Heme Oxygenase: Inhibitors and Inducers

A broad spectrum of molecules has currently been identified with the capacity to modulate the expression or functionality of HO-1. These molecules not only include HO-1 inhibitors, such as metalloporphyrins and imidazole-dioxolanes, but also HO-1 inducers including lipid-modulating, anti-proliferative, anti-inflammatory and anti-diabetic drugs (see Table 3).

Metalloporphyrins

Synthetic metalloporphyrins, structurally similar to heme, have been developed as first generation competitive inhibitors of HO-1 which include heavy metal chelates of deuteroporphyrin (DP), mesoporphyrin (MP), and protoporphyrin (PP), respectively ⁵¹⁷. Amongst them, tin mesoporphyrin (SnMP) could be identified as the most potent inhibitor for both HO isoenzymes ^{517, 518}. Several investigations approved the efficacy of metalloporphyrins in blocking HO activity. It has been shown previously that tin protoporphyrin IX (SnPPIX) markedly improved the efficacy of photodynamic therapy in cultured melanoma tumors ⁵¹⁹ and reduced Kaposi sarcoma growth in vivo ⁵²⁰. However, the applicability of metalloporphyrins is limited due to their poor solubility, lack of isoform selectivity and pleiotropic off-target effects including phototoxicity, influence on other heme-dependent enzymes, and, importantly, up-regulation of HO-1 expression ^521-526. Particularly with regard to its photosensitizing and phototoxic effects, SnPPIX has been abandoned for use in human infants, even though the photosensitizing properties of SnPPIX might be of advantage, such as in the photodynamic treatment of psoriasis⁵²⁷. The naturally occurring zinc protoporphyrin IX (ZnPPIX) has attracted much attention as it turns relatively dormant upon light exposure and thus does not show phototoxic effects in vivo ^{528, 529}. Previous investigations by Maines and colleagues revealed that the subcutaneous (s.c.) application of ZnPPIX efficiently suppressed HO activity in neonatal rats and neonatal rhesus monkeys ^{530, 531}. More recently, ZnPPIX was reported to suppress the angiogenesis of pancreatic and lung cancer and to block the metastatic potential of gastric cancer by suppressing pro-angiogenic VEGF ⁴⁹⁷. The insolubility of metalloporphyrins in water could be overcome by conjugation of the polyethylene-glycol or amphiphilic styrene-maleic acid co-polymer to ZnPPIX, thereby generating water-soluble compounds (PEG-ZnPPIX, SMA-ZnPPIX) with potent HO inhibitory capacity ^{532, 533}. Further attractive candidates comprise tin mesoporphyrin (SnMP) ⁵³⁴, chromium mesoporphyrin (CrMP) ⁵¹⁷, and zinc deuteroporphyrin IX bis-glycol (ZnDP-BG) ^{517, 535-537}. ZnDP-BG, a modified derivative of DP with a better oral absorption, has been shown to suppress both basal and heme-induced HO activity in the liver and spleen of Friend leukemia virus B (FVB) mice, and also to diminish in vivo bilirubin production ⁵²⁶. Since SnMP has been found as being 10-fold more potent than SnPP in blocking HO-1 activity ⁵³⁴, several clinical trials were conducted demonstrating a reduction of total bilirubin after SnMP application in neonates (reviewed by Schulz et al., 2012) ⁵²¹ Recently, a multicenter clinical trial evaluating the long-term effects of SnMP revealed that the early, predischarge SnMP administration decreased the duration of phototherapy, reversed total bilirubin trajectory and reduced the severity of concomitant hyperbilirubinemia in neonates ⁵³⁸.

Imidazole-dioxolanes

Imidazole-based molecules have been proven as second generation of HO-1 inhibitors with a better specificity towards HO-1, thereby increasing the options of developing new therapeutic drugs ^{539, 540}. These molecules function as non-competitive HO-1 inhibitors by preventing the oxidation of ferrous iron and oxygen binding, in case heme is coupled to the HO-1 binding site ^{539, 541}. Amongst them, azalanstat (QC-1) was about the first imidazole-dioxalane compound reported to inhibit the activity of HO-1 ⁵⁴². Several analogs based on the lead structure of QC-1 were synthesized and evaluated as novel inhibitors of heme oxygenase, including QC-15, QC-80, QC-82, QC-86, and QC-308, with QC-15 identified as being highly selective for HO-1 ^{540, 543}. Most recently, QC-15 was found to abrogate the suppressive effect of hemin on increased senescence in fibroblasts from patients with chronic obstructive pulmonary disease (COPD). COPD fibroblast showed mitochondrial dysfunction even at non-senescent passage which is preventable by hemin treatment. The protective effects of hemin against mitochondrial dysfunction is attributable to the increased activity of HO-1 as these effects were abolished by QC-15 and ZnPPIX, respectively ⁵⁴⁴. Moreover, QC-15 has been reported to reverse the suppressive capacity of the HO-1 induction on Aβ_1-42 toxicity in primary cultures of rat astrocytes ⁵⁴⁵. However, only limited information is available on the in vivo function of these novel inhibitors. Recent investigations by Csongradi et al. revealed that the systemic administration of QC-13 preferentially inhibits renal cortical HO-1 activity without inducing endogenous HO-1, thus identifying QC-13 as being a useful pharmacological tool to study the role of renal HO in kidney physiology and pathophysiology⁵⁴⁶. Intrarenal medullary interstitial infusion (IRMI) of QC-13 in C57BL/6J mice was noted to exacerbates angiotensin II-induced hypertension in these animals⁵⁴⁷, while angiotensin II-treated mice receiving IRMI infusions of QC-13 and the superoxide dismutase mimetic tempol had a significantly lower blood pressure than mice receiving QC-13 alone⁵⁴⁸. From this result the authors hypothesize that the renal medullary interstitial blockade of HO-1 aggravates angiotensin II-induced hypertension via an up-regulated formation of superoxide, thereby outlining the pivotal anti-oxidant function of HO-1 in the renal medulla⁵⁴⁸. OB-28, a novel azole-based competitive and reversible inhibitor of HO-1, has been demonstrated to significantly counteract behavioral deficits and neuropathological alterations in a transgenic mouse model of Alzheimer’s disease (AD)⁵⁴⁹. Recently, a novel class of potent and selective imidazole-based HO-1 inhibitors have been described leading to the identification of compound 1, a phenyl ethanolicazole-based inhibitor bearing a bromine atom in the phenyl ring, which could be characterized as being one of the most potent HO-1 inhibitors to date⁵⁵⁰. Compound 1 shows a favorable toxicity profile as well as non-tumorigenic or non-irritant effects, without negatively affecting the reproductive system⁵⁵⁰. Incubation of B16 melanoma cells with compound 1 and the DNA intercalator doxorubicin has been shown to exert a synergistic cytotoxic effect⁵⁵¹. Encapsulation of compound 1 into a nanodelivery system based styrene-maleic acid (SMA) micelles was carried out in order to improve compound 1’s water solubility. As a result thereof, encapsulation of compound 1 into SMA micelles attenuated the cytotoxic properties of the drug in accordance with the commonly reduced activity demonstrated in vitro by the nanoformulations⁵⁵².

HO-1 Inducers

Various pharmaceutical and natural agents are known to induce HO-1 (Table 3). A major group of HO-1 inducers is represented by lipid-modulating drugs, such as fibrates, phenolics, and statins. The PPAR-α agonist fenofibrate has been found to up-regulate the expression of HO-1 in human umbilical venous endothelial cells (ECs) and human vascular smooth muscle cells (SMCs)⁵⁵³. In line with this finding, the pleiotropic drug niacin that is known to decelerate the progression of coronary artery disease and to increase serum levels of bilirubin was reported to increase HO-1 expression in cultured human coronary artery ECs by activating the Nrf-2/p38 MAPK signaling pathway and inhibiting TNF-induced endothelial inflammation⁵⁵⁴.

Statins such as artovastatin, fluvastatin, mevastatin, lovastatin, pravastatin, and simvastatin have been noted to up-regulate HMOX1 expression in murine macrophages via protein kinase G, ERK, and p38MAPK signaling pathways ⁵⁵⁵. An increased expression of HO-1 is also observed in vascular ECs ⁵⁵⁶ and renal ECs ⁵⁵⁷ after exposure to simvastatin and pravastatin, respectively. The PI3K/Akt pathway obviously plays a crucial role in the simvastatin-mediated up-regulation of HO-1 in human ECs ⁵⁵⁸ and in human and rat vascular SMCs ⁵⁵⁹. Accordingly, fluvastatin protects coronary artery SMCs against oxidative stress by enhancing HO-1 expression via the PI3K/Akt/Nrf-2 signaling cascade ⁵⁶⁰.

Amongst phenolics, probucol has been demonstrated to up-regulate HO-1 expression and activity in vitro and in animal models of atherosclerosis ^561-563. In a study conducted by Zhou and collaborators, probucol significantly increased the expression of Nrf-2, NQO-1 and HO-1 in damaged astrocytes, while diminishing the expression of pro-inflammatory IL-1β, IL-6 and TNF, that are crucially involved in the protective effects of Nrf-2 against the inflammatory component of spinal cord injury (SCI) ⁵⁶⁴. As these effects were associated with a reduction in neural cell apoptosis and promotion of nerve function recovery, one can postulate that activation of the Nrf-2 signaling pathway obviously triggers the neuroprotective effects of probucol after SCI. Probucol exerts anti-restenotic and anti-thrombotic properties in rabbits subjected to endothelial denudation and stenting of the iliac artery ⁵⁶². These effects are likely related to its ability to promote in-stent re-endothelialization after femoral stenting ⁵⁶². In rabbit aortic balloon injury, a model of restenosis, probucol was reported to induce HO-1 and to inhibit atherosclerotic vascular disease by blocking macrophage accumulation, stimulating re-endothelialization, and inhibiting vascular SMC proliferation ⁵⁶⁵. In the same model, the mono-succinate probucol derivative succinobucol, characterized by an enhanced solubility in water, has been found to up-regulate HO-1 expression and to diminish neointimal hyperplasia in injured vessels ⁵⁶⁶. Probucol as well as succinobucol stimulate the number of endothelial progenitor cells and their adherence to the luminal surface of the injured vessel ⁵⁶⁶. In contrast to probucol, succinobucol obviously does not mediate its effects through HMOX1 up-regulation but rather via apoptosis induction ⁵⁶⁷. Meanwhile, clinical studies have been conducted to evaluate the effectiveness of probucol and succinobucol in atherosclerosis and restenosis, but with unsatisfactory outcomes. While, on the one hand, probucol had no effect on the degree of aorto-femoral atherosclerosis in human subjects ^{568, 569}, the agent has been noted to diminish cholesterol levels and to stabilize plaques, leading to a lower incidence of cardiac events in hypercholesterolemic patients ⁵⁷⁰. Also succinobucol failed to affect cardiovascular outcomes in patients with recent acute coronary syndromes, already managed with conventional treatments ⁵⁷¹. A multicenter, randomized, prospective study has been designed aiming at clarifying the safety of long-term probucol treatment in patients with prior coronary heart disease, and determining whether the addition of probucol to other lipid-lowering drugs improves cerebro- and cardiovascular outcomes ⁵⁷².

Anti-proliferative drugs such as paclitaxel and sirolimus are also able to induce the expression of HO-1. While paclitaxel has been noted to block the proliferation of vascular SMCs by up-regulating the expression of HO-1 via the JNK pathway ⁵⁷³, the mammalian target of rapamycin (mTOR) inhibitor sirolimus (rapamycin) suppresses the proliferation of vascular ECs and SMCs through the PI3K-dependent induction of HO-1 ⁵⁷⁴. Sirolimus was also found to induce HO-1 expression in renal cancer cells, thereby promoting tumor cell growth by blocking apoptosis and autophagy ⁵⁷⁵. In contrast to its cytoprotective effects in several tumor cells, sirolimus has been reported to induce the expression of HMOX1 in normal hepatocytes and to down-regulate HMOX1 in malignant liver cells implying a different mechanism of action for rapamycin in hepatocytes ⁵⁷⁶. Chromatin immunoprecipitation assays performed by Finn and collaborators demonstrated that sirolimus interferes with binding of PPAR-γ to its response elements in the HMOX1 promoter ⁵⁷⁷. Activation of PPAR-γ has been illustrated to induce the expression of HO-1 in vascular ECs and SMCs, suggesting HO-1 as being a down-stream target of PPAR-γ and HO-1 induction conferring the protective role of PPAR-γ activation against numerous stressors ^{553, 578}. Studies by Krönke et al. identified a direct transcriptional regulation of HO-1 by PPAR-α and PPAR-γ through two PPAR responsive elements within the HMOX1 promoter ⁵⁵³.

The anti-diabetic drug and PPAR-γ agonist rosiglitazone has been reported to protect rat cardiomyoblast cells H9c2(2-1) from oxidative stress by up-regulating the expression of HO-1 and also to exert cardioprotection in a murine model of myocardial I/R ⁵⁷⁹. Interestingly, rosiglitazone stimulated HO-1 as well as p21^Waf-1 expression in rat pulmonary artery SMCs (PASMCs) and thus suppressed the proliferation of PASMCs ⁵⁷⁸. This study also outlined a potential benefit of rosiglitazone administration for patients with pulmonary hypertension. In line with this notion, rosiglitazone ameliorated pulmonary arterial hypertension (PAH) through an up-regulation of HO-1 and p21^Waf-1in a rat model of PAH in vivo ⁵⁸⁰. Rosiglitazone administration significantly increased plasma HO-1 levels, ameliorated hypertension, improved vascular function, and reduced the elevated microalbumin : creatinine ratio in a reduced uterine perfusion pressure (RUPP) rat model of pre-eclampsia thereby preventing several of the pathophysiological characteristics associated with this pre-eclampsia model ⁵⁸¹. More recently, up-regulation of HO-1 in vitro or in vivo induced by rosiglitazone was found to attenuate VCAM1 gene expression and monocyte adhesion to human pulmonary alveolar epithelial cells (HPAECs) challenged with lipopolysaccharide (LPS) ⁵⁸². Rosiglitazone-induced HO-1 expression obviously occurs through PKCα/AMPKα/p38 MAPKα/SIRT1-dependent deacetylation of Ac-PGC1α and fragmentation of NCoR/PPAR-γ activation in HPAECs ⁵⁸².

The anti-inflammatory drug aspirin has been noted to up-regulate HO-1 protein levels and activity in ECs derived from human umbilical vein in vitro via an NO-dependent pathway ⁵⁸³. Aspirin protects human primary melanocytes against H₂O₂-induced oxidative stress by an Nrf-2-driven transcriptional activation of HMOX1 ⁵⁸⁴. In a spinal cord contusion model in Sprague-Dawley rats, intra-peritoneal administration of aspirin up-regulated protein expression of HO-1, Nrf-2 and NQO-1 in animals after spinal cord injury ⁵⁸⁵. Aspirin also reduced the levels of pro-apoptotic caspase-3 and Bax, suppressed tissue inflammation and restrained astrocyte activation by stimulating the Nrf-2/HO-1 signaling pathway ⁵⁸⁵. It is of note that a randomized, double-blind, placebo-controlled study revealed that aspirin at therapeutic doses did not enhance plasma HO-1 protein concentrations or venous monocyte HO-1 activity in healthy humans ⁵⁸⁶.

Among the pharmaceuticals compounds that are able to activate the Nrf-2/Keap-1 pathway, fumarates such as dimethyl fumarate (DMF) have attracted much attention due to their high capacity to induce HO-1 ⁵⁸⁷. In BV12 microglia cells, DMF exerts anti-inflammatory effects by up-regulating HO-1 mRNA and protein levels and concomitantly down-regulating pro-inflammatory mediators ⁵⁸⁷. Neuroprotective effects of DMF can be seen in mouse models of chronic experimental autoimmune encephalomyelitis in which the drug up-regulated anti-inflammatory IL-10 in the blood, blocked macrophage infiltration in the spinal cord ⁵⁸⁸, and stimulated the Keap-1-dependent expression of Nrf-2 in neurons of the motor cortex and the brainstem as well as in oligodendrocytes and astrocytes ⁵⁸⁹. In addition, DMF has been shown to abolish intimal hyperplasia in a model of vascular injury triggered by the Nrf-2 signaling pathway and concomitant up-regulation of p21^Cip-1 ⁵⁹⁰. Clinically, fumaric acid esters have been used in the medication of psoriasis ⁵⁹¹ whereas DMF has been evaluated in clinical trials on multiple sclerosis (MS) ¹⁶. Phase III clinical trials evaluating the safety and efficacy of DMF in patients with relapsing-remitting MS identified DMF as being an orally available agent with a favorable safety profile and thus placing it as an attractive first-line therapy option for the treatment of relapsing variants of MS ^{592, 593}. Meanwhile, oral DMF (BG-12) has been approved for the treatment of MS in various countries including Europe and the United States. The neuroprotective properties of DMF have been attributed, amongst others, to the modification of a critical cysteinyl residue in Keap-1 leading to stabilization of Nrf-2 and subsequent up-regulation of the down-stream effector HO-1 ^{589, 594}.

A subgroup of HO-1-inducing agents comprises electrophilic anti-oxidants such as plant-derived polyphenols. This naturally occurring phytochemicals have been shown to induce HO-1 expression via Nrf-2 activation and include, e.g., caffeic acid phenetyl ester ⁵⁹⁵, carnosol ⁵⁸⁷, curcumin ⁵⁹⁶, epigallocatechin-3 gallate (EGCG) ⁵⁹⁷, ethyl ferulate ⁵⁹⁸, quercetin ⁵⁹⁹, resveratrol ⁶⁰⁰, and sulforaphane ⁵⁸⁷. Amongst them, curcumin from the golden spice turmeric (Curcuma longa) has been evaluated extensively in clinical trials over the past years with respect to pharmacokinetics, safety, and efficacy against numerous human disorders with some promising effects observed in a variety of pro-inflammatory diseases including cancer (for a review see Gupta et al., 2013) ⁶⁰¹. However, in an open label uncontrolled phase-1 pilot study curcumin failed to induce HO-1 expression in PBMNCs from male subjects due to its low bioavailability ⁶⁰². This is somewhat surprising as a randomized placebo-controlled trial demonstrated enhanced urinary levels of HO-1 in dialysis-dependent cadaveric kidney recipients after curcumin and quercetin administration ⁶⁰³. Apart from its low bioavailability, curcumin shows poor absorption, biodistribution, and metabolism. In order to increase the bioavailability, permeability and resistance to metabolization of curcumin, several formulations have been prepared such as nanoparticles, liposomes, micelles, and phospholipid complexes ⁶⁰⁴.

The catechin (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and powerful catechin in cancer prevention and treatment ⁶⁰⁵. Pleiotropic effects of EGCG include anti-oxidant activities, cell signaling modulation, apoptosis induction, cell cycle arrest as well as inhibition of matrix metalloproteinases (MMPs), urokinase-plasminogen activator, telomerase, DNA methyltransferase, and blockage of the proteasome ⁶⁰⁶. In human vascular ECs, EGCG induces HO-1 expression and increases HO-1 activity through the p38MAPK/Nrf-2 signaling pathway ⁵⁹⁷. This is accompanied by the blockage of TNF-stimulated expression of vascular cell adhesion molecule 1 (VCAM-1) and decreased adhesion of monocytes to vascular ECs ⁵⁹⁷. EGCG protects human umbilical vein endothelial cells (HUVECs) from oxidative stress injury by up-regulating Nrf-2/HO-1 via activation of the p38MAPK and the ERK-1/-2 signaling pathways ⁶⁰⁷. Several investigations reveal neuroprotective properties of EGCG by blocking ROS formation in neurons, as well as through iron chelating and anti-apoptotic functions ^608-610. HO-1 expression has been found as being up-regulated in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) ⁶¹¹ which can be regulated by EGCG in vitro ⁶¹². Interestingly, Janssen and colleagues demonstrated that EGCG monotherapy ameliorates chronic EAE in mice by down-regulating HMOX1 expression in the cerebellum and spinal cord while the combinatorial treatment of EGCG and glatiramer acetate (GA), an immunomodulatory drug approved for relapsing-remitting MS, increased HMOX1 expression in cerebellum and spinal cord without affecting EAE progression ⁶¹². From these findings one can speculate that stress-induced HO-1 may initially exert protective properties, whereas chronic HO-1 up-regulation might be implicated in oligodendroglial cell death and disease progression ⁶¹³. In mice, EGCG was noted to increase nuclear translocation of Nrf-2 as well as HMOX1 expression and to suppress ROS formation in isolated pancreatic islets, accompanied by a decrease of blood glucose in transplanted animals ⁶¹⁴. Noteworthy, EGCG significantly up-regulated HO-1 expression through the PI3K/Akt pathway in HepG2 cells under insulin resistance conditions and improved high-fat and high-fructose diet (HFFD)-triggered insulin resistance and oxidative stress by activating the IRS-1/Akt and Keap-1/Nrf-2 transcriptional pathways in vivo ⁶¹⁵. Thus, administration of EGCG might be considered as being a promising approach as an adjuvant therapy in patients with obesity.

The phytoalexin resveratrol, a biologically active compound present in various plant species, exerts anti-inflammatory, anti-tumorigenic, anti-oxidant, neuroprotective, and anti-obesity effects. In neuron-like PC12 cells, resveratrol has been shown to up-regulate the expression of HMOX1 via the Nrf2-StRE/ARE signaling pathway thereby protecting PC12 cells from oxidative stress ⁶⁰⁰. Moreover, resveratrol has a protective effect on oligodendroglial functionality against LPS-induced cytotoxicity and this glioprotective mechanism is mediated by the HO-1/Nrf-2 signaling pathway ⁶¹⁶. In neural stem cells (NSCs), resveratrol treatment was noted to alleviate injury and to stimulate proliferation of NSCs, at least in part, by up-regulating the protein levels of HO‑1, Nrf-2, and NAD(P)H:quinone oxidoreductase 1 (NQO-1) following oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro ⁶¹⁷. It is also apparent that resveratrol increased the levels of HO-1 and Nrf-2, down-regulated the H. pylori-induced mRNA transcription and protein expression levels of IL-8 and iNOS, suppressed H. pylori-induced phosphorylation of IκBα in gastric mucosal tissues of mice after H. pylori infection ⁶¹⁸. These data clearly demonstrate that resveratrol attenuates H. pylori-associated gastritis by blocking the microbe-induced production of pro-inflammatory mediators and up-regulating HO-1 and Nrf-2 expression in inflamed tissues.

A growing body of evidence indicates anti-diabetic effects of resveratrol in animal models of diabetes and diabetic humans (summarized by Szkudelski and Szkudelska, 2015) ⁶¹⁹. Preliminary clinical trials show that resveratrol is effective in patients with type 2 diabetes mellitus (T2DM) ⁶²⁰. In a randomized, placebo-controlled, double-blind clinical trial, resveratrol was found to modulate the expression of HO-1 in PBMCs from T2DM patients by increasing Nrf-2 expression and down-regulating ROS formation ⁶²⁰. Most recently, resveratrol prevented cognitive deficits by reducing oxidative damage and inflammation in an experimental rat model of diabetes-induced vascular dementia, as evidenced by altered expression levels of HO-1, superoxide dismutase (SOD), NADPH oxidase, TNF-α and IL-1β; serum SOD and NADPH oxidase and hippocampal BDNF, TNF and IL-1β ⁶²¹. Thus, the vasculoprotective and neuroprotective effects of resveratrol may have an impact on the treatment of diabetic patients.

Hassania and co-workers described the capacity of the natural anti-cancer agent withaferin A (WA), a steroidal lactone extracted from the roots and leaves of Withania somnifera (ashwagandha, Indian ginseng), to directly target Keap-1 and to release Nrf-2, followed by HO-1 up-regulation, iron accumulation, and death of neuroblastoma cells via ferroptosis induction ³¹². A growing body of evidence indicates that WA abolishes experimentally induced tumorigenesis, essentially caused by its powerful anti-inflammatory, anti-oxidative, anti-proliferative and pro-apoptotic features ³²⁸. It is noteworthy that withaferin A sensitizes resistant tumor cells to existing chemotherapeutic agents ^622-624. WA is also able to eradicate high-risk neuroblastoma tumors in mice through lipid peroxidation-induced cell death which subsequently triggers the infiltration of a high number of immune cells ³¹². Of note, nanotargeting of WA avoided systemic side effects and markedly blocked growth of neuroblastoma tumors, thus rendering WA as being an auspicious aspirant for further therapeutic application using novel targeting approaches such as nanomedicine ³¹².

Sulforaphane (SFN), an isothiocyanate present in the species of the Brassicaceae, especially in broccoli sprouts, represents a potent activator of Nrf-2 which counteracts with oxidative stress by inducing detoxifying enzymes such as HO-1 ^625-628. In human HepG2 hepatoma cells, SFN markedly up-regulated Nrf-2 protein expression and StRE/ARE-mediated transcription activation, delayed Nrf-2 degradation through blockage of Keap-1, and thus stimulating HO-1 expression ⁶²⁵. SFN treatment of mdx mice, a mouse model of Duchenne muscular dystrophy (DMD), has been identified to improve muscle function via Nrf-2 activation and Nrf2-mediated up-regulation of HO-1 and NQO-1 rendering SFN as being an auspicious tool to target Nrf-2/HO-1 in DMD ⁶²⁷. Furthermore, SFN protected rodent retinas against I/R injury by enhancing nuclear accumulation of Nrf-2 and HO-1 expression in the I/R retinas ⁶²⁶. SFN increased the expression of anti-inflammatory Nrf-2 and HO-1 as well as anti-inflammatory IL-10 and IL-4 in BV2 microglial cells ⁶²⁹. The drug also significantly suppressed JNK-dependent signaling transduction, culminating in the down-regulation of NF-κB/AP-1-triggered expression of inflammatory mediators (iNOS, PGHS-2, NO, PGE₂) and pro-inflammatory cytokines (IL-1β, IL-6, TNF) ⁶²⁹. These data encourage further investigations in vivo and clinical trials aiming at evaluating the therapeutic potential of SFN in regulating the inflammatory response in neuroinflammatory diseases. In this regard, an in vivo study clearly emphasized the protective effects of SFN on testis damage and spermatogenic functions in Kunming mice exposed to cadmium through an up-regulation of Nrf-2, HO-1, and NQO-1 protein levels ⁶³⁰. This is in line with current observations in mouse Leydig cells eliciting a protective role of SFN against cadmium-induced oxidative damage by up-regulating the mRNA expression of NRF2, HMOX1, GSHPX1, NQO1, and GCLC ⁶³¹.

Table 3: Representative Inducers of Heme Oxygenase 1 (HO-1)

Agent	References
Anti-diabetic drugs
Rosiglitazone	Mersmann et al. (2008) ⁵⁷⁹, Li et al. (2010) ⁵⁷⁸, Zhang et al. (2014) ⁵⁸⁰, Cho et al. (2018) ⁵⁸²
Anti-inflammatory drugs
Aspirin	Grosser et al. (2003) ⁵⁸³, Bharucha et al. (2014) ⁵⁸⁶, Jian et al. (2016) ⁵⁸⁴, Wei et al. (2018) ⁵⁸⁵
Anti-proliferative drugs
Paclitaxel	Choi et al. (2004) ⁵⁷³
Sirolimus (rapamycin)	Visner et al. (2003) ⁵⁷⁴, Finn et al. (2009) ⁵⁷⁷, Banerjee et al. (2012) ⁵⁷⁵, Afroz et al. (2018) ⁵⁷⁶
Lipid-modulating drugs
Fenofibrate	Krönke et al. (2007) ⁵⁵³
Probucol	Walldius et al. (1994) ⁵⁶⁹, Sawayama et al. (2002) ⁵⁷⁰, Tardiff et al. (2003) ⁵⁶⁸, Lau et al. (2003) ⁵⁶⁵, Tanous et al. (2006) ⁵⁶², Wu et al. (2009) ⁵⁶⁶, Yamashita et al. (2016) ⁵⁷², Zhou et al. (2017) ⁵⁶⁴
Succinobucol	Tardiff et al. (2003) ⁵⁶⁸, Tardiff et al. (2008) ⁵⁷¹, Wu et al. (2009) ⁵⁶⁶, Midwinter et al. (2012) ⁵⁶⁷
Atorvastatin	Chen et al. (2006) ⁵⁵⁵, Ali et al. (2007) ⁶³²
Fluvastatin	Chen et al. (2006) ⁵⁵⁵, Makabe et al. (2010) ⁵⁶⁰
Pravastatin	Chen et al. (2006) ⁵⁵⁵, Chen et al. (2010) ⁵⁵⁷
Mevastatin	Chen et al. (2006) ⁵⁵⁵
Simvastatin	Lee et al. (2004) ⁵⁵⁹, Chen et al. (2006) ⁵⁵⁵, Uchiyama et al. (2006) ⁵⁵⁶, Hinkelmann et al. (2010) ⁵⁵⁸
Polyphenols
Curcumin	Balogun et al. (2003) ⁵⁹⁶, Shoskes et al. (2005) ⁶⁰³, Gupta et al. (2013) ⁶⁰¹, Klickovic et al. (2014) ⁶⁰², Prasad et al. (2014) ⁶⁰⁴
Epigallocatechin-3 gallate (EGCG)	Aktas et al. (2004) ⁶¹⁰, Stahnke et al. (2007) ⁶¹³, van Horssen et al. (2008) ⁶¹¹, Schroeder et al. (2009) ⁶⁰⁹, Pullikotil et al. (2012) ⁵⁹⁷, Mähler et al. (2013) ⁶⁰⁸, Yang et al. (2015) ⁶⁰⁷, Janssen et al. (2015) ⁶¹², Mi et al. (2018) ⁶¹⁵, Wada et al. (2019) ⁶¹⁴
Quercetin	Shoskes et al. (2005) ⁶⁰³, Yao et al. (2007) ⁵⁹⁹
Resveratrol	Chen et al. (2005) ⁶⁰⁰, Zhang et al. (2015) ⁶¹⁸, Shen et al. (2016) ⁶¹⁷, Rosa et al. (2018) ⁶¹⁶, Seyyedebrahimi et al. (2018) ⁶²⁰, Gocmez et al. (2019) ⁶²¹
Isothiocyanates
Sulforaphane	Dinkova-Kostava et al. (2004) ⁶²⁸, Jeong et al. (2005) ⁶²⁵, Pan et al. (2014) ⁶²⁶, Sun et al. (2015) ⁶²⁷, Yang et al. (2016) ⁶³⁰, Yang et al. (2019) ⁶³¹
Vitamines
Niacin	Wu et al. (2012) ⁵⁵⁴
Withanolides
Withaferin A	Hassania et al. (2018) ³¹²
Fumarates
Dimethyl fumarate (DMF)	Altmeyer et al. (1994) ⁵⁹¹, Schilling et al. (2006) ⁵⁸⁸, Linker et al. (2011) ⁵⁸⁹, Takaya et al. (2012) ⁵⁹⁴, Foresti et al. (2013) ⁵⁸⁷, Oh et al. (2014) ⁵⁹⁰, Nicholas et al. (2014) ⁵⁹², Dubey et al. (2015) ⁵⁹³, Ryter and Choi (2016) ¹⁶
Metalloporphyrine
Heme	Braggins et al. (1986) ³, Trakshel et al. (1986) ², Alam et al. (1989) ⁶³³
Heavy Metals
Cadmium chloride (CdCl₂)	Maines et al. (1986) ⁴, Alam et al. (1989) ⁶³³

Heme Oxygenase: Inhibitors and Inducers

Heme Oxygenase Research Products