Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

Adiponectin, an abundant adipose tissue-derived protein, exerts protective effect against cardiovascular disease. Adiponectin receptors (AdipoR1 and AdipoR2) mediate the beneficial effects of adiponectin on the cardiovascular system. However, the alteration of AdipoRs in cardiac remodeling is not fully elucidated. Here, we investigated the effect of angiotensin II (AngII) on cardiac AdipoRs expression and explored the possible molecular mechanism. AngII infusion into rats induced cardiac hypertrophy, reduced AdipoR1 but not AdipoR2 expression, and attenuated the phosphorylations of adenosine monophosphate-activated protein kinase and acetyl coenzyme A carboxylase, and those effects were all reversed by losartan, an AngII type 1 (AT1) receptor blocker. AngII reduced expression of AdipoR1 mRNA and protein in cultured neonatal rat cardiomyocytes, which was abolished by losartan, but not by PD123319, an AT2 receptor antagonist. The antioxidants including reactive oxygen species (ROS) scavenger NAC, NADPH oxidase inhibitor apocynin, Nox2 inhibitor peptide gp91 ds-tat, and mitochondrial electron transport chain complex I inhibitor rotenone attenuated AngII-induced production of ROS and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. AngII-reduced AdipoR1 expression was reversed by pretreatment with NAC, apocynin, gp91 ds-tat, rotenone, and an ERK1/2 inhibitor PD98059. Chromatin immunoprecipitation assay demonstrated that AngII provoked the recruitment of c-Myc onto the promoter region of AdipoR1, which was attenuated by PD98059. Moreover, AngII-induced DNA binding activity of c-Myc was inhibited by losartan, NAC, apocynin, gp91 ds-tat, rotenone, and PD98059. c-Myc small interfering RNA abolished the inhibitory effect of AngII on AdipoR1 expression. Our results suggest that AngII inhibits cardiac AdipoR1 expression in vivo and in vitro and AT1 receptor/ROS/ERK1/2/c-Myc pathway is required for the downregulation of AdipoR1 induced by AngII.     

Reactive oxygen species mediate lysophosphatidic acid induced signaling in ovarian cancer cells

Lysophosphatidic acid (LPA) is produced by tumor cells and is present in the ascites fluid of ovarian cancer patients. To determine the role of endogenous LPA in the ovarian cancer cell line SKOV3, we treated cells with the LPA receptor antagonist VPC32183 and found that it inhibited cell growth and induced apoptosis. Exogenous LPA further stimulated ERK and Akt phosphorylation and NF-κB activity. To determine if reactive oxygen species (ROS), which have been implicated as second messengers in cell signaling, were also involved in LPA signaling, we treated cells with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), and antioxidants N-acetyl cysteine, EUK-134 and curcumin, and showed that all blocked LPA-dependent NF-κB activity and cell proliferation. DPI and EUK-134 also inhibited Akt and ERK phosphorylation. LPA was shown to stimulate dichlorofluorescein fluorescence, though not in the presence of DPI, apocynin (an inhibitor of NADPH oxidase), VPC32183, or PEG-catalase. Akt phosphorylation was also inhibited by PEG-catalase and apocynin. These data indicate that NADPH oxidase is a major source of ROS and H(2)O(2) is critical for LPA-mediated signaling. Thus, LPA acts as a growth factor and prevents apoptosis in SKOV3 cells by signaling through redox-dependent activation of ERK, Akt, and NF-κB-dependent signaling pathways.     

Cadmium induction of reactive oxygen species activates the mTOR pathway, leading to neuronal cell death

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd induces neuronal apoptosis in part through activation of the mammalian target of rapamycin (mTOR) pathway. However, the underlying mechanism is unknown. Here we show that Cd induces the generation of reactive oxygen species (ROS) by upregulating the expression of NADPH oxidase 2 and its regulatory proteins (p22(phox), p67(phox), p40(phox), p47(phox), and Rac1) in PC12 and SH-SY5Y cells. Cd induction of ROS contributed to the activation of mTOR signaling, as pretreatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, prevented this event. Further studies reveal that Cd induction of ROS increased phosphorylation of the type I insulin-like growth factor receptor (IGFR) β subunit, which was abrogated by NAC. Wortmannin, a phosphoinositide 3'-kinase (PI3K) inhibitor, partially attenuated Cd-induced phosphorylation of Akt, p70 S6 kinase 1, and eukaryotic initiation factor 4E-binding protein 1, as well as apoptosis of the neuronal cells. In addition, overexpression of wild-type phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or pretreatment with aminoimidazole carboxamide ribonucleotide, an AMP-activated protein kinase (AMPK) activator, partially prevented Cd-induced ROS and activation of the mTOR pathway, as well as cell death. The results indicate that Cd induction of ROS activates mTOR signaling, leading to neuronal cell death, in part by activating the positive regulators IGFR/PI3K and by inhibiting the negative regulators PTEN/AMPK. The findings suggest that inhibitors of PI3K and mTOR, activators of AMPK, or antioxidants may be exploited for the prevention of Cd-induced neurodegenerative diseases.     

Urotensin-II-Mediated Reactive Oxygen Species Generation via NADPH Oxidase Pathway Contributes to Hepatic Oval Cell Proliferation

Urotensin II (UII), a somatostatin-like cyclic peptide, is involved in tumor progression due to its mitogenic effect. Our previous study demonstrated that UII and its receptor UT were up-regulated in human hepatocellular carcinoma (HCC), and exogenous UII promoted proliferation of human hepatoma cell line BEL-7402. Hepatic progenitor cell (HPCs) are considered to be one of the origins of liver cancer cells, but their relationship with UII remains unclear. In this work, we aimed to investigate the effect of UII on ROS generation in HPCs and the mechanisms of UII-induced ROS in promoting cell proliferation. Human HCC samples were used to examine ROS level and expression of NADPH oxidase. Hepatic oval cell line WB-F344 was utilized to investigate the underlying mechanisms. ROS level was detected by dihydroethidium (DHE) or 2’, 7’-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. For HCC samples, ROS level and expression of NADPH oxidase were significantly up-regulated. In vitro, UII also increased ROS generation and expression of NADPH oxidase in WB-F344 cells. NADPH oxidase inhibitor apocynin pretreatment partially abolished UII-increased phosphorylation of PI3K/Akt and ERK, expression of cyclin E/cyclin-dependent kinase 2. Cell cycle was then analyzed by flow cytometry and UII-elevated S phase proportion was inhibited by apocynin pretreatment. Finally, bromodeoxyuridine (Brdu) incorporation assay showed that apocynin partially abolished UII induced cell proliferation. In conclusion, this study indicates that UII-increased ROS production via the NADPH oxidase pathway is partially associated with activation of the PI3K/Akt and ERK cascades, accelerates G1/S transition, and contributes to cell proliferation. These results showed that UII plays an important role in growth of HPCs, which provides novel evidence for the involvement of HPCs in the formation and pathogenesis of HCC.     

Neutral sphingomyelinase, NADPH oxidase and reactive oxygen species. Role in acute hypoxic pulmonary vasoconstriction

The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium-denuded PA segments and their inhibition prevented hypoxia-induced voltage-gated potassium channel (K(V) ) inhibition and pulmonary vasoconstriction. Consistently, H(2) O(2) , or its analogue t-BHP, decreased K(V) currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia-induced ROS production, K(V) channel inhibition and vasoconstriction. Hypoxia induced p47(phox) phosphorylation and its interaction with caveolin-1. Inhibition of nSMase (GW4869) or PKCζ prevented p47(phox) phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase-PKCζ-NADPH oxidase as a necessary step required for ROS production and vasoconstriction.     

PGF2alpha-associated vascular smooth muscle hypertrophy is ROS dependent and involves the activation of mTOR, p70S6k, and PTEN

Prostaglandin F2alpha (PGF2alpha) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF2alpha-induced VSMC hypertrophy we examined the ability of the PGF2alpha analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6k), glycogen synthase kinase-3beta (GSK-3beta), phosphatase and tensin homolog (PTEN), extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) in growth arrested A7r5 VSMC. Fluprostenol-induced hypertrophy was associated with increased ROS, mTOR translocation from the nucleus to the cytoplasm, along with Akt, mTOR, GSK-3beta, PTEN and ERK1/2 but not JNK phosphorylation. Whereas inhibition of phosphatidylinositol 3-kinase (PI3K) by LY-294002 blocked fluprostenol-induced changes in total protein content, pre-treatment with rapamycin or with the MEK1/2 inhibitor U0126 did not. Taken together, these findings suggest that fluprostenol-induced changes in A7r5 hypertrophy involve mTOR translocation and occur through PI3K-dependent mechanisms.     

Role of reactive oxygen species in zinc deficiency-induced hepatic stellate cell activation

We previously reported that zinc deficiency caused a reduction in intracellular glutathione at 8 h after the addition of zinc chelator, diethylenetriamine pentaacetic acid (DTPA), compared with control levels in rat hepatic stellate cells. In this study, we investigated the role of reactive oxygen species and glutathione on the mechanism of zinc deficiency-induced hepatic stellate cell activation, via assessing collagen synthesis. Isolated hepatic stellate cells were incubated with or without DTPA. Type I collagen expression in hepatic stellate cells was detected by immunohistochemistry, and then quantification of the intensity of type I collagen expression was analyzed using a computer with NIH image. Intracellular glutathione was measured using HPLC. H(2)O(2) release from hepatic stellate cells into the overlying medium was assayed using a fluorimetric method. H(2)O(2) release by DTPA-treated hepatic stellate cells significantly increased from 4 h, but returned to control levels after zinc supplementation. When catalase was added to the culture at 6 h after the addition of DTPA, the staining for type I collagen was as weak as at control levels. Diphenyliodonium chloride, the inhibitor of NADPH oxidase, produced a marked reduction in zinc deficiency-induced H(2)O(2) release. The results of this study show that the depletion of intracellular glutathione levels triggers a progression of collagen synthesis in zinc deficient-hepatic stellate cells and this depletion may be induced by the stimulation of cellular production of H(2)O(2).     

Electromagnetic fields induce neural differentiation of human bone marrow derived mesenchymal stem cells via ROS mediated EGFR activation

Even though the inducing effect of electromagnetic fields (EMF) on the neural differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) is a distinctive, the underlying mechanism of differentiation remains unclear. To find out the signaling pathways involved in the neural differentiation of BM-MSCs by EMF, we examined the CREB phosphorylation and Akt or ERK activation as an upstream of CREB. In hBM-MSCs treated with ELF-EMF (50 Hz, 1 mT), the expression of neural markers such as NF-L, MAP2, and NeuroD1 increased at 6 days and phosphorylation of Akt and CREB but not ERK increased at 90 min in BM-MSCs. Moreover, EMF increased phosphorylation of epidermal growth factor receptor (EGFR) as an upstream receptor tyrosine kinase of PI3K/Akt at 90 min. It has been well documented that ELF-MF exposure may alter cellular processes by increasing intracellular reactive oxygen species (ROS) concentrations. Thus, we examined EMF-induced ROS production in BM-MSCs. Moreover, pretreatment with a ROS scavenger, N-acetylcystein, and an EGFR inhibitor, AG-1478, prevented the phosphorylation of EGFR and downstream molecules. These results suggest that EMF induce neural differentiation through activation of EGFR signaling and mild generation of ROS.     

The phosphorylation status of extracellular-regulated kinase 1/2 in astrocytes and neurons from rat hippocampus determines the thrombin-induced calcium release and ROS generation

Challenge of protease-activated receptors induces cytosolic Ca(2+) concentration ([Ca(2+) ](c)) increase, mitogen-activated protein kinase activation and reactive oxygen species (ROS) formation with a bandwidth of responses in individual cells. We detected in this study in situ the thrombin-induced [Ca(2+) ](c) rise and ROS formation in dissociated hippocampal astrocytes and neurons in a mixed culture. In identified cells, single cell responses were correlated with extracellular-regulated kinase (ERK)1/2 phosphorylation level. On average, in astrocytes, thrombin induced a transient [Ca(2+) ](c) rise with concentration-dependent increase in amplitude and extrusion rate and high ERK1/2 phosphorylation level. Correlation analysis of [Ca(2+) ](c) response characteristics of single astrocytes reveals that astrocytes with nuclear phosphoERK1/2 localization have a smaller Ca(2+) amplitude and extrusion rate compared with cells with a cytosolic phosphoERK1/2 localization. In naive neurons, without thrombin challenge, variable ERK1/2 phosphorylation patterns are observed. ROS were detected by hydroethidine. Only in neurons with increased ERK1/2 phosphorylation level, we see sustained intracellular rise in fluorescence of the dye lasting over several minutes. ROS formation was abolished by pre-incubation with the NADPH oxidase inhibitor apocynin. Additionally, thrombin induced an immediate, transient hydroethidine fluorescence increase. This was interpreted as NADPH oxidase-mediated O(2) (?-) -release into the extracellular milieu, because it was decreased by pre-incubation with apocynin, and could be eluted by superfusion. In conclusion, the phosphorylation status of ERK1/2 determines the thrombin-dependent [Ca(2+) ](c) increase and ROS formation and, thus, influences the capacity of thrombin to regulate neuroprotection or neurodegeneration.     

Oxidative Stress Inhibits Insulin-like Growth Factor-I Induction of Chondrocyte Proteoglycan Synthesis through Differential Regulation of Phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK Signaling Pathways

The ability of insulin-like growth factor I (IGF-I) to stimulate cartilage matrix synthesis is reduced in aged and osteoarthritic cartilage. Aging and osteoarthritis are associated with an increase in reactive oxygen species, which we hypothesized would interfere with normal IGF-I signaling. We compared IGF-I signaling in normal and osteoarthritic human articular chondrocytes and investigated the effects of oxidative stress induced by tert-butylhydroperoxide (tBHP). In normal human chondrocytes, IGF-I initiated a strong and sustained phosphorylation of IRS-1 (Tyr-612) and Akt (Ser-473) and transient ERK phosphorylation. In contrast, in osteoarthritic chondrocytes, which possessed elevated basal IRS-1 (Ser-312) and ERK phosphorylation, IGF-I failed to stimulate IRS-1 (Tyr-612) or Akt phosphorylation. In normal human chondrocytes, tBHP triggered strong IRS-1 (Ser-312 and Ser-616) and ERK phosphorylation and inhibited IGF-I-induced IRS-1 (Tyr-612) and Akt phosphorylation. Lentivirus-mediated overexpression of constitutively active (CA) Akt significantly enhanced proteoglycan synthesis, whereas both dominant negative Akt and CA MEK inhibited proteoglycan synthesis. CA Akt also promoted type II collagen and Sox9 expression, whereas tBHP treatment and CA MEK inhibited aggrecan, collagen II, and Sox9 mRNA expression. In osteoarthritic chondrocytes, the antioxidants Mn(III) tetrakis(4-benzoic acid)porphyrin and N-acetylcysteine increased the ratio of Akt to ERK phosphorylation and promoted IGF-I-mediated proteoglycan synthesis. Chemical inhibition of ERK significantly enhanced IGF-I phosphorylation of Akt and alleviated tBHP inhibition of Akt phosphorylation. These results demonstrate opposing roles for phosphatidylinositol 3-kinase-Akt and MEK-ERK in cartilage matrix synthesis and suggest that elevated levels of reactive oxygen species cause chondrocyte IGF-I resistance by altering the balance of Akt to ERK activity.     

Extracellular ATP-induced proliferation of adventitial fibroblasts requires phosphoinositide 3-kinase, Akt, mammalian target of rapamycin, and p70 S6 kinase signaling pathways

Extracellular nucleotides are increasingly recognized as important regulators of growth in a variety of cell types. Recent studies have demonstrated that extracellular ATP is a potent inducer of fibroblast growth acting, at least in part, through an ERK1/2-dependent signaling pathway. However, the contributions of additional signaling pathways to extracellular ATP-mediated cell proliferation have not been defined. By using both pharmacologic and genetic approaches, we found that in addition to ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), and p70 S6K-dependent signaling pathways are required for ATP-induced proliferation of adventitial fibroblasts. We found that extracellular ATP acting in part through G(i) proteins increased PI3K activity in a time-dependent manner and transient phosphorylation of Akt. This PI3K pathway is not involved in ATP-induced activation of ERK1/2, implying activation of independent parallel signaling pathways by ATP. Extracellular ATP induced dramatic increases in mTOR and p70 S6K phosphorylation. This activation of the mTOR/p70 S6 kinase (p70 S6K) pathway in response to ATP is because of independent contributions of PI3K/Akt and ERK1/2 pathways, which converge on the level of p70 S6K. ATP-dependent activation of mTOR and p70 S6K also requires additional signaling inputs perhaps from pathways operating through Galpha or Gbetagamma subunits. Collectively, our data demonstrate that ATP-induced adventitial fibroblast proliferation requires activation and interaction of multiple signaling pathways such as PI3K, Akt, mTOR, p70 S6K, and ERK1/2 and provide evidence for purinergic regulation of the protein translational pathways related to cell proliferation.     

Leucine stimulates HGF production by hepatic stellate cells through mTOR pathway

Branched chain amino acids modulate various cellular functions in addition to providing substrates for the production of proteins. We examined the mechanism underlying the stimulation by leucine of hepatocyte growth factor (HGF) production by hepatic stellate cells. Both p70 S6 kinase activity and phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) were up-regulated rapidly after leucine treatment of a rat hepatic stellate cell clone. No such activation was observed following treatment with valine or isoleucine. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), suppressed leucine-induced activation of p70 S6 kinase and 4E-BP1 and negated the stimulatory effect of leucine on HGF production. An mTOR-dependent signaling pathway mediates the stimulatory effect of leucine on the production of HGF by hepatic stellate cells.     

Angiotensin II-induced NADPH oxidase activation impairs insulin signaling in skeletal muscle cells

The renin-angiotensin system (RAS) and reactive oxygen species (ROS) have been implicated in the development of insulin resistance and its related complications. There is also evidence that angiotensin II (Ang II)-induced generation of ROS contributes to the development of insulin resistance in skeletal muscle, although the precise mechanisms remain unknown. In the present study, we found that Ang II markedly enhanced NADPH oxidase activity and consequent ROS generation in L6 myotubes. These effects were blocked by the angiotensin II type 1 receptor blocker losartan, and by the NADPH oxidase inhibitor apocynin. Ang II also promoted the translocation of NADPH oxidase cytosolic subunits p47phox and p67phox to the plasma membrane within 15 min. Furthermore, Ang II abolished insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), activation of protein kinase B (Akt), and glucose transporter-4 (GLUT4) translocation to the plasma membrane, which was reversed by pretreating myotubes with losartan or apocynin. Finally, small interfering RNA (siRNA)-specific gene silencing targeted specifically against p47phox (p47siRNA), in both L6 and primary myotubes, reduced the cognate protein expression, decreased NADPH oxidase activity, restored Ang II-impaired IRS1 and Akt activation as well as GLUT4 translocation by insulin. These results suggest a pivotal role for NADPH oxidase activation and ROS generation in Ang II-induced inhibition of insulin signaling in skeletal muscle cells.     

NOX4-Derived Reactive Oxygen Species Drive Apelin-13-Induced Vascular Smooth Muscle Cell Proliferation via the ERK Pathway

Apelin is the endogenous ligand of the G-protein-coupled receptor, apelin–angiotensin receptor-like 1 (APJ). Vascular smooth muscle cells express both apelin and APJ, which are important regulatory factors in the cardiovascular system. Apelin-13 significantly stimulated vascular smooth muscle cell proliferation. However, little is known about the precise cellular mechanisms responsible for vascular smooth muscle cell proliferation induced by apelin-13. Here, we present novel data that indicate the key role of NADPH oxidase 4-derived reactive oxygen species in proliferation of vascular smooth muscle cells treated with apelin-13. Apelin-13 stimulated reactive oxygen species production in a concentration- and time-dependent manner. Furthermore, DPI impaired apelin-13-induced reactive oxygen species generation and vascular smooth muscle cell proliferation. Apelin-13-treatment increased the expression of NADPH oxidase 4 in a dose-dependent manner. Down-regulation of NADPH oxidase 4 using siRNA prevented apelin-13-induced reactive oxygen species generation and vascular smooth muscle cell proliferation. An increase in reactive molecules can trigger the activation of ERK stress-sensitive signaling pathways. Additionally, siRNA-NOX4 and DPI reversed the phosphorylation of ERK induced by apelin-13. Apelin-13 induced vascular smooth muscle cell proliferation by NOX4-derived ROS via the ERK signaling pathway.     

Signaling pathway of nitric oxide production induced by ginsenoside Rb1 in human aortic endothelial cells: a possible involvement of androgen receptor

Ginsenosides have been shown to stimulate nitric oxide (NO) production in aortic endothelial cells. However, the signaling pathways involved have not been well studied in human aortic endothelial cells. The present study was designed to examine whether purified ginsenoside Rb1, a major active component of ginseng could actually induce NO production and to clarify the signaling pathway in human aortic endothelial cells. NO production was rapidly increased by Rb1. The rapid increase in NO production was abrogated by treatment with nitric oxide synthetase inhibitor, L-NAME. Rb1 stimulated rapid phosphorylation of Akt (Ser473), ERK1/2 (Thr202/Thr204) and eNOS (Ser1177). Rapid phosphorylation of eNOS (Ser1177) was prevented by SH-5, an Akt inhibitor or wortmannin, PI3-kinase inhibitor and partially attenuated by PD98059, an upstream inhibitor for ERK1/2. Interestingly, NO production and eNOS phosphorylation at Ser1177 by Rb1 were abolished by androgen receptor antagonist, nilutamide. The results suggest that PI3kinase/Akt and MEK/ERK pathways and androgen receptor are involved in the regulation of acute eNOS activation by Rb1 in human aortic endothelial cells.     

<Emphasis Type="Italic">S</Emphasis>-Propargyl-cysteine (SPRC) attenuated lipopolysaccharide-induced inflammatory response in H9c2 cells involved in a hydrogen sulfide-dependent mechanism

The present study attempts to investigate the effects of S-propargyl-cysteine (SPRC), a sulfur-containing amino acid, on lipopolysaccharide (LPS)-induced inflammatory response in H9c2 cardiac myocytes. We found that SPRC prevented nuclear factor-κB (NF-κB) activation assessed by NF-κB p65 phosphorylation and IκBα degradation, suppressed LPS-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and intracellular reactive oxygen species (ROS) production. Furthermore, incubation of H9c2 cells with SPRC induced phosphorylation of Akt in a time- and concentration-dependent manner. In addition, SPRC attenuated LPS-induced mRNA and protein expression of tumor necrosis factor-α (TNF-α), and mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS). The effects of SPRC were abolished by cystathionine γ-lyase [CSE-an enzyme that synthesizes hydrogen sulfide (H₂S)] inhibitor, dl-propargylglycine (PAG), SPRC-induced Akt phosphorylation and TNF-α release was also abolished by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Furthermore, SPRC also increased LPS-induced down-regulation expression of CSE and H₂S level in H9c2 cells. PAG abolished SPRC-induced up-regulation of H₂S level. Therefore, we concluded that SPRC produced an anti-inflammatory effect in LPS-stimulated H9c2 cells partly through the CSE/H₂S pathway by impairing IκBα/NF-κB signaling and by activating PI3K/Akt signaling pathway.