Gymnemic Acid Ameliorates Pancreatic β-Cell Dysfunction by Modulating Pdx1 Expression: A Possible Strategy for β-Cell Regeneration

BACKGROUND@#Endogenous pancreatic β-cell regeneration is a promising therapeutic approach for enhancing β-cell function and neogenesis in diabetes. Various findings have reported that regeneration might occur via stimulating β-cell proliferation, neogenesis, or conversion from other pancreatic cells to b-like cells. Although the current scenario illustrates numerous therapeutic strategies and approaches that concern endogenous β-cell regeneration, all of them have not been successful to a greater extent because of cost effectiveness, availability of suitable donors and rejection in case of transplantation, or lack of scientific evidence for many phytochemicals derived from plants that have been employed in traditional medicine. Therefore, the present study aims to investigate the effect of gymnemic acid (GA) on β-cell regeneration in streptozotocin-induced type 1 diabetic rats and high glucose exposed RIN5-F cells. @*METHODS@#The study involves histopathological and immunohistochemical analysis to examine the islet’s architecture.Quantitative polymerase chain reaction (qPCR) and/or immunoblot were employed to quantify the β-cell regeneration markers and cell cycle proliferative markers. @*RESULTS@#The immunoexpression of E-cadherin, β-catenin, and phosphoinositide 3-kinases/protein kinase B were significantly increased in GA-treated diabetic rats. On the other hand, treatment with GA upregulated the pancreatic regenerative transcription factor viz. pancreatic duodenal homeobox 1, Neurogenin 3, MafA, NeuroD1, and β-cells proliferative markers such as CDK4, and Cyclin D1, with a simultaneous downregulation of the forkhead box O, glycogen synthase kinase-3, and p21 cip1 in diabetic treated rats. Adding to this, we noticed increased nuclear localization of Pdx1 in GA treated high glucose exposed RIN5-F cells. @*CONCLUSION@#Our results suggested that GA acts as a potential therapeutic candidate for endogenous β-cell regeneration in treating type 1 diabetes.

Morinda citrifolia mitigates rotenone-induced striatal neuronal loss in male Sprague-Dawley rats by preventing mitochondrial pathway of intrinsic apoptosis.

OBJECTIVES: Parkinson disease (PD) is a neurodegenerative disorder affecting mainly the motor system, as a result of death of dopaminergic neurons in the substantia nigra pars compacta. The present scenario of research in PD is directed to identify novel molecules that can be administered individually or co-administered with L-Dopa to prevent the L-Dopa-Induced Dyskinesia (LID) like states that arise during chronic L-Dopa administration. Hence, in this study, we investigated whether Morinda citrifolia has therapeutic effects in rotenone-induced Parkinson's disease (PD) with special reference to mitochondrial dysfunction mediated intrinsic apoptosis. METHODS: Male Sprague-Dawley rats were stereotaxically infused with rotenone (3 µg in both SNPc and VTA) and co-treated with the ethyl acetate extract of Morinda citrifolia and levodopa. RESULTS: The results revealed that rotenone-induced cell death was reduced by MCE treatment as measured by decline in the levels of pro-apoptotic proteins. Moreover, MCE treatment significantly augmented the levels of anti-apoptotic Bcl2 and blocks the release of cytochrome c, thereby alleviating the rotenone-induced dopaminergic neuronal loss, as evidenced by tyrosine hydroxylase (TH) immunostaining in the striatum. DISCUSSION: Taken together, the results suggest that Morinda citrifolia may be beneficial for the treatment of neurodegenerative diseases like PD.

Beneficial antioxidative effect of the homeopathic preparation of Berberis vulgaris in alleviating oxidative stress in experimental urolithiasis.

BACKGROUND: Oxidative stress is a major mediator in the pathophysiology of several kidney diseases. The cellular damage is mediated by an alteration in the antioxidant status, which increases the concentration of reactive oxygen species (ROS) in the stationary state (oxidative stress). Therefore, interventions favoring the scavenging and/or depuration of ROS should attenuate or prevent the oxidative stress, thereby safeguarding the kidneys against damage. In this sense, this study attempts to evaluate the extent of oxidative stress in experimental urolithiasis by measuring some parameters of oxidant stress and antioxidant defenses in rat kidneys, before and after Berberis vulgaris homeopathic preparation supplementation, and to assess the role, if any, of homeopathic treatment in mitigating free radical toxicity in kidney stone disease. METHODS: Rat model of urolithiasis was established by administering 0.75% ethylene glycol (EG) in drinking water, and the effects of a homeopathic preparation of B. vulgaris root bark (HPBV) on the renal antioxidative defense system as well as on potent markers of free radical activities were investigated. RESULTS: HPBV brought about an augmentation in the activities of enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase and improved the nonenzymatic antioxidants, e.g., tocopherol, ascorbic acid, and glutathione. HPBV ameliorated the malondialdehyde and protein carbonyl levels and restored renal thiols almost completely. CONCLUSION: Thus, it is shown that HPBV acts as a renoprotective remedy in alleviating the renal calculi-associated oxidative damage by upregulating the antioxidant status.

Preliminary investigation on ultra high diluted B. vulgaris in experimental urolithiasis.

PURPOSE: The study focuses on the anti-urolithiasis potential of ultra-diluted homeopathic potency of Berberis vulgaris (B. vulgaris) root bark, commonly used in homeopathic system to treat renal calculi. METHODOLOGY: B. vulgaris root bark (200c, 20 µl/100 g body weight/day, p.o, for 28 days) was tested in an animal model of urolithiasis. Urolithiasis was induced in male Wistar rats by adding 0.75% ethylene glycol (EG) to drinking water. Urine and serum samples were analyzed for calcium, magnesium, phosphorus, uric acid and creatinine. Enzymic makers of renal damage (alkaline phosphatase, lactate dehydrogenase, leucine aminopeptidase and γ-glutamyl transpeptidase) were assessed in kidney and urine. Renal tissues were analyzed for oxalate content. RESULTS: Administration of EG to rats increased the levels of the stone-forming constituents calcium, phosphorus and uric acid, in urine. Levels were normalized by B. vulgaris treatment. The decrease in the urolithiasis inhibitor magnesium in urine was prevented by treatment with B. vulgaris. Serum creatinine levels were largely normalized by B. vulgaris treatment. Hyperoxaluria induced renal damage was evident from the decreased activities of tissue marker enzymes and an apparent escalation in their activity in the urine in control animals; this was prevented by B. vulgaris treatment. CONCLUSION: Homeopathic B. vulgaris root bark has strong anti-urolithiasis potential at ultra-diluted dose.

Attenuation of the inflammatory changes and lipid anomalies by epigallocatechin-3-gallate in hypercholesterolemic diet fed aged rats.

Epidemiological studies suggest that even in the absence of other risk factors advanced age itself significantly increases cardiovascular morbidity. Age aggravated inflammatory activity further plays a central role in the pathogenesis of atherosclerosis and its complications. EGCG, a major flavonoid present in green tea extract has been proved to be useful in lowering cholesterol levels thereby slowing down the progression of cardiovascular diseases in young animals. Thus, the endeavor of this study was to assess the impact of high-cholesterol diet on aging and vice versa and to exploit the potential of EGCG to combat age-associated hypercholesterolemia and mitigate inflammation. Male albino rats of Wistar strain (3 months-young and 24 months old-aged) were used in this study. Hypercholesterolemia was induced by the diet comprising of the normal rat chow supplemented with 4% cholesterol and 1% cholic acid. EGCG (100 mg/kg body weight/day) was given orally for 30 days. The results revealed abnormally elevated lipid levels, marker enzymes and inflammatory markers in serum of aged hypercholesterolemic rats when compared to young hypercholesterolemic rats, while treatment with EGCG partially reversed these aberrations. The present work demonstrates the inflammatory responses in hypercholesterolemic atherogenesis during aging and further underscores the salubrious role played by the EGCG in attenuating the inflammatory and lipid anomalies.

Senescence mediated redox imbalance in cardiac tissue: Antioxidant rejuvenating potential of green tea extract.

OBJECTIVE: The activities and capacities of antioxidant systems of tissue cells are declined during aging, leading to the gradual loss of pro-oxidant/antioxidant balance and accumulation of oxidative damage. Hence, the present study evaluated the role of green tea extract (GTE), rich in polyphenols, in combating age-associated macromolecular damage in rat cardiac tissue. METHODS: The antioxidant defense system, lipid peroxidation, protein oxidation, and redox status in heart tissue were studied using young and aged rats. RESULTS: Significant increases in lipid peroxidation, protein carbonyls, and marked decreases in glutathione redox status, protein thiols, and activities of enzymatic and non-enzymatic antioxidants were observed in aged rats compared with young rats. Supplementation of GTE (100 mg/kg of body weight per day) orally for 30 days ameliorated these changes significantly. CONCLUSION: This study accredits GTE's antioxidant rejuvenating potency and its role in the amelioration of senescence-mediated redox imbalance in aged rat cardiac tissue.

Mitochondrial alterations in aging rat brain: effective role of (-)-epigallo catechin gallate.

Aging is a multi-factorial process which involves deprivation in body's metabolism. Brain mitochondria are prone to oxidative damage owing to their high metabolic rate. The decline in antioxidant system during aging augments the neuronal damage to mitochondrial components like antioxidant system, Kreb's cycle enzymes and electron transport chain complexes. Since brain is an organ rich in fatty acids, lipid peroxidation products like hydroxynonenal are predominant. Those lipid peroxidation products conjugate with amino acids to form adducts which alter their structural and functional properties. Epigallo catechin gallate is a potent antioxidant which is rich in green tea extract. This study elucidated the antioxidant potential of epigallo catechin gallate to counteract the mitochondrial oxidative damage in brain. The study comprised of young (3-4 months old; 150+/-20 g) and aged (above 24 months; 420+/-20 g) male albino rats of Wistar strain in Groups I and II. Groups III and IV comprised of young and aged rats supplemented with epigallo catechin gallate (2mg/kg body weight) for 30 days. Antioxidants, Kreb's cycle enzymes and electron transport chain complexes were assayed in the mitochondrial fraction. Hydroxynonenal expression was carried out using immunohistochemical analysis. Epigallo catechin gallate supplementation decreased the expression of hydroxynonenal in aged brain, up-regulated the antioxidant system and augmented the activities of Kreb's cycle enzymes and electron transport chain complexes in aged brain mitochondria thus proving its antioxidant potential at the level of mitochondria.

Attenuation of senescence-induced oxidative exacerbations in aged rat brain by (-)-epigallocatechin-3-gallate.

Aging is a complex biological phenomenon which involves free radicals and oxidative stress. Brain is more susceptible and vulnerable to oxidative damage due to its high-polyunsaturated fatty acid content and high rate of aerobic metabolism. Since the antioxidant defense system is diminished during aging, antioxidant supplementation might be a protective strategy against age-associated oxidative damage. The present study evaluates the antioxidant potential of (-)-epigallocatechin-3-gallate (EGCG), a major polyphenol present in green tea against age-associated oxidative damage in rat brain. Male albino rats of Wistar strain were used in the study. Group I (young) and Group II (aged) rats received saline alone orally for 30 days. Group III (young) and Group IV (aged) rats received EGCG (2mg/kg body weight/day) orally for 30 days. Antioxidant status and oxidative damage were assessed. EGCG brought about an augmentation in the activities of enzymic antioxidants like superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and improved the non-enzymic antioxidants like tocopherol, ascorbic acid and glutathione. EGCG ameliorated the malondialdehyde and protein carbonyl levels. Thus, EGCG has emerged out as a good antioxidant neutraceutical and a neuroprotective agent in alleviating the age-associated oxidative damage in aged rat brain.

Repletion of antioxidant status by EGCG and retardation of oxidative damage induced macromolecular anomalies in aged rats.

Ageing is defined as the loss of ability to maintain cellular homeostasis of an organism associated with the free radical-elicited oxidative damage to cellular macromolecules. The aim of this study was to evaluate the efficacy of epigallocatechin-3-gallate (EGCG), the key component of green tea catechins on attenuating the age associated oxidative perturbances by assessing the redox status in mitotic (liver) and post-mitotic (skeletal muscle) tissues of young and aged rats. From the results, we observed an increase in the marker for oxidative damage such as lipid peroxidation and protein carbonyl content in aged rats, when compared to young rats. This was accompanied with decreased levels of both enzymic and non-enzymic antioxidants and redox index. On supplementation with EGCG (100 mg/kg body weight by oral gavage for 30 days), the levels of lipid peroxidation and protein carbonyl content were significantly decreased in aged rats, possibly by enhancing the GSH redox status, and both enzymic and non-enzymic antioxidants status. In conclusion, this study supports the beneficial effect of EGCG in alleviating oxidative disturbances in ageing and retard the age associated derangements in both mitotic and post-mitotic tissues.

Oral L-arginine supplementation ameliorates urinary risk factors and kinetic modulation of Tamm-Horsfall glycoprotein in experimental hyperoxaluric rats.

BACKGROUND: Oral supplementation of l-arginine (l-arg) is found to be beneficial in many kidney disorders. We determined whether l-arg supplementation safeguards the renal epithelial cell damage induced by hyperoxaluria with excretion of urinary marker enzymes and lithogenic salts with special reference to Tamm-Horsfall glycoprotein (THP). METHODS: Hyperoxaluria was induced by 0.75% ethylene glycol (EG) in drinking water. l-Arg was co-supplemented at the dose of 1.25 g/kg b.w. orally for 28 days. At the end of experimental period, 24-h urine samples were collected in all the experimental groups. Isolation and purification of THP was carried in rat urine and were subjected to spectrophotometric crystallization assay and calcium-(14)C-oxalate binding studies. Determination of the lithogenic risk factors like calcium, oxalate, phosphorus, citrate, and marker enzymes such as lactate dehydrogenase (LDH) and gamma-glutamyltransferase (gamma-GT) were carried out in the collected urine sample. RESULTS: Urinary excretion of calcium and oxalate was significantly increased in EG-treated rats. In l-arg supplemented hyperoxaluric rats, these concentrations were significantly (p<0.001) decreased when compared to that of hyperoxaluric rats, and were moderately elevated from that of control rats. The activities of urinary marker enzymes, both LDH and gamma-GT were 2-fold increased in EG-treated rats, when compared to control rats, but these values were maintained near normal in l-arg supplemented EG-treated rats. Citrate excretion was enhanced in the l-arg co-supplemented hyperoxaluric rats. In spectrophotometric crystallization assay system, l-arg supplemented rat THP showed inhibition in nucleation and aggregation phases, whereas EG-treated rat THP showed promotion of both calcium oxalate nucleation and aggregation phases. In calcium-(14)C-oxalate binding assay, THP derived from hyperoxaluric rats exhibited 2-fold increase (p<0.001) in the Ca*Ox binding when compared to control and l-arg supplemented animals. CONCLUSIONS: l-Arg could act as a potent antilithic agent, by increasing the level of citrate in the hyperoxaluria-induced rats and decreasing calcium oxalate binding to the THP. l-Arg also effectively prevents the deposition of calcium oxalate crystals by curtailing the renal epithelial damage and protein oxidation as evidenced by the normal activities of urinary marker enzymes in l-arg supplemented hyperoxaluric rats.

Beneficial effect of vitamin E supplementation on the biochemical and kinetic properties of Tamm-Horsfall glycoprotein in hypertensive and hyperoxaluric patients.

BACKGROUND: This study aimed to assess the therapeutic efficacy of oral vitamin E supplementation on the biochemical and kinetic properties of Tamm-Horsfall glycoprotein (THP) in hypertensive and hyperoxaluric patients. METHODS: Newly detected hypertensives (n = 200) and stone formers (n = 200) were each subdivided into two groups. One group (n = 100) was administered the antioxidant vitamin E at 400 mg/day given as an oral supplement along with standard therapeutic drugs for hypertension and hyperoxaluria and the patients were followed for a period of 9 months. The other group (n = 100) did not receive vitamin E (placebo controls). Age and sex-matched controls (n = 100) were monitored simultaneously. THP was isolated from 24 h urine samples before and at the end of every third month during a period of 9 months from the vitamin E-treated hypertensive and hyperoxaluric groups. THP samples were also collected from control subjects, and at the end of the ninth month from placebo controls. The isolated protein was assessed for purity by SDS-PAGE. The purity-checked proteins were subjected to spectrophotometric crystallization assay, calcium oxalate (CaOx) crystal interaction studies, and biochemical analysis of sialic acid, thiol and carbonyl content. Plasma superoxide, hydroxyl radical, hydrogen peroxide and vitamin E levels as well as superoxide dismutase and catalase activities were also monitored. RESULTS: The THP from the hypertensive and hyperoxaluric subjects exhibited a significant promoting effect on the nucleation and aggregation phases and caused a concomitant increase in CaOx crystal interaction. The altered kinetic properties of THP in these subjects were strongly associated with increased carbonyl content and with decreased thiol and sialic acid contents. Oral administration of vitamin E to these patients caused near normalization of these biochemical alterations and satisfactorily restored the kinetic properties of THP to near normal activity. At the end of 9 months, THP isolated from placebo controls (hypertensive and hyperoxaluric) showed highly aggregated calcium oxalate monohydrate crystals as observed by light microscopy. In contrast, vitamin E-supplemented patients showed CaOx dihydrate crystals that were similar to control THP. There was an imbalance in the oxidant and antioxidant levels. For the oxidants, superoxide, hydrogen peroxide and hydroxyl radical levels were increased, and for the antioxidants, there was loss of antioxidant enzyme activities and a decline in plasma vitamin E level in both hypertensive and hyperoxaluric patients. Supplementary antioxidant (vitamin E) corrected this imbalance to near normal conditions. CONCLUSION: We hypothesize that the loss of THP inhibitory activity in the hypertensive and hyperoxaluric patients in a crystallizing medium is mediated primarily by oxidative damage to this protein. The possible occurrence of renal stones in essential hypertensive subjects, and the risk of recurrence in hyperoxaluric subjects, may be explained by oxidative damage to renal tissues that remained unchecked by standard drug therapies. The normalization of the kinetic properties of THP following vitamin E supplementation is in support of our hypothesis.

Counteraction of oxalate induced nitrosative stress by supplementation of l-arginine, a potent antilithic agent.

BACKGROUND: Our understanding of nitrosative stress in the process of urolithiasis is far from complete. Earlier studies carried out in our laboratory demonstrate the presence of nitrated THP in stone formers, l-arginine (l-arg) a precursor of nitric oxide (NO), attenuates the endothelial dysfunction caused by reactive nitrogen species. We investigated the role of l-arg in ethylene glycol (EG)-induced urolithic rat model and observed its antilithic and antioxidative properties. METHODS: Hyperoxaluria was induced using 0.75% EG in drinking water. l-arg [1.25 g/kg body weight] was given orally for a period of 28 days. RESULTS: EG-treated rats showed significant loss in body weight and increase in the activities of oxalate synthesizing enzymes such as glycollic acid oxidase in liver. Lactate dehydrogenase activity in liver and kidney was increased. The activity of the free radical producing enzyme xanthine oxidase, tissue oxalate and calcium levels were significantly increased in EG-treated rats. Depletion in the antioxidant enzymes, membrane bound ATPases and thiol status was observed in these rats. l-arg co-supplementation to EG-treated rats maintained the activities of the oxalate synthesizing enzymes and free radical producing enzymes with in the normal range. Tissue oxalate and calcium levels were also maintained near normal in l-arg treated hyperoxaluric rats. l-arg, by its cytoprotective effect, maintained the thiol status, thereby preserving the activities of the membrane bound ATPases and preventing proteinuria and subsequent weight loss in EG-treated rats. CONCLUSION: l-arg feeding prevents the retention of calcium oxalate crystals in hyperoxaluric rats by way of protecting the renal cells from oxidative injury and also by providing a second line of defense through the normalization of the oxalate metabolism. It reduces the risk of stone formation, by curtailing free radicals and hyperoxaluria as both of them have to work in close association to form stones.