虎杖苷通过TLR4/NF-κB信号通路调控糖尿病肾病大鼠肾脏炎症作用的研究

目的：研究虎杖苷通过TLR4/NF-κB信号通路调控糖尿病肾病大鼠肾脏炎症作用的影响。方法：通过尾静脉注射链脲佐菌素,高脂饲料喂养建立糖尿病肾病大鼠模型。将成模的大鼠随机分为模型组、缬沙坦组（20 mg·kg^-1）、虎杖苷组（75 mg·kg^-1）、虎杖苷组（150 mg·kg^-1）,每组10只,日常进行高脂饮食喂养,连续灌胃给药90 d,每日1次。于第90天采集尿液,测量尿量及终点法测24 h尿白蛋白（UP）含量。通过HE染色观察大鼠肾脏组织的病理学变化。采用ELISA法检测肾组织中TGF-β1、FN纤维化指标表达及肾脏组织中炎症因子肿瘤坏死因子（TNF-α）、白介素-1β（IL-1β）、白介素-6（IL-6）的含量。Western blot法检测肾组织中Toll样受体4（TLR4）、髓样分化因子（Myd88）、核转录因子kappa B（NF-κB）的表达。结果：与模型组相比,虎杖苷组大鼠Scr、BUN、UP含量显著降低（P<0.05）。TGF-β1、FN纤维化指标明及炎症因子TNF-α、IL-1β、IL-6含量显减少（P<0.05）,TLR4、Myd88、NF-κB蛋白表达显著降低（P<0.05）。结论：虎杖苷对糖尿病肾病所引起的肾脏炎症有一定的保护作用,其作用机制可能与调节TLR4/NF-κB信号通路,降低肾脏组织的炎症因子含量,而起到抗炎的保护作用有关。     

普罗布考对甲基胍诱导纤维化相关因子在HK-2细胞表达的影响

目的研究降脂药普罗布考对小分子尿毒素甲基胍诱导纤维化相关因子在人肾小管上皮细胞表达的影响。方法 HK-2细胞分3组培养,A组（正常对照,不加干预因素）;B组培养基中加入0.5mmol.L-1甲基胍;C组培养基中加入0.5mmol.L-1甲基胍和20μmol.L-1普罗布考。各组细胞培养48h后用免疫细胞化学、RT-PCR检测TGF-β1、CTGF和FN,并用流式细胞仪对以上因子作定量检测,以观察其在HK-2细胞中的表达。结果在甲基胍刺激后,细胞免疫化学染色,HK-2细胞质中着色,显示TGF-β1、CTGF和FN均有表达。正常对照组HK-2细胞质中无着色。测定结果显示甲基胍组TGF-β1、CTGF和FN显著高于正常对照组（P〈0.01）,加入普罗布考后,表达显著下降（P〈0.01）。结论甲基胍能促进纤维化相关因子在肾小管上皮细胞中表达;普罗布考能抑制甲基胍诱导纤维化相关因子在肾小管上皮细胞的表达。     

Hederagenin inhibits high glucose-induced fibrosis in human renal cells by suppression of NLRP3 inflammasome activation through reducing cathepsin B expression

Diabetic nephropathy is a major complication of diabetes mellitus and is related to dysfunction of renal cells. Hederagenin is a triterpenoid saponin from some Chinese herbs with anti-inflammatory and anti-diabetic activities. However, its role in diabetic nephropathy progression is still obscure. This study aimed to explore the effects of hederagenin on renal cell dysfunction in vitro. Human renal mesangial cells (HRMCs) and human renal proximal tubular epithelial cells (HRPTEpiCs) were cultured under high glucose (HG) conditions to mimic diabetic nephropathy-like injury. Cell proliferation was evaluated by CCK-8. mRNA and protein levels were determined by qRT-PCR and western blotting, respectively. The secretion levels of fibrosis-related biomarkers were analyzed by ELISA. Results showed that hederagenin reduced HG-induced proliferation increase in HRMCs and HRPTEpiCs. Hederagenin attenuated HG-induced increase in mRNA and protein expression of NLRP3, ASC, and IL-1β. Hederagenin also suppressed HG-induced increase in mRNA and secretion levels of FN, Col. IV, PAI-1, and TGF-β1. NLRP3 inhibitor MCC950 attenuated HG-induced fibrosis of renal cells, and its activator nigericin reversed the suppressive effect of hederagenin on HG-induced fibrosis. Bioinformatics analysis predicted cathepsin B (CTSB) as a target of hederagenin to modulate NOD-like receptor (NLR) pathway. Hederagenin decreased CTSB level, and CTSB overexpression reversed the suppressive effect of hederagenin on HG-induced NLRP3 inflammasome activation and fibrosis in HRMCs and HRPTEpiCs. In conclusion, hederagenin attenuates HG-induced fibrosis of renal cells by inhibiting NLRP3 inflammasome activation via reducing CTSB expression, indicating a therapeutic potential of hederagenin in diabetic nephropathy.     

Simultaneous angiotensin receptor blockade and glucagon-like peptide-1 receptor activation ameliorate albuminuria in obese insulin-resistant rats

Insulin resistance increases renal oxidant production by upregulating NADPH oxidase 4 (Nox4) expression contributing to oxidative damage and ultimately albuminuria. Inhibition of the renin-angiotensin system (RAS) and activation of glucagon-like peptide-1 (GLP-1) receptor signalling may reverse this effect. However, whether angiotensin receptor type 1 (AT1) blockade and GLP-1 receptor activation improve oxidative damage and albuminuria through different mechanisms is not known. Using insulin-resistant Otsuka Long-Evans Tokushima Fatty (OLETF) rats, we tested the hypothesis that simultaneous blockade of AT1 and activation of GLP-1r additively decrease oxidative damage and urinary albumin excretion (U_albV) in the following groups: (a) untreated, lean LETO (n = 7), (b) untreated, obese OLETF (n = 9), (c) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan/kg/d; n = 9), (d) OLETF + GLP-1 mimetic (EXE; 10 µg exenatide/kg/d; n = 7) and (e) OLETF + ARB +exenatide (Combo; n = 6). Mean kidney Nox4 protein expression and nitrotyrosine (NT) levels were 30% and 46% greater, respectively, in OLETF compared with LETO. Conversely, Nox4 protein expression and NT were reduced to LETO levels in ARB and EXE, and Combo reduced Nox4, NT and 4-hydroxy-2-nonenal levels by 21%, 27% and 27%, respectively. At baseline, U_albV was nearly double in OLETF compared with LETO and increased to nearly 10-fold greater levels by the end of the study. Whereas ARB (45%) and EXE (55%) individually reduced U_albV, the combination completely ameliorated the albuminuria. Collectively, these data suggest that AT1 blockade and GLP-1 receptor activation reduce renal oxidative damage similarly during insulin resistance, whereas targeting both signalling pathways provides added benefit in restoring and/or further ameliorating albuminuria in a model of diet-induced obesity.     

Preventive effects and mechanisms of rhein on renal interstitial fibrosis in obstructive nephropathy

Renal interstitial fibrosis is a common outcome of a variety of chronic renal diseases. Here we evaluated the therapeutic efficacy of rhein on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO) and investigated the potential mechanisms. Mice underwent UUO, followed by orally administrated rhein (150 mg/kg/d) or control vehicle. Renal interstitial injury and the degree of fibrosis were evaluated by pathological staining and Western blot. The possible mechanisms were studied by Western blot, indirect immune-fluorescence and enzyme-linked immunosorbent assay. Our results showed that rhein therapy markedly ameliorated renal interstitial fibrotic lesions, reduced α-smooth muscle actin (α-SMA) expression, attenuated deposition of fibronectin (FN). Rhein also suppressed transforming growth factor-β1 (TGF-β1) and its type I receptor expression in obstructed kidneys. In vitro, rhein abolished the α-SMA and fibronectin expression of rat kidney interstitial fibroblasts cells (NRK-49F) induced by TGF-β1. These observations strongly suggest that rhein is a potent inhibitor of renal interstitial fibrosis, and its therapeutic mechanism is, at least in part, blocking interstitial fibroblasts cells activation.     

Effect of N-acetylcysteine on plasma adiponectin and renal adiponectin receptors in streptozotocin-induced diabetic rats

This study investigated the effect of N-acetylcysteine on plasma adiponectin, renal adiponectin receptors, lipid metabolism and oxidative stress in streptozotocin-induced diabetic rats. Metabolic parameters, plasma adiponectin level, renal protein expression of adiponectin receptors were analyzed in controls and diabetic rats treated with or without N-acetylcysteine in drinking water for 8 weeks. Plasma lipid, creatinine and free 5-F(2t)-isoprostane levels, urine protein excretion rate, mesangial matrix expansion index, and protein expression of renal connective tissue growth factor (CTGF) were increased in diabetic rats. The decreased plasma adiponectin levels and renal protein expression of adiponectin receptor 1 were accompanied by the decreased renal phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK)-alpha (Thr172) and protein expression of phospho-acetyl coenzyme A carboxylase (ACC) (Ser79) which led to the increased renal triglyceride levels in diabetic rats. There was no difference in the protein expression of renal adiponectin receptor 2 between control and diabetic rats. N-acetylcysteine treatment attenuated the increased oxidative stress, plasma and renal lipids, urine protein excretion rate, mesangial matrix expansion index, and protein expression of renal CTGF, but did not affect plasma adiponectin levels, renal protein expression of adiponectin receptor 1, phosphorylation of AMPK-alpha (Thr172) and renal protein expression of phospho-ACC (Ser79) in diabetic rats. These results suggested that the decreased plasma adiponectin and renal adiponectin receptor 1 result in the increased renal triglyceride that stimulates renal CTGF expression leading to the renal hypertrophy and the deteriorated renal function in the diabetic rats. N-acetylcysteine treatment attenuates the increased oxidative stress, but has no effect on the decreased plasma adiponectin and renal adiponectin receptor 1 in diabetic rats, indicating that oxidative stress may not contribute to the decreased plasma adiponectin and renal adiponectin receptor 1 protein expression in diabetic rats.     

Inhibition of arsenic-induced rat liver injury by grape seed exact through suppression of NADPH oxidase and TGF-β/Smad activation

Chronic arsenic exposure induces oxidative damage to liver leading to liver fibrosis. We aimed to define the effect of grape seed extract (GSE), an antioxidant dietary supplement, on arsenic-induced liver injury. First, Male Sprague-Dawley rats were exposed to a low level of arsenic in drinking water (30 ppm) with or without GSE (100 mg/kg, every other day by oral gavage) for 12 months and the effect of GSE on arsenic-induced hepatotoxicity was examined. The results from this study revealed that GSE co-treatment significantly attenuated arsenic-induced low antioxidant defense, oxidative damage, proinflammatory cytokines and fibrogenic genes. Moreover, GSE reduced arsenic-stimulated Smad2/3 phosphorylation and protein levels of NADPH oxidase subunits (Nox2, Nox4 and p47phox). Next, we explored the molecular mechanisms underlying GSE inhibition of arsenic toxicity using cultured rat hepatic stellate cells (HSCs). From the in vitro study, we found that GSE dose-dependently reduced arsenic-stimulated ROS production and NADPH oxidase activities. Both NADPH oxidases flavoprotein inhibitor DPI and Nox4 siRNA blocked arsenic-induced ROS production, whereas Nox4 overexpression suppressed the inhibitory effects of GSE on arsenic-induced ROS production and NADPH oxidase activities, as well as expression of TGF-β1, type I procollagen (Coll-I) and α-smooth muscle actin (α-SMA) mRNA. We also observed that GSE dose-dependently inhibited TGF-β1-induced transactivation of the TGF-β-induced smad response element p3TP-Lux, and that forced expression of Smad3 attenuated the inhibitory effects of GSE on TGF-β1-induced mRNA expression of Coll-I and α-SMA. Collectively, GSE could be a potential dietary therapeutic agent for arsenic-induced liver injury through suppression of NADPH oxidase and TGF-β/Smad activation.     

Inhibition of PKC-θ preserves cardiac function and reduces fibrosis in streptozotocin-induced diabetic cardiomyopathy

Background and Purpose

T-cell infiltration, interstitial fibrosis and cardiac dysfunction have been observed in diabetic patients with cardiovascular diseases. PKC-θ is crucial for the activation of mature T-cells. We hypothesized that inhibition of PKC-θ might protect diabetic hearts through inhibition of T-cell stimulation and maintenance of tight junction integrity.

Experimental Approach

A model of type 1 diabetes was induced by streptozotocin (STZ) (50 mg kg^–1 for 5 days) in male C57BL/6J wild-type (WT) mice and Rag1 knockout (KO) mice which lack mature lymphocytes. A cell-permeable selective PKC-θ peptide inhibitor (PI) was administered i.p. (0.2 mg kg^–1·day^–1) for 4 weeks (first phase) and 2 weeks (second phase). At the end of the 11th week, cardiac contractile force was measured in isolated perfused hearts. Cardiac morphology and fibrosis were determined. Phosphorylation of PKC-θ at Tyr³⁵⁸, infiltrated T-cells and tight junction protein ZO-1 within the hearts were detected, using immunohistochemcial techniques.

Key Results

PI did not affect high blood glucose level in both WT and Rag1 KO diabetic mice. Diabetes induced cardiac fibrosis in WT mice but not in Rag1 KO mice. PI attenuated cardiac fibrosis and improved cardiac contractility of WT diabetic hearts. PI decreased expression of phosphorylated PKC-θ, reduced the infiltration of T-cells and increased ZO-1 expression within WT diabetic hearts.

Conclusion and Implications

Inhibition of PKC-θ improves cardiac function and reduces cardiac fibrosis in WT mice with streptozotocin-induced diabetes. Mature T-cells play a key role in pathophysiology of diabetic cardiomyopathy.     

Cyclophilin D promotes tubular cell damage and the development of interstitial fibrosis in the obstructed kidney

Cyclophilin D (CypD) is an important component in mitochondrial‐dependent tubular cell death in acute kidney injury. However, it is not known whether CypD contributes to tubular cell damage in chronic interstitial fibrosis. We investigated this question in the unilateral ureter obstruction (UUO) model of renal interstitial fibrosis. Groups of CypD^?/? and wild type (WT) mice were killed 7 or 12 days after UUO surgery. The significant tubular cell apoptosis seen in WT UUO was significantly reduced in CypD^?/? UUO based on TUNEL and cleaved caspase 3 staining. Other markers of tubular cell damage; loss of E‐cadherin and AQP1 expression, were also reduced in the CypD^?/? UUO kidney. This reduced tubular damage was associated with less inflammation and a partial protection against loss of peritubular capillaries. The prominent accumulation of α‐SMA+ myofibroblasts and interstitial collagen deposition seen in WT UUO was significantly reduced in CypD^?/? UUO on day 12, but not day 7. Activation of several pro‐fibrotic signalling pathways (p38 MAPK, JNK and Smad3) was unaltered in CypD^?/? UUO, arguing that CypD acts independently to promote renal fibrosis. CypD deletion in cultured tubular cells attenuated oxidative stress‐induced pro‐inflammatory, pro‐fibrotic and apoptotic responses; however, responses to angiotensin II and LPS were unaffected. In contrast, CypD deletion in cultured renal fibroblasts did not affect PDGF‐induced proliferation or TGF‐β1‐induced collagen I expression, suggesting no direct role of CypD in the fibroblast response. In conclusion, we have identified a role for CypD in chronic tubular cell damage and in the development of renal interstitial fibrosis.     

Salviolone protects against high glucose-induced proliferation,oxidative stress,inflammation, and fibrosis of human renal mesangial cells by upregulating membrane metalloendopeptidase expression

As one of complications of diabetes mellitus, diabetic nephropathy is related to renal dysfunction. Membrane metalloendopeptidase (MME) is associated with the pathogenesis of diabetic nephropathy and exerts a protective function in high glucose (HG)-treated podocytes. Salviolone, one of important bioactive components from Salvia miltiorrhiza, possesses an anti-inflammatory activity. However, the roles of salviolone in renal mesangial cell dysfunction under HG condition remain unknown. The targets of salviolone in diabetic nephropathy were predicted by bioinformatics analysis. Relative mRNA level of MME was detected by qPCR in HG-treated human renal mesangial cells (HRMCs). Cell viability was analyzed using CCK-8 assay. Cell proliferation was investigated by EdU staining. Oxidative stress was evaluated by detection of ROS generation and levels of oxidative stress-related biomarkers. The inflammatory cytokines and fibrosis-related biomarkers were examined by ELISA. Our results showed that MME expression was decreased in diabetic nephropathy and HG-treated HRMCs. Salviolone increased MME level in HG-treated HRMCs. Salviolone mitigated HG-induced HRMC proliferation by increasing MME expression. Salviolone attenuated HG-induced ROS generation, MDA level increase, and SOD activity decrease through upregulating MME expression. Moreover, salviolone suppressed HG-induced increase of levels of TNF-α, IL-1β, IL-6, fibronectin, and collagen IV through upregulating MME expression. In conclusion, s alviolone attenuates proliferation, oxidative stress, inflammation, and fibrosis in HG-treated HRMCs through upregulating MME expression.     

姜黄素对糖尿病大鼠肾脏TGF-β1影响及保护性研究

目的观察糖尿病大鼠肾脏TGF—β1表达及姜黄素胶囊的治疗作用。方法采用STZ注射法制作糖尿病（DM）大鼠模型,随机分为正常组,DM组,姜黄素组。灌胃给药8周后,采用免疫组化等方法测定24h尿白蛋白排泄率（UAER）、肾小球滤过率（GFR）、肾皮质TGF-β1、FN、Col—Ⅳ的表达。结果（1）功能学变化：姜黄素组大鼠的24hUAER和GFR显著低于DM、对照组,差异有统计学意义（P〈0．05）。（2）免疫组化：姜黄素组大鼠肾皮质TGF—β1、FN、Col—Ⅳ的表达显著低于DM组,差异有统计学意义（P〈0．05）。结论姜黄素胶囊有降低早期糖尿病肾病大鼠尿白蛋白排泄率及减少过高的GFR、抑制‘肾组织增生、抑制。肾皮质TGF-β1、FN、Col—Ⅳ表达等保护作用。     

Tyrphostin AG490 reduces NAPDH oxidase activity and expression in the aorta of hypercholesterolemic apolipoprotein E-deficient mice

Oxidative stress-induced vascular injury represents a major contributor to the pathoetiology of atherosclerosis. Elevated NADPH oxidase (Nox) activity promotes oxidative injury of the cardiovascular cells. Janus-tyrosine-kinase (Jak) family regulate various aspects of the atherosclerotic process e.g., inflammation, cellular growth, proliferation, and migration. Here, we investigated the potential of Jak2 inhibition to counteract Nox-dependent O₂^?? formation in atherogenesis in hypercholesterolemic apolipoprotein E-deficient (ApoE^?/?) mice. Male ApoE^?/? mice fed a high-fat, cholesterol-rich diet were treated for 5 weeks with either vehicle or tyrphostin AG490 (1 mg/kg), a specific Jak2 inhibitor. Lucigenin-enhanced-chemiluminescence assay, real-time PCR and Western blot analysis revealed that Nox-derived O₂^?? generation, Nox1, Nox2, and Nox4 mRNA and protein levels were significantly elevated in the aortas of ApoE^?/? mice fed a high-fat diet compared to ApoE^?/? mice fed a normal diet. Treatment with tyrphostin AG490 significantly reduced the up-regulated Nox activity, the expression of each Nox subtype, as well as the protein level of CD68, a macrophage-specific marker. Morphometric analysis showed a marked reduction of atherosclerotic lesions in the aorta of AG490-treated animals. These data provide new insights into the regulation of vascular Nox by tyrphostins in the cardiovascular system. Since Jak2 transduces the signals of various cardiovascular risk factors, pharmacological manipulation of this signaling pathway may represent a novel strategy to reduce oxidative stress in atherosclerosis.     

BMP-7对TGF-β_1诱导人肾小管上皮细胞外基质表达的影响

目的观察骨形态发生蛋白-7(BMP-7)对TGF-β1诱导人近端肾小管上皮细胞(HK-2)细胞外基质(ECM)成分表达的影响,以探讨其延缓肾间质纤维化病变的作用机制。方法将体外培养的HK-2细胞分为空白对照组,3μg.L-1TGF-β1组,BMP-7组,TGF-β1加不同浓度BMP-7组。免疫荧光检测FN的表达;RT-PCR和Western blot分别检测FN、ColⅠα1mRNA及蛋白的表达。结果免疫荧光检测结果显示,3μg.L-1TGF-β1刺激HK-2细胞48h后,FN表达明显增强;加入200μg.L-1BMP-7干预后荧光明显减弱。RT-PCR和Western blot结果显示,TGF-β1刺激HK-2细胞48h后,FN、ColⅠα1mRNA及蛋白表达均明显高于对照组(P<0.01);BMP-7(100～400μg.L-1)可在mRNA及蛋白水平呈剂量依赖性下调TGF-β1诱导的FN、ColⅠα1的表达。结论BMP-7能抑制TGF-β1诱导的HK-2细胞FN、ColⅠα1的合成。表明BMP-7抑制肾小管间质纤维化的进展、改善肾功能的作用,部分是通过抑制肾小管上皮细胞分泌细胞外基质实现的。     

大黄酚对单侧输尿管梗阻模型大鼠肾组织CTGF、α-SMA和FN的影响

目的探讨大黄酚对大鼠单侧输尿管梗阻模型(UUO)肾间质纤维化的影响。方法 27只SD大鼠随机分为假手术组、模型组、大黄酚组。用单侧输尿管梗阻术建立肾间质纤维化模型,大黄酚治疗14 d后检测血肌酐、尿素氮水平;采用免疫组织化学方法观察肾组织结缔组织生长因子(CTGF)、α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)的表达情况。结果与假手术组相比,模型组血肌酐、尿素氮的水平以及CTGF、α-SMA、FN的表达均显著增高。与模型组相比,大黄酚组血肌酐、尿素氮水平降低,CTGF、α-SMA、FN的阳性表达显著下调。结论大黄酚可能通过抑制UUO大鼠肾组织CTGF、α-SMA、FN的表达,从而缓解肾间质纤维化的发生发展。     

First in class, potent, and orally bioavailable NADPH oxidase isoform 4 (Nox4) inhibitors for the treatment of idiopathic pulmonary fibrosis

We describe the design, synthesis, and optimization of first-in-class series of inhibitors of NADPH oxidase isoform 4 (Nox4), an enzyme implicated in several pathologies, in particular idiopathic pulmonary fibrosis, a life-threatening and orphan disease. Initially, several moderately potent pyrazolopyridine dione derivatives were found during a high-throughput screening campaign. SAR investigation around the pyrazolopyridine dione core led to the discovery of several double-digit nanomolar inhibitors in cell free assays of reactive oxygen species (ROS) production, showing high potency on Nox4 and Nox1. The compounds have little affinity for Nox2 isoform and are selective for Nox4/1 isoforms. The specificity of these compounds was confirmed in an extensive in vitro pharmacological profile, as well as in a counterscreening assay for potential ROS scavenging. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo, allowing further clinical trials for the potential treatment of fibrotic diseases, cancers, and cardiovascular and metabolic diseases.     

血竭素高氯酸盐对早期糖尿病肾病肾脏损伤防治作用的研究

目的探讨血竭素高氯酸盐对早期糖尿病肾病的肾脏保护作用。方法采用链脲佐菌素(streptozotocin,STZ)150mg·kg-1单次腹腔注射C57BL/6小鼠,建立小鼠早期糖尿病肾病模型,设正常对照组、糖尿病肾病模型组、胰岛素对照组及血竭素高氯酸盐5、10、20mg·kg-1.d-13种剂量治疗组(血竭素高氯酸盐溶于生理盐水中灌胃)。治疗4wk后检测各组小鼠肾重指数、肾小球细胞外基质(extracellular ma-trix,ECM)、24h尿蛋白含量、肌酐清除率,用RT-PCR方法检测纤连蛋白(fibronectin,FN)mRNA表达,免疫组化方法检测FN蛋白的表达及分布。结果与糖尿病肾病模型组比较,血竭素高氯酸盐治疗组的肌酐清除率及尿蛋白均降低(P<0.05);各治疗组均能减少肾皮质中肾小球ECM的积聚(P<0.05)、FN mRNA和蛋白的表达(P<0.01)。结论血竭素高氯酸盐能保护早期糖尿病肾病的肾损伤。     

Apocynin attenuates oxidative stress and cardiac fibrosis in angiotensin II-induced cardiac diastolic dysfunction in mice

Aim:

To investigate whether apocynin, a NADPH oxidase inhibitor, produced cardioproteictive effects in Ang II-induced hypertensive mice, and to elucidate the underlying mechanisms.

Methods:

C57BL/6 mice were subcutaneously infused Ang II for 4 weeks to mimic cardiac remodeling and fibrosis. Concomitantly the mice were administered apocynin (100 mg·kg⁻¹·d⁻¹) or/and the aldosterone receptor blocker eplerenone (200 mg·kg⁻¹·d⁻¹) via gavage for 4 weeks. Systolic blood pressure (SBP) and heart rate were measured, and transthoracic echocardiography was performed. For in vitro study, cardiac fibroblasts were treated with Ang II (10⁻⁷ mol/L) in the presence of apocynin (10⁻⁵ mol/L) or/and eplerenone (10⁻⁵ mol/L). Immunohistochemistry and Western blotting were used to quantify the expression levels of NADPH oxidase and osteopontin (OPN) proteins in the cells.

Results:

Both apocynin and eplerenone significantly decreased SBP, and markedly improved diastolic dysfunction in Ang II-induced hypertensive mice, accompanied with ameliorated oxidative stress and cardiac fibrosis. In the Ang II-treated cardiac fibroblasts, the expression levels of NOX4 and OPN proteins were markedly upregulated. Both Apocynin and eplerenone significantly suppressed the increased expression levels of NOX4 and OPN proteins in the Ang II-treated cells. In all the experiments, apocynin and eplerenone produced comparable effects. Co-administration of the two agents did not produce synergic effects.

Conclusion:

Apocynin produces cardioproteictive effects comparable to those of eplerenone. The beneficial effects of apocynin on myocardial oxidative stress and cardiac fibrosis might be mediated partly through a pathway involving NADPH oxidase and OPN.