The present study aimed to explore the effect of nuclear factor erythroid 2 related factor 2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway in intestinal protection by Sishen Pills against ulcerative colitis(UC). After the UC model was induced by 3% dextran sodium sulfate(DSS), experimental animals were randomly divided into control group, model group, salazosulfapyridine(SASP) group, and low-and high-dose Sishen Pills groups. Drug intervention(ig) was performed for seven consecutive days during modeling. On the 7 th day, the mice were euthanized. The body weight and colon length were recorded, and the histopathological changes of the colon were observed by HE staining. Serum interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), total antioxidant capacity(T-AOC), malondialdehyde(MDA), and reactive oxygen species(ROS) were detected by ELISA. The protein and mRNA expression of Nrf2, HO-1, and NADPH quinine oxidoreductase-1(NQO-1) was determined by Western blot and reverse transcription-polymerase chain reaction(RT-PCR). Compared with the normal group, the model group exhibited reduced body weight, colon length, and T-AOC, increased IL-6, TNF-α, MDA, and ROS, and diminished protein and mRNA expression of Nrf2, HO-1, and NQO-1 in the colon tissues. Compared with the model group, the SASP group and high-dose Sishen Pills group showed elevated body weight, colon length, and T-AOC, lowered IL-6, TNF-α, MDA, and ROS levels, and increased protein and mRNA expression of Nrf2, HO-1, and NQO-1 in the colon tissues. As assessed by HE staining, Sishen Pills could improve the pathological changes of the colon. The findings suggested that Sishen Pills could protect the colon against UC induced by 3% DSS. The specific mechanism of action may be related to the anti-inflammatory and anti-oxidative stress effects by the activation of the Nrf2/HO-1 signaling pathway.
Animals ; Colitis, Ulcerative/genetics* ; Dextran Sulfate ; Heme Oxygenase-1/metabolism* ; Mice ; NF-E2-Related Factor 2/metabolism* ; Signal Transduction

Periodontal disease is associated with chronic oxidative stress and inflammation. Caffeic acid phenethyl ester (CAPE), which is a potent inducer of heme oxygenase 1 (HO1), is a central active component of propolis, and the application of propolis improves periodontal status in diabetic patients. Here, primary murine macrophages were exposed to CAPE. Target gene expression was assessed by whole-genome microarray, RT-PCR and Western blotting. The antioxidative and anti-inflammatory activities of CAPE were examined by exposure of the cells to hydrogen peroxide, saliva and periodontal pathogens. The involvement of HO1 was investigated with the HO1 inhibitor tin protoporphyrin (SnPP) and knockout mice for Nrf2, which is a transcription factor for detoxifying enzymes. CAPE increased HO1 and other heat shock proteins in murine macrophages. A p38 MAPK inhibitor and Nrf2 knockout attenuated CAPE-induced HO1 expression in macrophages. CAPE exerted strong antioxidative activity. Additionally, CAPE reduced the inflammatory response to saliva and periodontal pathogens. Blocking HO1 decreased the antioxidative activity and attenuated the anti-inflammatory activity of CAPE. In conclusion, CAPE exerted its antioxidative effects through the Nrf2-mediated HO1 pathway and its anti-inflammatory effects through NF-κB inhibition. However, preclinical models evaluating the use of CAPE in periodontal inflammation are necessary in future studies.
Animals ; Caffeic Acids ; pharmacology ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Inflammation ; drug therapy ; Mice ; NF-kappa B ; antagonists & inhibitors ; genetics ; metabolism ; Oxidative Stress ; drug effects ; Phenylethyl Alcohol ; analogs & derivatives ; pharmacology

To explore the effect of epigallocatechin gallate (EGCG) on oxidative stress and Nrf2/HO-1 pathway in neurons subjected to oxygen-glucose deprivation/reperfusion (OGD/R).
 Methods: Primary cultured cerebral cortical neurons were prepared from Sprague-Dawley rats, and the OGD/R cell model was established. After pretreatment with EGCG at different concentrations (12.5, 25.0, 50.0 or 100.0 μmol/L), the neurons were subjected to OGD/R. The cell viability, reactive oxygen species (ROS) level and malondialdehyde (MDA) content were assessed after reperfusion. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were measured. The expression of Nrf2 protein in nucleus, HO-1 mRNA and protein were detected.
 Results: OGD/R treatment reduced the cell viability, elevated ROS level and MDA content, decreased SOD and GSH-Px activities. The expression of Nrf2 protein in nucleus, HO-1 mRNA and protein were increased (P<0.01). Pretreatment with EGCG promoted the survival of neurons exposed to OGD/R, decreased ROS level and MDA content while increased SOD and GSH-Px activities. The levels of Nrf2 protein in nucleus, HO-1 mRNA and protein were upregulated (P<0.01).
 Conclusion: EGCG can reduce the oxidative stress of neurons subjected to OGD/R, which may be related to activation of Nrf2/HO-1 signal pathway and enhancement of the antioxidant ability of neurons.
Animals ; Catechin ; analogs & derivatives ; pharmacology ; Cell Survival ; drug effects ; Cells, Cultured ; Gene Expression Regulation ; drug effects ; Glucose ; Heme Oxygenase-1 ; genetics ; metabolism ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Neurons ; drug effects ; Neuroprotective Agents ; pharmacology ; Oxidative Stress ; drug effects ; Oxygen ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control

The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 μmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling.
8,11,14-Eicosatrienoic Acid ; analogs & derivatives ; metabolism ; pharmacology ; Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Apoptosis ; drug effects ; Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Atherosclerosis ; genetics ; metabolism ; pathology ; Catalase ; genetics ; metabolism ; Cytochrome P-450 CYP2C8 ; genetics ; metabolism ; Gene Expression Regulation ; Heme Oxygenase-1 ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Models, Biological ; NADPH Oxidase 2 ; NADPH Oxidases ; genetics ; metabolism ; NF-E2-Related Factor 2 ; antagonists & inhibitors ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Reactive Oxygen Species ; antagonists & inhibitors ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism ; pharmacology

Heme oxygenase-1 (HO-1) plays important roles in anti-oxidant, anti-inflammatory and immunoregulative activities. The aim of this study was to observe if HO-1 transfection could inhibit the damage of osteoblasts induced by ethanol. HO-1 was transfected into osteoblasts via constructed plasmid. After exposure to ethanol for 24 h, cytoactivity and apoptosis of osteoblasts were measured by MTT assay and flow cytometry, respectively. Furthermore, the oxidative stress and inflammatory factors in osteoblasts were measured. Compared to positive control group, the cytoactivity of transfected osteoblasts was significantly increased, and the apoptosis rate was significantly decreased (P<0.05). At the same time, the levels of reactive oxygen species (ROS), methane dicarboxylic aldehyde (MDA), tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) were significantly decreased (P<0.05), and superoxide dismutase (SOD) level was increased (P<0.05) in the transfected osteoblasts as compared with positive controls. These results suggest that HO-1 plays a protective role in osteoblasts, and HO-1 transfection can effectively inhibit bone damage induced by ethanol.
Cells, Cultured ; Ethanol ; toxicity ; Gene Expression Regulation ; drug effects ; Genetic Vectors ; pharmacology ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Osteoblasts ; cytology ; drug effects ; Oxidative Stress ; drug effects ; Transfection

Bucillamine is used for the treatment of rheumatoid arthritis. This study investigated the protective effects of bucillamine against cisplatin-induced damage in auditory cells, the organ of Corti from postnatal rats (P2) and adult Balb/C mice. Cisplatin increases the catalytic activity of caspase-3 and caspase-8 proteases and the production of free radicals, which were significantly suppressed by pretreatment with bucillamine. Bucillamine induces the intranuclear translocation of Nrf2 and thereby increases the expression of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GSS), which further induces intracellular antioxidant glutathione (GSH), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2). However, knockdown studies of HO-1 and SOD2 suggest that the protective effect of bucillamine against cisplatin is independent of the enzymatic activity of HO-1 and SOD. Furthermore, pretreatment with bucillamine protects sensory hair cells on organ of Corti explants from cisplatin-induced cytotoxicity concomitantly with inhibition of caspase-3 activation. The auditory-brainstem-evoked response of cisplatin-injected mice shows marked increases in hearing threshold shifts, which was markedly suppressed by pretreatment with bucillamine in vivo. Taken together, bucillamine protects sensory hair cells from cisplatin through a scavenging effect on itself, as well as the induction of intracellular GSH.
Animals ; Antioxidants/*metabolism/*pharmacology ; Apoptosis/drug effects ; Caspase 3/metabolism ; Caspase 8/metabolism ; Cell Line ; Cisplatin/*toxicity ; Cysteine/*analogs & derivatives/pharmacology ; Gene Expression Regulation/*drug effects ; Gene Knockdown Techniques ; Glutathione/*metabolism ; Heme Oxygenase-1/genetics ; Intracellular Space/metabolism ; Male ; Metabolic Detoxication, Phase II/genetics ; Mice ; NF-E2-Related Factor 2/genetics ; Nitric Oxide/biosynthesis ; Organ of Corti/*drug effects/*metabolism ; RNA Interference ; Rats ; Reactive Oxygen Species/metabolism ; Superoxide Dismutase/genetics

OBJECTIVETo investigate whether emodin exerts protective effects on mouse with allergic asthma.

METHODSA mouse model of allergic airway inflflammation was employed. The C57BL/6 mice sensitized and challenged with ovalbumin (OVA) were intraperitoneally administered 10 or 20 mg/kg emodin for 3 days during OVA challenge. Animals were sacrificed 48 h after the last challenge. Inflammatory cell count in the bronchoalveolar lavage fluid (BALF) was measured. The levels of interleukin (IL)-4, IL-5, IL-13 and eotaxin in BALF and level of immunoglobulin E (IgE) in serum were measured with enzyme-linked immuno sorbent assay kits. The mRNA expressions of IL-4, IL-5, heme oxygenase (HO)-1 and matrix metalloproteinase-9 (MMP-9) were determined by real-time quantitative polymerase chain reaction.

RESULTSEmodin induced significant suppression of the number of OVA-induced total inflammatory cells in BALF. Treatment with emodin led to significant decreases in the levels of IL-4, IL-5, IL-13 and eotaxin in BALF and total IgE level in serum. Histological examination of lung tissue revealed marked attenuation of allergen-induced lung eosinophilic inflammation. Additionally, emodin suppressed IL-4, IL-5 and MMP-9 mRNA expressions and induced HO-1 mRNA expression.

CONCLUSIONEmodin exhibits anti-inflammatory activity in the airway inflammation mouse model, supporting its therapeutic potential for the treatment of allergic bronchial asthma.

Animals ; Bronchoalveolar Lavage Fluid ; cytology ; Chemokines ; metabolism ; Disease Models, Animal ; Emodin ; chemistry ; pharmacology ; therapeutic use ; Female ; Gene Expression Regulation ; drug effects ; Heme Oxygenase-1 ; genetics ; metabolism ; Immunoglobulin E ; blood ; Interleukins ; genetics ; metabolism ; Leukocytes ; drug effects ; metabolism ; Lung ; drug effects ; metabolism ; pathology ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mice, Inbred C57BL ; Ovalbumin ; Pneumonia ; blood ; drug therapy ; pathology ; Protective Agents ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism

Paeoniflorin is the main active ingredient of Chinese herbaceous peony. This study is to investigate the protective effect of paeoniflorin (Pae) on acute brain damage induced by lipopolysaccharide (LPS) in mice. The mice were randomly assigned to the normal control, model control (LPS), as well as groups of paeoniflorin and lipopolysaccharide (Pae + LPS). Then the mice were administered intraperitioneally with normal saline or Pae (10, 30 mg · kg(-1)) once daily for 6 d. One hour after intrapertioneally treatment on the seventh day, each group were injected LPS (5 mg · kg(-1)) to establish the endotoxin lipopolysaccharide inflammation model except the normal group. The mice were sacrificed after 6 h and the brain homogenates were prepared and measured. The malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), hydrogen peroxide (H2O2), succinatedehydrogenase (SDH), Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase were dectected by the colorimetric method. The levels of HO-1 and Nrf2 protein in subcellular fractions of brain tissue were detected by Western blot. The results demonstrated that the administration with paeoniflorin reduced the levels of the MDA production; significantly increase the activities of antioxidant enzyme (SOD and GSH-PX). In addition, paeoniflorin could enhance the total antioxidant capacity, decrease the level of H2O2, and increase the activities of SDH, Na(+)-K(+)-ATPase and Ca(2+)-Mg(2+)-ATPase. Furthermore, paeoniflorin can increase the expression of HO-1 and activate the nuclear transfer of Nrf2. Taking together, these findings suggest that paeoniflorin alleviate the acute inflammation in mice brain damage induced by LPS, which is related with its antioxidant effect and improvement of energy metabolism.
Animals ; Energy Metabolism ; drug effects ; Glucosides ; pharmacology ; Heme Oxygenase-1 ; genetics ; Lipopolysaccharides ; pharmacology ; Male ; Membrane Proteins ; genetics ; Mice ; Mice, Inbred BALB C ; Monoterpenes ; pharmacology ; Oxidative Stress ; drug effects ; Sodium-Potassium-Exchanging ATPase ; metabolism

We evaluated the effect of cobalt chloride (CoCl2) on TNF-alpha and IFN-gamma-induced-inflammation and reactive oxygen species (ROS) in renal tubular epithelial cells (HK-2 cells). We treated HK-2 cells with CoCl2 before the administration of TNF-alpha/IFN-gamma. To regulate hemeoxygenase-1 (HO-1) expression, the cells were treated CoCl2 or HO-1 siRNA. CoCl2 reduced the generation of ROS induced by TNF-alpha/IFN-gamma. TNF-alpha/IFN-gamma-treated-cells showed an increase in the nuclear translocation of phosphorylated NF-kappaBp65 protein, the DNA-binding activity of NF-kappaBp50 and NF-kappaB transcriptional activity and a decrease in IkappaBalpha protein expression. These changes were restored by CoCl2. We noted an intense increase in monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES) production in TNF-alpha/IFN-gamma-treated cells. We demonstrated that this effect was mediated through NF-kappaB signaling because an NF-kappaB inhibitor significantly reduced MCP-1 and RANTES production. CoCl2 effectively reduced MCP-1 and RANTES production. The expression of HO-1 was increased by CoCl2 and decreased by HO-1 siRNA. However, knockdown of HO-1 by RNA interference did not affect MCP-1 or RANTES production. We suggest that CoCl2 has a protective effect on TNF-alpha/IFN-gamma-induced inflammation through the inhibition of NF-kappaB and ROS in HK-2 cells. However, CoCl2 appears to act in an HO-1-independent manner.
Cell Line ; Chemokine CCL2/metabolism ; Chemokine CCL5/metabolism ; Cobalt/*pharmacology ; Epithelial Cells/cytology/metabolism ; Heme Oxygenase-1/antagonists & inhibitors/genetics/metabolism ; Humans ; *Inflammation ; Interferon-gamma/pharmacology ; Kidney Tubules, Proximal/cytology ; NF-kappa B/antagonists & inhibitors/genetics/*metabolism ; NF-kappa B p50 Subunit/genetics/metabolism ; Oxidative Stress/*drug effects ; Phosphorylation ; Protein Binding ; RNA Interference ; RNA, Small Interfering/metabolism ; Transcription Factor RelA/metabolism ; Tumor Necrosis Factor-alpha/pharmacology

OBJECTIVETo assess the effect of atorvastatin on lipopolysaccharide (LPS)-induced TNF-α production in RAW264.7 macrophages.

METHODSRAW264.7 macrophages were treated in different LPS concentrations or at different time points with or without atorvastatin. TNF-α level in supernatant was measured. Expressions of TNF-α mRNA and protein and heme oxygenase-1 (HO-1) were detected by ELISA, PCR, and Western blot, respectively. HO activity was assayed.

RESULTSLPS significantly increased the TNF-α expression and secretion in a dose- and time-dependent manner. The HO-1 activity and HO-1 expression level were significantly higher after atorvastatin treatment than before atorvastatin treatment and attenuated by SB203580 and PD98059 but not by SP600125, suggesting that the ERK and p38 mitogen-activated protein kinase (MAPK) pathways participate in regulating the above-mentioned effects of atorvastatin. Moreover, the HO-1 activity suppressed by SnPP or the HO-1 expression inhibited by siRNA significantly attenuated the effect of atorvastatin on TNF-α expression and production in LPS-stimulated macrophages.

CONCLUSIONAtorvastatin can attenuate LPS-induced TNF-α expression and production by activating HO-1 via the ERK and p38 MAPK pathways, suggesting that atorvastatin can be used in treatment of inflammatory diseases such as sepsis, especially in those with atherosclerotic diseases.

Adjuvants, Immunologic ; pharmacology ; Animals ; Atorvastatin Calcium ; Enzyme Activation ; drug effects ; Heme Oxygenase-1 ; genetics ; metabolism ; Heptanoic Acids ; pharmacology ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; Membrane Proteins ; genetics ; metabolism ; Mice ; Pyrroles ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism