Haem oxygenase-1 (HO-1) is a ubiquitously expressed gene involved in cellular homoeostasis, and its imbalance in expression results in various disorders. To alleviate such disorders, HO-1 gene expression needs to be modulated. Codon usage bias results from evolutionary forces acting on any nucleotide sequence and determines the gene expression. Like codon usage bias, codon pair bias also exists, playing a role in gene expression. In the present study, HO-1 gene was recoded by manipulating codon and codon pair bias, and four such constructs were made through codon/codon pair deoptimization and codon/codon pair optimization to reduce and enhance the HO-1 gene expression. Codon usage analysis was done for these constructs for four tissues brain, heart, pancreas and liver. Based on codon usage in different tissues, gene expression of these tissues was determined in terms of the codon adaptation index. Based on the codon adaptation index, minimum free energy, and translation efficiency, constructs were evaluated for enhanced or decreased HO-1 expression. The analysis revealed that for enhancing gene expression, codon pair optimization, while for reducing gene expression, codon deoptimization is efficacious. The recoded constructs developed in the study could be used in gene therapy regimens to cure HO-1 over or underexpression-associated disorders.

UNASSIGNED: Acute kidney injury (AKI), the common complication after cardiopulmonary resuscitation (CPR), seriously affects the prognosis of cardiac arrest (CA) patients. However, there are limited studies on post-resuscitation AKI. In addition, it has been demonstrated that N-acetylcysteine (N-AC) as an ROS scavenger, has multiorgan-protective effects on systemic and regional ischaemia-reperfusion injuries. However, no studies have reported its protective effects against post-resuscitation AKI and potential mechanisms. This study aimed to clarify the protective effects of N-AC on post-resuscitation AKI and investigate whether its potential mechanism was mediated by activating Nrf-2/HO-1 pathway in the kidney.
UNASSIGNED: We established cardiac arrest models in rats. All animals were divided into four groups: the sham, control, N-AC, and ZnPP groups. Animals in each group except for the ZnPP group were assigned into two subgroups based on the survival time: 6 and 48 h. The rats in the control, N-AC, and ZnPP groups underwent induction of ventricular fibrillation (VF), 8 min untreated VF and cardiopulmonary resuscitation. Renal function indicators, were detected using commercial kits. Renal pathologic changes were assessed by haematoxylin-eosin (HE) staining. Oxidative stress and inflammatory responses were measured using the corresponding indicators. Apoptosis was evaluated using terminal uridine nick-end labeling (TUNEL) staining, and expression of proteins associated with apoptosis and the Nrf-2/HO-1 pathway was measured by western blotting.
UNASSIGNED: N-AC inhibited post-resuscitation AKI. We observed that N-AC reduced the levels of biomarkers of renal function derangement; improved renal pathological changes; and suppressed apoptosis, oxidative stress, and inflammatory response. Additionally, the production of ROS in the kidneys markedly decreased by N-AC. More importantly, compared with the control group, N-AC further upregulated the expression of nuclear Nrf2 and endogenous HO-1 in N-AC group. However, N-AC-determined protective effects on post-resuscitation AKI were markedly reversed after pretreatment of the HO-1 inhibitor zinc protoporphyrin (ZnPP).
UNASSIGNED: N-AC alleviated renal dysfunction and prolonged survival in animal models of CA. N-AC partially exerts beneficial renal protection via activation of the Nrf-2/HO-1 pathway. Altogether, all these findings indicated that N-AC as a common clinical agent, may have the potentially clinical utility to improve patients the outcomes in cardiac arrest.

Curcumin displays anticancer properties; however, some issues with the drug delivery mode limit its therapeutic use. Although reformulation and derivatization of curcumin have improved its bioavailability, curcumin derivatives may not retain the same anticancer properties as the parent compound. The present study investigated the anticancer properties of two curcumin complexes, the iron‑curcumin [Fe(Cur)3] and boron‑curcumin [B(Cur)2] complexes, in the MDA‑MB‑231 breast cancer cell line. The cellular localization of curcumin, B(Cur)2 and Fe(Cur)3 was determined by fluorescence microscopy. Cell proliferation, migration and invasion were also analysed. Furthermore, apoptosis‑associated proteins were detected by using a proteome profiler array, and ion channel gene expression was analysed by reverse transcription‑quantitative PCR. The results demonstrated that the three compounds were localized in the perinuclear and cytoplasmic regions of the cell, and displayed cytotoxicity with IC50 values of 25, 35 and 8 µM for curcumin, B(Cur)2 and Fe(Cur)3, respectively. In addition, the three compounds inhibited cell invasion, whereas only curcumin and B(Cur)2 inhibited cell migration. Furthermore, cell exposure to curcumin resulted in an increase in the relative expression of the two key proapoptotic proteins, cytochrome c and cleaved caspase‑3, as well as the antiapoptotic protein haem oxygenase‑1. In addition, curcumin increased the expression levels of the voltage‑gated potassium channels Kv2.1 and Kv3.2. Similarly, the expression levels of the chloride channel bestrophin‑1 and the calcium channel coding gene calcium voltage‑gated channel auxiliary subunit γ4 were increased following exposure to curcumin. Taken together, these results indicated that Fe(Cur)3 and B(Cur)2 may display similar anticancer properties as curcumin, suggesting that chemical complexation may be considered as a strategy for improving the potency of curcumin in the treatment of breast cancer.

UNASSIGNED: High-dose glucocorticoid (GC) therapy always causes osteoporosis partly by inducing osteoblast apoptosis. However, the underlying mechanisms of GC-induced apoptosis remain elusive. Haem oxygenase-1 (HO-1) is a cytoprotective protein that rescues cells from H2O2 or high glucose-induced apoptosis. In bone metabolism, HO-1 also participates in osteoclast and osteoblast differentiation.
UNASSIGNED: The present study aimed to investigate the protective role of HO-1 against GC-induced osteoblast apoptosis and to elucidate the underlying mechanism.
UNASSIGNED: Mouse osteoblastic MC3T3-E1 cells were treated with dexamethasone (Dex) for 24 h in the presence or absence of cobalt (III) protoporphyrin IX chloride (CoPP, an inducer of HO-1). In some experiments, U0126 was added to the culture 1 h before CoPP treatment. The induction of apoptosis was determined by flow cytometry. Cell viability was evaluated using a cell counting kit-8 (CCK-8) assay. The expression levels of Bax and bcl-2 were measured by real-time polymerase chain reaction and Western blot. HO-1, extracellular signal-regulated kinase (ERK)-1/2 and pERK1/2 protein levels were measured by Western blot analysis.
UNASSIGNED: Dex promoted apoptosis and inhibited cell viability in MC3T3-E1 cells. In addition, Dex significantly increased Bax expression and reduced Bcl-2 expression. The expression of HO-1 was also reduced after Dex treatment. HO-1 induction by CoPP significantly attenuated Dex-induced apoptosis as evidenced by Annexin V/PI staining. The mRNA expression level of antiapoptotic gene Bcl-2 was also increased after CoPP treatment. Moreover, CoPP treatment increased the phosphorylation of ERK1/2. U0126, an inhibitor of ERK activation, significantly abrogated the protective effects of CoPP.
UNASSIGNED: Our results demonstrate that HO-1 induction by CoPP can attenuate Dex-induced apoptosis of mouse osteoblastic MC3T3-E1 cells. The antiapoptotic effect of HO-1 induction may be correlated with the activation of ERK1/2 signalling pathway. The translational potential of this article: HO-1 induction by CoPP can prevent GC-induced osteoblast apoptosis. Our findings will highlight the therapeutic potential of HO-1 induction in GC-induced osteoporosis.

The cardiac protection of mesenchymal stem cell (MSC) transplantation for myocardial infarction (MI) is largely hampered by low cell survival. Haem oxygenase 1 (HO-1) plays a critical role in regulation of cell survival under many stress conditions. This study aimed to investigate whether pre-treatment with haemin, a potent HO-1 inducer, would promote the survival of MSCs under serum deprivation and hypoxia (SD/H) and enhance the cardioprotective effects of MSCs in MI. Bone marrow (BM)-MSCs were pretreated with or without haemin and then exposed to SD/H. The mitochondrial morphology of MSCs was determined by MitoTracker staining. BM-MSCs and haemin-pretreated BM-MSCs were transplanted into the peri-infarct region in MI mice. SD/H induced mitochondrial fragmentation, as shown by increased mitochondrial fission and apoptosis of BM-MSCs. Pre-treatment with haemin greatly inhibited SD/H-induced mitochondrial fragmentation and apoptosis of BM-MSCs. These effects were partially abrogated by knocking down HO-1. At 4 weeks after transplantation, compared with BM-MSCs, haemin-pretreated BM-MSCs had greatly improved the heart function of mice with MI. These cardioprotective effects were associated with increased cell survival, decreased cardiomyocytes apoptosis and enhanced angiogenesis. Collectively, our study identifies haemin as a regulator of MSC survival and suggests a novel strategy for improving MSC-based therapy for MI.

Intracerebral haemorrhage (ICH) is a severe neurological disorder caused by bleeding within the brain tissue. Inflammation has been implicated in ICH pathogenesis and is a potential therapeutic target for ICH. Haemin, an activator of haem oxygenase-1 (HO-1), rapidly increases HO-1 protein expression and activity and has been shown to distinctly affect anti-inflammatory functions after central nervous system (CNS) injury. However, less is known about the mechanisms that underlie the anti-inflammatory effects of haemin in aged rats post-ICH. Here, we performed microarray analysis to identify miRNAs that respond strongly to HO-1 regulation in ICH rats and found that miR-21-5p induced the most significant change. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and Gene Ontology (GO) analysis, we focused on dual-specificity phosphatase 8 (DUSP8) from the predicted miR-21-5p targets. Luciferase reporter assays confirmed that miR-21-5p bound directly to DUSP8. MiR-21-5p upregulation in vitro downregulated DUSP8 expression. Importantly, intracerebroventricularly injecting antagomir for miR-21-5p (A-miR-21-5p), which was used to inhibit miR-21-5p in aged ICH rats, significantly reduced the neurological defects, repaired cognitive impairment, alleviated blood-brain barrier (BBB) permeability, inhibited neuronal apoptosis posthaemorrhage and accelerated haematoma absorption. In addition, serum miR-21-5p levels were notably elevated in patients relative to healthy individuals and were correlated with National Institutes of Health Stroke Scale (NIHSS) scores and clinical outcomes. In summary, A-miR-21-5p increased HO-1 expression in cerebral haematomas, thus eliciting the DUSP8-modulated perifocal neuroprotective effect of haemin. MiR-21-5p with haemin therapy may be a potential therapy post-ICH.

Several biological effects of haem oxygenase (HO)-1, including anti-inflammatory, antiapoptotic and antioxidative properties were reported; however, the role of HO-1 in apoptosis is still unclear. In the presence of stimulation by cobalt protoporphyrin (CoPP), an HO-1 inducer, apoptotic characteristics were observed, including DNA laddering, hypodiploid cells, and cleavages of caspase (Casp)-3 and poly(ADP) ribose polymerase (PARP) proteins in human colon carcinoma COLO205, HCT-15, LOVO and HT-29 cells in serum-free (SF) conditions with increased HO-1, but not heat shock protein 70 (HSP70) or HSP90. The addition of 10% foetal bovine serum (FBS) or 1% bovine serum albumin accordingly inhibited CoPP-induced apoptosis and HO-1 protein expression in human colon cancer cells. CoPP-induced apoptosis of colon cancer cells was prevented by the addition of the pan-caspase inhibitor, Z-VAD-FMK (VAD), and the Casp-3 inhibitor, Z-DEVD-FMK (DEVD). N-Acetyl cysteine inhibited reactive oxygen species-generated H2 O2 -induced cell death with reduced intracellular peroxide production, but did not affect CoPP-induced apoptosis in human colorectal carcinoma (CRC) cells. Two CoPP analogs, ferric protoporphyrin and tin protoporphyrin, did not affect the viability of human CRC cells or HO-1 expression by those cells, and knockdown of HO-1 protein expression by HO-1 small interfering (si)RNA reversed the cytotoxic effect elicited by CoPP. Furthermore, the carbon monoxide (CO) donor, CORM, but not FeSO4 or biliverdin, induced DNA ladders, and cleavage of Casp-3 and PARP proteins in human CRC cells. Increased phosphorylated levels of the endoplasmic reticular (ER) stress proteins, protein kinase R-like ER kinase (PERK), and eukaryotic initiation factor 2α (eIF2α) by CORM and CoPP were identified, and the addition of the PERK inhibitor, GSK2606414, inhibited CORM- and CoPP-induced apoptosis. Increased GRP78 level and formation of the HO-1/GRP78 complex were detected in CORM- and CoPP-treated human CRC cells. A pro-apoptotic role of HO-1 against the viability of human CRC cells via induction of CO and ER stress was firstly demonstrated herein.

MicroRNAs are small noncoding RNAs that are central factors between hepatitis C virus (HCV) and host cellular factors for viral replication and liver disease progression, including liver fibrosis, cirrhosis and hepatocellular carcinoma. In the present study, we found that overexpressing miR-let-7c markedly reduced HCV replication because it induced haem oxygenase-1 (HO-1) expression by targeting HO-1 transcriptional repressor Bach1, ultimately leading to stimulating an antiviral interferon response and blockade of HCV viral protease activity. In contrast, the antiviral actions of miR-let-7c were attenuated by miR-let-7c inhibitor treatment, exogenously expressing Bach1 or suppressing HO-1 activity and expression. A proposed model indicates a key role for miR-let-7c targeting Bach1 to transactivate HO-1-mediated antiviral actions against HCV. miR-let-7c may serve as an attractive target for antiviral development.

Ultraviolet A (UVA) irradiation is a potential environmental stressor, which contributes to inflammation, photoaging, and carcinogenesis. UVA causes endoplasmic reticulum stress, hence phosphorylates the α subunit of eIF2. Meanwhile, UVA also induces expression of haem oxygenase-1 (HO-1) and nuclear factor erythroid-derived two related factor 2 (Nrf2) in human skin cells. In mouse JB6 cell, we found high dose UVA could change cell morphology, cause cell viability loss. UVA irradiation activated phosphorylation of eIF2α and Nrf2-HO-1 pathway in a dose-dependent manner. Besides, modulation of eIF2α phosphorylation status could alter expression pattern of Nrf2-HO-1 signalling. Salubrinal, a selective inhibitor of eIF2α dephosphorylation, increased the S phase in cell cycle of JB6 cells after UVA irradiation, suggesting phosphorylation status of eIF2α may affect cellular homeostasis under UVA irradiation. The study directed to further acknowledge about the relationship of UVA-induced eIF2α phosphorylation and Nrf2-HO-1 pathway, which may play a role in phototherapy and photo protection.

This study investigated the effects of high haem oxygenase-1 (HO-1) expression on oxidative injury and the biological behaviours of rat dermal fibroblasts, under high glucose conditions.
Rat dermal fibroblasts were cultured in normal glucose (1.0g/l), high glucose (4.5g/l) or haemin (5μm). A bilirubin kit, real-time polymerase chain reaction (RT-PCR) and Western blotting measured the protease activity, mRNA, and protein levels of HO-1, respectively. An enzyme-linked immunosorbent assay (ELISA) kit measured media levels of 8-hydroxydeoxyguanosine (8-OHdG), reactive oxygen species (ROS) and collagen (hydroxyproline) secretion. Cell proliferation was measured using flow cytometry. Cell apoptosis was measured using Hoechst 33258 staining and flow cytometry. The transwell method and scratch test evaluated cell migration.
HO-1 expression exhibited a time-dependent change that was lowest in the high glucose (HG) group at 96 hours compared with the normal glucose (NG) group. In the HG group, the 8-OHdG, ROS and cell apoptosis were increased, and collagen secretion, cell proliferation and cell migration (horizontal and vertical) were decreased compared with the NG group at 96 hours. Haemin treatment sustained high HO-1 expression for at least 96 hours, and the cells exhibited decreased 8-OHdG and ROS, increased collagen synthesis, improved proliferation and migration ability, and decreased apoptosis in the NG and haemin (NH) group/HG and haemin (HH) group compared with the NG/HG groups. These cells recovered from oxidative injury and biological behaviours dysfunction.
Haemin induces HO-1 expression in fibroblasts and it may influence the oxidative injury and biological behaviours of fibroblasts. These findings suggest that HO-1 may accelerate the healing of diabetic wounds via alleviation of oxidative injury and improvement of biological behaviours of fibroblasts.