Yuan-Hua Chen

Melatonin modulates TLR4-mediated inflammatory genes through MyD88- and TRIF-dependent signaling pathways in lipopolysaccharide-stimulated RAW264.7 cells.

Abstract:  Increasing evidence demonstrates that melatonin has an anti‐inflammatory effect. Nevertheless, the molecular mechanisms remain obscure. In this study, we investigated the effect of melatonin on toll‐like receptor 4 (TLR4)‐mediated molecule myeloid differentiation factor 88 (MyD88)‐dependent and TRIF‐dependent signaling pathways in lipopolysaccharide (LPS)‐stimulated macrophages. RAW264.7 cells were incubated with LPS (2.0 μg/mL) in the absence or presence of melatonin (10, 100, 1000 μm). As expected, melatonin inhibited TLR4‐mediated tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐1β, IL‐6, IL‐8, and IL‐10 in LPS‐stimulated macrophages. In addition, melatonin significantly attenuated LPS‐induced upregulation of cyclooxygenase (COX)‐2 and inducible nitric oxide synthase (iNOS) in macrophages. Further analysis showed that melatonin inhibited the expression of MyD88 in LPS‐stimulated macrophages. Although it had no effect on TLR4‐mediated phosphorylation of c‐Jun N‐terminal kinase (JNK), p38, and extracellular regulated protein kinase (ERK), melatonin significantly attenuated the activation of nuclear factor kappa B (NF‐κB) in LPS‐stimulated macrophages. In addition, melatonin inhibited TLR4‐mediated Akt phosphorylation in LPS‐stimulated macrophages. Moreover, melatonin significantly attenuated the elevation of interferon (IFN)‐regulated factor‐3 (IRF3), which was involved in TLR4‐mediated TRIF‐dependent signaling pathway, in LPS‐stimulated macrophages. Correspondingly, melatonin significantly alleviated LPS‐induced IFN‐β in macrophages. In conclusion, melatonin modulates TLR4‐mediated inflammatory genes through MyD88‐dependent and TRIF‐dependent signaling pathways.

Melatonin alleviates cadmium‐induced cellular stress and germ cell apoptosis in testes

Abstract:  Increasing evidence demonstrates that melatonin has an anti‐apoptotic effect in somatic cells. However, whether melatonin can protect against germ cell apoptosis remains obscure. Cadmium (Cd) is a testicular toxicant and induces germ cell apoptosis. In this study, we investigated the effects of melatonin on Cd‐evoked germ cell apoptosis in testes. Male ICR mice were intraperitoneally (i.p.) injected with melatonin (5 mg/kg) every 8 hr, beginning at 8 hr before CdCl2 (2.0 mg/kg, i.p.). As expected, acute Cd exposure resulted in germ cell apoptosis in testes, as determined by terminal dUTP nick‐end labeling (TUNEL) staining. Melatonin significantly alleviated Cd‐induced testicular germ cell apoptosis. An additional experiment showed that spliced form of XBP‐1, the target of the IRE‐1 pathway, was significantly increased in testes of mice injected with CdCl2. GRP78, an endoplasmic reticulum (ER) chaperone, and CHOP, a downstream target of the PERK pathway, were upregulated in testes of Cd‐treated mice. In addition, acute Cd exposure significantly increased testicular eIF2α and JNK phosphorylation, indicating that the unfolded protein response (UPR) pathway was activated by CdCl2. Interestingly, melatonin almost completely inhibited Cd‐induced ER stress and the UPR in testes. In addition, melatonin obviously attenuated Cd‐induced heme oxygenase (HO)‐1 expression and protein nitration in testes. Taken together, these results suggest that melatonin alleviates Cd‐induced cellular stress and germ cell apoptosis in testes. Melatonin may be useful as pharmacological agents to protect against Cd‐induced testicular toxicity.

Cadmium-induced teratogenicity: association with ROS-mediated endoplasmic reticulum stress in placenta.

The placenta is essential for sustaining the growth of the fetus. An increased endoplasmic reticulum (ER) stress has been associated with the impaired placental and fetal development. Cadmium (Cd) is a potent teratogen that caused fetal malformation and growth restriction. The present study investigated the effects of maternal Cd exposure on placental and fetal development. The pregnant mice were intraperitoneally injected with CdCl(2) (4.5mg/kg) on gestational day 9. As expected, maternal Cd exposure during early limb development significantly increased the incidences of forelimb ectrodactyly in fetuses. An obvious impairment in the labyrinth, a highly developed tissue of blood vessels, was observed in placenta of mice treated with CdCl(2). In addition, maternal Cd exposure markedly repressed cell proliferation and increased apoptosis in placenta. An additional experiment showed that maternal Cd exposure significantly upregulated the expression of GRP78, an ER chaperone. Moreover, maternal Cd exposure induced the phosphorylation of placental eIF2α, a downstream molecule of PERK signaling. In addition, maternal Cd exposure significantly increased the level of placental CHOP, another target of PERK signaling, indicating that the unfolded protein response (UPR) signaling was activated in placenta of mice treated with CdCl(2). Interestingly, alpha-phenyl-N-t-butylnitrone, a free radical spin-trapping agent, significantly alleviated Cd-induced placental ER stress and UPR. Taken together, these results suggest that reactive oxygen species (ROS)-mediated ER stress might be involved in Cd-induced impairment on placental and fetal development. Antioxidants may be used as pharmacological agents to protect against Cd-induced fetal malformation and growth restriction.

Maternal vitamin D deficiency during pregnancy elevates the risks of small for gestational age and low birth weight infants in Chinese population.

CONTEXT Vitamin D deficiency is common in pregnant women. Nevertheless, the association between maternal vitamin D status during pregnancy and the risk of having small for gestational age (SGA) and low birth weight (LBW) infants is uncertain. OBJECTIVE The objective of this study was to investigate whether there is a correlation between maternal vitamin D deficiency during pregnancy and the risk of having SGA and LBW infants in a Chinese population. DESIGN AND PARTICIPANTS This was a population-based birth cohort study that recruited 3658 eligible mother-and-singleton-offspring pairs. MAIN OUTCOME MEASURES Serum 25-hydroxyvitamin D was measured by RIA. The rate and relative risk (RR) for SGA and LBW infants were calculated among subjects with vitamin D deficiency and insufficiency during pregnancy. RESULTS There was a positive correlation between maternal serum 25-hydroxyvitamin D level and offspring birth weight (r = 0.477; P < .001). Further analysis showed that 4.98% of neonates were LBW infants among the subjects with vitamin D deficiency (RR, 12.00; 95% confidence interval [CI], 4.37, 33.00) and 1.32% among the subjects with vitamin D insufficiency (RR, 3.18; 95% CI, 1.07, 9.48). After adjustment for confounders, the RR for LBW infants was 12.31 (95% CI, 4.47, 33.89) among subjects with vitamin D deficiency and 3.15 (95% CI, 1.06, 9.39) among subjects with vitamin D insufficiency. Moreover, 16.01% of neonates were SGA infants among subjects with vitamin D deficiency (RR, 5.72; 95% CI, 3.80, 8.59) and 5.59% among subjects with vitamin D insufficiency (RR, 1.99; 95% CI, 1.27, 3.13). After adjustment for confounders, the RR for SGA infants was 6.47 (95% CI, 4.30, 9.75) among subjects with vitamin D deficiency and 2.01 (95% CI, 1.28, 3.16) among subjects with vitamin D insufficiency. CONCLUSION Maternal vitamin D deficiency during pregnancy elevates the risk of SGA and LBW infants in a Chinese population.

Reactive Oxygen Species Contribute to Lipopolysaccharide-Induced Teratogenesis in Mice

Lipopolysaccharide (LPS) has been associated with adverse developmental outcome, including embryonic resorption, fetal death and growth retardation, and preterm delivery. In the present study, we showed that an ip injection with LPS daily from gestational day (gd) 8 to gd 12 resulted in the incidence of external malformations. The highest incidence of malformed fetuses was observed in fetuses from dams exposed to 20 microg/kg LPS, in which 34.9% of fetuses per litter were externally malformed. In addition, 17.4% of fetuses per litter in 30 microg/kg group and 12.5% of fetuses per litter in 10 microg/kg group were externally malformed. Importantly, external malformations were also observed in fetuses from dams exposed to only two doses of LPS (20 microg/kg, ip) on gd 8, in which 76.5% (13/17) of litters and 39.1% of fetuses per litter were affected. LPS-induced teratogenicity seemed to be associated with oxidative stress in fetal environment, measured by lipid peroxidation, nitrotyrosine residues, and glutathione (GSH) depletion in maternal liver, embryo, and placenta. alpha-Phenyl-N-t-butylnitrone (PBN, 100 mg/kg, ip), a free radical spin-trapping agent, abolished LPS-induced lipid peroxidation, nitrotyrosine residues, and GSH depletion. Consistent with its antioxidant effects, PBN decreased the incidence of external malformations. Taken together, these results suggest that reactive oxygen species might be, at least partially, involved in LPS-induced teratogenesis.

Melatonin protects against lipopolysaccharide-induced intra-uterine fetal death and growth retardation in mice

Abstract:  Lipopolysaccharide (LPS) has been associated with adverse developmental outcomes, including intra‐uterine fetal death (IUFD) and intra‐uterine growth retardation (IUGR). However, the exact mechanism for LPS‐induced IUFD and IURD remains unclear. LPS stimulates macrophages to generate reactive oxygen species (ROS). Therefore, we hypothesize that ROS may be involved in LPS‐induced IUFD and IURD. Melatonin is a powerful endogenous antioxidant. In this study, we investigated the protective effects of melatonin on LPS‐induced IUFD and IURD in ICR mice. All pregnant mice except controls received an intraperitoneal (75 μg/kg, i.p.) injection of LPS on gestational day (gd) 15–17. The experiment was carried out in two different modes. In mode A, the pregnant mice received two doses of melatonin within 24 hr, one (5 or 10 mg/kg) injected immediately after LPS and the other (5 or 10 mg/kg) injected at 3 hr after LPS. In mode B, the pregnant mice were pretreated with 10 mg/kg of melatonin 18 hr before LPS and then received two doses of melatonin in 24 hr, one (10 mg/kg) injected immediately after LPS and the other (10 mg/kg) injected 3 hr after LPS. The number of live fetuses, dead fetuses and resorption sites were counted on gd 18. Live fetuses in each litter were weighed. Crown‐rump and tail lengths were examined and skeletal development was evaluated. Results showed that post‐treatments with melatonin significantly attenuated LPS‐induced IUFD in a dose‐dependent manner. Surprisingly, pre‐ plus post‐treatments with melatonin almost blocked LPS‐induced IUFD. In addition, both post‐treatments and pre‐ plus post‐treatments with melatonin significantly alleviated LPS‐induced decreases in crown‐rump and tail lengths and reversed LPS‐induced skeletal developmental retardation. However, melatonin had little effect on LPS‐induced decrease in fetal weight. Furthermore, pre‐ plus post‐treatments with melatonin significantly attenuated LPS‐induced lipid peroxidation in maternal liver. These results indicate that melatonin protects against LPS‐induced IURD and IUGR via counteracting LPS‐induced oxidative stress.

Endoplasmic reticulum stress is involved in hepatic SREBP-1c activation and lipid accumulation in fructose-fed mice.

A link between fructose drinking and nonalcoholic fatty liver disease (NAFLD) has been demonstrated in human and rodent animals. The aim of the present study was to investigate whether endoplasmic reticulum (ER) stress is mediated in the development of fructose-induced NAFLD. Female CD-1 mice were fed with 30% fructose solution for eight weeks. Hepatic lipid accumulation was assessed. Hepatic nuclear sterol regulatory element-binding protein (SREBP)-1c was measured. Results showed that hepatic SREBP-1c was activated in mice fed with fructose solution. Fatty acid synthase (fas) and acetyl-CoA carboxylase (acc), two target genes of SREBP-1c, were up-regulated. Fructose-evoked hepatic SREBP-1c activation seemed to be associated with insulin-induced gene (Insig)-1 depletion. An ER stress and unfolded protein response (UPR), as determined by an increased glucose-regulated protein (GRP78) expression and an increased eIF2α and PERK phosphorylation, were observed in liver of mice fed with fructose solution. Phenylbutyric acid (PBA), an ER chemical chaperone, not only significantly attenuated ER stress, but also alleviated fructose-induced hepatic Insig-1 depletion. PBA inhibited fructose-evoked hepatic SREBP-1c activation and the expression of SREBP-1c target genes, and protected against hepatic lipid accumulation. In conclusion, ER stress contributes, at least in part, to hepatic SREBP-1c activation and lipid accumulation in fructose-evoked NAFLD.

Melatonin inhibits endoplasmic reticulum stress and epithelial-mesenchymal transition during bleomycin-induced pulmonary fibrosis in mice.

Several reports indicate that melatonin alleviates bleomycin (BLM)-induced pulmonary fibrosis in rodent animals. Nevertheless, the exact mechanism remains obscure. The present study investigated the effects of melatonin on endoplasmic reticulum (ER) stress and epithelial-mesenchymal transition (EMT) during BLM-induced lung fibrosis. For the induction of pulmonary fibrosis, mice were intratracheally injected with a single dose of BLM (5.0 mg/kg). Some mice were intraperitoneally injected with melatonin (5 mg/kg) daily for a period of 3 wk. Twenty-one days after BLM injection, lung fibrosis was evaluated. As expected, melatonin significantly alleviated BLM-induced pulmonary fibrosis, as evidenced by Sirius red staining. Moreover, melatonin significantly attenuated BLM-induced EMT to myofibroblasts, as determined by its repression of α-SMA expression. Further analysis showed that melatonin markedly attenuated BLM-induced GRP78 up-regulation and elevation of the cleaved ATF6 in the lungs. Moreover, melatonin obviously attenuated BLM-induced activation of pulmonary eIF2α, a downstream target of the PERK pathway. Finally, melatonin repressed BLM-induced pulmonary IRE1α phosphorylation. Correspondingly, melatonin inhibited BLM-induced activation of XBP-1 and JNK, two downstream targets of the IRE1 pathway. Taken together, these results suggest that melatonin alleviates ER stress and ER stress-mediated EMT in the process of BLM-induced pulmonary fibrosis.

Melatonin alleviates lipopolysaccharide-induced placental cellular stress response in mice.

Abstract:  Melatonin protects mice from lipopolysaccharide (LPS)‐induced fetal death and intra‐uterine growth retardation. Nevertheless, its molecular mechanism remains obscure. In the present study, we investigated the effects of melatonin on LPS‐induced cellular stress in placenta. Pregnant mice were given with melatonin [5.0 mg/kg, intraperitoneal (i.p.)] 30 min before and 150 min after LPS (300 μg/kg, i.p.) on gestational day 15. Oxidative stress, endoplasmic reticulum (ER) stress, hypoxic stress, and heat stress in placenta were analyzed at 4 hr after LPS. As expected, maternal LPS administration resulted in placental glutathione (GSH) depletion and up‐regulated the expression of placental antioxidative enzymes. In addition, LPS significantly increased the level of inducible nitric oxide synthase (iNOS) and enhanced the intensity of placental 3‐nitrotyrosine residues. An ER stress, as determined by a decreased GRP78 expression, an obvious eIF2α and JNK phosphorylation, and an increased CHOP expression, were observed in placenta of pregnant mice injected with LPS. In addition, LPS significantly increased mRNA level of placental HIF‐1α, VEGF, and ET‐1, the markers of hypoxic stress. Heme oxygenase (HO)‐1, a marker of heat stress, was also up‐regulated in placenta of LPS‐treated pregnant mice. Interestingly, LPS‐induced placental oxidative stress, hypoxic stress, and ER stress were significantly alleviated when pregnant mice were given with melatonin, whereas melatonin had little effect on LPS‐evoked placental HO‐1 expression. In conclusion, maternally administered melatonin alleviates LPS‐induced cellular stress in the placenta. Melatonin may be useful as pharmacological agents to protect the fetuses against LPS‐induced intra‐uterine fetal death and intra‐uterine growth restriction.

Maternally administered melatonin differentially regulates lipopolysaccharide-induced proinflammatory and anti-inflammatory cytokines in maternal serum, amniotic fluid, fetal liver, and fetal brain

Abstract:  Lipopolysaccharide (LPS) has been associated with adverse developmental outcome, including intra‐uterine fetal death and intra‐uterine growth retardation. In the LPS model, tumor necrosis factor alpha (TNF‐α) is the major mediator leading to intra‐uterine fetal death and intra‐uterine growth retardation. Interleukin (IL)‐10 protects rodents against LPS‐induced intra‐uterine fetal death and intra‐uterine growth retardation. Melatonin is an immunomodulator. In the present study, we investigated the effect of maternally administered melatonin on LPS‐induced proinflammatory and anti‐inflammatory cytokines in maternal serum, amniotic fluid, fetal liver and fetal brain. The time pregnant mice were injected with melatonin [5.0 mg/kg, intraperitoneal (i.p.)] 30 min before LPS (500 μg/kg, i.p.) on gestational day 17. As expected, TNF‐α, IL‐1β, IL‐6 and IL‐10 were obviously increased in maternal serum and amniotic fluid in response to LPS. In addition, maternal LPS exposure significantly increased the levels of TNF‐α, IL‐1β, IL‐6 and IL‐10 in fetal liver, and TNF‐α and IL‐10 in fetal brain. Melatonin pretreatment significantly attenuated LPS‐evoked elevation of TNF‐α in maternal serum. On the contrary, melatonin aggravated LPS‐induced increase in IL‐10 in maternal serum. Melatonin had no effect on LPS‐evoked IL‐1β and IL‐6 in maternal serum and amniotic fluid. Interestingly, maternally administered melatonin also significantly attenuated LPS‐evoked elevation of TNF‐α in fetal brain, whereas the indole aggravated LPS‐induced increase in IL‐10 in fetal liver. Taken together, these results indicate that maternally administered melatonin differentially regulates LPS‐induced proinflammatory and anti‐inflammatory cytokines in maternal serum, amniotic fluid, fetal liver, and fetal brain.