Troxerutin downregulates C/EBP‐β gene expression via modulating the IFNγ‐ERK1/2 signaling pathway to ameliorate rotenone‐induced retinal neurodegeneration

Troxerutin, a natural flavonoid guards against oxidative stress and apoptosis with a high capability of passing through the blood‐brain barrier. Our aim was to investigate the role of troxerutin in experimentally induced retinal neurodegeneration by modulating the interferon‐gamma (IFNγ)‐extracellular signal‐regulated kinases 1/2 (ERK1/2)‐CCAAT enhancer‐binding protein β (C/EBP‐β) signaling pathway. Three groups of rats (10 each group) were included. Group I (control group), group II (rotenone treated group): the rats were injected subcutaneously with a single rotenone dosage of 3 mg/kg repeated every 48 hours for 60 days to trigger retinal neurodegeneration. Group III (troxerutin‐treated group): rats received troxerutin (150 mg/kg/day) by oral gavage 1 hour before rotenone administration. A real‐time polymerase chain reaction technique was applied to measure messenger RNA (mRNA) levels of retinal C/EBP‐β. Enzyme‐linked immunosorbent assay technique was utilized to assay tumor necrosis factor‐α (TNF‐α), IFNγ, and ERK1/2 levels. Finally, reactive oxygen species (ROS), as well as carbonylated protein (CP) levels, were assessed spectrophotometrically. Improved retinal neurodegeneration by downregulation of C/EBP‐β mRNA gene expression, also caused a significant reduction of TNF‐α, IFNγ, ERK1/2 as well as ROS and CP levels compared with the diseased group. These findings could hold promise for the usage of troxerutin as a protective agent against rotenone‐induced retinal neurodegeneration.     

Tumour necrosis factor‐α promotes liver ischaemia‐reperfusion injury through the PGC‐1α/Mfn2 pathway

Tumour necrosis factor (TNF)‐α has been considered to induce ischaemia‐reperfusion injury (IRI) of liver which is characterized by energy dysmetabolism. Peroxisome proliferator–activated receptor‐γ co‐activator (PGC)‐1α and mitofusion2 (Mfn2) are reported to be involved in the regulation of mitochondrial function. However, whether PGC‐1α and Mfn2 form a pathway that mediates liver IRI, and if so, what the underlying involvement is in that pathway remain unclear. In this study, L02 cells administered recombinant human TNF‐α had increased TNF‐α levels and resulted in down‐regulation of PGC‐1α and Mfn2 in a rat liver IRI model. This was associated with hepatic mitochondrial swelling, decreased adenosine triphosphate (ATP) production, and increased levels of reactive oxygen species (ROS) and alanine aminotransferase (ALT) activity as well as cell apoptosis. Inhibition of TNF‐α by neutralizing antibody reversed PGC‐1α and Mfn2 expression, and decreased hepatic injury and cell apoptosis both in cell culture and in animals. Treatment by rosiglitazone sustained PGC‐1α and Mfn2 expression both in IR livers, and L02 cells treated with TNF‐α as indicated by increased hepatic mitochondrial integrity and ATP production, reduced ROS and ALT activity as well as decreased cell apoptosis. Overexpression of Mfn2 by lentiviral‐Mfn2 transfection decreased hepatic injury in IR livers and L02 cells treated with TNF‐α. However, there was no up‐regulation of PGC‐1α. These findings suggest that PGC‐1α and Mfn2 constitute a regulatory pathway, and play a critical role in TNF‐α‐induced hepatic IRI. Inhibition of the TNF‐α or PGC‐1α/Mfn2 pathways may represent novel therapeutic interventions for hepatic IRI.     

Antioxidant and antiapoptotic effects of proanthocyanidin and ginkgo biloba extract against doxorubicin‐induced cardiac injury in rats

Grape seed proanthocyanidins (GSPE) and ginkgo biloba extract (EGb761) are considered to have protective effects against several diseases. The cardiotoxicity of doxorubicin (DOX) has been reported to be associated with oxidative damage. This study was conducted to evaluate the cardioprotective effects of GSPE and EGb761 against DOX‐induced heart injury in rats. DOX was administered as a single i.p. dose (20 mg kg^–1) to adult male rats. DOX‐intoxicated rats were orally administered GSPE (200 mg kg^–1 day^–1) or EGb761 (100 mg kg^–1 day^–1) for 15 consecutive days, starting 10 days prior DOX injection. DOX‐induced cardiotoxicity was evidenced by a significant increase in serum aspartate transaminase (AST), creatine phosphokinase isoenzyme (CK‐MB), lactate dehydrogenase (LDH), total cholesterol (TC) and triglyceride (TG) activities and levels. Increased oxidative damage was expressed by the depletion of cardiac reduced glutathione (GSH), elevation of cardiac total antioxidant (TAO) level and accumulation of the lipid peroxidation product, malondialdehyde (MDA). Significant rises in cardiac tumour necrosis factor‐alpha (TNF‐α) and caspase‐3 levels were noticed in DOX‐intoxicated rats. These changes were ameliorated in the GSPE and EGb761‐treated groups. Histopathological analysis confirmed the cardioprotective effects of GSPE and EGb761. In conclusion, GSPE and EGb761 mediate their protective effect against DOX‐induced cardiac injury through antioxidant, anti‐inflammatory and antiapoptotic mechanisms. Copyright © 2012 John Wiley & Sons, Ltd.     

HYDAMTIQ,a selective PARP‐1 inhibitor,improves bleomycin‐induced lung fibrosis by dampening the TGF‐β/SMAD signalling pathway

Idiopathic pulmonary fibrosis is a severe disease characterized by excessive myofibroblast proliferation, extracellular matrix and fibrils deposition, remodelling of lung parenchyma and pulmonary insufficiency. Drugs able to reduce disease progression are available, but therapeutic results are unsatisfactory; new and safe treatments are urgently needed. Poly(ADP‐ribose) polymerases‐1 (PARP‐1) is an abundant nuclear enzyme involved in key biological processes: DNA repair, gene expression control, and cell survival or death. In liver and heart, PARP‐1 activity facilitates oxidative damage, collagen deposition and fibrosis development. In this study, we investigated the effects of HYDAMTIQ, a potent PARP‐1 inhibitor, in a murine model of lung fibrosis. We evaluated the role of PARP on transforming growth factor‐β (TGF‐β) expression and TGF‐β/SMAD signalling pathway in lungs. Mice were intratracheally injected with bleomycin and then treated with either vehicle or different doses of HYDAMTIQ for 21 days. Airway resistance to inflation and lung static compliance, markers of lung stiffness, were assayed. Histochemical and biochemical parameters to evaluate TGF‐β/SMAD signalling pathway with alpha‐smooth muscle actin (αSMA) deposition and the levels of a number of inflammatory markers (tumour necrosis factor‐α, interleukin‐1β, iNOS and COX‐2) were performed. Bleomycin administration increased lung stiffness. It also increased lung PARP activity, TGF‐β levels, pSMAD3 expression, αSMA deposition and content of inflammatory markers. HYDAMTIQ attenuated all the above‐mentioned physiological, biochemical and histopathological markers. Our findings support the proposal that PARP inhibitors could have a therapeutic potential in reducing the progression of signs and symptoms of the disease by decreasing TGF‐β expression and the TGF‐β/SMAD transduction pathway.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

A novel molecule ‘shati’ increases dopamine uptake via the induction of tumor necrosis factor‐α in pheochromocytoma‐12 cells

The psychostimulant properties of methamphetamine (METH) are associated with an increase in extracellular dopamine (DA) levels in the brain, via facilitation of DA’s release from pre‐synaptic nerve terminals and inhibition of its reuptake through DA transporter. Recently, we have demonstrated that tumor necrosis factor‐α (TNF‐α) increases DA uptake and inhibits METH dependence. Moreover, we have clarified ‘shati’ identified in the nucleus accumbens of mice treated with METH is involved in METH dependence. In the present study, we investigated the effects of TNF‐α on DA uptake in PC12 cells and established a PC12 cell line transfected with a vector containing shati cDNA to examine the precise mechanism behind the role of shati in DA uptake. Moreover, we examined the relationship between shati and TNF‐α. TNF‐α increased DA uptake via the mitogen‐activated protein kinase kinase pathway and inhibited the METH‐induced decrease in DA uptake in PC12 cells. Transfection of the vector containing shati cDNA into PC12 cells, induced the expression of shati and TNF‐α mRNA, accelerated DA uptake, and inhibited the METH‐induced decrease in DA uptake. These results suggest that the functional roles of shati in METH‐regulated behavioral changes are mediated through inhibition of the METH‐induced decrease in DA uptake via TNF‐α.     

PPAR‐β facilitating maturation of hepatic‐like tissue derived from mouse embryonic stem cells accompanied by mitochondriogenesis and membrane potential retention

Relatively little is known about mitochondria metabolism in differentiating embryonic stem (ES) cells. Present research focused on several elements of cellular energy metabolism in hepatic‐like tissue derived from mouse ES cells. We demonstrated that mitochondrial location patterns and mitochondrial membrane potential (ΔΨ_m) existed in subsequent differentiation of the tissue. Mitochondriogenesis appeared at the early stage and kept a normal ΔΨ_m in differentiated mature hepatocytes. Peroxisome proliferator‐activated receptor‐α (PPAR‐α) expression was transitorily increased at the beginning, and kept a relatively low level later, which accompanied by expression of PPAR‐γ coactivator (PGC)‐1α, a master regulator of mitochondrial biogenesis. PPAR‐β expression showed robust up‐regulation in the late differentiation course. Enhanced co‐expressions of PPAR‐β and albumin with catalysis of UDP‐glucuronosyltransferases (UGTs) were observed at mature stage. While PPAR‐γ expression changed little before and after differentiation. Mitochondriogenesis could be accelerated by PPAR‐α specific agonist WY14643 and abolished by its antagonist GW6471 at the early stage. Neither of them affected mitochondrial ΔΨ_m and albumin generation in the differentiated hepatocytes. Furthermore, maturation of hepatic‐like tissue and mitochondriogenesis in hepatocyte could be efficiently stimulated by PPAR‐β specific agonist L165041 and abolished by PPAR‐β specific antagonist GSK0660, but not affected by PPAR‐γ specific agonist GW1929. In conclusion, the derived hepatic tissue morphologically possessed cellular energy metabolism features. PPAR‐α seemed only necessary for early mitochondriogenesis, while less important for ΔΨ_m retention in the mature tissue derived. The stimulation of PPAR‐β but not ‐γ enhanced hepatogenesis, hepatocytes maturation, and mitochondriogenesis. PPAR‐β took an important role in cellular energy metabolism of hepatogenesis. J. Cell. Biochem. 109: 498–508, 2010. © 2009 Wiley‐Liss, Inc.     

Hesperidin modulates dextran sulfate sodium‐induced ulcerative colitis in rats: Targeting sphingosine kinase‐1‐ sphingosine 1 phosphate signaling pathway,mitochondrial biogenesis,inflammation, and apoptosis

Ulcerative colitis (UC) is a chronic gastrointestinal disorder interfering with life quality. A total of 60 male Wistar rats were divided into four equal groups: Control (group I), hesperidin only (group II), UC untreated (group III), and UC treated with hesperidin (group IV). Hesperidin had modulatory effects on UC pathogenesis, which might be through alleviating colonic sphingosine phosphate phosphatase 2 messenger RNA expression and sphingosine kinase‐1 levels, thus suppressing the subsequent downstream inflammatory and apoptotic cascades represented by decreased macrophage inflammatory protein‐1α and enhancement of B‐cell lymphoma 2 immunohistochemistry expression. Also, it improved mitochondrial biogenesis by increasing the peroxisome proliferator‐activated receptor‐gamma‐coactivator 1‐α level. It successfully restored redox potential as evidenced by marked alleviations of the nitric oxide and peroxynitrite levels, increasing total antioxidant capacity, and activating the superoxide dismutase enzyme. Also, hesperidin alleviated the UC disease activity index and improved the histopathological picture. These findings may offer a new therapeutic strategy for UC treatment.     

Perivascular adipose tissue‐derived stromal cells contribute to vascular remodeling during aging

Aging is an independent risk factor for vascular diseases. Perivascular adipose tissue (PVAT), an active component of the vasculature, contributes to vascular dysfunction during aging. Identification of underlying cell types and their changes during aging may provide meaningful insights regarding the clinical relevance of aging‐related vascular diseases. Here, we take advantage of single‐cell RNA sequence to characterize the resident stromal cells in the PVAT (PVASCs) and identified different clusters between young and aged PVASCs. Bioinformatics analysis revealed decreased endothelial and brown adipogenic differentiation capacities of PVASCs during aging, which contributed to neointimal hyperplasia after perivascular delivery to ligated carotid arteries. Mechanistically, in vitro and in vivo studies both suggested that aging‐induced loss of peroxisome proliferator‐activated receptor‐γ coactivator‐1 α (PGC1α) was a key regulator of decreased brown adipogenic differentiation in senescent PVASCs. We further demonstrated the existence of human PVASCs (hPVASCs) and overexpression of PGC1α improved hPVASC delivery‐induced vascular remodeling. Our finding emphasizes that differentiation capacities of PVASCs alter during aging and loss of PGC1α in aged PVASCs contributes to vascular remodeling via decreased brown adipogenic differentiation.     

Rac1 activation induces tumour necrosis factor‐α expression and cardiac dysfunction in endotoxemia

Induction of tumour necrosis factor‐α (TNF‐α) expression leads to myocardial depression during sepsis. However, the underlying molecular mechanisms are not fully understood. The aim of this study was to investigate the role of Rac1 in TNF‐α expression and cardiac dysfunction during endotoxemia and to determine the involvement of phosphoinositide‐3 kinase (PI3K) in lipopolysaccharide (LPS)‐induced Rac1 activation. Our results showed that LPS‐induced Rac1 activation and TNF‐α expression in cultured neonatal mouse cardiomyocytes. The response was inhibited in Rac1 deficient cardiomyocytes or by a dominant‐negative Rac1 (Rac1N17). To determine whether PI3K regulates Rac1 activation, cardiomyocytes were treated with LY294002, a PI3K selective inhibitor. Treatment with LY294002 decreased Rac1 activity as well as TNF‐α expression stimulated by LPS. Furthermore, inhibition of PI3K and Rac1 activity decreased LPS‐induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation. To investigate the role of Rac1 in myocardial depression during endotoxemia in vivo, wild‐type and cardiomyocyte‐specific Rac1 deficient mice were treated with LPS (2 mg/kg, i.p.). Deficiency in Rac1 significantly decreased myocardial TNF‐α expression and improved cardiac function during endotoxemia. We conclude that PI3K‐mediated Rac1 activation is required for induction of TNF‐α expression in cardiomyocytes and cardiac dysfunction during endotoxemia. The effect of Rac1 on TNF‐α expression seems to be mediated by increased NADPH oxidase activity and ERK1/2 phosphorylation.     

Neuroprotection by quercetin via mitochondrial function adaptation in traumatic brain injury: PGC‐1α pathway as a potential mechanism

The aim of this study was to investigate the neuroprotective effects of quercetin in mouse models of traumatic brain injury (TBI) and the potential role of the PGC‐1α pathway in putative neuroprotection. Wild‐type mice were randomly assigned to four groups: the sham group, the TBI group, the TBI+vehicle group and the TBI+quercetin group. Quercetin, a dietary flavonoid used as a food supplement, significantly reduced TBI‐induced neuronal apoptosis and ameliorated mitochondrial lesions. It significantly accelerated the translocation of PGC‐1α protein from the cytoplasm to the nucleus. In addition, quercetin restored the level of cytochrome c, malondialdehyde and superoxide dismutase in mitochondria. Therefore, quercetin administration can potentially attenuate brain injury in a TBI model by increasing the activities of mitochondrial biogenesis via the mediation of the PGC‐1α pathway.     

Protective effects of cilengitide on inflammation in chondrocytes under excessive mechanical stress

Chondrocytes constantly receive external stimuli, which regulates remodeling. An optimal level of mechanical stress is essential for maintaining chondrocyte homeostasis, however, excessive mechanical stress induces inflammatory cytokines and protease, such as matrix metalloproteinases (MMPs). Therefore, excessive mechanical stress is considered to be one of the main causes to cartilage destruction leading to osteoarthritis (OA). Integrins are well‐known as cell adhesion molecules and act as receptors for extracellular matrix (ECM), and are believed to control intracellular signaling pathways both physically and chemically as a mechanoreceptor. However, few studies have focused on the roles and functions of integrins in inflammation caused by excessive mechanical stress. In this study, we examined the relationship between integrins (αVβ3 and αVβ5) and the expression of inflammatory factors under mechanical loading in chondrocytes by using an integrin receptor antagonist (cilengitide). Cilengitide suppressed the gene expression of interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), matrix metalloproteinase‐3 (MMP‐3), and MMP‐13 induced by excessive mechanical stress. In addition, the protein expression of IL1‐β and MMP‐13 was also inhibited by the addition of cilengitide. Next, we investigated the involvement of intracellular signaling pathways in stress‐induced integrin signaling in chondrocytes by using western blotting. The levels of p‐FAK, p‐ERK, p‐JNK, and p‐p38 were enhanced by excessive mechanical stress and the enhancement was suppressed by treatment with cilengitide. In conclusion, this study revealed that excessive mechanical stress may activate integrins αVβ3 and αVβ5 on the surface of chondrocytes and thereby induce an inflammatory reaction by upregulating the expression of IL‐1β, TNF‐α, MMP‐3, and MMP‐13 through phosphorylation of FAK and MAPKs.     

Modification of the detrimental effect of TNF‐α on human endothelial progenitor cells by fasudil and Y27632

Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor‐α (TNF‐α) reduced their number and biological activity. Yet, signal transduction events linked to TNF‐α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in‐ flammation. Results demonstrated that incubation with fasudil starting from 50 μM but not Y27632 determined a dose‐dependent improvement of EPC number during exposure to TNF‐α (P < 0.05 vs. TNF‐α alone). Analysis of the signal transduction pathway activated by TNF‐α revealed that the increased expression of p‐p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF‐α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF‐α) as well as the inhibitor of Erk1/2‐specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF‐α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D‐DIGE and MALDI‐TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin‐related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:351–360, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20345     

Pinitol attenuates LPS‐induced pneumonia in experimental animals: Possible role via inhibition of the TLR‐4 and NF‐κB/IκBα signaling cascade pathway

Pneumonia is a chronic disorder of the respiratory system associated with worsening quality of life and a significant economic burden. Pinitol, a plant cyclic polyol, has been documented for immune‐inflammatory potential. The aim of present investigation was to evaluate the potential and possible mechanism of action of pinitol against lipopolysaccharide (LPS)‐induced pneumonia in the experimental animal model. Pneumonia was induced in Sprague‐Dawley rats by intratracheal administration of LPS (2 mg/kg). Animals were treated with either vehicle or dexamethasone or pinitol (5 or 10 or 20 mg/kg). Potential of pinitol against LPS‐induced pulmonary insult was assessed based on behavioral, biochemical, molecular, and ultrastructural studies. Intratracheal instillation of LPS induced significant (P < .05) inflammatory infiltration in bronchoalveolar lavage fluid (BALF) and lung tissue reflected by elevated pleural effusion volume, lung edema, BALF polymorphonuclear leukocytes count and lung myeloperoxidase levels, which was attenuated by pinitol (10 and 20 mg/kg) administration. Pinitol also markedly (P < .05) inhibited LPS‐induced alterations in electrocardiographic, hemodynamic changes, right ventricular, and lung function tests. The LPS‐induced downregulated nuclear factor erythroid 2–related factor 2 (Nrf‐2) and heme oxygenase‐1 (HO‐1), whereas upregulated transforming growth factor‐β (TGF‐β), tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3), and inducible nitric oxide synthase (iNOs) lung messenger RNA expressions were significantly (P < .05) inhibited by pinitol. Western blot analysis suggested pinitol markedly (P < .05) decreased nuclear factor‐κB (NF‐κB), inhibitor of nuclear factor κB (IkBα), toll‐like receptor 4 (TLR‐4), and cyclooxygenase‐II (COX‐II) protein expressions in the lung. These findings were further supported by histological and ultrastructural analyses of lung tissue that show pinitol significantly (P < .05) ameliorates LPS‐induced aberrations in lung tissue. In conclusion, pinitol attenuated LPS‐induced pneumonia via inhibition of TLR‐4 to downregulate the NF‐κB/IκBα signaling cascade and thus ameliorated the production of proinflammatory cytokines (TNF‐α, ILs, NLRP3, and TGF‐β), inflammatory mediators (COX‐II and iNOs) and elevated oxidative stress (Nrf‐2 and HO‐1).