Upregulation Of eNOS Expression Research Articles

Beneficial effects of Hawthorn extract on metabolic hypertensive rats by regulating Treg/Th17 immune balance

There is an increasing global trend toward unhealthy lifestyles and dietary decisions, like the overconsumption of alcohol and high-sugar and high-fat diets (ACHSFDs). Poor eating habits can cause an imbalance of the Treg/Th17 immune axis, thereby leading to an imbalance of anti-inflammatory and pro-inflammatory factors, which damages target organs and the vascular endothelium causing MH. Hawthorn has been shown to improve gastrointestinal function, lower blood pressure, and regulate metabolic abnormalities. However, whether it acts on MH by regulating the Treg/Th17 immune axis remains unclear. In this study, the hawthorn extract reduced blood pressure[systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MBP)] and improved dyslipidemia in rats with ACHSFD-induced MH. In addition, the extract reduced serum AST and ALT levels and liver coefficient and improved liver fat deposition and fibrosis. On the other hand, the hawthorn extract downregulated RORγt expression, upregulated Foxp3 expression, increased IL-10 levels in the aorta, and reduced IL-17, IL-6, and TNF-α content. Moreover, the extract upregulated eNOS expression in the aorta and increased NO content in the serum. These findings indicate that hawthorn extract can inhibit aortic inflammatory injury and improve vascular endothelial function by regulating the Treg/Th17 immune imbalance.

Effect of electroacupuncture on haemodynamic changes during intubation for general anaesthesia is mediated by nitric oxide synthase‑3 via the regulation of microRNA‑155, microRNA‑335 and microRNA‑383.

Intubation for general anaesthesia is a life‑threatening risk because it can cause haemodynamic changes. Electroacupuncture (EA) has been reported to alleviate the risk of intubation. In the present study, haemodynamic changes were measured at different time points before and after EA. Reverse transcription‑quantitative PCR was performed to measure the expression of micro (mi)RNAs and endothelial NO synthase (eNOS) mRNA. Western blotting was performed to evaluate the expression of eNOS protein. A luciferase assay was used to explore the inhibitory role of miRNAs in eNOS expression. The transfection of miRNA precursors and antagomirs was performed to assess their effect on eNOS expression. The systolic blood pressure, diastolic blood pressure and mean arterial pressure of patients were significantly decreased by EA, while the heart rate of patients was markedly increased. The expression of micro RNA (miR)‑155, miR‑335 and miR‑383 was effectively inhibited by EA in the plasma and peripheral blood monocytes of patients, whereas eNOS expression and NOS production were markedly elevated by EA. The luciferase activity of the eNOS vector was significantly inhibited by miR‑155, miR‑335 and miR‑383 mimics but activated by miR‑155, miR‑335 and miR‑383 antagomirs. miR‑155, miR‑335 and miR‑383 precursors suppressed the expression of eNOS, while miR‑155, miR‑335 and miR‑383 antagomirs enhanced the expression of eNOS. The present study demonstrated that EA may exert a vasodilative effect during intubation for general anaesthesia by promoting NO production and upregulating eNOS expression. The effect of EA on upregulating eNOS expression may be mediated by its inhibitory effect on the expression of miRNA‑155, miRNA‑335 and miRNA‑383.

Metformin Prevents Endothelial Dysfunction in Endometriosis through Downregulation of ET-1 and Upregulation of eNOS.

This study aimed to evaluate if the treatment with metformin affects the morphologic structure, endothelial function, angiogenesis, inflammation and oxidation-responsive pathways in the heart of mice with surgically induced endometriosis. B6CBA/F1 mice (n = 37) were divided into four groups; Sham (S), Metformin (M), Endometriosis (E) and Metformin/Endometriosis (ME). The cross-sectional area of cardiomyocytes was assessed after Hematoxylin-Eosin staining and fibrosis after Picrosirius-Red staining. ET-1, nitric oxide synthases-iNOS and eNOS, and VEGF and VEGFR-2 were detected by immunofluorescence. Semi-quantification of ET-1, eNOS, VEGF, NF-kB, Ikβα and KEAP-1 was performed by Western blotting. MIR199a, MIR16-1, MIR18a, MIR20a, MIR155, MIR200a, MIR342, MIR24-1 and MIR320a were quantified by Real-Time qPCR. The interaction of endometriosis and metformin effects was assessed by a two-way ANOVA test. Compared with the other groups, M-treated mice presented a higher cross-sectional area of cardiomyocytes. Heart fibrosis increased with endometriosis. Treatment of endometriosis with metformin in the ME group downregulates ET-1 and upregulates eNOS expression comparatively with the E group. However, metformin failed to mitigate NF-kB expression significantly incremented by endometriosis. The expression of MIR199a, MIR16-1 and MIR18a decreased with endometriosis, whereas MIR20a showed an equivalent trend, altogether reducing cardioprotection. In summary, metformin diminished endometriosis-associated endothelial dysfunction but did not mitigate the increase in NF-kB expression and cardiac fibrosis in mice with endometriosis.

Renoprotection and Its Mechanism of Fasudil in Nitric Oxide Synthase-Deficient Hypertensive Rats

Abstract Background To investigate whether fasudil can improve renal microcirculation and protect the kidney in NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive rats by inhibiting Rho/Rho associated coiled-coil containing protein kinase (ROCK) signal pathway. Methods The nitric oxide-deficient hypertension was induced by l-NAME (60 mg/l·d) which was provided in drinking water for 8 weeks in Wistar rats. Then they were randomly divided into l-NAME and l-NAME + fasudil groups. Blood, urine, and left kidney samples were collected after fasudil intervention for 6 weeks. Renal artery blood flow, renal function markers in serum and urine, morphological changes of renal interlobular artery, and expression levels of related proteins were detected. Results Compared with normal control rats, the levels of serum creatinine (SCr) and blood urea nitrogen (BUN) increased while creatinine clearance (CCr) decreased in l-NAME rats. Fasudil treatment significantly decreased SCr and BUN, and increased CCr compared with l-NAME group. Compared with normal controls, the intima of renal interlobular artery increased, the lumen narrowed, the expression of ROCK-1, phosphorylated myosin phosphatase 1/myosin phosphatase 1 (p-MYPT1/MYPT1) increased while endothelial nitric oxide synthase (eNOS) protein levels decreased in l-NAME group (all P < 0.05). Fasudil treatment reduced intimal hyperplasia and lumen stenosis of renal interlobular artery, downregulated ROCK-1 and p-MYPT protein levels and upregulated eNOS expression in l-NAME rats (all P < 0.05). Conclusions Fasudil treatment improves renal function and structure of renal interlobar artery in nitric oxide-deficient hypertensive rats induced by l-NAME.

Exercise‐Induced Shear Stress Upregulates eNOS Protein in vitro

BackgroundEndothelial cell dysfunction has been shown to be the first step in the pathogenesis of cardiovascular disease. A risk factor in the development of cardiovascular disease is physical inactivity. Endothelial shear stress (ESS) involves mechanoreceptors on the surface of endothelial cells that are capable of sensing different forces, triggering a biochemical response leading to cellular adaptations. Exercise‐induced ESS might act as a protective mechanism in preventing endothelial cell dysfunction. Endothelial nitric oxide synthase (eNOS), a key enzyme involved maintaining vascular homeostasis has been shown to be upregulated in response to higher shear stress levels. Although exercise has been shown to improve endothelial cell function, the role of varying exercise intensities on endothelial cell homeostasis has not been explored. The purpose of this study was to determine the effects of in vitro exercise‐induced ESS during 3 different exercise intensities on eNOS expression.MethodsCommercially available human umbilical vein endothelial cells (HUVEC; Sigma‐Aldrich, St Louis, MO) were cultured until 90‐100% confluence. Exercise‐induced ESS in vivo was obtained from our previous reports. Cultured cells were exposed to pulsatile resting ESS (18 dynes/cm2) for 2 hours, followed by 1 hour at resting ESS, low‐intensity exercise‐induced ESS (35 dynes/cm2), moderate‐intensity exercise‐induced ESS (50 dynes/cm2), or high‐intensity exercise‐induced ESS (75 dynes/cm2) on a closed circuit pump and channeled slide (Ibidi pump system (PumpControl Software 1.5.4) and slides, Ibidi Inc., Fitchburg, WI). A set of cells were exposed to physiologically low shear stress levels (10 dynes/cm2) for 3 hours as a control. Following ESS conditions, total protein was extracted, and western immunoblotting was performed to measure eNOS protein expression with GAPDH used as the normalizing protein. Statistical analysis was performed using graphpad prism 8 software (Graphpad Software.). Significance was considered at p<0.05.ResultsLow and moderate intensity ESS increased eNOS protein expression compared to physiologically low conditions (P<0.05). There was a trend showing high intensity ESS increasing eNOS expression compared to physiologically low conditions (p<0.10). Additionally, moderate intensity ESS showed a significant increase in eNOS compared to resting conditions (P<0.05).ConclusionsOur data shows that exercise‐induced ESS upregulates eNOS expression. Moderate intensity exercise was shown to produce the highest levels of eNOS expression. Our results from previous studies also show that moderate intensity exercise is capable of activating eNOS, which may act as a protective mechanism to prevent atherosclerosis development.

The Mechanism Actions of Astragaloside IV Prevents the Progression of Hypertensive Heart Disease Based on Network Pharmacology and Experimental Pharmacology.

Astragaloside IV (AS-IV) has been used to treat cardiovascular disease. However, whether AS-IV exerts a protective effect against hypertensive heart disease has not been investigated. This study aimed to investigate the antihypertensive and cardioprotective effects of AS-IV on L-NAME-induced hypertensive rats via network pharmacology and experimental pharmacology. The network pharmacology and bioinformatics analyses were performed to obtain the potential targets of AS-IV and hypertensive heart disease. The rat hypertension model was established by administrated 50 mg/kg/day of L-NAME for 5 weeks. Meanwhile, hypertension rats were intragastrically administrated with vehicle or AS-IV or fosinopril for 5 weeks. Cardiovascular parameters (systolic blood pressure, diastolic blood pressure, mean arterial pressure, heart rates, and body weight), cardiac function parameters (LVEDd, LVEDs, and fractional shortening), cardiac marker enzymes (creatine kinase, CK-MB, and lactate dehydrogenase), cardiac hypertrophy markers (atrial natriuretic peptide and brain natriuretic peptide), endothelial function biomarkers (nitric oxide and eNOS), inflammation biomarkers (IL-6 and TNF-α) and oxidative stress biomarkers (SOD, MDA, and GSH) were measured and cardiac tissue histology performed. Network pharmacological analysis screened the top 20 key genes in the treatment of hypertensive heart disease treated with AS-IV. Besides, AS-IV exerted a beneficial effect on cardiovascular and cardiac function parameters. Moreover, AS-IV alleviated cardiac hypertrophy via down-regulating the expression of ANP and BNP and improved histopathology changes of cardiac tissue. AS-IV improved endothelial function via the up-regulation of eNOS expression, alleviated oxidative stress via increasing antioxidant enzymes activities, and inhibited cardiac inflammation via down-regulating IL-6 and TNF-α expression. Our findings suggested that AS-IV is a potential therapeutic drug to improve L-NAME-induced hypertensive heart disease partly mediated via modulation of eNOS and oxidative stress.

440-P: Dual Long-Term Effects of Free Fatty Acid on Human Endothelial Barrier In Vitro Are Mediated by NO, Mitochondrial ROS, and Dicarbonyl Stress

In diabetes-associated hyperlipidemia, free fatty acids (FFAs) are thought to induce insulin resistance and endothelial dysfunction leading to vascular injury and cardiovascular T2DM complications. However, evidence is scarce regarding the effects of FFA on vascular endothelial barrier. We used human umbilical vein endothelial cells (HUVEC) to model, in vitro, the endothelial barrier and reveal its responses to experimental hyperlipidemia induced by long-term treatment with palmitic acid in complex with albumin. Palmitate was continuously present in the growth medium at 0.8 mM to reproduce FFA levels typical for T2DM. We developed conditions to maintain the HUVEC monolayers in the presence of the high palmitate concentration for up to 2 weeks by adding exogenous carnitine to facilitate mitochondrial β-oxidation of FFA. In this model system, palmitate readily augmented the transendothelial electric resistance (TER) of HUVEC via increased NO production by endothelial NO-synthase (eNOS). Western blots showed that palmitate neither disturbed insulin signaling cascade towards eNOS nor upregulated eNOS expression. After the initial 3-4-day rise in TER, further treatment with palmitate led to barrier stabilization and attenuation as compared to FFA-free conditions. This later phase was associated with increased ROS and malondialdehyde (MDA) accumulation, both prevented by the mitochondria-targeted antioxidant SkQ1. Treatment of HUVEC with exogenous MDA impaired the TER and barrier suggesting that accumulation of MDA-modified proteins may contribute to the barrier dysfunction upon long-term experimental hyperlipidemia. Altogether, obtained evidence suggests that prolonged exposure of vascular endothelium to high levels of palmitate compromises endothelium barrier capacity and insulin-mediated eNOS regulation turning the enzyme permanently active. Disclosure M. Samsonov: None. V. P. Shirinsky: None. N. Podkuychenko: None. A. Y. Khapchaev: None. T. Vlasik: None. V. Lankin: None. M. Skulachev: Other Relationship; Self; Mitotech LLC. I. Stafeev: None. A. V. Vorotnikov: None. M. V. Shestakova: None. Funding Russian Science Foundation (19-15-00361)

Protective effects of rice bran hydrolysates on heart rate variability, cardiac oxidative stress, and cardiac remodeling in high fat and high fructose diet-fed rats

Objective: To examine the ameliorative effect of rice bran hydrolysates (RBH) on metabolic disorders, cardiac oxidative stress, heart rate variability (HRV), and cardiac structural changes in high fat and high fructose (HFHF)-fed rats. Methods: Male Sprague-Dawley rats were daily fed either standard chow diet with tap water or an HFHF diet with 10% fructose in drinking water over 16 weeks. RBH (500 and 1 000 mg/kg/day) was orally administered to the HFHF-diet-fed rats during the last 6 weeks of the study period. At the end of the treatment, metabolic parameters, oxidative stress, HRV, and cardiac structural changes were examined. Results: RBH administration significantly ameliorated metabolic disorders by improving lipid profiles, insulin sensitivity, and hemodynamic parameters. Moreover, RBH restored HRV, as evidenced by decreasing the ratio of low-frequency to high-frequency power of HRV, a marker of autonomic imbalance. Cardiac oxidative stress was also mitigated after RBH supplementation by decreasing cardiac malondialdehyde and protein carbonyl, upregulating eNOS expression, and increasing catalase activity in the heart. Furthermore, RBH mitigated cardiac structural changes by reducing cardiac hypertrophy and myocardial fibrosis in HFHF-diet-fed rats. Conclusions: The present findings suggest that consumption of RBH may exert cardioprotective effects against autonomic imbalances, cardiac oxidative stress, and structural changes in metabolic syndrome.

Human Tissue Kallikrein 1 Is Downregulated in Elderly Human Prostates and Possesses Potential In Vitro Antioxidative and Antifibrotic Effects in Rodent Prostates.

Objective The aim of the present study was to investigate the protective effects and mechanisms of KLK1 on aging-related prostate alterations and search clues about the application of KLK1 to the treatment of human BPH. Methods Thirty-six rats including 26 male wild-type SD rats and 10 transgenic rats were fed to 3- or 18-month-old and divided into three groups: young WTR (yWTR) as the control (n = 16), aged WTR (aWTR) (n = 10), and aged TGR (aTGR) (n = 10). The prostates of the three groups of rats (10 rats per group) were harvested to evaluate the levels of KLK1 expression, oxidative stress, fibrosis, and involved signaling pathways, such as NO/cGMP, COX-2/PTGIS/cAMP, and TGF-β1/RhoA/ROCK1, via quantitative PCR, Western blot, histological examinations, and ELISA. Moreover, the remaining 6 yWTRs were sacrificed to obtain primary prostate fibroblast and aortic endothelial cells, and a coculture system was built with the cells for the verification of above signaling pathways in vitro. And the direct effects of bradykinin on prostate cells were detected by MTT experiment. Prostate specimens of 47 patients (age from 48 to 92 years) undergoing BPH surgery were collected after approval. Histological examinations and KLK1 IHC were preformed to analyze the relationship between KLK1 expression and age and prostate fibrosis. Results The human KLK1 gene only existed and was expressed in aTGR. The prostate of young rats expressed more KLK1 than the aged and the expression of KLK1 in prostate decreased with age in humans (r = −0.347, P = 0.018). Compared to the aWTR group, the yWTR and aTGR groups showed milder fibrosis, less oxidative stress, upregulated NO/cGMP, and COX-2/PTGIS/cAMP signaling pathways and inhibited TGF-β1/RhoA/ROCK1 signaling pathway. In the coculture system, KLK1 suppressed TGF-β1-mediated fibroblast-to-myofibroblast transdifferentiation via cleaving LMWK to produce the BK which upregulate eNOS expression and NO production in endothelial cells. BK not only slightly stimulated the proliferation ability of prostatic stromal cells but also upregulated iNOS and inhibited TGF-β1 expression in them. Conclusion KLK1 protects prostate from oxidative stress and fibrosis via amplified NO/cGMP signal in aged rats. The decrease of KLK1 expression with aging is laying the groundwork for the application of KLK1 to the treatment of human BPH. The current experimental data showed that the side effects of KLK1 on the prostate cell were not obvious.

An Enriched Environment Enhances Angiogenesis Surrounding the Cingulum in Ischaemic Stroke Rats

An enriched environment (EE) has been demonstrated to improve functional recovery in animal models of ischaemic stroke through enhancing vascular endothelial growth factor- (VEGF-) mediated neuroprotection accompanied by angiogenesis in the ischaemic hemisphere. Whether EEs also promote VEGF-mediated neuroprotection and angiogenesis in the contralateral hemisphere remains unclear. Here, we explored the effect of EEs on VEGF expression and angiogenesis within the contralateral cerebral cortex in a rat middle cerebral artery occlusion/reperfusion (MCAO/r) model. We assessed the expression levels of platelet endothelial cell adhesion molecule-1 (CD31), VEGF, and endothelial nitric oxide synthase (eNOS) in the whole contralateral cerebral cortex using Western blotting assay but did not find an increase in the expression of CD31, VEGF, or eNOS in MCAO/r rats housed in EEs, which suggested that EEs did not enhance the overall expression of VEGF and eNOS or angiogenesis in the entire contralateral cortex. We further analysed the local effect of EEs by immunohistochemistry and found that in and around the bilateral cingulum in MCAO/r rats housed in EEs, haematopoietic progenitor cell antigen- (CD34-) positive endothelial progenitor cells were significantly increased compared with those of rats housed in standard cages (SCs). Further experiments showed that EEs increased neuronal VEGF expression surrounding the cingulum in MCAO/r rats and robustly upregulated eNOS expression. These results revealed that EEs enhanced angiogenesis, VEGF expression, and activation of the VEGF-eNOS pathway in and/or around the cingulum in MCAO/r rats, which were involved in the functional recovery of MCAO/r rats.

Empagliflozin treatment does not affect the hypertensive response to Ang II administration to rats but decreases oxidative stress in the arterial wall, and endothelial and cardiac dysfunction

Abstract Background Selective sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown cardiovascular protection in type 2 diabetes patients with established cardiovascular disease independently of glycemic control. Angiotensin II (Ang II) and H2O2 have been shown to be strong inducers of the expression of SGLT2 and 1 in endothelial cells promoting oxidative stress and endothelial dysfunction. Purpose This study examined the cardiovascular protective effect of empagliflozin (empa) in a normoglycemic experimental model of hypertension in the rat. Methods Male Wistar rats received empa (30 mg/kg/day) provided in the diet for 5 weeks. After 1 week, rats underwent sham surgery (sham rats) or surgery with implantation of an osmotic mini-pump infusing Ang II (0.4 mg/kg/d) for 4 weeks. Systolic blood pressure (SBP) was assessed by sphygmomanometry, the cardiac function using echocardiography, the expression level of target proteins by immunofluorescence staining, and the level of oxidative stress using dihydroethidium staining. Results Angiotensin II administration increased systolic blood pressure from about 130 to 180 mmHg, which was not affected by the empa treatment. The 4-week Ang II treatment did not significantly affect the systolic cardiac function (cardiac output, left ventricle ejection fraction) but impaired the diastolic function as indicated by a reduced E' and IVRT values, and an increased E/E' value. The Ang II treatment increased significantly the heart and right ventricle weight whereas the left ventricle + septum weight was slightly but not significantly increased. No such functional and structural changes were observed in the Ang II + empa treatment group. An increased immunofluorescence eNOS signal in the endothelium, and a higher level of ROS throughout the aorta wall were observed in the Ang II-treated group, both of which were significantly reduced in the empa + Ang II-treated group. In the Ang II-treated group, the high level of oxidative stress in the aorta was significantly reduced by the AT1 receptor antagonist losartan, the NADPH oxidase inhibitor VAS-2871, the eNOS inhibitor NG-nitro-L-arginine and also to a greater extent by the selective SGLT2 inhibitor empa compared to the dual SGLT1/2 inhibitor sotagliflozin. Conclusion(s) The present findings indicate that although the empa treatment did not affect the hypertensive response of rats to Ang II, the SGLT2 inhibitor prevented the deleterious impact of Ang II on the diastolic cardiac function and remodeling, and the upregulation of eNOS expression and oxidative stress in the aorta wall. Thus, these findings highlight the protective potential of empa on the cardiovascular system in a normoglycemic hypertensive experimental model. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Boehringer Ingelheim Pharma GmbH & Co KG (Biberach an der Riss, Germany)

Inhibition of ferroptosis alleviates atherosclerosis through attenuating lipid peroxidation and endothelial dysfunction in mouse aortic endothelial cell

Atherosclerosis (AS) is the fundamental pathological state of many serious vascular diseases, characterized by disorders of lipid metabolism. Ferroptosis is a type of regulated cell death that is mainly mediated by iron-dependent lipid peroxidation. In this study, whether ferroptosis has occurred in AS and the potential effects of ferroptosis on AS were investigated. Ferroptosis inhibitor ferrostatin-1 (Fer-1) was administered to high-fat diet (HFD)-induced AS in ApoE−/- mice. The results showed that Fer-1 could alleviate AS lesion in HFD-fed ApoE−/- mice. Additionally, Fer-1 partially inhibited the iron accumulation, lipid peroxidation and reversed the expressions of ferroptosis indicators SLC7A11 and glutathione peroxidase 4 (GPX4) in HFD-fed ApoE−/- mice. Next, we evaluated the effects of inhibition of ferroptosis on oxidized-low density lipoprotein (ox-LDL)-induced mouse aortic endothelial cells (MAECs). Results showed that Fer-1 increased cell viability and reduced cell death in ox-LDL-treated MAECs. Moreover, Fer-1 decreased iron content and lipid peroxidation and up-regulated the levels of SLC7A11 and GPX4. Additionally, Fer-1 down-regulated the expressions of adhesion molecules and up-regulated eNOS expression. Iron chelator deferoxamine was used to demonstrate ferroptosis could be partially inhibited by iron complexation in ox-LDL-treated MAECs. Our results indicated that ferroptosis might occur during the initiation and development of AS. More importantly, inhibition of ferroptosis could alleviate AS through attenuating lipid peroxidation and endothelial dysfunction in AECs. Our findings might contribute to a deeper understanding regarding the pathological process of AS and provide a therapeutic target for AS.

Tangeretin ameliorates erectile and testicular dysfunction in a rat model of hypertension

Tangeretin is a polymethoxyflavone concentrated in citrus peels and has several biological activities. This study examined whether tangeretin improved reproductive dysfunction in Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertensive rats. Male Sprague-Dawley rats received L-NAME to induce hypertension and reproductive dysfunction for 5 w and were treated with tangeretin (15 or 30 mg/kg) or sildenafil citrate (10 mg/kg) for the final two weeks. Mean arterial pressure (MAP), intracavernosal pressure (ICP) response to cavernous nerve stimulation, endothelial nitric oxide synthase (eNOS), Angiotensin II receptor type 1 (AT1R) and gp91phox protein expressions and malondialdehyde (MDA) level in penile tissues were measured. Sperm concentrations and motility, seminiferous tubule morphology, serum testosterone, testicular eNOS and steroidogenic acute regulatory protein (StAR) expression were evaluated. Aortic superoxide generation, plasma and testicular MDA and plasma nitrate/nitrite levels were determined. Tangeretin reduced blood pressure and increased the maximum ICP/MAP associated with suppression of AT1R/gp91phox and upregulation of eNOS expression in hypertensive rats (P < 0.05). Furthermore, improvement of sperm quality relevant to increased testicular eNOS and StAR expression was found in tangeretin treated rats (P < 0.05). Changes in seminiferous tubule morphology in hypertensive rats were recovered by tangeretin (P < 0.05). It increased testosterone levels and reduced oxidative stress biomarkers and raised plasma nitrate/nitrite levels in L-NAME rats (P < 0.05). In conclusion, tangeretin improved maximum ICP/MAP and testicular dysfunction and morphology in rats treated with L-NAME. The molecular mechanisms are mediated by modulations of penile eNOS and AT1R/gp91phox expressions and testicular eNOS and StAR expression.

Protective role of poly(lactic‐co‐glycolic) acid nanoparticle loaded with resveratrol against isoproterenol‐induced myocardial infarction

Our study is aimed at evaluating the effects of pretreatment with Poly(lactic-co-glycolic) acid nanoparticle loaded with resveratrol (RSV PLGA NPs) compared to conventional resveratrol (RSV) on isoproterenol (ISO) induced myocardial infarction (MI) in rats. Sixty rats were randomly divided into six groups of 10 rats each. RSV and RSV PLGA NPs were given by gavage in two different doses (50 mg/kg body weight [BW] and 100 mg/kg BW) for 3 weeks. RSV and RSV PLGA NPs were given for 2 weeks starting 1 week before ISO administration. The blood samples were taken 24 hr after the last dose of ISO. The antioxidant, anti-inflammatory, and cardioprotective effects were evaluated in all groups. Only 100 mg/kg dose of RSV and both doses of RSV PLGA NPs offered a cardioprotective effect by preventing cardiac troponin T (cTnT) levels, lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) activities leakage from cardiomyocytes, with the best result for RSV PLGA NPs. All the oxidative stress parameters were significantly improved after RSV PLGA NPs compared to RSV pretreatment. RSV PLGA NPs were more efficient than RSV in limiting the increase in inflammatory cytokine expressions such as tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), and NF-kappaB (NF-kB) expression. In addition, RSV PLGA NPs significantly upregulated eNOS expression and downregulated iNOS expression. RSV PLGA NPs better prevented myocardial necrosis and reduced interstitial edema and neutrophil infiltration than RSV, on histopathological examination. Therefore, improving the bioactivity of RSV by nanotechnology may help limit cardiac injury after myocardial infarction.

Luhong Formula Has a Cardioprotective Effect on Left Ventricular Remodeling in Pressure-Overloaded Rats.

Background Luhong formula (LHF)—a traditional Chinese medicine containing Cervus nippon Temminck, Carthamus tinctorius L., Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, Codonopsis pilosula (Franch.) Nannf., Cinnamomum cassia Presl, and Lepidium apetalum Willd—is used in the treatment of heart failure, but little is known about its mechanism of action. We have investigated the effects of LHF on antifibrosis. Methods Forty-eight SD male rats were randomly assigned into six groups (n = 8), model group, sham-operation group, perindopril group (0.036 mg/ml), LHF high doses (LHF-H, 1.44 g/mL), LHF middle doses (LHF-M, 0.72 g/mL), and LHF low doses (LHF-L, 0.36 g/mL). Except the sham-operation group, the other groups were received an abdominal aorta constriction to establish a model of myocardial hypertrophy. The HW and LVW were measured to calculate the LVW/BW and HW/BW. ELISA was used to detect the serum concentration of BNP. The expressions of eNOS, TGF-β1, caspase-3, VEGF, and VEGFR2 in heart tissues were assessed by western blot analysis. mRNA expressions of eNOS, Col1a1, Col3a1, TGF-β1, VEGF, and VEGFR2 in heart tissues were measured by RT-PCR. The specimens were stained with hematoxylin-eosin (HE) and picrosirius red staining for observing the morphological characteristics and collagen fibers I and III of the myocardium under a light microscope. Results LHF significantly lowered the rat's HW/BW and LVM/BW, and the level of BNP in the LHF-treated group compared with the model group. Histopathological and pathomorphological changes of collagen fibers I and III showed that LHF inhibited myocardial fibrosis in heart failure rats. Treatment with LHF upregulated eNOS expression in heart tissue and downregulated Col1a1, Col3a1, TGF-β1, caspase-3, VEGF, and VEGFR2 expression. Conclusion LHF can improve left ventricular remodeling in a pressure-overloaded heart failure rat model; this cardiac protective ability may be due to cardiac fibrosis and attenuated apoptosis. Upregulated eNOS expression and downregulated Col1a1, Col3a1, TGF-β1, caspase-3, VEGF, and VEGFR2 expression may play a role in the observed LHF cardioprotective effect.

Fibroblast growth factor 21 enhances angiogenesis and wound healing of human brain microvascular endothelial cells by activating PPARγ

Angiogenesis of brain microvascular endothelial cells (BMECs) is required in the functional restoration of brain injury, such as traumatic brain injury (TBI) and ischemic stroke. Fibroblast growth factor 21 (FGF21) is an angiogenic molecule that functions through the formation of the FGF21/FGFR1/β-klotho complex but does not cause carcinogenic events. The current study was to determine whether recombinant human FGF21 (rhFGF21) could promote angiogenesis and scratch wound healing of human brain microvascular endothelial cells (HBMECs) and the possible underlying mechanism. rhFGF21 promoted angiogenesis and migration of HBMECs. The FGFR1 inhibitor PD173074 was applied to demonstrate that rhFGF21 functions through the formation of FGF21/FGFR1/β-klotho complexes. In addition, the specific PPARγ inhibitor GW9662 and PPARγ activator rosiglitazone were applied to determine that the role of rhFGF21 in increasing angiogenesis is through the PPARγ pathway. In addition, we revealed that the effect of rhFGF21 acts partially through upregulating eNOS expression. In conclusion, our study provides novel evidence that rhFGF21 can enhance the angiogenesis and migration of HBMECs through the formation of the FGF21/FGFR1/β-klotho complex via PPARγ activation and eNOS upregulation, indicating that FGF21 is a potential therapeutic angiogenic agent for the treatment of human brain injury.

The role of total flavone of Camellia on cerebrovascular vasopasm after subarachnoid hemorrhage in rats

Background: This study was undertaken to explore the role of total flavones of Camellia (TFC) on the cerebrovascular dysfunction in rats after experimental subarachnoid hemorrhage (SAH). Materials and Methods: The contraction and dilation of cerebral basilar artery (BA), nitric oxide (NO) level, and nitric oxide synthase (NOS) activity in rat serum and the expression of endothelial NOS (eNOS) in cerebral vessels were measured. Furthermore, the effect of potassium channel blockers, endothelium removal, L-NG-nitroarginine methyl ester (L-NAME), or prostacyclin I2 (PG I2) production inhibitor on the response of isolated BA derived from normal rats was also evaluated to explore the underlying mechanism of vasodilation induced by total flavones. Results: The contraction of rat BA to U46619 markedly increased and the vasodilation to acetylcholine remarkably reduced after SAH. Interestingly, these vascular dysfunctions were profoundly ameliorated by pretreatment of TFC. Moreover, total flavones could induce a concentration-dependent relaxation in isolated BA from normal rats, which was obviously eliminated by co-application of potassium channel blockers, ChTx and Apamin, application of L-NAME, or endothelial removal. In addition, total flavones pretreatment obviously improved the expression of eNOS in BA, serum NO level, and NOS activity at 48 h after SAH. Conclusion: These findings revealed that TFC has protective effect on cerebrovascular dysfunction after SAH and demonstrated that the protection could be due to its upregulation of eNOS expression and activation of potassium channel.

Oxymatrine ameliorates diabetes-induced aortic endothelial dysfunction via the regulation of eNOS and NOX4.

Oxymatrine (OMT) is the major quinolizidine alkaloid extracted from the root of Sophora flavescens Ait (the Chinese herb Kushen) and exhibits diverse pharmacological actions. In this study, we investigated the effects of OMT on diabetes-associated aortic endothelial dysfunction in a rat model of diabetes and its mechanisms. Male Sprague-Dawley rats were randomly divided into five groups: control, diabetic rats, diabetic rats treated with OMT (60, 120 mg/kg per day, by gavage), and diabetic rats treated with metformin (20 mg/kg per day, by gavage). The serum fasting blood glucose, insulin, total cholesterol, triglyceride, and nitric oxide (NO) levels were determined with commercial kits. Biochemical indices reflecting oxidative stress, such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were analyzed with commercial kits. Mitochondrial reactive oxygen species 2',7'-dichlorofluorescein diacetate (DCFH-DA) was measured by fluorescence microscopy. Histological analyses were conducted to observe morphological changes. Western blot analysis was applied to detect the expression levels of eNOS and NOX4. Reverse transcription polymerase chain reaction was used to detect the expressions of eNOS and NOX4 messenger RNA (mRNA). The diabetic rats exhibited markedly reduced body weight and increased plasma glucose levels. Moreover, the diabetic rats showed oxidative stress (significantly increased MDA and decreased SOD, CAT, GSH-Px, and serum NO levels). Hyperglycemia caused significant endothelial injury and dysfunction, including vasodilative and histologic changes in the diabetic rats. The expressions of phospho-eNOS protein and mRNA were significantly decreased, while the NOX4 protein expression was increased in the aortas of the diabetic rats. All of these diabetes-induced effects were reversed by OMT in the diabetic rats. The OMT treatment ameliorates diabetic endothelial dysfunction through enhanced NO bioavailability by upregulating eNOS expression and downregulating expression of NOX4.

Berbamine protects the heart from isoproterenol induced myocardial infarction by modulating eNOS and iNOS expressions in rats

AimThe current study was designed to investigate the effect of berbamine (BBM) on isoproterenol (ISO) induced changes in cardiac marker enzymes, myocardial oxidative stress, lipid profile and expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in male Wistar rats. Rats were pretreated with BBM (25mg/kg) through intraperitoneal injection for 7 days followed by induction of myocardial infarction (MI) by subcutaneous injection of ISO (85mg/kg) for last two days. Key findings: In the present study, the histopathological findings of the heart tissue showed that BBM treatment significantly minimized the damage induced by ISO. BBM pretreatment showed a significant decrease in heart weight, serum marker enzymes, lipid peroxidation and significant increase in cardiac endogenous enzymatic and non-enzymatic antioxidants compared to the ISO-treated group. In addition, we observed significantly upregulated eNOS expression and downregulated iNOS expression in BBM pretreated group. Thus, BBM protected the rat’s heart from ISO-induced myocardial infarction by its antioxidant, and antilipidemic properties. Significance: The results of the present investigation suggested that BBM efficiently ameliorated the ISO-induced myocardial infarction in rats.

The AGP-PPARγ axis promotes oxidative stress and diabetic endothelial cell dysfunction.

Alkyl-glycerophosphate (AGP) accumulates in atherogenic oxidized-LDL and human atherosclerotic plaques and is a potent agonist of peroxisome-proliferator-activated receptor-gamma (PPARγ). Recent studies suggest a potential regulatory role for PPARγ in endothelial nitric oxide synthase (eNOS) expression/activation and nitrogen oxide (NO) generation in the vascular endothelium. Importantly, eNOS-induced NO and advanced glycation end-products (AGEs) are involved in blood-vessel damage, and diabetic patients exhibit high serum NO and AGE levels; however, the effect of AGP on NO- and AGE-mediated endothelium dysfunction remains unknown. Investigation of the AGP-specific effects on NO- and AGE-mediated dysfunction and the underlying molecular mechanisms revealed that AGP upregulated eNOS expression and NO production, and that eNOS silencing and PPARγ antagonism inhibited AGP-mediated eNOS upregulation and NO production. Moreover, AGP-PPARγ-axis-mediated NO production promoted the generation of reactive oxygen species and AGE formation. These results suggested that AGP plays a significant role in the initiation/progression of diabetes-related atherosclerosis through PPARγ activation.