Tin Protoporphyrin Research Articles

Effects of Resveratrol and trans-3,5,4'-Trimethoxystilbene on Glutamate-Induced Cytotoxicity, Heme Oxygenase-1, and Sirtuin 1 in HT22 Neuronal Cells

Resveratrol (trans-3,5,4’-trihydroxystilbene) has received considerable attention recently for the potential neuroprotective effects in neurodegenerative disorders where heme oxygenase-1 (HO-1) and sirtuin 1 (SIRT1) represent promising therapeutic targets. Resveratrol has been known to increase HO-1 expression and SIRT1 activity. In this study, the effects of resveratrol and trans-3,5,4’-trimethoxystilbene (TMS), a resveratrol derivative, on cytotoxicity caused by glutamate-induced oxidative stress, HO-1 expression, and SIRT1 activation have been investigated by using murine hippocampal HT22 cells, which have been widely used as an in vitro model for investigating glutamate-induced neurotoxicity. Resveratrol protected HT22 neuronal cells from glutamateinduced cytotoxicity and increased HO-1 expression as well as SIRT1 activity in a concentration-dependent manner. Cytoprotec-tion afforded by resveratrol was partially reversed by the specific inhibition of HO-1 expression by HO-1 small interfering RNA and the nonspecific blockage of HO-1 activity by tin protoporphyrin IX, but not by SIRT1 inhibitors. Surprisingly, TMS, a resveratrol derivative with methoxyl groups in lieu of the hydroxyl groups, and trans-stilbene, a non-hydroxylated analog, failed to protect HT22 cells from glutamate-induced cytotoxicity and to increase HO-1 expression and SIRT1 activity. Taken together, our findings suggest that the cytoprotective effect of resveratrol was at least in part associated with HO-1 expression but not with SIRT1 activation and, importantly, that the presence of hydroxyl groups on the benzene rings of resveratrol appears to be necessary for cytoprotection against glutamate-induced oxidative stress, HO-1 expression, and SIRT1 activation in HT22 neuronal cells.

Neuroprotective Effects of Dehydroglyasperin C through Activation of Heme Oxygenase-1 in Mouse Hippocampal Cells

Licorice, the root of the Glycyrrhiza species ( Glycyrrhiza uralensis Fisher), is known to have antioxidant, anti-inflammatory, antiviral, and antitumor properties. The objective of this study is to explore the neuroprotective effect of dehydroglyasperin C (DGC) against glutamate-induced oxidative stress in mouse hippocampal HT22 cells. DGC significantly reduced cytotoxicity and reactive oxygen species (ROS) generation induced by glutamate in HT22 cells, whereas DGC did not restore glutathione depletion caused by glutamate. In addition, it was further investigated whether DGC affected the expression of heme oxygenase (HO)-1, one of the major cellular antioxidant defense systems, and it was found that DGC dose-dependently increased HO-1 expression. DGC-mediated cytoprotection of HT22 neuronal cells from glutamate insult was abrogated by either HO-1 inhibitor (Tin protoporphyrin, SnPP) or AKT inhibitor (LY294002). In conclusion, the present results demonstrate for the first time that DGC protects neuronal cells against glutamate-induced oxidative injury through the induction of HO-1 expression, which is, in turn, activated maybe through Nrf2-Keap1 and PI3K/AKT signaling pathways.

Santamarin, a sesquiterpene lactone isolated from Saussurea lappa, represses LPS-induced inflammatory responses via expression of heme oxygenase-1 in murine macrophage cells

Saussurea lappa C.B. Clarke (Compositae) is indigenous to India and Pakistan. The dried root of S. lappa has been traditionally used for alleviating pain in abdominal distention and tenesmus, indigestion with anorexia, dysentery, nausea, and vomiting. Santamarin is a sesquiterpene lactone isolated from S. lappa. In the present study, santamarin inhibited inducible nitric oxide synthase (iNOS) protein, reduced iNOS-derived nitric oxide (NO), suppressed COX-2 protein and reduced COX-derived PGE2 production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and murine peritoneal macrophages. Similarly, santamarin reduced tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production. In addition, santamarin suppressed the phosphorylation and degradation of IκB-α as well as the nuclear translocation of p65 in response to LPS in RAW264.7 cells. Furthermore, santamarin induced heme oxygenase (HO)-1 expression mRNA and protein level that plays a cytoprotective role against inflammation. The induction of HO-1 is primarily regulated at the transcriptional level, and its induction by various agents is mediated by the nuclear transcription factor E2-related factor 2 (Nrf2), master regulator of antioxidant responses. Unbound Nrf2 translocates into the nucleus and binds to the antioxidant response element (ARE) in the upstream promoter region of many antioxidative genes, where it initiates their transcription. The effects of santamarin on LPS-induced NO, PGE2, TNF-α, and IL-1β production were partially reversed by the HO-1 inhibitor, tin protoporphyrin (SnPP). Therefore, our data suggest that the anti-inflammatory effect of santamarin in macrophages may be exerted through a novel mechanism that involves HO-1 expression.

Antioxidant roles of heme oxygenase, carbon monoxide and bilirubin in cerebral circulation during epileptic seizures

Epileptic seizures cause sustained loss of endothelium‐dependent regulation of cerebral blood flow. Heme oxygenase (HO) is cytoprotective against seizure‐induced cerebral endothelial dysfunction. Oxidative stress is a potential cause of vascular damage. Using a model of bicuculline‐induced epileptic seizures in newborn pigs, we addressed the hypotheses that: 1) seizures increase production of reactive oxygen species (ROS) in cerebral vasculature, 2) endogenous HO activity exerts antioxidant effects, and 3) CO and bilirubin exhibit antioxidant properties in cerebral circulation. As assessed by dihydroethidium (DHE) oxydation, seizures elevate ROS in cerebral vessels. Burst of ROS during seizures was blocked by tiron, a ROS scavenger, and reduced by apocynin, a NADPH oxidase inhibitor. A HO inhibitor, tin protoporphyrin, potentiated whereas a HO‐1 inducer, cobalt protoporphyrin, blocked the ROS increases in cerebral vessels during seizures. The products of HO activity, CO (as CORM‐A1, 2 mg/kg ip) and bilirubin (3–5 mg/kg, iv) administered before or during a 20‐min window after seizure onset attenuated ROS elevation. These data indicate that: 1) constitutive HO‐2 and inducible HO‐1 have antioxidant properties in cerebral vasculature; 2) antioxidant properties of CO and bilirubin could contribute to the HO‐mediated endothelial survival mechanism in cerebral circulation during epileptic seizures.

Anti-inflammatory Effects of Amomum compactum on RAW 264.7 cells via induction of heme oxygenase-1

Amomum compactum is commonly used in Korean traditional medicine. In this study, we demonstrate that A. compactum ethanolic extract (ACEE) has anti-inflammatory effects in a lipopolysaccharide-induced RAW 264.7 cell model of inflammation. In this system, ACEE prominently inhibited the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), interleukin (IL)-6 and tumor necrosis factor (TNF)-α, and inhibited the protein expression of inducible nitric oxide synthase and cyclooxygenase-2. Furthermore, ACEE treatment inhibited the translocation of nuclear factor-kappaB (NF-κB) and the degradation of inhibitory factor-kappaB alpha, but enhanced the expression of heme oxygenase (HO)-1 and the nuclear translocation of nuclear factor-erythroid 2 (Nrf2). Treatment with tin protoporphyrin IX dichloride (SnPP), a selective HO-1 inhibitor, reversed the ACEE-induced suppression of NO production, suggesting that the induction of HO-1 is involved in the suppression of NO, TNF-α, and IL-6 production by ACEE. Taken together, these results suggest that ACEE have anti-inflammatory effects occurring through HO-1 induction, which leads to suppression of the blocking NF-κB.

Concomitant Induction of Heme Oxygenase‐1 Attenuates the Cytotoxicity of Arsenic Species from Lumbricus Extract in Human Liver HepG2 Cells

Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme that degrades heme to three products, biliverdin, carbon monoxide (CO), and iron ion. The present study was originally designed to characterize the HO-1 induction by Lumbricus extract as a potential cytoprotective mechanism. Through bioactivity-guided fractionation, with human HepG2 cells as the cellular detector, surprisingly, we found that arsenic was enriched in the active fractions isolated from Lumbricus extract. Arsenic speciation was further carried out by liquid chromatography with inductively coupled plasma mass spectrometry (LC/ICP-MS). Our results showed that Lumbricus extract contained two major arsenic species, arsenite (As(III) ; 53.7%) and arsenate (As(V) ; 34.2%), and six minor arsenic species. Commercial sodium arsenite (NaAsO(2) ) was used to verify the effects of Lumbricus extract on HO-1 expression and related intracellular signaling pathways. Both p38 MAP kinase and NF-E2-related factor 2 (Nrf2) pathways were found to modulate HO-1 induction by Lumbricus extract and NaAsO(2) . The cytotoxicity of arsenite was augmented by p38 MAP kinase inhibitor SB202190 and HO-1 inhibitor tin protoporphyrin IX (SnPP), whereas p38 MAP kinase inhibitor SB202190 also inhibited HO-1 induction by NaAsO(2) . These results suggest that arsenic-containing compounds are responsible for HO-1 induction by Lumbricus extract. Although the exact role of toxic arsenic compounds in the treatment of oxidative injury remains unclear, concomitant HO-1 induction may be a key mechanism to antagonize the cytotoxicity of arsenic compounds in human cells.

Pharmacological Induction of Heme Oxygenase-1 by a Triterpenoid Protects Neurons Against Ischemic Injury

Heme oxygenase-1 (HO-1) is an inducible Phase 2 enzyme that degrades toxic heme; its role in cerebral ischemia is not fully understood. We hypothesize that chemically induced HO-1 upregulation with the novel triterpenoid CDDO-Im (2-cyano-3,12 dioxooleana-1,9 dien-28-oyl imidazoline), a robust inducer of Phase 2 genes, protects neurons against ischemic injury. Using 3 different models of ischemia, including oxygen-glucose deprivation in neuronal cultures, global ischemia in rats, and focal ischemia in mice, we determined (1) whether CDDO-Im induces HO-1 expression and protects against ischemic injury; and (2) whether HO-1 inhibition disrupts the neuroprotective effect of CDDO-Im. CDDO-Im treatment (50-300 nmol/L) resulted in 8-fold HO-1 upregulation in cultured neurons and protected against oxygen-glucose deprivation. The protection was abolished when the cultures were transfected with nuclear factor (erythroid-derived 2) like-2-shRNA or coincubated with tin protoporphyrin IX, a specific HO-1 inhibitor. In the rat model of global ischemia, intracerebroventricular infusion of CDDO-Im (0.5-1.5 μg) augmented HO-1 expression in hippocampal neurons and resulted in significant increases in CA1 neuronal survival after global ischemia. To further strengthen the clinical relevance of the CDDO-Im treatment, we tested its effects in the mouse model of temporary focal ischemia (60 minutes). Postischemic intraperitoneal injection of CDDO-Im (10-100 μg) enhanced HO-1 expression and significantly reduced neurological dysfunction and infarct volume. Intracerebroventricular infusion of tin protoporphyrin IX reduced the neuroprotective effect of CDDO-Im against global and focal ischemia. CDDO-Im confers neuroprotection against ischemic injury by upregulating HO-1, suggesting that enhance of HO-1 expression may be a legitimate strategy for therapeutic intervention of stroke.

Analysis of early biochemical markers and regulation by tin protoporphyrin IX in a model of spontaneous osteoarthritis

Age-related changes in joint tissues lead to osteoarthritis (OA). Detection of early changes in OA patients may help to initiate treatments before the establishment of irreversible joint destruction. STR/ort mice develop with age a severe degenerative joint disease that resembles human OA thus allowing the investigation of biochemical markers as well as new treatments in an accelerated time frame. We have analyzed the changes in serum levels of different mediators during the early phases of idiopathic OA in STR/ort mice. Serum levels of matrix metalloproteinase-3 (MMP-3) but not those of tumor necrosis factor-α, interleukin(IL)-1β, IL-17 or prostaglandin E2 correlated with histopathological changes in knees of STR/ort mice at 9weeks. Treatment of animals with tin protoporphyrin IX (SnPP, 12mg/kg/dayi.p.) for 4weeks significantly reduced the progression of OA. Our data suggest that MMP-3 is a sensitive biomarker to detect early OA alterations and that SnPP could be a protective agent in OA.

Antioxidant Roles of Heme Oxygenase, Carbon Monoxide, and Bilirubin in Cerebral Circulation during Seizures

Postictal cerebrovascular dysfunction is an adverse effect of seizures in newborn piglets. The brain heme oxygenase (HO) provides protection against cerebrovascular dysfunction. We investigated the contribution of reactive oxygen species (ROS) to seizure-induced vascular damage and the mechanism of HO vasoprotection. In a bicuculline model of seizures, we addressed the hypotheses: (1) seizures increase brain ROS; (2) ROS contribute to cerebral vascular dysfunction; (3) ROS initiate a vasoprotective mechanisms by activating endogenous HO; and (4) HO products have antioxidant properties. As assessed by dihydroethidium oxidation (ox-DHE), seizures increased ROS in cerebral vessels and cortical astrocytes; ox-DHE elevation was prevented by tiron and apocynin. An HO inhibitor, tin protoporphyrin, potentiated, whereas an HO-1 inducer, cobalt protoporphyrin, blocked seizure-induced increase in DHE oxidation. Heme oxygenase products carbon monoxide (CO) (CORM-A1) and bilirubin attenuated ox-DHE elevation during seizures. Antioxidants tiron and bilirubin prevented the loss of postictal cerebrovascular dilations to bradykinin, glutamate, and sodium nitroprusside. Tiron and apocynin abrogated activation of the brain HO during seizures. Overall, these data suggest that long-term adverse cerebrovascular effects of seizures are attributed to oxidative stress. On the other hand, seizure-induced ROS are required for activation of the endogenous antioxidant HO/CO/bilirubin system that alleviates oxidative stress-induced loss of postictal cerebrovascular function in piglets.

The Modulatory Role of Heme Oxygenase on Subpressor Angiotensin II-Induced Hypertension and Renal Injury

Angiotensin II (AngII) causes hypertension (HTN) and promotes renal injury while simultaneously inducing reno-protective enzymes like heme oxygenase-1 (HO-1). We examined the modulatory role of HO on sub-pressor angiotensin II (SP-AngII) induced renal inflammation and injury. We first tested whether the SP-AngII-induced renal dysfunction, inflammation and injury are exacerbated by either preventing (chronic HO-1 inhibition) or reversing (late HO-1 inhibition) SP-AngII-induced HO (using tin protoporphyrin; SnPP). We next examined whether additional chronic or late induction of SP-AngII-induced HO (using cobalt protoporphyrin; CoPP), prevents or ameliorates renal damage. We found that neither chronic nor late SnPP altered blood pressure. Chronic SnPP worsened SP-AngII-induced renal dysfunction, inflammation, injury and fibrosis, whereas late SnPP worsened renal dysfunction but not inflammation. Chronic CoPP prevented HTN, renal dysfunction, inflammation and fibrosis, but surprisingly, not the NGAL levels (renal injury marker). Late CoPP did not significantly alter SP-AngII-induced HTN, renal inflammation or injury, but improved renal function. Thus, we conclude (a) endogenous HO may be an essential determining factor in SP-AngII induced renal inflammation, injury and fibrosis, (b) part of HO's renoprotection may be independent of blood pressure changes; and (c) further induction of HO-1 protects against renal injury, suggesting a possible therapeutic target.

Developmental delay in hypoxia-induced HO-1 expression predisposes to gut injury

Necrotizing enterocolitis (NEC) is an often fatal disease that affects 5-8% of preterm newborn infants but does not occur in older infants and children. As carbon monoxide (CO) may exert protective effects against NEC, we assessed patterns of intestinal injury and investigated the expression of the CO-producing enzyme heme oxygenase-1 (HO-1) in mature and immature rat guts in response to hypercapnia and reoxygenation (H/R). Gut barrier failure (increased permeability for dextran) was assessed in immature (newborn rats) and mature rats (weanling rats) subjected to H/R. Their guts were assayed for apoptosis (caspase-3 activity), expression of inducible NO synthase (iNOS) and HO-1 [quantitative polymerase chain reaction (PCR) and immunoblot]. The role of HO-1 was investigated in experiments involving HO-1 induction by hemin or HO-1 inhibition by tin protoporphyrin IX. In the mature gut, H/R induced the expression of intestinal HO-1 within 48 h, whereas in the immature gut HO-1 up-regulation was delayed by 48 h. Immature, but not mature, rats exhibited gut barrier failure, apoptosis and increased iNOS expression upon H/R. After the induction of HO-1 by hemin, gut barrier failure and apoptosis were abrogated in the immature gut, while the inhibition of HO-1 by tin protoporphyrin IX significantly aggravated gut injury. These experiments point to an immaturity-dependent lag in HO-1 expression upon H/R in the immature gut and link low HO-1 to gut barrier failure induced by H/R in a non-infectious dam-fed animal model of gut injury.

Neuroprotective effects of dehydroglyasperin C through increased expression of heme oxygenase-1 in mouse hippocampal cells

. The objective of this study was to explore the neuroprotective effect of dehydroglyasperin C (DGC) againstglutamate-induced oxidative stress in the mouse hippocampal HT22 cells. DGC signiÞcantly reduced cytotoxicity and reactive oxygenspecies (ROS) generation induced by glutamate in HT22 cells, whereas DGC did not restore glutathione depletion caused by glutamate. Inaddition, we investigated further whether DGC affected the expression of heme oxygenase (HO)-1, one of major cellular antioxidantdefense systems, and found that DGC dose-dependently increased HO-1 expression. DGC-mediated cytoprotection of HT22 neuronal cellsfrom glutamate insult was abrogated by either HO-1 inhibitor (Tin protoporphyrin, SnPP) or AKT inhibitor (LY294002). In conclusion,the present results demonstrate for the Þrst time that DGC protects neuronal cells against glutamate-induced oxidative injury through theinduction of HO-1 expression which is, in turn, activated maybe through Nrf2-Keap1 and PI3K/AKT signaling pathways.

Malaria impairs resistance to Salmonella through heme- and heme oxygenase–dependent dysfunctional granulocyte mobilization

In sub-Saharan Africa, invasive non-Typhoid Salmonella (NTS) is a common and often fatal complication of Plasmodium falciparum infection. Induction of heme oxygenase-1 (HO-1) mediates tolerance to the cytotoxic effects of heme during malarial hemolysis but might impair resistance to NTS by limiting production of bactericidal reactive oxygen species. We show that co-infection of mice with Plasmodium yoelii 17XNL (Py17XNL) and S. typhimurium causes acute, fatal bacteremia with increased bacterial load; features reproduced by phenylhydrazine hemolysis or hemin administration. S. typhimurium localized predominantly in granulocytes. Py17XNL, phenylhydrazine and hemin caused premature mobilization of granulocytes from bone marrow with a quantitative defect in the oxidative burst. Inhibition of HO by tin protoporphyrin abrogated the impairment of resistance to S. typhimurium by hemolysis. Thus a mechanism of tolerance to one infection, malaria, impairs resistance to another, NTS. Furthermore, HO inhibitors may be useful adjunctive therapy for NTS infection in the context of hemolysis.

Quercetin protects against pulmonary oxidant stress via heme oxygenase-1 induction in lung epithelial cells

The lung is a primary target for oxygen toxicity because of its constant exposure to high oxygen levels and environmental oxidants. Quercetin is one of the most commonly found dietary flavonoids, and it provides cytoprotective actions via activation of specific transcriptional factors and upregulation of endogenous defensive pathways. In the present study, we showed that quercetin increased the levels of heme oxygenase (HO)-1 expression and protected against hydrogen peroxide (H2O2)-induced cytotoxicity in lung epithelial cell lines. Quercetin suppressed H2O2-induced apoptotic events, including hypodiploid cells, activation of caspase 3 enzyme activity and lactate dehydrogenase release. This cytoprotective effect was attenuated by the addition of the HO inhibitor, tin protoporphyrin IX. In addition, the end products of heme metabolites catalyzed by HO-1, carbon monoxide and bilirubin, protect against H2O2-induced cytotoxicity in LA-4 cells. Quercetin may well be one of the promising substances to attenuate oxidative epithelial cell injury in lung inflammation.

Inhibition of Nitric Oxide Production by Ethyl Digallates Isolated from Galla Rhois in RAW 264.7 Macrophages

Galla Rhois and its components are known to possess anti-infl ammatory properties. In the present study, we prepared equilibrium mixture of ethyl m-digallate and ethyl p-digallate isomers (EDG) from Galla Rhois and examined its effect on nitric oxide (NO) production in murine macrophage cell line. Treatment of RAW264.7 macrophages with EDG signifi cantly inhibited NO production and inducible nitric oxide synthase (iNOS) expression stimulated by LPS, as assessed by Western blot and quantitative RTPCR analyses. We also demonstrated that EDG treatment led to an increase in heme oxygenase-1 (HO-1) mRNA and protein expression. EDG treatment also enhanced expression level of nuclear factor-erythroid 2-related factor 2 (Nrf2) in nucleus, which is critical for transcriptional induction of HO-1. Treatment with SnPP (tin protoporphyrin IX), a selective HO-1 inhibitor, reversed EDG-mediated inhibition of nitrite production, suggesting that HO-1 plays an important role in the suppression of NO production by EDG. Taken together, these results indicate that EDG isolated from Galla Rhois suppresses LPS-stimulated NO production in RAW 264.7 macrophages via HO-1 induction.

Heme arginate potentiates latent HIV-1 reactivation while inhibiting the acute infection

Human immunodeficiency virus-1 (HIV-1) successfully escapes from host immune surveillance, vaccines and antiretroviral agents. The available antiretroviral compounds can only control viremia, but it is impossible to eliminate the virus from the organism, namely because HIV-1 provirus persists in the reservoir cells from which the virus repeatedly disseminates into new cells. Current therapeutic approaches, however, do not specifically address the stage of virus reactivation.Heme has been demonstrated as very efficient in inhibiting HIV-1 reverse transcription, while its derivative hemin ameliorated HIV-1 infection via induction of heme oxygenase-1. Normosang (heme arginate; HA) is a human hemin-containing compound used to treat acute porphyria. In this work, we studied the effects of HA in HIV-1-acutely infected T-cell lines, and in cell lines harboring either a complete HIV-1 provirus (ACH-2 cells) or an HIV-1 “mini-virus” (Jurkat clones expressing EGFP under control of HIV LTR). We demonstrate that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA alone stimulated the reactivation of HIV-1 “mini-virus” and synergized with phorbol ester or TNF-α in the reactivation of HIV-1 provirus. The stimulatory effects of HA were inhibited by N-acetyl cysteine, suggesting an increased redox stress and activation of NF-κB. Further, HA induced expression of heme oxygenase-1 (HO-1) in ACH-2 cells, while HO-1 was found expressed in untreated Jurkat clones. Inhibitor of HO-1 activity, tin protoporphyrin IX, further increased HA-mediated reactivation of HIV-1 “mini-virus” in Jurkat clones, and this effect was also inhibited by N-acetyl cysteine. The stimulatory effects of HA on HIV-1 reactivation thus seem to involve HO-1 and generation of free radicals. Additionally, the effective concentrations of HA did neither affect normal T-cell activation with PMA nor induce activation of the unstimulated cells.In conclusion, HA appears to possess a combination of unique properties that could help to decrease the pool of latently infected reservoir cells, while simultaneously inhibiting HIV-1 replication in newly infected cells. Our results thus suggest a new direction to explore in treatment of HIV/AIDS disease.

Simvastatin induces the expression of hemeoxygenase-1 against ischemia–reperfusion injury on the testes in rats

We evaluate the protective role of simvastatin-induced HO-1 in remote preconditioning against testis ischemia–reperfusion (IR) injury in vivo. Simvastatin was intraperitoneally (i.p.) injected 24h before IR injury. Testis was occluded in the right testis for 40min and followed by 30min of reperfusion to induce IR injury. Tin protoporphyrin (Snpp), a competitive inhibitor of hemeoxygenase, was i.p. injected 1h before the IR injury in separate groups of rats. The rat testes were harvested 24h later. Induction of HO-1 expression by simvastatin was significantly increased at 24 and 48h. Rats pre-treated with simvastatin showed higher expression of HO-1 protein by Western blotting and immunohistochemistry (IHC), and presented lower caspases-3 activity by caspase-3 activity assay. TUNEL staining analysis revealed simvastatin pretreatment significantly reduced IR induced cellular apoptosis. Contrarily, the simvastatin-induced cytoprotective effect was entirely abolished by administrations of Snpp. Further, lower caspase-3 activities were also noted in simvastatin plus Snpp (SS) group than the control plus Snpp (CS) group. After IR injury, eNOS immunoreactivity was markedly increased in the germ cell and Leydig cell of testicular tissues. Pretreatment of simvastatin significantly decreased eNOS immunoreactivity in the germ cell of the tubules in the rat testes. In conclusion, we suggest HO-1 plays a protective role in IR-induced injury in the testes of rats.

Anti-inflammatory effects of lindenenyl acetate via heme oxygenase-1 and AMPK in human periodontal ligament cells

The molecular basis for the anti-inflammatory effects of lindenenyl acetate (LA) was investigated in the lipopolysaccharide (LPS)-stimulated human periodontal ligament (HPDL) cell model. LA concentration-dependently inhibited LPS-induced inducible nitric oxide synthase (iNOS) derived nitric oxide (NO) and cyclooxygenase-2 (COX-2) derived prostaglandin E2 (PGE(2)) production in HPDL cells. LA also attenuated the production of LPS-induced tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-12. LA stimulated heme oxygenase-1 (HO-1) protein expression and enzyme activity of HPDL cells in a dose-dependent manner. Pretreatment with the HO-1 inhibitor, tin protoporphyrin (SnPP), attenuated the inhibitory activities of LA on LPS-induced inflammatory NO, PGE(2), IL-1β, TNF-α, IL-6 and IL-12 production. LA induced translocation of Nrf-2. Furthermore, an inhibitor of JNK MAPK abolished LA-induced HO-1 expression. LA exposure up-regulated the levels of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and its upstream kinase activators, including LKB1 and Ca2+/calmodulin-dependent protein kinase kinase-II. Furthermore, compound C, a specific AMPK inhibitor, partially blocked the LA-induced anti-inflammatory effect. Taken together, these results indicate that LA has anti-inflammatory activity in HPDL cells that might be mediated by the HO-1, AMPK, JNK MAPK, and Nrf-2 pathways. Thus, LA may serve as a potential therapeutic agent in periodontal disease.

Involvement of heme oxygenase-1 induction in inhibitory effect of ethyl gallate isolated from Galla Rhois on nitric oxide production in RAW 264.7 macrophages

In the present study, we investigated an anti-inflammatory effect of ethyl gallate (EG) isolated from Galla Rhois as evaluated by inhibition of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and a potential role of heme oxygenase-1 (HO-1) in the inhibition of NO production elicited by EG. Treatment of RAW264.7 macrophages with EG significantly inhibited the production of NO and iNOS expression stimulated by lipopolysaccharide (LPS). We also demonstrated that EG treatment increased HO-1 mRNA and protein expression, as assessed by quantitative RT-PCR and Western blot analysis. EG treatment also increased the levels of nuclear factor-erythroid 2-related factor 2, which is critical for transcriptional induction of HO-1. In addition, treatment with SnPP (tin protoporphyrin IX), a selective HO-1 inhibitor, counteracted the inhibitory effect of EG on nitrite production, suggesting that HO-1 is, at least in part, implicated in the inhibition of NO production induced by EG treatment. Taken together, these results indicate that EG isolated from Galla Rhois suppresses NO production in LPS-stimulated RAW 264.7 macrophages via HO-1 induction.

Antecedent hydrogen sulfide elicits an anti-inflammatory phenotype in postischemic murine small intestine: role of heme oxygenase-1

We recently demonstrated that preconditioning with an exogenous hydrogen sulfide donor (NaHS-PC) 24 h before ischemia and reperfusion (I/R) causes postcapillary venules to shift to an anti-inflammatory phenotype in C57BL/6J wild-type (WT) mice such that these vessels fail to support increases in postischemic leukocyte rolling (LR) and leukocyte adhesion (LA). The objective of the present study was to determine whether heme oxygenase-1 (HO-1) is a mediator of these anti-inflammatory effects noted during I/R in mice preconditioned with NaHS. Intravital fluorescence microscopy was used to visualize LR and LA in single postcapillary venules of the murine small intestine. I/R induced marked increases in LR and LA, effects that were prevented by NaHS-PC. Treatment with the HO inhibitor tin protoporphyrin IX, but not the inactive protoporphyrin CuPPIX, just before reperfusion prevented the anti-inflammatory effects of antecedent NaHS. The anti-inflammatory effects of NaHS-PC were mimicked by preconditioning with hemin, an agent that induces HO-1 expression. We then evaluated the effect of NaHS as a preconditioning stimulus in mice that were genetically deficient in HO-1 (HO-1(-/-) on an H129 background with appropriate WT strain controls). NaHS-PC was ineffective in HO-1(-/-) mice. Our work indicates that HO-1 serves as an effector of the anti-inflammatory effects of NaHS-PC during I/R 24 h later.