Altered regulation of renal nitric oxide,atrial natriuretic peptide and cyclooxygenase systems in aldosterone escape in rats

The present study was aimed to determine whether there is an altered role of local nitric oxide (NO), atrial natriuretic peptide (ANP) and cyclooxygenase (COX) systems in the kidney in association with the aldosterone escape. Male Sprague–Dawley rats were used. Aldosterone (200 μg/day) was infused through entire time course. The control group was kept on a low sodium diet (0.02 mEq/day), and the experimental group was supplied with a higher sodium diet (2.0 mEq/day). Four days after beginning the regimen, the kidneys were taken. The protein expression of NO synthase (NOS) and COX isoforms was determined by semiquantitative immunoblotting. The mRNA expression of components of ANP system was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclases were determined by the amount of cGMP generated in responses to sodium nitroprusside and ANP, respectively. There developed aldosterone escape in the experimental group. Accordingly, the renal content and the urinary excretion of NO increased. The expression of nNOS was increased in the inner medulla. Neither the expression of eNOS nor that of iNOS was changed. The expression and the catalytic activity of soluble guanylyl cyclase remained unaltered. The mRNA expression of ANP was increased. Neither the expression of NPR-A or NPR-C nor the activity of particulate guanylyl cyclase was altered in the papilla. The protein expression of COX-2 was increased in the inner medulla, while that of COX-1 remained unchanged. In conclusion, the upregulation of nNOS, ANP, and COX-2 may be causally related with the aldosterone escape.     

Neuroprotective effect of allicin against traumatic brain injury via Akt/endothelial nitric oxide synthase pathway-mediated anti-inflammatory and anti-oxidative activities

Allicin, one of the main biologically active compounds derived from garlic, has been shown to exert various anti-oxidative and anti-inflammatory activities in in vitro and in vivo studies. Here, we sought to investigate the potential neuroprotective effects of allicin against traumatic brain injury (TBI) in rats. We found that allicin treatment (10 and 50 mg/kg, not 1 mg/kg) significantly reduced brain edema and motor functional deficits, as well as apoptotic neuronal cell death in injured cortex. These protective effects could be observed even if the administration was delayed to 4 h after injury. Moreover, allicin treatment decreased the expression levels of MDA and protein carbonyl, preserved the endogenous antioxidant enzyme activities, and suppressed the expression of inflammatory cytokines. The results of Western blot analysis showed that allicin increased the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Blocking Akt/eNOS pathway activation by specific inhibitor LY294002 (10 μL, 10 mmol/L) or L-NIO (0.5 mg/kg) partly reversed the protective effects of allicin and its anti-inflammatory activities. The allicin induced anti-oxidative activity was partly prevented by LY294002, but not L-NIO. In summary, our data strongly suggested that allicin treatment at an appropriate dose can exert protective effect against TBI through Akt/eNOS pathway-mediated anti-inflammatory and anti-oxidative activities.     

Inhibition of nNOS reduces ischemic cell death through down-regulating calpain and caspase-3 after experimental stroke

In vitro nitric oxide (NO) regulates calpain and caspase-3 activation, and in vivo neuronal nitric oxide synthase (nNOS), calpain and caspase-3 participate in the ischemic brain injury. Our objective was to investigate whether nNOS was involved in the ischemic brain injury through activating calpain and caspase-3 during experimental stroke. Rats received 1-h ischemia by intraluminant filament, and then reperfused for 23 h (R 23 h). nNOS inhibitor 7-nitroindozale (7-NI, 50 mg/kg) was administrated intraperitoneally 5 min before ischemia. Our data showed that treatment with 7-NI markedly reduced neurological deficits, the brain swelling, and the infarct volume at R 23 h. Enzyme studies revealed significant suppression of the activities of m-calpain and caspase-3 in penumbra and core, and the activities of μ-calpain in penumbra, but not in core, in 7-NI-treated rats versus vehicle-treated rats. Western blot analysis demonstrated that 7-NI markedly increased the levels of MAP-2 and spectrin in penumbra and core compared with vehicle-treated rats. Histopathological studies displayed that 7-NI significantly reduced the necrotic cell death in penumbra and core, and apoptotic cell death in penumbra, but not in core. These data demonstrate the involvement of NO produced by nNOS in the ischemic neuronal injury through affecting the activation of calpain and caspase-3 in penumbra and core after experimental stroke, which provides a new perspective on possible mechanisms of action of nNOS inhibition in cerebral ischemia.     

Ghrelin protects human pulmonary artery endothelial cells against hypoxia-induced injury via PI3-kinase/Akt

Endothelial injury and diminished NO release induced by hypoxia is thought to be a critical factor in the development of pulmonary artery hypertension (PAH). Ghrelin (Ghr) is a well-characterized hormone and has protective effects on the cardiovascular system, specifically by promoting the vascular endothelial cell function. The aim of this study was to investigate the effect of the Ghr on the hypoxia-induced injury in human pulmonary artery endothelial cells (HPAECs) and on the involved transduction pathway. Effects were investigated by treating cells with varying concentrations of Ghr in the absence or presence of inhibitors that target phosphoinositide 3-kinase (PI3K), in normoxic or hypoxic conditions for 24 h. Our results indicated that the treatment with 10⁻⁷ mol/l Ghr significantly enhanced cell viability (P < 0.05, n = 5) and upregulated the ratio of Bcl-2/Bax under hypoxic condition (P < 0.05, n = 4), as compared with the hypoxic condition alone. However, an addition of the PI3K/Akt inhibitor LY294002 inhibited these Ghr-mediated effects. Moreover, the Ghr (10⁻⁷ mol/l) significantly increased NO secretion and eNOS phosphorylation in comparison with the hypoxia or normoxia alone group (P < 0.05, n = 4). Nevertheless, the treatment with LY294002 (20 μ mol/l) decreased the Ghr-induced NO release as well as the eNOS activity. In conclusion, the Ghr could inhibit hypoxia-mediated HPAECs dysfunction via the PI3K/Akt pathway, and the bcl-2/bax ratio was also involved in the protective action of the Ghr in HPAECs. As such, the Ghr demonstrates a significant potential to prevent and treat PAH affected by the endothelial dysfunction.     

Quercetin intake during lactation modulates the AMP-activated protein kinase pathway in the livers of adult male rat offspring programmed by maternal protein restriction

Quercetin, a naturally occurring flavonoid, has been reported to possess numerous biological activities including activation of adenosine-5’-monophosphate-activated protein kinase (AMPK). We investigated the effects of quercetin intake during lactation on the AMPK activation in the livers of adult offspring programmed by maternal protein restriction during gestation. Pregnant Wistar rats were fed control and low-protein diets during gestation. Following delivery, each dam received a control or 0.2% quercetin-containing control diet during lactation as follows: control on control (CC), control on restricted (LPC) and 0.2% quercetin-containing control on restricted (LPQ). At weaning (week 3), some of the pups from each dam were killed, and the remaining pups (CC, n= 8; LPC, n= 10; LPQ, n= 13) continued to receive a standard laboratory diet and were killed at week 23. Blood chemistry and phosphorylation levels of AMPKα, acetyl-CoA carboxylase (ACC), endothelial nitric oxide synthase (eNOS) and mammalian target of rapamycin (mTOR) in the livers of male offspring were examined. At week 3, the level of phosphorylated AMPK protein in LPQ increased about 1.5- and 2.1-fold compared with LPC and CC, respectively, and the level in LPQ at week 23 increased about 1.9- and 2.9-fold, respectively. A significant increase in phosphorylated ACC and eNOS levels was found in LPQ. There was no significant difference among the three groups in the level of phosphorylated mTOR protein. In conclusion, quercetin intake during lactation up-regulates AMPK activation in the adult offspring of protein-restricted dams and modulates the AMPK pathway in the liver.     

Niacin bound chromium treatment induces myocardial Glut-4 translocation and caveolar interaction via Akt,AMPK and eNOS phosphorylation in streptozotocin induced diabetic rats after ischemia-reperfusion injury

Diabetes, one of the major risk factors of metabolic syndrome culminates in the development of Ischemic Heart Disease (IHD). Refined diets that lack micronutrients, mainly trivalent chromium (Cr³⁺) have been identified as the contributor in the rising incidence of diabetes. We investigated the effect of niacin-bound chromium (NBC) during ischemia/reperfusion (IR) injury in streptozotocin induced diabetic rats. Rats were randomized into: Control (Con); Diabetic (Dia) and Diabetic rats fed with NBC (Dia + NBC). After 30 days of treatment, the isolated hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. NBC treatment demonstrated significant increase in left ventricular functions and significant reduction in infarct size and cardiomyocyte apoptosis in Dia + NBC compared with Dia. Increased Glut-4 translocation to the lipid raft fractions was also observed in Dia + NBC compared to Dia. Reduced Cav-1 and increased Cav-3 expression along with phosphorylation of Akt, eNOS and AMPK might have resulted in increased Glut-4 translocation in Dia + NBC. Our results indicate that the cardioprotective effect of NBC is mediated by increased activation of AMPK, Akt and eNOS resulting in increased translocation of Glut-4 to the caveolar raft fractions thereby alleviating the effects of IR injury in the diabetic myocardium.     

Polymorphisms of the NOS3 gene and risk of myocardial infarction in the Tunisian population

Controversial results regarding the association of eNOS gene (NOS3) polymorphisms with myocardial infarction (MI) have been reported. This study investigated the relationship of the −786T>C (rs2070744), 894G>T (rs1799983) and 4a4b polymorphisms of the NOS3 gene with the presence of MI in the Tunisian population. In addition, we also examined the association of NOS3 gene haplotypes with MI in Tunisian subjects.A total of 303 patients with MI and 225 controls were included in the study. The 894G>T and −786T>C single nucleotide polymorphisms were analyzed by PCR-RFLP, and 4a4b polymorphism just for PCR.There was significant linkage disequilibrium between the three NOS3 polymorphisms (p < 0.0001). The genotype distribution and allele frequency of NOS3 4a4b, but not −786T>C and 894G>T, polymorphism was significantly different between MI patients and controls. The univariate logistic regression analysis showed a significant association of the 4a4b polymorphism and MI according to co-dominant, dominant and recessive models (co-dominant model OR: 4.38, 95%CI: 1.24–15.41; p = 0.021, dominant model OR: 1.66, 95%CI: 1.14–2.42); p = 0.007, and recessive model OR: 3.85, 95%CI: 1.10–13.47; p = 0.035). The multivariate analysis, adjusted for traditional cardiovascular risk factors, revealed that the NOS3 4a4a genotype was an independent predisposing factor to MI, according to the models considered. In addition, a haplotype 7 (C-T-4a), (OR = 12.05, p = 0.010) was a risk factor of MI after controlling for classical risk factors.These finding suggest that the 4a4b polymorphism of the NOS3 gene was associated with MI in Tunisian patients.     

Intermittent-hypoxia induced autophagy attenuates contractile dysfunction and myocardial injury in rat heart

Sleep apnea syndrome (SAS) is considered to be associated with heart failure (HF). It is known that autophagy is induced in various heart diseases thereby promotes survival, but its excess may be maladaptive. Intermittent hypoxia (IH) plays pivotal role in the pathogenesis of SAS. We aimed to clarify the relationships among IH, autophagy, and HF. Rats underwent IH at a rate of 20 cycles/h (nadir of 4% O2 to peak of 21% O2 with 0% CO2) or normal air breathing (control) for 8 h/d for 3 weeks. IH increased the cardiac LC3II/LC3I ratio. The IH induced upregulation of LC3II was attenuated by the administration of an inhibitor of autophagosome formation 3-methyladenine (3-MA), but enhanced by an inhibitor of autophagosome–lysosome fusion chloroquine (CQ), showing enhanced autophagic flux in IH hearts. Electron microscopy confirmed an increase in autophagosomes and lysosomes in IH. With 3-MA or CQ, IH induced progressive deterioration of fractional shortening (FS) on echocardiography over 3 weeks, although IH, 3-MA, or CQ alone had no effects. With CQ, IH for 4 weeks increased serum troponin T levels, reflecting necrosis. Western blotting analyses showed dephosphorylation of Akt and mammalian target of rapamycin (mTOR) at Akt (Ser2448, 2481) sites, suggesting the activation of autophagy via Akt inactivation. Conclusions. IH-mediated autophagy maintains contractile function, whereas when autophagy is inhibited, IH induces systolic dysfunction due to myocyte necrosis. General significance. This study highlighted the potential implications of autophagy in cardio-protection in early SAS patients without comorbidity, reproduced in normal rats by 3 ~ 4 weeks of IH.     

dl-trans-3,4-Dihydroxy-1-selenolane (DHSred) heals indomethacin-mediated gastric ulcer in mice by modulating arginine metabolism

BackgroundThe importance of the arginine metabolism in gastric ulcer-healing is given relatively less attention. Hence the role of controlling this pathway by dl-trans-3,4-dihydroxy-1-selenolane (DHS_red) and omeprazole against indomethacin-induced stomach ulceration in mouse was investigated.MethodsSwiss albino mice were ulcerated with indomethacin followed by treatment with the test samples, and the activities of myeloperoxidase (MPO), total nitric oxide synthase (NOS) and arginase, the expressions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), and the pro-/anti-inflammatory cytokine levels were assayed. NOS-inhibitors were also used to establish the biochemical mechanism.ResultsIndomethacin induced maximum ulceration in mice on the 3rd day, associated with reduced arginase activity, eNOS expression, along with increased MPO and total NOS activities, nitric oxide (NO) generation, iNOS expression, and pro-/anti-inflammatory (Th₁/Th₂₎ cytokine ratio. Treatment with DHS_red (2.5 mg kg^− 1 × 3 days) restored the cytokine balance to shift the iNOS/NO axis to the arginase/polyamine axis as revealed from the increased arginase activity and eNOS expression, and reduced iNOS expression, total NOS activity and NO level.ConclusionsThe ulcer-healing property of DHS_red, but not of omeprazole was due to a favorable pro-/anti-inflammatory cytokine ratio that shifted the arginine metabolism to the polyamine pathway and increased the eNOS/iNOS ratio. The healing action of omeprazole was not significantly associated with these parameters.General significanceThe shift in the ariginine-metabolism from the iNOS/NO axis to the arginase/polyamine axis is guided by Th_1/Th₂ cytokines ratio and plays an important role in gastric ulcer-healing. The favourable effects of the non-toxic and water-soluble compound, DHS_red on these pathways and other COX-dependent and antioxidative parameters suggested it to be a promising anti-ulcer formulation for further studies.     

Angiotensin-converting enzyme 2 ameliorates renal fibrosis by blocking the activation of mTOR/ERK signaling in apolipoprotein E-deficient mice

Angiotensin-converting enzyme 2 (ACE2) has been shown to prevent atherosclerotic lesions and renal inflammation. However, little was elucidated upon the effects and mechanisms of ACE2 in atherosclerotic kidney fibrosis progression. Here, we examined regulatory roles of ACE2 in renal fibrosis in the apolipoprotein E (ApoE) knockout (KO) mice. The ApoEKO mice were randomized to daily deliver either angiotensin (Ang) II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. Downregulation of ACE2 and upregulation of phosphorylated Akt, mTOR and ERK1/2 levels were observed in ApoEKO kidneys. Ang II infusion led to increased tubulointerstitial fibrosis in the ApoEKO mice with greater activation of the mTOR/ERK1/2 signaling. The Ang II-mediated renal fibrosis and structural injury were strikingly rescued by rhACE2 supplementation, associated with reduced mRNA expression of TGF-β1 and collagen I and elevated renal Ang-(1–7) levels. In cultured mouse kidney fibroblasts, exposure with Ang II (100 nmol L⁻¹) resulted in obvious elevations in superoxide generation, phosphorylated levels of mTOR and ERK1/2 as well as mRNA levels of TGF-β1, collagen I and fibronectin 1, which were dramatically prevented by rhACE2 (1 mg mL⁻¹) or mTOR inhibitor rapamycin (10 μmol L⁻¹). These protective effects of rhACE2 were eradicated by the Ang-(1–7)/Mas receptor antagonist A779 (1 μmol L⁻¹). Our results demonstrate the importance of ACE2 in amelioration of kidney fibrosis and renal injury in the ApoE-mutant mice via modulation of the mTOR/ERK signaling and renal Ang-(1–7)/Ang II balance, thus indicating potential therapeutic strategies by enhancing ACE2 action for preventing atherosclerosis and fibrosis-associated kidney disorders.     

Acidic pH increases cGMP accumulation through the OGR1/phospholipase C/Ca2 +/neuronal NOS pathway in N1E-115 neuronal cells

Neuronal NO synthase (nNOS)-mediated cGMP accumulation has been shown to affect a variety of neuronal cell activities, regardless of whether they are detrimental or beneficial, depending on the amount of their levels, under the physiological and pathological situations. In the present study, we examined the role of proton-sensing G protein-coupled receptors (GPCRs), which have been identified as new pH sensors, in the acidic pH-induced nNOS/cGMP activity in N1E-115 neuronal cells. In this cell line, ovarian cancer G protein-coupled receptor 1 (OGR1) and G protein-coupled receptor 4 (GPR4) mRNAs are expressed. An extracellular acidic pH increased cGMP accumulation, which was inhibited by nNOS-specific inhibitors. Acidic pH also activated phospholipase C/Ca^2 + pathways and Akt-induced phosphorylation of nNOS at S1412, both of which have been shown to be critical regulatory mechanisms for nNOS activation. The acidic pH-induced activation of the phospholipase C/Ca^2 + pathway, but not Akt/nNOS phosphorylation, was inhibited by small interfering RNA specific to OGR1 and YM-254890, an inhibitor of G_q/11 proteins, in association with the inhibition of cGMP accumulation. Moreover cGMP accumulation was inhibited by 2-aminoethoxydiphenyl borate, an inhibitor of inositol 1,4,5-trisphosphate channel; however, it was not by wortmannin, a phosphatidylinositol 3-kinase inhibitor, which inhibited Akt/nNOS phosphorylation. In conclusion, acidic pH stimulates cGMP accumulation preferentially through the OGR1/G_q/11 proteins/phospholipase C/Ca^2 +/nNOS in N1E-115 neuronal cells. Akt-mediated phosphorylation of nNOS, however, does not appreciably contribute to the acidification-induced accumulation of cGMP.     

Cardioprotective effects of lipoic acid,quercetin and resveratrol on oxidative stress related to thyroid hormone alterations in long-term obesity

This study investigated possible mechanisms for cardioprotective effects of lipoic acid (LA), quercetin (Q) and resveratrol (R) on oxidative stress related to thyroid hormone alterations in long-term obesity. Female C57BL/6 mice were fed on high-fat diet (HFD), HFD + LA, HFD + R, HFD + Q and normal diet for 26 weeks. Body weight, blood pressure, thyroid hormones, oxidative stress markers, angiotensin converting enzyme (ACE), nitric oxide synthase (NOS) and ion pump activities were measured, and expression of cardiac genes was analyzed by real-time polymerase chain reaction. HFD induced marked increase (P < .05) in body weight, blood pressure and oxidative stress, while plasma triidothyronine levels reduced. ACE activity increased (P < .05) in HFD mice (0.69 ± 0.225 U/mg protein) compared with controls (0.28 ± 0.114 U/mg protein), HFD + LA (0.231 ± 0.02 U/mg protein) and HFD + Q (0.182 ± 0.096 U/mg protein) at 26 weeks. Moreover, Na⁺/K⁺-ATPase and Ca^2 +-ATPase activities increased in HFD mice whereas NOS reduced. A 1.5-fold increase in TRα1 and reduction in expression of the deiodinase iodothyronine DIO1, threonine protein kinase and NOS3 as well as up-regulation of AT1α, ACE, ATP1B1, GSK3β and Cja1 genes also occurred in HFD mice. Conversely, LA, Q and R inhibited weight gain; reduced TRα1 expression as well as increased DIO1; reduced ACE activity and AT1α, ATP1B1 and Cja1 gene expression as well as inhibited GSK3β; increased total antioxidant capacity, GSH and catalase activity; and reduced blood pressure. In conclusion, LA, resveratrol and quercetin supplementation reduces obesity thereby restoring plasma thyroid hormone levels and attenuating oxidative stress in the heart and thus may have therapeutic potential in heart diseases.     

Nonspecific inhibition of nitric oxide synthesis evokes endothelin-dependent increases in myocardial contractility

The role of endogenous nitric oxide (NO) in modulating myocardial contractility is still unclear, in part because of unknown, secondary effects of blocking NO release. We hypothesized that the nonspecific inhibition of nitric oxide synthase (NOS) enhances endothelin-1 (ET-1) effects, which can play a role in ET-A receptor-dependent myocardial contractile responses. The myocardial contractility was estimated from the slope of the left ventricular end-systolic pressure–diameter relationship in closed-chest, pentobarbital-anesthetized dogs. Group 1 (n = 7) was the saline-treated control, while in groups 2 (n = 7) and 3 (n = 7) N-nitro-l-arginine (NNA, 4 mg kg^?1), a nonselective NOS blocker, was administered with or without pretreatment with the ET-A receptor antagonist ETR-P1/fl peptide (100 nmol kg^?1 iv). Plasma ET-1, nitrite/nitrate (NO_x) and blood superoxide levels were measured, and myocardial ET-1 content and xanthine oxidoreductase (XOR) activity were determined from myocardial biopsies. The infusion of NNA over 120 min decreased the plasma NO_x, significantly elevated the plasma ET-1 and blood superoxide levels, and in parallel greatly increased the left ventricular contractility as compared with the untreated controls [47.5 vs 30 mm Hg mm^?1]. The myocardial ET-1 content decreased simultaneously, while the XOR activity and blood superoxide level were significantly elevated. These effects, including NNA-induced positive inotropy, were significantly suppressed by pretreatment with ETR-P1/fl peptide. These results demonstrate that a diminished NO synthesis leads to a preponderant ET-1 effect, which increases myocardial contractility through an ET-A receptor-dependent mechanism.     

Chiral linkers to improve selectivity of double-headed neuronal nitric oxide synthase inhibitors

To develop potent and selective nNOS inhibitors, new double-headed molecules with chiral linkers that derive from natural amino acids or their derivatives have been designed. The new structures contain two ether bonds, which greatly simplifies the synthesis and accelerates structure optimization. Inhibitor (R)-6b exhibits a potency of 32 nM against nNOS and is 475 and 244 more selective for nNOS over eNOS and iNOS, respectively. Crystal structures show that the additional binding between the aminomethyl moiety of 6b and the two heme propionates in nNOS, but not eNOS, is the structural basis for its high selectivity. This work demonstrates the importance of stereochemistry in this class of molecules, which significantly influences the potency and selectivity of the inhibitors. The structure–activity information gathered here provides a guide for future structure optimization.     

Activation of cardiac hypertrophic signaling pathways in a transgenic mouse with the human PRKAG2 Thr400Asn mutation

Human mutations in PRKAG2, the gene encoding the γ2 subunit of AMP activated protein kinase (AMPK), cause a glycogen storage cardiomyopathy. In a transgenic mouse with cardiac specific expression of the Thr400Asn mutation in PRKAG2 (TG^T400N), we previously reported initial cardiac hypertrophy (ages 2–8 weeks) followed by dilation and failure (ages 12–20 weeks). We sought to elucidate the molecular mechanisms of cardiac hypertrophy. TG^T400N mice showed significantly increased cardiac mass/body mass ratios up to ～ 3-fold beginning at age 2 weeks. Cardiac expression of ANP and BNP were ～ 2- and ～ 5-fold higher, respectively, in TG^T400N relative to wildtype (WT) mice at age 2 weeks. NF-κB activity and nuclear translocation of the p50 subunit were increased ～ 2- to 3-fold in TG^T400N hearts relative to WT during the hypertrophic phase. Phosphorylated Akt and p70S6K were elevated ～ 2-fold as early as age 2 weeks. To ascertain whether these changes in TG^T400N mice were a consequence of increased AMPK activity, we crossbred TG^T400N with TG^α2DN mice, which express a dominant negative, kinase dead mutant of the AMPK α2 catalytic subunit and have low myocardial AMPK activity. Genetic reversal of AMPK overactivity led to a reduction in hypertrophy, nuclear translocation of NF-κB, phosphorylated Akt, and p70S6K. We conclude that inappropriate activation of AMPK secondary to the T400N PRKAG2 mutation is associated with the early activation of NF-κB and Akt signaling pathway, which mediates cardiac hypertrophy.     

MicroRNA-224 upregulation and AKT activation synergistically predict poor prognosis in patients with hepatocellular carcinoma

Background and aimPrevious evidence has shown that microRNA (miR)-224 may function as an onco-miRNA in hepatocellular carcinoma (HCC) cells by activating AKT signaling. However, little is known about the clinical significance of the combined expression of miR-224 and phosphorylated-AKT (pAKT) on human HCC. The aim of this study was to investigate the synergistical influence of miR-224 and pAKT on clinical characteristics and prognosis in patients with HCC.MethodsOne-hundred and thirty HCC patients who had undergone curative liver resection were selected. In situ hybridization and immunohistochemistry were respectively performed to detect the expression of miR-224 and pAKT in the respective tumors.ResultsCompared with the adjacent nonneoplastic liver tissues, the expression levels of miR-224 and pAKT protein in HCC tissues were both significantly increased (both P < 0.001). In addition, the combined upregulation of miR-224 and pAKT protein was significantly associated with serum AFP (P = 0.01), tumor stage (P = 0.002) and tumor grade (P = 0.008). Moreover, HCC patients highly expressing both miR-224 and pAKT protein had worse 5-year disease-free survival and 5-year overall survival (both P < 0.001). Furthermore, the Cox proportional hazards model showed that the combined upregulation of miR-224 and pAKT protein (miR-224-high/pAKT-high) may be independent poor prognostic factors for both 5-year disease-free survival (P = 0.008) and 5-year overall survival (P = 0.01) in HCC.ConclusionThese results indicate for the first time that miR-224 upregulation and AKT activation may synergistically associate with tumor progression of HCC. The combined high expression of miR-224 and pAKT may be a potential indicator for predicting unfavorable prognosis in HCC patients.     

Analysis of smoking and LPO in ALS

Smoking has been suggested as one of the risk factor for amyotrophic lateral sclerosis (ALS) development. In order to investigate whether adverse effects of cigarette smoke in ALS have any association with increase in oxidative stress, disease severity, lipid hydroperoxides (LPO) and superoxide dismutase-1 (SOD1) levels were measured in biofluids of smoker and never smoker ALS patients and clinically correlated. Serum and CSF from sporadic ALS patients (n = 50) diagnosed with El Escorial criteria were collected in the study. Serum (n = 50) and CSF (n = 42) were also collected from normal healthy controls. The LPO levels were estimated using commercially available kits. Enzyme-linked immunosorbent assays (ELISAs) were used to quantitate SOD1. Their levels were further analyzed among smoker and never smoker subjects. Significantly elevated LPO in sera and CSF of ALS patients were observed (p < 0.05). There was considerably increased LPO in sera and CSF of smoker ALS subjects matched with disease severity as compared to never smoker ALS (p < 0.05). ALS group did not show any alteration in SOD1 when compared to controls (p > 0.05). In addition, no change has been observed in SOD1 levels in ALS subjects who smoke (p > 0.05). Increased LPO and unaltered SOD1 in ALS patients may suggest the neuro-pathological association of LPO with ALS disease independent of SOD1. With current findings, it may be proposed that LPO levels might constitute as probable biomarker for smoker ALS patients, however, it cannot be concluded without larger gender matched studies. Additional investigations are needed to determine whether LPO upregulation is primary or secondary to motor neuron degeneration in ALS.     

Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor

The continuing threat to biodiversity lends urgency to the need of identification of sustainable source of natural products. This is not so much trouble if there is a microbial source of the compound. Herein, violacein, a natural indolic pigment extracted from Chromobacterium violaceum, was evaluated for its antitumoral potential against the Ehrlich ascites tumor (EAT) in vivo and in vitro. Evaluation of violacein cytotoxicity using different endpoints indicated that EAT cells were twofold (IC₅₀ = 5.0 μM) more sensitive to the compound than normal human peripheral blood lymphocytes. In vitro studies indicated that violacein cytotoxicity to EAT cells is mediated by a rapid (8–12 h) production of reactive oxygen species (ROS) and a decrease in intracellular GSH levels, probably due to oxidative stress. Additionally, apoptosis was primarily induced, as demonstrated by an increase in Annexin-V positive cells, concurrently with increased levels of DNA fragmentation and increased caspase-2, caspase-9 and caspase-3 activities up to 4.5-, 6.0- and 5.5-fold, respectively, after 72 h of treatment. Moreover, doses of 0.1 and 1.0 μg kg^?1 violacein, administered intraperitoneally (i.p.) to EAT-bearing mice throughout the lifespan of the animals significantly inhibited tumor growth and increased survival of mice. In view of these results, a 35-day toxicity study was conducted in vivo. Complete hematology, biochemistry (ALT, AST and creatinine levels) and histopathological analysis of liver and kidney indicated that daily doses of violacein up to 1000 μg kg^?1 for 35 days are well tolerated and did not cause hematotoxicity nor renal or hepatotoxicity when administered i.p. to mice. Altogether, these results indicate that violacein causes oxidative stress and an imbalance in the antioxidant defense machinery of cells culminating in apoptotic cell death. Furthermore, this is the first report of its antitumor activity in vivo, which occurs in the absence of toxicity to major organs.     

Effect of peppermint (Mentha piperita) oil on in vitro methanogenesis and fermentation of feed with buffalo rumen liquor

The effect of inclusion of peppermint (Mentha piperita) oil (at 0, 0.33, 1.0 and 2.0 μl/ml of incubation medium) on gas and methane production, fermentation of feed and microbial profile was studied in in vitro gas production test, using 200 mg of wheat straw and concentrate mixture in equal proportion as substrate in a 100 ml graduated syringe. The buffalo rumen liquor was used as inoculum and the observations were recorded at 24 h of incubation. Methane emission was reduced (P<0.001) by 19.9%, 46.0% and 75.6% at 0.33, 1.0 and 2.0 μl levels, respectively. The concentration (mM/100 ml) of total volatile fatty acids was reduced (P<0.01) by inclusion of peppermint oil at higher levels (1.0 and 2.0 μl) whereas at 0.33 μl level there was no effect. The proportion of acetate increased (P<0.05) and that of propionate decreased (P<0.001) at 1.0 and 2.0 μl levels of peppermint oil. There was a fall (P<0.001) in carboxymethylcellulase and xylanase activities and the inhibition increased with the increasing level of peppermint oil which resulted in a dose dependent decrease (P<0.05) in in vitro true digestibility of feed. At 0.33 μl level of peppermint oil, the population density of total bacteria was similar to that of control but fungi, Ruminococcus flavefaciens and methanogens increased by 4-, 6- and 2-folds, respectively, as determined with real-time PCR. At 1.0 and 2.0 μl levels the population density of total bacteria, fungi, Fibrobacter succinogens and methanogens decreased drastically and fell below the control values. The numbers of holotrichs and spirotrichs were reduced (P<0.001) by increasing dose of peppermint oil. The higher doses of peppermint oil were toxic for the rumen microbes but the lower levels could be further explored in in vivo experiments as rumen modifier to reduce methanogenesis.