NADPH氧化酶与男性勃起功能障碍

勃起功能障碍是男性的常见病和多发病。目前认为氧化应激是引起阴茎勃起功能障碍的重要机制之一。NADPH氧化酶广泛存在于机体多个系统(包括阴茎组织),发挥重要的生理功能。在多种病理情况下,NADPH氧化酶可以在阴茎组织中催化合成大量活性氧,导致过度氧化应激,从而影响阴茎勃起功能。本文就NADPH氧化酶的组成、同源物、活性调节、生理功能、在勃起功能障碍中的作用以及在勃起功能障碍治疗中的应用作一综述。     

Intermedin inhibits unilateral ureteral obstruction-induced oxidative stress via NADPH oxidase Nox4 and cAMP-dependent mechanisms

NADPH oxidase Nox4-derived reactive oxygen species (ROS) play important roles in renal fibrosis. Our previous study demonstrated that intermedin (IMD) alleviated unilateral ureteral obstruction (UUO)-induced renal fibrosis by inhibition of ROS. However, the precise mechanisms remain unclear. Herein, we investigated the effect of IMD on Nox4 expression and NADPH oxidase activity in rat UUO model, and explored if these effect were achieved through cAMP-PKA pathway, the important post-receptor signal transduction pathway of IMD, in TGF-β1-stimulated rat proximal tubular cell (NRK-52E). Renal fibrosis was induced by UUO. NRK-52E was exposed to rhTGF-β1 to establish an in vitro model of fibrosis. IMD was overexpressed in the kidney and in NRK-52E by IMD gene transfer. We studied UUO-induced ROS by measuring dihydroethidium levels and lipid peroxidation end-product 4-hydroxynonenal expression. Nox4 expression in the obstructed kidney of UUO rat or in TGF-β1-stimulated NRK-52E was measured by quantitative RT-PCR and Western blotting. We analyzed NADPH oxidase activity using a lucigenin-enhanced chemiluminescence system. We showed that UUO-stimulated ROS production was remarkably attenuated by IMD gene transfer. IMD overexpression inhibited UUO-induced up-regulation of Nox4 and activation of NADPH oxidase. Consistent with in vivo results, TGF-β1-stimulated increase in Nox4 expression and NADPH oxidase activity was blocked by IMD. In NRK-52E, these beneficial effects of IMD were abolished by pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89), a PKA inhibitor, and mimicked by a cell-permeable cAMP analog dibutyl-cAMP. Our results indicate that IMD exerts anti-oxidant effects by inhibition of Nox4, and the effect can be mediated by cAMP-PKA pathway.     

Antioxidants ameliorate the expression of vascular endothelial growth factor mediated by protein kinase C in diabetic podocytes.

BACKGROUND: The increased production of reactive oxygen species (ROS) may be involved in the onset or development of diabetic vascular complications. The release of ROS from podocytes plays a role in the pathogenesis of glomerular damage in various experimental glomerular diseases. Although it is assumed that the podocyte injury also plays an important role in diabetic nephropathy, the mechanism is still unknown. METHODS: Using a differentiated mouse podocyte cell line, we investigated: (1) whether a high level of ambient glucose increases the level of ROS, (2) whether the protein kinase C (PKC) pathway is involved in a high-glucose-induced generation of ROS and vascular endothelial growth factor (VEGF) and (3) whether antioxidants ameliorate PKC-mediated VEGF expression in diabetic milieu. RESULTS: Intracellular ROS generation was significantly higher in high glucose than in control conditions in cultured podocytes. High ambient glucose also increased VEGF mRNA and protein expression. The high-glucose-induced increases in ROS and VEGF mRNA and protein by podocytes were effectively inhibited by pretreatment with various antioxidants and were completely restored by PKC inhibition. The results show that cultured mouse podocytes produce ROS in response to high glucose, and that PKC is involved in high-glucose-induced ROS and VEGF production by podocyte. CONCLUSION: Increased ROS in podocytes may play a role in the pathogenesis of podocyte injury in diabetic nephropathy.     

Disruption of Renal Tubular Mitochondrial Quality Control by Myo-Inositol Oxygenase in Diabetic Kidney Disease

Diabetic kidney disease (DKD) is associated with oxidative stress and mitochondrial injury. Myo-inositol oxygenase (MIOX), a tubular-specific enzyme, modulates redox imbalance and apoptosis in tubular cells in diabetes, but these mechanisms remain unclear. We investigated the role of MIOX in perturbation of mitochondrial quality control, including mitochondrial dynamics and autophagy/mitophagy, under high-glucose (HG) ambience or a diabetic state. HK-2 or LLC-PK1 cells subjected to HG exhibited an upregulation of MIOX accompanied by mitochondrial fragmentation and depolarization, inhibition of autophagy/mitophagy, and altered expression of mitochondrial dynamic and mitophagic proteins. Furthermore, dysfunctional mitochondria accumulated in the cytoplasm, which coincided with increased reactive oxygen species generation, Bax activation, cytochrome C release, and apoptosis. Overexpression of MIOX in LLC-PK1 cells enhanced the effects of HG, whereas MIOX siRNA or d-glucarate, an inhibitor of MIOX, partially reversed these perturbations. Moreover, decreasing the expression of MIOX under HG ambience increased PTEN-induced putative kinase 1 expression and the dependent mitofusin-2–Parkin interaction. In tubules of diabetic mice, increased MIOX expression and mitochondrial fragmentation and defective autophagy were observed. Dietary supplementation of d-glucarate in diabetic mice decreased MIOX expression, attenuated tubular damage, and improved renal functions. Notably, d-glucarate administration also partially attenuated mitochondrial fragmentation, oxidative stress, and apoptosis and restored autophagy/mitophagy in the tubular cells of these mice. These results suggest a novel mechanism linking MIOX to impaired mitochondrial quality control during tubular injury in the pathogenesis of DKD and suggest d-glucarate as a potential therapeutic agent for the amelioration of DKD.     

兔髂动脉再狭窄与NADPH氧化酶的关系

目的研究NADPH氧化酶与血管壁活性氧(ROS)生成及再狭窄的关系。方法构建兔髂动脉2次损伤后再狭窄模型,逆转录-聚合酶链反应(RT-PCR)检测2次损伤后不同时段兔髂动脉NADPH氧化酶催化亚基gp91phox和p22phox mRNA表达的变化;原位杂交观察gp91phox和p22phox mRNA表达在血管壁的定位。结果gp91phox mRNA的表达在2次损伤后逐渐上升,在14 d(1．554±0．105)、28 d(1．444±0．360)达到峰值,与术后即刻组(0．572±0．018)比较,差异有统计学意义(P<0．01);p22phox mRNA的表达在2次损伤即刻(1．514±0．036)即处于高水平,术后1 d(0．832±0．059)略有下降后又逐渐升高,并在14 d(1．714±0．249)、28 d(1．564±0．151)达峰值。在2次损伤后,各时段血管壁gp91phox和p22phox mRNA均可见阳性表达,其表达主要位于新生内膜和外膜。结论NADPH氧化酶各个亚基在再狭窄过程中可能行使不同功能;氧化酶主要在新生内膜与外膜发挥作用。     

NADPH氧化酶在转化生长因子β1诱导大鼠肾小管上皮细胞转分化中的作用

目的探讨NADPH氧化酶在转化生长因子β1（TGF-β1）诱导大鼠肾小管上皮细胞（NRK-52E）转分化中的作用。方法用TGF-β1（10μg/L）刺激NRK-52E细胞不同时间,观察α-平滑肌肌动蛋白（α-SMA）、E-钙黏蛋白（E-cadherin）、纤溶酶原激活物抑制剂1（PAI- 1）及Ⅰ型胶原（Col-Ⅰ）的表达。部分实验中细胞在TGF-β1刺激前用NADPH氧化酶抑制剂DPI预处理1 h。用激光共聚焦显微镜观察细胞内活性氧（ROS）的产生。用RT-PCR方法检测NADPH氧化酶p22phox、gp91phox、p47phox和p67phox亚单位mRNA的表达。α-SMA、E-cadherin、PAI-1及Col-ⅠmRNA及蛋白的表达分别采用RT-PCR、Western印迹和细胞免疫化学检测。结果TGF-β1可显著上调NADPH氧化酶p67phox亚单位mRNA的表达,8 h及24 h时分别为对照组的2.43倍及3.59倍（P〈0.01）。TGF-β1可显著促进细胞ROS的产生,5 min时已是对照组的2.5倍（P〈0.05）。DPI预处理同时可显著逆转TGF-β1诱导NRK-52E细胞ROS的产生（P〈0.05）、α-SMA的表达上调、E-cadherin的表达下调以及PAI-1和Col-Ⅰ的表达上调。结论TGF-β1可促进NRK-52E细胞增加ROS的产生。ROS介导了TGF-β1诱导NRK-52E细胞的转分化,促进肾脏纤维化。     

Four-and-a-Half LIM Domains Protein 2 Is a Coactivator of Wnt Signaling in Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a microvascular complication that leads to kidney dysfunction and ESRD, but the underlying mechanisms remain unclear. Podocyte Wnt-pathway activation has been demonstrated to be a trigger mechanism for various proteinuric diseases. Notably, four-and-a-half LIM domains protein 2 (FHL2) is highly expressed in urogenital systems and has been implicated in Wnt/β-catenin signaling. Here, we used in vitro podocyte culture experiments and a streptozotocin-induced DKD model in FHL2 gene-knockout mice to determine the possible role of FHL2 in DKD and to clarify its association with the Wnt pathway. In human and mouse kidney tissues, FHL2 protein was abundantly expressed in podocytes but not in renal tubular cells. Treatment with high glucose or diabetes-related cytokines, including angiotensin II and TGF-β1, activated FHL2 protein and Wnt/β-catenin signaling in cultured podocytes. This activation also upregulated FHL2 expression and promoted FHL2 translocation from cytosol to nucleus. Genetic deletion of the FHL2 gene mitigated the podocyte dedifferentiation caused by activated Wnt/β-catenin signaling under Wnt-On, but not under Wnt-Off, conditions. Diabetic FHL2^+/+ mice developed markedly increased albuminuria and thickening of the glomerular basement membrane compared with nondiabetic FHL2^+/+ mice. However, FHL2 knockout significantly attenuated these DKD-induced changes. Furthermore, kidney samples from patients with diabetes had a higher degree of FHL2 podocyte nuclear translocation, which was positively associated with albuminuria and progressive renal function deterioration. Therefore, we conclude that FHL2 has both structural and functional protein-protein interactions with β-catenin in the podocyte nucleus and that FHL2 protein inhibition can mitigate Wnt/β-catenin–induced podocytopathy.     

紫草素对糖尿病小鼠中性粒细胞胞外诱捕网表达的影响

目的:探讨紫草素对佛波酯(PMA)诱导的糖尿病小鼠骨髓来源的中性粒细胞胞外诱捕网（NETs）的影响,为其治疗糖尿病足溃疡提供依据。方法:采用链脲佐菌素腹腔注射构建糖尿病小鼠模型,密度梯度离心法提取小鼠骨髓来源的中性粒细胞,并进行体外培养,PMA刺激其诱导NETs模型,采用CCK-8法筛选紫草素的安全剂量;采用Sytox Green核酸荧光染料法定量检测NETs水平;PicoGreen荧光染料法定量检测dsDNA/NETs含量;荧光探针定量检测中性粒细胞活性氧(ROS)水平;荧光免疫组化法检测NETs标志物瓜氨酸化组蛋白H3(Cit-H3)的表达水平。结果：紫草素在0.125～1μg/mL浓度范围内对中性粒细胞活性无影响。与模型组比较,紫草素1、0.5、0.25μg/mL浓度组中性粒细胞NETs产生的荧光强度显著降低（P <0.01）,细胞外dsDNA水平显著降低（P <0.01）,细胞内ROS水平显著降低（P<0.01）,细胞Cit-H3表达显著下调（P <0.01）。结论:紫草素通过抑制糖尿病小鼠骨髓来源的中性粒细胞ROS水平和Cit-H3表达,从而抑制NET...     

Effect of Pentoxifylline on Renal Function and Urinary Albumin Excretion in Patients with Diabetic Kidney Disease: The PREDIAN Trial

Diabetic kidney disease (DKD) is the leading cause of ESRD. We conducted an open-label, prospective, randomized trial to determine whether pentoxifylline (PTF), which reduces albuminuria, in addition to renin-angiotensin system (RAS) blockade, can slow progression of renal disease in patients with type 2 diabetes and stages 3–4 CKD. Participants were assigned to receive PTF (1200 mg/d) (n=82) or to a control group (n=87) for 2 years. All patients received similar doses of RAS inhibitors. At study end, eGFR had decreased by a mean±SEM of 2.1±0.4 ml/min per 1.73 m² in the PTF group compared with 6.5±0.4 ml/min per 1.73 m² in the control group, with a between-group difference of 4.3 ml/min per 1.73 m² (95% confidence interval [95% CI], 3.1 to 5.5 ml/min per 1.73 m²; P<0.001) in favor of PTF. The proportion of patients with a rate of eGFR decline greater than the median rate of decline (0.16 ml/min per 1.73 m² per month) was lower in the PTF group than in the control group (33.3% versus 68.2%; P<0.001). Percentage change in urinary albumin excretion was 5.7% (95% CI, −0.3% to 11.1%) in the control group and −14.9% (95% CI, −20.4% to −9.4%) in the PTF group (P=0.001). Urine TNF-α decreased from a median 16 ng/g (interquartile range, 11–20.1 ng/g) to 14.3 ng/g (interquartile range, 9.2–18.4 ng/g) in the PTF group (P<0.01), with no changes in the control group. In this population, addition of PTF to RAS inhibitors resulted in a smaller decrease in eGFR and a greater reduction of residual albuminuria.