Purpose. To investigate whether hyperosmolar stress stimulates production of inflammatory mediators and activates the mitogen-activated protein kinase (MAPK) signaling pathways, c-jun n-terminal kinases (JNKs), extracellular-regulated kinases (ERKs), and p38 on the mouse ocular surface. Methods. 129SvEv/CD-1 mixed mice were treated with a balanced salt solution (BSS) (305 mOsM) or a hyperosmotic saline solution (HOSS) (500 mOsM). Untreated age-matched mice were used as controls. The concentrations of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were measured by enzyme-linked immunosorbent assay. Gelatinase activity was determined by in situ zymography. Corneal and conjunctival epithelia were lysed for Western blot with MAPK antibodies or used for semiquantitative reverse transcription and polymerase chain reaction and gene array. Results. Compared with age-matched controls and mice treated with BSS, the concentration of IL-1β in tear fluid washings and the concentrations of IL-1β and TNF-α and gelatinolytic activity in the corneal and conjunctival epithelia were significantly increased in mice treated with HOSS for 2 days. The expressions of IL-1β, TNF-α, and matrix metalloproteinase 9 (MMP-9) messenger RNA by the corneal and conjunctival epithelia were also notably stimulated in mice treated with HOSS. The levels of phosphorylated JNK1/2, ERK1/2, and p38 MAPKs in the corneal and conjunctival epithelia were slightly increased in mice treated with BSS, but markedly increased in mice treated with HOSS. Conclusions. These results show that the hyperosmolarity stimulates expression and production of IL-1β, TNF-α, and MMP-9 and activates JNK, ERK, and p38 MAPK signaling pathways on the mouse ocular surface. These findings suggest that hyperosmolar stress, as it may occur in dry eye, promotes ocular surface inflammation.
To investigate the role of interferon (IFN)-γ in dry eye-associated conjunctival apoptosis.Desiccating stress (DS) was created in C57BL/6 (B6) and C57BL/6 IFN-γ-knockout (B6γKO) mice. A separate group of mice of both strains also received subconjunctival injections of exogenous IFN-γ or vehicle control (BSA) at days 0, +2, and +4 after DS. Immunoreactivity to active (Ac)-caspase-3, -8, and -9 and terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) were evaluated in cryosections. Goblet cell apoptosis was assessed by MUC5AC and TUNEL double staining. Levels of caspase-3, -8, -9, Fas, and Fas-associated protein with Death Domain (FADD) mRNA in conjunctiva were measured by real-time PCR. The activity of caspase-3, -8, or -9 was measured using fluorometric assay.Increased Ac-caspase-3 and -8 and TUNEL immunoreactivity were noted in conjunctival epithelia in B6 mice compared with B6γKO mice after DS, and exogenous IFN-γ administration further increased these parameters. DS-induced conjunctival apoptosis was greatest in the goblet cell area and was accompanied by a decrease in MUC5AC expression in the B6 and B6-IFN-γ-injected groups compared with the B6γKO and B6-BSA-injected groups. B6γKO mice were resistant to DS-induced apoptosis; however, B6γKO receiving IFN-γ yielded results similar to those for B6 wild-type. Caspase-9 production and activity were not increased with DS in B6 or B6γKO mice; however, the administration of IFN-γ significantly increased caspase-9 production and activity in both strains compared with vehicle-injected mice.IFN-γ plays a pivotal role in exacerbating conjunctival apoptosis through dual apoptotic pathways with DS.
we conducted a case-control study to investigate the association between dietary folate, vitamin B6 and vitamin B12 intake, MTHFR and MTR genotype, and breast cancer risk.Genotyping for MTHFR C677T and A1298C and MTR A2756G polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) method. The intake of folate, vitamin B6 and vitamin B12 were calculated by each food item from questionnaire.Subjects with breast cancer tended to have more first-degree relatives (χ(2) =30.77, P<0.001) and have high intake of folate (t=2.42, P=0.008) and Vitamin B6 (t=2.94, P=0.002). Compared to the reference group, women with MTHFR 677 TT genotype and T allele had a significantly increased risk of breast cancer, with ORs (95%CI) of 1.8(1.08-2.27) and 1.39(1.02-1.92), respectively. For those who had folate intake＜450 ug/day, MTHFR 667TT genotype was associated with a higher risk of breast cancer (OR=2.45, 95% CI=1.09-5.82, P=0.02). Similarly, subjects with Vitamin B6 intake＜0.84 mg/day and MTHFR 667T allele genotype was correlated with a marginally increased risk of breast cancer. A significant interaction was observed between MTHFR C667T polymorphism and folate intake on the risk of breast cancer (P for interaction was 0.025).This case-control study found a significant association between MTHFR C667T polymorphism, folate intake and vitamin B6 and breast cancer risk, and a significant interaction was observed between MTHFR C667T polymorphism and folate intake on the risk of breast cancer.
Purpose: L-carnitine suppresses inflammatory responses in human corneal epithelial cells (HCECs) exposed to hyperosmotic stress. In this study, we determined if L-carnitine induces this protective effect through suppression of reactive oxygen species (ROS)-induced oxidative damage in HCECs. Methods: Primary HCECs were established from donor limbal explants. A hyperosmolarity dry-eye model was used in which HCECs are cultured in 450 mOsM medium with or without L-carnitine for up to 48 hours. Production of reactive oxygen species (ROS), oxidative damage markers, oxygenases and antioxidative enzymes were analyzed by 2′,7′-dichlorofluorescein diacetate (DCFDA) kit, semiquantitative PCR, immunofluorescence, and/or Western blotting. Results: Reactive oxygen species production increased in HCECs upon substitution of the isotonic medium with the hypertonic medium. L-carnitine supplementation partially suppressed this response. Hyperosmolarity increased cytotoxic membrane lipid peroxidation levels; namely, malondialdehyde (MDA) and hydroxynonenal (HNE), as well as mitochondria DNA release along with an increase in 8-OHdG and aconitase-2. Interestingly, these oxidative markers were significantly decreased by coculture with L-carnitine. Hyperosmotic stress also increased the mRNA expression and/or protein production of heme oxygenase-1 (HMOX1) and cyclooxygenase-2 (COX2), but inhibited the levels of antioxidant enzymes, superoxide dismutase-1 (SOD1), glutathione peroxidase-1 (GPX1), and peroxiredoxin-4 (PRDX4). However, L-carnitine partially reversed this altered imbalance between oxygenases and antioxidant enzymes induced by hyperosmolarity. Conclusions: Our findings demonstrate for the first time that L-carnitine protects HCECs from oxidative stress by lessening the declines in antioxidant enzymes and suppressing ROS production. Such suppression reduces membrane lipid oxidative damage markers and mitochondrial DNA damage.
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