Hirsutenone reduces deterioration of tight junction proteins through EGFR/Akt and ERK1/2 pathway both converging to HO-1 induction

Oxidative stress-induced disruption of epithelial tight junctions (TJ) plays a critical role in the pathogenesis of intestinal disorders, including inflammatory bowel disease (IBD). The current study investigated the protective effect of hirsutenone against disruption of the intestinal barrier in vitro and in a mouse model of colitis. Caco-2 cells were stimulated with tert-butyl hydroperoxide (t-BH). Hirsutenone prevented the t-BH-induced increase in permeability by inhibiting the reduction in zonula occludens-1 (ZO-1) expression, and rapidly stimulated tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Hirsutenone-mediated protection against the loss of ZO-1 depends on the activation of both ERK1/2 and Akt signaling pathways. Interestingly, hirsutenone-mediated activation of Akt, but not ERK1/2, signaling was EGFR-dependent. Hirsutenone increased heme oxygenase-1 (HO-1) expression through both EGFR/Akt- and ERK1/2-dependent pathways, contributing to the protective effects against TJ dysfunction. Colitis was induced in mice by intrarectal administration of 2,4,6,-trinitrobenzene sulfonic acid (TNBS). Hirsutenone administration improved the clinical parameters and tissue histological appearance, increased HO-1 expression, attenuated reduction of ZO-1 and occludin mRNA, and promoted BrdU incorporation in the colonic epithelium of TNBS-treated mice. Taken together, our results demonstrate that hirsutenone reverse disordered intestinal permeability by activating EGFR/Akt and ERK1/2 pathways, which are involved in the regulation of HO-1 expression. These findings highlight the potential of hirsutenone for clinical applications in the treatment of IBD.     

Heme oxygenase-1 counteracts contrast media-induced endothelial cell dysfunction

Endothelial cell (EC) dysfunction is involved in the pathogenesis of contrast-induced acute kidney injury, which is a major adverse event following coronary angiography. In this study, we evaluated the effect of contrast media (CM) on human EC proliferation, migration, and inflammation, and determined if heme oxygenase-1 (HO-1) influences the biological actions of CM. We found that three distinct CM, including high-osmolar (diatrizoate), low-osmolar (iopamidol), and iso-osmolar (iodixanol), stimulated the expression of HO-1 protein and mRNA. The induction of HO-1 was associated with an increase in NF-E2-related factor-2 (Nrf2) activity and reactive oxygen species (ROS). CM also stimulated HO-1 promoter activity and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the CM-mediated induction of HO-1 and activation of Nrf2 was abolished by acetylcysteine. Finally, CM inhibited the proliferation and migration of ECs and stimulated the expression of intercellular adhesion molecule-1 and the adhesion of monocytes on ECs. Inhibition or silencing of HO-1 exacerbated the anti-proliferative and inflammatory actions of CM but had no effect on the anti-migratory effect. Thus, induction of HO-1 via the ROS-Nrf2 pathway counteracts the anti-proliferative and inflammatory actions of CM. Therapeutic approaches targeting HO-1 may provide a novel approach in preventing CM-induced endothelial and organ dysfunction.     

血红素加氧酶-1在大鼠矽肺纤维化中的作用

目的观察血红素加氧酶-1在大鼠矽肺纤维化中的作用。方法 Wistar大鼠60只,随机分为4组,分别为对照组(A组)和染尘模型组(B组)、B组+HO-1抑制剂(锌原卟啉)组(C组)、B组+HO-1诱导剂(钴原卟啉)组(D组)。采用气管暴露法滴注二氧化硅悬液制造大鼠矽肺模型。C、D组分别腹腔注射锌原卟啉12.5 mg/kg、钴原卟啉5 mg/kg,隔日一次,连续4周后,取肺,HE染色观察各组肺组织病理结构,Western blot检测各组肺组织HO-1蛋白含量,荧光定量PCR检测各组肺组织HO-1mRNA的表达。结果与对照组比较,B组出现典型的矽肺纤维化的病理改变,C组较B组加重,D组较B组改善;对照组中HO-1蛋白表达明显低于其他各组(P0.05),抑制剂C组较B组HO-1蛋白表达明显降低,而诱导剂D组较B组HO-1蛋白表达量明显升高(P0.05)。RT-PCR检测A、B、C、D组HO-1 mRNA相对表达量分别为1、2.25±0.38、1.81±0.74、7.75±0.56,抑制剂C组较B组表达明显降低,而诱导剂D组较B组表达显著升高(P0.05)。结论 HO-1参与了大鼠矽肺纤维化的形成过程,通过对其干预可以改变大鼠矽肺纤维化的程度,可为矽肺纤维化的诊断与治疗提供新途径。     

血红素加氧酶-1在前列腺癌中的表达

目的探讨血红素加氧酶-1(HO-1)在人前列腺癌组织中的表达,以及HO-1活性对体外培养的前列腺癌细胞(PC3)的影响。方法　收集我院2000年1月至2007年4月前列腺癌根治性手术标本30例,前列腺增生开放手术大体标本30例。行RT-PCR检测HO-1的mRNA表达,免疫组化检测HO-1的表达。应用ZnPP、CoPP干预体外培养的PC3细胞,24h后,RT-PCR检测HO-1 mRNA的表达,MTT法酶标检测仪检测细胞A570nm值,流式细胞术检测细胞凋亡。结果人前列腺癌组织HO-1的mRNA表达明显高于良性增生前列腺组织;免疫组化前列腺癌中HO-1表达阳性率为76.9%,良性前列腺组织仅少许有微弱表达。应用ZnPP、CoPP干预PC3细胞后,RT-PCR可见ZnPP抑制PC3细胞HO-1表达,且随浓度增加抑制作用增加;CoPP可诱导HO-1表达,随浓度增加表达增强。MTT检测显示,ZnPP组PC3细胞A值随浓度增加而减少,而CoPP组随浓度增加A值增加,呈现浓度依赖性(P<0.05)。流式细胞技术结果显示,ZnPP可诱导PC3细胞凋亡,CoPP可保护细胞减少凋亡。结论人前列腺癌组织中HO-1基因表达及蛋白水平均显著高于良性前列腺组织;体外细胞实验证实,ZnPP可抑制前列腺癌PC3细胞系HO-1活性,抑制其增殖,诱导其凋亡。而CoPP可诱导前列腺癌PC3细胞系HO-1表达,抑制其凋亡,诱导其增殖。     

Carbon monoxide modulates the response of human basophils to FcepsilonRI stimulation through the heme oxygenase pathway

We report the effects of exogenous and endogenous carbon monoxide (CO) on the immunological activation of human basophils. Hemin (1-100 microM), a heme oxygenase substrate analogue, significantly increased the formation of bilirubin from partially purified human basophils, thus indicating that these cells express heme oxygenase. This effect was reversed by preincubating the cells for 30 min with Zn-protoporphyrin IX (100 microM), a heme oxygenase inhibitor. Hemin (100 microM) also decreased immunoglobulin G anti-Fcepsilon (anti-IgE)-induced activation of basophils, measured by the expression of a membrane granule-associated protein, identified as cluster differentiation protein 63 (CD63), and by histamine release. These effects were reversed by Zn-protoporphyrin IX (100 microM), by oxyhemoglobin (HbO(2)), a CO scavenger (100 microM), and by 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ), an inhibitor of the soluble guanylyl cyclase (100 microM). Exposure of basophils to exogenous CO (10 microM for 30 min) also decreased their activation, while nitrogen (N(2)) was ineffective. HbO(2) and ODQ reversed the inhibition, reversing both membrane protein CD63 expression and histamine release to basal values. Both hemin and exogenous CO significantly raised cGMP levels in basophils and blunted the rise of calcium levels caused by immunological activation. This study suggests that CO increases cGMP formation, which in turn induces a fall in intracellular Ca(2+) concentration, thereby resulting in the inhibition of human basophil activation.     

Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7 cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals

High mobility group box 1 (HMGB1) is now recognized as a late mediator of sepsis. We tested hypothesis that ascorbic acid (AscA) induces heme oxygenase (HO)-1 which inhibits HMGB1 release in lipopolysaccharide (LPS)-stimulated cells and increases survival of septic mice. AscA increased HO-1 protein expression in a concentration- and time-dependent manner via Nrf2 activation in RAW 264.7 cells. HO-1 induction by AscA was significantly reduced by Nrf2 siRNA-transfected cells. Mutation of cysteine to serine of keap-1 proteins (C151S, C273S, and C288S) lost the ability of HO-1 induction by AscA, due to failure of translocation of Nrf-2 to nucleus. The PI3 kinase inhibitor, LY294002, inhibited HO-1 induction by AscA. Oxyhemoglobin (HbO₂), LY294002, and ZnPPIX (HO-1 enzyme inhibitor) reversed effect of AscA on HMGB1 release. Most importantly, administration of AscA (200 mg/kg, i.p.) significantly increased survival in LPS-induced endotoxemic mice. In cecal ligation and puncture (CLP)-induced septic mice, AscA reduced hepatic injury and serum HMGB1 and plasminogen activator inhibitor (PAI)-1 in a ZnPPIX-sensitive manner. In addition, AscA failed to increase survival in Nrf2 knockout mice by LPS. Thus, we concluded that high dose of AscA may be useful in the treatment of sepsis, at least, by activation of Nrf2/HO-1 signals.     

促红细胞生成素对慢性肾功能衰竭大鼠肾功能的影响

目的:研究促红细胞生成素(EPO)对慢性肾功能衰竭(CRF)大鼠肾功能的影响及其作用机制.方法:将5/6肾切除大鼠随机分为3组:Ⅰ组为假手术组;Ⅱ组为CRF组;Ⅲ组为给予促红细胞生成素的CRF组.第2次术后8周检测各组血压、尿蛋白、血清尿素氮、血肌酐、血红蛋白;观察肾组织病理改变,检测血清及肾组织中血红蛋白氧合酶 1(HO 1)活性;用免疫组化方法检测HO 1在肾脏中的表达.结果:Ⅲ组与Ⅱ组比较,血压、尿蛋白、血肌酐及尿素氮水平明显降低(P＜0.05),肾小球系膜增生及间质纤维化程度明显减轻(P＜0.05);HO 1活性检测显示,Ⅲ组大鼠血清中HO 1活性明显高于Ⅱ组(P＜0.05),免疫组化显示Ⅲ组大鼠肾组织中HO 1表达明显高于Ⅱ组(面密度、平均光度)(P＜0.05).结论:EPO使CRF大鼠肾功能得到改善,并使CRF大鼠血清及肾组织中HO 1表达及活性明显升高.     

从非抗凝猪血中制备原卟啉钠方法的建立及优化研究

目的:建立并优化从非抗凝猪血中制备原卟啉钠的方法。方法:先以非抗凝猪血制得血红素,再加入硫化亚铁等经酯化、皂化反应制得原卟啉钠;采用紫外吸收光谱与红外光谱对其进行定性检测、紫外分光光度法对其进行定量检测;通过正交试验对原卟啉钠的制备方法进行优化,确定最佳工艺参数。结果:建立了从非抗凝猪血中制备原卟啉钠的方法,每千克非抗凝猪血可以制得10.6g血红素,每5g血红素可以制得3.46g原卟啉钠,所得原卟啉钠经紫外和红外光谱鉴定证实,纯度为88.8%;最佳制备工艺是酯化时甲醇-氯仿加入量为70mL∶120mL、皂化时甲苯-1mo·lL-1氢氧化钠甲醇溶液用量为50mL∶80mL、90℃加热回流2.0h。结论:所建立的制备原卟啉钠的方法操作简便,原料易得,周期短,成本低,产量较高,是一种较理想的制备原卟啉钠的方法。