Tanshinone IIA protects against myocardial ischemia reperfusion injury by activating the PI3K/Akt/mTOR signaling pathway

ObjectiveTo determine the mechanism by which Tanshinone IIA (Tan IIA) relieves myocardial ischemia reperfusion injury (MIRI) in rats via the PI3K/Akt/mTOR signaling pathway.MethodsSprague-Dawley (SD) rats received an intravenous injection of Tan IIA and LY294002 and were divided into the sham, control (myocardial ischemia reperfusion), Tan-L (low-dose Tan IIA), Tan-H (high-dose Tan IIA), Tan-L + LY (low-dose Tan IIA + LY294002), Tan-H + LY (high-dose Tan IIA + LY294002) and LY (LY294002) groups. Cardiomyocytes obtained from neonatal rats were treated with hypoxia reoxygenatin, Tan IIA and LY294002 and divided into the blank, control, Tan-L, Tan-H, Tan-L + LY, Tan-H + LY and LY groups. Creatine kinase MB isoenzyme (CK-MB) and lactic dehydrogenase (LDH) levels in serum and cardiomyocytes were measured. Area of necrosis/area at risk (AN/AAR) was determined with double staining of TTC and Evan’s blue; viability and apoptosis of cardiomyocytes with MTT and TUNEL assays; SOD, MDA, H₂O₂, SDH and COX levels in heart mitochondria together with PI3K/Akt/mTOR and eNOS expressions and phosphorylation with Western blotting.ResultsThe Tan-L and Tan-H groups showed a remarkable decrease in AN/AAR, serum CK-MB and LDH, mitochondrial MDA and H₂O₂ levels but an increase in SOD activity, SDH and COX levels compared with the control group. However, compared with the Tan-L and Tan-H groups, the Tan-L + LY, Tan-H + LY and LY groups indicated an inverse tendency of those indicators. As shown by MTT and TUNEL, the control group had more severe cell damage than the blank group. Furthermore, cell damage and apoptosis were less severe in the Tan-L and Tan-H groups than in the control group, while the Tan-L + LY, Tan-H + LY and LY groups showed an opposite tendency when compared with the Tan-L and Tan-H groups. Meanwhile, the Tan-L and Tan-H groups showed significantly higher expression levels of PI3K, p-Akt/Akt, mTOR and p-eNOS/eNOS than the control group, whereas the Tan-L + LY, Tan-H + LY and LY groups had lower expression levels than the Tan-L and Tan-H groups.ConclusionOur study provided evidence that Tan IIA could activate the PI3K/Akt/mTOR signaling pathway to relieve MIRI in rats.     

The role of feed regulating peptides on weight loss in patients with pulmonary tuberculosis

PurposeMalnutrition is a prominent feature of tuberculosis (TB). The aim of our study was to explore the function of plasma regulatory proteins in pulmonary TB and to investigate the relationship between these parameters and loss of body weight.MethodsPlasma levels of fasting insulin, leptin, ghrelin, adiponectin and orexin-A were measured in 23 pulmonary TB patients, 39 patients with pulmonary sarcoidosis, 22 patients with different diffuse interstitial lung diseases and 21 healthy patients serving as controls.ResultPlasma leptin (p < 0.001) and orexin-A (p < 0.01) levels were significantly decreased in TB patients compared with those of the other study subjects. TB patients also had higher levels of plasma ghrelin compared with those of the other study subjects, while sarcoidosis patients had higher plasma adiponectin levels than the other study subjects. Glucose levels were similar in all groups, yet, insulin and Homeostasis Model of Assessment—Insulin Resistance (HOMA-IR) levels were significantly higher in the TB group compared to the other study groups. There was no correlation between leptin, ghrelin, adiponectin and orexin-A and other parameters.ConclusionsThese data suggest that leptin and orexin-A levels have effects on weight loss in patients with pulmonary tuberculosis. Particularly, leptin may play a role in the early immune response to pulmonary TB and prolonged inflammation may further suppress leptin production. Measurement of HOMA-IR can indeed be used as a marker for the risk of activated TB. Further clinical studies are needed to better understand the role of feed regulating proteins in pulmonary tuberculosis.     

The influence of lipoic acid on caveolin-1-regulated antioxidative enzymes in the mouse model of acute ulcerative colitis

AimThis study was undertaken to verify if two-weeks treatment of lipoic acid (LA) influence colon damage and pro-inflammatory cytokine synthesis during DSS-induced acute colitis. Moreover, as LA has anti-oxidative properties, we analyzed its influence on the level of antioxidative enzymes, HO-1 and eNOS, and their regulator- caveolin-1.MethodsLA was administrated to male C57/BALBc mice at a dose of 25 or 50 mg/kg/day (i.p.) for 21 days. Acute colitis was induced by administration of 4% DSS (w/v) in drinking water for 5 days, followed by 2 days of normal drinking water. Mice in LA + DSS groups were treated with LA (25 or 50 mg/kg/day; i.p.) starting 14 days prior to 4% DSS. Control group received saline for 21 days. In the colon tissue we measured myeloperoxidase activity (MPO), IL-1β, IL-6, IL-17A, IL-23 (ELISA method), and tissue level of cav-1, phospho-eNOS, total eNOS and HO-1 (Western blot).ResultsAdministration of DSS significantly increased total colon damage (p < 0.001), myeloperoxidase (MPO) activity (p < 0.05) and pro-inflammatory IL-6 (p < 0.05). There was also a tendency towards higher IL-1β, IL-17A, and IL-23 in the colon. LA alone did not influence total colon damage, MPO activity, and pro-inflammatory cytokines concentration compared to control (p < 0.05). Notably, mice treated with LA and DSS had significantly decreased total colon damage score (p < 0.001), despite augmented colon MPO activity (p < 0.01), but similar (IL-17A) or even significantly higher level (IL-1β, IL-23) as compared to the DSS group (p < 0.05). IL-6 was insignificantly decreased after LA treatment at a dose of 50 mg/kg.In acute colitis there was a tendency towards an increase in cav-1 and HO-1 and a decrease p-eNOS/total eNOS ratio. Moreover, the LA + DSS groups had higher expression of HO-1 and p-eNOS/total eNOS (p < 0.05) compared to the DSS group, and a tendency towards higher cav-1 level. The changes did not depend on LA dose.ConclusionOur study indicated that LA, at lower doses, may influence cav-1-regulated antioxidative enzyme levels (HO-1 and p-eNOS/total eNOS) despite an increase in colon pro-inflammatory cytokine levels during acute colitis. Hence, LA treatment may be – to some extent – beneficial in attenuation of acute colitis.     

Lactobacillus rhamnosus induced epithelial cell apoptosis,ameliorates inflammation and prevents colon cancer development in an animal model

Background/AimProbiotics have been suggested as prophylactic measure in colon carcinogenesis. This study aimed at determining the potential prophylactic activity of Lactobacillus rhamnosus GG CGMCC 1.2134 (LGG) strain on colorectal carcinogenesis via measuring its effect on Nuclear factor kappa B (NFκB) inflammatory pathway and apoptosis.Materials and methods64 Sprague Dawley rats were grouped into four as follows; Group 1 (Healthy control), Group 2 (LGG), Group 3 (cancer control Dimethyl hydrazine (DMH)) and Group 4 (LGG + DMH). LGG was administered orally to LGG and LGG + DMH groups. Colon carcinogenesis was chemically induced in LGG + DMH and DMH groups by weekly injection of 40 mg/kg DMH. Animals were sacrificed after 25 weeks of experiment and tumor characteristics assessed. The change in expression of NFκB-p65, COX-2, TNFα, Bcl-2, Bax, iNOS, VEGFα, β-catenin, Casp3 and p53 were evaluated by western blotting and qRT-PCR.ResultsLGG treatment significantly reduced tumor incidence, multiplicity and volume in LGG + DMH treatment group compared to DMH cancer control group. Also, LGG treatment reduced the expression of β-catenin and the inflammatory proteins NFκB-p65, COX-2 and TNFα; the anti-apoptotic protein Bcl-2, but increased the expression of the pro-apoptotic proteins Bax, casp3 and p53 compared with DMH group.ConclusionLGG have a potential protection effect against colon carcinogenesis; inducing apoptosis and ameliorating inflammation, and may hold a promise as bio-therapeutic dietary agent.     

Effects of activin A and its downstream ERK1/2 in oxygen and glucose deprivation after isoflurane-induced postconditioning

BackgroundIsoflurane postconditioning (ISPOC) plays a neuroprotection role in the brain. Previous studies confirmed that isoflurane postconditioning can provide better protection than preconditioning in acute hypoxic–ischemic brain damage, such as acute craniocerebral trauma and ischemic stroke. Numerous studies have reported that activin A can protect rat’s brain from cell injury. However, whether activin A and its downstream ERK1/2 were involved in isoflurane postconditioning-induced neuroprotection is unknown.MethodsA total of 80 healthy Sprague–Dawley rats weighing 50–70 g were randomly divided into 10 groups of 8: normal control, oxygen and glucose deprivation (OGD), 1.5% ISPOC, 3.0% ISPOC, 4.5% ISPOC, blocker of activin A (SB431542), blocker of ERK1/2 (U0126), 3.0% ISPOC + SB431542, 3.0% ISPOC + U0126, and vehicle (dimethyl sulfoxide(DMSO)) group. Blockers (SB431542 and U0126) were used in each concentration of isoflurane before OGD. Hematoxylin–eosin staining, 2,3,5-triphenyl tetrazolium chloride staining, and propidium iodide (PI) staining were conducted to assess the reliability in the brain slices. Immunofluorescence, Western blot, and quantitative real-time PCR(Q-PCR) were performed to validate the protein expression levels of activin A, Smad2/3, P-Smad2/3, ERK1/2, and phosphorylation ERK1/2 (P-ERK1/2).ResultsThe number of damaged neurons and mean fluorescence intensity(MFI) of PI staining increased, but formazan generation, expression levels of activin A and P-ERK1/2 protein, and mRNA synthesis level of activin A decreased in the OGD group compared with the normal control group (p < 0.05). The number of damaged neurons and MFI of PI staining decreased, but formazan production, expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A increased significantly in the 1.5% ISPOC and 3.0% ISPOC groups (p < 0.05) compared with the OGD group. The result in the 4.5% ISPOC group, was completely opposite to the 1.5% ISPOC and 3.0% ISPOC groups. The number of damage neuron and MFI of PI staining increased, but formazan production, expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A decreased in the 4.5% ISPOC group. However, the expression levels of activin A, P-Smad2/3, and P-ERK1/2, and mRNA synthesis level of activin A in the 4.5% ISPOC group were higher than the OGD group (p < 0.05). The other results were compared between the SB431542 group/the U0126 group and 3.0% ISPOC group. The MFI of PI staining increased, but the expression levels of activin A, P-Smad2/3, and P-ERK1/2 decreased (p < 0.05). The expression level of ERK1/2 protein in all groups exhibited no change (p > 0.05).ConclusionResults of this study showed that 3.0% concentration of isoflurane postconditioning provided better neuroprotection than 1.5% and 4.5% concentrations of isoflurane. Activin A/Smad 2/3 and activin A/ERK1/2 signaling pathway may be involved in ISPOC-induced neuroprotection.     

Effect of notch1,2,3 genes silicing on NF-κB signaling pathway of macrophages in patients with atherosclerosis

BackgroundNotch and NF-κB signaling pathways both play important roles in the regulation of atherosclerosis (AS). However, the mechanisms of notch and NF-κB signaling pathways on AS are still unclear. In this study, we aimed to investigate the effects of notch1,2,3 genes silicing by siRNA on notch and NF-κB signaling pathways of macrophages in patients with atherosclerosis (AS), so as to seek the treatment of AS from genetic perspective.MethodsPeripheral blood mononuclears of 31 patients with AS were isolated by density gradient centrifugation and transformed by PMA to macrophages. Then macrophages were transfected with notch1-siRNA (notch1-siRNA group), notch2-siRNA (notch2-siRNA group), notch3-siRNA (notch3-siRNA group), negative control siRNA (NC group) and none (control group). RT-PCR and Western blot analysis were applied to assess the expression level of Delta-like-4 (DLL4), Jagged-1 (JAG1), IκBα and P52. Electrophoretic mobility shift assay (EMSA) was used to observe the NF-κB DNA binding activity. Subcellular distributions of NF-κB/P52 were detected through immunofluorescence. mRNA expression levels of TNF-α, IL-6 and IL-6 in macrophages were also determined with RT- PCR. The expression of 20S proteasome was detected by Western blot.ResultsAfter transfected with siRNA, there was no difference in the expression of DLL4, JAG1, IκBα and P52 between NC group and control group (p > 0.05). Compared with NC group and control group, the expression of DLL4, P52 and JAG1 in notch1-siRNA group, notch2-siRNA group and notch3-siRNA group was significantly downregulated (p < 0.05 or p < 0.01, respectively), whereas the expression of IκBα was significantly increased (P < 0.05 or p < 0.01, respectively), especially in notch1-siRNA group. The binding activity of NF-κB DNA was lower in notch1- siRNA group, notch2-siRNA group and notch3-siRNA group compared with NC group and control group (p < 0.05), especially in notch1-siRNA group. The fluorescence intensity of p52 was decreased significantly both in the nucleus and cytoplasm in notch1-siRNA group, notch2-siRNA group and notch3-siRNA group compared with NC group and control group (p < 0.05), which decreased more obviously in the nucleus, especially in notch1-siRNA group. The TNF-α, IL-1 and IL-6 expression of notch1-siRNA group, notch2-siRNA group and notch3-siRNA group was lower compared to NC group and control group (p < 0.05 or p < 0.01, respectively), also especially in notch1-siRNA group. 20S proteasome level was significantly lower in notch1-siRNA group, notch2-siRNA group and notch3-siRNA group than in NC group and control group (p < 0.05 or p < 0.01, respectively), especially in notch1-siRNA group.ConclusionsThere was a positive regulation between Notch and NF-κB signaling pathway in patients with AS. Notch1 may play a more important role than notch2 and notch 3 in the regulation of NF-κB signaling pathway in AS.     

Endothelial nitric oxide synthetase,methylenetetrahydrofolate reductase polymorphisms,and cardiovascular complications in Tunisian patients with nondiabetic renal disease

ObjectivesThe relationship between endothelial nitric oxide synthase (eNOS), methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and cardiovascular disease (CVD) in Tunisian patients with chronic renal disease (CRD) has not been examined. We investigated (a) the relationship of these gene polymorphisms with the presence and the severity of renal disease, and (b) their relationships with CVD in these patients.Design and methodsWe used PCR-RFLP analysis to detect the eNOS G894T, MTHFR C677T and A1298C variants in 100 patients with CRD and in 120 healthy controls.ResultsMTHFR C677T and A1298C polymorphisms were not associated with the presence of renal disease. However, we found that eNOS G894T polymorphism was associated with the presence and severity of renal disease and with CVD in CRD patients (P = 0.028, P = 0.018, P = 0.016 respectively). We showed that 894T allele was an independent risk factor of severity of renal disease and the incidence of CVD (P = 0.01 and P < 0.01 respectively).ConclusionThe G894T polymorphism of the eNOS gene is associated with severity of renal disease. Presence of the 894T allele aggravated renal damage and increased the incidence of CVD in Tunisian CRD patients.     

Correlation between concentrations of 8-oxo-7,8-dihydro-2′-deoxyguanosine in urine,plasma and saliva measured by on-line solid-phase extraction LC-MS/MS

Background8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is the most frequently measured biomarker of oxidative stress. Chromatographic-based methods for 8-oxodGuo in urine are well established; however, the 8-oxodGuo measurement in plasma and saliva has been problematic.MethodsWe firstly and successfully applied an on-line solid-phase extraction (SPE) LC-MS/MS following manual SPE pretreatment to quantify the 8-oxodGuo both in plasma and saliva. Urine, plasma and saliva specimens were simultaneously collected from 50 healthy adults and measured for 8-oxodGuo.ResultsMean baseline levels of 8-oxodGuo in plasma and saliva were 21.7 ± 9.2 and 5.1 ± 2.6 pg/ml, respectively, being far lower than that in urine (6.2 ± 4.8 ng/ml). The 8-oxodGuo levels obtained in this study for plasma and saliva were, however, up to several hundred times lower than those reported by commercial ELISA kit in the literature. Furthermore, the 8-oxodGuo levels in plasma and saliva were significantly correlated with the 8-oxodGuo levels in urine (Spearman correlation coefficients, r = 0.33, P = 0.02 for plasma and r = 0.56, P = 0.0015 for saliva). 8-OxodGuo in plasma was also correlated with the 8-oxodGuo in saliva (r = 0.52, P = 0.0041).ConclusionsSignificantly correlations were observed between plasma, saliva and urine, giving the possibility of using other body fluids in addition to urine for assessing whole body oxidative stress.     

Significant peri-operative reduction in plasma osteopontin levels after coronary artery by-pass grafting

ObjectivesOsteopontin (OPN) is a multifunctional protein associated with vascular injury and has been linked to atherosclerosis and inflammation. We sought to investigate whether OPN changes in relation to coronary artery by-pass grafting (CABG) surgery.Design and methodsWe studied 50 consecutive patients (63 ± 10 years old, 6 women and 44 men) undergoing elective CABG. Plasma OPN levels were determined by an enzyme-linked immunosorbent assay at baseline and in 24 and 72 h, post-operatively. Cardiac enzymes — creatine kinase, the MB isoenzyme of creatine kinase, troponin-I- and C-reactive protein (CRP) were also determined at all three time points.ResultsOPN levels 72 h post-op decreased significantly compared to pre-op and 24 h post-op levels (p < 0.001) whereas there was no difference between the pre-op and first post-op values (p = 0.57). The relative change in OPN levels between pre-op and 72 h post-op correlated negatively with absolute troponin-I levels at 72 h post-op (? 0.51, p = 0.005). OPN levels 72 h post-op correlated significantly with CRP at baseline (r = 0.73, p = 0.002).ConclusionsOPN plasma concentrations decreased after CABG surgery in the early post-operative period. The significance of this observation needs further investigation.     

Profiles of inflammatory markers and lipoprotein subclasses in patients undergoing continuous ambulatory peritoneal dialysis

BackgroundPatients undergoing continuous ambulatory peritoneal dialysis (CAPD) often have inflammation and dyslipidemia that accelerate to atherosclerosis. This study aimed to evaluate chronic inflammation and dyslipidemia in CAPD patients.MethodsWe measured inflammatory markers and lipoprotein subclasses in 20 CAPD patients (12 men and 8 women, aged 59.5 ± 9.9 y) and 20 gender-matched controls. Lipoproteins were separated by high-performance liquid chromatography (HPLC) using an anion-exchange column.ResultsHigh-sensitivity C-reactive protein and serum amyloid A protein (SAA) were higher among CAPD patients vs. controls (1.6 ± 2.2 vs. 0.8 ± 1.2 mg/l, p < 0.05; 11.9 ± 12.8 vs. 4.5 ± 2.4 mg/l). HPLC analysis revealed that chylomicron, VLDL, and IDL cholesterol levels were higher among CAPD vs. controls. In contrast, HDL cholesterol was lower among CAPD patients vs. controls. In the subgroup analysis, SAA levels were significantly lower among patients receiving CAPD for > 3 y than among controls. However, IDL cholesterol was consistently higher among CAPD patients vs. controls.ConclusionsCAPD patients have chronic inflammation and dyslipidemia. IDL cholesterol is the only lipoprotein subclass that is consistently elevated regardless of CAPD duration. More attention should be paid to dyslipidemia in the management of the CAPD patients.     

Hydrogen sulfide inhibits development of atherosclerosis through up-regulating protein S-nitrosylation

Hydrogen sulfide (H₂S) is an important gaseous signaling molecule that serves many important regulatory roles in physiological and pathophysiological conditions. H₂S exerts an anti-atherosclerotic effect through mediating the biological functions of nitric oxide (NO). However, its mechanism of action is unclear. The purpose of this study is to explore the effect mechanism of H₂S on the development of atherosclerosis with regard to protein S-nitrosylation. A total of 45 male apoE^−/− mice were randomly divided into three groups. Atherosclerosis was induced by Western diet (21% fat and 0.15% cholesterol) with/without administration of a H₂S donor (NaHS) or an endogenous cystathionine γ-lyase inhibitor (d, l-propargylglycine) for 12 weeks. After 12 weeks, plasma lipid and plasma NO levels were measured. Aortic gross lesion area and histopathological features of aortic lesion were determined. Additionally, the level of S-nitrosylated proteins in vascular smooth muscle cells (VSMCs) was detected using immunofluorescence in aorta. Rat VSMCs were performed in an in vitro experiment. Inducible nitric oxide synthase (iNOS) protein expression, NO generation, protein S-nitrosylation, and cell proliferation and migration were measured. We found that H₂S significantly reduced the aortic atherosclerotic lesion area (P = 0.006) and inhibited lipid and macrophage accumulation (P = 0.004, P = 0.002) and VSMC proliferation (P = 0.019) in apoE^−/− mice. H₂S could up-regulate levels of plasma NO and protein S-nitrosylation in aorta VSMCs. However, d, l- propargylglycine had the opposite effect, increasing the lesion area and the content of lipids and macrophages in the lesions of apoE^−/− mice and down-regulating plasma NO levels and protein S-nitrosylation in aorta VSMCs. In vitro experiments, H₂S could significantly reverse the reduction of iNOS expression and NO generation induced by oxidized low-density lipoprotein in VSMCs. Moreover, H₂S could increase the protein S-nitrosylation level of VSMCs in a dose-dependent manner, and the effect could be inhibited by iNOS inhibitors. In addition, proliferation and migration of VSMCs could be inhibited by H₂S in a dose-dependent manner, which could be blocked by an iNOS inhibitor or protein S-nitrosylation removal agent. Our data suggest that H₂S could inhibit the development of atherosclerosis by up-regulating plasma NO and protein S-nitrosylation, thereby inhibiting the proliferation and migration of VSMCs.     

Protein oxidation biomarkers in plasma of type 2 diabetic patients

ObjectivesTo evaluate oxidative stress and the extent of oxidation of plasma proteins in type 2 diabetic patients.Design and methodsStudy was carried out on blood from 31 diabetic patients of both sexes (mean age = 58 ± 7; duration of diabetes 12 ± 5 years) and healthy age and sex matched normal subjects. Biomarkers of protein oxidation; plasma protein carbonyls (PCO), advanced oxidation protein products (AOPPs) and –SH group and free radical scavenging capacity of plasma was measured.ResultsPCO and AOPPS levels were significantly (P < 0.005) higher in diabetic patients in comparison to healthy volunteers. Reduced free radical scavenging capacity (P < 0.001) and –SH group (P < 0.05) was observed in plasma of type 2 diabetic patients.ConclusionsOur data suggest that diabetics are susceptible to protein oxidation. Oxidative modulation of proteins due to reduced radical scavenging activity of plasma patients may be one of the reasons of altered physiological processes in type 2 diabetic patients.     

Association of genetic variations in aromatase gene with serum estrogen and estrogen/testosterone ratio in Chinese elderly men

BackgroundSingle nucleotide polymorphism (SNP) rs2470152 of the gene CYP19A1 is associated with serum estradiol (E2) levels in Caucasian men. However, it remains to be verified if rs2470152 is the sole determinant accounting for the association. We determined whether 2 CYP19A1 SNPs tagging different haploblocks (rs2470152 and rs2899470) are associated with sex steroid levels in Chinese men.MethodSerum sex steroid level including E2, estrone (E1) and testosterone (T), of 1402 Chinese men aged ≥ 65 years were analyzed. Genotyping of the two CYP19A1 SNPs was performed using T_m-shift allele-specific PCR.ResultsSNP rs2899470 was significantly associated with serum E2, E1 levels and E2/T ratio (p < 0.001). However, SNP rs2470152 was only modestly associated with E2/T ratio (p = 0.023). Analysis of haplotype showed a significant association between C-G, T-T haplotype with serum E2/T ratio (p = 0.019 and p = 1 × 10^? 5, respectively). Similarly, E2 levels was also associated the T-T and T-G haplotypes (p = 1 × 10^? 5).ConclusionThe genetic variation of CYP19A1 was associated with circulating estrogen levels in Chinese elderly men. In addition, it revealed that haplotype of rs2899470 and rs2470152, rather than rs2899470 alone, was a better indicator for the serum E2/T ratio and E2 levels.     

Amelioratory effect of flavonoids rich Pergularia daemia extract against CFA induced arthritic rats

PurposePergularia daemia Forsk. (Asclepiadaceae) is a traditionally reported medicinal herb used to treat joint pain and arthritis. However, there are no scientific reports about anti-arthritic activity of P. daemia methanolic extract on rats as animal model. This study identifies bioactive compounds present in the P. daemia methanolic extract and evaluates its anti-arthritic potential in CFA induced arthritic rats.Methods and resultsPhytoconstituents of P. daemia extract were examined using LC-ESI/MS method. Anti-arthritic activity of P. daemia extract was determined by various biochemical experiments (RF, ESR and CRP), ultrasonography and histological analysis. LC-ESI/MS analysis resulted in the identification of major flavonoids compounds such as formononetin, qurecetin, chrysoeriol, taxifolin and naringenin. Serum biomarker analysis, after the treatment with PDME (500 mg/kg b.w.) revealed that the hemoglobin (11.84 ± 0.42 g/dL) and RBC (8.38 ± 0.67 million/mm³) levels were significantly increased whereas WBC (8.91 ± 0.38 thousands/mm³), RF (17.94 ± 0.45 IU/mL), ESR (7.91 ± 0.12 mm/h) and CRP (22.56 ± 0.26 mg/L) levels were decreased when compared with the CFA induced arthritic control group. Histology results revealed that treatment with PDME has resulted in significant prevention against bony destruction by decreasing soft tissue swelling and narrowing of joint spaces (250 and 500 mg/kg b.w.).ConclusionAnti-arthritic effect of P. daemia might be due to the presence of these bioactive flavonoids. These findings lend pharmacological support to the reported folkloric use of P. daemia in the treatment and management of painful, arthritic inflammatory conditions.     

A novel and automated assay for thiol/disulphide homeostasis

ObjectivesTo develop a novel and automated assay determining plasma thiol/disulphide homeostasis, which consists of thiol–disulphide exchanges.Design and methodsNative thiol and total thiol concentrations were synchronously measured as a paired test. In the first vessel, the amount of native thiol groups was measured by a modified Ellman reagent. At the parallel run, first, dynamic disulphide bonds were reduced to free thiol groups by NaBH₄. The unused reductant remnants were completely removed by formaldehyde. Thus, the total thiol amount could be accurately measured. Mercaptoethanol solutions were used as calibrators. The half value of the difference between total thiol and native thiol amounts gave the disulphide bond amount.ResultsNo separation step for the assay was needed. All processes were performed using an automated analyser within about 10 min. Plasma disulphide levels were 17.29 ± 5.32 μmol/L, native thiol levels were 397 ± 62 μmol/L and disulphide/native thiol per cent ratios were 4.32 ± 1.49 in healthy subjects. Plasma disulphide levels were higher in patients with degenerative diseases and lower in patients with proliferative diseases.ConclusionAn easy, inexpensive, practical, fully automated and also optionally manual spectrophotometric assay can be used to determine plasma dynamic thiol/disulphide homeostasis.     

MUC1 568 A/G genotype-dependent Cancer Antigen 15-3 levels in breast cancer patients

ObjectivesCA 15-3 is a widely used tumor marker for breast cancer. We have investigated whether the MUC1 568 A/G polymorphism can influence CA 15-3 levels in healthy women and patients with breast tumors.Design and methodsCA 15-3 was measured in 208 healthy women, in 67 with benign disease, and in 162 women with breast cancer. All subjects were genotyped for the MUC1 568 A/G polymorphism.ResultsSignificant differences were observed between mean CA 15-3 levels of control subjects grouped according to the MUC1 568 genotype (mean ± SD): AA (10.3 ± 3.8), AG (15.9 ± 5.0) and GG (19.0 ± 5.6) U/mL, p < 0.0001. Similar (median) results were observed in women with benign breast disease: AA (10.2), AG (14.2) and GG (16.6) U/mL, p < 0.0001, and those with breast cancer: AA (10.4), AG (17.1) and GG (23.9) U/mL, p < 0.0001.ConclusionsThe MUC1 568 A/G polymorphism strongly influences CA 15-3 levels in healthy women and women with either benign or malignant breast tumors.     

MTHFR C677T and eNOS G894T variants in preeclamptic women: Contribution to lipid peroxidation and oxidative stress

ObjectivesWe aimed to investigate the association between methylenetetrahydrofolate reductase (MTHFR) C677T and endothelial nitric oxide synthase (eNOS) G894T polymorphisms with lipid peroxidation, total antioxidant capacity (TAC) and the risk of preeclampsia in preeclamptic women.Design and MethodsWe screened a sample of 198 unrelated women with mild and severe forms of preeclampsia and 101 unrelated women with normal pregnancy with the eNOS and MTHFR variants using PCR-RFLP method. Also, the serum malondialdehyde (MDA) and TAC levels were determined using HPLC and commercial kits, respectively.ResultsThe frequency of combined genotypes of MTHFR CT and TT (CT + TT) and T allele tended to be higher in severe preeclamptic women compared to controls. There was no significant difference for eNOS G894T genotype and allele frequencies between patients and controls. A significantly higher level of triglycerides was observed in the presence of combined genotypes of MTHFR CT and TT and also eNOS GT and TT (GT + TT) in preeclamptic women compared to controls with the same genotype. Also, the presence of MTHFR TT genotype in severe preeclamptic women was significantly associated with the increased serum MDA level compared to CC genotype. In severe preeclamptic women the presence of CT and combined genotypes of CT and TT was significantly associated with the decreased TAC level compared to CC genotype. Also, a higher MDA level was observed in mild preeclamptic women with eNOS TT genotype compared to those with GG genotype but the difference was not significant.ConclusionThe present study indicates that MTHFR C677T polymorphism through affecting on TG level, lipid peroxidation and oxidative stress might be involved in the pathogenesis of severe preeclampsia.     

Caspase-cleaved fragments of cytokeratin 18 in patients with chronic hepatitis B

BackgroundDuring hepatocyte apoptosis, intermediate filament protein cytokeratin 18 is cleaved by caspases at Asp396 which can be specifically detected by the monoclonal antibody M30 (M30-antigen). In this study, we sought to determine whether serum M30-antigen levels can serve as a useful biomarker of liver injury in the clinical spectrum of HBV infection.MethodsSerum M30-antigen levels were measured in inactive HBV carriers (n = 54), patients with HBeAg-negative chronic hepatitis B (CHB, n = 47), patients with HBeAg-positive CHB (n = 42) and healthy controls (n = 29). All subjects were treatment-naïve.ResultsThere were significant differences in serum M30-antigen levels across the study groups (P < 0.001; Kruskal–Wallis test). Post hoc analyses revealed that M30-antigen levels did not differ significantly between inactive HBV carriers (median 109.6 U/L) and healthy controls (median 106.1 U/L). However, both patients with HBeAg-negative (CHB, median 182.9 U/L, P < 0.001) and HBeAg-positive CHB (median 158.3 U/L, P < 0.001) had significantly higher levels of M30-antigen compared with inactive HBV carriers.ConclusionsHepatocyte apoptotic activity – as reflected by serum M30-antigen levels – is increased in chronic active hepatitis B, but is not associated with the HBeAg status. In contrast, apoptosis does not appear to be a prominent feature of inactive HBV carriers.