Overexpression of heme oxygenase-1 protects smooth muscle cells against oxidative injury and inhibits cell proliferation

To investigate whether the expression of exogenous heme oxygenase-1 (HO-1) gene within vascular smooth muscle cells (VSMC) could protect the cells from free radical attack and inhibit cell proliferation, we established an in vitro transfection of human HO-1 gene into rat VSMC mediated by a retroviral vector. The results showed that the profound expression of HO-1 protein as well as HO activity was 1.8- and 2.0-fold increased respectively in the transfected cells compared to the non-transfected ones. The treatment of VSMC with different concentrations of H2O2 led to the remarkable cell damage as indicated by survival rate and LDH leakage. However, the resistance of the HO-1 transfected VSMC against H2O2 was significantly raised. This protective effect was dramatically diminished when the transfected VSMC were pretreated with ZnPP-IX, a specific inhibitor of HO, for 24 h. In addition, we found that the growth potential of the transfected cells was significantly inhibited directly by increased activity of HO-1, and this effect might be related to decreased phosphorylation of MAPK. These results suggest that the overexpression of introduced hHO-1 is potentially able to reduce the risk factors of atherosclerosis, partially due to its cellular protection against oxidative injury and to its inhibitory effect on cellular proliferation.

Region-dependent effects of diabetes and insulin-replacement on neuronal nitric oxide synthase-and heme oxygenase-immunoreactive submucous neurons

AIM To investigate the intestinal segment-specific effects of diabetes and insulin replacement on the density of different subpopulations of submucous neurons. METHODS Ten weeks after the onset of type 1 diabetes samples were taken from the duodenum, ileum and colon of streptozotocin-induce diabetic, insulin-treated diabetic and sex-and age-matched control rats. Whole-mount preparations of submucous plexus were prepared from the different gut segments for quantitative fluorescent immunohistochemistry. The following double-immunostainings were performed: neuronal nitric oxide synthase(n NOS) and Hu C/D, heme oxygenase(HO) 1 and peripherin, as well as HO2 and peripherin. The density of n NOS-, HO1-and HO2-immunoreactive(IR) neurons was determined as a percentage of the total number of submucous neurons. RESULTS The total number of submucous neurons and the proportion of n NOS-, HO1-and HO2-IR subpopulations were not affected in the duodenal ganglia of control, diabetic and insulin-treated rats. While the total neuronal number did not change in either the ileum or the colon, the density of nitrergic neurons exhibited a 2-and 3-fold increase in the diabetic ileum and colon, respectively, which was further enhanced after insulin replacement. The presence of HO1-and HO2-IR submucous neurons was robust in the colon of controls(38.4%-50.8%), whereas it was significantly lower in the small intestinal segments(0.0%-4.2%, P < 0.0001). Under pathophysiological conditions the only alteration detected was an increase in the ileum and a decrease in the colon of the proportion of HO-IR neurons in insulin-treated diabetic animals. CONCLUSION Diabetes and immediate insulin replacement induce the most pronounced region-specific alterations of n NOS-, HO1-and HO2-IR submucous neuronal density in the distal parts of the gut.

Hepatic steatosis prevents heme oxygenase-1 induction by isoflurane in the rat liver

AIM:To characterize the inductive effects of isoflurane(ISO) on hepatic heme oxygenase-1(HO-1) in an animal model of hepatic steatosis.METHODS:Lean(LEAN) and obese(FAT) Zucker rats were randomized into 4 groups:1:LEAN + pentobarbital sodium(PEN);2:LEAN + ISO;3:FAT + PEN;4:FAT + ISO.The animals were mechanically ventilated for 6 h.In vitro analyses of liver tissue included determination of HO-1 mRNA and protein expression as well as measurement of HO enzyme activity and immunohistochemical analyses.RESULTS:Compared to PEN treatment,ISO administration profoundly induced hepatic HO-1 mRNA and protein expression and significantly increased HO enzyme activity in lean Zucker rats.In contrast,no difference in HO-1 gene expression was observed after ISO or PEN anesthesia in obese Zucker rats.CONCLUSION:The present study demonstrates that ISO is an inducer of hepatic HO-1 gene expression in non-steatotic organs but failed to upregulate HO-1 in steatotic livers.

Heme oxygenase-1 as a therapeutic target in inflammatory disorders of the gastrointestinal tract

Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. HO-1 not only protects against oxidative stress and apoptosis, but has received a great deal of attention in recent years because ofits potent anti-inflammatory functions. Studies with HO-1 knockout animal models have led to major advances in the understanding of how HO-1 might regulate inflammatory immune responses, although little is known on the underlying mechanisms. Due to its beneficial effects the targeted induction of this enzyme is considered to have major therapeutic po- tential for the treatment ofinflammatory disorders. This review discusses current knowledge on the mechanisms that mediate anti-inflammatory protection by HO-1. More specifically, the article deals with the role of HO-1 in the pathophysiology of viral hepatitis, inflammatorybowel disease, and pancreatitis. The effects of specific HO-1 modulation as a potential therapeutic strategy in experimental cell culture and animal models of these gastrointestinal disorders are summarized. In conclusion, targeted regulation of HO-1 holds major promise for future clinical interventions in inflammatory diseases of the gastrointestinal tract.

Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats

AIM： To test the hypothesis that enhancement of the activity of heine oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heine oxygense-1 in a CCl4-induced micronodular cirrhosis model. METHODS： Recombinant adeno-associated viruses carrying rat HO-1 or GFP gene were generated, 1×10^12 vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fibrosis. RESULTS： Conditioning the rat liver with over-expression of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The development of micronodular cirrhosis was significantly inhibited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to controls, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with improved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells （HSCs） in rAAV/ HO-1-transduced livers. CONCLUSIONS： Enhancement of HO activity in the livers suppresses the development of cirrhosis.

Rosmarinic acid elicits neuroprotection in ischemic stroke via Nrf2 and heme oxygenase 1 signaling

Rosmarinic acid（RA） can elicit a neuroprotective effect against ischemic stroke, but the precise molecular mechanism remains poorly understood. In this study, an experimental ischemic stroke model was established in CD-1 mice（Beijing Vital River Laboratory Animal Technology, Beijing, China） by occluding the right middle cerebral artery for 1 hour and allowing reperfusion for 24 hours. After intraperitoneally injecting model mice with 10, 20, or 40 mg/kg RA, functional neurological deficits were evaluated using modified Longa scores. Subsequently, cerebral infarct volume was measured using TTC staining and ischemic brain tissue was examined for cell apoptosis with TUNEL staining. Superoxide dismutase activity and malondialdehyde levels were measured by spectrophometry. Expression of heme oxygenase-1（HO-1）, nuclear factor erythroid 2-related factor 2（Nrf2）, Bcl-2, Bax, Akt, and phospho-Ser473 Akt proteins in ischemic brain tissue was detected by western blot, while mRNA levels of Nrf2, HO-1, Bcl-2, and Bax were analyzed using real time quantitative PCR. In addition, HO-1 enzyme activity was measured spectrophotometrically. RA（20 and 40 mg/kg） greatly improved neurological function, reduced infarct volume, decreased cell apoptosis, upregulated Bcl-2 protein and mRNA expression, downregulated Bax protein and mRNA expression, increased HO-1 and Nrf2 protein and mRNA expression, increased superoxide dismutase activity, and decreased malondialdehyde levels in ischemic brain tissue of model mice. However, intraperitoneal injection of a HO-1 inhibitor（10 mg/kg zinc protoporphyrin IX） reversed the neuroprotective effects of RA on HO-1 enzyme activity and Bcl-2 and Bax protein expression. The PI3 K/Akt signaling pathway inhibitor LY294002（10 mM） inhibited Akt phosphorylation, as well as Nrf2 and HO-1 expression. Our findings suggest that RA has anti-oxidative and anti-apoptotic properties that protect against ischemic stroke by a mechanism involving upregulation of Nrf2 and HO-1 expression via the PI3 K/Akt signaling pathway.

Assessment of heme oxygenase-1 （HO-1） activity in the cavernous tissues of sildenafil citrate-treated rats

Aim： To assess heine oxygenase-1 （HO-1） activity in the cavemous tissue of sildenafil citrate-treated rats. Methods： One hundred and ninety-two Sprague-Dawley male rats, divided into four equal groups, were investigated. Group 1, the control group, received regular animal chow; group 2 received sildenafil citrate by intragastric tube; group 3 received sildenafil and HO inhibitor （zinc protoporphyrin, ZnPP）; and group 4 received sildenafil and nitric oxide synthase （NOS） inhibitor L-nitroarginine methyl ester （L-NAME）. Twelve rats from each group were killed after 0.5 h, 1 h, 2 h and 3 h of drug administration. Then HO-1 activity, cGMP levels and NOS enzymatic activity in the cavernous tissues were estimated. Results： In cavemous tissue, HO-1 activity, NOS enzymatic activity and cGMP concentration increased significantly in sildenafil-treated rats compared to other groups throughout the experiment. Rats receiving either HO or NOS inhibitors showed a significant decrease in these parameters. HO- 1 cavemous tissue activity and NOS enzymatic activity demonstrated a positive significant correlation with cGMP levels （r = 0.646, r = 0.612 respectively; P 〈 0.001）. Conclusion： The actions of PDE5 inhibitor sildenafil citrate in the cavernous tissue are partly mediated through the interdependent relationship between both HO-1 and NOS activities.

Existence of Heme Oxygenase-carbon Monoxide-cyclic Guanosine Monophosphate Pathway in Human Trabecular Meshwork Cells In Vitro

To confirm the existence of heme oxygenase (HO)-carbon monoxide (CO)- cyclic guanosine monophosphate (cGMP) pathway in the cultured human trabecular meshwork cells (HTMCs) in vitro, and to evaluate the inductive role of hemin on this pathway, HTMCs of the third to fourth generation were cultured in vitro. Reverse transcripase-polymerase chain reaction (RT-PCR) was employed for detection of HO-1 and HO-2 mRNA. Immunohistochemical staining was used to detect HO-1 and HO-2 proteins. Hemin was added into the culture solution. The HO-1 mRNA levels were quantified by RT-PCR. The relative amount of carbon monoxide released into the media was measured with the quantifying carbon monoxide hemoglobin (HbCO) by spectrophotometry. Radioimmunoassay was used to determine changes of cGMP in HTMCs. The results showed that cultured cells had the specific characteristics of HTMCs. Both HO-1 and HO-2 genes were expressed in HTMCs, as well as HO-1 and HO-2 proteins in HTMCs. Hemin induced HO-1 mRNA, HbCO and cGMP in a dose-dependent manner. In conclusion, HO-CO-cGMP pathway exists in the cultured HTMCs and can be induced by hemin. Pharmacological stimulation of HO-CO-cGMP pathway may constitute a novel therapeutic approach to rescuing glaucoma.

Natural heme oxygenase-1 inducers in hepatobiliary function

Many physiological effects of natural antioxidants, their extracts or their major active components, have been reported in recent decades. Most of these compounds are characterized by a phenolic structure, similar to that of o-tocopherol, and present antioxidant properties that have been demonstrated both in vitro and in vivo. Polyphenors may increase the capacity of endogenous antioxidant defences and modulate the cellular redox state. Changes in the cellular redox state may have wide-ranging consequences for cellular growth and differentiation. The majority of in vitro and in vivo studies conducted so far have attributed the protective effect of bioactive polyphenols to their chemical reactivity toward free radicals and their capacity to prevent the oxidation of important intracellular components. However, in recent years a possible novel aspect inthe mode of action of these compounds has been suggested; that is, the ultimate stimulation of the heme oxygenase-1 （HO-1） pathway is likely to account for the established and powerful antioxidant/anti-inflammatory properties of these polyphenols. The products of the HO-catalyzed reaction, particularly carbon mon- oxide （CO） and biliverdin/bilirubin have been shown to exert protective effects in several organs against oxidative and other noxious stimuli. In this context, it is interesting to note that induction of HO-1 expression by means of natural compounds contributes to protection against liver damage in various experimental models. The focus of this review is on the significance of targeted induction of HO-1 as a potential therapeutic strategy to protect the liver against various stressors in several pathological conditions.

Heme oxygenase 1-mediated ferroptosis in Kupffer cells initiates liver injury during heat stroke

With the escalating prevalence of global heat waves,heat stroke has become a prominent health concern,leading to substantial liver damage.Unlike other forms of liver injury,heat strokeinduced damage is characterized by heat cytotoxicity and heightened inflammation,directly contributing to elevated mortality rates.While clinical assessments have identified elevated bilirubin levels as indicative of Kupffer cell dysfunction,their specific correlation with heat stroke liver injury remains unclear.Our hypothesis proposes the involvement of Kupffer cell ferroptosis during heat stroke,initiating IL-1bmediated inflammation.Using single-cell RNA sequencing of murine macrophages,a distinct and highly susceptible Kupffer cell subtype,Clec4Ft/CD206t,emerged,with heme oxygenase 1(HMOX-1)playing a pivotal role.Mechanistically,heat-induced HMOX-1,regulated by early growth response factor 1,mediated ferroptosis in Kupffer cells,specifically in the Clec4F t/CD206 t subtype(KC2),activating phosphatidylinositol 4-kinase beta and promoting PI4P production.This cascade triggered NLRP3 inflammasome activation and maturation of IL-1b.These findings underscore the critical role of targeted therapy against HMOX-1 in ferroptosis within Kupffer cells,particularly in Clec4F t/CD206 t KCs.Such an approach has the potential to mitigate inflammation and alleviate acute liver injury in the context of heat stroke,offering a promising avenue for future therapeutic interventions.

Heme oxygenase 1 linked to inactivation of subchondral osteoclasts in osteoarthritis

Osteoarthritis(OA)is a chronic progressive osteoarthropathy in the elderly.Osteoclast activation plays a crucial role in the occurrence of subchondral bone loss in early OA.However,the specific mechanism of osteoclast differentiation in OA remains unclear.In our study,gene expression profiles related to OA disease progression and osteoclast activation were screened from the Gene Expression Omnibus(GEO)repository.GEO2R and Funrich analysis tools were employed to find differentially expressed genes(DEGs).Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses demonstrated that chemical carcinogenesis,reactive oxygen species(ROS),and response to oxidative stress were mainly involved in osteoclast differentiation in OA subchondral bone.Furthermore,fourteen DEGs that are associated with oxidative stress were identified.The first ranked differential gene,heme oxygenase 1(HMOX1),was selected for further validation.Related results showed that osteoclast activation in the pathogenesis of OA subchondral bone is accompanied by the downregulation of HMOX1.Carnosol was revealed to inhibit osteoclastogenesis by targeting HMOX1 and upregulating the expression of antioxidant protein in vitro.Meanwhile,carnosol was found to alleviate the severity of OA by inhibiting the activation of subchondral osteoclasts in vivo.Our research indicated that the activation of osteoclasts due to subchondral bone redox dysplasia may serve as a significant pathway for the advancement of OA.Targeting HMOX1 in subchondral osteoclasts may offer novel insights for the treatment of early OA.

Auxin induces lateral root formation in Bupleurum: A heme oxygenase dependent approach

Objective: The content of saikosaponins in genus Bupleurum is increased with numbers of lateral root, but the genetic mechanisms are largely unknown. This study aims to identify the heme oxygenase(HO) gene family members of B. chinense and B. scorzonerifolium, and assess their role in the root development in Bupleurum.Methods: The gene sequences of HO family were selected from iso-seq full-length transcriptome data of B. chinense and B. scorzonerifolium, and were analyzed in physicochemical properties, conserved domains,motifs and phylogenetic relationship. In addition, the expression patterns of HO gene in different parts of roots were compared via transcriptome sequencing and qRT-PCR in the two species.Results: Five Bupleurum HO genes(BcHO1-BcHO5) belonging to the HO1 subfamily were identified from the transcriptome data, whereas the HO_(2) subfamily member was not identified. The expression levels of BcHO1 and BcHO_(2) were significantly higher than those of other three HO members in the transcriptome analysis. In addition, the expression profile of BcHO1 showed consistency with lateral root development in B. chinense and B. scorzonerifolium.Conclusion: Hos might participate in the auxin-induced morphogenesis of lateral roots. The yield of saikosaponin may be improved by manipulating expression of these genes.

The preparation of rat heme oxygenase 1 mutant to reduce the level of bilirubin

Objective To prepare rat heme oxygenase 1 (HO 1) mutants and to determine the activity and inhibition of this mutated enzyme Methods pcDNA3HO1 containing truncated native rat HO 1 cDNA and pcDNA3HO1Δ25 carrying mutated rat HO 1 cDNA (His25Ala) were constructed, respectively COS 1 cells transfected with pcDNA3HO1 and pcDNA3HO1Δ25 were collected and their activities were analyzed Results Native rat HO 1 was highly expressed in transfected cells and its activity was 13?688-15?600?U/mg protein per hour However, the enzyme activity of mutated HO 1 declined and the value was 1948-2160?U/mg protein per hour When an equal amount of mutant was added to the enzyme reaction system, the level of bilirubin decreased by 42% Conclusion The His25Ala mutant reduced the formation of bilirubin, suggesting that the mutant could competely bind the heme with native enzyme

Endogenous Hydrogen Peroxide Plays a Positive Role in the Upregulation of Heme Oxygenase and Acclimation to Oxidative Stress in Wheat Seedling Leaves

Pretreatment of lower H2O2 doses （0.05, 0.5 and 5 mM） for 24 h was able to dose-dependently attenuate lipid peroxidation in wheat seedling leaves mediated by further oxidative damage elicited by higher dose of H2O2 （150 mM） for 6 h, with 0.5 mM H2O2 being the most effective concentrations. Further results illustrated that 0.5 mM H2O2 pretreatment triggered the biphasic production of H2O2 during a 24 h period. We also noticed that only peak Ⅰ （0.25 h） rather than peak Ⅱ （4 h） was approximately consistent with the enhancement of heme oxygenase （HO） activity, HO-1 gene expression. Meanwhile, enhanced superoxide dismutase （SOD） activity, Mn-SOD and Cu, Zn-SOD transcripts might be a potential source of peak Ⅰ of endogenous H2O2. Further results confirmed that 0.5 mM H2O2 treatment for 0.5 h was able to upregulate HO gene expression, which was detected by enzyme activity determination, semi-quantitative reverse transcription-polymerase chain reaction and western blotting. Meanwhile, the application of N,N＇-dimethylthiourea, a trap for endogenous H2O2, not only blocked the upregulation of HO, but also reversed the corresponding oxidation attenuation. Together, the above results suggest that endogenous H2O2 production （peak Ⅰ） plays a positive role in the induction of HO by enhancing its mRNA level and protein expression, thus leading to the acclimation to oxidative stress.

Heme oxygenase-1 polymorphism associated with severity of chronic obstructive pulmonary disease

Background Recent studies have suggested that susceptibility to chronic obstructive pulmonary disease （COPD） might be related to the length polymorphism of （GT）n repeat in the 5＇-flanking region of heme oxygenase-1 （HOX-1） gene. However, there has been no research about the relationship between the polymorphism of HOX-1 gene and severity of COPD. Methods The polymorphism of HOX-1 gene in 452 patients with COPD from Han population in Southwest China was analysed by fragment analysis. The frequencies of the HOX-1 genotype were compared with the stage of COPD of each patient. Results The HOX-1 genotypes were classified into two groups： group I were individuals with class L allele （the number of GT 〉32 repeats）, and group II were those without class L allele （the number of GT 〈32 repeats）. The genotypic frequency of the HOX-1 group I was significantly higher than group II in the very severe COPD patients （36.8% vs 22.4%, P〈0.01, OR=2.0, 95% CI 1.3-3.1）, while the genotypic frequency of the HOX-1 group II was lower in the mild COPD （16.0% vs 26.0%, P=-0.02, OR=0.5, 95% CI 0.3-0.9）. However, in moderate and severe stages COPD, there were similar genotypic frequencies between HOX-1 group Ⅰ and group Ⅱ. Conclusions Genetic polymorphism in HOX-1 is associated with the severity of COPD in Southwest China. COPD patients with class L allele may be susceptible to develop very severe COPD. Conversely, the COPD patients without class L allele may be more easily stabilized on mild COPD.

Effect of fusion protein TAT and heme oxygenase-1 on liver sinusoidal endothelial cells apoptosis during preservation injury

Background Proteins or peptides can be directly transferred into cells when covalently linked to protein transduction domains （PTDs）. TAT is one of the most widely studied PTDs. The effect of fusion protein TAT and heme oxygenase-1 （HO-1） on liver sinusoidal endothelial cells （SECs） apoptosis during cold storage is unknown. The present study aimed to determine whether fusion protein TAT-HO-1 would transduce efficiently into liver during cold storage, and, if so, to determine whether TAT-HO-1 would attenuate SECs apoptosis during preservation injury in rat. Methods Livers of Sprague-Dawley rats were harvested and randomly assigned to group 1 （HTK solution） and group 2 （HTK solution containing TAT-HO-1 fusion protein） according to the type of the preservation solution. The transduction efficiency of TAT-HO-1 was examined and the impairment of SECs was assessed during the period of cold storage followed by 1 hour of reperfusion. Results TAT-HO-1 can transduce efficiently into liver during cold storage. A significantly lower apoptotic index of SECs was observed in group 2, at 6, 12 and 18 hours of cold storage after 1 hour reperfusion, when compared with group 1. TAT-HO-1 reduced HA and ET levels in liver at each time point. Both Bcl-2 and Bax protein were expressed in hepatocytes and SECs at the periphery of the sinusoidal space. Moreover, higher Bcl-2 expression and lower Bax expression were observed in group 2. Conclusions TAT-HO-1 can transduce efficiently into rat livers and shows a protective effect on SECs by attenuating apoptosis during cold ischemia/reperfusion injury. Protein transduction will be a novel therapeutic strategy to reduce the risk of preservation injury in liver transplantation.

Chinese herbal medicine compound Yi-Zhi-Hao pellet inhibits replication of influenza virus infection through activation of heme oxygenase-1

As a leading cause of respiratory disease, influenza A virus(IAV) presents a pandemic threat in annual seasonal outbreaks. Given the limitation of existing anti-influenza therapies, there remains to be a requirement for new drugs. Compound Yi-Zhi-Hao pellet(CYZH) is a famous traditional Chinese medicine(TCM) used in the clinic, whose formula has been recorded in Complication of National Standard for Traditional Chinese Medicine to treat common cold. In this study, we found that CYZH exhibited a broad-spectrum anti-influenza activity and inhibited the expression of viral RNA and proteins in vitro. Mechanistically, CYZH had no inhibitory activities against viral protein hemagglutinin and IAV RNA-dependent RNA polymerase. Instead, it induced activation of erythroid 2-related factor 2(Nrf2) and nuclear factor kappa B(NF-κB), which subsequently upregulated heme oxygenase-1(HO-1) expression.Also, CYZH protected cells from oxidative damage induced by reactive oxygen series. In conclusions,CYZH inhibits IAV replication in vitro, at least partly by activating expression of the Nrf2/HO-1 pathway.

Change and role of heme oxygenase-1 in injured lungs following limb ischemia/reperfusion in rats

Objective: To study the change and role of heme oxygenase-1 (HO-1) in injured lungs following limb ischemia/reperfusion in rats. Methods: A total of 96 healthy male Sprague-Dawley rats, weighing 250-300 g, were used in this study. Hind limb ischemia was made on 40 rats through clamping the infrarenal aorta for 2 hours with a microvascular clip, then limb reperfusion for 0, 4, 8, 16 and 24 hours (n=8 in each time point) was performed, respectively. Other 8 rats undergoing full surgical operation including isolation of the infrarenal aorta without occlusion were taken as the sham operation group. Lung tissues were obtained from the 48 animals and Northern blotting and Western blotting were employed to measure the changes of HO-1 mRNA and protein expression, respectively. Immunohistochemistry technique was used to determine the cell types responsible for HO-1 expression after limb ischemia/reperfusion. Then hind limb ischemia was made on other 12 rats through clamping the infrarenal aorta for 2 hours with a microvascular clip, among whom, 6 rats were given zinc protoporphyrin (ZnPP), an inhibitor of HO. Then limb reperfusion for 16 hours was performed on all the 12 rats. And other 12 rats underwent full surgical operation including isolation of the infrarenal aorta without occlusion, among whom, 6 rats were then given ZnPP. Then lung tissues were obtained from the 24 animals and lung injury markers, lung histology, polymorphonuclear leukocyte (PMN) count and malondialdehyde (MDA) content were detected, respectively. HO activity was determined through measuring the carboxyhemoglobin (COHb) level in artery blood with a CO-oximeter after limb ischemia/reperfusion. And the animal mortality was observed on the other 24 rats. Results: Northern blotting analysis showed that HO-1 mRNA increased significantly at 4 hours after reperfusion, peaked at 16 hours, and began to decrease at 24 hours. In contrast, no positive signal was observed in the sham and simple ischemia animals. Increased HO-1 mRNA levels were accompanied by similar increases in HO-1 protein. Lung PMNs and MDA content increased significantly at 4, 8, 16 and 24 hours after reperfusion, compared with the sham controls (P< 0.001), while they decreased in rats with reperfusion for 16 hours when compared with rats with reperfusion for 4 hours (P< 0.001). Immunohistochemical studies showed that HO-1 was expressed in a variety of cell types, including the airway epithelia, alveolar macrophages and vascular smooth muscular cells. The blood COHb level and animal mortality increased significantly after limb ischemia/reperfusion compared with the sham controls (P< 0.001). ZnPP administrated to the ischemia/reperfusion animals led to a decrease in the COHb level and an increase in lung PMN number, MDA content and animal mortality (P< 0.001 compared with ischemia/reperfusion group), and the lung injury was aggravated. Conclusions: Limb ischemia/reperfusion up-regulates pulmonary HO-1 expression, which serves as a compensatory protective response to the ischemia/reperfusion-induced lung injury in rats.

Protective effect of hemoglobin-induced heme oxygenase-1 on injured lungs caus ed by limb ischemia-reperfusion in rats

To determine the role of hemoglobin( HB) induced heme oxygenase 1 (HO 1) in injured lungs caused by limb ischemia reperfusion (I/R) in rats. Methods: A rat model of ischemia in the hind limbs was made by clamping the infrarenal aorta with a microvascular clip, and lung injury occurre d after reperfusion. To induce the expression of HO 1 in the lungs, Hb was admi nistrated intraperitoneally at 16 hours before reperfusion. Northern blotting an d Western blotting were used to detect the expression of HO 1 in the lungs, and the carboxyhemoglobin (COHb) level in arterial blood was assayed. The effect of h emoglobin (Hb) on the injured lungs after limb I/R was determined by measuring t he changes of lung histology, polymorphonuclear (PMN) count, malondialdehyde (MD A) content and wet to dry weight ratio (W/D). Zinc protoporphyrin (ZnPP), an i nhibitor of HO, was used to determine whether HO 1 was induced by Hb after lun g injury. Results: Hb led to a significant increase in HO 1 mRNA and pro tein expression in the lungs, accompanied by the increase of COHb level in arter ial blood. Compared with the sham controls, the lung PMN count, MDA content and W/D significantly increased at 4 hours after limb I/R, which reversed by th e pretreatment with Hb at 16 hours before reperfusion. ZnPP blocked this protect ive role of Hb in the injured lungs. Conclusions: Hb can induce the lung HO 1 expression, which pla ys an important role in the defense against I/R induced lung injury in rats.