Central role of endogenous Toll-like receptor-2 activation in regulating inflammation, reactive oxygen species production, and subsequent neointimal formation after vascular injury

Background

It is now evident that inflammation after vascular injury has significant impact on the restenosis after revascularization procedures such as angioplasty, stenting, and bypass grafting. However, the mechanisms that regulate inflammation and repair after vascular injury are incompletely understood. Here, we report that vascular injury-mediated cytokine expression, reactive oxygen species (ROS) production, as well as subsequent neointimal formation requires Toll-like receptor-2 (TLR-2) mediated signaling pathway in vivo.

Methods and results

Vascular injury was induced by cuff-placement around the femoral artery in non-transgenic littermates (NLC) and TLR-2 knockout (TLR-2KO) mice. After cuff-placement in NLC mice, expression of TLR-2 was significantly increased in both smooth muscle medial layer and adventitia. Interestingly, we found that inflammatory genes expression such as tumor necrosis factor-α, interleukin-1β (IL-1β), IL-6, and monocyte chemoattractant protein-1 were markedly decreased in TLR-2KO mice compared with NLC mice. In addition, ROS production after vascular injury was attenuated in TLR-2KO mice compared with NLC mice. Since we observed the significant role of endogenous TLR-2 activation in regulating inflammatory responses and ROS production after vascular injury, we determined whether inhibition of endogenous TLR-2 activation can inhibit neointimal proliferation after vascular injury. Neointimal hyperplasia was markedly suppressed in TLR-2KO mice compared with WT mice at both 2 and 4 weeks after vascular injury.

Conclusions

These findings suggested that endogenous TLR-2 activation might play a central role in the regulation of vascular inflammation as well as subsequent neointimal formation in injured vessels.     

RANTES gene mRNA expression and its -403 G/A promoter polymorphism in coronary artery disease

Objective

Increased RANTES expression has been described to have a role in atherosclerosis plaque formation. Functional polymorphisms within RANTES promoter region have shown association with increased risk of coronary atherosclerosis (CAD). The aim of this study was to examine the RANTES mRNA expression in patients with CAD compared to patients without CAD and its association with RANTES − 403 G/A polymorphism in an Iranian population.

Methods

The study was performed on 319 patients who underwent coronary artery angiography and patients with > 50% stenosis in vessels considered as case groups (CAD+) N = 191 and normal vessels group as control (CAD−) N = 128. In each group 20 patients were examined for RANTES mRNA expression.RANTES mRNA expression was examined using quantitative real-time PCR. Genotyping of − 403 polymorphism was performed using PCR-RFLP technique.

Results

We found that RANTES mRNA expression was increased to 1.37 fold in CAD patients compared to the controls but the difference was not statistically significant. Also comparing the RANTES mRNA expression in patients with different RANTES − 403 G/A polymorphism showed that in patients carrying AA genotype RANTES mRNA expression was increased to 1.74 fold compared to patients carrying GG genotype and to 1.51 fold compared to patients carrying GA genotype. No significant difference for allele and genotype frequencies of RANTES − 403 polymorphism was found between cases and controls.

Conclusion

More studies on larger number of samples are required to further evaluate role of RANTES in pathogenesis of CAD.     

WT1 CpG islands methylation in human lung cancer: A pilot study

Background

CpG island hypermethylation of gene promoters and regulatory regions is a well-known mechanism of epigenetic silencing of tumor suppressors and is directly linked to carcinogenesis. Wilm’s tumor gene (WT1) is a tumor suppressor protein involved in the regulation of human cell growth and differentiation and a modulator of oncogenic K Ras signaling in lung cancer. Changes in the pattern of methylation of the WT1 gene have not yet been studied in detail in human lung cancer. In this study we compared the methylation profile of WT1 gene in samples of neoplastic and non-neoplastic lung tissue taken from the same patients.

Methods

DNA was extracted from neoplastic and normal lung tissue obtained from 16 patients with non small cell lung cancer (NSCLC). The methylation status of 29 CpG islands in the 5′ region of WT1 was determined by pyrosequencing. Statistical analysis was carried out by T test and Mann Whitney test.

Results

The mean percentage of methylation, considering all CpG islands of WT1 in the neoplastic tissues of the 16 NSCLC patients, was 16.2 ± 3.4, whereas in the normal lung tissue from the same patients it was 5.6 ± 1.7 (p < 0.001). Adenocarcinomas presented higher methylation levels than squamous cell carcinomas (p < 0,001).

Conclusions

Methylation of WT1 gene is significantly increased in NSCLC. Both histotype and exposure to cigarette smoke heavily influence the pattern of CpG islands which undergo hypermethylation.     

Relación entre el rendimiento en la prueba de estación unipodal y la velocidad del centro de presión en adultos mayores

Introduction

Frequent falls are one of the most important health problems in the elderly population. The unipedal stance test (UPST), asses postural stability and is used in fall risk measures. Despite this, there is little information about its relationship with posturographic parameters (PP) that characterizes postural stability. Center of pressure velocity (CoPV) is one of the best PP that describes postural stability. The aim of this study was to analyze the relation between UST score and CoPV in elderly population.

Materials and methods

A sample of 38 healthy elderly subjects where divided in two groups according to their UPST score, low performance (LP, n = 11) and high performance (HP, n = 27). The correlation between UPST score and COP mean velocity (CoPmV), recorded from a posturographic test, was analyzed between both groups.

Results

An inverse correlation between UPST score and CoPmV was found in both groups. However, this was higher in the LP group (r = −0.69, P = .02) compared to the HP (r = −0.39, P = .04).

Conclusions

Based on the results of this investigation, it may be concluded that the achievement on UPST has an inverse relationship with CoPmV, especially in subjects with low performance in the UPST.     

Protective effects of allopurinol against acute liver damage and cirrhosis induced by carbon tetrachloride: Modulation of NF-κB, cytokine production and oxidative stress

Background

The aim of this work was to evaluate the hepatoprotective ability of allopurinol to prevent the liver injury induced by carbon tetrachloride (CCl₄).

Methods

Acute liver damage was induced with CCl₄ (4 g/kg, by gavage); allopurinol (50 mg/kg, by gavage) was given 1 h before and 1 h after CCl₄ intoxication and two daily doses for the previous three days. Cirrhosis was established by CCl₄ administration (0.4 g/kg, i. p. three times a week, eight weeks); allopurinol was administered (100 mg/kg, by gavage, daily) during the long-term of CCl₄ treatment. Alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), xanthine oxidase (XO), lipid peroxidation, reduced and oxidized glutathione (GSH, GSSG, respectively), hydroxyproline and histopathologycal analysis were performed. Nuclear factor-κB (NF-κB), pro-inflammatory and anti-inflammatory cytokines, transforming growth factor-β (TGF-β) and metalloproteinase-13 (MMP-13) were analyzed by Western blots.

Results

Acute injury increased ALT and γ-GTP activities, additionally enhanced NF-κB nuclear translocation and cytokines production such as tumor necrosis factor-α, interleukine-1β, and interleukine-6. Allopurinol partially prevented these effects, while increased interleukine-10. Acute and chronic CCl₄ treatments altered the levels of XO activity, lipid peroxidation, and GSH/GSSG ratio, while these remained within normal range with allopurinol administration. Necrosis, fibrosis and TGF-β production induced in chronic injury were partially prevented by allopurinol, interestingly, this drug induced MMP-13 activity.

Conclusions

Allopurinol possesses antioxidant, anti-inflammatory and antifibrotic properties, probably by its capacity to reduce NF-κB nuclear translocation and TGF-β expression, as well as to induce MMP-13.General significanceAllopurinol might be effective treatment of liver diseases.     

Relation between 3435C>T multidrug resistance 1 gene polymorphism with high dose methylprednisolone treatment of childhood acute idiopathic thrombocytopenic purpura

The current study was conducted to assess 3435C>T multidrug resistance 1 gene polymorphism and the efficacy of high dose methylprednisolone (HDMP) in childhood acute idiopathic thrombocytopenic purpura patients.

Methods

A total of 31 childhood acute Idiopathic thrombocytopenic purpura patients (17 females, 14 males) between the ages of 2 and 16 years of age were included in the study. High-dose methylprednisolone was given at a dose of 30 mg/kg/day for 3 days and 20 mg/kg/day for 4 days, consecutively and intravenously. Polymerase chain reaction-restriction fragment length polymorphism was used for the detection of C3435T single nucleotide polymorphism. Fragments obtained were 238 bp to T/T genotype, 172 bp and 60 bp fragments to the C/C genotype, and 238 bp, 172 bp and 60 bp to the C/T genotype.

Results

The distribution of CC, CT, and TT genotypes were 19.0%, 61.3%, and 19.4%, respectively. Both allele frequencies of C and T were the same — 50%. There was no significant difference in genotype and allele distribution between the patients with ITP and the control group (χ² = 0.84 p = 0.65, χ² = 0.2 p = 0.63, respectively). There were no significant differences in age, gender, and pre- and post-treatment platelet counts between CC, CT, and TT genotypes of the MDR gene. Response to treatment shows no significant difference between genotype and allele groups.

Conclusion

In our study, there was no difference in the HDMP treatment response between MDR1 gene genotypes. However, it should be noted that this study includes a small group of patients. Our data should therefore be considered preliminary, awaiting further confirmatory studies on an expanded patient base.     

A re-investigation of saliva collection procedures that highlights the risk of potential positive interference in cortisol immunoassay

Background

The use of saliva for measurement of cortisol permits non-invasive study of adrenal function, but collection can be technically difficult, particularly in small infants. Saliva collection can be assisted by citric acid to increase saliva flow, or by the use of cotton or polyester swabs in the mouth.

Aim

To determine whether different methods of saliva collection affect cortisol radioimmunoassay (RIA) performance.

Experimental

Cortisol was measured in saliva collected from 16 adults using intra-oral cotton swabs or polyester swabs, compared with saliva dribbled directly into a pot either alone (plain saliva) or after citric acid had been placed on the tongue. An in-house RIA, without prior extraction, was used to measure cortisol with an encapsulated sheep antibody.

Results

Mean (median) salivary cortisol was 10.9 (10.5) nmol L⁻¹ in plain saliva, 10.4 (8.4) nmol L⁻¹ in citric acid stimulated saliva; 25.3 (25.1) nmol L⁻¹ in saliva collected on cotton swabs, and 27.9 (27.3) nmol L⁻¹ collected on polyester swabs. Cortisol in saliva collected using citric acid was not significantly different from plain saliva (p = 0.997), but cortisol in saliva collected using cotton and polyester swabs was significantly higher than that of plain saliva (p < 0.01).

Conclusion

The use of cotton or polyester swabs for collection of saliva can result in spuriously high levels of cortisol when measured by RIA.     

Resultados de la endarterectomía carotídea en pacientes ancianos

Introduction

Forty-five per cent of stoke patients have a surgically accessible stenosis. The objective of our study is to describe the response to carotid endarterectomy (EA) in patients of advanced age compared to younger ones.

Material and method

Retrospective evaluation of the clinical history of all patients who underwent an endarterectomy in a tertiary hospital between January 1995 and December 2006. The patients were grouped into those 75 years or older and those less than this age. The incidence of peri-operative complications in the first month after surgery, and the long-term mortality was evaluated using a survival analysis.

Results

Data were collected on 147 EA in 134 patients of 75 years or more, and on 201 EA in 177 patients less than 75 years-old. The incidence of peri-operative complications was similar in both groups, with a mortality of 2% in the older age group and a stroke incidence of 2.6% (half transient ischaemic accidents). The older patients had a mean follow-up of 4.1 years, with a survival of 86% at one year and 54% at 5 years and with the main cause of death being heart disease.

Conclusions

Carotid EA is a safe and effective technique for the treatment of extracranial carotid stenosis in the elderly, having the same peri-operative morbidity and mortality as younger ones. Age must not affect our therapeutic attitude, although an exhaustive cardiology study must be made in the elderly prior to the operation.     

Efectos del entrenamiento en estrategias de regulación emocional en el bienestar de cuidadores de enfermos de Alzheimer

Introduction

The present research shows the results of a psychoeducational intervention programme centered on the regulation of the emotion among Alzheimer patients’ caregivers.

Materials and methods

52 informal caregivers of Alzheimer's patients participated. These caregivers were distributed into two groups: the experimental group (n = 20) and the control group (n = 32). All the participants were evaluated before and after the intervention programme through the application of different measurement tools measuring variables related to the care giving process; stressors, modulation variables and care giving consequences.

Results

In the inter group contrast, the experimental group, when compared with the control condition, obtained higher scores in positive affect, subjective well-being, regulation of emotions, and satisfaction with caregiving. However, the experimental group recorded lower values in perceived stress and negative affect. With reference to the intragroup contrast, the experimental group showed a significant decrease in dysfunctional thoughts and emotional attention. The control group registered higher levels of psychosocial support and lower satisfaction with caregiving.

Conclusions

The training programme, that we both developed and conducted, has contributed to a greater feeling of emotional well-being amongst the its participant caregivers, who now take more adequate care of their emotions and suffer fewer dysfunctional thoughts in relation to caregiving. In future studies, the stability of the results presented in this investigation should be established due to the progressive character of the skills learned during the programme, and the changing needs associated with the caregiving process.     

Study of the initial phase of biofilm formation using a biofomic approach

Aims

The purpose of this work was to study the initial steps of formation of a biofilm using the BioFilm Ring Test® and the Crystal violet staining technique.

Methods and results

Eight strains of Pseudomonas aeruginosa were studied. The two methods revealed that four strains formed a rapid biofilm. The biofilm formed by these strains was detected after only 45 min with the BioFilm Ring Test® and after 6 h with the Crystal violet method. The enumeration of bacteria of the PA01 strain confirmed that, after 30 min, a significant amount of bacteria had attached on the bottom of the culture wells. After 48 h the Crystal violet method detected a biofilm with all strains. The four strains which rapidly formed a biofilm did not differ from the slow-forming strains by their mucoid character or their swarming motility or their synthesis of rhamnose. They showed higher swimming mobility.

Conclusions

Our results show that the BioFilm Ring Test® is a method specially suited for the study of the initial phase of the formation of a biofilm.

Significance and impact of study

The BioFilm Ring Test® is an easy and rapid alternative to the Crystal violet staining and the enumeration methods.     

Common polymorphisms in the P-selectin gene in women with recurrent spontaneous abortions

Background

To investigate possible associations of P-selectin polymorphisms with idiopathic recurrent pregnancy loss (RPL).

Methods

Study subjects comprised 270 consecutive RPL cases attending outpatient maternity services, and 322 multi-parous control women. P-selectin genotyping was done by PCR-RFLP and PCR-ASA methods.

Results

The P-selectin variants rs1800807, rs1800805, and rs6127, were in Hardy Weinberg equilibrium, and low linkage disequilibrium was noted between the three studied SNPs. The frequency of rs6127 A allele (P < 0.001I), but not rs1800807 C allele (P = 0.957) or rs1800805 A allele (P = 0.760), was higher in RPL cases than in control women. Significant differences in the distribution of rs6127 (P < 0.001), but not rs1800807 (P = 0.444) or rs1800805 (P = 0.391) genotypes were seen between cases and controls, and only rs6127 showed a significant association with RPL, with increments of 2.65 and 4.96 in disease risk seen for heterozygous and homozygous carriers, respectively. Among the 8 three-locus Pselectin haplotypes constructed (rs1800807/rs1800805/rs6127), increased frequency of GGG (Pc = 0.0249), CGG (Pc = 0.0256), and CAG (Pc = 0.0174) haplotypes, and lower frequency of CGA haplotype (Pc = 0.0091) were seen in RPL cases, thus conferring disease susceptibility and protective nature to these haplotypes, respectively.

Conclusions

P-selectin gene polymorphisms and haplotypes contribute to RPL development.     

Pharmacological mechanism responsible for the Atractylodes japonica-induced distal colonic contraction in rats

Background and aim

Atractylodes japonica Koidz (Compositae) has been commonly used to treat the gastrointestinal (GI) disorders in Korean traditional medicine, but its pharmacological roles in the regulation of GI motility have not been clarified yet.

Methods

Atractylodes japonica was sequentially partitioned with MeOH, n-hexane, CHCl₃, EtOAc and n-BuOH saturated with H₂O, and the effects of Atractylodes japonica extracts on the spontaneous contractility of GI muscle strips prepared from rats were measured.

Results

Among five different fractionations, EtOAc extracts of Atractylodes japonica (AJEA) dose-dependently increased the low frequency contraction of distal colon longitudinal muscles (DCLM), and the ED₅₀ values were revealed to be 1.71 × 10⁻⁹ g/ml. Among GI tracts, a prominent contractile response to AJEA was observed only in the DCLM. The contractile patterns produced by AJEA remarkably differed from those caused by acetylcholine and 5-HT. 4-DAMP and methoctramine at 0.5 μM significantly blocked the AJEA (1.0 μg/ml)-induced contraction of DCLM, but ondansetron, GR113808 and methysergide at 1.0 μM in combination did not change the AJEA-induced DCLM contractions. Acetylethylcholine mustard (5.0 μM) significantly diminished the AJEA-induced DCLM contractions, whereas p-chlorophenyl alanine (1.0 μM) did not affect the stimulatory effects of AJEA on the DCLM contractions.

Conclusion

The present results suggest that AJEA may specifically act on the DCLM among GI smooth muscles, and AJEA-induced DCLM contraction is likely mediated, at least, by activation of ChAT and acetylcholinergic muscarinic receptors.     

Effects of recombinant adeno-associated viral vectors on angiopoiesis and osteogenesis in cultured rabbit bone marrow stem cells via co-expressing hVEGF and hBMP genes: A preliminary study in vitro

Objective

VEGF and BMP play important roles in angiogenesis and osteogenesis. Combining these two factors may be a promising therapeutic strategy for avascular necrosis of the femoral head (ANFH).

Methods

Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and purified by density gradient centrifugation combined with attachment culture methods. The purity and characteristics of the BMSCs were detected by cell surface antigen identification. The best MOI of BMSCs transfected with rAAV was detected by fluorescent cell counting, and cell viability was determined by MTT assay. Expression of the genes of interest was detected by GFP gene expression, RT-PCR assay, and ELISA assay. The biological activities of VEGF and BMP were detected by angiogenic and osteogenic assays.

Results

The best MOI of BMSCs transfected with rAAV was 5 × 10⁴ v.g./cell. Cell growth curves showed vigorous cell viability. Expressions of the GFP, VEGF165, and BMP₇ genes were detected 1 day post-transfection and peaked 14 days post-transfection. Expression of the genes of interest was sustained over 1 month. VEGF and BMP proteins secreted from BMSCs transfected with rAAV-hVEGF₁₆₅-IRES-hBMP₇ enhanced angiogenesis and osteogenesis in vitro.

Conclusion

Recombinant adeno-associated viral vectors co-expressing the hVEGF₁₆₅ and hBMP₇ genes showed efficient gene expression ability. The VEGF₁₆₅ and BMP₇ proteins expressed from the vector have efficient biological activity in vitro.     

Serum miR-146a and miR-223 as potential new biomarkers for sepsis

Objective

Current biomarkers cannot completely distinguish sepsis from systemic inflammatory response syndrome (SIRS) caused by other non-infectious diseases. Circulating microRNAs (miRNAs) are promising biomarkers for several diseases, but their correlation with sepsis is not totally clarified.

Methods

Seven miRNAs related to inflammation or infection were included in the present study. Serum miRNA expression was investigated in 50 patients diagnosed with sepsis, 30 patients with SIRS and 20 healthy controls to evaluate the diagnostic and prognostic value. Expression levels of serum miRNAs were determined by quantitative PCR using the Qiagen miScript system. Serum CRP and IL-6 levels were determined by enzyme linked immunosorbent assay.

Results

Serum miR-146a and miR-223 were significantly reduced in septic patients compared with SIRS patients and healthy controls. The areas under the receiver operating characteristic curve of miR-146a, miR-223 and IL-6 were 0.858, 0.804 and 0.785, respectively.

Conclusion

Serum miR-146a and miR-223 might serve as new biomarkers for sepsis with high specificity and sensitivity. (ClinicalTrials.gov number, NCT00862290.)     

Semaphorin-plexin signalling genes associated with human breast tumourigenesis

Introduction

Gene expression profiling has enabled us to demonstrate the heterogeneity of breast cancers. The potential of a tumour to grow and metastasise is partly dependant on its ability to initiate angiogenesis or growth and remodelling of new blood vessels, usually from a pre-existing vascular network, to ensure delivery of oxygen, nutrients, and growth factors to rapidly dividing transformed cells along with access to the systemic circulation. Cell-cell signalling of semaphorin ligands through interaction with their plexin receptors is important for the homeostasis and morphogenesis of many tissues and has been widely studied for a role in neural connectivity, cancer, cell migration and immune responses. This study investigated the role of four semaphorin/plexin signalling genes in human breast cancers in vivo and in vitro.

Materials and methods

mRNA was extracted from formalin fixed paraffin embedded archival breast invasive ductal carcinoma tissue samples of progressive grades (grades I-III) and compared to tissue from benign tumours. Gene expression profiles were determined by microarray using the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays and validated by Q-PCR using a Corbett RotorGene 6000. Following validation, the gene expression profile of the identified targets was correlated with those of the human breast cancer cell lines MCF-7 and MDA-MD-231.

Results

The array data revealed that 888 genes were found to be significantly (p ≤ 0.05) differentially expressed between grades I and II tumours and 563 genes between grade III and benign tumours. From these genes, we identified four genes involved in semaphorin-plexin signalling including SEMA4D which has previously been identified as being involved in increased angiogenesis in breast cancers, and three other genes, SEMA4F, PLXNA2 and PLXNA3, which in the literature were associated with tumourigenesis, but not directly in breast tumourigenesis. The microarray analysis revealed that SEMA4D was significantly (P = 0.0347) down-regulated in the grade III tumours compared to benign tumours; SEMA4F, was significantly (P = 0.0159) down-regulated between grades I and II tumours; PLXNA2 was significantly (P = 0.036) down-regulated between grade III and benign tumours and PLXNA3 significantly (P = 0.042) up-regulated between grades I and II tumours. Gene expression of SEMA4D was validated using Q-PCR, demonstrating the same expression profile in both data sets. When the sample set was increased to incorporate more cases, SEMA4D continued to follow the same expression profile, including statistical significance for the differences observed and small standard deviations. In vitro the same pattern was present where expression for SEMA4D was significantly higher in MDA-MB-231 cells when compared to MCF-7 cells. The expression of SEMA4F, PLXNA2 and PLXNA3 could not be validated using Q-PCR, however in vitro analysis of these three genes revealed that both SEMA4F and PLXNA3 followed the microarray trend in expression, although they did not reach significance. In contrast, PLXNA2 demonstrated statistical significance and was in concordance with the literature.

Discussion

We, and others, have proposed SEMA4D to be a gene with a potentially protective effect in benign tumours that contributes to tumour growth and metastatic suppression. Previous data supports a role for SEMA4F as a tumour suppressor in the peripheral nervous system but our data seems to indicate that the gene is involved in tumour progression in breast cancer. Our in vitro analysis of PLXNA2 revealed that the gene has higher expression in more aggressive breast cancer cell types. Finally, our in vitro analysis on PLXNA3 also suggest that this gene may have some form of growth suppressive role in breast cancer, in addition to a similar role for the gene previously reported in ovarian cancer. From the data obtained in this study, SEMA4D may have a role in more aggressive and potentially metastatic breast tumours.

Conclusions

Semaphorins and their receptors, the plexins, have been implicated in numerous aspects of neural development, however their expression in many other epithelial tissues suggests that the semaphorin-plexin signalling system also contributes to blood vessel growth and development. These findings warrant further investigation of the role of semaphorins and plexins and their role in normal and tumour-induced angiogenesis in vivo and in vitro. This may represent a new front of attack in anti-angiogenic therapies of breast and other cancers.     

Large enhancement of skeletal muscle cell glucose uptake and suppression of hepatocyte glucose-6-phosphatase activity by weak uncouplers of oxidative phosphorylation

Background

Perturbation of energy homeostasis in skeletal muscle and liver resulting from a transient inhibition of mitochondrial energy transduction can produce effects of relevance for the control of hyperglycemia through activation of the AMP-activated protein kinase, as exemplified by the antidiabetic drug metformin. The present study focuses on uncoupling of oxidative phosphorylation rather than its inhibition as a trigger for such effects.

Methods

The reference weak uncoupler 2,4-dinitrophenol, fourteen naturally-occurring phenolic compounds identified as uncouplers in isolated rat liver mitochondria, and fourteen related compounds with little or no uncoupling activity were tested for enhancement of glucose uptake in differentiated C2C12 skeletal muscle cells following 18 h of treatment at 25-100 μM. A subset of compounds were tested for suppression of glucose-6-phosphatase (G6Pase) activity in H4IIE hepatocytes following 16 h at 12.5-25 μM. Metformin (400 μM) was used as a standard in both assays.

Results

Dinitrophenol and nine of eleven compounds that induced 50% or more uncoupling at 100 μM in isolated mitochondria enhanced basal glucose uptake by 53 to 269%; the effect of the 4′-hydroxychalcone butein was more than 6-fold that of metformin; negative control compounds increased uptake by no more than 25%. Dinitrophenol and four 4′-hydroxychalconoids also suppressed hepatocyte G6Pase as well as, or more effectively than metformin, whereas the unsubstituted parent compound chalcone, devoid of uncoupling activity, had no effect.

Conclusions

Activities key to glycemic control can be induced by a wide range of weak uncouplers, including compounds free of difficult-to-metabolize groups typically associated with uncouplers.

General significance

Uncoupling represents a valid and possibly more efficient alternative to inhibition for triggering cytoprotective effects of therapeutic relevance to insulin resistance in both muscle and liver. Identification of actives of natural origin and the insights into their structure-activity relationship reported herein may lead to alternatives to metformin.     

American ginseng (Panax quinquefolius) prevents glucose-induced oxidative stress and associated endothelial abnormalities

Purpose

Ginseng (Araliaceae), demonstrates widespread biological effects because of its purported antioxidant and other properties. The present study was undertaken to investigate the effects of American ginseng root extract on glucose-induced oxidative stress and associated oxidative damage to human umbilical vein endothelial cells (HUVECs).

Methods

Following pretreatment with various concentrations of ginseng (alcoholic extract), HUVECs were incubated with various concentrations of d-glucose ranging from 5 to 25 mmol/l for 24 h. l-Glucose was used at a concentration of 25 mmol/l as a control.

Results

Glucose-induced oxidative stress detected by intracellular reactive oxygen species accumulation, superoxide anion generation and DNA damage in HUVECs were significantly prevented by ginseng. Treatment of HUVECs with ginseng further led to significant prevention of glucose-induced NF-κB activation. Glucose-induced increase in fibronectin (FN), EDB⁺FN (a splice variant of FN), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) mRNAs and protein levels were also prevented by ginseng treatment.

Conclusion

These data indicate that American ginseng prevented glucose-induced damage in the HUVECs through its antioxidant properties.