This project aimed to explore the protective effect of ginsenoside Rg_1 on hypoxia/reoxygenation(H/R)-induced H9 c2 cardiomyocyte injury and its underlying signaling pathway. The H/R model of H9 c2 cardiomyocytes was established and then the cells were divided into different treatment groups. CCK-8(cell counting kit-8) was used to detect the activity of cardiomyocytes; Brdu assay was used to detect the proliferation of H9 c2 cells; the caspase-3 activity was tested, and then the protein expression was assessed by Western blot. Flow cytometry was used to evaluate the apoptosis level of cardiomyocytes. Ginsenoside Rg_1 inhibited H/R-induced cardiomyocyte apoptosis and caspase-3 activity, promoted nuclear transcription of nuclear factor erythroid-2 related factor 2(Nrf2), and enhanced the expression of the downstream heme oxygenase-1(HO-1). Ginsenoside Rg_1 could increase Nrf2 nuclear transcription and HO-1 expression with the increase of concentration(10, 20, 40, 60 μmol·L~(-1)). However, the protective effect of ginsenoside Rg_1 on cardiomyocytes was significantly weakened after the transfection of Nrf2-siRNA. Ginsenoside Rg_1 could protect cardiomyocytes by activating the Nrf2/HO-1 pathway.
Apoptosis ; Ginsenosides/pharmacology* ; Heme Oxygenase-1/genetics* ; Humans ; Hypoxia ; Myocytes, Cardiac ; NF-E2-Related Factor 2/genetics*

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

To investigate the effect of sinomenine on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophages and the underlying mechanisms.
 Methods: The mouse RAW264.7 macrophages were treated with sinomenine and/or LPS with or without heme oxygenase-1 (HO-1) inhibitor Znpp. Real-time PCR, ELISA, immunofluenscence, and Western blot were used to detect the mRNA expression of TNF-α and IL-6, the release of TNF-α and IL-6, the protein expression of HO-1 and autophagy, respectively.
 Results: Compared with the control group, the mRNA expression and release of inflammatory cytokines TNF-α and IL-6 were increased, the green fluorescence of autophagy-related protein LC3 was accumulated and the protein expression of HO-1 was increased in RAW264.7 cells after LPS treatment (P<0.05). Compared with the LPS group, sinomenine treatment could reduce the mRNA expression and release of TNF-α and IL-6, accompanied by increasess in green fluorescence aggregation of LC3 and HO-1 production (P<0.05). HO-1 inhibitor Znpp could weaken the ability of sinomenine through suppressing TNF-α and IL-6 expression and decreasing the aggregation of LC3 green fluorescence (P<0.05).
 Conclusion: Sinomenine could alleviate LPS-induced inflammation in RAW264.7 macrophages, which might be related to HO-1 mediated autophagy. This study provides an experimental and theoretical basis for the clinical application of sinomenine in prevention and treatment of inflammation.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Autophagy ; drug effects ; Gene Expression Regulation, Enzymologic ; drug effects ; Heme Oxygenase-1 ; genetics ; Inflammation ; chemically induced ; Lipopolysaccharides ; Macrophages ; drug effects ; Mice ; Morphinans ; pharmacology

Excessive microglial cell activation is related to the progression of chronic neuro-inflammatory disorders. Heme oxygenase-1 (HO-1) expression mediated by the NFE2-related factor (Nrf-2) pathway is a key regulator of neuro-inflammation. Nardostachys chinensis is used as an anti-malarial, anti-nociceptive, and neurotrophic treatment in traditional Asian medicines. In the present study, we examined the effects of an ethyl acetate extract of N. chinensis (EN) on the anti-neuro-inflammatory effects mediated by HO-1 up-regulation in Salmonella lipopolysaccharide (LPS)- or Staphylococcus aureus lipoteichoic acid (LTA)-stimulated BV2 microglial cells. Our results indicated that EN suppressed pro-inflammatory cytokine production and induced HO-1 transcription and translation through Nrf-2/antioxidant response element (ARE) signaling. EN markedly inhibited LPS- and LTA-induced activation of nuclear factor-kappa B (NF-κB) as well as phosphorylation of mitogen-activated protein kinases (MAPKs) and signal transducer and activator of transcription (STAT). Furthermore, EN protected hippocampal HT22 cells from indirect neuronal toxicity mediated by LPS- and LTA-treated microglial cells. These results suggested that EN impairs LPS- and LTA-induced neuro-inflammatory responses in microglial cells and confers protection against indirect neuronal damage to HT22 cells. In conclusion, our findings indicate that EN could be used as a natural anti-neuro-inflammatory and neuroprotective agent.
Anti-Inflammatory Agents ; pharmacology ; Cell Line ; Heme Oxygenase-1 ; genetics ; immunology ; Humans ; Lipopolysaccharides ; adverse effects ; Microglia ; cytology ; drug effects ; immunology ; Mitogen-Activated Protein Kinases ; genetics ; immunology ; NF-kappa B ; genetics ; immunology ; Nardostachys ; chemistry ; Neuroprotective Agents ; pharmacology ; Plant Extracts ; pharmacology ; Teichoic Acids ; adverse effects

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 the relationship between (GT)n polymorphism and esophageal cancer by analyzing the connection between microsatellite polymorphisms in the promoter of heme oxygenase-1 and the clinicopathological characteristics of esophageal squamous cell carcinoma (ESCC) in Han chinese population.

METHODSThe (GT)n repeats in HO-1 gene in 83 male and 43 female hospital-based patients with ESCC (aged between 40 and 79 years with a mean of (61 ± 8) years) and 134 healthy control individuals were obtained by DNA sequencing. Polymorphisms of the (GT)n repeats were generally grouped into three classes based on allele frequencies: class S alleles (<25 repeats), class M alleles (25 to 29 repeats), and class L alleles (≥30 repeats). The correlation between susceptibility and clinicopathological characteristics of ESCC were analyzed by χ2 test. For in vitro experiments, the transient-transfection assay was performed to explore the correlation between different lengths of (GT)n repeats and promoter activity by assessing the promoter activities of HO-1 gene in cultured Ecal09 cells treated with H2O2 by analysis of cariance.

RESULTSHigher frequencies of L-allele (25. 8% vs. 14. 9%, χ2 = 9. 520, P = 0. 002), L-allele carrier (41. 3% vs. 27. 6%, χ2 = 5. 381 , P = 0. 020) were found in patients with ESCC. Furthermore, the lymphatic metastasis rate (63. 5% vs. 41. 8%, χ = 5. 685, P = 0. 017) and the detection rate of poorly differentiated ESCC cell (53. 8% vs. 28. 4%, χ2 = 8. 335, P = 0. 004) was significantly higher in L-allele carriers compared to non-L-allele carriers. In transfection experiments, promoter activities of 5'-flanking regions of the HO-1 gene in Eca109 cells transfected with the recombinant gene carrying (GT)16 repeat after treatment with H2O2 increased (F = 23. 615,P = 0. 008). In H2O treated control group, compared to (GT)26 and (GT)36, the basal promoter activities of HO-1 gene carrying (GT)16 repeat increased (F =41. 376, P = 0. 003; F = 50. 761, P = 0. 002).

CONCLUSIONThe long (GT)n repeats of HO-1 gene promoter can increase the susceptibility of esophageal squamous cell carcinoma and the risk of lymphatic metastasis.

Alleles ; Asian Continental Ancestry Group ; Carcinoma, Squamous Cell ; etiology ; pathology ; Esophageal Neoplasms ; etiology ; pathology ; Female ; Gene Frequency ; Heme Oxygenase-1 ; genetics ; Humans ; Hydrogen Peroxide ; Lymphatic Metastasis ; Male ; Microsatellite Repeats ; Polymorphism, Genetic ; Promoter Regions, Genetic ; Risk Factors ; Transfection

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

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