骨髓间充质干细胞介导的血红素氧合酶-1促进大鼠心肌梗死后血管再生

目的探讨骨髓间充质干细胞(MSCs)介导的血红素氧合酶-1(HO-1)对心肌梗死后心脏血管再生及左心室功能的影响。方法取大鼠骨髓,体外分离扩增培养MSCs,HO-1腺病毒转染。结扎左前降支1 h后,分别将HO-1-MSCs、MSCs多点注射到大鼠心肌梗死区周边,对照组注射等量PBS。结果 MSCs介导的HO-1能在体外及体内获得稳定表达;HO-1-MSCs组促血管生长因子VEGF、FGF2的表达及毛细血管密度明显高于MSCs组和对照组(P<0.01);但促血管再生的作用可被HO抑制剂阻断。HO-1-MSCs组心肌细胞凋亡及纤维化明显低于MSCs组和对照组(P<0.01);HO-1-MSCs组左室收缩功能各项指标明显优于其他两组(P<0.01)。HO-1-MSCs组心室壁变厚,心室腔明显缩小。结论 MSCs介导的血红素氧合酶-1能促进心肌梗死后心脏血管再生,改善左心室功能。

Influence of heme oxygenase-1 expression on immune liver fibrosis induced by cobalt protoporphyrin in rats

AIM:To investigate the effect of heme oxygenase-1 (HO-1)expression on immune liver fibrosis induced by cobalt protoporphyrin(CoPP)in rats. METHODS:An immune liver fibrosis model of rat was established by administering human serum albumin (HSA).The rats were divided into CoPP,liver fibrosis and normal control groups.Rats in the CoPP group received intraperitoneal CoPP concurrently with HSA. Expression of HO-1 protein was observed by Western blotting and immunohistochemistry.Hematoxylin and eosin(HE)staining was performed to assess fibrosis proliferation and distribution,proliferation extent of fibroblasts,and alterations in hepatocytes and inflammatory cells.TypeⅠandⅢcollagens were detected with Van Gieson’s(VG)staining and Foot’s reticular fiber staining,respectively.In addition, spindle-shaped cells existing at perisinusoidal locations beyond portal and septa areas were investigated with HE staining. RESULTS:Western blotting and immunohistochemistry showed that the expression of HO-1 protein was higher in the CoPP group than in the liver fibrosis group(P<0.05).Compared with the liver fibrosis group,the serological index of hepatic fibrosis in the CoPP group decreased significantly(P<0.05).HE,VG and Foot’s staining revealed that administration of CoPP reduced the extent of hepatic fibrosis.The levels of serological indicators and the number of spindle-shaped cells at perisinuous locations beyond the portal and septa areas were reduced in the CoPP group.Only a few inflammatory cells were seen around the portal areas and central veins in the CoPP group. CONCLUSION:Increased endogenous HO-1 may suppress liver fibrosis by protecting liver cells, inhibiting inflammatory cell infiltration and hepatic stellate cell transformation.

Heme oxygenase-1 prevents liver fibrosis in rats by regulating the expression of PPAR_γ and NF-_κB

AIM:To investigate the effects of heme oxygenase(HO)-1 on liver fibrosis and the expression of peroxisome proliferator-activated receptor gamma(PPARγ) and nuclear factor-kappa B(NF-κB) in rats.METHODS:Sixty Wistar rats were used to construct liver fibrosis models and were randomly divided into 5 groups:group A(normal,untreated),group B(model for 4 wk,untreated),group C(model for 6 wk,untreated),group D [model for 6 wk,treated with zinc protoporphyrin Ⅸ(ZnPP-Ⅸ) from week 4 to week 6],group E(model for 6 wk,treated with hemin from week 4 to week 6).Next,liver injury was assessed by measuring serum alanine aminotransferase(ALT),aspartate aminotransferase(AST) and albumin levels.The degree of hepatic fibrosis was evaluated by measuring serum hyaluronate acid(HA),type Ⅳ collagen(Ⅳ-C) and by histological examination.Hydroxyproline(Hyp) content in the liver homogenate was determined.The expres-sion levels of alpha-smooth muscle actin(α-SMA) in liver tissue were measured by real-time quantitative polymerase chain reaction(RT-PCR).The expression levels of PPARγ and NF-κB were determined by RT-PCR and Western blotting.RESULTS:The expression of HO-1 increased with the development of fibrosis.Induction of HO-1 by hemin significantly attenuated the severity of liver injury and the levels of liver fibrosis as compared with inhibition of HO-1 by ZnPP-Ⅸ.The concentrations of serum ALT,AST,HA and Ⅳ-C in group E decreased compared with group C and group D(P < 0.01).Amount of Hyp and α-SMA in the liver tissues in group E decreased compared with group C(0.62 ± 0.14 vs 0.84 ± 0.07,1.42 ± 0.17 vs 1.84 ± 0.17,respectively,P < 0.01) and group D(0.62 ± 0.14 vs 1.11 ± 0.16,1.42 ± 0.17 vs 2.56 ± 0.37,respectively,P < 0.01).The expression of PPARγ at levels of transcription and translation decreased with the development of fibrosis especially in group D;and it increased in group E compared with groups C and D(0.88 ± 0.15 vs 0.56 ± 0.19,0.88 ± 0.15 vs 0.41 ± 0.11,respectively,P < 0.01).The expression of NF-κB increased with the development of fibrosis especially in group D;and it decreased in group E compared with groups C and D(1.43 ± 0.31 vs 1.89 ± 0.29,1.43 ± 0.31 vs 2.53 ± 0.54,respectively,P < 0.01).CONCLUSION:Our data demonstrate a potential mechanism that HO-1 can prevent liver fibrosis by enhancing the expression of PPARγ and decreasing the expression of NF-κB in liver tissues.

Protective effect of heme oxygenase-1 on Wistar rats with heart failure through the inhibition of inflammation and amelioration of intestinal micro- circulation

Background Myocardial infarction （MI） has likely contributed to the increased prevalence of heart failure （HF）. As a result of re- duced cardiac function, splanchnic blood flow decreases, causing ischemia in villi and damage to the intestinal barrier. The induction of heme oxygenase-1 （HO-1） could prevent, or lessen the effects of stress and inflammation. Thus, the effect and mechanism thereof of HO-1 on the intestines of rats with HF was investigated. Methods Male Wistar rats with heart failure through ligation of the left coronary artery were identified with an left ventricular ejection fraction of 〈 45% through echocardiography and then divided into various experimental groups based on the type of peritoneal injection they received [MI： saline; MI ＋ Cobalt protoporphyrin （CoPP）： CoPP solution; and MI ＋ Tin mesoporphyrin IX dichloride （SnMP）： SnMP solution]. The control group was comprised of rats without coronary ligation. Echocardiogra- phy was performed before ligation for a baseline and eight weeks after ligation in order to evaluate the cardiac function of the rats. The bac- terial translocation （BT） incidence, mesenteric microcirculation, amount of endotoxins in the vein serum, ileum levels of HO- 1, carbon oxide （CO）, nitric oxide （NO）, intedeuldn （IL）-10, turnour necrosis factor-et （TNF-ct）, and the ileum morphology were determined eight weeks after the operation. Results The rats receiving MI ＋ CoPP injections exhibited a recovery in cardiac function, an amelioration of mesenteric microcirculation and change in morphology, a lower BT incidence, a reduction in serum and ileac NO and TNF-ct levels, and an elevation in ileac HO-1, CO, and interleukin-10 （[L-10） levels compared to the MI group （P 〈 0.05）. The rats that received the MI ＋ SnMP injections exhibited results inverse to the MI （P 〈 0.05） group. Conclusions HO-1 exerted a protective effect on the intestines of rats with HF by inhibiting the inflammation and amelioration of microcirculation through the CO pathway. This protective effect could be independent from the recovery of cardiac function.

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

AIM. To investigate the influence of heme oxygenase-1 （HO-1） gene transfer on the viability and function of cultured rat islets in vitro. METHODS： Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene （Ad- HO-1） or enhanced green fluorescent protein gene （Ad- EGFP）, and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index （SI） was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS： After seven days culture, the viability of cultured rat islets decreased significantly （92% ± 6% vs 52% ± 13%, P 〈 0.05）, and glucose-stimulated insulin release also decreased significantly （6.47 ± 0.55 mIU/ L/30IEO vs 4.57 ± 0.40 mIU/L/3OIEO., 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. Transfection of rat islets with adenoviral vectors at an 1±10 of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets（71% ± 15% vs 52% ± 13%, P 〈 0.05）. There was no significant difference in insulin release upon low glucose stimulation （2.8 mmol/L） among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group （P 〉 0.05）, while when stimulated by high glucose （16.7 mmol/L） solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively （12.50 ±2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ, 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively （2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P 〈 0.05）. CONCLUSION： The viability and function of rat islets decrease over time in in vitro culture, and heine oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.

Diazoxide attenuates ischemia/reperfusion injuryvia upregulation of heme oxygenase-1 after liver transplantation in rats

AIM:To evaluate the effects of diazoxide on ischemia/reperfusion(I/R)-injured hepatocytes and further elucidate its underlying mechanisms.METHODS:Male Sprague-Dawley rats were randomized(8 for donor and recipient per group)into five groups:I/R group(4 h of liver cold ischemia followed by 6 h of reperfusion);heme oxygenase-1(HO-1)small interfering RNA(siRNA)group(injection of siRNA via donor portal vein 48 h prior to harvest);diazoxide(DZ) group(injection of DZ via donor portal vein 10 min prior to harvest);HO-1 siRNA+DZ group;and siRNA control group.Blood and liver samples were collected at 6 h after reperfusion.The mRNA expressions and protein levels of HO-1 were determined by reverse transcription polymerase chain reaction and Western blotting,and tissue morphology was examined by light and transmission electron microscopy.Serum transaminases level and cytokines concentration were also measured.RESULTS:We observed that a significant reduction of HO-1 mRNA and protein levels in HO-1 siRNA and HO-1 siRNA+DZ group when compared with I/R group,while the increases were prominent in the DZ group.Light and transmission electron microscopy indicated severe disruption of tissue with lobular distortion and mitochondrial cristae damage in the HO-1 siRNA and HO-1 siRNA+DZ groups compared with DZ group.Serum alanine aminotransferase,aspartate transaminase,tumor necrosis factor-αand interleukin-6 levels increased in the HO-1 siRNA and HO-1 siRNA+DZ groups,and decreased in the DZ group.CONCLUSION:The protective effect of DZ may be induced by upregulation of HO-1.By inhibiting expression of HO-1,this protection pretreated with DZ was abolished.

Heme oxygenase-1 system and gastrointestinal inflammation:A short review

Heme oxygenase-1(HO-1) system catalyzes heme to biologically active products:carbon monoxide,biliverdin/bilirubin and free iron.It is involved in maintaining cellular homeostasis and many physiological and pathophysiological processes.A growing body of evidence indicates that HO-1 activation may play an important protective role in acute and chronic inflammation of gastrointestinal tract.This review focuses on the current understanding of the physiological significance of HO-1 induction and its possible roles in the gastrointestinal inflammation studied to date.The ability to upregulate HO-1 by pharmacological means or using gene therapy may offer therapeutic strategies for gastrointestinal inflammation in the future.

Iron increases HMOX1 and decreases hepatitis C viral expression in HCV-expressing cells

AIM： To investigate effects of iron on oxidative stress, heme oxygenase-1 （HMOX1） and hepatitis C viral （HCV） expression in human hepatoma ceils stably expressing HCV proteins. METHODS： Effects of iron on oxidative stress, HMOX1, and HCV expression were assessed in CON1 cells. Measurements included mRNA by quantitative reverse transcription-polymerase chain reaction, and protein levels by Western blots. RESULTS： Iron, in the form of ferric nitrilotriacetate,increased oxidative stress and upegulated HMOX1 gene expression. Iron did not affect mRNA or protein levels of Bach1, a repressor of HMOXl. Silencing the up-regulation of HMOXl nuclear factor-erythroid 2-related factor 2 （Nrf2） by Nrf2-siRNA decreased FeNTA-mediated up-regulation of HMOXl mRNA levels. These iron effects were completely blocked by deferoxamine （DFO）. Iron also significantly decreased levels of HCV core mRNA and protein by 80%-90%, nonstructural 5A mRNA by 90% and protein by about 50% in the Con1 full length HCV replicon cells, whereas DFO increased them. CONCLUSION： Excess iron up-regulates HMOXl and down-regulates HCV gene expression in hepatoma cells. This probably mitigates liver injury caused by combined iron overload and HCV infection.

Protective effect of ischemic postconditioning on lung ischemia-reperfusion injury in rats and the role of heme oxygenase-1

Objective： To investigate the effect of ischemic postconditioning （1PO） on acute lung ischemia-reperfusion （I/R） injury and the protein expression of haeme oxygenase-1 （HO-1）, a cytoprotective defense against oxidative injury. Methods： After being anesthetized with chloralhydrate, forty-eight healthy SD rats were randomly divided into 6 groups （8 in each）： sham operation group （S group）; I/R group： left lung hilum was clamped for 40 minutes followed by 105 minutes of reperfusion; IPO group： left lung hilum was clamped for40 minutes and postconditioned by 3 cycles of 30 seconds of reperfusion and 30 seconds of reocclusion; Heroin （HM）＋ I/R group： heroin, an inducer of HO-1 was injected intraperitoneally at 40 μmol·kg^-1·day^-1 for two consecutive days prior to 40 minutes clamping of left lung hilum; ZnPPIX＋IPO group： zinc protoporphyrin IX, an inhibitor of HO-1 was injected intraperitoneally at 20 mg·kg^-1 24 hours prior to 40 minutes clamping of left lung hilum; and HM＋S group： HM was administered as in the HM＋I/R group without inducing lung I/R. Arterial partial pressure of oxygen （PaO2） and malondialdehyde （MDA） content in serum were assessed. The left lung was removed for determination of wet/dry lung weight ratio and expression of HO-1 protein by immuno-histochemical technique and for light microscopic examination. Results： The PaO2 was significantly lower in all the experimental groups compared with sham group （90 roan Hg ±11 mmHg）. However, the values of PaO2in IPO （81 mm Hg±7 mm Hg） and HM＋I/R （80 mm Hg±9 mm Hg） were higher than that in I/R （63 mm Hg±9 mm Hg） and ZnPPIX＋IPO （65 mm Hg±8 mm Hg） groups （P〈0.01）. The protein expression of HO- 1 in lung tissue was significantly increased in I/R group compared with S group （P〈0.01）. While the HO-1 protein expression was higher in IPO and HM＋I/R groups as compared with I/R group （P〈0.05, P〈0.01 ）. The lung wet/ dry （W/D） weight ratio and MDA content in serum were significantly increased in I/R group as compared with S or HM＋S groups （P〈0.01）, accompanied by severe lung tissue histological damage, which was attenuated either by IPO or by HM pretreatment （P〈0.01, IPO or HM＋I/R vs. I/R）. The protective effect of IPO was abolished by ZnPPIX. Conclusion： Ischemic postconditioning can attenuate the lung ischemia-reperfusion injury through upregulating the protein expression of HO-I that leads to reduced postischemic oxidative damage.

Effect of moxibustion on expressions of HO-1 and MCP-3 protein in colon of rats with Crohn's disease

Objective： To observe the effect of moxibustion therapy on heme oxygenase-1（HO-1） and monocyte chemoattractant protein-3（MCP-3） protein expressions in the colonic mucosa of rats with Crohn＇s disease（CD）, and to explore the intestinal mucosal immune mechanism of moxibustion therapy in treating CD. Methods： The CD rat model was established using the internationally accepted Morris method. The rats were randomly divided into a model group, a herbal cake-partitioned moxibustion group, a mild moxibustion group, a cigarette moxibustion group and a hot compress group, which were compared with the normal group. Except the normal group and the model group, rats in the other groups accepted different moxibustion therapies on bilateral Tianshu（ST 25）. Hematoxylin-eosin（HE） staining was conducted and the pathological changes of the colon were observed under light microscope; the expressions of HO-1 and MCP-3 protein in rat＇s colonic mucosa were determined by immunohisto-chemistry. Results： Compared with the normal group, rats in the model group showed mucosal defect, villus destruction or loss, submucosal congestion and edema, glandular destruction or disappearance, reduced goblet cells, ulcer formation, significantly increased positive target area and positive target integral optical density of HO-1 and MCP-3 protein expression（all P〈0.01）. After treatment, compared with the model group, colonic mucosa was significantly improved in the herbal cake-partitioned moxibustion group and the mild moxibustion group, which mainly showed that the intestinal glands were arranged regularly, ulcer surfaces were covered by the neoformative epitheliums, or intestinal ulcers were replaced by the nascent granulation tissue, and submucosal edema was alleviated, with a small amount of inflammatory cell infiltration. The total areas and the integral optical densities of the positive targets for rat＇s colonic mucosa HO-1 and MCP-3 protein expressions were decreased（all P〈0.01）. Compared with the cigarette moxibustion group and the hot compress group, the total areas and the integral optical densities of the positive targets for rat＇s colonic mucosa HO-1 and MCP-3 protein expressions were significantly decreased（all P〈0.01） in the herbal cake-partitioned moxibustion group and the mild moxibustion group. Conclusion： Herbal cake-partitioned moxibustion and mild moxibustion can significantly improve the inflammatory response of colonic mucosa in CD rats. It can down-regulate the expressions of HO-1 and MCP-3 proteins in the colonic mucosa of CD rats, which may be one of the mechanism in intestinal mucosal immunity caused by moxibustion therapy.

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.

人参皂苷Rb1对肠缺血再灌注致肾损伤的影响及其机制

目的 观察人参皂苷Rb1对肠缺血再灌注致肾损伤的影响及机制.方法 将48只成年雄性C57BL/6J小鼠随机分为4组（n=12）：假手术组（S组）、肠缺血再灌注组（ⅡR组）、Rb1低剂量组（Rb1-30组）、Rb1高剂量组（Rb1-60组）.采用小鼠肠缺血再灌注模型.Rb1-30组和Rb1-60组分别于再灌注前腹腔给予30、60 mg/kg的Rb1.再灌注2h时心脏采血检测血尿素氮（BUN）、肌酐（Cr）和中性粒细胞明胶酶相关脂质运载蛋白（NGAL）,观察肾脏病理学改变,检测肾组织超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量及Western blot检测血红素氧合酶-1（HO-1）、NF-E2相关因子（Nrf2）蛋白表达.结果 ⅢR组（HO-1：4.03±0.60,Nrf2：3.42 ±0.47）高于S组（HO-1：1.50±0.16,Nrf2：0.61 ±0.14）（P＜0.01）,而Rh1-30组（HO-1：8.78±1.12,Nrf2：6.02±0.55）和Rb1-60组（HO-1：15.76±1.33,Nrf2：8.29±0.66）较ⅢR组进一步增高（P＜0.05）.与S组（16.70 ±4.06）比较,ⅡR组（198.60±9.71）中肾损伤程度增高（P＜0.01）,血清BUN（21.55±1.50）、Cr（20.65±1.53）和NGAL（155.93±8.07）水平、肾组织MDA（5.00±1.53）含量升高而SOD（9.49±1.39）活性降低（P＜0.01）.与ⅡR组比较,Rb1-60组（94.60±9.24）中肾损伤程度减轻（P＜0.05）,BUN（Rb1-30：15.52±1.23;Rb1-60：12.91±1.41）、Cr（Rb1-30： 16.00±1.50;Rb1-60： 13.14±1.22）和NGAL（Rb1-30：124.44±6.52;Rb1-60：101.76±12.20）水平、肾组织MDA（Rb1-30：4.12±0.47;Rb1-60：3.22±0.54）含量降低而SOD（Rb1-30：14.77±1.87;Rb1-60：18.91±1.56）活性升高（P＜0.01）.结论 肠缺血再灌注可致肾损伤,人参皂苷Rb1通过激活Nrf2/ARE通路,诱导HO-1表达,从而减轻肠缺血再灌注所致肾损伤.