Mi-Zhen Xia

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.

Folic Acid Protects against Lipopolysaccharide-Induced Preterm Delivery and Intrauterine Growth Restriction through Its Anti-Inflammatory Effect in Mice

Increasing evidence demonstrates that maternal folic acid (FA) supplementation during pregnancy reduces the risk of neural tube defects, but whether FA prevents preterm delivery and intrauterine growth restriction (IUGR) remains obscure. Previous studies showed that maternal lipopolysaccharide (LPS) exposure induces preterm delivery, fetal death and IUGR in rodent animals. The aim of this study was to investigate the effects of FA on LPS-induced preterm delivery, fetal death and IUGR in mice. Some pregnant mice were orally administered with FA (0.6, 3 or 15 mg/kg) 1 h before LPS injection. As expected, a high dose of LPS (300 μg/kg, i.p.) on gestational day 15 (GD15) caused 100% of dams to deliver before GD18 and 89.3% of fetuses dead. A low dose of LPS (75 μg/kg, i.p.) daily from GD15 to GD17 resulted in IUGR. Interestingly, pretreatment with FA prevented LPS-induced preterm delivery and fetal death. In addition, FA significantly attenuated LPS-induced IUGR. Further experiments showed that FA inhibited LPS-induced activation of nuclear factor kappa B (NF-κB) in mouse placentas. Moreover, FA suppressed LPS-induced NF-κB activation in human trophoblast cell line JEG-3. Correspondingly, FA significantly attenuated LPS-induced upregulation of cyclooxygenase (COX)-2 in mouse placentas. In addition, FA significantly reduced the levels of interleukin (IL)-6 and keratinocyte-derived cytokine (KC) in amniotic fluid of LPS-treated mice. Collectively, maternal FA supplementation during pregnancy protects against LPS-induced preterm delivery, fetal death and IUGR through its anti-inflammatory effects.

Vitamin D3 pretreatment alleviates renal oxidative stress in lipopolysaccharide-induced acute kidney injury.

Increasing evidence demonstrates that reactive oxygen species plays important roles in sepsis-induced acute kidney injury. This study investigated the effects of VitD3 pretreatment on renal oxidative stress in sepsis-induced acute kidney injury. Mice were intraperitoneally injected with lipopolysaccharide (LPS, 2.0mg/kg) to establish an animal model of sepsis-induced acute kidney injury. In VitD3+LPS group, mice were orally pretreated with three doses of VitD3 (25 μg/kg) at 1, 24 and 48 h before LPS injection. As expected, oral pretreatment with three daily recommended doses of VitD3 markedly elevated serum 25(OH)D concentration and efficiently activated renal VDR signaling. Interestingly, LPS-induced renal GSH depletion and lipid peroxidation were markedly alleviated in VitD3-pretreated mice. LPS-induced serum and renal nitric oxide (NO) production was obviously suppressed by VitD3 pretreatment. In addition, LPS-induced renal protein nitration, as determined by 3-nitrotyrosine residue, was obviously attenuated by VitD3 pretreatment. Further analysis showed that LPS-induced up-regulation of renal inducible nitric oxide synthase (inos) was repressed in VitD3-pretreated mice. LPS-induced up-regulation of renal p47phox and gp91phox, two NADPH oxidase subunits, were normalized by VitD3 pretreatment. In addition, LPS-induced down-regulation of renal superoxide dismutase (sod) 1 and sod2, two antioxidant enzyme genes, was reversed in VitD3-pretreated mice. Finally, LPS-induced tubular epithelial cell apoptosis, as determined by TUNEL, was alleviated by VitD3 pretreatment. Taken together, these results suggest that VitD3 pretreatment alleviates LPS-induced renal oxidative stress through regulating oxidant and antioxidant enzyme genes.

Vitamin D Deficiency Attenuates High-Fat Diet-Induced Hyperinsulinemia and Hepatic Lipid Accumulation in Male Mice

It is increasingly recognized that vitamin D deficiency is associated with increased risks of metabolic disorders among overweight children. A recent study showed that vitamin D deficiency exacerbated inflammation in nonalcoholic fatty liver disease through activating toll-like receptor 4 in a high-fat diet (HFD) rat model. The present study aimed to further investigate the effects of vitamin D deficiency on HFD-induced insulin resistance and hepatic lipid accumulation. Male ICR mice (35 d old) were randomly assigned into 4 groups as follows. In control diet and vitamin D deficiency diet (VDD) groups, mice were fed with purified diets. In HFD and VDD+HFD groups, mice were fed with HFD. In VDD and VDD+HFD groups, vitamin D in feed was depleted. Feeding mice with vitamin D deficiency diet did not induce obesity, insulin resistance, and hepatic lipid accumulation. By contrary, vitamin D deficiency markedly alleviated HFD-induced overweight, hyperinsulinemia, and hepatic lipid accumulation. Moreover, vitamin D deficiency significantly attenuated HFD-induced up-regulation of hepatic peroxisome proliferator-activated receptor γ, which promoted hepatic lipid uptake and lipid droplet formation, and its target gene cluster of differentiation 36. In addition, vitamin D deficiency up-regulated carnitine palmitoyltrans 2, the key enzyme for fatty acid β-oxidation, and uncoupling protein 3, which separated oxidative phosphorylation from ATP production, in adipose tissue. These data suggest that vitamin D deficiency is not a direct risk factor for obesity, insulin resistance, and hepatic lipid accumulation. Vitamin D deficiency alleviates HFD-induced overweight, hyperinsulinemia, and hepatic lipid accumulation through promoting fatty acid β-oxidation and elevating energy expenditure in adipose tissue.

Supplementation With Vitamin D3 During Pregnancy Protects Against Lipopolysaccharide-Induced Neural Tube Defects Through Improving Placental Folate Transportation

Several reports demonstrated that maternal lipopolysaccharide (LPS) exposure at middle gestational stage caused neural tube defects (NTDs). This study investigated the effects of supplementation with vitamin D3 (VitD3) during pregnancy on LPS-induced NTDs. Pregnant mice except controls were ip injected with LPS (25 μg/kg) daily from gestational day (GD)8 to GD12. In LPS+VitD3 group, pregnant mice were orally administered with VitD3 (25 μg/kg) before LPS injection. As expected, a 5-day LPS injection resulted in 62.5% (10/16) of dams and 20.3% of fetuses with NTDs. Additional experiment showed that a 5-day LPS injection downregulated placental proton-coupled folate transporter (pcft) and reduced folate carrier 1 (rfc1), 2 major folate transporters in placentas. Consistent with downregulation of placental folate transporters, folate transport from maternal circulation into embryos was disturbed in LPS-treated mice. Interestingly, VitD3 not only inhibited placental inflammation but also attenuated LPS-induced downregulation of placental folate transporters. Correspondingly, VitD3 markedly improved folate transport from maternal circulation into the embryos. Importantly, supplementation with VitD3 during pregnancy protected mice from LPS-induced NTDs. Taken together, these results suggest that supplementation with VitD3 during pregnancy prevents LPS-induced NTDs through inhibiting placental inflammation and improving folate transport from maternal circulation into the embryos.

Cadmium Selectively Induces MIP-2 and COX-2 Through PTEN-Mediated Akt Activation in RAW264.7 Cells

Increasing evidence demonstrates that cadmium (Cd) induces inflammation, but its mechanisms remain obscure. The present study showed that treatment with CdCl₂ selectively upregulates macrophage inflammatory protein (MIP)-2 and cyclooxygenase (COX)-2 in RAW264.7 cells. Concomitantly, Cd²⁺ markedly elevated the level of phosphorylated Akt in dose- and time-dependent manners. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), blocked Cd²⁺-evoked Akt phosphorylation. Correspondingly, LY294002 significantly repressed Cd²⁺-induced upregulation of MIP-2 and COX-2 in RAW264.7 cells. Further experiments showed that treatment with Cd²⁺ significantly reduced the level of PTEN protein in RAW264.7 cells. MG132, a specific proteasome inhibitor, blocked Cd²⁺-induced reduction in PTEN protein as well as Akt phosphorylation, implicating the involvement of proteasome-mediated PTEN degradation. Of interest, Cd²⁺-induced degradation of PTEN protein appears to be associated with PTEN ubiquitination. N-acetylcysteine, a glutathione (GSH) precursor, blocked Cd²⁺-evoked PTEN degradation as well as Akt phosphorylation. By contrast, L-buthionine-S,R-sulfoximine, an inhibitor of cellular GSH synthesis, exacerbated Cd²⁺-induced PTEN degradation and Akt phosphorylation. Alpha-phenyl-N-tert-butylnitrone and vitamin C, two antioxidants, did not prevent from Cd²⁺-induced PTEN degradation and Akt phosphorylation. In conclusion, Cd²⁺ selectively induces MIP-2 and COX-2 through PTEN-mediated PI3K/Akt activation. Cellular GSH depletion mediates Cd²⁺-induced PTEN degradation and subsequent PI3K/Akt activation in macrophages.

Low vitamin D status is associated with inflammation in patients with prostate cancer

Vitamin D deficiency has been associated with increased risks of prostate cancer. Nevertheless, the mechanisms remain unclear. The aim of this study was to analyze the association among prostate cancer, vitamin D status and inflammation. Sixty patients with newly diagnosed prostate cancer and 120 age-matched controls were recruited for this study. Vitamin D status was evaluated and serum inflammatory molecules were measured. Serum 25-(OH)D was lower in patients with prostate cancer. Moreover, serum 25(OH)D was lower in patients with severe prostate cancer than patients with mild and moderate prostate cancer. By contrast, serum C-reactive protein (CRP) and interleukin (IL)-8, two inflammatory molecules, were elevated in patients with prostate cancer. Serum 25-(OH)D was negatively correlated with serum CRP and IL-8 in patients with prostate cancer. Additional analysis showed that the percentage of vitamin D receptor positive nucleus in the prostate was reduced in patients with prostate cancer. By contrast, the percentage of nuclear factor kappa B p65-positive nucleus was elevated in patients with prostate cancer. Our results provide evidence that there is an association among prostate cancer, vitamin D deficiency and inflammatory signaling. Inflammation may be an important mediator for prostate cancer progression in patients with low vitamin D status.

Orally administered melatonin prevents lipopolysaccharide-induced neural tube defects in mice.

Lipopolysaccharide (LPS) has been associated with adverse pregnant outcomes, including fetal demise, intra-uterine growth restriction (IUGR), neural tube defects (NTDs) and preterm delivery in rodent animals. Previous studies demonstrated that melatonin protected against LPS-induced fetal demise, IUGR and preterm delivery. The aim of the present study was to investigate the effects of melatonin on LPS-induced NTDs. All pregnant mice except controls were intraperitoneally injected with LPS (25 µg/kg) daily from gestational day (GD)8 to GD12. Some pregnant mice were orally administered with melatonin (MT, 50 mg/kg) before each LPS injection. A five-day LPS injection resulted in 27.5% of fetuses with anencephaly, exencephaly or encephalomeningocele. Additional experiment showed that maternal LPS exposure significantly down-regulated placental proton-coupled folate transporter (pcft) and disturbed folate transport from maternal circulation through the placentas into the fetus. Interestingly, melatonin significantly attenuated LPS-induced down-regulation of placental pcft. Moreover, melatonin markedly improved the transport of folate from maternal circulation through the placentas into the fetus. Correspondingly, orally administered melatonin reduced the incidence of LPS-induced anencephaly, exencephaly or encephalomeningocele. Taken together, these results suggest that orally administered melatonin prevents LPS-induced NTDs through alleviating LPS-induced disturbance of folate transport from maternal circulation through the placenta into the fetus.

N-acetylcysteine alleviates cadmium-induced placental endoplasmic reticulum stress and fetal growth restriction in mice

Cadmium (Cd) is a developmental toxicant that induces fetal growth restriction (FGR). Placental endoplasmic reticulum (ER) stress is associated with FGR. This study investigated the effects of N-acetylcysteine (NAC) on Cd-induced placental ER stress and FGR. Pregnant mice were intraperitoneally injected with CdCl2 daily from gestational day (GD)13 to GD17. As expected, Cd reduced fetal weight and crown-rump length. Cd decreased the internal space of blood vessels in the placental labyrinth layer and inhibited placental cell proliferation. Several genes of growth factors, such as Vegf-a, placental growth factor, Igf1 and Igf1r, and several genes of nutrient transport pumps, such as Glut1, Fatp1 and Snat2, were down-regulated in placenta of Cd-treated mice. Moreover, Cd evoked placental ER stress. Of interest, NAC alleviated Cd-induced FGR. Additional experiment showed that NAC inhibited Cd-induced impairment of placental development and placental ER stress. Therefore, NAC may be exploited for prevention of Cd-induced placental insufficiency and FGR.

Vitamin D3 pretreatment protects against lipopolysaccharide-induced early embryo loss through its anti-inflammatory effects.

Increasing evidence demonstrates that inflammatory cytokines are involved in LPS‐induced adverse pregnant outcomes including early embryo loss. Vitamin D3 (VitD3) has anti‐inflammatory activity. We aimed to investigate the effects of vitamin D3 (VitD3) on LPS‐induced early embryo loss in mice.