Inhibition of aldehyde dehydrogenase 2 by oxidative stress is associated with cardiac dysfunction in diabetic rats

Left ventricular (LV) dysfunction is a common comorbidity in diabetic patients, although the molecular mechanisms underlying this cardiomyopathic feature are not completely understood. Aldehyde dehydrogenase 2 (ALDH2) has been considered a key cardioprotective enzyme susceptible to oxidative inactivation. We hypothesized that hyperglycemia-induced oxidative stress would influence ALDH2 activity, and ALDH2 inhibition would lead to cardiac functional alterations in diabetic rats. Diabetes was induced by intraperitoneal (i.p.) injection of 60 mg/kg streptozotocin. Rats were divided randomly into four groups: control, untreated diabetic, diabetic treated with N-acetylcysteine (NAC) and diabetic treated with α-lipoic acid (α-LA). Cardiac contractile function, oxidative stress markers and reactive oxygen species (ROS) levels were assessed. ALDH2 activity and expression also were determined. The role of ALDH2 activity in change in hyperglycemia-induced mitochondrial membrane potential (Δψ) was tested in cultured neonatal cardiomyocytes. Myocardial MDA content and ROS were significantly higher in diabetic rats than in controls, whereas GSH content and Mn-SOD activity were decreased in diabetic rats. Compared with controls, diabetic rats exhibited significant reduction in LV ejection fraction and fractional shortening, accompanied by decreases in ALDH2 activity and expression. NAC and α-LA attenuated these changes. Mitochondrial Δψ was decreased greatly with hyperglycemia treatment, and high glucose combined with ALDH2 inhibition with daidzin further decreased Δψ. The ALDH2 activity can be regulated by oxidative stress in the diabetic rat heart. ALDH2 inhibition may be associated with LV reduced contractility, and mitochondrial impairment aggravated by ALDH2 inhibition might reflect an underlying mechanism which causes cardiac dysfunction in diabetic rats.     

Myocardial dysfunction and neurohumoral activation without remodeling in left ventricle of monocrotaline-induced pulmonary hypertensive rats

In monocrotaline (MCT)-induced pulmonary hypertension (PH), only the right ventricle (RV) endures overload, but both ventricles are exposed to enhanced neuroendocrine stimulation. To assess whether in long-standing PH the left ventricular (LV) myocardium molecular/contractile phenotype can be disturbed, we evaluated myocardial function, histology, and gene expression of autocrine/paracrine systems in rats with severe PH 6 wk after subcutaneous injection of 60 mg/kg MCT. The overloaded RV underwent myocardial hypertrophy (P < 0.001) and fibrosis (P = 0.014) as well as increased expression of angiotensin-converting enzyme (ACE) (8-fold; P < 0.001), endothelin-1 (ET-1) (6-fold; P < 0.001), and type B natriuretic peptide (BNP) (15-fold; P < 0.001). Despite the similar upregulation of ET-1 (8-fold; P < 0.001) and overexpression of ACE (4-fold; P < 0.001) without BNP elevation, the nonoverloaded LV myocardium was neither hypertrophic nor fibrotic. LV indexes of contractility (P < 0.001) and relaxation (P = 0.03) were abnormal, however, and LV muscle strips from MCT-treated compared with sham rats presented negative (P = 0.003) force-frequency relationships (FFR). Despite higher ET-1 production, BQ-123 (ET(A) antagonist) did not alter LV MCT-treated muscle strip contractility distinctly (P = 0.005) from the negative inotropic effect exerted on shams. Chronic daily therapy with 250 mg/kg bosentan (dual endothelin receptor antagonist) after MCT injection not only attenuated RV hypertrophy and local neuroendocrine activation but also completely reverted FFR of LV muscle strips to positive values. In conclusion, the LV myocardium is altered in advanced MCT-induced PH, undergoing neuroendocrine activation and contractile dysfunction in the absence of hypertrophy or fibrosis. Neuroendocrine mediators, particularly ET-1, may participate in this functional deterioration.     

Ascorbic acid and N-acetyl cysteine prevent uncoupling of nitric oxide synthase and increase tolerance to ischemia/reperfusion injury in diabetic rat heart

Abstract

Oxidative stress may cause a loss of tetrahydrobiopterin (BH4), a co-factor of nitric oxide synthase (NOS), decrease the bioavailability of NO and aggravate ischemia/reperfusion (I/R) injury in diabetic heart. We hypothesized that ascorbic acid (AA) and N-acetyl cysteine (NAC) protect the diabetic heart from I/R injury by increasing BH4/dihydrobiopterin (BH2) ratio and inhibiting uncoupling of NOS. Diabetes mellitus was induced in rats by streptozotocin treatment, and the hearts were isolated and perfused. BH4 and BH4/BH2 ratio decreased in the diabetic heart associated with increased production of superoxide and nitrotyrosine (NT). Treatment with AA or NAC significantly increased BH4/BH2 ratio in the diabetic heart associated with decreased production of superoxide and NT and increased generation of nitrate plus nitrite (NOx). Pre-treatment with AA or NAC before 30 min ischemia followed by 120 min reperfusion improved left ventricular (LV) function and reduced infarct size in the diabetic but not non-diabetic hearts. The NOS inhibitor, L-NAME, inhibited the increase in the generation of superoxide, NT and NOx, but aggravated LV function and increased infarct size in the diabetic heart. L-NAME also abrogated the increase in NOx and improvement of LV function and the infarct size-limiting effect induced by AA or NAC in the diabetic heart. These results suggest that AA and NAC increase BH4/BH2 ratio and prevent NOS uncoupling in the diabetic heart. Resultant increase in the bioavailability of NO renders the diabetic heart toleratant to I/R injury.     

Chronic kidney disease-induced cardiac fibrosis is ameliorated by reducing circulating levels of a non-dialysable uremic toxin, indoxyl sulfate

Cardiovascular death commonly occurs in patients with chronic kidney disease. Indoxyl sulfate (IS), a uremic toxin, has been demonstrated in vitro as a contributory factor in cardiac fibrosis, a typical pathological finding in uremic cardiomyopathy. This study aimed to determine if cardiac fibrosis is reversible by lowering serum IS levels using an oral charcoal adsorbent, AST-120. Subtotal-nephrectomized (5/6-STNx) Sprague-Dawley rats were randomized to receive either AST-120 (AST-120, n=13) or no treatment (vehicle, n=17) for 12 weeks. Sham operated rats (n=12) were used as controls. Early left ventricular (LV) diastolic dysfunction was demonstrated by an increase in peak velocity of atrial filling [A and A' waves] and a decrease of E/A and E'/A' ratios obtained by echocardiography. This was accompanied by a 4.5-fold increase in serum IS (p<0.001) as well as elevated tail-cuff blood pressure (p<0.001) and heart weight (p<0.001). Increased LV fibrosis (p<0.001), gene expression of pro-fibrotic (TGF-β, CTGF) and hypertrophic (ANP, β-MHC and α-skeletal muscle actin) markers, as well as TGF-β and phosphorylated NF-κB protein expression were observed in STNx + vehicle rats. Treatment with AST-120 reduced serum creatinine (by 54%, p<0.05) and urine total protein (by 27%, p<0.05) vs vehicle whilst having no effect on blood pressure (AST-120=227 ± 11 vs vehicle =224 ± 8 mmHg, ns) and heart weight. The increase in serum IS was prevented with AST-120 (by 100%, p<0.001) which was accompanied by reduced LV fibrosis (68%, p<0.01) and TGF-β and phosphorylated NF-κB protein expression (back to sham levels, p<0.05) despite no significant change in LV function. In conclusion, STNx resulted in increased cardiac fibrosis and circulating IS levels. Reduction of IS with AST-120 normalizes cardiac fibrosis, in a blood pressure independent manner.     

Involvement of endogenous ouabain-like compound in the cardiac hypertrophic process in vivo

The Na(+)/K(+)-ATPase inhibitor ouabain has been shown to trigger hypertrophic growth of cultured cardiomyocytes; however, the significance of endogenous ouabain-like compound (OLC) in the hypertrophic process in vivo is unknown. Here we characterized the involvement of OLC in left ventricular (LV) hypertrophy induced by norepinephrine (NE) and angiotensin II (Ang II) infusions in rats. Administration of NE (300 microg/kg/h) via subcutanously implanted osmotic minipumps for 72 h resulted in a significant increase in left ventricular weight to body weight (LVW/BW) ratio (P<0.001) and a substantial up-regulation of atrial natriuretic peptide (ANP) gene expression (13.2-fold, P<0.001). NE infusion induced a transient increase in plasma OLC levels at 12 h (P<0.05), which returned to control levels by 72 h. Adrenalectomy markedly reduced both basal and NE-induced increase in plasma OLC levels. LVW/BW ratio was not modulated by adrenalectomy; however, ANP gene expression was blunted by 44% (P<0.01) and 47% (P<0.05) at 12 and 72 h, respectively. In agreement, adrenalectomy reduced up-regulation of ANP without affecting LV mass in rats infused with Ang II (33 microg/kg/h). Administration of exogenous ouabain (1 nM to 100 microM) for 24 h had no effect on ANP gene expression in cultured neonatal rat ventricular myocytes. However, the up-regulation of ANP mRNA levels induced by the alpha-adrenergic agonist phenylephrine (1 microM) was markedly enhanced by ouabain (100 microM) (5.6-fold vs. 9.6-fold, P<0.01). These data show that OLC as an adrenal-derived factor may be required for the induction LV ANP gene expression during the hypertrophic process.     

Angiotensin preconditioning of the heart

Angiotensin II (Ang II) has been found to exert preconditioning-like effect on mammalian hearts. Diverse mechanisms are known to exist to explain the cardioprotective abilities of Ang II preconditioning. The present study hypothesized, based on the recent report that Ang II generates reactive oxygen species (ROS) through NADPH oxidase, that Ang II preconditioning occurs through redox cycling. To test this hypothesis, a group of rat hearts was treated with Ang II in the absence or presence of an NADPH oxidase inhibitor, apocynin; or a cell-permeable ROS scavenger, N-acetyl cysteine (NAC). Ang II pretreatment improved postischemic ventricular recovery; reduced myocardial infarction; and decreased the number of cardiomyocyte apoptosis, indicating its ability to precondition the heart against ischemic injury. Both apocynin and NAC almost abolished the preconditioning ability of Ang II. Ang II resulted in increase in ROS activity in the heart, which was reduced by either NAC or apocynin. Ang II also increased both the NADPH oxidase subunits gp91 phox and p22phox mRNA expression, which was abolished with apocynin and NAC. Our results thus demonstrate that the Ang II preconditioning was associated with enhanced ROS activities and increased NADPH oxidase subunits p22phox and gp91phox expression. Both NAC and apocynin reduced ROS activities simultaneously abolishing preconditioning ability of Ang II, suggesting that Ang II preconditioning occurs through redox cycling. That both NAC and apocynin reduced ROS activities and abolished Ang II-mediated increase in p22phox and gp91phox activity further suggest that such redox cycling occurs via both NADPH oxidase-dependent and -independent pathways.     

Ascorbic acid and N-acetyl cysteine prevent uncoupling of nitric oxide synthase and increase tolerance to ischemia/reperfusion injury in diabetic rat heart

Oxidative stress may cause a loss of tetrahydrobiopterin (BH4), a co-factor of nitric oxide synthase (NOS), decrease the bioavailability of NO and aggravate ischemia/reperfusion (I/R) injury in diabetic heart. We hypothesized that ascorbic acid (AA) and N-acetyl cysteine (NAC) protect the diabetic heart from I/R injury by increasing BH4/dihydrobiopterin (BH2) ratio and inhibiting uncoupling of NOS. Diabetes mellitus was induced in rats by streptozotocin treatment, and the hearts were isolated and perfused. BH4 and BH4/BH2 ratio decreased in the diabetic heart associated with increased production of superoxide and nitrotyrosine (NT). Treatment with AA or NAC significantly increased BH4/BH2 ratio in the diabetic heart associated with decreased production of superoxide and NT and increased generation of nitrate plus nitrite (NOx). Pre-treatment with AA or NAC before 30 min ischemia followed by 120 min reperfusion improved left ventricular (LV) function and reduced infarct size in the diabetic but not non-diabetic hearts. The NOS inhibitor, L-NAME, inhibited the increase in the generation of superoxide, NT and NOx, but aggravated LV function and increased infarct size in the diabetic heart. L-NAME also abrogated the increase in NOx and improvement of LV function and the infarct size-limiting effect induced by AA or NAC in the diabetic heart. These results suggest that AA and NAC increase BH4/BH2 ratio and prevent NOS uncoupling in the diabetic heart. Resultant increase in the bioavailability of NO renders the diabetic heart toleratant to I/R injury.     

The protective effect of ApolipoproteinA-I on myocardial ischemia-reperfusion injury in rats

It is well established that reperfusion of heart is the optimal method for salvaging ischemic myocardium, however, the success of this therapy could be limited by reperfusion injury, which is involved in inflammatory responses. High density lipoprotein (HDL) has an anti-inflammatory function and can protect the heart from ischemia-reperfusion (I/R) injury. In this study, we investigated the cardioprotective role of apolipoprotein A-I (ApoA-I), the major apolipoprotein of HDL, in I/R injury. Using rats subjected to myocardial I/R by ligation of left anterior descending coronary artery (LAD), we found that administration of ApoA-I (20 mg/kg, iv) before the onset of reperfusion of myocardial infarction can significantly reduce serum creatine kinase (CK) levels (62.1+/-13.8%, p<0.01) and heart TNF-alpha as well as IL-6 levels, compared with saline controls (40.4+/-14.7%, 44+/-9.8%, p<0.01 respectively). Moreover, ApoA-I treatment suppresses the expression of ICAM-1 on endothelium, thus diminishing neutrophil adherence, transendothelial migration, and the subsequent myocyte injury. We concluded that ApoA-I could effectively protect rat heart from I/R injury.     

Exercise protects against diabetic cardiomyopathy by the inhibition of the endoplasmic reticulum stress pathway in rats

To explore the protective effect of exercise training on the injury of myocardium tissues induced by streptozotocin (STZ) in diabetic rats and the relationship with endoplasmic reticulum stress (ERS), the male sprague-dawley (SD) rats were fed with high-fat and high-sugar diet for 4 weeks, followed by intraperitoneal injection of STZ, 40 mg/kg, to establish a diabetes model, and then 10 rats were randomly selected as diabetes mellitus (DM) controls and 20 eligible diabetic rats were randomized into two groups: low-intensity exercise training (n = 10) and high-intensity exercise training (n = 10). After 12 weeks of exercise training, rats were killed and serum samples were used to determine cardiac troponin-I (cTn-I). Myocardial tissues were sampled for morphological analysis to detect myocardial cell apoptosis, and to analyze protein expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and caspase-12. Different intensities (low and high) significantly reduced serum cTn-I levels compared with the DCM group (p < 0.01), and significantly reduced the percentage of apoptotic myocardial cells and improved the parameters of cardiac function. Hematoxylin and eosin and Masson staining indicated that exercise training could attenuate myocardial apoptosis. Additionally, exercise training significantly reduced GRP78, CHOP, and cleaved caspase-12 protein expression in an intensity-dependent manner. These findings suggest that exercise appeared to ameliorate diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress-induced apoptosis in diabetic rats.     

Left ventricular dysfunction and associated cellular injury in rats exposed to chronic intermittent hypoxia.

Obstructive sleep apnea (OSA) increases cardiovascular morbidity and mortality. We have reported that chronic intermittent hypoxia (CIH), a direct consequence during OSA, leads to left ventricular (LV) remodeling and dysfunction in rats. The present study is to determine LV myocardial cellular injury that is possibly associated with LV global dysfunction. Fifty-six rats were exposed either to CIH (nadir O(2) 4-5%) or sham (handled normoxic controls, HC), 8 h/day for 6 wk. At the end of the exposure, we studied LV global function by cardiac catheterization, and LV myocardial cellular injury by in vitro analyses. Compared with HC, CIH animals demonstrated elevations in mean arterial pressure and LV end-diastolic pressure, but reductions in cardiac output (CIH 141.3 +/- 33.1 vs. HC 184.4 +/- 21.2 ml x min(-1) x kg(-1), P < 0.01), maximal rate of LV pressure rise in systole (+dP/dt), and maximal rate of LV pressure fall in diastole (-dP/dt). CIH led to significant cell injury in the left myocardium, including elevated LV myocyte size, measured by cell surface area (CIH 3,564 +/- 354 vs. HC 2,628 +/- 242 microm(2), P < 0.05) and cell length (CIH 148 +/- 23 vs. HC 115 +/- 16 microm, P < 0.05), elevated terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-stained positive cell number (CIH 98 +/- 45 vs. HC 15 +/- 13, P < 0.01), elevated caspase-3 activity (906 +/- 249 vs. 2,275 +/- 1,169 pmol x min(-1) x mg(-1), P < 0.05), and elevated expression of several remodeling gene markers, including c-fos, atrial natriuretic peptide, beta-myosin heavy chain, and myosin light chain-2. However, there was no difference between groups in sarcomere contractility of isolated LV myocytes, or in LV collagen deposition on trichrome-stained slices. In conclusion, CIH-mediated LV global dysfunction is associated with myocyte hypertrophy and apoptosis at the cellular level.     

Ischemic preconditioning attenuates mitochondrial localization of PTEN induced by ischemia-reperfusion

Although the induction of myocyte apoptosis by ischemia-reperfusion (I/R) is attenuated by ischemic preconditioning (IPC), the underlying mechanism is not fully understood. Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) promotes apoptosis through Akt-dependent and -independent mechanisms. We tested the hypothesis that IPC attenuates the mitochondrial localization of PTEN in the myocardium induced by I/R. Isolated hearts from wild-type mice were exposed to IPC or normal perfusion followed by 30 min of ischemia and reperfusion. IPC attenuated myocardial infarct size and apoptosis after I/R. Heart fractionation showed that mitochondrial PTEN and Bax protein levels and the physical association between them were increased by 30 min of I/R and that IPC attenuated all of these effects of I/R. Muscle-specific PTEN knockout decreased mitochondrial Bax protein levels in the reperfused myocardium and increased cell survival. To determine whether PTEN relocalization to mitochondria was influenced by I/R-induced production of ROS, hearts were perfused with N-acetylcysteine (NAC) to scavenge ROS or H(2)O(2) to mimic I/R-induced ROS. Mitochondrial PTEN protein levels were decreased by NAC and increased by H(2)O(2). PTEN protein overexpression was generated in mouse hearts by adenoviral gene transfer. PTEN overexpression increased mitochondrial PTEN and Bax protein levels and ROS production, whereas muscle-specific PTEN knockout produced the opposite effects. In conclusion, myocardial I/R causes PTEN localization to the mitochondria, related to the generation of ROS; IPC attenuates the mitochondrial localization of PTEN after I/R, potentially inhibiting the translocation of Bax to the mitochondria and resulting in improved cell viability.     

Differential Phenotypes of Tissue-Infiltrating T Cells during Angiotensin II-Induced Hypertension in Mice

Hypertension remains the leading risk factor for cardiovascular disease (CVD). Experimental hypertension is associated with increased T cell infiltration into blood pressure-controlling organs, such as the aorta and kidney; importantly in absence of T cells of the adaptive immune system, experimental hypertension is significantly blunted. However, the function and phenotype of these T cell infiltrates remains speculative and undefined in the setting of hypertension. The current study compared T cell-derived cytokine and reactive oxygen species (ROS) production from normotensive and hypertensive mice. Splenic, blood, aortic, kidney and brain T cells were isolated from C57BL/6J mice following 14-day vehicle or angiotensin (Ang) II (0.7 mg/kg/day, s.c.) infusion. T cell infiltration was increased in aorta, kidney and brain from hypertensive mice. Cytokine analysis in stimulated T cells indicated an overall Th1 pro-inflammatory phenotype, but a similar proportion (flow cytometry) and quantity (cytometric bead array) of IFN-γ, TNF-α, IL-4 and IL-17 between vehicle- and Ang II- treated groups. Strikingly, elevated T cell-derived production of a chemokine, chemokine C-C motif ligand 2 (CCL2), was observed in aorta (∼6-fold) and kidney in response to Ang II, but not in brain, spleen or blood. Moreover, T cell-derived ROS production in aorta was elevated ∼3 -fold in Ang II-treated mice (n = 7; P<0.05). Ang II-induced hypertension does not affect the overall T cell cytokine profile, but enhanced T cell-derived ROS production and/or leukocyte recruitment due to elevated CCL2, and this effect may be further amplified with increased infiltration of T cells. We have identified a potential hypertension-specific T cell phenotype that may represent a functional contribution of T cells to the development of hypertension, and likely several other associated vascular disorders.     

Alarin alleviated cardiac fibrosis via attenuating oxidative stress in heart failure rats

The present study was to explore whether alarin could alleviate heart failure (HF) and attenuate cardia fibrosis via inhibiting oxidative stress. The fibrosis of cardiac fibroblasts (CFs) was induced by angiotensin (Ang) II. HF models were induced by ligation of the left anterior descending artery to cause ischemia myocardial infarction (MI) in Sprague–Dawley rats. Alarin (1.0 nM/kg/d) was administrated by intraperitoneal injection for 28 days. The decreases of left ventricular (LV) ejection fraction (EF), fractional shortening (FS), the maximum of the first differentiation of LV pressure (LV ± dp/dt_max) and LV systolic pressure (LVSP), and the increases of LV volume in systole (LVVS), LV volume in diastole (LVVD), LV end-systolic diameter (LVESD) and LV end-diastolic diameter (LVEDD) in MI rats were improved by alarin treatment. The increases in the expression levels of collagen I, collagen III, and transforming growth factor (TGF)-β were inhibited by alarin treatment in CFs and in the hearts of MI rats. The levels of NADPH oxidase (Nox) activity, superoxide anions and malondialdehyde (MDA) levels were increased, and the level of superoxide dismutase (SOD) activity was reduced in Ang II-treated CFs, which were reversed by alarin. Nox1 overexpression reversed the effects of alarin on attenuating the increases of collagen I, collagen III and TGF-β expression levels induced by Ang II in CFs. These results indicated that alarin improved HF and cardiac fibrosis via inhibiting oxidative stress in HF rats. Nox1 played important roles in the regulation of alarin effects on attenuating CFs fibrosis induced by Ang II.

     

Alterations in vascular endothelial function in the aorta and mesenteric artery in type II diabetic rats

We used the partial protection exerted by suitable dosages of nicotinamide against the beta-cytotoxic effect of streptozotocin (STZ) to create an experimental diabetic syndrome in adult rats that appears closer to type II diabetes mellitus than other available animal models. The dosage of 230 mg/kg of nicotinamide given intraperitoneally 15 min before STZ administration (65 mg/kg i.v.) yielded animals with hyperglycemia (187.8 +/- 17.8 vs. 103.8 +/- 2.8 mg/dL in controls; P < 0.001) and preservation of plasma insulin levels. This study assessed the relationship between endothelial dysfunction and agonist-induced contractile responses in such rats. In the thoracic aorta, the acetylcholine (ACh) induced relaxation was significantly reduced and the noradrenaline (NA) induced contractile response was significantly increased in diabetic rats compared with age-matched control rats. In the superior mesenteric artery, the ACh-induced relaxation was similar in magnitude between diabetic and age-matched control rats; however, the ACh-induced endothelium-derived hyperpolarizing factor (EDHF) type relaxation was significantly weaker in diabetic rats than in the controls. The phenylephrine (PE) induced contractile response was not different between the two groups. The plasma concentration of NOx (NO2- + NO3-) was significantly lower in diabetic rats than in control rats. We conclude that vasomotor activities in conduit arteries are impaired in this type II diabetes model.     

阿托伐他汀通过RGS6改善糖尿病心肌病大鼠心功能的作用及其机制研究

目的:探讨阿托伐他汀通过调节RGS6/NAD(P)H氧化酶/活性氧生成通路保护糖尿病心肌病大鼠心功能的药理作用机制。方法:40只6周龄雄性Wistar大鼠按随机数字表法随机分为对照组,糖尿病心肌病模型组,低剂量阿托伐他汀组,高剂量阿托伐他汀组,每组10只。实验过程中动态监测大鼠体质量及血脂水平;实验结束后脉冲多普勒检测各组大鼠心功能指标;组织活性氧检测试剂盒检测心肌组织中活性氧的水平;免疫组化法检测大鼠心肌组织中RGS6的表达;Western blot法检测大鼠心肌组织中RGS6及NAD (P)H氧化酶活性亚单位p47phox和p67phox的水平。结果:与对照组相比,糖尿病心肌病模型大鼠体质量明显减少(P0.01),血脂水平明显升高(P0.01),心脏E/A、LVEF、FS值降低(P0.01),心肌组织活性氧生成明显增多(P0.01),心肌组织RGS6及p47phox、p67phox表达明显上调(P0.01),而不同剂量阿托伐他汀干预均可有效逆转上述指标的改变。结论:阿托伐他汀对糖尿病心肌病大鼠的心脏具有明显保护作用,其机制可能与对RGS6/NAD(P)H氧化酶/活性氧生成通路的抑制有关。     

Dynamics of progestogenic activity in serum following administration of Ligusticum chuanxiong

Many women are using botanical alternatives for menopausal hormone replacement therapy (HRT) because current progestins, compounds with progesterone activity, have adverse risk profiles. However the development of phyto-progestins for HRT is hampered by the absence of basic pharmacokinetic/pharmacodynamic (PK/PD) data due to the lack of methods to capture summated effects of the numerous compounds that contribute to bioactivity in vivo. In this study, we explored the utility of progesterone receptor (PR)-driven bioassays to track changes in serum progestogenic activity following administration of traditional Chinese medicinal herb, Ligusticum chuanxiong, with potent progestogenic activity. Sensitive and specific (>300-fold) increases in progestogenic activity were observed when HeLa cells transfected with PR and a PR-driven promoter were exposed to the progestogenic drug, medroxy-progesterone acetate (MPA), suggesting the utility of the bioassay to measure progestogenic effects for PK/PD studies. Progestogens were administered to male Sprague-Dawley rats and serum extracted for measurement of progestogenic activity. Effect-time studies indicate that injection of MPA and L. chuanxiong extract raised area-under-curve of progestogenic activity in sera by 8.2-fold (p<0.001) and 4.5-fold (p<0.01) respectively, compared to sera from rats administered vehicle only. Administration of MPA and L. chuanxiong extract by the oral route resulted in a 5.4 (p<0.001) and 2.3-fold (p=0.07) increase respectively. Our data suggest that PR-responsive reporter gene bioassays can measure bioavailability of compounds, known and unknown, of complex botanicals for hormone replacement therapy. L. chuanxiong extracts exert progestogenic activity in vivo, and may have utility for progesterone-replacement therapy.     

NAD(P)H oxidase inhibitor prevents blood pressure elevation and cardiovascular hypertrophy in aldosterone-infused rats

Increased bioavailability of reactive oxygen species (ROS) has been implicated in the pathogenesis of mineralocorticoid hypertension. To find out the source of ROS, we evaluated the role of NAD(P)H oxidase in blood pressure (BP) elevation, cardiovascular hypertrophy, and fibrosis in aldosterone-salt rats. Aldosterone infusion (0.75 microg/h) significantly increased BP, which is attenuated by apocynin (1.5 mmol/L). Cardiac hypertrophy developed by aldosterone infusion was also normalized with apocynin. Greater mRNA for p22phox and NAD(P)H oxidase activity (more than twofold) in aorta of aldosterone-infused rats was reduced in apocynin-treated rats. Aldosterone infusion increased marginally procollagen I and III expression in LV compared to controls and apocynin decreased procollagen. Masson's Trichrome stain showed increased cardiac perivascular fibrosis, which was reduced by apocynin. These results suggest that NAD(P)H oxidase plays an important role in cardiovascular damage associated with mineralocorticoid hypertension.     

Extracellular superoxide dismutase protects the heart against oxidative stress and hypertrophy after myocardial infarction

Extracellular superoxide dismutase (EC-SOD) contributes only a small fraction to total SOD activity in the heart but is strategically located to scavenge free radicals in the extracellular compartment. EC-SOD expression is decreased in myocardial-infarction (MI)-induced heart failure, but whether EC-SOD can abrogate oxidative stress or modify MI-induced ventricular remodeling has not been previously studied. Consequently, the effects of EC-SOD gene deficiency (EC-SOD KO) on left ventricular (LV) oxidative stress, hypertrophy, and fibrosis were studied in EC-SOD KO and wild-type mice under control conditions, and at 4 and 8 weeks after permanent coronary artery ligation. EC-SOD KO had no detectable effect on LV function in normal hearts but caused small but significant increases of LV fibrosis. At 8 weeks after MI, EC-SOD KO mice developed significantly more LV hypertrophy (LV mass increased 1.64-fold in KO mice compared to 1.35-fold in wild-type mice; p<0.01) and more fibrosis and myocyte hypertrophy which was more prominent in the peri-infarct region than in the remote myocardium. EC-SOD KO mice had greater increases of nitrotyrosine in the peri-infarct myocardium, and this was associated with a greater reduction of LV ejection fraction, a greater decrease of sarcoplasmic or endoplasmic reticulum calcium2+ ATPase, and a greater increase of atrial natriuretic peptide in the peri-infarct zone compared to wild-type mice. EC-SOD KO was associated with more increases of phosphorylated p38 (p-p38(Thr180/Tyr182)), p42/44 extracellular signal-regulated kinase (p-Erk(Thr202/Tyr204)), and c-Jun N-terminal kinase (p-JNK(Thr183/Tyr185)) both under control conditions and after MI, indicating that EC-SOD KO increases activation of mitogen-activated protein kinase signaling pathways. These findings demonstrate that EC-SOD plays an important role in protecting the heart against oxidative stress and infarction-induced ventricular hypertrophy.     

Evaluation of glycated insulin in diabetic animals using immunocytochemistry and radioimmunoassay

Glycated insulin was evaluated in plasma and biological tissues of diabetic animal models by immunocytochemistry (ICC) and a novel radioimmunoassay. Glycated insulin circulated at 0.10 +/- 0.04 ng/ml and 2.20 +/- 0.14 ng/ml in lean and diabetic obese (ob/ob) mice, corresponding to 12.5 and 9.8% total plasma insulin, respectively. The concentration of glycated insulin was elevated 22-fold in obese mice compared to controls (P < 0.001). In the pancreas, glycated insulin was 48 +/- 10 and 83 +/- 4 ng/g wt (P < 0.05) in lean and obese mice, respectively, representing approximately 2% total insulin in the diabetic pancreas (4.60 +/- 0.17 microg/g wt). ICC revealed fluorescent positively stained cells in pancreatic islets from hydrocortisone (HC)-treated diabetic rats. Fasting of HC-treated rats, resulted in 3-fold and 15-fold reductions in plasma glycated insulin (P < 0.01) and insulin (P < 0.001), respectively. Following a 30 min feeding period in these insulin resistant rats, plasma glucose, insulin, and glycated insulin increased (P < 0.001) rapidly with 1.4-, 1.6-, and 2.9-fold elevations, respectively. Injection of HC-treated rats with insulin (50 U/kg) resulted in a rapid 33% decrease of plasma glucose (P < 0.001) and a marked 4-fold increase in plasma insulin (P < 0.01), whereas glycated insulin concentrations remained unchanged. Since glycation of insulin impairs biological activity, physiologically regulated secretion of glycated insulin into the circulation in diabetic animal models suggests a role in the pathogenesis of diabetes.     

PPARδ coordinates angiotensin II-induced senescence in vascular smooth muscle cells through PTEN-mediated inhibition of superoxide generation

Cellular senescence-associated changes in blood vessels have been implicated in aging and age-related cardiovascular disorders. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR) δ coordinates angiotensin (Ang) II-induced senescence of human vascular smooth muscle cells (VSMCs). Activation of PPARδ by GW501516, a specific ligand for PPARδ, significantly attenuated Ang II-induced generation of superoxides and suppressed senescence of VSMCs. A marked increase in the levels of p53 and p21 induced by Ang II was blunted by the treatment with GW501516. Ligand-activated PPARδ up-regulated expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and suppressed the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Knockdown of PTEN with siRNA abrogated the effects of PPARδ on cellular senescence, on PI3K/Akt signaling, and on generation of ROS in VSMCs treated with Ang II. Finally, administration of GW501516 to apoE-deficient mice treated with Ang II significantly reduced the number of senescent cells in the aorta, where up-regulation of PTEN with reduced levels of phosphorylated Akt and ROS was demonstrated. Thus, ligand-activated PPARδ confers resistance to Ang II-induced senescence by up-regulation of PTEN and ensuing modulation of the PI3K/Akt signaling to reduce ROS generation in vascular cells.