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Endothelial cell senescence is the main risk factor contributing to vascular dysfunction and the progression of aging-related cardiovascular diseases. However, the relationship between endothelial cell metabolism and endothelial senescence remains unclear. The present study provides novel insight into fatty acid metabolism in the regulation of endothelial senescence. In the replicative senescence model and H2O2-induced premature senescence model of primary cultured human umbilical vein endothelial cells (HUVECs), fatty acid oxidation (FAO) was suppressed and fatty acid profile was disturbed, accompanied by downregulation of proteins associated with fatty acid uptake and mitochondrial entry, in particular the FAO rate-limiting enzyme carnitine palmitoyl transferase 1A (CPT1A). Impairment of fatty acid metabolism by silencing CPT1A or CPT1A inhibitor etomoxir facilitated the development of endothelial senescence, as implied by the increase of p53, p21, and senescence-associated β-galactosidase, as well as the decrease of EdU-positive proliferating cells. In the contrary, rescue of FAO by overexpression of CPT1A or supplement of short chain fatty acids (SCFAs) acetate and propionate ameliorated endothelial senescence. In vivo, treatment of acetate for 4 weeks lowered the blood pressure and alleviated the senescence-related phenotypes in aortas of Ang II-infused mice. Mechanistically, fatty acid metabolism regulates endothelial senescence via acetyl-coenzyme A (acetyl-CoA), as implied by the observations that suppression of acetyl-CoA production using the inhibitor of ATP citrate lyase NDI-091143 accelerated senescence of HUVECs and that supplementation of acetyl-CoA prevented H2O2-induced endothelial senescence. Deficiency of acetyl-CoA resulted in alteration of acetylated protein profiles which are associated with cell metabolism and cell cycle. These findings thus suggest that improvement of fatty acid metabolism might ameliorate endothelial senescence-associated cardiovascular diseases.

Numerous studies reveal that metabolism dysfunction contributes to the development of pathological cardiac hypertrophy. While the abnormal lipid and glucose utilization in cardiomyocytes responding to hypertrophic stimuli have been extensively studied, the alteration and implication of glutaminolysis are rarely discussed. In the present work, we provide the first evidence that glutamate dehydrogenase (GDH), an enzyme that catalyzes conversion of glutamate into ɑ-ketoglutarate (AKG), participates in isoprenaline (ISO)-induced cardiac hypertrophy through activating mammalian target of rapamycin (mTOR) signaling. The expression and activity of GDH were enhanced in cultured cardiomyocytes and rat hearts following ISO treatment. Overexpression of GDH, but not its enzymatically inactive mutant, provoked cardiac hypertrophy. In contrast, GDH knockdown could relieve ISO-triggered hypertrophic responses. The intracellular AKG level was elevated by ISO or GDH overexpression, which led to increased phosphorylation of mTOR and downstream effector ribosomal protein S6 kinase (S6K). Exogenous supplement of AKG also resulted in mTOR activation and cardiomyocyte hypertrophy. However, incubation with rapamycin, an mTOR inhibitor, attenuated hypertrophic responses in cardiomyocytes. Furthermore, GDH silencing protected rats from ISO-induced cardiac hypertrophy. These findings give a further insight into the role of GDH in cardiac hypertrophy and suggest it as a potential target for hypertrophy-related cardiomyopathy.

Ischemic stroke is one of the leading causes of mortality worldwide. The available treatments are not effective. Phosphodiesterase 9A (PDE9A) is an intracellular cyclic guanosine monophosphate (cGMP) hydrolase considered to be a promising therapeutic target for brain diseases. This study explored neuroprotective effects and the underlying mechanism of LW33, a novel PDE9A inhibitor, on ischemic stroke in vitro and in vivo.A middle cerebral artery occlusion (MCAO) model was established in adult male Sprague-Dawley rats and an oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in human SH-SY5Y cells to mimic ischemia-reperfusion injury in vitro.LW33 increased cell viability, reduced lactate dehydrogenase activity, and OGD/R-induced apoptosis of SH-SY5Y cells. The protective effects of LW33 against stroke occurred in the recovery phase. LW33 administration significantly reduced cerebral infarction volume in MCAO rats, without causing significant deformation or necrosis of neurons in the cortex. LW33 also improved learning and cognitive dysfunction and reduced other pathological changes in MCAO rats in the recovery period. Moreover, LW33 stimulated the cGMP/PKG/CREB pathway and up-regulated the expression of the apoptosis-related proteins, and this effect was reversed by KT5823 treatment.LW33 inhibited cell apoptosis and promoted neuronal repair to alleviate OGD/R and MCAO induced pathological alterations via the cGMP/PKG/CREB pathway, indicating that LW33 may be a promising therapeutic target for ischemic stroke.

The extracts from the roots of Salvia miltiorrhiza Bunge (Danshen) are widely and traditionally used in the treatment of angina pectoris, acute myocardial infarct, hyperlipidemia and stroke in China and other Asian countries. In this study, we have investigated the role of P-glycoprotein (P-gp) in the intestinal absorption of tanshinone IIA (TSA), a major active constituent of Danshen, using several in vitro and in vivo models. The oral bioavailability of TSA was about 2.9-3.4% in rats, with non-linear pharmacokinetics when its dosage increased. In a single pass rat intestinal perfusion model, the permeability coefficients (P(app)) based on TSA disappearance from the luminal perfusates (P(lumen)) were 6.2- to 7.2-fold higher (P0.01) than those based on drug appearance in mesenteric venous blood (P(blood)). The P(blood), but not P(lumen), was significantly increased when co-perfused with verapamil, or quinidine (both P-gp inhibitors). The uptake and efflux of TSA in confluent Caco-2 cells were significantly altered in the presence of verapamil, quinidine, MK-571, or probenecid. The transport of TSA across Caco-2 monolayers was pH-, temperature- and ATP-dependent. Furthermore, the transport from the apical (AP) to basolateral (BL) side of the Caco-2 monolayers was 3.3- to 8.5-fold lower than that from the BL to AP side, but such a polarized transport was attenuated by co-incubated verapamil or quinidine. A polarized transport was also observed in the control MDCKII cells and more apparent in MDR1-MDCKII monolayers, with the P(app) values of TSA in the BL-AP direction being 7- to 9-fold higher in MDR1-MDCKII monolayers than those in the control MDCKII cells. Moreover, TSA significantly inhibited P-gp-mediated transport of digoxin in P-gp-overexpressing membrane vesicles with an IC(50) of 2.6 microM, but stimulated vanadate-sensitive P-gp ATPase activity with estimated K(m) and V(max) values of 10.70 +/- 0.69 microM and 67.65 +/- 1.31 nmol/min/mg protein, respectively. TSA was extensively metabolized to tanshinone IIB (TSB), and two other oxidative metabolites in rat liver microsomes, but the formation rate of TSB in rat intestinal microsomes was only about 1/10 of that in liver microsomes. These findings indicate that TSA is a substrate and reversing agent for P-gp; and P-gp-mediated efflux of TSA into the gut lumen and the first-pass metabolism contribute to the low oral bioavailability. Further studies are needed to explore the role of other drug transporters and first-pass metabolism in the low bioavailability of TSA.

To study the prevention effect of Huoluotongnao tablet on stroke.Bilateral common carotid artery ligation and reperfusion injury model and reperfusion injury in focal cerebral ischemia-induced thrombosis line method rat model were used.Huoluotongnao tablet could significantly reduce the pathological injury of rat brain tissue changes of these two models, and increase the activity of SOD and decrease the content of MDA in the brain tissue and plasma of rats. The brain water content of treatment groups were significantly reduced. The behavioral index and cerebral infarction range index were effectively improved in the middle cerebral artery occlusion reperfusion model rats.Huoluotongnao tablet has certain prevention effect on stroke.

To study the prevention effect of Huoluotongnao tablet on stroke.Thrombosis on arteriovenous shunt rats model, platelet aggregation and hypertension combined high cholesterol rats model were used.Huoluotongnao tablet high and low dosage could inhibit the formation of arteriovenous thrombosis and platelet aggregation significantly ,the inhibition rate was 17.71%, 22.69%, 20.34% and 24.43%, respectively. Pretreatment of Huoluotongnao tablet could inhibit the formation of arteriovenous thrombosis significantly; The levels of CHOz in all treatment groups of hypertension combined high cholesterol rats model were decreased significantly,the levels of TGz and LDL-C were decreased in the high dosage group,the blood pressure was decreased in the middle dosage group. eta bL, eta P and eta r (B/P) were decreased in the middle and high dosage groups. eta bM, AI and CY were decreased in the middle and high dosage groups. Huoluotongnao tablet had effect on blood lipid,blood pressure and hemorheology and in a dose-dependence manner. Its minimal effecting dose was the middle dose. g/kg (crude drug) and has certain prevention effect on stroke.Huoluotongnao tablet's minimal effecting dose is 1.28

The orphan nuclear receptor NOR1 belongs to the NR4A subfamily of the nuclear hormone receptor superfamily, and is involved in glucose and fat metabolism. However, its potential contribution to cardiovascular diseases remains to be assessed. Here, the roles of NOR1 in cardiac hypertrophy induced by isoprenaline and the underlying molecular mechanisms were investigated.NOR1 was expressed in cardiomyocytes treated with isoprenaline. After NOR1 overexpression or knockdown in neonatal rat cardiomyocytes, cellular hypertrophy was monitored by measuring cell surface area and the mRNA of hypertrophic biomarkers. Interactions between NOR1 and PARP-1 were investigated by co-immunoprecipitation. NOR1 expression and PARP-1 activity were measured in rats with cardiac hypertrophy induced by isoprenaline.Treatment with isoprenaline significantly up-regulated NOR1 expression and PARP-1 activity both in vivo and in vitro. Specific gene silencing of NOR1 attenuated isoprenaline-induced cardiomyocyte hypertrophy, whereas NOR1 overexpression exacerbated cardiac hypertrophy. We identified a physical interaction between NOR1 and PARP-1, which was enhanced by NOR1 transfection and thereby led to PARP-1 activation. Overexpression of NOR1, but not C293Y, a NOR1 mutant lacking the PARP-1 binding activity, increased cellular surface area and the mRNA levels of atrial natriuretic factor and brain natriuretic polypeptide, effects blocked by the PARP-1 inhibitor 3-aminobenzamide or siRNA for PARP-1.This is the first evidence that NOR1 was involved in isoprenaline-induced cardiac hypertrophy. The pro-hypertrophic effect of NOR1 can be partly attributed to its regulation of PARP-1 enzymic activity.

Sirtuin 6 (SIRT6) is involved in regulation of glucose and fat metabolism. However, its possible contribution to cardiac dysfunction remains to be determined. In the present study, the effect of SIRT6 on cardiac hypertrophy induced by angiotensin II (AngII) and the underlying molecular mechanisms were investigated.The expression and deacetylase activity of SIRT6 were measured in hypertrophic cardiomyocytes induced by AngII. After SIRT6 overexpression by transfection, or depletion by RNA interference in neonatal rat cardiomyocytes, cellular hypertrophy was monitored by measuring cell surface area and the mRNA levels of hypertrophic biomarkers. Further, the interaction between SIRT6 and the transcription factor NF-κB was investigated by co-immunoprecipitation, confocal immunofluorescence microscopy and luciferase reporter gene assay. The expression and deacetylase activity of SIRT6 were measured in vivo, using the abdominal aortic constriction (AAC) model of cardiac hypertrophy in rats.In AngII-induced hypertrophic cardiomyocytes and also in AAC-induced hypertrophic hearts, the expression of SIRT6 protein was upregulated, while its deacetylase activity was decreased. Overexpression of wild-type SIRT6 but not its catalytically inactive mutant, attenuated AngII-induced cardiomyocyte hypertrophy. We further demonstrated a physical interaction between SIRT6 and NF-κB catalytic subunit p65, whose transcriptional activity could be repressed by SIRT6 overexpression.Our findings suggest that SIRT6 suppressed cardiomyocyte hypertrophy in vitro via inhibition of NF-κB-dependent transcriptional activity and that this effect was dependent on its deacetylase activity.

This study aimed to investigate the possible modification of the neuroprotective effect of sodium ferulate, when orally co-administered with borneol, in transient global cerebral ischaemia-induced functional, histological and cellular alterations in mice.The bilateral common carotid artery occlusion was conducted in C57 BL/6J mice for 25 min. The mice were then subjected to a water maze test over an extended recovery period, followed by an assessment of neuronal loss in the CA1 region of the hippocampus (haematoxylin and eosin staining). The blood-brain barrier permeability (Evans blue tracing), brain oedema and oxidative stress were assayed and histological sections were also immunostained for gliofibrillar acid protein (GFAP) expression.The ischaemia reperfused mice were associated with long-lasting spatial learning deficits in the absence of other behavioural impairments and with neurodegeneration in the hippocampal CA1 region. However, the histological injuries were significantly attenuated by oral co-administration of sodium ferulate and borneol. Furthermore, combined treatment with sodium ferulate and borneol resulted in a significant reduction in brain oedema, GFAP-positive cells, malonaldialdehyde levels and blood-brain barrier permeability, but an increase in superoxide dismutase activity.Borneol may have benefits for the neuroprotective effect of sodium ferulate against injury induced in the brain by ischaemia/reperfusion.

Berberine is one of the main constituents of Coptidis rhizoma (CR) and Cortex phellodendri. In this study, we investigated the beneficial effects of berberine on renal function and its possible mechanisms in rats with diabetic nephropathy (DN).Male Wistar rats were divided into three groups: normal, diabetic model, and berberine treatment groups. Rats in the diabetic model and berberine treatment groups were induced to diabetes by intraperitonal injection with streptozotocin (STZ). Glomerular area, glomerular volume, fasting blood glucose (FBG), blood urea nitrogen (BUN), serum creatinine (Cr) and urine protein for 24 hours (UP24h) were measured using commercially available kits. Meanwhile, the activity of superoxide dismutase (SOD), content of malondialdehyde (MDA) in serum, activity of aldose reductase (AR) and the expression of AR mRNA and protein in kidney were detected by different methods.The results showed that oral administration of berberine (200 mg x kg(-1) x d(-1)) significantly ameliorated the ratio of kidney weight to body weight. Glomerular area, glomerular volume, FBG, BUN, Cr and UP24h were significantly decreased in the berberine treatment group compared with the diabetic model group (P0.05). Berberine treatment significantly increased serum SOD activity and decreased the content of MDA compared with diabetic model group (P0.05). AR activity as well as the expression of AR mRNA and protein in the kidney was markedly decreased in the berberine treatment group compared with diabetic model group (P0.05).These results suggested that berberine could ameliorate renal dysfunction in DN rats through controlling blood glucose, reduction of oxidative stress and inhibition of the activation of the polyol pathway.

To observe the effects of antioxidation and ceramide content of improved prescription of Didang-tang (IPDT) on exprimental atherosclerosis(AS) rabbits.Plasm Superoxide Dismutase(SOD) activity was detected with micro-content fast detecting method, Plasm Malondialdehyde(MDA) content with improved BaMuGuoFu method, and Aortic Ceramide (CER) content with thinlayer scanning.IPDT could effectivly improve plasma SOD activity and decrease plasma MDA content and decrease aortic CER content.IPDT on exprimental AS is related to the improvement of antioxidation and decrease of CER content.

We examined the effect of endogenous and exogenous nitric oxide (NO) on protein kinase C (PKC) activity induced by angiotensin II (Ang II) in cultured neonatal rat cardiomyocytes. The results are as follows. The activity of PKC was increased by Ang II (0.01-10 micromol/L) in a dose-dependent manner, but decreased by NO precursor L-arginine (L-Arg) (10 micromol/L-10 mmol/L) in a dose-dependent manner in cultured neonatal rat cardiomyocytes. Pretreatment with L-Arg (100 micromol/L) decreased significantly Ang II -activated PKC activity and PKC activity induced by phorbol 12-myristate 13-acetate (PMA) ( 10 micromol/L), a PKC activator. Pretreatment with N(G)-nitro-L-argingie methyl ester (L-NAME), a nitric oxide synthase (NOS) blocker, may inhibit significantly the role of L-Arg on Ang II - and PMA-activated PKC activity. The activity of PKC was also decreased by NO donor sodium nitroprusside (SNP) (10 micromol/L-1 mmol/L) in a dose-dependent manner in cultured neonatal rat cardiomyocytes. Pretreatment with SNP (10 micromol/L) decreased significantly Ang II - and PMA-activated PKC activity. These results indicate that PKC was controlled by both NO and Ang II. PKC may be a cross talk between Ang II and NO in cardiomyocytes. NO abolished the activity of PKC and impaired PKC downstream signaling transduction pathway cascades.

The aim of this study was to determine the molecular mechanism of nitric oxide (NO) in preventing cardiomyocytes from hypertrophic response induced by angiotensin II (Ang II). Hypertrophic response of neonatal rat cardiomyocytes was assayed by protein synthesis rate and expression of atrial natriuretic peptide (ANP) mRNA. The level of NO was shown by the content of nitrate and nitrite in cardiac myocytes. The protein expression of MKP-1 and the gene expression of eNOS were measured with Western blotting and RT-PCR, respectively. The results are as follows. (1) L-arginine (L-Arg) induced a dose-dependent increase in NO by 16% and 31% at the concentrations of 10 micromol/L and 100 micromol/L, respectively. L-Arg also increased the gene expression of eNOS. However, these effects were inhibited by L-NAME, the inhibitor of NOS. (2) The gene expression and the protein synthesis of ANP induced by Ang II (0.1 micromol/L) were inhibited by L-Arg (100 micromol/L). The inhibitory action of L-Arg was abolished after pretreatment with antisense oligoneucleotide against MKP-1. (3) L-Arg (100 micromol/L) increased the protein expression of MKP-1 by 225%, which was inhibited by L-NAME, an NOS inhibitor, and KT-5823, a cGMP-dependent protein kinase (PKG) inhibitor. However, Ang II enhanced the effect induced by L-Arg. The above results show that NO may activate PKG, and thereby promote the protein expression of MKP-1 and inactivate MAPK, resulting in an inhibition of cardiomyocyte hypertrophic response induced by Ang II.