To understand the underlying mechanism(s) for the effect of exercise at different intensities on T cell and DNA vaccination responses, we treated mice in a training protocol with regular moderate-intensity exercise (MIE) or prolonged, exhaustive high-intensity exercise (HIE). After 6 weeks of training, splenocytes were isolated to evaluate cytokine expression and T-regulatory (Treg) cell proportion by RT-PCR and FACS, respectively. Another set of mice that completed the same training protocol were used to determine DNA vaccination responses. These mice were immunized three times with HBV DNA vaccine at 2-week intervals and euthanized on day 14 after the last immunization. Serum and splenocytes were isolated to determine humoral and cell-mediated immunity (CMI). Results showed that HIE increased anti-inflammatory cytokine expression and CD4(+) CD25(+) Treg cell proportion. Further, HIE decreased IFN-γ expression, T-lymphocyte proliferation, and antigen-specic cytotoxic response in HBV DNA vaccine-immunized mice. MIE did not change anti-inflammatory cytokine expression or CD4(+) CD25(+) Treg cell proportion but increased pro-inflammatory cytokine expression and augmented antigen-specific CMI. Thus, MIE lower the risk of cancer and infectious illness through enhancing the pro-inflammatory responses. By contrast, HIE might increase the risk of common infections, such as upper respiratory tract infection, due to an up-regulation of CD4(+) CD25(+) Treg cells and anti-inflammatory responses.

To elucidate the effects of maternal dietary betaine supplementation on hepatic expression of cholesterol metabolic genes in newborn piglets and the involved epigenetic mechanisms, we fed gestational sows with control or betaine-supplemented diets (3 g/kg) throughout pregnancy. Neonatal piglets born to betaine-supplemented sows had higher serum methionine concentration and hepatic content of betaine, which was associated with significantly up-regulated hepatic expression of glycine N-methyltransferase. Prenatal betaine exposure increased hepatic cholesterol content and modified the hepatic expression of cholesterol metabolic genes in neonatal piglets. Sterol regulatory elementbinding protein 2 was down-regulated at both mRNA and protein levels, while 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) was down-regulated at the mRNA level, but up-regulated at the protein level, in betaine-exposed piglets. The transcriptional repression of HMGCR was associated with CpG island hypermethylation and higher repressive histone mark H3K27me3 (histone H3 lysine 27 trimethylation) on the promoter, whereas increased HMGCR protein content was associated with significantly decreased expression of miR-497. Furthermore, LDL receptor was significantly down-regulated at both mRNA and protein levels in the liver of betaine-exposed piglets, which was associated with promoter CpG hypermethylation. In addition, the expression of cholesterol-27a-hydroxylase (CYP27a1) was up-regulated at both mRNA and protein levels, while the expression of cholesterol-7a-hydroxylase (CYP7a1) was increased at the mRNA level, but unchanged at the protein level associated with increased expression of miR-181. These results indicate that maternal betaine supplementation increases hepatic cholesterol content in neonatal piglets through epigenetic regulations of cholesterol metabolic genes, which involve alterations in DNA and histone methylation and in the expression of microRNA targeting these genes.Key words: Betaine: Epigenetic regulation: Cholesterol metabolism: Maternal diet: PigletsCholesterol is an essential component of cell membranes and also serves as a precursor for life-sustaining steroid hormones and bile acids (1) . It is well known that deregulation of cholesterol metabolism contributes to obesity, diabetes and CVD (2,3) . Moreover, cholesterol is particularly essential for embryogenesis (4,5) , and low plasma cholesterol level is usually correlated with low body weight at birth (6 -8) .Hepatic cholesterol homeostasis is maintained through the coordinated regulation of three relevant processes: biosynthesis; transportation; transformation (4,9) . In particular, sterol regulatory element-binding protein-2 (SREBP2) (10) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (11) are key factors/enzymes for cholesterol biosynthesis, LDL receptor (LDLR) and HDL receptor (scavenger receptor class B type I (SR-BI)) are responsible for transportation (12) , while cholesterol-7a-hydroxylase (CYP7a1) and cholesterol-27a-hydroxylase (CYP27a1) (13...

In this study, gestational sows were fed control or betaine-supplemented diets (3 g/kg) throughout the pregnancy, and the newborn piglets were used to elucidate whether maternal dietary betaine affected offspring hepatic gluconeogenic genes through epigenetic mechanisms. Neonatal piglets born to betaine-supplemented sows had significantly higher serum and hepatic betaine contents, together with significantly greater expression of methionine metabolic enzymes in the liver. Interestingly, significantly higher serum concentrations of lactic acid and glucogenic amino acids, including serine, glutamate, methionine and histidine, were detected in the piglets born to betaine-supplemented sows, which were coincident with higher hepatic glycogen content and PEPCK1 enzyme activity, as well as greater protein expression of gluconeogenic enzymes, pyruvate carboxylase (PC), cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK1), mitochondrional phosphoenolpyruvate carboxykinase (PEPCK2) and fructose-1, 6-bisphosphatase (FBP1). Moreover, maternal betaine significantly changed the methylation status of both CpGs and histones on the promoter of gluconeogenic genes. The lower PEPCK1 mRNA was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3, while the up-regulated PEPCK2 and FBP1 mRNA was associated with DNA hypomethylation and more enriched activation histone mark H3K4me3. Furthermore, the expression of two miRNAs predicted to target PC and 6 miRNAs predicted to target PEPCK1 was dramatically suppressed in the liver of piglets born to betaine-supplemented sows. Our results provide the first evidence that maternal betaine supplementation affects hepatic gluconeogenic genes expression in newborn piglets through enhanced hepatic methionine metabolism and epigenetic regulations, which involve DNA and histone methylations, and possibly miRNAs-mediated post-transcriptional mechanism.

Several studies have shown that combination treatment with natural products and chemotherapy agents can improve the sensitivity and cytotoxicity of chemotherapy agents. Resveratrol, a natural product, has many biological effects including antitumor and antiviral activities, as well as vascular protective effect. The aim of this study is to investigate the synergistic anticancer effect of resveratrol in combination with cisplatin and the potential anticancer mechanisms involved in A549 cells. The results obtained from Cell Counting Kit-8 and isobolographic analysis demonstrated that combination of resveratrol and cisplatin resulted in synergistic cytotoxic effects in A549 cells. Results from Hoechst staining, flow cytometry and western blot analysis suggested that resveratrol enhanced cisplatin-mediated apoptosis. Meanwhile, the changes of LC3-II and P62 levels and formation of autophagosome suggested that resveratrol in combination with cisplatin triggered autophagy. More importantly, inhibiting autophagy by 3-methyladenine markedly attenuated the apoptosis caused by combination of resveratrol and cisplatin in A549 cells. Taken together, our study provides the first evidence that resveratrol combined with cisplatin synergistically induce apoptosis via modulating autophagic cell death in A549 cells. These findings also help us to understand the role of natural products in combination with chemotherapy agents in lung cancer.

Betaine has been widely used in animal and human nutrition to promote muscle growth and performance, yet it remains unknown whether maternal betaine supplementation during gestation affects the metabolic characteristics of neonatal skeletal muscles. In the present study, feeding sows with betaine-supplemented diets throughout gestation significantly upregulated the expression of mtDNA-encoded OXPHOS genes (p < 0.05), including COX1, COX2, and ND5, in the muscle of newborn piglets, which was associated with enhanced mitochondrial COX enzyme activity (p < 0.05). Concurrently, maternal betaine supplementation increased the plasma betaine concentration and muscle expression of methyl transfer enzymes (p < 0.05), BHMT and GNMT, in offspring piglets. Nevertheless, Dnmt3a was downregulated at the level of both mRNA and protein, which was associated with a hypomethylated mtDNA D-loop region (p < 0.05). These results suggest that maternal betaine supplementation during gestation enhances expression of mtDNA-encoded genes through D-loop DNA hypomethylation in the skeletal muscle of newborn piglets.