血红素氧合酶-1减轻大鼠移植肝缺血再灌注损伤的作用及机制

目的 探讨血红素氧合酶-1(HO-1)减轻大鼠移植肝缺血再灌注损伤的作用及其机制.方法 选择近交系雄性SD大鼠作为肝移植的供、受者;采用单纯随机方法将48只SD大鼠随机分为对照组、抑制组和诱导组(每组供、受者各8只).对照组:供肝不用任何药物处理;抑制组:在获取供肝前24 h,经供者腹腔注射HO-1抑制剂锌原卟啉20 mg/kg进行预处理;诱导组:在获取供肝前24 h,经供者腹腔注射HO-1诱导剂钴原卟啉5 mg/kg进行预处理.获取供肝后,在4℃UW液中冷保存24 h.肝移植前检测供肝HO-1的表达水平;肝移植后6 h采血并获取移植肝标本,分离培养枯否细胞;检测受者的肝功能;检测枯否细胞培养上清中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的含量;观察移植肝组织病理学表现以及枯否细胞CD14 mRNA的表达水平和蛋白含量测定.结果 移植前诱导组供肝HO-1的表达水平明显增高.移植后诱导组血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)含量明显降低;移植肝组织病理学损伤减轻;枯否细胞培养上清中TNF-α和IL-6含量减少;而且枯否细胞上的CD14 mRNA表达水平和蛋白含量也明显低于抑制组.结论 诱导供肝HO-1表达上调可能抑制了枯否细胞的激活,从而减轻大鼠移植肝缺血再灌注损伤.     

Heme Oxygenase-1 Overexpression Protects Rat Livers from Ischemia/Reperfusion Injury with Extended Cold Preservation

This study analyzes the effects and mechanisms of heme oxygenase-1 (HO-1)-mediated cytoprotection in rat livers exposed to cold preservation. In the first series, rats were pretreated with cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), HO-1 inducer and antagonist, respectively. Livers were stored at 4 degrees C for 24 h, and then perfused ex vivo for 2 h. Livers pretreated with CoPP had significantly higher portal venous blood flow and increased total bile production, as compared with the ZnPP group. This correlated with histologic (Banff) criteria of hepatocyte injury/liver function. In the second series, rat livers were stored at 4 degrees C for 24 h or 40 h, and then transplanted into syngeneic recipients. After 24 h of preservation, 80% of rats bearing CoPP-pretreated liver grafts survived 21 days (vs. 50% in controls). After 40h of cold preservation, liver transplant survival at day 1, 7 and 21 for the CoPP group was: 100%, 71% and 57%, respectively (vs. 50%, 50% and 33% in controls). This correlated with improved hepatic function/histologic (Suzuki) criteria of hepatocyte injury after HO-1 overexpression (immunohistology/Western blots) by infiltrating macrophages. This study documents the potential utility of HO-1-inducing agents in preventing ischemia/reperfusion injury resulting from prolonged storage of liver transplants.     

乌司他丁对大鼠原位肝脏移植供肝的保护作用

目的：探讨乌司他丁对大鼠原位肝脏移植供肝的保护作用及机制。 方法：分别用单纯UW液（模型组）或含乌司他丁（乌司他丁组）、HO-1诱导剂CoPP（CoPP组）、HO-1抑制剂ZnPP（ZnPP组）的UW液灌注切取的供体大鼠肝脏并保留灌注液1 h后,原位移植受体大鼠。移植后24 h取移植肝脏与受体大鼠血标本,行肝脏病理学检查及评分;分别用real-time PCR和Western bolt法检测肝组织HO-1 mRNA与蛋白的表达;用Elisa法检测大鼠血清中IL-2和IL-10的含量。 结果：与模型组比较,乌司他丁组与CoPP组供肝的损伤明显减轻、Suzuki评分降低,而ZnPP组损伤加重、Suzuki评分升高（均P<0.05）;乌司他丁组与CoPP组HO-1 mRNA与蛋白的表达明显上调,而ZnPP组明显下调（均P<0.05）;乌司他丁组与CoPP组大鼠血清中IL-2水平明显降低、IL-10水平明显升高,而ZnPP组IL-2水平明显升高、IL-10水平明显降低（均P<0.05）。 结论：乌司他丁可能通过上调移植大鼠肝脏的HO-1水平,减轻再灌注损伤、抑制排斥反应而发挥保护作用。

     

上调肝脏HO-1抑制肥大细胞脱颗粒减轻大鼠肝脏缺血再灌注损伤

目的 探索HO-1对肝脏缺血再灌注损伤中肥大细胞脱颗粒的影响。方法 将20只SD大鼠随机分成4组：假手术组（Sham组）,缺血再灌注损伤组（I/RI组）,HO-1诱导剂钴原卟啉组(CoPP组,术前24h给予CoPP,5 mg/kg)及HO-1抑制剂锌原卟啉组(ZnPP组,术前24h给予ZnPP,20 mg/kg)。建立大鼠缺血再灌注损伤模型,各组于再灌注后2h收集标本。RT-PCR检测肝脏组织HO-1 mRNA表达,Western blot检测肝脏组织HO-1蛋白表达;测定血清中ALT、AST水平;肝脏组织甲苯胺蓝染色检测肥大细胞脱颗粒数量,HE染色评价肝脏组织损伤情况。结果 与Sham组相比,I/RI组、CoPP组、ZnPP组大鼠组织HO-1 RNA和蛋白表达增加,血清ALT、AST水平升高,肥大细胞脱颗粒数量增多,肝脏细胞损伤加重。CoPP组与I/RI组相比,HO-1 mRNA和蛋白表达增加,血清ALT、AST水平减低,肥大细胞脱颗粒数量减少,肝细胞损伤减轻。ZnPP组与I/RI组相比,HO-1 mRNA和蛋白表达减少,血清ALT、AST水平升高,肥大细胞脱颗粒数量增多、肝细胞损伤严重。组间比较差异具有统计学意义(P＜0.05)。结论 HO-1过表达能减轻肝脏I/RI,其机制可能与抑制肝脏组织中肥大细胞脱颗粒有关。     

Improved Long-Term Graft Survival after HO-1 Induction in Brain-Dead Donors

Brain death (BD) of the donor, a risk factor uniquely relevant for organs derived from cadaver donors, influences organ quality by induction of various inflammatory events. Consequently ischemia/reperfusion injury is deteriorated and acute and chronic rejections accelerated. Donor treatment might be an approach to improve the quality of the graft. The induction of heme oxygenase 1 (HO-1) has been shown to exert beneficial effects in living-donor transplantation models. Therefore, we examined the impact of donor treatment with the selective inducer of HO-1, cobalt protoporphyrin (CoPP), on organ quality and transplant outcome in a standardized BD model in a F344-->LEW kidney transplant rat model. Immediately after BD induction, donor animals were administered a single dose of CoPP (5 mg/kg) and in control groups, HO-1 activity was blocked with zinc protoporphyrin (ZnPP, 20 mg/kg). Recipients of organs from brain-dead donors treated with CoPP survived significantly better than those from untreated brain-dead donors (p < 0.05) and intra-graft analysis showed improved histology (p < 0.05). Blockade of HO-1 with ZnPP decreased the survival rates (p < 0.05) comparable to untreated brain-dead donors. Our results demonstrate that HO-1 induction by one single treatment of CoPP in brain-dead donors leads to enhanced allograft survival.     

血红素氧合酶-1对移植的异种心脏的保护作用

目的探讨血红素氧合酶-1(HO-1)对移植的异种心脏的保护作用及其可能机制。方法应用NI H小鼠到Wistar大鼠颈部异位心脏移植模型,术前对供者分别用HO-1的诱导剂CoPP或HO-1的阻滞剂ZnPP进行处理,术后受者相应接受CoPP或ZnPP处理,部分受者同时接受环孢素A(CsA)处理,并设空白对照组和单用CsA组。分别用免疫组化、逆转录聚合酶链反应、Westernblot蛋白印迹杂交等检测移植心组织中HO-1、HO-1mRNA、凋亡蛋白酶-3以及信号传导与转录因子-3(STAT-3)的表达,测定心肌组织中HO-1的活性,采用原位末端标记法检测心肌细胞凋亡情况,比较不同处理因素之间的差异。结果单一接受CoPP者(CoPP组)移植心脏存活时间长于空白对照组及单一接受ZnPP者(P<0.01),但短于同时接受CoPP和CsA处理者(CoPP CsA组,P<0.01);CoPP组及CoPP CsA组HO-1、HO-1mRNA的表达及HO-1的活性均高于其余各组(P<0.01),其心肌细胞凋亡数和凋亡蛋白酶-3低于其余各组(P<0.05),而STAT-3高于其余各组(P<0.01)。结论CoPP诱导产生的HO-1可延长NI H小鼠到Wistar大鼠异种移植心脏的存活时间,该作用可能与STAT-3激活、凋亡蛋白酶-3受抑制而导致心肌细胞凋亡发生率下降有关。     

Pharmacological preconditioning with simvastatin protects liver from ischemia-reperfusion injury by heme oxygenase-1 induction

BACKGROUND: The protective role of heme oxygenase-1 (HO-1) against liver ischemia-reperfusion (I/R) injury in models of hypoxic and remote preconditioning has been proved. The feasible candidates who induce HO-1 and thorough which exert the protective effects are under investigation. The aim was to study the role of HO-1 in pharmacological preconditioning by simvastatin in a rat model. METHODS: Pharmacological preconditioning by intraperitoneal injection of simvastatin (5 mg/kg) was tested on a partial liver I/R model on rats. The expression of HO-1 protein and enzyme activities in livers, serum alanine transaminase (ALT) levels, and TUNEL staining of liver after I/R injury were measured in rats with and without simvastatin preconditioning. RESULTS: HO-1 was induced and persistently overexpressed in the hepatocytes 24 hr after simvastatin treatment. Simvastatin preconditioning diminished the elevation of serum ALT levels 4 hr after I/R injury (69.6+/-26.3 U/L) (P<0.05 vs. other groups) when compared with control (403.8+/-261.9 U/L) and zinc protoporphyrin (ZnPP)-pretreated (717.5+/-205.6 U/L) groups. Simvastatin preconditioning diminished the apoptosis after I/R injury as well (apoptosis index: 26.4+/-8 for Simvastatin, 78+/-7 for control, and 85.3+/-2 for ZnPP group; P<0.05). The addition of ZnPP negated the protective effects of simvastatin as evidenced in the ALT level (406.2+/-243.0 U/L) and apoptosis index (75.6+/-6). The heme oxygenase activity in treated rats correlated with these results. CONCLUSIONS: The induction of HO-1 by simvastatin preconditioning played a protective role against hepatic I/R injury.     

HO-1诱导剂体外灌注对大鼠移植肝脏保护作用的研究

探讨在移植肝脏低温保存期间灌注血红素加氧酶-1(heme oxygenase-1,HO-1)诱导剂钴原卟啉(cobalt protoporphyrin,CoPP)对大鼠移植肝脏的保护作用.方法 以Wistar大鼠为供体,SD大鼠为受体,根据供肝体外灌注低温保存期间所灌注的液体不同,将其分为4组:空白对照组、CoPP组、nodosin组和锌原卟啉(zinc protoporphyrin,ZnPP)组.每组各建立12对大鼠原位肝移植模型,各组分别在移植手术后1 d、3 d、7 d、21 d处死3只大鼠切取肝脏组织标本,进行肝脏组织病理检查与Suzuki评分.结果 原位肝移植术后1 d、3 d、7 d,CoPP组和nodosin组大鼠的肝脏病理损伤比空白对照组和ZnPP组轻,但Suzuki评分差异无统计学意义(P>0.05).原位肝移植术后21 d,nodosin组与CoPP组的病理损伤均较空白对照组轻,两组的Suzuki评分与空白对照组比较差异有统计学意义(P<0.01,P<0.05).ZnPP组的移植肝脏损伤较重,ZnPP组的Suzuki评分与nodosin组比较差异有统计学意义(P<0.01).结论 在移植肝低温保存期间灌注HO-1诱导剂可以减轻大鼠肝移植术后肝脏组织的病理损伤,而且在术后晚期对移植肝保护效果更加明显.     

Contributions of heme oxygenase-1 in postconditioning-protected ischemia-reperfusion injury in rat liver transplantation

Background

Heme oxygenase-1 (HO-1), an oxidative stress-response gene up-regulated by various physiological and exogenous stimuli, has cytoprotective activities. Ischemic postconditioning (Postcon) can protect an organ from ischemia-reperfusion (I/R) injury. In the present study, we investigated the potential contributions of HO-1 to Postcon-dependent protection against I/R injury in rat liver transplantation models.

Materials and methods

Adult male Sprague-Dawley rats were randomly divided into four groups: sham group with laparotomy for liver exposure; I/R group with 24-hour cold ischemia of the donor liver; Postcon group with the same treatment as the I/R group plus ischemic Postcon; and zinc protoporphyrin (ZnPP HO-1 inhibitor) + Postcon group treated the same as the Postcon cohort with donors pretreated using ZnPP 24 hours before the I/R injury. We measured liver tissue and peripheral blood samples collected at 6 hours after reperfusion and serum transaminase levels, histopathology, liver tissue malondialdehyde (MDA) content, superoxide dismutase (SOD) activity and HO-1 expression in the liver.

Results

Postcon significantly diminished the elevation of serum transaminases levels after I/R injury when compared with I/R and ZnPP+Postcon groups. Postcon treated rats showed significantly lower MDA production and higher SOD activity. HO-1 was induced in rat livers exposed to Postcon; its levels were obviously overexpressed after 6 hours in Postcon rats. Inhibiting the expression of HO-1, negated the protective effects of Postcon.

Conclusions

Induction of HO-1 in the Postcon condition played a protective role against hepatic I/R injury and enhanced the early antioxidative activity. The protective effects of Postcon were significantly associated with greater intrahepatic HO-1 expression.     

Transient limb ischemia induces remote preconditioning in liver among rats: the protective role of heme oxygenase-1

BACKGROUND: We have reported the protective role of heme oxygenase-1 (HO-1) in the mechanism of hypoxic preconditioning. We wish to investigate the role of HO-1 in remote preconditioning (RP) against hepatic ischemia/reperfusion (I/R) injury in rats. METHODS: The remote preconditioning was produced by four cycles of 10-min ischemia-reperfusion of the hind limb of rats. Partial hepatic ischemia was produced in the left lobes for 45 min followed by 240 min of reperfusion. Zinc-protoporphyrin IX (ZnPP), a specific inhibitor of HO enzymatic activity, was intra-peritoneally injected 1 hr before the ischemia-reperfusion injury in separate groups of RP rats. Serum alanine transaminase (ALT) levels, expression of hepatic HO-1 protein and mRNA, immunohistochemical staining and HO enzymatic activity were measured. RESULTS: HO-1 was induced in the livers of rats 4 hr after the RP stimuli, and the overexpression persisted for 24 hr. Immunohistochemical staining demonstrated induction of HO-1 in the hepatocytes. The peripheral lymphocytes did not express HO-1 after RP. RP diminished the elevation of serum ALT levels 4 hr after I/R injury (283.7+/-167.4 U L) when compared with controls (1297.7+/-729.3 U L) and RP+ ZnPP pretreated groups (1429.9+/-750.9 U L). The heme oxygenase activity in treated rats also correlated these results (286.8+/-34.3 pmol mg protein hr for the RP group, 156.3+/-27.5 pmol mg protein hr for the RP+ ZnPP pretreated group, and 170.6+/-19.4 pmol mg protein hr for the control group, 144.8+/-7.8 pmol mg protein hr for the control+ ZnPP pretreated group). CONCLUSION: Our results indicated that the induction of HO-1 in remote preconditioning played a protective role against hepatic I/R injury.     

Heme oxygenase-1 attenuates ischemia/reperfusion-induced apoptosis and improves survival in rat renal allografts

BACKGROUND: Kidneys can be preserved only for a limited time without jeopardizing graft function and survival. Induction of heat shock proteins (HSPs) can protect against ischemia/reperfusion (I/R) injury. Therefore, we investigated whether the induction of the HSP, heme oxygenase-1 (HO-1), improves outcome following isotransplantation after an extended period of cold storage. METHODS: Rats were subjected to heat preconditioning (HP; 42 degrees C for 20 minutes). Kidneys harvested after 24 hours, were preserved in cold University of Wisconsin (UW) solution at 4 degrees C for 45 hours and transplanted into bilateral nephrectomized rats. Cobalt protoporphyrin (CoPP) was administered in another group of animals in order to induce HO-1 pharmacologically, while other groups of animals received the HO-1 inhibitor, tin protophorphyrine (SnPP), following HP or CoPP. RESULTS: Cold ischemia caused a complete attenuation of graft function within 3 days following transplantation and subsequent death of all animals, whereas HP protected graft function and five of nine rats survived for 3 weeks. HP inhibited the induction of osteopontin and induced the expression of HO-1, HSP 70 and 90, and the antiapoptotic factor Bcl-XL. Grafts exposed to HP were protected against structural I/R injuries as revealed by histologic assessment using a semiquantitative score. Furthermore, induction of apoptosis was attenuated and activation of caspase-3 was inhibited. Comparable results were observed after administration of CoPP, whereas SnPP inhibited the effects of HP and CoPP. CONCLUSION: HP or administration of CoPP induced both HO-1, preserved kidney graft function, and prevented postreperfusion apoptosis after cold preservation.     

锌原卟啉对种植性乳腺癌细胞凋亡的影响

目的探讨锌原卟啉（ZnPP）对种植性乳腺癌细胞凋亡及信号传导和转录因子-3（STAT-3）的影响。方法应用种植性乳腺癌模型，结合ZnPP、钴原卟啉（CoPP）处理，分别用逆转录．聚合酶链反应（RT-PCR）检测乳腺癌组织中的血红素氧合酶-1（HO-1）mRNA并HO-1酶活性，免疫组织化学、Western blot蛋白印迹杂交检测癌组织中HO-1、CAS-3、bel-XL、STAT-3蛋白表达。TUNEL法评价肿瘤细胞凋亡。结果ZnPP组肿瘤平均直径明显低于其余各组（t=3．6324，P〈0．01）；CoPP组最大（t=3．5546，P〈0．01）。ZnPP组肿瘤组织内的HO-1 mRNA、HO-1蛋白及HO-1酶活性均低于其余各组，而CoPP组高于其余各组。ZnPP组STAT-3及bel-XL蛋白表达低于CoPP组（t=2．4216，P〈0．05）（t=4．3706，P〈0．01），CoPP＋ZnPP组STAT-3高于空白对照组（t=2．7965，P〈0．05），其余各组间差异无统计学意义（P〉0．05）。ZnPP组CAS-3蛋白表达及肿瘤细胞凋亡指数与其他组相比较明显增高（t=3．6856，P〈0．01）（t=3．5969，P〈0．01）。结论ZnPP可能通过抑制HO-1蛋白的表达来降低STAT-3和bcl-XL蛋白表达，从而增加CAS3的表达。并可能由此增加了肿瘤细胞凋亡的发生。     

Peritransplant treatment with cobalt protoporphyrin attenuates chronic renal allograft rejection

Allogen-independent injury contributes to chronic rejection in renal allografts and heme oxygenase-1 (HO-1) has been shown to be protective in a number of settings. This study evaluated the effect of renal allograft recipient HO-1 up-regulation on chronic rejection in a rat model. Rat (F344 to Lewis) renal transplantation recipients were grouped: (i) cyclosporine (CsA) alone (0.75 mg/kg s.c. x 10 day; n = 5); (ii) CsA + low dose cobalt protoporphyrin (CoPP) an HO-1 inducer (0.5 mg/kg i.p. on days -5,0,5; n = 13) and (iii) CsA + high dose CoPP (5.0 mg/kg i.p. on days -5,0,5; n = 8). Renal function was assessed by serum creatinine levels on day 140. Histopathologic changes in allografts were graded. Morphometric analyses performed to objectively quantify the vascular changes and glomerulosclerosis. HO-1 expression quantified by Western blot and both HO-1 and endothelin (ET-1) localized using immunohistochemistry. Recipients treated with CsA + high dose CoPP had significantly decreased cortical scarring, vascular hyalinization and intimal thickness. They also had a significant, dose-dependent, reduction in luminal obliteration and glomerulosclerosis by morphometric analyses. This freedom from chronic rejection in recipients treated with CoPP translated into quiescent grafts at postoperative day 140 with immunostaining and Western blot demonstrating decreased level of HO-1 versus controls (P = 0.012). In summary, the peritransplant up-regulation of HO-1 in renal allograft recipients significantly attenuates chronic rejection in rat renal allografts by inhibiting transplant vasculopathy.     

The protective role of heme oxygenase-1 on the liver after hypoxic preconditioning in rats

BACKGROUND: Hypoxic preconditioning (HP) confers cytoprotection against ischemia/reperfusion (I/R) injury. This effect is in part attributable to the induction of heme oxygenase (HO)-1. This experiment evaluates liver cell damage after I/R injury in HP rats. METHODS: HP rats were prepared by exposure (15 hr/day) to an altitude chamber (5500 m) for 2 weeks. Partial hepatic ischemia was produced in the left lobes for 45 min followed by 180 min of reperfusion. Zinc (Zn) protoporphyrin (PP), a specific inhibitor of HO enzymatic activity, was subcutaneously injected 1 hr before the I/R injury into separate groups of sea-level (SL) control and HP rats. Serum alanine aminotransferase (ALT) levels, liver HO-1 mRNA and protein, and HO enzymatic activity were measured. RESULTS: HO-1 was induced in the livers of rats exposed to HP. The levels of HO-1 mRNA and protein were obviously overexpressed after 2 weeks of HP. HP diminished the elevation of serum ALT levels after I/R injury (83.7+/- 4.9 U/L) when compared with SL controls (280.8+/-19.4 U/L) and HP+ZnPP-pretreated groups (151.3+/-4.4 U/L). The HO activity in treated rats also was correlated with these results (237.9+/-19.8 pmol/mg of protein per hour for the HP group, 164.3+/-12.7 pmol/mg of protein per hour for the HP+ZnPP group, and 182.6+/-8 pmol/mg of protein per hour for the SL controls). CONCLUSIONS: The authors' results indicated that the induction of HO-1 in hypoxic preconditioning played a protective role against hepatic I/R injury.     

Cytoprotective and Antiapoptotic Effects of IL-13 in Hepatic Cold Ischemia/Reperfusion Injury Are Heme Oxygenase-1 Dependent

Liver injury caused by ischemia/reperfusion (I/R) insult represents the major problem following orthotopic liver transplantation (OLT). I/R damage has been linked to Th1-like cytokine producers. This study evaluates putative cytoprotective effects/mechanisms of Th2-type IL-13 gene transfer. IL-13 overexpression prevented hepatic insult in a rat model of 24 h cold ischemia followed by OLT, as assessed: (i) profoundly decreased hepatocellular damage (sGOT levels), and ameliorated histological signs of I/R injury (Suzuki criteria), consistent with long-term OLT survival; (ii) prevented hepatic apoptosis (TUNEL stains) and up-regulated expression of antiapoptotic (A20, Bcl-2/Bcl-xl)/antioxidant (HO-1) genes. However, inhibition of HO-1 with tin protoporphyrin reversed cytoprotective/antiapoptotic effects of IL-13. In conclusion, cytoprotection rendered by virally induced IL-13 against hepatic I/R injury in this clinically relevant rat hepatic cold I/R injury model was accomplished via decreased apoptosis and induction of antiapoptotic/antioxidant molecules. HO-1 neutralization studies suggest that HO-1 represents one of putative IL-13 downstream effectors. This study provides the rationale for novel approaches to maximize organ donor pool through the safer use of OLTs despite prolonged periods of cold ischemia.     

血红素加氧酶-1对乳鼠心肌细胞缺氧复氧损伤的影响

目的 探讨血红素加氧酶-1(HO-1)对乳鼠心肌细胞缺氧复氧损伤的影响.方法 新生SD大鼠8只,日龄1～3 d,原代培养心肌细胞,随机分为4组:正常对照组(C组)常规培养8 h;缺氧复氧组(HR组)采用缺氧2 h,复氧6 h的方法制备心肌细胞缺氧复氧模型;血晶素组(Hemin组)缺氧前24 h及缺氧即刻,培养基中加入Hemin,终浓度为20 μmol/L;血晶素+锌原卟啉组(Hemin+ZnPP组)缺氧前24 h及缺氧即刻培养基中同时加入Hemin及ZnPP,终浓度均为20 μmol/L.各组细胞均接种于35 mm培养皿(2 ml/皿)或50 ml培养瓶(3 ml/瓶),每组45皿和3瓶.于复氧结束后采用蛋白印迹法测定心肌细胞HO-1表达,台盼蓝染色法测定心肌细胞存活率,应用全自动生化分析仪测定细胞培养液乳酸脱氧酶(LDH)活性,超声破碎细胞离心后取上清,采用硫代巴比妥酸法测定细胞MDA水平,黄嘌呤氧化酶法测定细胞SOD活性.结果 与C组比较,其余3组培养液LDH活性、心肌细胞MDA水平及HO-1表达升高,心肌细胞存活率及SOD活性降低(P＜0.05).与HR组比较,Hemin组培养液LDH活性、心肌细胞MDA水平降低,HO-1表达、心肌细胞存活率及SOD活性升高(P＜0.05),Hemin+ZnPP组上述指标差异无统计学意义(P＞0.05).HR组和Hemin+ZnPP组细胞缺氧复氧损伤明显,Hemin组细胞缺氧复氧损伤减轻.结论 HO-1可减轻乳鼠心肌细胞缺氧复氧损伤.     

Induction of heme oxygenase-1 in the donor reduces graft immunogenicity

There is increasing evidence that the induction of the enzyme heme oxygenase-1 (HO-1) improves both graft function and survival. Although it has been shown that HO-1 promotes graft protection, it remains unknown whether it reduces graft immunogenicity by modulating dendritic cells. In the current experiment, we investigated the impact of HO-1 induction on frequencies and trafficking of donor-derived dendritic cells (DCs). Kidneys from DA rats were transplanted into untreated Lewis recipients. Donor animals were treated with cobalt protoporphyrin (CoPP; 5 mg/kg IP) 24 hours prior to organ harvesting to induce HO-1. Controls remained untreated or received zinc protoporphyrin (ZnPP; 20 mg/kg, IP) to block HO-1 induction. Analyses of grafts, spleens, lymph nodes and blood of Lewis recipients were performed at days 1 and 3 posttransplantation. Donor-specific DCs were determined by flow cytometry using haplotype-specific mAb against RT1(ab) and mAb against OX62(+) antigens. Cell markers (CD4/CD8(+) T cells, ED1(+) monocytes, MHC class II(+) CD86(+) DC) were measured by immunohistochemical staining. T-cell alloreactivity of recipient splenocytes was measured by ELISPOT. Induction of HO-1 reduced frequencies of donor-derived DCs in the graft and recipient compartments, which was associated with reduced frequencies of CD4(+) T cells and CD8(+) T cells and alloreactivity. Expression of costimulatory molecule CD86 and MHC class II antigens were also reduced, although not significantly. Thus, induction of HO-1 reduced graft immunogenicity. These mechanisms may explain the protective effects of HO-1 induction.     

Protective effect of hemoglobin—induced heme oxygenase—1 on injured lungs caused by limb ischemia—reperfusion in rats

OBJECTIVE: 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 occurred after reperfusion. To induce the expression of HO-1 in the lungs, Hb was administrated intraperitoneally at 16 hours before reperfusion. Northern blotting and 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 hemoglobin (Hb) on the injured lungs after limb I/R was determined by measuring the changes of lung histology, polymorphonuclear (PMN) count, malondialdehyde (MDA) content and wet-to-dry weight ratio (W/D). Zinc protoporphyrin (ZnPP), an inhibitor of HO, was used to determine whether HO-1 was induced by Hb after lung injury. RESULTS: Hb led to a significant increase in HO-1 mRNA and protein expression in the lungs, accompanied by the increase of COHb level in arterial 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 the pretreatment with Hb at 16 hours before reperfusion. ZnPP blocked this protective role of Hb in the injured lungs. CONCLUSIONS: Hb can induce the lung HO-1 expression, which plays an important role in the defense against I/R induced lung injury in rats.     

HO-1 upregulation suppresses type 1 IFN pathway in hepatic ischemia/reperfusion injury

Upregulation of heme oxygenase (HO)-1, a heat shock protein 32, protects against hepatic ischemia/reperfusion (I/R) injury. Activation of "innate" toll-like receptor (TLR) 4 system triggers the I/R injury cascade. This study explores cytoprotective functions of HO-1 overexpression following exogenous administration of cobalt protoporphyrin (CoPP), and its relationship with the TLR4 pathway in a model of mouse partial hepatic warm I/R injury. CoPP treatment markedly improved hepatic function and histology, and suppressed pro-inflammatory cytokine elaboration profile, as compared with untreated controls. Although administration of CoPP did not affect intrahepatic TLR4, it downregulated IFN-inducible protein 10 (IP-10) expression. As IP-10 is the major product of type-1 IFN pathway downstream of TLR4, we then infused recombinant IFN-beta (rIFN-beta) directly into mouse livers. Interestingly, infusion of rIFN-beta upregulated hepatic IP-10 expression. In contrast, adjunctive CoPP treatment decreased IP-10 levels in mouse livers infused with rIFN-beta. Thus, CoPP-induced HO-1 upregulation suppresses type-1 IFN pathway downstream of TLR4 system in hepatic warm I/R injury model.