Dimethyl fumarate protects thioacetamide‐induced liver damage in rats: Studies on Nrf2, NLRP3, and NF‐κB

The present study was designed to investigate the hepatoprotective potential of dimethyl fumarate (DMF) against thioacetamide (TAA)‐induced liver damage. Wistar rats were treated with DMF (12.5, 25, and 50 mg/kg/day, orally) and TAA (200 mg/kg intraperitoneally, every third day) for 6 consecutive weeks. TAA exposure significantly reduced body weight, increased liver weight and index, and intervention with DMF did not ameliorate these parameters. DMF treatment significantly restored TAA‐induced increase in the levels of aspartate aminotransferase, alanine aminotransferase, γ‐glutamyl transferase, total bilirubin, uric acid, malondialdehyde, reduced glutathione, and histopathological findings such as inflammatory cell infiltration, deposition of collagen, necrosis, and bridging fibrosis. DMF treatment significantly ameliorated TAA‐induced hepatic stellate cell activation, increase in inflammatory cascade markers (NACHT, LRR, and PYD domains‐containing protein 3; NLRP3, apoptosis‐associated speck like protein containing a caspase recruitment domain; ASC, caspase‐1, nuclear factor‐kappa B; NF‐κB, interleukin‐6), fibrogenic makers (α‐smooth muscle actin; ɑ‐SMA, transforming growth factor; TGF‐β1, fibronectin, collagen 1) and antioxidant markers (nuclear factor (erythroid‐derived 2)‐like factor 2; Nrf2, superoxide dismutase‐1; SOD‐1, catalase). The present findings concluded that DMF protects against TAA‐induced hepatic damage mediated through the downregulation of inflammatory cascades and upregulation of antioxidant status.     

Hepatoprotective effect of Herpetospermum caudigerum Wall. on carbon tetrachloride‐induced hepatic fibrosis in rats

Tibetan medicine Herpetospermum caudigerum Wall. (HCW) has long been employed to treat hepatitis, inflammatory diseases and jaundice according to the records of “The Four Medical Tantras” in China. This study was investigated to explore the protective effects of HCW on hepatic fibrosis and the possible mechanism in a rat model. Hepatic fibrosis was established by intragastric administration of 3 ml/kg carbon tetrachloride (CCl₄) twice a week for 6 weeks. CCl₄‐treated rats were received HCW (1 and 3 g/kg/d) and silymarin (0.1 g/kg/d) from 3 to 6 weeks. The results showed that HCW could significantly decrease the levels of AST, ALT, HA, LN, PCIII, Col IV, TNF‐α, IL‐1β and IL‐6. Moreover, HCW could effectively inhibit collagen deposition and reduce the pathological damage. Analysis experiments finally exhibited that HCW was able to markedly inhibit hepatic fibrosis by modulating the expressions of NF‐κB p65, IκBα, Samd3 and TGF‐β1 proteins. Therefore, our results suggest that HCW has hepatoprotective activity against CCl₄‐induced hepatic fibrosis in rats by regulating the inflammatory responses.     

Lophirones B and C halt acetaminophen hepatotoxicity by upregulating redox transcription factor Nrf‐2 through Akt,PI3K,and PKC pathways

We investigated the mechanism of lophirones B‐ and C‐mediated protection against acetaminophen hepatotoxicity. Mice were pretreated with 20 mg/kg body weight lophirones B and C for 7 days and challenged with acetaminophen on day 7. Acetaminophen raised nuclear factor‐κB (NF‐κB) in the liver of mice but lowered protein kinase B (Akt). Although, acetaminophen produced no significant alteration on nuclear erythroid related factor‐2 (Nrf‐2), phosphoinositide 3‐kinase (PI3K) and protein kinase C (PKC), lophirones B and C raised the level of these proteins and Akt. The acetaminophen‐mediated increase in NF‐κB was significantly reversed by lophirones B and C. Lophirones B and C prevented acetaminophen‐mediated alterations in serum biomarkers of hepatic injury. Similarly, lophirones B and C lowered the biomarkers of oxidative stress in the liver of acetaminophen‐treated mice. It can be inferred from this study that lophirones B and C prevent acetaminophen‐induced liver injury by enhancing Nrf‐2 through Akt, PI3K, and PKC pathways.     

Critical role of Toll‐like receptor 2 in Bacteroides fragilis‐mediated immune responses in murine peritoneal mesothelial cells

In this study, the role of Toll‐like receptor 2 (TLR2) in immune responses of murine peritoneal mesothelial cells against Bacteroides fragilis was investigated. Enzyme linked immunosorbent assay was used to measure cytokines and chemokines. Activation of nuclear factor κB (NF‐κB‐α) and mitogen‐activated protein kinases (MAP kinases) was investigated by western blot analysis. B. fragilis induced production of interleukin‐6, chemokine (C‐X‐C motif) ligand 1 (CXCL1) and chemokine (C‐C motif) ligand 2 (CCL2) in wild type peritoneal mesothelial cells; this was impaired in TLR2‐deficient cells. In addition, in response to B. fragilis, phosphorylation of inhibitory NF‐κB‐α and c‐Jun N‐terminal kinase mitogen‐activated protein kinase (MAPK) was induced in wild type mesothelial cells, but not in TLR2‐deficient cells,. Inhibitor assay revealed that NF‐κB and MAPKs are essential for B. fragilis‐induced production of CXCL1 and CCL2 in mesothelial cells. These findings suggest that TLR2 mediates immune responses in peritoneal mesothelial cells in response to B. fragilis.     

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.     

Dl‐3‐n‐butylphthalide attenuates acute inflammatory activation in rats with spinal cord injury by inhibiting microglial TLR4/NF‐κB signalling

In this study, we examined the neuroprotective effects and anti‐inflammatory properties of Dl‐3‐n‐butylphthalide (NBP) in Sprague‐Dawley (SD) rats following traumatic spinal cord injury (SCI) as well as microglia activation and inflammatory response both in vivo and in vitro. Our results showed that NBP improved the locomotor recovery of SD rats after SCI an significantly diminished the lesion cavity area of the spinal cord, apoptotic activity in neurons, and the number of TUNEL‐positive cells at 7 days post‐injury. NBP inhibited activation of microglia, diminished the release of inflammatory mediators, and reduced the upregulation of microglial TLR4/NF‐κB expression at 1 day post‐injury. In a co‐culture system with BV‐2 cells and PC12 cells, NBP significantly reduced the cytotoxicity of BV‐2 cells following lipopolysaccharide (LPS) stimulation. In addition, NBP reduced the activation of BV‐2 cells, diminished the release of inflammatory mediators, and inhibited microglial TLR4/NF‐κB expression in BV‐2 cells. Our findings demonstrate that NBP may have neuroprotective and anti‐inflammatory properties in the treatment of SCI by inhibiting the activation of microglia via TLR4/NF‐κB signalling.     

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).     

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.     

Reactive oxygen species are involved in the IFN‐γ‐stimulated production of Th2 chemokines in HaCaT keratinocytes

The increased generation of reactive oxygen species (ROS) induces inflammation in different cell types. However, it is unclear whether ROS play an essential role in the production of thymus and activation‐regulated chemokine (TARC/CCL17) and macrophage‐derived chemokine (MDC/CCL22) in keratinocytes. Here, we investigated the function of ROS in the production of these two Th2 chemokines in interferon‐gamma (IFN‐γ)‐treated HaCaT keratinocytes. We found that IFN‐γ‐induced production of both chemokines in parallel with the increased generation of intracellular ROS. A ROS scavenger, N‐acetyl cysteine (NAC), significantly inhibited the IFN‐γ‐induced production of chemokines as well as the activation of I kappa‐B (IκB)–nuclear factor‐kappa B (NF‐κB). Inhibitors of Janus family kinases (JAKs), p38 mitogen‐activated kinase (MAPK), and NF‐κB suppressed IFN‐γ‐induced production of TARC and MDC. NF‐κB activation was inhibited by both inhibitors of JAKs and p38 MAPK. Importantly, IFN‐γ‐stimulated phosphorylation of p38 MAPK was significantly suppressed by JAKs inhibitors, but not significantly affected by NAC or L ‐buthionine sulfoximine (L‐BSO). However, IFN‐γ‐stimulated activation of IκB and NF‐κB was suppressed by NAC but enhanced by BSO. Furthermore, inhibition of p38 MAPK and JAKs did not affect ROS generation in IFN‐γ‐stimulated HaCaT cells. These results indicate that intracellular ROS and JAKs/p38 MAPK both contribute independently to IFN‐γ‐stimulated production of TARC and MDC in HaCaT keratinocytes, by increasing NF‐κB activation. J. Cell. Physiol. 226: 58–65, 2010. © 2010 Wiley‐Liss, Inc.     

RTP801 Regulates Maneb‐ and Mancozeb‐Induced Cytotoxicity via NF‐κB

Environmental factors have been implicated in the pathogenesis of neurodegenerative diseases. Maneb (MB) and mancozeb (MZ) have been extensively used as pesticides. Exposure to MB lowers the threshold for dopaminergic damage triggered by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine. MB and MZ potentiate 1‐methyl‐4‐phenylpyridium (MPP⁺)‐induced cytotoxicity in rat pheochromocytoma (PC12) cells partially via nuclear factor kappa B (NF‐κB) activation. RTP801 dramatically increased by oxidative stresses and DNA damage is the possible mechanism of neurotoxins‐induced cell death in many studies. This study demonstrated that MB and MZ induced DNA damage as seen in comet assay. The expressions of RTP801 protein and mRNA were elevated after MB and MZ exposures. By knocking down RTP801 using shRNA, we demonstrated that NF‐κB activation by MB and MZ was regulated by RTP801 and cell death triggered by MB and MZ was associated with RTP801 elevation. This revealed that the toxic mechanisms of dithiocarbamates are via the cross talk between RTP801 and NF‐κB.     

Naringin prevents ultraviolet‐B radiation‐induced oxidative damage and inflammation through activation of peroxisome proliferator‐activated receptor γ in mouse embryonic fibroblast (NIH‐3T3) cells

The present study, we investigate the preventive role of naringin, a dietary flavonoid, against ultraviolet‐B (UVB) radiation (280‐320 nm) induced oxidative damage and inflammatory responses in mouse embryonic fibroblast cell lines (NIH‐3T3). In this study, 20 mJ/cm ² of UVB radiation induces cell cytotoxicity, reactive oxygen species (ROS) generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Treatment with naringin (60 µM) prior UVB exposure prevented the cell cytotoxicity, ROS generation, DNA damage, and antioxidants depletion in NIH‐3T3 cells. Furthermore, naringin prevents UVB‐induced mitogen‐activated protein kinase families and nuclear factor‐κB (NF‐κB)‐mediated activation of inflammatory factors, that is TNF‐α, IL‐6, IL‐10, and COX‐2 in NIH‐3T3 cells. Peroxisome proliferator‐activated receptor γ (PPARγ) is an anti‐inflammatory agent and it suppressed the UVB‐mediated oxidative and inflammatory responses. In this study, naringin activates PPARγ and prevents inflammatory biomarkers in NIH‐3T3 cells. Thus, naringin prevents UVB‐mediated inflammation and oxidative damage in NIH‐3T3 cells probably over controlling NF‐κB expression and activation of PPARγ.     

Hepatoprotective effect of bis(4‐methylbenzoyl) diselenide against CCl4‐induced oxidative damage in mice

From a pharmacological point of view, organoseleniums are compounds with important and interesting antioxidant and biological activities. The aim of this study was to evaluate the hepatoprotective effect of bis(4‐methylbenzoyl) diselenide (BMD) against carbon tetrachloride (CCl₄)–induced oxidative damage in mice. The animals received BMD (25 mg/kg p.o., for 3 days), and after 1 day, CCl₄ (1 mg/kg body weight) was administered by intraperitoneal route. One day after the CCl₄ exposure, the animals were euthanized for biochemical and histological analysis. Treatment with BMD (25 mg/kg p.o.) protected against aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma‐glutamyl transferase and lactate dehydrogenase activity increases induced by CCl₄ plasma exposure. Treatment with BMD (25 mg/kg) protected against increases in thiobarbituric reactive species and decreasing non‐protein thiols and ascorbic acid levels in liver of mice. Catalase and superoxide dismutase activity inhibition in the liver caused by CCl₄ were protected by treatment with BMD (25 mg/kg). Glutathione S‐transferase activity was inhibited by CCl₄ and remained unaltered even after treatment with BMD. Sections of liver from CCl₄‐exposed mice presented an intense infiltration of inflammatory cells and loss of the cellular architecture. BMD (25 mg/kg) attenuated CCl₄‐induced hepatic histological alterations. The results demonstrated the hepatoprotective effects of BMD in the mouse liver, possibly by modulating the antioxidant status. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Aldehyde dehydrogenase 2 activation ameliorates CCl4‐induced chronic liver fibrosis in mice by up‐regulating Nrf2/HO‐1 antioxidant pathway

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is critical in the pathogenesis of alcoholic liver cirrhosis. However, the effect of ALHD2 on liver fibrosis remains to be further elucidated. This study aimed to demonstrate whether ALDH2 regulates carbon tetrachloride (CCl₄)‐induced liver fibrosis and to investigate the efficacy of Alda‐1, a specific activator of ALDH2, on attenuating liver fibrosis. ALDH2 expression was increased after chronic CCl₄ exposure. ALDH2 deficiency accentuated CCl₄‐induced liver fibrosis in mice, accompanied by increased expression of collagen 1α1, α‐SMA and TIMP‐1. Moreover, ALDH2 knockout triggered more ROS generation, hepatocyte apoptosis and impaired mitophagy after CCl₄ treatment. In cultured HSC‐T6 cells, ALDH2 knockdown by transfecting with lentivirus vector increased ROS generation and α‐SMA expression in an in vitro hepatocyte fibrosis model using TGF‐β1. ALDH2 overexpression by lentivirus or activation by Alda‐1 administration partly reversed the effect of TGF‐β1, whereas ALDH2 knockdown totally blocked the protective effect of Alda‐1. Furthermore, Alda‐1 administration protected against liver fibrosis in vivo, which might be mediated through up‐regulation of Nrf2/HO‐1 cascade and activation of Parkin‐related mitophagy. These findings indicate that ALDH2 deficiency aggravated CCl₄‐induced hepatic fibrosis through ROS overproduction, increased apoptosis and mitochondrial damage, whereas ALDH2 activation through Alda‐1 administration alleviated hepatic fibrosis partly through activation of the Nrf2/HO‐1 antioxidant pathway and Parkin‐related mitophagy, which indicate ALDH2 as a promising anti‐fibrotic target and Alda‐1 as a potential therapeutic agent in treating CCl₄‐induced liver fibrosis.