De-Xiang Xu

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.

Melatonin attenuates lipopolysaccharide (LPS)-induced apoptotic liver damage in d-galactosamine-sensitized mice

D-Galactosamine (GalN) depletes UTP primarily in liver, resulting in decreased RNA synthesis in hepatocytes. When given together with a sublethal dose of lipopolysaccharide (LPS), GalN highly sensitizes animals to produce apoptotic liver injury with severe hepatic congestion, resulting in rapid death. Melatonin is a cytokine modulator, antioxidant and anti-apoptotic agent. In the present study, we investigated the effect of melatonin on LPS-induced apoptotic liver damage in GalN-sensitized mice. Female CD-1 mice were intraperitoneally (i.p.) injected with melatonin (5.0mg/kg) 30min before GalN/LPS (700mg10microg/kg, i.p.), another two doses of melatonin (2.5mg/kg, i.p.) being administered 1 and 2h after GalN/LPS. Results showed that serum alanine aminotransferase (ALT) activities were markedly increased 8h after GalN/LPS treatment, massive hemorrhage being observed in histological sections of liver from GalN/LPS-treated mice. Melatonin significantly attenuated GalN/LPS-induced elevation of serum ALT. In parallel, melatonin distinctly improved GalN/LPS-induced congestion. Additional experiment showed that melatonin significantly attenuated GalN/LPS-induced hepatic apoptosis, measured by inhibition of caspase-3 activities and attenuation of DNA laddering. Furthermore, melatonin markedly increased hepatic Se-dependent glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) activities and attenuated hepatic glutathione (GSH) depletion in GalN/LPS-treated mice. Increases in serum tumor necrosis factor alpha (TNF-alpha), which were observed in GalN/LPS-treated mice, were significantly reduced by melatonin. However, melatonin had no effect on LPS-evoked nitric oxide production in GalN-sensitized mice. Taken together, these results indicate that melatonin protected against LPS-induced liver damage in GalN-sensitized mice through its strong ROS-scavenging, antiinflammatory and antiapoptotic effects.

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.

Role of receptor interacting protein (RIP)1 on apoptosis-inducing factor-mediated necroptosis during acetaminophen-evoked acute liver failure in mice.

Acetaminophen (APAP) overdose induces apoptosis-inducing factor (AIF)-dependent necroptosis, but the mechanism remains obscure. The present study investigated the role of receptor interacting protein (RIP)1, a critical mediator of necroptosis, on AIF-dependent necroptosis during APAP-induced acute liver failure. Mice were intraperitoneally injected with APAP (300 mg/kg). As expected, hepatic RIP1 was activated as early as 1 h after APAP, which is earlier than APAP-induced hepatic RIP3 upregulation. APAP-evoked RIP1 activation is associated with hepatic glutathione (GSH) depletion. Either pretreatment or post-treatment with Nec-1, a selective inhibitor of RIP1, significantly alleviated APAP-induced acute liver failure. Moreover, Nec-1 improved the survival and prevented APAP-induced necroptosis, as determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Further analysis showed that Nec-1 significantly inhibited APAP-induced hepatic c-Jun N-terminal kinase (JNK) phosphorylation and mitochondrial Bax translocation. In addition, Nec-1 blocked APAP-induced translocation of AIF from the mitochondria to the nucleus. Of interest, no changes were induced by Nec-1 on hepatic CYP2E1 expression. In addition, Nec-1 had little effect on APAP-induced hepatic GSH depletion at early stage. Taken together, these results suggest that RIP1 is involved in APAP-induced necroptosis. Nec-1 is an effective antidote for APAP-induced acute liver failure.

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.

Mitochondrial signaling pathway is also involved in bisphenol A induced germ cell apoptosis in testes

Bisphenol A (BPA) is a potential endocrine disruptor and testicular toxicant. An earlier study showed that BPA-induced germ cell apoptosis through the Fas/FasL apoptotic pathway. In the present study, we aimed to investigate whether the mitochondrial pathway is also involved in the process of BPA-mediated germ cell apoptosis in testes. Male mice were administered with BPA (160 or 480 mg/kg) by gavage daily from postnatal day 35 (PND35) to PND49. Germ cell apoptosis in testes was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL). As expected, the number of TUNEL+ germ cells per tubule and the percentage of tubules with TUNEL+ germ cells were significantly increased in testes of mice treated with BPA during puberty. TUNEL+ germ cells were observed mainly in stages VII-VIII seminiferous tubules in testes. An increase in the level of Fas and FasL was observed in testes of mice exposed to BPA during puberty. In addition, pubertal BPA exposure evoked the activation of caspase-8 and caspase-3 in testes. Interestingly, pubertal BPA exposure also caused the translocation of cytochrome c from mitochondria into cytosol. In addition, pubertal BPA exposure upregulated the level of Bax and active caspase-9 in testes. Taken together, these results suggest that pubertal BPA exposure induces germ cell apoptosis in testes through not only the Fas/FasL signaling pathway but also the mitochondrial apoptotic pathway.

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.