Rosmarinic acid protects mice from lipopolysaccharide/d-galactosamine-induced acute liver injury by inhibiting MAPKs/NF-κB and activating Nrf2/HO-1 signaling pathways

Rosmarinic acid (RA) has antioxidation, anticancer, antibacterial, anti-inflammatory and various biological functions. In our study, we aim to evaluate effects of RA on acute liver injury caused by LPS and d-galactosamine (d-GalN) and its underlying molecular mechanism in mice. Our findings showed that RA could protect C57BL/6 mice from LPS/d-GalN-induced acute liver injury, which not only reflected on declining aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of the serum, but also restrained the phosphorylation of nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK1/2) and p38 protein expression and the content of tissue myeloperoxidase (MPO) elevation. Moreover, RA could enhance the level of glutathione-dependent peroxidase (GSH-PX). Furthermore, RA promoted that nuclear factor erythroid-2-related factor 2 (Nrf2) transported into nucleus, and then up-regulated heme oxygenase 1 (HO-1), glutamate-cysteine ligase catalytic (GCLC), glutamate cysteine ligase modifier (GCLM) and quinone oxidoreductase (NQO1). These results indicated that RA could protect the mice from acute liver injury induced by LPS/d-GalN.     

β-Patchoulene from patchouli oil protects against LPS-induced acute lung injury via suppressing NF-κB and activating Nrf2 pathways

β-Patchoulene (β-PAE), a tricyclic sesquiterpene isolated from the essential oil of the leaves and stems of Pogostemon cablin (Blanco) Benth., has been reported to have potent anti-inflammatory activity. The aim of this study was to evaluate the potential protective effect of β-PAE on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and to illuminate the underlying mechanisms. ALI was induced by intracheal instillation of LPS into lung, and dexamethasone (DEX) was used as a positive control. Results indicated that pretreatment with β-PAE significantly decreased the mortality rate of mice and lung W/D weight ratio, ameliorated lung pathological changes as compared to model group. Meanwhile, β-PAE pretreatment markedly inhibited the increase of TNF-α, IL-6 and IL-1β secretions in the bronchoalveolar lavage fluid, and prevented LPS-induced elevations of MPO activity and MDA level in the lung. Additionally, β-PAE pretreatment significantly elevated miR-146a expression and suppressed the LPS-induced activation of NF-κB and expression of its mediated genes (TNF-α, IL-6 and IL-1β). β-PAE was also observed to markedly upregulate the Nrf2 and HO-1 expression and activate the antioxidant genes (NQO-1, GCLC and HO-1). Taken together, β-PAE possessed protective effect against LPS-induced ALI, which might be associated with its differential regulation of NF-κB and Nrf2 activities and up-regulation of expression of miR-146a. The results rendered β-PAE a promising anti-inflammatory agent worthy of further development into a pharmaceutical drug for the treatment of ALI.     

Chlorogenic acid prevents diabetic nephropathy by inhibiting oxidative stress and inflammation through modulation of the Nrf2/HO-1 and NF-ĸB pathways

Oxidative and inflammatory damage have been suggested to play important roles in the pathogenesis of diabetic nephropathy (DN). Chlorogenic acid (CGA) is one of the most abundant polyphenols and has known immunoprotective, antioxidant and anti-inflammatory properties. In the present study, animal experiments were designed to examine the protective effects of CGA in DN, and cellular experiments were designed to explore the underlying mechanisms. CGA significantly attenuated diabetic renal damage based on histological pathology and molecular biological methods. Pre-treatment with CGA increased the nuclear translocation of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) and reduced the phosphorylation of IĸB and subsequent nuclear translocation of nuclear factor kappa beta (NF-ĸB). Nrf2 small interfering RNA (siRNA) and the HO-1 inhibitor zinc protoporphyrin (ZnPPIX) significantly increased the nuclear translocation of NF-ĸB and the production of pro-inflammatory cytokines in HBZY-1 cells. The NF-ĸB inhibitor pyrrolidine dithiocarbamate (PDTC) increased Nrf2 nuclear translocation and HO-1 expression and antioxidant levels. Thus, these results suggest that CGA is a potential therapeutic agent in the treatment of DN due to its antioxidant and anti-inflammatory effects which are mediated via the modulation of the Nrf2/HO-1 and NF-ĸB pathways. Moreover, CGA-induced the activation of Nrf2/HO-1,which interacts with the inhibition of NF-ĸB, as each begets and amplifies the other.     

Ginsenoside Rg1 protects primary cultured rat hippocampal neurons from cell apoptosis induced by β-amyloid protein

Objectives: Estrogen is known to prominently benefit neuronal syndromes and neurodegenerative diseases. Ginsenoside Rg1, an active ingredient found in a Chinese plant, ginseng root, was previously demonstrated to exert estrogen-like activity. This study was performed to assess the neuroprotective effect of ginsenoside Rg1 against apoptosis induced by β-amyloid protein 25–35 (Aβ_25–35) in primary cultured rat hippocampal neuronal cells as well as in the underlying mechanisms.

Methods: We first measured cell viability and lactate dehydrogenase (LDH) release from primary cultured rat hippocampal neurons. After that, the inhibition effects of ginsenoside Rg1 on neuronal cell apoptosis were evaluated with flow cytometric analysis. Furthermore, western blot analysis was used for detecting the expression of apoptosis-related proteins Bcl-2, Bax, and active caspase 3.

Results: The results show that ginsenoside Rg1 could increase neuronal viability and reduce LDH release; rescue cell apoptosis induced by Aβ_25–35; decrease the expression of caspase 3, increase the ratio of Bcl-2/Bax at the protein levels compared with the cells only treated with Aβ_25–35.

Conclusions: Taken together, our results indicate that the apoptosis induced by Aβ_25–35 could be reversed by ginsenoside Rg1. Furthermore, this neuroprotective effect is probably mediated by up-regulating the ratio of Bcl-2/Bax that activates caspase 3.     

Protective effects of morin on lipopolysaccharide/d-galactosamine-induced acute liver injury by inhibiting TLR4/NF-κB and activating Nrf2/HO-1 signaling pathways

Morin, a bioactive flavonoid extracted from the bark of Moraceae plants and many medicinal herbs, has anti-inflammatory and antioxidative effects. In this research, we explored the protective effects of morin against lipopolysaccharide (LPS) and d-galactosamine (D-GalN) induced acute liver injury in mice. Mice were given an intraperitoneal injection of morin before LPS and D-GalN treatment and the HepG2 cells were only given morin to investigate its effects. The results showed that morin markedly inhibited the production of serum alanine transaminase (ALT), aspartate aminotransferase (AST), interleukin-6 (IL-6), tumor necrosis factor (TNF-α) and hepatic TNF-α, IL-6, and myeloperoxidase (MPO) induced by LPS/D-GalN. In order to evaluate morin effect in the future, we investigated the expression of nuclear factor E2 related factor 2 (Nrf2), nuclear factor-kappaB (NF-κB), toll like receptor 4 (TLR4) on liver injury. Taken together, these results suggested that morin could exert the anti-inflammatory and anti-oxidative effects against LPS/D-GalN-induced acute liver injury by activating Nrf2 signal pathways and inhibiting NF-κB activation.     

2,3,4′,5-tetrahydroxystilbene-2-O-β-d-glucoside exerts anti-inflammatory effects on lipopolysaccharide-stimulated microglia by inhibiting NF-κB and activating AMPK/Nrf2 pathways

2,3,4′,5-Tetrahydroxystilbene-2-O-β-d-glucoside (THSG) affects neuroinflammation-related neurodegenerative diseases and inhibits neuroinflammatory mediators. However, the detailed impacts and underlying mechanisms of THSG on neuroinflammatory responses are still unclear. The aim of this study was to investigate the anti-neuroinflammatory mechanism of THSG via AMPK/Nrf2 signaling pathways. This study showed that THSG attenuated LPS-induced iNOS, COX-2, TNF-α, and IL-6 activation in microglia. Furthermore, it was observed that activation of IκBα and NF-κB was significantly increased upon LPS stimulation, and suppressed by THSG treatment in a dose-dependent manner. The expression of HO-1 and NQO1, as well as Nrf2 activation, was induced by THSG in microglia. The promoter activity of ARE and HO-1 also increased in a dose-dependent manner following THSG treatment. Nrf2/HO-1/NQO1 has anti-inflammatory properties; the knock-down of Nrf2/HO-1/NQO1 by specific siRNA prevented the THSG-mediated inhibition of iNOS and COX-2 promoter activity. Consistent with this concept, the phosphorylation of LKB1, CaMKII, and AMPK were elevated after THSG treatment. The blockade of AMPK by a pharmacological inhibitor prevented THSG-induced HO-1 and NQO1 expression. The anti-inflammatory properties of THSG were also reversed by treatment with an AMPK inhibitor. In conclusion, we demonstrated that THSG attenuates the LPS-induced neuroinflammatory response mediated by AMPK/Nrf2 signaling pathways.     

Amelioration of cyclosporine-induced testicular toxicity by carvedilol and/or alpha-lipoic acid: Role of TGF-β1, the proinflammatory cytokines,Nrf2/HO-1 pathway and apoptosis

Cyclosporine is an immunosuppressive agent that is used to prevent organ rejection after organ transplantation. Due to the widespread use of this type of surgery, the effect of cyclosporine on reproduction and fertility should have a specific interest. Our aim was to assess the effect of carvedilol and/or alpha-lipoic acid on cyclosporine-induced testicular toxicity in rats. Sixty male Wistar rats were divided into six equal groups: Control; cyclosporine; cyclosporine + carvedilol; cyclosporine + alpha-lipoic acid; cyclosporine + carboxymethyl cellulose; and cyclosporine + carvedilol +alpha-lipoic acid. Food intake, testis weight, testicular functions, serum testosterone, luteinizing hormone and follicle-stimulating hormone were measured. Also, testicular tissue 3 β-hydroxysteroid dehydrogenase, 17 β- hydroxysteroid dehydrogenase, paroxonase-1, proinflammatory cytokines, transforming growth factor beta-1, nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Heme oxygenase-1 (HO-1) content and sperm characteristics were determined. Parts of the testes were subjected to histopathological and electron microscopic examination. The carvedilol/alpha-lipoic acid combination restored the food intake, testicular weight and functions, sperm characteristics, hormonal profile and the antioxidant defences compared to the use of each of these drugs alone. Also, this combination significantly ameliorated inflammation (P < .05) and induced significant increase in tissue Nrf2/HO-1 content (P < .05) and significant improvement of the histopathological and electron microscopic picture (P < .05) compared to the use of each of these drugs alone. So, carvedilol/alpha-lipoic acid combination might represent a novel therapeutic strategy to ameliorate testicular damage induced by cyclosporine.     

Synergistic protection of Schizandrin B and Glycyrrhizic acid against bleomycin-induced pulmonary fibrosis by inhibiting TGF-β1/Smad2 pathways and overexpression of NOX4

Pulmonary fibrosis, a progressive and lethal lung disease, is a major therapeutic challenge for which new therapeutic strategies are warranted. Schisandrin B (Sch B) and Glycyrrhizic acid (GA) are the principal active ingredients of Schisandra chinensis and Glycyrrhiza glabra respectively, which have been reported to protect against lung injures. The present study was aimed at exploring the combinatorial therapeutic effects on bleomycin-induced pulmonary fibrosis. Lung fibrotic injuries were induced in mice by a single intratracheal instillation of 5 mg/kg bleomycin (BLM). Then, these mice were administered with Sch B (100 mg/kg) or/and GA (75 mg/kg) for 28 days. BLM-triggered structure distortion, collagen overproduction, excessive inflammatory infiltration, pro-inflammatory cytokine release, and oxidative stress damages in lung tissues were attenuated to a higher degree by combinatorial treatment than by treatment of the individual agents. The expression of TGF-β1 and the phosphorylation of its downstream target, Smad2 were enhanced by BLM, but weakened by Sch B or/and GA. Furthermore, the significant overexpression of NADPH oxidase 4 (NOX4) was observed in BLM-induced pulmonary fibrosis, which was inhibited by Sch B or/and GA. Our study reveals that the synergistic protection by Sch B and GA against BLM-induced pulmonary fibrosis is correlated to its anti-inflammatory, anti-oxidative and anti-fibrotic properties, involving inhibition of TGF-β1/Smad2 signaling pathways and overexpression of NOX4.     

SP protects cerebellar granule cells against β-amyloid-induced apoptosis by down-regulation and reduced activity of Kv4 potassium channels

The tachykinin endecapeptide substance P (SP) has been demonstrated to exert a functional role in neurodegenerative disorders, including Alzheimer's disease (AD). Aim of the present study was to evaluate the SP neuroprotective potential against apoptosis induced by the neurotoxic beta-amyloid peptide (Aβ) in cultured rat cerebellar granule cells (CGCs). We found that SP protects CGCs against both Aβ_25-35- and Aβ_1-42-induced apoptotic CGCs death as revealed by live/dead cell assay, Hoechst staining and caspase(s)-induced PARP-1 cleavage, through an Akt-dependent mechanism.Since in CGCs the fast inactivating or A-type K⁺ current (I_KA) was potentiated by Aβ treatment through up-regulation of Kv4 subunits, we investigated whether I_KA and the related potassium channel subunits could be involved in the SP anti-apoptotic activity.Patch-clamp experiments showed that the Aβ-induced increase of I_KA current amplitude was reversed by SP treatment. In addition, as revealed by Western blot analysis and immunofluorescence studies, SP prevented the up-regulation of Kv4.2 and Kv4.3 channel subunits expression.These results indicate that SP plays a role in the regulation of voltage-gated potassium channels in Aβ-mediated neuronal death and may represent a new approach in the understanding and treatment of AD.     

Ghrelin protects H9c2 cells from hydrogen peroxide-induced apoptosis through NF-κB and mitochondria-mediated signaling

Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. Herein we investigate the protective effects of ghrelin in H₂O₂-induced apoptosis of H9c2 cells, as well as the possible molecular mechanisms involved. To study apoptosis, the cells were assessed by morphologic examination, MTS assay, Annexin V–propidium iodide dual staining and TUNEL analysis. Intracellular reactive oxygen species (ROS) production and mitochondrial membrane potential were also measured. To investigate the underlying molecular mechanisms, the expression of Bcl-2, Bax, active caspase-9 and NF-κB were assessed by Western blotting, and caspase-3 activity was determined by a colorimetric activity assay kit. After stimulation with H₂O₂ for 18 h, H9c2 cells viability decreased significantly; a large fraction of cells underwent apoptosis. We observed a dose-dependent rescue of H9c2 cells from H₂O₂-induced apoptosis in the presence of different ghrelin concentrations. Preincubation with ghrelin also restored the ROS and mitochondrial membrane potential levels that had been altered by H₂O₂ treatment. Moreover, ghrelin decreased H₂O₂-induced Bax production and caspase-9 activation, and increased Bcl-2 levels. NF-κB phosphorylation was also significantly inhibited by ghrelin in H₂O₂-treated cells. Caspase-3 activation was suppressed by ghrelin in H₂O₂-treated H9c2 cells in a dose-dependent manner. In summary, ghrelin protects H9c2 cells from oxidative stress-induced apoptosis through downregulation of Bax expression, caspase-9 activation and NF-κB phosphorylation, and upregulation of Bcl-2 expression. Caspase-3 activation was also reduced in a dose-dependent manner. These data suggest that ghrelin might protect against cardiovascular disease by protecting the mitochondria.     

Procyanidin B2 protects H2O2-induced oxidative damage in PC12 cells by regulating PI3K/Akt and Nrf2/HO-l signaling pathways北大核心CSCD

Aim To explore the protective effect of proanthocyanidin B2 (PC-B2) on oxidative damage of PC 12 cells induced by hydrogen peroxide (H2 O2 ) and the related mechanism. Methods PC12 cells were treated with H2 O2 (200 μmol • L -1) for 24 h to establish oxidative damage model in vitro, and were randomly divided into normal group, normal + PC-B2 group, H2O2-induced model group and H2 O2 +PC-B2 group. Cell proliferation and cytotoxicity in each group were detected by CCK-8 and LDH assays, respectively. The expressions of MDA, SOD, CAT and GSH-Px were detected by ELISA kit. Cell apoptosis was observed by TUNEL staining. The expressions of apoptosis-related factors Bax, Bcl-2, caspase-3, p-PI3 K, p-Akt, PI3 K/Akt and Nrf2/HO-l pathway were detected by RT-PCR and Western blot. Results Compared with the normal group, the cell proliferation ability of the H2 O2 model group decreased, the contents of LDH and MDA increased, the contents of SOD, CAT and GSH-Px decreased, and the apoptosis was aggravated. After PCB2 intervention, the proliferation ability of PC 12 cells increased, the contents of LDH and MDA decreased, and the contents of SOD, CAT and GSH-Px increased. PC-B2 effectively inhibited the apoptosis of PC12 cells and up-regulated the expression of PI3K/Akt and Nrf2/HO-l proteins. Conclusions PC-B2 can protect PC12 cells from oxidative damage induced by H2 O2 and improve the apoptosis of PC 12 cells. The mechanism may be related to the activation of PI3K/Akt and Nrf2/HO-l signaling pathways. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Downregulation of NO and PGE2 in LPS-stimulated BV2 microglial cells by trans-isoferulic acid via suppression of PI3K/Akt-dependent NF-κB and activation of Nrf2-mediated HO-1

Little is known about whether trans-isoferulic acid (TIA) regulates the production of lipopolysaccharide (LPS)-induced proinflammatory mediators. Therefore, we examined the effect of TIA isolated from Clematis mandshurica on LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in BV2 microglial cells. We found that TIA inhibited the production of LPS-induced NO and PGE₂ without accompanying cytotoxicity in BV2 microglial cells. TIA also downregulated the expression levels of specific regulatory genes such as inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) by suppressing LPS-induced NF-κB activity via dephosphorylation of PI3K/Akt. In addition, we demonstrated that a specific NF-κB inhibitor PDTC and a selective PI3K/Akt inhibitor, LY294002 effectively attenuated the expression of LPS-stimulated iNOS and COX-2 mRNA, while LY294002 suppressed LPS-induced NF-κB activity, suggesting that TIA attenuates the expression of these proinflammatory genes by suppressing PI3K/Akt-mediated NF-κB activity. Our results showed that TIA suppressed NO and PGE₂ production through the induction of nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent heme oxygenase-1 (HO-1). Taken together, our data indicate that TIA suppresses the production of proinflammatory mediators such as NO and PGE₂, as well as their regulatory genes, in LPS-stimulated BV2 microglial cells, by inhibiting PI3K/Akt-dependent NF-κB activity and enhancing Nrf2-mediated HO-1 expression.     

Regulation of Nrf2 and NF-κB activities may contribute to the anti-inflammatory mechanism of xylopic acid

Inflammopharmacology - Xylopic acid (XA) is a kaurene diterpene which naturally exists in African plants such as Xylopia aethiopica. It has been established to exhibit acute and chronic...     

Differential regulation of somatostatin receptor dephosphorylation by β-arrestin1 and β-arrestin2

Signaling of G protein-coupled receptors (GPCRs) is tightly regulated by coordinated phosphorylation of intracellular serine and threonine residues. Although the mechanisms of agonist-induced phosphorylation have been deciphered for many GPCRs, the regulation of their dephosphorylation remains poorly understood. Using a combination of siRNA knockdown screening and phosphosite-specific antibodies, we have recently identified the catalytic subunit β of protein phosphatase 1 (PP1β) as major constituent of the GPCR phosphatase responsible for dephosphorylation of the sst₂ somatostatin receptor. However, PP1-targeting subunits specifically required for GPCR dephosphorylation have not been identified so far. Here, we show that siRNA knockdown of β-arrestin1 strongly inhibits sst₂ receptor dephosphorylation. Co-immunoprecipitation experiments demonstrate that β-arrestin1 and PP1β exist as constitutive complex that mediates rapid dephosphorylation of sst₂ receptors at or near the plasma membrane. By contrast, β-arrestin2 is not essential for rapid sst₂ receptor dephosphorylation. Together, these findings reveal a novel scaffolding function of β-arrestin1 that facilitates efficient targeting of PP1β to phosphorylated GPCRs.     

In vitro inhibition of CYP2B1 monooxygenase by β-myrcene and other monoterpenoid compounds

β-myrcene (MYR) is an acyclic monoterpene found in the essential oils of several useful plants such as lemongrass (Cymbopogon citratus), hop, bay, verbena and others. Recently it has been reported that MYR as well as lemongrass oil blocked the metabolic activation of some promutagens (e.g., cyclophosphamide and aflatoxin B1) in in vitro genotoxicity assays. The present study was performed to evaluate the inhibitory effects of MYR and some other monoterpenoid compounds on microsomal enzymes involved in the activation of genotoxic substances. The effects of MYR and other monoterpenes on the activity of pentoxyresorufin-O-depenthylase (PROD), a selective marker for CYP2B1, was determined in a pool of liver microsomes prepared from phenobarbital-treated rats. The effect of MYR on the activity of ethoxyresorufin-O-deethylase (EROD), a marker for CYP4501A1, was investigated in liver microsomes of untreated rats. Results revealed that MYR had almost no effect on EROD (IC₅₀>50 μM), but produced a concentration-dependent inhibition of PROD activity (IC₅₀=0.14 μM). The analysis of alterations produced by MYR on PROD kinetic parameters (Lineweaver-Burk plot) suggested that inhibition is competitive (K_i=0.14 μM). The inhibitory effects of seven other monoterpenes on PROD activity (pentoxyresorufin 5 μM) were also studied and the IC₅₀ were as follows: (−)-α-pinene, 0.087 μM; (+)-α-pinene, 0.089 μM; d-limonene, 0.19 μM; α-terpinene, 0.76 μM; citral, 1.19 μM; citronellal, 1.56 μM, and (±) camphor, 7.89 μM. The potent inhibitory effects on CYP4502B1 suggest that MYR, and other monoterpenes, interfere with the metabolism of xenobiotics which are substrates for this isoenzyme.     

Long-term in vitro treatment of INS-1 rat pancreatic β-cells by unsaturated free fatty acids protects cells against gluco- and lipotoxicities via activation of GPR40 receptors

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