Amphiregulin and ocular axial length.

PURPOSE: To assess the potential role of amphiregulin as messenger molecule in ocular axial elongation. METHODS: The experimental study included guinea pigs (total n = 78) (age: 3-4 weeks) which underwent bilateral lens-induced myopization and received 15 days later three intraocular injections in weekly intervals of amphiregulin antibody (doses:5 µg, 10 µg, 20 µg) into their right eyes, and three phosphate-buffered saline injections into their left eyes; and guinea pigs without lens-induced myopization and which received three unilateral intraocular injections of amphiregulin antibody (dose: 20 µg) or amphiregulin (doses: 1 ng; 10 ng; 20 ng) into their right eyes, and three phosphate-buffered saline injections into their left eyes. Seven days later, the animals were sacrificed. Intravitally, we performed biometry, and histology and immunohistochemistry post-mortem. RESULTS: In animals with bilateral lens-induced myopization, the right eyes receiving amphiregulin antibody showed reduced axial elongation in a dose-dependent manner (dose: 5 µg: side difference: 0.14 ± 0.05 mm;10 µg: 0.22 ± 0.06 mm; 20 µg: 0.32 ± 0.06 mm; p < 0.001), thicker sclera (all p < 0.05) and higher cell density in the retinal nuclear layers and retinal pigment epithelium (RPE) (all p < 0.05). In animals without lens-induced myopia, the right eyes with amphiregulin antibody application (20 µg) showed reduced axial elongation (p = 0.04), and the right eyes with amphiregulin injections experienced increased (p = 0.02) axial elongation in a dose-dependent manner (1 ng: 0.04 ± 0.06 mm; 10 ng: 0.10 ± 0.05 mm; 20 ng: 0.11 ± 0.06 mm). Eyes with lens-induced axial elongation as compared to eyes without lens-induced axial elongation revealed an increased visualization of amphiregulin upon immunohistochemistry and higher expression of mRNA of endogenous amphiregulin and epidermal growth factor receptor, in particular in the outer part of the retinal inner nuclear layer and in the RPE. CONCLUSION: Amphiregulin may be associated with axial elongation in young guinea pigs.

Elaidic acid induces cell apoptosis through induction of ROS accumulation and endoplasmic reticulum stress in SH­SY5Y cells.

Elaidic acid, which is a major trans fatty acid, has been reported to be involved in neurotoxicity; however, the underlying molecular mechanisms underlying its neurotoxic effects remain largely unknown. Therefore, the present study aimed to investigate the potential mechanisms underlying elaidic acid­induced neuronal damage in vitro. The SH­SY5Y neuroblastoma cell line was used as a model in the present study. Following treatment of cells with various concentrations of elaidic acid or with vehicle for 24 h, cell viability was measured using the MTT assay. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) release were measured using flow cytometry. Cell apoptosis was measured by Annexin V­fluorescein isothiocyanate/propidium iodide double staining, and cellular redox status was determined using ELISA analysis. Furthermore, western blotting was used to detect the protein expression levels of factors associated with oxidative damage and components of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) signaling pathways. The results demonstrated that elaidic acid treatment inhibited cell viability, elevated cell apoptosis and resulted in a loss of MMP. In addition, elaidic acid induced marked alterations in cellular redox status. Treatment with high doses of elaidic acid treatment also enhanced the release of ROS, and upregulated lipid peroxide and malondialdehyde levels; however, it reduced superoxide dismutase and glutathione peroxidase activities. Furthermore, elaidic acid resulted in upregulation of nuclear factor erythroid 2­related factor 2 and downregulation of heme oxygenase 1, which are two key antioxidative factors. Elaidic acid treatment also induced or inhibited the expression of numerous ER stress/UPR­associated molecules. It induced glucose­regulated protein 78 (GRP78) expression, whereas the expression levels of activating transcription factor 4 (ATF4) and CCAAT/enhancer­binding protein homologous protein (CHOP) were upregulated and then downregulated following treatment with various doses of elaidic acid. These results indicated that elaidic acid inhibited SH­SY5Y cell growth and induced apoptosis by enhancing oxidative stress and activating the ER stress/UPR signaling pathway and the GRP78/ATF4/CHOP pathway.

Beta amyloid peptide (25-35) leading to inflammation through Toll-like receptors and the anti-inflammatory effect of genistein in BV-2 cells.

Genistein, the main soy isoflavone component, has received much attention for its potential multifunction. Here, we reported that in BV-2 cells, genistein significantly inhibited beta amyloid peptides 25-35 (Aß25-35)-induced inflammatory response. The results indicated that Aß25-35-stimulated BV-2 cells upregulated Toll-like receptors 2 and 4, Myd88, and IKK gene expression with the increasing expression of IL-6 and decreasing expression of TGF-ß and IL-10. Further, inhibiting TLR4 expression with small interfering RNA prevented the inflammatory response induced by Aß25-35, indicating the key role of TLRs in Aß-mediated inflammation. Genistein pre-treated BV-2 cells showed less inflammatory response when exposed to Aß25-35. These results suggested that Aß induced BV-2 cells inflammation though TLRs and genistein has an anti-inflammatory effect in vitro.

Soy isoflavone alleviates Aß1-42-induced impairment of learning and memory ability through the regulation of RAGE/LRP-1 in neuronal and vascular tissue.

The neuroprotective properties of soy isoflavone (SIF) have been demonstrated by our previous studies and others, but its potential mechanism is not clear. Because of the key role of neurovascular dysfunction in the pathogenesis of Alzheimer's disease (AD), we hypothesized neurovascular tissue might be one neuroprotective target of SIF. In the present study, learning and memory ability, ß-amyloid (Aß) expressions both in neurovascular tissue and plasma, the receptor for advanced glycation end products (RAGE), low-density lipoprotein receptor-related protein (LRP)-1, nuclear factor-κB p65 (NF-κB p65), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) expressions in neurovascular tissue were measured in Wistar rats following lateral cerebral ventricle administration of Aß1-42 by miniosmotic pump with or without intragastric administration of SIF from 14 days before surgery to the end of experiment. The results showed that SIF could improve the impairment of learning and memory of rats induced by Aß1-42, maintain Aß homeostasis in brain, regulate the disordered expressions of RAGE/LRP-1 and restrain RAGE related NF-κB and inflammatory cytokines activation in neurovascular structure. These results suggested that SIF could protect Aß-impaired learning and memory in rats, and its mechanism might be associated with the regulation of vascular Aß transportation and vascular inflammatory reaction.

Soy isoflavone attenuates brain mitochondrial oxidative stress induced by ß-amyloid peptides 1-42 injection in lateral cerebral ventricle.

The aim of this study is to investigate whether soy isoflavone (SIF) reduces oxidative stress and improves the antioxidant ability in mitochondria of rat brain damaged by injection of beta-amyloid peptides 1-42 (Aß1-42). Forty Wistar rats were randomly divided into control, Aß1-42, SIF + Aß1-42, and SIF groups according to body weight. The rats in the SIF + Aß1-42 group and SIF group were intragastrically administered SIF suspension in 0.5% CMC-Na for 28 days, whereas the rats in control group and Aß1-42 group were administered the same volume of 0.5% CMC-Na. On day 14, the rats in the Aß1-42 group and SIF + Aß1-42 group were injected with Aß1-42 into the lateral cerebral ventricle with physiological saline. The rat brains were then sampled, and brain mitochondria were isolated. After this, the mitochondrial membrane potential (MMP) and mitochondrial redox state were measured. The contents of brain nuclear factor E2-related factor (Nrf2) and heme oxygenase-1 (HO-1) protein in brain tissue were quantitated by Western blot. The results showed that SIF maintained the MMP, elevated the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio, and increased glutathione peroxidase (GPx) and manganese superoxide dismutase (MnSOD) protein expression in brain mitochondria. Additionally, SIF reversed the Aß1-42-induced downregulation of the protein expression of Nrf2 and HO-1 in brain tissue. These results indicated that SIF could alleviate the oxidative damage and maintain the redox imbalance in brain mitochondria damaged by Aß1-42. This might result from regulation of the Nrf2/HO-1 pathway.

The role of glutathione S-transferase M1 and T1 gene polymorphisms and fruit and vegetable consumption in antioxidant parameters in healthy subjects.

The correlation of glutathione S-transferase (GST) M1/T1 genetic polymorphisms with oxidative stress-related chronic diseases was proved recently. The aim of the present study was to investigate the association of GSTM1/T1 genetic polymorphisms with antioxidant biomarkers and consumption of fruits and vegetables (F&V) in healthy subjects. In this study, for conducting a 3 d dietary survey, 190 healthy adults were recruited. After DNA extraction, a multiple PCR method was used for GSTM1/T1 genotyping. A spectrophotometer method was applied for the determination of plasma total antioxidant capacity (T-AOC), vitamin C level and erythrocyte GST enzyme activity. A general linear model was used to compare the mean values of antioxidant parameters for different GSTM1/T1 genotypes and consumption of F&V. Polymorphisms of GSTM1/T1 had no effects on plasma T-AOC and vitamin C levels. Deletion of the GSTM1 gene decreased the erythrocyte GST activity. There was correlation between plasma T-AOC and consumption of F&V in the GSTM1⁻ or GSTT1⁺ subjects. A similar pattern was evident for erythrocyte GST activity in the GSTM1⁻ subjects. No association was found among consumption of F&V and GSTM1/T1 genotypes and plasma vitamin C level. Different consumption of F&V had no impact on plasma T-AOC and vitamin C levels in the GSTM1⁻/GSTT1⁺ or GSTM1⁻/GSTT1⁻ subjects. The erythrocyte GST activity was more sensitive to consumption of F&V in the individuals with the GSTM1⁻/GSTT1⁺ genotype. Association was found among GSTM1/T1 genotypes, antioxidant parameters and consumption of F&V. Large-scale and multiple ethnic studies are needed to further evaluate the relationship.

Genistein inhibits mitochondrial-targeted oxidative damage induced by beta-amyloid peptide 25-35 in PC12 cells.

The antioxidative properties of genistein (Gen) have been demonstrated by our previous studies and others, but its potential mechanism was not very clear. Because of the key role of mitochondria in oxidant production, we wondered if mitochondria were one of Gen's neuroprotective targets. In the present study we investigated whether Gen has protective effects on mitochondria damaged by Aß25-35. PC12 cells were pre-incubated with or without Gen for 2 h followed by the incubation with 20 µM Aß25-35 for another 24 h before mitochondrial membrane fluidity (MMF), mitochondrial membrane potential (MMP) , and mitochondrial redox state were measured. The results showed that Gen alleviated the decrease of MMF induced by Aß25-35, and maintained the MMP. Additionally, Gen promoted the mitochondrial antioxidative capability through increasing the GSH/GSSG ratio, GPx activity and MnSOD protein expression in mitochondria. Moreover, Gen reversed the changes of ChAT mRNA and AChE mRNA expression in cells induced by Aß25-35. These results suggested that Gen can protect the mitochondrial membrane and maintain redox state in mitochondria damaged by Aß25-35.

A peptide conjugate of vitamin E succinate targets breast cancer cells with high ErbB2 expression.

Overexpression of erbB2 is associated with resistance to apoptosis. We explored whether high level of erbB2 expression by cancer cells allows their targeting using an erbB2-binding peptide (LTVSPWY) attached to the proapoptotic alpha-tocopheryl succinate (alpha-TOS). Treating erbB2-low or erbB2-high cells with alpha-TOS induced similar levels of apoptosis, whereas alpha-TOS-LTVSPWY induced greater levels of apoptosis in erbB2-high cells. alpha-TOS rapidly accumulated in erbB2-high cells exposed to alpha-TOS-LTVSPWY. The extent of apoptosis induced in erbB2-high cells by alpha-TOS-LTVSPWY was suppressed by erbB2 RNA interference as well as by inhibition of either endocytotic or lysosomal function. alpha-TOS-LTVSPWY reduced erbB2-high breast carcinomas in FVB/N c-neu transgenic mice. We conclude that a conjugate of a peptide targeting alpha-TOS to erbB2-overexpressing cancer cells induces rapid apoptosis and efficiently suppresses erbB2-positive breast tumors.

Inhibitory effects of apigenin on the growth of gastric carcinoma SGC-7901 cells.

AIM: To explore the growth inhibition and apoptosis-inducing effect of apigenin on human gastric carcinoma SGC-7901 cells. METHODS: The effects of apigenin on the growth, clone formation and proliferation of human gastric carcinoma SGC-7901 cells were observed by MTT, clone-forming assay, and morphological observation. Fluorescent staining and flow cytometry analysis were used to detect apoptosis of cells. RESULTS: Apigenin obviously inhibited the growth, clone formation and proliferation of SGC-7901 cells in a dose-dependent manner. Inhibition of growth was observed on d 1 at the concentration of 80 micromol/L, while after 4 d, the inhibition rate (IR) was 90%. The growth IRs at the concentration of 20, 40, and 80 micromol/L were 38%, 71%, and 99% respectively on the 7th d. After the cells were treated with apigenin for 48 h, the number of clone-forming in control, 20, 40, and 80 micromol/L groups was 217+/-16.9, 170+/-11.1 (P < 0.05), 98+/-11.1 (P < 0.05), and 25+/-3.5 (P < 0.05) respectively. Typical morphological changes of apoptosis was found by fluorescent staining. The cell nuclei had lost its smooth boundaries, chromatin was condensed, and cell nuclei were broken. Flow cytometry detected typical apoptosis peak. After the cells were treated with apigenin for 48 h, the apoptosis rates were 5.76%, 19.17%, and 29.30% respectively in 20, 40, and 80 micromol/L groups. CONCLUSION: Apigenin shows obvious inhibition on the growth and clone formation of SGC-7901 cells by inducing apoptosis.