Mechanism of Jiming Powder in ameliorating heart failure with preserved ejection fraction based on metabolomics / 中国中药杂志

In this study, untargeted metabolomics was conducted using the liquid chromatography-tandem mass spectrometry(LC-MS/MS) technique to analyze the potential biomarkers in the plasma of mice with heart failure with preserved ejection fraction(HFpEF) induced by a high-fat diet(HFD) and nitric oxide synthase inhibitor(Nω-nitro-L-arginine methyl ester hydrochloride, L-NAME) and explore the pharmacological effects and mechanism of Jiming Powder in improving HFpEF. Male C57BL/6N mice aged eight weeks were randomly assigned to a control group, a model group, an empagliflozin(10 mg·kg~(-1)·d~(-1)) group, and high-and low-dose Jiming Powder(14.3 and 7.15 g·kg~(-1)·d~(-1)) groups. Mice in the control group were fed on a low-fat diet, and mice in the model group and groups with drug intervention were fed on a high-fat diet. All mice had free access to water, with water in the model group and Jiming Powder groups being supplemented with L-NAME(0.5 g·L~(-1)). Drugs were administered on the first day of modeling, and 15 weeks later, blood pressure and cardiac function of the mice in each group were measured. Heart tissues were collected for hematoxylin-eosin(HE) staining to observe pathological changes and Masson's staining to observe myocardial collagen deposition. Untargeted metabolomics analysis was performed on the plasma collected from mice in each group, and metabolic pathway analysis was conducted using MetaboAnalyst 5.0. The results showed that the blood pressure was significantly lower and the myocardial concentric hypertrophy and left ventricular diastolic dysfunction were significantly improved in both the high-dose and low-dose Jiming Powder groups as compared with those in the model group. HE and Masson staining showed that both high-dose and low-dose Jiming Powder significantly alleviated myocardial fibrosis. In the metabolomics experiment, 23 potential biomarkers were identified and eight strongly correlated metabolic pathways were enriched, including linoleic acid metabolism, histidine metabolism, alpha-linolenic acid metabolism, glycerophospholipid metabolism, purine metabolism, porphyrin and chlorophyll metabolism, arachidonic acid metabolism, and pyrimidine metabolism. The study confirmed the pharmacological effects of Jiming Powder in lowering blood pressure and ameliorating HFpEF and revealed the mechanism of Jiming Powder using the metabolomics technique, providing experimental evidence for the clinical application of Jiming Powder in treating HFpEF and a new perspective for advancing and developing TCM therapy for HFpEF.

Effects of nitric oxide on C2C12 cell inflammatory responses and notexin-induced muscle injury / Efectos del óxido nítrico en las respuestas inflamatorias de las células C2C12 y la lesión muscular inducida por notexina

SUMMARY: Skeletal muscle injury is an acute inflammatory condition caused by an inflammatory response. To reduce inflammatory cell infiltration and relieve skeletal muscle injury, efficient treatment is urgently needed. Nitric oxide is a free radical molecule reported to have anti-inflammatory effects. In this study, we showed that NO could inhibit the inflammatory response of C2C12 cells in vitro and protect rat skeletal muscle injury from notexin in vivo. NO synthase inhibitor (L-NG-Nitroarginine Methyl Este?L-NAME) and NO donor (sodium nitroprusside dehydrate ?SNP) were used to explore the vital role of lipopolysaccharides (LPSs) in LPS-stimulated C2C12 myoblasts.The expression of IL-18 and IL-1b was upregulated by L-NAME and downregulated by SNP, as indicated by the ELISA results. NO can reduce ASC, Caspase-1, and NLRP3 mRNA and protein levels. Furthermore, NO was detected in the rat model. The results of immunohistochemical staining showed that the production of DMD decreased. We conducted qRT-PCR and western blotting to detect the expression of Jo-1, Mi-2, TLR2, and TLR4 on day 6 post injury following treatment with L-NAME and SNP. The expression of Jo-1, Mi-2, TLR2, and TLR4 was upregulated by L-NAME and significantly reversed by SNP. NO can alleviate C2C12 cell inflammatory responses and protect rat skeletal muscle injury from notexin.

RESUMEN: La lesión del músculo esquelético es una afección inflamatoria aguda causada por una respuesta inflamatoria. Para reducir la infiltración de células inflamatorias y aliviar la lesión del músculo esquelético es necesario un tratamiento eficaz. El óxido nítrico es una molécula de radicales libres que tiene efectos antiinflamatorios. En este estudio, demostramos que el ON podría inhibir la respuesta inflamatoria de las células C2C12 in vitro y proteger la lesión del músculo esquelético de rata de la notexina in vivo. El inhibidor de ON sintasa (L-NG-nitroarginina metil este, L-NAME) y el donante de ON (nitroprusiato de sodio deshidratado, SNP) se utilizaron para explorar el papel vital de los lipopolisacáridos (LPS) en los mioblastos C2C12 estimulados por LPS. La expresión de IL- 18 e IL-1b fue regulada positivamente por L-NAME y regulada negativamente por SNP, como indican los resultados de ELISA. El ON puede reducir los niveles de proteína y ARNm de ASC, Caspasa-1 y NLRP3. Además, se detectó ON en el modelo de rata. Los resultados de la tinción inmunohistoquímica mostraron que disminuyó la producción de DMD. Realizamos qRT-PCR y transferencia Western para detectar la expresión de Jo-1, Mi-2, TLR2 y TLR4 el día 6 después de la lesión después del tratamiento con L-NAME y SNP. La expresión de Jo-1, Mi-2, TLR2 y TLR4 fue regulada positivamente por L- NAME y significativamente revertida por SNP. El ON puede aliviar las respuestas inflamatorias de las células C2C12 en ratas, y proteger la lesión del músculo esquelético de la notexina.

Canagliflozin reduces the contractile response of isolated heart of normal adult rats / Canagliflozin reduce la respuesta contráctil del corazón aislado de ratas adultas normales

SUMMARY: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) represent a unique class of glucose-declining renal-targeted drugs. The SGLT2i Canagliflozin (CANA) is an anti-hyperglycemic drug that reduces various cardiovascular and renal outcomes in patients with type 2 diabetes mellitus. This study aimed to explore the potential effects of CANA on the isolated healthy adult rat hearts to show if CANA has positive inotropic or cardiac depressant effects via analyzing the amplitude and frequency of cardiac contractions. In isolated normal adult rat hearts, the effects of CANA on cardiac contractility were examined. In a dose-response curve, CANA led to a significant cardiac depressant effect in a dose-dependent manner. This cardiac depressant effect of CANA (10-6 M) was not prevented by atropine. However, this cardiac depressant effect was partially antagonized by both Isoproterenol (10-5 M) and Calcium chloride (10-6 M), suggesting beta-adrenoceptor and calcium channel blocking actions. In addition, the cardiac depressant effect of CANA (10-6 M) was mitigated in part by Nitric oxide synthase inhibitor, L-NAME, suggesting that its action probably depends to some extent on the accumulation of nitric oxide, which decreases the rise of intracellular Calcium. Data from this study demonstrate that CANA has a significant cardiac relaxant effect in isolated hearts of healthy adult rats by different possible mechanisms. This inhibitory effect on cardiac contractility may help improve the diastolic ventricular filling providing a therapeutic potential to help the other cardioprotective mechanisms of CANA in the prevention and treatment of heart failure.

RESUMEN: Los inhibidores del cotransportador de sodio- glucosa 2 (SGLT2i) representan una clase única de fármacos dirigidos a los riñones que disminuyen la glucosa. El SGLT2i Canagliflozin (CANA) es un fármaco antihiperglucémico que reduce varios resultados cardiovasculares y renales en pacientes con diabetes mellitus tipo 2. Este estudio tuvo como objetivo explorar los efectos potenciales de CANA en corazones aislados de ratas adultas sanas para indicar si CANA tiene efectos inotrópicos o depresores cardíacos positivos mediante el análisis de la amplitud y la frecuencia de las contracciones cardíacas. En corazones aislados de ratas adultas normales, se examinaron los efectos de CANA sobre la contractilidad cardíaca. En una curva de dosis-respuesta, CANA condujo a un efecto depresor cardíaco significativo de manera dependiente de la dosis. Este efecto depresor cardíaco de CANA (10-6 M) no fue impedido por la atropina. Sin embargo, este efecto depresor cardíaco fue parcialmente antagonizado tanto por el isoproterenol (10-5 M) como por el cloruro de calcio (10-6 M), lo que sugiere acciones bloqueadoras de los receptores beta adrenérgicos y de los canales de calcio. Además, el efecto depresor cardíaco de CANA (10-6 M) fue mitigado en parte por el inhibidor de la sintasa de óxido nítrico, L-NAME, lo que sugiere que su acción probablemente depende en cierta medida de la acumulación de óxido nítrico, lo que disminuye el aumento de calcio intracelular. Los datos de este estudio demuestran que CANA tiene un efecto relajante cardíaco significativo en corazones aislados de ratas adultas sanas por diferentes mecanismos posibles. Este efecto inhibitorio sobre la contractilidad cardíaca puede ayudar a mejorar el llenado ventricular diastólico proporcionando un potencial terapéutico para ayudar a los otros mecanismos cardioprotectores de CANA en la prevención y tratamiento de la insuficiencia cardíaca.

Electrocardiographic Abnormalities in Hypertension Models

Abstract Background: Hypertensive condition can lead to abnormalities in heart structure and electrical activity. The electrocardiogram (ECG) is a recording of the electrical activity of the heart and widely used to diagnose and detect heart problem. Objective: We conducted a comparative ECG analysis between two hypertension models (L-NAME and SHR) and their controls (Wistar and Wistar-Kyoto) at six and 15 th week of age. Methods: Blood pressure was measured at the end of the 15 th week, and electrocardiography was performed at six and 15 weeks of age in anaesthetized rats. Data normality was confirmed by Kolmogorov-Smirnov test followed by unpaired Student's t-test and the Mann-Whitney for parametric and non-parametric data, respectively. Results are expressed as mean ± SD. The accepted level of significance was set at p < 0.05. Results: L-NAME exhibited prolongation of JT and QT intervals and SHR showed a decrease in heart rate when compared to Wistar-Kyoto and L-NAME. Wistar-Kyoto exhibited short PR interval with increased QRS complex, and only QT prolongation at 15 weeks compared to Wistar. Conclusions: All the hypertension models used in this study featured an increase in blood pressure. However, while SHR showed cardiac dysfunction, L-NAME exhibited changes in ventricular performance. These results may guide future studies on different types and models of hypertension.

Effects of oxidative stress on liver, brain and spinal cord of rats using L-NAME and treated with hydroxyurea. A model of sickle cell complication

Abstract Purpose: To analyze the serum levels of nitric oxide and correlate them with the levels of thiobarbituric acid reactive substances (TBARS) in liver, brain and spinal cord of animals using L-NAME and treated with hydroxyurea. Methods: Eighteen male albino Wistar rats were divided into three groups. NG-nitro-L-arginine methyl ester (L-NAME) was intraperitoneally administered to induce oxidative stress. TBARS and plasma nitric oxide levels were analyzed in all groups. Histopathology of the liver and vascular tissue was performed. Results: Statistically significant differences were seen in liver, brain and spinal cord TBARS levels. Conclusions: Following the use of L-NAME, hepatic tissue increased the number of Kupffer cells as oxidative stress and inflammatory response increased. The use of L-NAME caused an increase in lipid peroxidation products and, consequently, in oxidative stress in animals. Hydroxyurea doses of 35 mg / kg / day reduced TBARS values in liver, brain and spinal cord.

Effects of NO/cGMP inhibitors in a rat model of anaphylactoid shock

Anaphylactic shock can be defined as an acute syndrome, and it is the most severe clinical manifestation of allergic diseases. Anaphylactoid reactions are similar to anaphylactic events but differ in the pathophysiological mechanism. Nitric oxide (NO) inhibitors during anaphylaxis suggest that NO might decrease the signs and symptoms of anaphylaxis but exacerbate associated vasodilation. Therefore, blocking the effects of NO on vascular smooth muscle by inhibiting the guanylate cyclase (GC) would be a reasonable strategy. This study aimed to investigate the effects of NO/cGMP pathway inhibitors methylene blue (MB), Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), and indigo carmine (IC) in shock induced by compound 48/80 (C48/80) in rats. The effect was assessed by invasive blood pressure measurement. Shock was initiated by C48/80 intravenous bolus injection 5 min before (prophylactic) or after (treatment) the administration of the inhibitors MB (3 mg/kg), L-NAME (1 mg/kg), and IC (3 mg/kg). Of the groups that received drugs as prophylaxis for shock, only the IC group did not present the final systolic blood pressure (SBP) better than the C48/80 group. Regarding shock treatment with the drugs tested, all groups had the final SBP similar to the C48/80group. Altogether, our results suggested that inhibition of GC and NO synthase in NO production pathway was not sufficient to revert hypotension or significantly improve survival.

Chronic aerobic exercise associated to low-dose L-NAME improves contractility without changing calcium handling in rat cardiomyocytes

Nitric oxide (NO) inhibition by high-dose NG-nitro-L-arginine methyl ester (L-NAME) is associated with several detrimental effects on the cardiovascular system. However, low-dose L-NAME increases NO synthesis, which in turn induces physiological cardiovascular benefits, probably by activating a protective negative feedback mechanism. Aerobic exercise, likewise, improves several cardiovascular functions in healthy hearts, but its effects are not known when chronically associated with low-dose L-NAME. Thus, we tested whether the association between low-dose L-NAME administration and chronic aerobic exercise promotes beneficial effects to the cardiovascular system, evaluating the cardiac remodeling process. Male Wistar rats were randomly assigned to control (C), L-NAME (L), chronic aerobic exercise (Ex), and chronic aerobic exercise associated to L-NAME (ExL). Aerobic training was performed with progressive intensity for 12 weeks; L-NAME (1.5 mg·kg-1·day-1) was administered by orogastric gavage. Low-dose L-NAME alone did not change systolic blood pressure (SBP), but ExL significantly increased SBP at week 8 with normalization after 12 weeks. Furthermore, ExL promoted the elevation of left ventricle (LV) end-diastolic pressure without the presence of cardiac hypertrophy and fibrosis. Time to 50% shortening and relaxation were reduced in ExL, suggesting a cardiomyocyte contractile improvement. In addition, the time to 50% Ca2+ peak was increased without alterations in Ca2+ amplitude and time to 50% Ca2+ decay. In conclusion, the association of chronic aerobic exercise and low-dose L-NAME prevented cardiac pathological remodeling and induced cardiomyocyte contractile function improvement; however, it did not alter myocyte affinity and sensitivity to intracellular Ca2+ handling.

Glabridin alleviates ischemia/reperfusion-induced functional failure of smooth muscle of rat ileum by upregulating the cAMP / Glabridin alivia la falla funcional inducida por isquemia/reperfusión del músculo liso de ileum murino regulando el incremento de cAMP

Despite the development of modern medicine, alternative medicine, which has not lost its timeliness, remains attractive for the treatment of various diseases. Glabridin, a major flavonoid of Glycyrrhiza glabra, is known for its antioxidant and anti-inflammatory activity. The aim of this study was: 1) to determine the possible protective role of glabridin against ischemia/reperfusion (I/R) injury of the intestine; 2) to evaluate the in vitrocontractile responses of ileum smooth muscles to acetylcholine after an intestinal I/R; and 3) to explain the underlying molecular mechanism of its effect. Rats were assigned to groups of six rats each; 1) I/R, 2) gla10, 3) gla20, 4) gla40, 5) N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester monohydrochloride (L-NAME)+gla40, and 6) Sham group. The healing effect of glabridin was abolished by L-NAME. Glabridin did not cause contractility of the smooth muscles to acetylcholine-induced contractile responses in intestinal I/R. Yet, it increased to spontaneous basal activity.

A pesar del desarrollo de la medicina moderna, la medicina alternativa, sin perder su vigencia, sigue siendo atractiva para el tratamiento de varias enfermedades. Glabradina, el flavonoide mayoritario de Glycyrrhiza glabra, es conocido por su actividad antioxidante y antiinflamatoria. Los propósitos de este estudio fueron: 1) Determinar el posible rol protector de glabradina ante daños intestinales por isquemia/reperfusion (I/R) 2) Evaluar in vitrolas respuestas de contracción de los músculos lisos del ileum ante acetilcolina después de I/R intestinal; y 3) Explicar el mecanismo molecular subyacente de este efecto. Se asignaron grupos de seis ratas: 1) I/R, 2) gla10, 3) gla20, 4) gla40, 5) N5-[imino(nitroamino)metil]-L-ornithina, metil ester monohidrochloruro (L-NAME)+gla40, y 6) Grupo testigo. El efecto curativo de glabridina fue abolido por L-NAME. Glabridina no causó contracción en el músculo liso como respuesta acetilcolina-inducida I/R. Además, incrementa la actividad basal expontánea.

Colonic Transit Disorder Mediated by Downregulation of Interstitial Cells of Cajal/Anoctamin-1 in Dextran Sodium Sulfate-induced Colitis Mice

BACKGROUND/AIMS: Interstitial cells of Cajal (ICC) and their special calcium-activated chloride channel, anoctamin-1 (ANO1) play pivotal roles in regulating colonic transit. This study is designed to investigate the role of ICC and the ANO1 channel in colonic transit disorder in dextran sodium sulfate (DSS)-treated colitis mice. METHODS: Colonic transit experiment, colonic migrating motor complexes (CMMCs), smooth muscle spontaneous contractile experiments, intracellular electrical recordings, western blotting analysis, and quantitative polymerase chain reaction were applied in this study. RESULTS: The mRNA and protein expressions of c-KIT and ANO1 channels were significantly decreased in the colons of DSS-colitis mice. The colonic artificial fecal-pellet transit experiment in vitro was significantly delayed in DSS-colitis mice. The CMMCs and smooth muscle spontaneous contractions were significantly decreased by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an ANO1 channel blocker, and NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of nitric oxide synthase activity, in DSS-colitis mice compared with that of control mice. Intracellular electrical recordings showed that the amplitude of NPPB-induced hyperpolarization was more positive in DSS-colitis mice. The electric field stimulation-elicited nitric-dependent slow inhibitory junctional potentials were also more positive in DSS-colitis mice than those of control mice. CONCLUSION: The results suggest that colonic transit disorder is mediated via downregulation of the nitric oxide/ICC/ANO1 signalling pathway in DSS-colitis mice.

Effect of co-administration of BRL-37344 and tadalafil on reduction of overactive bladder symptoms after induction of detrusor overactivity in mice

Abstract Purpose: To evaluate the impact of the combination of BRL 37344 and tadalafil (TDF) on the reduction of overactive bladder (OB) symptoms. Methods: Thirty mice were randomized into 5 groups (G) of 6 animals each. L-NAME was used to induce DO. G1: Control; G2: L-NAME; G3: L-NAME + TDF; G4: L-NAME + BRL 37344; G5: L-NAME + TDF + BRL 37344. After 30 days of treatment, the animals were submitted to cystometry to evaluate non-voiding contractions (NVC), threshold pressure (TP), baseline pressure (BP), frequency of micturition (FM) and threshold volume (TV). Differences between the groups were analyzed with ANOVA followed by the Tukey test. Results: NVC increased in G2 (4.33±2.58) in relation to G1 (1.50±0.55). NVC decreased in G3 (2.00±1.10), G4 (1.50±1.52) and G5 (2.00±1.26) compared to G2 (p<0.05). FM decreased in G3 (0.97±0.71), G4 (0.92±0.38) and G5 (1.05±0.44) compared to G2 (p<0.05). However, the combination of TDF and BRL37344 was not more effective at increasing NVC and improving FM than either drug alone. The five groups did not differ significantly with regard to TV. Conclusion: The combination of BRL 37344 and TDF produced no measurable additive effect on reduction of OB symptoms.

Potentiation of endothelium-dependent vasorelaxation of mesenteric arteries from spontaneously hypertensive rats by gemigliptin, a dipeptidyl peptidase-4 inhibitor class of anti-diabetic drug

Dipeptidyl peptidase4 (DPP4) inhibitors such as gemigliptin are anti-diabetic drugs elevating plasma concentration of incretins such as GLP-1. In addition to the DPP4 inhibition, gemigliptin might directly improve the functions of vessels under pathological conditions. To test this hypothesis, we investigated whether the acetylcholine-induced endothelium dependent relaxation (ACh-EDR) of mesenteric arteries (MA) are altered by gemigliptin pretreatment in Spontaneous Hypertensive Rats (SHR) and in Wistar-Kyoto rats (WKY) under hyperglycemia-like conditions (HG; 2 hr incubation with 50 mM glucose). ACh-EDR of WKY was reduced by the HG condition, which was significantly recovered by 1 µM gemigliptin while not by saxagliptin and sitagliptin up to 10 µM. The ACh-EDR of SHR MA was also improved by 1 µM gemigliptin while similar recovery was observed with higher concentration (10 µM) of saxagliptin and sitagliptin. The facilitation of ACh-EDR by gemigliptin in SHR was not observed under pretreatment with NOS inhibitor, L-NAME. In the endotheliumdenuded MA of SHR, sodium nitroprusside induced dose-dependent relaxation was not affected by gemigliptin. The ACh-EDR in WKY was decreased by treatment with 30 µM pyrogallol, a superoxide generator, which was not prevented by gemigliptin. Exendin-4, a GLP-1 analogue, could not enhance the ACh-EDR in SHR MA. The present results of ex vivo study suggest that gemigliptin enhances the NOS-mediated EDR of the HG-treated MA as well as the MA from SHR via GLP-1 receptor independent mechanism.

Beneficial Effect of Berberis amurensis Rupr. on Penile Erection / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate whether the methanol extract of Berberis amurensis Rupr. (BAR) augments penile erection using in vitro and in vivo experiments.</p><p><b>METHODS</b>The ex vivo study used corpus cavernosum strips prepared from adult male New Zealand White rabbits. In in vivo studies for intracavernous pressure (ICP), blood pressure, mean arterial pressure (MAP), and increase of peak ICP were continuously monitored during electrical stimulation of Sprague-Dawley rats.</p><p><b>RESULTS</b>Preconstricted with phenylephrine (PE) in isolated endotheliumintact rabbit corus cavernosum, BAR relaxed penile smooth muscle in a dose-dependent manner, which was inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one, a soluble guanylyl cclase inhibitor. BAR significantly relaxed penile smooth muscles dose-dependently in ex vivo, and this was inhibited by pretreatment with L-NAME H-[1,2,4]-oxadiazole-[4,3-α]-quinoxalin-1-one. BAR-induced relaxation was significantly attenuated by pretreatment with tetraethylammonium (TEA, P<0.01), a nonselective K channel blocker, 4-aminopyridine (4-AP, P<0.01), a voltage-dependent K channel blocker, and charybdotoxin (P<0.01), a large and intermediate conductance Ca sensitive-K channel blocker, respectively. BAR induced an increase in peak ICP, ICP/MAP ratio and area under the curve dose dependently.</p><p><b>CONCLUSION</b>BAR augments penile erection via the nitric oxide/cyclic guanosine monophosphate system and Ca sensitive-K (BK and IK) channels in the corpus cavernosum.</p>

Supplementing punicalagin reduces oxidative stress markers and restores angiogenic balance in a rat model of pregnancy-induced hypertension

Pre-eclampsia (PE) is a pregnancy disorder that is characterised by severe hypertension and increased risks of foetal and maternal mortality. The aetiology of PE not completely understood; however, maternal nutrition and oxidative stress play important roles in the development of hypertension. The treatment options for PE are currently limited to anti-hypertensive drugs. Punicalagin, a polyphenol present in pomegranate juice, has a range of bioactive properties. The effects of supplementation with punicalagin on angiogenesis and oxidative stress in pregnant rats with induced hypertension were investigated. The pregnant rats were randomly divided into five experimental groups (n=12 per group). Hypertension was induced using an oral dose of NG-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg/day) on days 14–19 of pregnancy. Punicalagin (25, 50 or 100 mg/kg) was given orally on days 14–21 of pregnancy. Punicalagin treatment at the tested doses significantly reduced diastolic, systolic, and mean arterial blood pressure in L-NAME treated rats from day 14. Punicalagin also restored angiogenic balance by increasing the expression of vascular endothelial growth factor and downregulating vascular endothelial growth factor receptor-1/fms-like tyrosine kinase-1. Punicalagin, significantly increased the placental nitric oxide levels as compared to PE group. The increased levels of oxidative stress in rats with PE were markedly decreased by treatment with punicalagin. Punicalagin at the tested doses markedly (p < 0.05) enhanced the placental antioxidant capacity in L-NAME-treated rats. The raised catalase activity observed following L-NAME induction was significantly (p < 0.05) and restored to normal activity levels in punicalagin treatment. Further, 100 mg dose of punicalagin exhibited higher protective effects as compared to lower doses of 25 and 50 mg. This study shows that supplementation with punicalagin decreased blood pressure and oxidative stress and restored angiogenic balance in pregnant rats with induced PE.

The Protective Effect of Melissa officinalis L. in Visceral Hypersensitivity in Rat Using 2 Models of Acid-induced Colitis and Stress-induced Irritable Bowel Syndrome: A Possible Role of Nitric Oxide Pathway

BACKGROUND/AIMS: The aim of present study is to estimate the effects of Melissa officinalis L. (MO) on visceral hypersensitivity (VH), defecation pattern and biochemical factors in 2 experimental models of irritable bowel syndrome (IBS) and the possible role of nitric oxide. METHODS: Two individual models of IBS were induced in male Wistar-albino rats. In the acetic acid model, the animals were exposed to rectal distension and abdominal withdrawal reflex, and the defecation patterns were determined. In the restraint stress model, the levels of TNF-α, myeloperoxidase, lipid peroxidation, and antioxidant powers were determined in the (removed) colon. Rats had been treated with MO, L-NG-nitroarginine methyl ester (L-NAME), aminoguanidine (AG), MO + AG, or MO + L-NAME in the mentioned experimental models. RESULTS: Hypersensitive response to rectal distension and more stool defecation in control rats have been observed in comparison to shams. MO-300 significantly reduced VH and defecation frequency in comparison to controls. VH and defecation pattern did not show significant change in AG + MO and L-NAME + MO groups compared to controls. Also, significant reduction in TNF-α, myeloperoxidase, thiobarbituric acid reactive substances (TBARS), and an increase in antioxidant power in MO-300 group was recorded compared to controls. AG + MO and L-NAME + MO groups showed a reverse pattern compared to MO-300 group. CONCLUSIONS: MO can ameliorate IBS by modulating VH and defecation patterns. Antioxidant and anti-inflammatory properties along with its effect on the nitrergic pathway seem to play important roles in its pharmacological activity.

Effects of Fengliao-Changweikang in Diarrhea-predominant Irritable Bowel Syndrome Rats and Its Mechanism Involving Colonic Motility

BACKGROUND/AIMS: This study was designed to investigate the effect of Fengliao-Changweikang (FLCWK) in diarrhea-predominant irritable bowel syndrome (IBS-D) rats and explore its underlying mechanisms. METHODS: IBS-D model rats were induced by neonatal maternal separation (NMS) combined with restraint stress (RS). In in vivo experiments, the model rats were randomly divided into 5 groups: NMS + RS, FLCWK (low dose, middle dose, and high dose), and pinaverium bromide. The normal control (no handling) rats were classified as the NH group. The therapeutic effect of FLCWK was evaluated by fecal characteristics, electromyographic response and abdominal withdrawal reflex scores. In in vitro experiments, the model rats were randomly divided into 2 groups: NMS + RS, FLCWK (middle dose), and no handling rats were used as the NH group. The differences in basic tension and ACh-induced tension of isolated colonic longitudinal smooth muscle strips (CLSMs) among the 3 groups were observed. In addition, different inhibitors (nifedipine, TMB-8, L-NAME, methylene blue, and 4-AP) were pretreated to explore the underlying mechanisms. RESULTS: In in vivo experiments, fecal characteristics, electromyographic response, and abdominal withdrawal reflex scores significantly improved in the FLCWK group, compared with the NMS + RS group. In in vitro experiments, the basic tension and ACh-induced tension of CLSMs in IBS-D rats were significantly inhibited by FLCWK. After pre-treatment with different inhibitors, the ACh-induced tension of CLSMs in each group showed no significant difference. CONCLUSIONS: FLCWK manifested curative effect in IBS-D rats by inhibiting colonic contraction. The underlying mechanisms may be related to regulatory pathway of nitric oxide/cGMP/Ca2+ and specific potassium channels.

Salvinorin A preserves cerebral pial artery autoregulation after forebrain ischemia via the PI3K/AKT/cGMP pathway

This study aimed to investigate the protective effect of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms. Thirty Sprague-Dawley rats received forebrain ischemia for 10 min. The dilation responses of the cerebral pial artery to hypercapnia and hypotension were assessed in rats before and 1 h after ischemia. The ischemia reperfusion (IR) control group received DMSO (1 µL/kg) immediately after ischemia. Two different doses of salvinorin A (10 and 20 µg/kg) were administered following the onset of reperfusion. The 5th, 6th, and 7th groups received salvinorin A (20 µg/kg) and LY294002 (10 µM), L-NAME (10 μM), or norbinaltorphimine (norBIN, 1 μM) after ischemia. The levels of cGMP in the cerebrospinal fluid (CSF) were also measured. The phosphorylation of AKT (p-AKT) was measured in the cerebral cortex by western blot at 24 h post-ischemia. Cell necrosis and apoptosis were examined by hematoxylin-eosin staining (HE) and TUNEL staining, respectively. The motor function of the rats was evaluated at 1, 2, and 5 days post-ischemia. The dilation responses of the cerebral pial artery were significantly impaired after ischemia and were preserved by salvinorin A treatment. In addition, salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis of the cerebral cortex and improved the motor function of the rats. These effects were abolished by LY294002, L-NAME, and norBIN. Salvinorin A preserved cerebral pial artery autoregulation in response to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway.

Nitric oxide modulates ATP-evoked currents in mouse Leydig cells

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

The role of potassium channels in the endothelial dysfunction induced by periodontitis

Abstract Objective: Periodontitis is associated with endothelial dysfunction, which is clinically characterized by a reduction in endothelium-dependent relaxation. However, we have previously shown that impairment in endothelium-dependent relaxation is transient. Therefore, we evaluated which mediators are involved in endothelium-dependent relaxation recovery. Material and methods: Rats were subjected to ligature-induced experimental periodontitis. Twenty-one days after the procedure, the animals were prepared for blood pressure recording, and the responses to acetylcholine or sodium nitroprusside were obtained before and 30 minutes after injection of a nitric oxide synthase inhibitor (L-NAME), cyclooxygenase inhibitor (Indomethacin, SC-550 and NS- 398), or calcium-dependent potassium channel blockers (apamin plus TRAM- 34). The maxilla and mandible were removed for bone loss analysis. Blood and gingivae were obtained for C-reactive protein (CRP) and myeloperoxidase (MPO) measurement, respectively. Results: Experimental periodontitis induces bone loss and an increase in the gingival MPO and plasmatic CRP. Periodontitis also reduced endothelium-dependent vasodilation, a hallmark of endothelial dysfunction, 14 days after the procedure. However, the response was restored at day 21. We found that endothelium-dependent vasodilation at day 21 in ligature animals was mediated, at least in part, by the activation of endothelial calcium-activated potassium channels. Conclusions: Periodontitis induces impairment in endothelial-dependent relaxation; this impairment recovers, even in the presence of periodontitis. The recovery is mediated by the activation of endothelial calcium-activated potassium channels in ligature animals. Although important for maintenance of vascular homeostasis, this effect could mask the lack of NO, which has other beneficial properties.

Effects of a Single Bout of Resistance Exercise in Different Volumes on Endothelium Adaptations in Healthy Animals / Efeitos de uma Sessão de Exercício Resistido em Diferentes Volumes sobre Adaptações Endoteliais em Animais Saudáveis

Abstract Background: Resistance exercise (RE) has been recommended for patients with cardiovascular diseases. Recently, a few studies have demonstrated that the intensity of a single bout of RE has an effect on endothelial adaptations to exercise. However, there is no data about the effects of different volumes of RE on endothelium function. Objective: The aim of the study was to evaluate the effects of different volumes of RE in a single bout on endothelium-dependent vasodilatation and nitric oxide (NO) synthesis in the mesenteric artery of healthy animals. Methods: Male Wistar rats were divided into three groups: Control (Ct); low-volume RE (LV, 5 sets x 10 repetitions) and high-volume RE (HV, 15 sets x 10 repetitions). The established intensity was 70% of the maximal repetition test. After the exercise protocol, rings of mesenteric artery were used for assessment of vascular reactivity, and other mesenteric arteries were prepared for detection of measure NO production by DAF-FM fluorescence. Insulin responsiveness on NO synthesis was evaluated by stimulating the vascular rings with insulin (10 nM). Results: The maximal relaxation response to insulin increased in the HV group only as compared with the Ct group. Moreover, the inhibition of nitric oxide synthesis (L-NAME) completely abolished the insulin-induced vasorelaxation in exercised rats. NO production showed a volume-dependent increase in the endothelial and smooth muscle layer. In endothelial layer, only Ct and LV groups showed a significant increase in NO synthesis when compared to their respective group under basal condition. On the other hand, in smooth muscle layer, NO fluorescence increased in all groups when compared to their respective group under basal condition. Conclusions: Our results suggest that a single bout of RE promotes vascular endothelium changes in a volume-dependent manner. The 15 sets x 10 repetitions exercise plan induced the greatest levels of NO synthesis.

Resumo Fundamentos: O exercício resistido (ER) tem sido recomendado para pacientes com doenças cardiovasculares. Recentemente, alguns estudos demonstraram que a intensidade de uma sessão de ER exerce um efeito sobre a disfunção endotelial. No entanto, não há dados sobre os efeitos de diferentes volumes de ER sobre a função endotelial. Objetivo: O objetivo deste estudo foi avaliar os efeitos de diferentes volumes de ER, realizados em uma única sessão, sobre a vasodilatação dependente do endotélio e síntese de óxido nítrico (NO) em artéria mesentérica de animais saudáveis. Métodos: Ratos Wistar machos foram divididos em três grupos: Controle (Ct); baixo volume (BV, 5 séries x 10 repetições) e alto volume de ER (AV, 15 séries x 10 repetições). Foi estabelecida a intensidade de 70% do teste de repetição máxima. Após o protocolo de exercício, anéis de artéria mesentérica foram utilizados na avaliação da reatividade vascular, e outras artérias mesentéricas foram preparadas para a detecção da produção de NO por fluorescência com para do DAF-FM. A resposta à insulina pela síntese de NO foi avaliada estimulando-se os anéis vasculares com insulina (10nM). Resultados: A resposta máxima do relaxamento induzido por insulina foi aumentada somente no grupo AV em comparação ao grupo Ct. Além disso, a inibição da síntese do NO (L-NAME), aboliu completamente o relaxamento vascular induzido por insulina em ratos exercitados. A produção de NO mostrou um aumento dependente do volume no endotélio e no músculo liso. No endotélio, apenas os grupos Ct e BV mostraram aumento significativo na síntese de NO quando comparado aos seus respectivos grupos sob condição basal. No entanto, no músculo liso, a fluorescência foi aumentada em todos os grupos quando comparados aos seus respectivos grupos sob a condição basal. Conclusões: Nossos resultados sugerem que uma única sessão de ER foi capaz de promover adaptações no endotélio vascular. Além disso, nós observamos que este efeito é volume-dependente e o volume de 15 séries x10 repetições induziu o maior aumento na síntese de NO.

Aerobic Swim Training Restores Aortic Endothelial Function by Decreasing Superoxide Levels in Spontaneously Hypertensive Rats

OBJECTIVE: We aimed to determine whether aerobic training decreases superoxide levels, increases nitric oxide levels, and improves endothelium-dependent vasodilation in the aortas of spontaneously hypertensive rats. METHODS: Spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were distributed into 2 groups: sedentary (SHRsd and WKYsd, n=10 each) and swimming-trained (SHRtr, n=10 and WKYtr, n=10, respectively). The trained group participated in training sessions 5 days/week for 1 h/day with an additional work load of 4% of the animal’s body weight. After a 10-week sedentary or aerobic training period, the rats were euthanized. The thoracic aortas were removed to evaluate the vasodilator response to acetylcholine (10-10 to 10-4 M) with or without preincubation with L-NG-nitro-L-arginine methyl ester hydrochloride (L-NAME; 10-4 M) in vitro. The aortic tissue was also used to assess the levels of the endothelial nitric oxide synthase and nicotinamide adenine dinucleotide oxidase subunit isoforms 1 and 4 proteins, as well as the superoxide and nitrite contents. Blood pressure was measured using a computerized tail-cuff system. RESULTS: Aerobic training significantly increased the acetylcholine-induced maximum vasodilation observed in the SHRtr group compared with the SHRsd group (85.9±4.3 vs. 71.6±5.2%). Additionally, in the SHRtr group, superoxide levels were significantly decreased, nitric oxide bioavailability was improved, and the levels of the nicotinamide adenine dinucleotide oxidase subunit isoform 4 protein were decreased compared to the SHRsd group. Moreover, after training, the blood pressure of the SHRtr group decreased compared to the SHRsd group. Exercise training had no effect on the blood pressure of the WKYtr group. CONCLUSIONS: In SHR, aerobic swim training decreased vascular superoxide generation by nicotinamide adenine dinucleotide oxidase subunit isoform 4 and increased nitric oxide bioavailability, thereby improving endothelial function.