Search

Ferulic acid plays a neuroprotective role in cerebral ischemia. The aim of this study was to identify the proteins that are differentially expressed following ferulic acid treatment during ischemic brain injury using a proteomics technique. Middle cerebral artery occlusion (MCAO) was performed to induce a focal cerebral ischemic injury in adult male rats, and ferulic acid (100 mg/kg) or vehicle was administered immediately after MCAO. Brain tissues were collected 24 hr after MCAO. The proteins in the cerebral cortex were separated using two-dimensional gel electrophoresis and were identified by mass spectrometry. We detected differentially expressed proteins between vehicle- and ferulic acid-treated animals. Adenosylhomocysteinase, isocitrate dehydrogenase [NAD(+)], mitogen-activated protein kinase kinase 1 and glyceraldehyde-3-phosphate dehydrogenase were decreased in the vehicle-treated group, and ferulic acid prevented the injury-induced decreases in these proteins. However, pyridoxal phosphate phosphatase and heat shock protein 60 were increased in the vehicle-treated group, while ferulic acid prevented the injury-induced increase in these proteins. It is accepted that these enzymes are involved in cellular metabolism and differentiation. Thus, these findings suggest evidence that ferulic acid plays a neuroprotective role against focal cerebral ischemia through the up- and down-modulation of specific enzymes.

Background Obesity is a health hazard that is associated with a number of diseases and metabolic abnormalities, such as type-2 diabetes, hypertension, dyslipidemia, and coronary heart disease. In the current study, we investigated the effects of Citrus aurantium flavonoids (CAF) on the inhibition of adipogenesis and adipocyte differentiation in 3T3-L1 cells. Methods During adipocyte differentiation, 3T3-L1 cells were treated with 0, 10, and 50 μg/ml CAF, and then the mRNA and protein expression of adipogenesis-related genes was assayed. We examined the effect of CAF on level of phosphorylated Akt in 3T3-L1 cells treated with CAF at various concentrations during adipocyte differentiation. Results The insulin-induced expression of C/EBPβ and PPARγ mRNA and protein were significantly down-regulated in a dose-dependent manner following CAF treatment. CAF also dramatically decreased the expression of C/EBPα, which is essential for the acquisition of insulin sensitivity by adipocytes. Moreover, the expression of the aP2 and FAS genes, which are involved in lipid metabolism, decreased dramatically upon treatment with CAF. Interestingly, CAF diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3β (Ser9), which may reduce glucose uptake in response to insulin and lipid accumulation. Furthermore, CAF not only inhibited triglyceride accumulation during adipogenesis but also contributed to the lipolysis of adipocytes. Conclusions In the present study, we demonstrate that CAF suppressed adipogenesis in 3T3-L1 adipocytes. Our results indicated that CAF down-regulates the expression of C/EBPβ and subsequently inhibits the activation of PPARγ and C/EBPα. The anti-adipogenic activity of CAF was mediated by the inhibition of Akt activation and GSK3β phosphorylation, which induced the down-regulation of lipid accumulation and lipid metabolizing genes, ultimately inhibiting adipocyte differentiation.

To understand the tissue-specific expression of the rat placental lactogen-I variant (rPL-Iv) gene, we investigated the methylation pattern of the 5'-flanking region of this gene in various rat tissues. We report that the 5'-flanking region of the rPL-Iv gene was hypomethylated in placenta that expressed the gene and hypermethylated in those tissues that did not express the gene. Moreover, the intron region of the rPL-Iv gene was hypomethylated in the placenta, but hypermethylated in the liver, kidney and pituitary. Although there are 5 CpG sites and the density of CpG dinucleotide is lower within 2 kb of the rPL-Iv 5'-flanking region, the methylated promoter reporter gene produced strong repression in the transcriptional activity of the gene. In addition, the 5'-flanking and intron regions of the rPL-Iv gene were hypomethylated on day 12 of gestation, and the methylation pattern in the placenta remained unchanged from mid-pregnancy until term. The entire genomic region of the rPL-Iv gene might be hypermethylated in tissues other than the placenta, within which its methylated status repress expression of the placenta-specific rPL-Iv gene. Interestingly, the methylation status of the intron region of the rPL-Iv in proliferating Rcho-1 cells was changed to the unmethylated status on day 8 and 12 of differentiation of Rcho-1 cells. These results demonstrate that demethylation in the rPL-Iv upstream region was induced at an early stage of placental development, and once the 5'-flanking region of the rPL-Iv had been demethylated, its status on the rPL-Iv genomic region was continued during pregnancy. Taken together, these results suggest that DNA methylation is responsible for the silencing of tissue-specific genes in non-expressing cells, while defined combinations of trophoblast factors dictate the expression of unmethylated rPL-Iv gene in placenta trophoblast cells.

EGb 761 is a standardized extract of Gingko biloba that exerts protective effects against ischemic brain injury. This study investigated whether EGb 761 modulates the neuroprotective effects through Akt and its downstream targets, Bad and FKHR. Adult male rats were treated with EGb 761 (100 mg/kg) or vehicle prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 hours after MCAO and infarct volumes were analyzed. EGb 761 significantly reduced infarct volume. Potential activation was mearsured by phosphorylation of Akt at Ser473, Bad at Ser136, and FKHR at Ser256 using Western blot analysis. EGb 761 prevented the injury-induced decrease of pAkt and its down stream targets, pBad and pFKHR. Furthermore, EGb 761 prevented the injury-induced increase of cleaved caspase-3 levels. In conclusion, this study suggests that EGb 761 prevents cell death due to brain injury and that EGb 761 protection is affected by preventing the injury-induce decrease of Akt phosphorylation.

We identified a 3.4-kb 5′-flanking region of the rPL-I gene and examined its promoter activity using rat trophoblast Rcho-1 cells. A regulatory element between base pairs (bp) −2,487 and −2,310 in the 5′-flanking region was essential for maximum promoter activity of the rPL-I gene. This regulatory element was further characterized between bp −2,443 to −2,415 and −2,374 to −2,345. Electrophoretic mobility shift analysis showed that the interaction of nuclear extract proteins from differentiated Rcho-1 cells was inhibited by competition with a GATA-like sequence in the promoter, but not by a mutated GATA sequence. Moreover, the promoter activity of 2487 eLuc containing two novel GATA sites was significantly elevated by co-transfection of a GATA-2 expression vector in proliferating Rcho-1 cells. Our results demonstrate that GATA-2 is involved in multiple promoter regions to activate the specific expression of the rPL-I gene in placental tissue.

Coenzyme Q_{10} (CoQ_{10}) is a naturally occurring antioxidant and a prominent component of mitochondrial electron transport chain. In the present study, we investigated the effect of CoQ_{10} nanoparticle against photoaging using immunohistochemistry and Western blot analysis in the hairless mouse skin induced by ultraviolet B (UVB) irradiation (300 mJ/cm;{2}, 3 min/day for 21 days). In the UVB-irradiated distilled water (DW)-treated group, manganese superoxide dismutase (SOD2) and glutathione peroxidase (GPx) immunoreactivity and their protein levels in the skin were significantly lower than those in the control group. However, SOD2 and GPx immunoreactivity and their protein levels in the skin of the UVB-irradiated CoQ_{10}-treated group were higher than those in the UVB-irradiated DW-treated group. GPx activity in the skin in the UVB-irradiated DW-treated group significantly decreased compared to that in the control group; whereas GPx activity in the UVB-irradiated CoQ_{10}-treated group was similar to that in the control group. These results suggest that CoQ_{10} strongly inhibits oxidative stress in the skin induced by UVB via increasing SOD2 and GPx.

We previously showed that estradiol prevents neuronal cell death through the activation of Akt and its downstream targets Bad and FKHR. This study investigated whether estradiol modulates the survival pathway through other downstream targets of Akt, including mammalian target of rapamycin (mTOR) and p70S6 kinase. It is known that mTOR is a downstream target of Akt and a central regulator of protein synthesis, cell growth, and cell cycle progression. Adult female rats were ovariectomied and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24h after MCAO and infarct volumes were analyzed. We confirmed that estradiol significantly reduces infarct volume and decreases the number of positive cells for TUNEL staining in the cerebral cortex. Brain injury-induced a decrease in phospho-mTOR and phospho-p70S6 kinase. Estradiol prevented the injury-induced decrease in Akt activation and phosphorylation of mTOR and p70S6 kinases, and the subsequent decrease in S6 phosphorylation. Our findings suggest that estradiol plays a potent protective role against brain injury by preventing the injury-induced decrease of mTOR and p70S6 kinase phosphorylation.

Estradiol acts as a neuroprotective factor against brain injury. This study investigated whether estradiol modulates the Bcl-2 family proteins in ischemic brain injury. Adult female rats were ovariectomized and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 hr after MCAO, and infarct volumes were analyzed. Estradiol significantly reduces the infarct volume and decreases the positive cells of TUNEL staining in cerebral cortex. In ischemic cerebral cortex, the level of Bcl-2 was decreased, and the level of Bax was significantly increased. Estradiol prevents the injury-induced decrease of Bcl-2 and increase of Bax. In conclusion, our findings suggest that estradiol plays a potent protective role in brain injury through the regulation of Bcl-2 family proteins.

Estradiol prevents neuronal cell death through the inhibition of apoptotic signals and the activation of cell survival signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the activation of Akt and its downstream targets, including Bad, Bcl-x(L), and 14-3-3. Adult female rats were ovariectomied and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. We confirmed that estradiol significantly reduces infarct volume and decreases the positive cells of TUNEL staining in the cerebral cortex. Potential activation was measured by phosphorylation of Akt at Ser473 and Bad at Ser136 using Western blot analysis. Estradiol prevents the injury-induced decrease of pAkt, pBad, and Bcl-x(L). Further, in the presence of estradiol, the interaction of pBad and 14-3-3 increased, compared to that of oil-treated animals. Our findings suggest that estradiol prevents cell death due to brain injury and that Akt activation and Bad phosphorylation by estradiol mediated these protective effects.

Here, we report the presence of two splice variants of the human epithelial sodium channel alpha subunit (h alpha ENaC) containing Alu cassette, namely h alpha ENaC+22 and h alpha ENaC+Alu, in various tissues. Functional expression of these splice variants with hENaC beta and gamma subunits produced loss-of-channel activity in the Xenopus oocyte expression system. Interestingly, coexpression of h alpha ENaC+22 or h alpha ENaC+Alu, respectively, with wild type hENaC alpha, beta, and gamma subunits enhanced the expression of amiloride-sensitive current in oocytes. The presence of Alu sequences in the 3'-untranslated region of h gamma ENaC was also identified.

It is well known that increased cAMP levels in cultured astrocytes can convert flat polygonal shaped astrocytes into process-bearing, stellate astrocytes. In this study, we have examined the possible existence of astrocyte regional heterogeneity in morphological changes in response to cAMP stimulation. Primary astrocyte cultures were prepared from six different regions of neonatal rat brains, including cerebral cortex, hippocampus, brain stem, mid brain, cerebellum, and hypothalamus. After about 2 weeks in culture, the astrocyte culture medium was changed to DMEM containing various concentrations of 8-CPT-cAMP, a membrane permeable cAMP analog, for 2 h. We found that 250 microM 8-CPT-cAMP produced a maximum effect causing95% stellation in all regional astrocytes except hypothalamic astrocytes (56% stellation). At lower cAMP concentrations, cell stellation most effectively occurred in cerebellar astrocytes. To examine further the regional heterogeneity of astrocyte morphological changes, glutamate was added together with 8-CPT-cAMP to block cAMP-induced astrocyte stellation. Interestingly, glutamate blockage on cAMP-induced astrocyte stellation was brain region-specific in that cerebral and hippocampal astrocytes were effectively blocked by glutamate when compared to other regional astrocytes. Furthermore, glutamate inhibited isoproterenol-induced astrocyte stellation in a region-specific manner similarly as in cAMP-induced stellation. The present study demonstrates that astrocytes derived from different regions of the neonatal rat brain maintain different levels of morphological plasticity in culture.

Activity-dependent changes of conduction velocity (CV) and conduction block in single A(delta) fibers of primary afferent neurons were characterized in a rat model of neuropathy (NP). Injured dorsal root (DR) fiber in NP rats exhibited profoundly greater decreases of CV following impulse activity than did DR fiber in normal rats. Activity-dependent conduction block was absent up to 100 Hz of activity rate in DR fiber of NP rats, but was present above 25 Hz in normal rats. Profiles of activity dependence in sciatic fibers were similar in both NP and normal rats. These results suggest that nerve injury may alter activity-dependent hypoexcitability of A(delta) DR fibers. Furthermore, this excitability change may be responsible for the elevated pain perception in neuropathy.

Single unit responses of the primary somatosensory (SI) cortical neurons to the stimulation of the forepaw single digit were monitored in anesthetized rats before and after subcutaneous injection of lidocaine to an ipsilateral homologous receptive field (IHRF). Quantitative determination of the temporal changes of afferent sensory transmission was done by analyzing poststimulus time histograms of unit responses. Temporary deafferentation to the IHRF induced immediate, but reversible suppression of afferent sensory transmission in the SI cortex and this suppression lasts up to 35 min post-deafferentation period (during 10-15 min, -21.81 +/- 5.9%, P < 0.01). This result suggests that temporary absence of afferent inflow from the digit to the SI cortex may exert interhemispheric modulation of afferent sensory transmission in the opposite somatosensory cortex of anesthetized rats.

The effect of topical application of interleukin 2 (IL-2) on afferent sensory transmission to the neurones in the primary somatosensory (SI) cortex was determined quantitatively in anaesthetized rats. IL-2 (0.1, 1.0, 5.0 units) significantly suppressed afferent sensory transmission in SI cortical neurones (n = 19) in a dose-dependent manner. IL-2-induced suppression fully recovered by 60 min after drug. In control experiments, saline solution containing 0.2% bovine serum albumin, used as a vehicle, did not affect afferent sensory transmission. Our results suggest that IL-2 and its receptor present in the SI cortex may be involved in the processing of afferent sensory information.

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and was known to stimulate the release of growth factor in various cells. Recently, we reported the cellular localization of PACAP and its type I (PAC1) receptor in rat placenta during pregnancy. Placenta is a critical organ that synthesizes several growth factors and angiogenic factors for the fetal development and its own growth. However, there is little information regarding the cellular localization of PACAP and its receptor in human placenta at various gestations. The aim of the present study was to define the expression and distribution of PACAP and PAC1 receptor mRNAs in the human placenta during the pregnancy period. PACAP and PAC1 receptor mRNAs were expressed in stroma cells of stem villi and terminal villi. At the early stage, on 7 and 14 weeks, PACAP and PAC1 receptor genes were moderately expressed in stroma cells surrounding the blood vessels within stem villi. These genes were strongly expressed in stroma cells of stem villi and terminal villi on 24 and 38 weeks. The expression of these genes was increased as gestation advanced, and localized in the same areas. Localization of PACAP and PAC1 receptor demonstrate the evidence that PACAP may play an important role, as an autoregulator or pararegulator via its PAC1 receptor. In conclusion, our findings strongly suggest that PACAP may have a critical role in physiological function of the placenta for gestational maintenance and fetal growth.

Unlike most other mammals, human bodies do not have the ability to synthesize vitamin C inside of their own bodies. Therefore, humans must obtain vitamin C through daily diet. Gulo(-/-) mice strain is known with deficiency, in which vitamin C intake can be controlled by diet like human, and would be valuable for investigating the molecular mechanism of various diseases. In the present study, we established Gulo(-/-) mice model and investigated the differentially expressed proteins in stomach tissue of Gulo(-)(/-) mice after Helicobacter pylori-infected, and followed by DENA, using immunohistochemistry and proteomic approach. The results of immunohistochemistry analysis of stomach tissue showed that the tumor suppressor, p53 protein, expression was significantly decreased (p0.05) but not messenger RNA (mRNA) transcriptional level, and 14-3-3 ε, 14-3-3 δ, Ki-67 and cleaved caspase 3 expressions were significantly increased (p0.05) by H. Pylori infection, and followed by DENA treatment in Gulo(-/-) mice. Moreover, knockdown of 14-3-3 isoforms (14-3-3 ε, 14-3-3 σ, 14-3-3 ζ and 14-3-3 η) were significantly increased sub-G1 phase (characteristics of apoptosis) in AGS cells and, phenotypic changes like cell shrinkage, density and cleaved nuclei were also observed. Proteome analyses showed that 14-3-3 σ, 14-3-3 η, and tropomyosin alpha-1 chain were down-regulated, and Hspd1 protein and HSC70 were up-regulated after H. Pylori-infection, and followed by DENA. The combined results of immunohistochemistry and proteomic analysis suggest that H. pylori altered the p53 and 14-3-3 isoforms expression and DENA further enhanced the H. pylori effect, which might be involved in carcinogenesis and metastasis of gastric cancer on Gulo(-/-) mice.

Background Obesity is one of the greatest public health problems and major risk factors for serious metabolic diseases and significantly increases the risk of premature death. The aim of this study was to determine the inhibitory effects of Rubus crataegifolius Bunge (RCB) on adipocyte differentiation in 3 T3-L1 cells and its anti-obesity properties in high fat diet (HFD)-induced obese rats. Methods 3 T3-L1 adipocytes and HFD-induced obese rats were treated with RCB, and its effect on gene expression was analyzed using RT-PCR and Western blotting experiments. Results RCB treatment significantly inhibited adipocyte differentiation by suppressing the expression of C/EBPβ, C/EBPα, and PPARγ in the 3 T3-L1 adipocytes. Subsequently, the expression of the PPARγ target genes aP2 and fatty acid synthase (FAS) decreased following RCB treatment during adipocyte differentiation. In uncovering the specific mechanism that mediates the effects of RCB, we demonstrated that the insulin-stimulated phosphorylation of Akt strongly decreased and that its downstream substrate phospho-GSK3β was downregulated following RCB treatment in the 3 T3-L1 adipocytes. Moreover, LY294002, an inhibitor of Akt phosphorylation, exerted stronger inhibitory effects on RCB-mediated suppression of adipocyte differentiation, leading to the inhibition of adipocyte differentiation through the downregulation of Akt signaling. An HFD-induced obesity rat model was used to determine the inhibitory effects of RCB on obesity. Body weight gain and fat accumulation in adipose tissue were significantly reduced by the supplementation of RCB. Moreover, RCB treatment caused a significant decrease in adipocyte size, associated with a decrease in epididymal fat weight. The serum total cholesterol (TC) and triglyceride (TG) levels decreased in response to RCB treatment, whereas HDL cholesterol (HDL-C) increased, indicating that RCB attenuated lipid accumulation in adipose tissue in HFD-induced obese rats. Conclusion Our results demonstrate an inhibitory effect of RCB on adipogenesis through the reduction of the adipogenic factors PPARγ, C/EBPα, and phospho-Akt. RCB had a potent anti-obesity effect, reducing body weight gain in HFD-induced obese rats. Electronic supplementary material The online version of this article (doi:10.1186/s12986-016-0091-0) contains supplementary material, which is available to authorized users.

Background Vitamin C (ascorbic acid) is an essential nutrient of most living tissues that readily acts as a strong reducing agent, which is abundant in fruits and vegetables. Although, it inhibits cell growth in many human cancer cells in vitro, treatment in cancer is still controversial. Hence, the purpose of this study was to investigate the molecular mechanism of the inhibitory effect of vitamin C on AGS cell growth, and protein profiles in AGS cells after exposure to vitamin C treatment, by using proteomic tools. Results Vitamin C showed a cytotoxic effect on AGS cells (IC50 300 μg/mL) and, 20 differentially expressed proteins (spot intensities which show ≥2 fold change and statistically significant, p Conclusions Our proteome results suggest that the apoptosis related proteins were involved in promoting and regulating cell death of AGS cells, and might be helpful to understand the molecular mechanism of vitamin C on AGS cell growth inhibition.