Ascorbic acid attenuates cadmium-induced myocardial hypertrophy and cardiomyocyte injury through Nrf2 signaling pathways comparable to resveratrol.

Chronic cadmium (Cd) exposure severely affects the structural integrity of the heart, leading to cardiovascular disease. This study investigates the protective role of ascorbic acid (AA) and resveratrol (Res) in cellular defense against Cd-induced cardiomyocyte damage and myocardial hypertrophy in H9c2 cardiomyocytes. Experimental results showed that AA and Res treatment significantly increased cell viability, reduced ROS production, attenuated lipid peroxidation, and increased antioxidant enzyme activity in Cd-induced H9c2 cells. AA and Res decreased the mitochondrial membrane permeability and protected the cells from Cd induced cardiomyocyte damage. This also suppressed the pathological hypertrophic response triggered by Cd, which increased the cell size of cardiomyocytes. Gene expression studies revealed that cells treated with AA and Res decreased the expression of hypertrophic genes ANP (two-fold), BNP (one-fold) and ß- MHC (two-fold) compared to Cd exposed cells. AA and Res promoted the nuclear translocation of Nrf2 and increased the expression of antioxidant genes (HO-1, NQO1, SOD and CAT) during Cd mediated myocardial hypertrophy. This study proves that AA and Res play a significant role in improving Nrf2 signaling, thereby reversing stress-induced injury, and facilitating the regression of myocardial hypertrophy.

Chrysin attenuates high-fat-diet-induced myocardial oxidative stress via upregulating eNOS and Nrf2 target genes in rats.

Hypercholesterolemia is one of the risk factors associated with increased morbidity and mortality in cardiovascular disorders. Chrysin (Chy) is reported to exhibit anti-inflammatory, anti-cancerous, anti-oxidative, anti-aging, and anti-atherogenic properties. In the present study, we aimed to investigate whether Chy would mediate the cardioprotective effect against hypercholesterolemia-triggered myocardial oxidative stress. Male Sprague Dawley rats were divided into different groups as control and fed with high-fat diet (HFD) followed by oral administration of Chy (100 mg/kg b.wt), atorvastatin (Atv) (10 mg/kg b.wt), and L-NAME (10 mg/kg b.wt) for 30 days. At the end of the experimental period, the rats were sacrificed and tissues were harvested. Biochemical results showed a significant increase of cardiac disease marker enzymes (ALT, AST, and CKMB), lipid peroxidation, and lipid profile (TC, TG, LDL, and VLDL) in HFD-fed rat tissues when compared to control, whereas oral administration of Chy significantly reduced the activities of these marker enzymes and controlled the lipid profile. qRT-PCR studies revealed that Chy administration significantly increased the expression of endothelial nitric oxide synthase (eNOS), and Nrf2 target genes such as SOD, catalase, and GCL3 in left ventricular heart tissue of HFD-challenged rats. Immunohistochemistry results also showed that Chy treatment increased myocardial protein expression of eNOS and Nrf2 in HFD-challenged rats. Concluding the results of the present study, the Chy could mediate the cardioprotective effect through the activation of eNOS and Nrf2 signaling against hypercholesterolemia-induced oxidative stress. Thus, the administration of Chy would provide a promising therapeutic strategy for the prevention of HFD-induced oxidative stress-mediated myocardial complications.

The -866G/A polymorphism in the promoter of the UCP2 gene is associated with risk for type 2 diabetes and with decreased insulin levels.

BACKGROUND AND AIM: Oxidative stress and impaired insulin secretion is an underlying major risk factor for the development of type 2 diabetes (T2D). Uncoupling protein-2 (UCP2) is involved in the regulation of reactive oxygen species production, insulin secretion, and lipid metabolism. Based on this we aimed to find an association of UCP2 (G-866A) polymorphism with the risk of T2D in South Indian population. METHODS: A total of 318 T2D patients and 312 controls were enrolled in this study. All the study subjects were genotyped for UCP2 (G-866A) polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Fasting blood glucose, HbA1c, serum lipid profile, systolic and diastolic blood pressure were measured by standard biochemical methods. Fasting serum insulin level was measured by ELISA. RESULTS: In UCP2 (G-866A) polymorphism, the distribution of GA (46%) and AA (14%) genotypes were significantly higher in T2D patients than the healthy controls. The frequency of GA and AA genotypes have high risk towards the development of T2D with an Odds Ratio (OR) of 1.55 (P = 0.01) and 2.04 (P = 0.01) respectively. Moreover, SNP-866 G>A allele was found to be significantly associated with T2D (OR = 1.48, P = 0.001, 95% CI = 1.16-1.88). Further, the UCP2 AA genotype showed significantly decreased level of insulin by the reduction in pancreatic ß-cell function in T2D patients. CONCLUSION: UCP2 (G-866A) polymorphism may play a crucial role in the pathogenesis of insulin secretion thus leads to the development of T2D.

Association of paraoxonase-1 gene polymorphisms with insulin resistance in South Indian population.

BACKGROUND AND AIM: Insulin resistance plays a crucial role in the pathogenesis of type 2 diabetes and cardiovascular diseases. Recently, paraoxonase-1(PON1) is reported to have an ability to reduce insulin resistance by promoting glucose transporter-4 (GLUT-4) expression in vitro. Single nucleotide polymorphism (SNP) in PON1 is associated with variability in enzyme activity and concentration. Based on this we aimed to investigate the association of PON1 (Q192R and L55M) polymorphisms with the risk of developing insulin resistance in adult South Indian population. METHODS: Two hundred and eighty seven (287) Type 2 diabetes patients and 293 healthy controls were enrolled in this study. All the study subjects were genotyped for PON1 (Q192R and L55M) missense polymorphisms using polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) method. Fasting serum insulin level was measured by ELISA. RESULTS: The distribution of QR/RR and LM/MM genotypes were significantly higher in type 2 diabetes patients compared with healthy controls. Moreover, the R and M alleles were significantly associated with type 2 diabetes with an Odds Ratio of 1.68 (P < 0.005) and 2.24 (P < 0.005) respectively. SNP 192 Q > R genotypes were found to be significantly associated with higher BMI, cholesterol, triglycerides, LDL, fasting serum insulin and HOMA-IR. Further, the mutant allele or genotypes of PON1 L55M were associated with higher BMI, triglycerides, VLDL, fasting serum insulin and HOMA-IR among adult type 2 diabetes patients. CONCLUSION: PON1 (Q192R and L55M) polymorphisms may play a crucial role in pathogenesis and susceptibility of insulin resistance thus leads to the development of type 2 diabetes in South Indian population.

Mobile group II intron based gene targeting in Lactobacillus plantarum WCFS1.

The usage of recombinant lactic acid bacteria for delivery of therapeutic proteins to the mucosa has been emerging. In the present study, an attempt was made to engineer a thyA mutant of Lactobacillus plantarum (L. plantarum) using lactococcal group II intron Ll.LtrB for the development of biologically contained recombinant L. plantarum for prevention of calcium oxalate stone disease. The 3 kb Ll.LtrB intron donor cassettes from the source vector pACD4C was PCR amplified, ligated into pSIP series of lactobacillus vector pLp_3050sAmyA, yielding a novel vector pLpACD4C (8.6 kb). The quantitative real-time PCR experiment shows 94-fold increased expression of Ll.LtrB intron and 14-fold increased expression of ltrA gene in recombinant L. plantarum containing pLpACD4C. In order to target the thyA gene, the potential intron RNA binding sites in the thyA gene of L. plantarum was predicted with help of computer algorithm. The insertion location 188|189s of thyA gene (lowest E-0.134) was chosen and the wild type intron Ll.LtrB was PCR modified, yielding a retargeted intron of pLpACDthyA. The retargeted intron was expressed by using induction peptide (sppIP), subsequently the integration of intron in thyA gene was identified by PCR screening and finally ThyA- mutant of L. plantarum (ThyA18) was detected. In vitro growth curve result showed that in the absence of thymidine, colony forming units of mutant ThyA18 was decreased, whereas high thymidine concentration (10 µM) supported the growth of the culture until saturation. In conclusion, ThyA- mutant of L. plantarum (ThyA18) constructed in this study will be used as a biologically contained recombinant probiotic to deliver oxalate decarboxylase into the lumen for treatment of hyperoxaluria and calcium oxalate stone deposition.

Regression of oxidative stress by targeting eNOS and Nrf2/ARE signaling: a guided drug target for cardiovascular diseases.

Cardiovascular diseases (CVDs) are the major health concern and the leading cause of death. Imbalance between free radicals and anti-oxidant defence is associated with cellular dysfunctions leading to the pathophysiology of various diseases including cardiac and vascular diseases. The stress responsive transcription factor NF-E2-related factor 2/antioxidant response element (Nrf2/ARE) regulates the expression of many detoxifying genes. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important regulator of vascular function. Involvement of NO in modulating Nrf2 signaling is well established. Thus, it is apparent that increasing NO bioavailability and antioxidant status in vascular and myocardial tissue can be considered as a potential strategy to prevent the onset of vascular dysfunction and CVDs and is therefore of therapeutical interest. Based on the marked protective effect of Nrf2/ARE signalling and intriguing links between antioxidant mechanism and endothelial derived NO, the aim of the present review is to compile conclusive evidence for the involvement of NO-Nrf2/ARE axis in the regulation of cardiovascular function. This review also discusses on improving eNOS and Nrf2 signalling by Nrf2 activators which holds promise for countering cardiac and vascular disorders.

Recombinant Lactobacillus plantarum expressing and secreting heterologous oxalate decarboxylase prevents renal calcium oxalate stone deposition in experimental rats.

BACKGROUND: Calcium oxalate (CaOx) is the major constituent of about 75% of all urinary stone and the secondary hyperoxaluria is a primary risk factor. Current treatment options for the patients with hyperoxaluria and CaOx stone diseases are limited. Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption. Thus, the aim of the present study is to examine the in vivo oxalate degrading ability of genetically engineered Lactobacillus plantarum (L. plantarum) that constitutively expressing and secreting heterologous oxalate decarboxylase (OxdC) for prevention of CaOx stone formation in rats. The recombinants strain of L. plantarum that constitutively secreting (WCFS1OxdC) and non-secreting (NC8OxdC) OxdC has been developed by using expression vector pSIP401. The in vivo oxalate degradation ability for this recombinants strain was carried out in a male wistar albino rats. The group I control; groups II, III, IV and V rats were fed with 5% potassium oxalate diet and 14th day onwards group II, III, IV and V were received esophageal gavage of L. plantarum WCFS1, WCFS1OxdC and NC8OxdC respectively for 2-week period. The urinary and serum biochemistry and histopathology of the kidney were carried out. The experimental data were analyzed using one-way ANOVA followed by Duncan's multiple-range test. RESULTS: Recombinants L. plantarum constitutively express and secretes the functional OxdC and could degrade the oxalate up to 70-77% under in vitro. The recombinant bacterial treated rats in groups IV and V showed significant reduction of urinary oxalate, calcium, uric acid, creatinine and serum uric acid, BUN/creatinine ratio compared to group II and III rats (P < 0.05). Oxalate levels in kidney homogenate of groups IV and V were showed significant reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V. CONCLUSION: The present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

Screening of indigenous oxalate degrading lactic acid bacteria from human faeces and South Indian fermented foods: assessment of probiotic potential.

Lactic acid bacteria (LAB) have the potential to degrade intestinal oxalate and this is increasingly being studied as a promising probiotic solution to manage kidney stone disease. In this study, oxalate degrading LAB were isolated from human faeces and south Indian fermented foods, subsequently assessed for potential probiotic property in vitro and in vivo. Based on preliminary characteristics, 251 out of 673 bacterial isolates were identified as LAB. A total of 17 strains were found to degrade oxalate significantly between 40.38% and 62.90% and were subjected to acid and bile tolerance test. Among them, nine strains exhibited considerable tolerance up to pH 3.0 and at 0.3% bile. These were identified as Lactobacillus fermentum and Lactobacillus salivarius using 16S rDNA sequencing. Three strains, Lactobacillus fermentum TY5, Lactobacillus fermentum AB1, and Lactobacillus salivarius AB11, exhibited good adhesion to HT-29 cells and strong antimicrobial activity. They also conferred resistance to kanamycin, rifampicin, and ampicillin, but were sensitive to chloramphenicol and erythromycin. The faecal recovery rate of these strains was observed as 15.16% (TY5), 6.71% (AB1), and 9.3% (AB11) which indicates the colonization ability. In conclusion, three efficient oxalate degrading LAB were identified and their safety assessments suggest that they may serve as good probiotic candidates for preventing hyperoxaluria.

Naringenin confers protection against oxidative stress through upregulation of Nrf2 target genes in cardiomyoblast cells.

Cardiovascular diseases are the major health concern and the leading cause of death. Numerous studies have shown that oxidative stress stimuli have been incriminated in the pathogenesis of both acute and chronic heart disease. Though it is well known that bioflavonoids protect cells against reactive oxygen species (ROS)-induced damage, the molecular mechanisms involved are uncertain. Understanding the possible intracellular signaling pathways triggered by flavonoids will help to overcome the cardiac diseases resulting from oxidative stress. In the present study, we investigated whether naringenin (NGN) supplementation would improve the antioxidant defence under oxidative stress through the activation of Nrf2 signaling in cultured cardiomyoblast. NGN pretreatment significantly reduced stress-mediated apoptotic cell death and lipid peroxidation and showed increased level of reduced glutathione in H2O2-treated cardiomyoblast. In addition, NGN inhibited the production of NO and trigged the synthesis of antioxidant marker enzymes. Gene expression studies revealed that NGN upregulated the transcription of Akt and downregulated NF-κB and Caspase 3 genes. Notably, transcription of Nrf2 and its target genes was also upregulated. Taken together, the present study revealed that NGN elicits potent cytoprotective effect against oxidative stress by regulating Nrf2 and its target genes. In conclusion, the present work suggests that improving Nrf2 signaling by NGN supplementation would be a rational approach to facilitate ROS detoxification by augmenting both expression and activity of phase II detoxification enzymes for the alleviation of cardiac complications.