Yue Gao

Ferulic acid protects lymphocytes from radiation-predisposed oxidative stress through extracellular regulated kinase

Purpose:u2003The objective of this study was to investigate the radioprotective effect of ferulic acid (FA) on irradiated lymphocytes and discover the possible mechanisms of protection. Materials and methods:u2003Lymphocytes were pretreated for 12u2009h with FA (0.001–0.1u2009μM) and then exposed to 3 Gy radiation. Cell apoptosis, intracellular reactive oxygen species (ROS), and signal pathway was analysed. Results:u2003Irradiation increased cell death, DNA fragmentation and intracellular ROS. Pretreatment with FA significantly reversed this tendency and attenuated the irradiation-induced ROS generation. Furthermore, several anti-apoptotic characteristics of FA were determined, including the ability to diminish cytosolic Ca2+ concentration, inhibit caspase-3 activation and cytochrome c translocation, upregulate B-cell lymphoma 2 (Bcl-2) and downregulate Bcl-2-associated X protein (Bax) in 3 Gy-irradiated lymphocytes. Signal pathway analysis showed FA decreased the activation of extracellular regulated kinase (ERK), which had been activated by radiation. Conclusion:u2003The results suggest that FA had a radioprotective effect through the ERK pathway to inhibit apoptosis and oxidation, and it may be an effective candidate for treating radiation diseases associated with oxidative stress.

β-sitosterol decreases irradiation-induced thymocyte early damage by regulation of the intracellular redox balance and maintenance of mitochondrial membrane stability†

Both radiation injury and oxidation toxicity occur when cells are exposed to ion irradiation (IR), ultimately leading to apoptosis. This study was designed to determine the effect of β‐sitosterol (BSS) on early cellular damage in irradiated thymocytes and a possible mechanism of effect on irradiation‐mediated activation of the apoptotic pathways. Thymocytes were irradiated (6 Gy) with or without BSS. Cell apoptosis and apoptosis‐related proteins were evaluated. BSS decreased irradiation‐induced cell death and nuclear DNA strand breaks while attenuating intracellular reactive oxygen species (ROS) and increasing the activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). BSS decreased the release of cytochrome c from mitochondria to the cytosol and the mitochondrio‐nuclear translocation of apoptosis‐inducing factor (AIF). Furthermore, BSS partially inhibited the radiation‐induced increase of cleaved caspase 3 and cleaved PARP, and attenuated the activation of JNK and AP‐1. In addition, evidence suggests that ROS generated by irradiation are involved in this course of cell damage. The results indicate that BSS confers a radioprotective effect on thymocytes by regulation of the intracellular redox balance which is carried out via the scavenging of ROS and maintenance of mitochondrial membrane stability. J. Cell. Biochem. 102: 748–758, 2007.

Ginsenoside Rg1 inhibits proliferation of vascular smooth muscle cells stimulated by tumor necrosis factor-α

AbstractAim:To investigate the proliferation of vascular smooth muscle cells (VSMC) affected by ginsenoside Rg1 and further explore the molecular mechanism of ginsenoside Rg1 using proteomics.Methods:The proliferation of VSMC was measured by MTS assay kit and flow cytometry. Proteomic alterations were analyzed using two-dimensional electrophoresis and peptide mass fingerprinting. Differential proteins found in proteomics were confirmed by RT-PCR.Results:The proliferation of VSMC was enhanced significantly after tumor necrosis factor-α (TNF-α) treatment, and ginsenoside Rg1 treatment inhibited proliferation in a dose-dependent manner. Proteomic analysis showed 24 protein spots were changed, including 17 spots that were increased and 7 spots that were decreased. Ginsenoside Rg1 could restore the expression levels of these proteins, at least partly, to basic levels of untreated cells. The expression of G-protein coupled receptor kinase, protein kinase C (PKC)-ζ, N-ras protein were decreased, while cycle related protein p21 was increased by ginsenoside Rg1 in TNF-α treated VSMC.Conclusion:PKC-ζ and p21 pathway might be the mechanism for inhibitory effects of ginsenoside Rg1 on proliferation of VSMC.

Effects of ferulic acid on hematopoietic cell recovery in whole-body gamma irradiated mice

Purpose:u2003The objective of this study was to investigate the mechanism for ferulic acid (FA)-induced radioprotection by evaluating the recovery of bone marrow cells and peripheral blood hematology. Materials and methods:u2003Balb/c mice were irradiated at a dose of 2.5 Gy using cobalt-60 gamma resources. Following irradiation, FA was administered intragastrically for seven consecutive days. Hematopoietic progenitor colony-forming cell assays were used to assess the reconstitution of bone marrow after radiation-induced myelosuppression. Cytokine levels were investigated using enzyme-linked immunosorbent assay and Western blot analysis. Results:u2003The results demonstrated that FA treatment enhanced hematopoietic progenitor cell activity resulting in accelerated blood cell recovery. FA administration increased levels of granulocyte-colony stimulating factor (G-CSF) and erythropoietin. Conclusion:u2003These results suggest radioprotective efficacy by FA may be a result of early recovery of hematopoietic cells due to enhanced production of G-CSF and erythropoietin.

Ophiopogonin D maintains Ca2＋ homeostasis in rat cardiomyocytes in vitro by upregulating CYP2J3/ EETs and suppressing ER stress

Aim:CYP2J3 in myocardium metabolizes arachidonic acid to 4 regioisomeric epoxyeicosatrienoic acids (EETs), which have diverse biological activities in rat heart. In this study we examined whether CYP2J3 was involved in cardioprotective effects of ophiopogonin D (OPD), a steroidal glycoside isolated from Chinese herb Radix ophiopogonis.Methods:Rat cardiomyoblast cell line (H9c2 cells) was tested. Intracellular Ca2+ concentrations ([Ca2+]i) were measured using Fluo-4/AM. The expression of calcium-regulating molecules and ER stress signaling molecules was measured with qRT-PCR and Western blot analyses. Cell apoptosis was quantified with Hoechst 33258 staining and TUNEL assay. The level of 14,15-DHET, a stable metabolite of 14,15-EET, was assessed with ELISA.Results:Angiotensin II (10−6 mol/L) significantly decreased the expression of calcium-regulating molecules (SERCA2a, PLB, RyR2 and FKBP12.6), and elevated [Ca2+]i in H9c2 cells. Furthermore, angiotensin II markedly increased the expression of ER stress signaling molecules (GRP78, CHOP, p-JNK and cleaved caspase-12) and ER stress-mediated apoptosis. OPD (100, 250 and 500 nmol/L) dose-dependently increased CYP2J3 expression and 14,15-DHET levels in normal H9c2 cells. Pretreatment of H9c2 cells with OPD suppressed angiotensin II-induced abnormalities in Ca2+ homeostasis, ER stress responses and apoptosis. Overexpression of CYP2J3 or addition of exogenous 14,15-EET also prevented angiotensin II-induced abnormalities in Ca2+ homeostasis, whereas transfection with CYP2J3 siRNA diminished the effects of OPD on Ca2+ homeostasis. Furthermore, the intracellular Ca2+ chelator BAPTA suppressed angiotensin II-induced ER stress responses and apoptosis in H9c2 cells.Conclusion:OPD is a novel CYP2J3 inducer that may offer a therapeutic benefit in treatment of cardiovascular diseases related to disturbance of Ca2+ homeostasis and ER stress.

Shenfu Injection (参附注射液) suppresses inflammation by targeting haptoglobin and pentraxin 3 in rats with chronic ischemic heart failure

ObjectiveTo investigate the regulatory effects of Shenfu Injection (SFI, 参附注射液) on hemodynamic parameters and serum proteins in rats with post-infarction chronic heart failure (CHF).MethodsForty-five healthy Wistar rats were randomized into three groups: sham, heart failure (model) and SFI group. The CHF was induced by left coronary artery ligation. Seven days after the surgical operation, animals in the sham group and the model group received saline (6.2 mL/kg/d), while animals in the SFI group received SFI (6.2 mL/kg d) intraperitoneally. Four weeks later, cardiac hemodynamic parameters were measured via the carotid route. The expression of serum proteins was analyzed by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS).ResultsRecording of hemodynamic parameters showed that left ventricular systolic pressure (LVSP), maximum rate of left ventricular pressure (+dp/dtmax) rise, and maximum rate of left ventricular pressure (−dp/dtmax) decrease, while the left ventricular end diastolic pressure (LVEDP) rose in the model group compared to those in the sham group (P <0.05). The results of the MALDI-TOF MS indicated that haptoglobin (HP), pentraxin 3 (PTX3) and alpha-1-antitrypsin were up-regulated, while serum albumin and 40S ribosomal protein were down-regulated in the model group (P <0.05). Compared with the model group, LVSP, +dp/dtmax and −dp/dtmax were higher, while LVEDP was lower in the SFI group (P<0.05). Expression levels of HP and PTX3 were lower than in the model group (P<0.05).ConclusionSFI could improve hemodynamic function and decrease inflammatory reactions in the pathophysiology of CHF. The serum proteins HP and PTX3 could be potential biomarkers for chronic ischemic heart failure, and they could also be the serum protein targets of SFI.

Tetramethylpyrazine protects lymphocytes from radiation-induced apoptosis through nuclear factor-κB.

AIMnRadiation induces an important apoptosis response in irradiated organs. The objective of this study was to investigate the radioprotective effect of tetramethylpyrazine (TMP) on irradiated lymphocytes and discover the possible mechanism of protection.nnnMETHODnLymphocytes were pretreated for 12 h with TMP (25-200 μmol·L(-1)) and then exposed to 4 Gy radiation. Cell apoptosis and the signaling pathway were analyzed.nnnRESULTSnIrradiation increased cell death, DNA fragmentation, activated caspase activation and cytochrome c translocation, downregulated B-cell lymphoma 2 (Bcl-2) and up-regulated Bcl-2-associated X protein (Bax). Pretreated with TMP significantly reversed this tendency. Several anti-apoptotic characteristics of TMP, including the ability to increase cell viability, inhibit caspase-9 activation, and upregulate Bcl-2 and down-regulate Bax in 4Gy-irradiated lymphocytes were determined. Signal pathway analysis showed TMP could translate nuclear factor-κB (NF-κB) from cytosol into the nucleus.nnnCONCLUSIONnThe results suggest that TMP had a radioprotective effect through the NF-κB pathway to inhibit apoptosis, and it may be an effective candidate for treating radiation diseases associated with cell apoptosis.

Investigating the in vitro metabolism of veratridine: Characterization of metabolites and involved cytochrome P450 isoforms

Veratridine is a lipid-soluble alkaloid extracted from Veratrum officinale and other species of the family Liliaceae. Veratridine prevents inactivation of Na(+) channel via binding the receptor site 2, causes influx of sodium ion and depolarization and induces apoptosis of neuronal cells. In the present study, we investigated the metabolism of veratridine and the effects of selective cytochrome P450 (CYP) inhibitors on the metabolism of veratridine in rat liver microsomes. The metabolites were separated and assayed by liquid-chromatography-electrospray ionization-ion trap tandem mass spectrometry (LC-ESI-QIT-MSn), and further identified by their mass spectra and chromatographic behaviors. Result showed that four CYP isoforms (CYP1A, CYP2B, CYP2E1, CYP3A) were involved in the metabolism of veratridine in vitro and seven metabolites of veratridine were detected incubating with rat liver microsomes. Some of the metabolites were presumed to be potential mediates of neurotoxicity via protein binging. Further research in vivo needs to link the metabolism of veratridine to its toxicity.

Ginkgolide B protects human umbilical vein endothelial cells against xenobiotic injuries via PXR activation

Aim:Pregnane X receptor (PXR) is a nuclear receptor that regulates a number of genes encoding drug metabolism enzymes and transporters and plays a key role in xeno- and endobiotic detoxification. Ginkgolide B has shown to increase the activity of PXR. Here we examined whether ginkgolide B activated PXR and attenuated xenobiotic-induced injuries in endothelial cells.Methods:Human umbilical vein endothelial cells (HUVECs) were treated with ginkgolide B. The expression of PXR, CYP3A4, MDR1, VCAM-1, E-selectin and caspase-3 were quantified with qRT-PCR and Western blot analysis. Cell apoptosis was analyzed with flow cytometry. Fluorescently labeled human acute monocytic leukemia cells (THP-1 cells) were used to examine cell adhesion.Results:Ginkgolide B (30–300 μmol/L) did not change the mRNA and protein levels of PXR in the cells, but dose-dependently increased nuclear translocation of PXR protein. Ginkgolide B increased the expression of CYP3A4 and MDR1 in the cells, which was partially reversed by pretreatment with the selective PXR signaling antagonist sulforaphane, or transfection with PXR siRNA. Functionally, ginkgolide B dose-dependently attenuated doxorubicin- or staurosporine-induced apoptosis, which was reversed by transfection with PXR siRNA. Moreover, ginkgolide B suppressed TNF-α-induced THP-1 cell adhesion and TNF-α-induced expression of vascular adhesion molecule 1 (VCAM-1) and E-selectin in the cells, which was also reversed by transfection with PXR siRNA.Conclusion:Ginkgolide B exerts anti-apoptotic and anti-inflammatory effects on endothelial cells via PXR activation, suggesting that a PXR-mediated endothelial detoxification program may be important for protecting endothelial cells from xeno- and endobiotic-induced injuries.

Ferulic acid prevents LPS-induced up-regulation of PDE4B and stimulates the cAMP/CREB signaling pathway in PC12 cells.

Aim:Phosphodiesterase 4 (PDE4) isozymes are involved in different functions, depending on their patterns of distribution in the brain. The PDE4 subtypes are distributed in different inflammatory cells, and appear to be important regulators of inflammatory processes. In this study we examined the effects of ferulic acid (FA), a plant component with strong anti-oxidant and anti-inflammatory activities, on lipopolysaccharide (LPS)-induced up-regulation of phosphodiesterase 4B (PDE4B) in PC12 cells, which in turn regulated cellular cAMP levels and the cAMP/cAMP response element binding protein (CREB) pathway in the cells.Methods:PC12 cells were treated with LPS (1 μg/mL) for 8 h, and the changes of F-actin were detected using laser scanning confocal microscopy. The levels of pro-inflammatory cytokines were measured suing ELISA kits, and PDE4B-specific enzymatic activity was assessed with a PDE4B assay kit. The mRNA levels of PDE4B were analyzed with Q-PCR, and the protein levels of CREB and phosphorylated CREB (pCREB) were determined using immunoblotting. Furthermore, molecular docking was used to identify the interaction between PDE4B2 and FA.Results:Treatment of PC12 cells with LPS induced thick bundles of actin filaments appearing in the F-actin cytoskeleton, which were ameliorated by pretreatment with FA (10–40 μmol/L) or with a PDE4B inhibitor rolipram (30 μmol/L). Pretreatment with FA dose-dependently inhibited the LPS-induced production of TNF-α and IL-1β in PC12 cells. Furthermore, pretreatment with FA dose-dependently attenuated the LPS-induced up-regulation of PDE4 activity in PC12 cells. Moreover, pretreatment with FA decreased LPS-induced up-regulation of the PDE4B mRNA, and reversed LPS-induced down-regulation of CREB and pCREB in PC12 cells. The molecular docking results revealed electrostatic and hydrophobic interactions between FA and PDE4B2.Conclusion:The beneficial effects of FA in PC12 cells might be conferred through inhibition of LPS-induced up-regulation of PDE4B and stimulation of cAMP/CREB signaling pathway. Therefore, FA may be a potential therapeutic intervention for the treatment of neuroinflammatory diseases such as AD.