energy intake increased the relative weight of breast tissue (p < 0.05) and the content of breast fat (p < 0.05). At the same time, the oil red staining of breast slices also showed an increase in breast fat content in high-energy treatment. High energy intake increased the DNA concentration in breast tissues (p < 0.05). In addition, high energy intake increased the concentration of triglycerides, free fatty acids, and total cholesterol in the blood of gilts (p < 0.05), and the supplementation of high fiber tended to reduce free fatty acids, total cholesterol, and estradiol (p < 0.1). Proteomic analysis suggested that there were notable differences in the cytoskeleton, intracellular non-membrane-bounded organelle, apoptosis, receptor activity, and endopeptidase inhibitor activity in molecular function between the energy and fiber effects (p < 0.05). High fiber intake also decreased the mRNA expression of 5-HT7, Bax, and caspase-3 in the breast tissue of gilts (p < 0.05), which further confirmed the importance of fiber in regulating breast development in gilt. Our results indicate that increasing gilt energy intake improved breast weight and fat deposition and increased breast cell apoptosis. Increased fiber intake reduced breast fat deposition and breast cell apoptosis at high energy intake in gilts. These results provide a potential strategy for dietary intervention against high energy intake in gilts and even in humans.