Diallyl sulfide ameliorates carbon tetrachloride‐induced hepatotoxicity in rats via suppressing stress‐activated MAPK signaling pathways

The underlined effects of diallyl sulfide (DAS) against CCL₄‐induced oxidative, inflammatory, and apoptotic acute hepatic damage were assessed. Administration of DAS (50, 100, and 200 mg/kg) along with CCL ₄ effectively mitigated serum aspartate aminotransferase, alanine aminotransferase activities, MDA, TNF‐α, IL‐1β, and MCP‐1 levels, as well as significantly restored HO‐1, GSH levels and SOD activity in liver tissues compared with those in rats treated with CCL ₄. Moreover, DAS inhibited CCL ₄‐induced increase of liver NF‐κB (p65), Bax, p38 MAPK, and JNK protein expression. In addition, DAS accelerated protein expression of Nrf2 and Bcl‐2. The hepatoprotective properties of DAS were further confirmed by the reduced severity of hepatic damage as demonstrated by histopathological findings. In conclusion, DAS achieved its protective potential against CCL4‐induced hepatotoxicity through antiapoptotic activity, as well as the synchronized modulation of NF‐κB and Nrf2 for the favor of antioxidant/anti‐inflammatory effects via suppression of the upstream stress‐activated MAPKs pathways.     

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).     

Embelin impact on paraquat‐induced lung injury through suppressing oxidative stress,inflammatory cascade,and MAPK/NF‐κB signaling pathway

The current examination was intended to observe the defensive impacts of embelin against paraquat‐incited lung damage in relationship with its antioxidant and anti‐inflammatory action. Oxidative stress marker, like malondialdehyde (MDA), antioxidative enzymes, for example, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px), inflammatory cytokines, such as interleukin‐1β (IL‐1β), tumor necrosis factor‐α, and IL‐6, histological examination, and nuclear factor kappa B/mitogen‐activated protein kinase (NF‐κB/MAPK) gene expression were evaluated in lung tissue. Embelin treatment significantly decreased MDA and increased SOD, CAT, and GSH Px. Embelin significantly reduced levels of inflammatory cytokines in paraquat‐administered and paraquat‐intoxicated rats. In addition, embelin suggestively decreased relative protein expression of nuclear NF‐κB p65, p‐NF‐κBp65, p38 MAPK, and p‐p38 MAPKs in paraquat‐intoxicated rats. The outcomes show the impact of embelin inhibitory action on NF‐κB and MAPK and inflammatory cytokines release, and the decrease of lung tissue damage caused by paraquat.     

Loss of PTEN induces lung fibrosis via alveolar epithelial cell senescence depending on NF‐κB activation

Idiopathic pulmonary fibrosis (IPF) is an aging‐associated disease with poor prognosis. Currently, there are no effective drugs for preventing the disease process. The mechanisms underlying the role of alveolar epithelial cell (AEC) senescence in the pathogenesis of IPF remain poorly understood. We aimed to explore whether PTEN/NF‐κB activated AEC senescence thus resulting in lung fibrosis. First, we investigated the association between the activation of PTEN/NF‐κB and cellular senescence in lung tissues from IPF patients. As a result, decreased PTEN, activated NF‐κB and increased senescent markers (P21^WAF1, P16^ink4a, and SA‐β‐gal) were found in AECs in fibrotic lung tissues detected by immunohistochemistry (IHC) and immunofluorescence (IF). In vitro experiments showed increased expression levels of senescent markers and augmented senescence‐associated secretory phenotype (SASP) in AECs treated with bleomycin (Blm); however, PTEN was reduced significantly following IκB, IKK, and NF‐κB activation after stimulation with Blm in AECs. AEC senescence was accelerated by PTEN knockdown, whereas senescence was reversed via NF‐κB knockdown and the pharmacological inhibition (BMS‐345541) of the NF‐κB pathway. Interestingly, we observed increased collagen deposition in fibroblasts cultured with the supernatants collected from senescent AECs. Conversely, the deposition of collagen in fibroblasts was reduced with exposure to the supernatants collected from NF‐κB knockdown AECs. These findings indicated that senescent AECs controlled by the PTEN/NF‐κB pathway facilitated collagen accumulation in fibroblasts, resulting in lung fibrosis. In conclusion, our study supports the notion that as an initial step in IPF, the senescence process in AECs may be a potential therapeutic target, and the PTEN/NF‐κB pathway may be a promising candidate for intervention.     

Arachidonic acid release by H2O2 mediated proliferation of mouse embryonic stem cells: Involvement of Ca2+/PKC and MAPKs‐induced EGFR transactivation

Reactive oxygen species (ROS) generated by a variety of endogenous factors and roles in embryonic stem (ES) cells has yet to be identified. Thus, we examined role of arachidonic acid (AA) in H₂O₂‐indued proliferation of mouse ES cells and its related signaling molecules. AA release was maximally increased in response to 10^?4 M H₂O₂ for 1 h. In addition, H₂O₂ increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the phosphorylation of protein kinase C (PKC), p44/42, p38 mitogen‐activated protein kinase (MAPK), and JNK/SAPK. Moreover, H₂O₂ induced an increase in the phosphorylation of epidermal growth factor receptor (EGFR), which was blocked by the inhibition of p44/42 or p38 MAPKs. The inhibition of each signal molecule with specific inhibitors blocked H₂O₂‐induced cytosolic phospholipase A₂ (cPLA₂) activation and AA release. H₂O₂ increased NF‐κB phosphorylation to induce an increase in the levels of cyclooxygenase (COX)‐2 proteins. Subsequently, H₂O₂ stimulated PGE₂ synthesis, which was reduced by the inhibition of NF‐κB activation. Moreover, each H₂O₂ or PGE₂ increased DNA synthesis and the number of cells. However, H₂O₂‐induced increase in DNA synthesis was inhibited by the suppression of cPLA₂ pathway. In conclusion, H₂O₂ increased AA release and PGE₂ production by the upregulation of cPLA₂ and COX‐2 via Ca²⁺/PKC/MAPKs and EGFR transactivation, subsequently proliferation of mouse ES cells. J. Cell. Biochem. 106: 787–797, 2009. © 2009 Wiley‐Liss, Inc.     

Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS‐dependent mitochondrial signalling pathway

Oxidative stress is an important molecular mechanism underlying lung fibrosis. The mitochondrion is a major organelle for oxidative stress in cells. Therefore, blocking the mitochondrial signalling pathway may be the best therapeutic manoeuver to ameliorate lung fibrosis. Astaxanthin (AST) is an excellent antioxidant, but no study has addressed the pathway of AST against pulmonary oxidative stress and free radicals by the mitochondrion‐mediated signalling pathway. In this study, we investigated the antioxidative effects of AST against H₂O₂‐ or bleomycin (BLM)‐induced mitochondrial dysfunction and reactive oxygen species (ROS) production in alveolar epithelial cells type II (AECs‐II) in vivo and in vitro. Our data show that AST blocks H₂O₂‐ or BLM‐induced ROS generation and dose‐dependent apoptosis in AECs‐II, as characterized by changes in cell and mitochondria morphology, translocation of apoptotic proteins, inhibition of cytochrome c (Cyt c) release, and the activation of caspase‐9, caspase‐3, Nrf‐2 and other cytoprotective genes. These data suggest that AST inhibits apoptosis in AECs‐II cells through the ROS‐dependent mitochondrial signalling pathway and may be of potential therapeutic value in lung fibrosis treatment.     

Role of Spm–Cer‐S1P signalling pathway in MMP‐2 mediated U46619‐induced proliferation of pulmonary artery smooth muscle cells: protective role of epigallocatechin‐3‐gallate

During remodelling of pulmonary artery, marked proliferation of pulmonary artery smooth muscle cells (PASMCs) occur s , which contributes to pulmonary hypertension. Thromboxane A₂ (TxA₂) has been shown to produce pulmonary hypertension. The present study investigates the inhibitory effect of epigallocatechin‐3‐gallate (EGCG) on the TxA₂ mimetic, U46619‐induced proliferation of PASMCs. U46619 at a concentration of 10 nM induces maximum proliferation of bovine PASMCs. Both pharmacological and genetic inhibitors of p³⁸MAPK, NF‐κB and MMP‐2 significantly inhibit U46619‐induced cell proliferation. EGCG markedly abrogate U46619‐induced p³⁸MAPK phosphorylation, NF‐κB activation, proMMP‐2 expression and activation, and also the cell proliferation. U46619 causes an increase in the activation of sphingomyelinase (SMase) and sphingosine kinase (SPHK) and also increase sphingosine 1 phosphate (S1P) level. U46619 also induces phosphorylation of ERK1/2, which phosphorylates SPHK leading to an increase in S1P level. Both pharmacological and genetic inhibitors of SMase and SPHK markedly inhibit U46619‐induced cell proliferation. Additionally, pharmacological and genetic inhibitors of MMP‐2 markedly abrogate U46619‐induced SMase activity and S1P level. EGCG markedly inhibit U46619‐induced SMase activity, ERK1/2 and SPHK phosphorylation and S1P level in the cells. Overall, Sphingomyeline–Ceramide–Sphingosine‐1‐phosphate (Spm–Cer–S1P) signalling axis plays an important role in MMP‐2 mediated U46619‐induced proliferation of PASMCs. Importantly, EGCG inhibits U46619 induced increase in MMP‐2 activation by modulating p³⁸MAPK–NFκB pathway and subsequently prevents the cell proliferation. Copyright © 2015 John Wiley & Sons, Ltd.     

Endogenous hydrogen sulfide reduces airway inflammation and remodeling in a rat model of asthma

Endogenous hydrogen sulfide (H₂S) is hypothesized to have an important role in systemic inflammation. We investigated if endogenous H₂S may be a crucial mediator in airway inflammation and airway remodeling in a rat model of asthma and if endogenous H₂S may exert its anti-inflammatory effect by inhibiting inducible nitric oxide synthase (iNOS)/NO pathway. Cystathionine-γ-lyase (CSE; a H₂S-synthesizing enzyme) was mainly expressed in airway and vascular smooth muscle cells in rat lung tissue. Levels of endogenous H₂S was decreased in pulmonary tissue in ovalbumin (OVA)-treated rats. Exogenous administration of NaHS alleviated airway inflammation and airway remodeling: peak expiratory flow (PEF) increased, goblet cell hyperplasia and collagen deposition score decreased, with decreased total cells recovered from bronchoalveolar fluid (BALF) and influx of eosinophils and neutrophils. The H₂S levels of serum and lung tissue were positively correlated with PEF and negatively correlated with the level of eosinophils and neutrophils in BALF, score of lung pathology. NaHS treatment significantly attenuated pulmonary iNOS activation in OVA-treated rats. These results suggest that the CSE/H₂S pathway plays an anti-inflammatory and anti-remodeling part in asthma pathogenesis and could be a novel target in prevention and treatment of asthma.     

Inhibition of inflammation and oxidative stress by an imidazopyridine derivative X22 prevents heart injury from obesity

Inflammation and oxidative stress plays an important role in the development of obesity‐related complications and cardiovascular disease. Benzimidazole and imidazopyridine compounds are a class of compounds with a variety of activities, including anti‐inflammatory, antioxidant and anti‐cancer. X22 is an imidazopyridine derivative we synthesized and evaluated previously for anti‐inflammatory activity in lipopolysaccharide‐stimulated macrophages. However, its ability to alleviate obesity‐induced heart injury via its anti‐inflammatory actions was unclear. This study was designed to evaluate the cardioprotective effects of X22 using cell culture studies and a high‐fat diet rat model. We observed that palmitic acid treatment in cardiac‐derived H9c2 cells induced a significant increase in reactive oxygen species, inflammation, apoptosis, fibrosis and hypertrophy. All of these changes were inhibited by treatment with X22. Furthermore, oral administration of X22 suppressed high‐fat diet‐induced oxidative stress, inflammation, apoptosis, hypertrophy and fibrosis in rat heart tissues and decreased serum lipid concentration. We also found that the anti‐inflammatory and anti‐oxidative actions of X22 were associated with Nrf2 activation and nuclear factor‐kappaB (NF‐κB) inhibition, respectively, both in vitro and in vivo. The results of this study indicate that X22 may be a promising cardioprotective agent and that Nrf2 and NF‐κB may be important therapeutic targets for obesity‐related complications.     

An Aza resveratrol–chalcone derivative 6b protects mice against diabetic cardiomyopathy by alleviating inflammation and oxidative stress

Inflammation and oxidative stress play a crucial role in the development of diabetic cardiomyopathy (DCM). We previously had synthesized an Aza resveratrol–chalcone derivative 6b, of which effectively suppressing lipopolysaccharide (LPS)‐induced inflammatory response in macrophages. This study aimed to investigate the potential protective effect of 6b on DCM and underlying mechanism. In H9c2 myocardial cells, 6b potently decreased high glucose (HG)‐induced cell fibrosis, hypertrophy and apoptosis, alleviating inflammatory response and oxidant stress. In STZ‐induced type 1 diabetic mice (STZ‐DM1), orally administration with 6b for 16 weeks significantly attenuated cardiac hypertrophy, apoptosis and fibrosis. The expression of inflammatory cytokines and oxidative stress biomarkers was also suppressed by 6b distinctly, without affecting blood glucose and body weight. The anti‐inflammatory and antioxidative activities of 6b were mechanistic associated with nuclear factor‐kappa B (NF‐κB) nucleus entry blockage and Nrf2 activation both in vitro and in vivo. The results indicated that 6b can be a promising cardioprotective agent in treatment of DCM via inhibiting inflammation and alleviating oxidative stress. This study also validated the important role of NF‐κB and Nrf2 taken in the pathogenesis of DCM, which could be therapeutic targets for diabetic comorbidities.     

Down‐regulation of mitogen‐activated protein kinases and nuclear factor‐κB signaling is involved in rapamycin suppression of TLR2‐induced inflammatory response in monocytic THP‐1 cells

Tripalmitoyl‐S‐glycero‐Cys‐(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen‐activated protein kinases (MAPKs) and nuclear factor‐κB (NF‐κB) signal pathway. Rapamycin can suppress TLR‐induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2‐induced inflammatory responses was investigated. It was found that Pam3CSK4‐induced pro‐inflammatory cytokines were significantly down‐regulated at both the mRNA and protein levels in THP‐1 cells pre‐treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3‐kinase/protein kinase‐B (PI3K/AKT) signaling did not suppress the expression of pro‐inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT‐PCR showed that Erk and NF‐κB signal pathways are related to the production of pro‐inflammatory cytokines. Inhibition of Erk or NF‐κB signaling significantly down‐regulated production of pro‐inflammatory cytokines. Additionally, western blot showed that pre‐treatment of THP‐1 cells with rapamycin down‐regulates MAPKs and NF‐κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4‐induced pro‐inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2‐induced inflammatory responses by down‐regulation of Erk and NF‐κB signaling.     

Hydrogen sulfide improves left ventricular function in smoking rats via regulation of apoptosis and autophagy

The present study was designed to investigate the protective effects of hydrogen sulfide (H₂S) against cigarette smoking-induced left ventricular dysfunction in rats. Left ventricular structure and function were assessed using two-dimensional echocardiography. Cardiomyocyte apoptosis was determined by Annexin V/PI and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Cardiac autophagy was evaluated by detection of autophagy-related protein expression and observation of autophagosomes. Our results indicated that administration of NaHS (a donor of H₂S) could protect against smoking-induced left ventricular systolic dysfunction. H₂S was found to exert anti-apoptotic effects in the myocardium of smoking rats by inhibiting JNK and P38 mitogen-activated protein kinases pathways and activating PI3K/Akt signaling. Moreover, H₂S could also reduce smoking-induced autophagic cell death via regulation of AMPK/mTOR signaling pathway. In conclusion, our study demonstrates that H₂S can improve left ventricular systolic function in smoking rats via regulation of apoptosis and autophagy.     

Effect of arachidonic acid on hypoxia‐induced IL‐6 production in mouse ES cells: Involvement of MAPKs,NF‐κB,and HIF‐1α

This study examined the role of arachidonic acid (AA) in hypoxia‐induced production of interleukin (IL)‐6 and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia with AA induced IL‐6 production, which was mediated by reactive oxygen species (ROS). In addition, hypoxia increased the levels of p38 mitogen‐activated protein kinases (MAPKs) and stress‐activated protein kinase/c‐jun NH₂‐terminal kinase (SAPK/JNK) phosphorylation, which were blocked by antioxidant (vitamin C). Inhibition of p38 MAPK and SAPK/JNK blocked hypoxia‐ or hypoxia with AA‐induced nuclear factor‐kappa B (NF‐κB) activation. Furthermore, hypoxia‐induced increase in hypoxia‐inducible factor‐1α (HIF‐1α) expression was regulated by NF‐κB activation. Consequently, the increased HIF‐1α expression induced activation of matrix metalloproteinase (MMP)‐2 and MMP‐9. The expression of each signaling molecule stimulated an increase in IL‐6 production that was greater in hypoxic conditions with AA than with hypoxia alone. Finally, inhibition of IL‐6 production using IL‐6 antibody or soluble IL‐6 receptor attenuated the hypoxia‐induced increases in DNA synthesis of mouse ES cells. In conclusion, AA potentiates hypoxia‐induced IL‐6 production through the MAPKs, NF‐κB, and HIF‐1α pathways in mouse ES cells. J. Cell. Physiol. 222: 574–585, 2010. © 2009 Wiley‐Liss, Inc.     

Activation and induction of cytosolic phospholipase A2 by IL‐1β in human tracheal smooth muscle cells: Role of MAPKs/p300 and NF‐κB

Cytosolic phospholipase A₂ (cPLA₂) plays a pivotal role in mediating agonist‐induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by IL‐1β. However, the mechanisms underlying IL‐1β‐induced cPLA₂ expression and PGE₂ synthesis in human tracheal smooth muscle cells (HTSMCs) remain unknown. IL‐1β‐induced cPLA₂ protein and mRNA expression, PGE₂ production, or phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, which was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs of MEK1, p42, p38, and JNK2. IL‐1β‐induced cPLA₂ expression was also inhibited by pretreatment with a NF‐κB inhibitor, helenalin or transfection with siRNA of NIK, IKKα, or IKKβ. IL‐β‐induced NF‐κB translocation was blocked by pretreatment with helenalin, but not U0126, SB202190, and SP600125. In addition, transfection with p300 siRNA blocked cPLA₂ expression induced by IL‐1β. Moreover, p300 was associated with the cPLA₂ promoter, which was dynamically linked to histone H4 acetylation stimulated by IL‐1β. These results suggest that in HTSMCs, activation of MAPKs, NF‐κB, and p300 are essential for IL‐1β‐induced cPLA₂ expression and PGE₂ secretion. J. Cell. Biochem. 109: 1045–1056, 2010. © 2010 Wiley‐Liss, Inc.     

Involvement of endogenous hydrogen sulfide in cigarette smoke-induced changes in airway responsiveness and inflammation of rat lung

Hydrogen sulfide (H₂S), recently considered the third endogenous gaseous transmitter, may have an important role in systemic inflammation. We investigated whether endogenous H₂S may be a crucial mediator in airway responsiveness and airway inflammation in a rat model of chronic exposure to cigarette smoke (CS). Rats randomly divided into control and CS-exposed groups were treated with or without sodium hydrosulfide (NaHS, donor of H₂S) or propargylglycine (PPG, inhibitor of cystathionine-γ-lyase [CSE], an H₂S-synthesizing enzyme) for 4-month exposure. Serum H₂S level and CSE protein expression in lung tissue were higher, by 2.04- and 2.33-fold, respectively, in CS-exposed rats than in controls (P < 0.05). Exogenous administration of NaHS to CS-exposed rats alleviated airway reactivity induced by acetylcholine (Ach) or potassium chloride (KCl) by 17.4% and 13.8%, respectively, decreased lung pathology score by 32.7%, inhibited IL-8 and TNF- α concentrations in lung tissue by 34.2% and 31.4%, respectively, as compared with CS-exposed rats (all P < 0.05). However, blocking endogenous CSE with PPG in CS-exposed rats increased airway reactivity induced by Ach or KCl, by 24.1% and 24.5%, respectively, and aggravated lung pathology score, by 44.8%, as compared with CS-exposed rats (all P < 0.01). Incubation in vitro with NaHS, 1–3 mmol/L, relaxed rat tracheal smooth muscle precontracted by Ach or KCl. However, the NaHS-induced relaxation was not blocked by glibenclamide (10^?4 mol/L), L-NAME (10^?4 mol/L), or ODQ (1 μmol/L) or denudation of epithelium. Endogenous H₂S may have a protective role of anti-inflammation and bronchodilation in chronic CS-induced pulmonary injury.