Shreesh Ojha

Up-regulation of PPARγ, heat shock protein-27 and -72 by naringin attenuates insulin resistance, β-cell dysfunction, hepatic steatosis and kidney damage in a rat model of type 2 diabetes

Naringin, a bioflavonoid isolated from grapefruit, is well known to possess lipid-lowering and insulin-like properties. Therefore, we assessed whether naringin treatment ameliorates insulin resistance (IR), β-cell dysfunction, hepatic steatosis and kidney damage in high-fat diet (HFD)-streptozotocin (STZ)-induced type 2 diabetic rats. Wistar albino male rats were fed a HFD (55 % energy from fat and 2 % cholesterol) to develop IR and on the 10th day injected with a low dose of streptozotocin (40 mg/kg, intraperitoneal (ip)) to induce type 2 diabetes. After confirmation of hyperglycaemia (>13·89 mmol/l) on the 14th day, different doses of naringin (25, 50 and 100 mg/kg per d) and rosiglitazone (5 mg/kg per d) were administered orally for the next 28 d while being maintained on the HFD. Naringin significantly decreased IR, hyperinsulinaemia, hyperglycaemia, dyslipidaemia, TNF-α, IL-6, C-reactive protein and concomitantly increased adiponectin and β-cell function in a dose-dependent manner. Increased thiobarbituric acid-reactive substances and decreased antioxidant enzyme activities in the serum and tissues of diabetic rats were also normalised. Moreover, naringin robustly increased PPARγ expression in liver and kidney; phosphorylated tyrosine insulin receptor substrate 1 in liver; and stress proteins heat shock protein (HSP)-27 and HSP-72 in pancreas, liver and kidney. In contrast, NF-κB expression in these tissues along with sterol regulatory element binding protein-1c and liver X receptor- expressions in liver were significantly diminished. In addition, microscopic observations validated that naringin effectively rescues β-cells, hepatocytes and kidney from HFD-STZ-mediated oxidative damage and pathological alterations. Thus, this seminal study provides cogent evidence that naringin ameliorates IR, dyslipidaemia, β-cell dysfunction, hepatic steatosis and kidney damage in type 2 diabetic rats by partly regulating oxidative stress, inflammation and dysregulated adipocytokines production through up-regulation of PPARγ, HSP-27 and HSP-72.

β-Caryophyllene, a CB2 receptor agonist produces multiple behavioral changes relevant to anxiety and depression in mice

Recent evidence suggests that the cannabinoid receptor subtype 2 (CB2) is implicated in anxiety and depression disorders, although few systematic studies in laboratory animals have been reported. The aim of the current experiments was to test the effects of the CB2 receptor potent-selective agonist β-caryophyllene (BCP) in animals subjected to models of anxiolytic- and antidepressant-like effects. Therefore effects of BCP (50mg/kg) on anxiety were assessed using the elevated plus maze (EPM), open field (OF), and marble burying test (MBT). However for depression, the novelty-suppressed feeding (NSF), tail suspension test (TST), and forced swim tests (FST) were used. Results indicated that adult mice receiving BCP showed amelioration of all the parameters observed in the EPM test. Also, BCP significantly increased the time spent in the center of the arena without altering the general motor activity in the OF test. This dose was also able to decrease the number of buried marbles and time spent digging in the MBT, suggesting an anti-compulsive-like effect. In addition, the systemic administration of BCP reduced immobility time in the TST and the FST. Finally, BCP treatment decreased feeding latency in the NSF test. Most importantly, pre-administration of the CB2 receptor antagonist AM630, fully abrogated the anxiolytic and the anti-depressant effects of BCP. Taken together, these preclinical results suggest that CB2 receptors may provide alternative therapeutic targets for the treatment of anxiety and depression. The possibility that BCP may ameliorate the symptoms of these mood disorders offers exciting prospects for future studies.

Cardiovascular friendly natural products: a promising approach in the management of CVD

Natural products play an important role as nutritional supplements and provide potential health benefits in cardiovascular diseases (CVD). Compiling data from experimental, epidemiological and clinical studies indicates that dietary nutrients have profound cardioprotective effects in the primary as well as secondary prevention of coronary heart disease, hence they are considered as cardiovascular friendly natural products. The mechanism of cardioprotection produced by dietary nutritional supplements such as flavonoids (citrus fruits, pulses, red wine, tea and cocoa), olive oil, omega-3 (ω-3) fatty acids (fish oil and fish-based products), lycopene (tomato and tomato-based products), resveratrol (grapes and red wine), coffee, and soy in the prevention and treatment of cardiovascular disorders have been discussed in the present review, with the emphasis of epidemiological and clinical studies. Based on the intriguing results of various studies, prophylactic and therapeutic potential of cardiovascular friendly natural products have been suggested. The supplementation of cardiovascular friendly natural products needs to be considered in all populations who have high prevalence of CVD.

The cannabinoid receptor 2 agonist, β-caryophyllene, reduced voluntary alcohol intake and attenuated ethanol-induced place preference and sensitivity in mice.

Several recent studies have suggested that brain CB2 cannabinoid receptors play a major role in alcohol reward. In fact, the implication of cannabinoid neurotransmission in the reinforcing effects of ethanol (EtOH) is becoming increasingly evident. The CB2 receptor agonist, β-caryophyllene (BCP) was used to investigate the role of the CB2 receptors in mediating alcohol intake and ethanol-induced conditioned place preference (EtOH-CPP) and sensitivity in mice. The effect of BCP on alcohol intake was evaluated using the standard two-bottle choice drinking method. The mice were presented with increasing EtOH concentrations and its consumption was measured daily. Consumption of saccharin and quinine solutions was measured following the EtOH preference tests. Finally, the effect of BCP on alcohol reward and sensitivity was tested using an unbiased EtOH-CPP and loss of righting-reflex (LORR) procedures, respectively. BCP dose-dependently decreased alcohol consumption and preference. Additionally, BCP-injected mice did not show any difference from vehicle mice in total fluid intake in a 24-hour paradigm nor in their intake of graded concentrations of saccharin or quinine, suggesting that the CB2 receptor activation did not alter taste function. More importantly, BCP inhibited EtOH-CPP acquisition and exacerbated LORR duration. Interestingly, these effects were abrogated when mice were pre-injected with a selective CB2 receptor antagonist, AM630. Overall, the CB2 receptor system appears to be involved in alcohol dependence and sensitivity and may represent a potential pharmacological target for the treatment of alcoholism.

Cardioprotective effect of lycopene in the experimental model of myocardial ischemia-reperfusion injury

The efficacy of lycopene to limit myocardial injury after ischemia and reperfusion was explored in the present study. Adult male albino Wistar rats were divided into three experimental groups and orally received olive oil as vehicle (sham and control I-R) or lycopene 1 mg/kg dissolved in olive oil (lycopene treated group) respectively for 31 days. On the 31st day, animals of the control I-R and lycopene treated groups were subjected to 45 min of occlusion of the LAD coronary artery and were thereafter reperfused for 1 h. The ischemia-reperfusion injury resulted in significant cardiac necrosis, depression in hemodynamics, decline in antioxidant status and rise in lipid peroxidation product levels in the control I-R group as compared to sham control. In histopathological examinations myocardial damage produced after I-R was significantly prevented in the lycopene treated group. Lycopene treatment resulted in preservation of the myocardial antioxidant status and altered hemodynamic parameters as compared to control I-R group. Furthermore, I-R-induced lipid peroxidation was significantly inhibited in the lycopene treated group. These beneficial cardioprotective effects also translated into the functional recovery of the heart. The beneficial effect of lycopene likely results from the suppression of oxidative stress, which results in the reduction of myocardial injury.

Challenges and issues with streptozotocin-induced diabetes – A clinically relevant animal model to understand the diabetes pathogenesis and evaluate therapeutics

Streptozotocin (STZ) has been extensively used over the last three decades to induce diabetes in various animal species and to help screen for hypoglycemic drugs. STZ induces clinical features in animals that resemble those associated with diabetes in humans. For this reason STZ treated animals have been used to study diabetogenic mechanisms and for preclinical evaluation of novel antidiabetic therapies. However, the physiochemical characteristics and associated toxicities of STZ are still major obstacles for researchers using STZ treated animals to investigate diabetes. Another major challenges in STZ-induced diabetes are sustaining uniformity, suitability, reproducibility and induction of diabetes with minimal animal lethality. Lack of appropriate use of STZ was found to be associated with increased mortality and animal suffering. During STZ use in animals, attention should be paid to several factors such as method of preparation of STZ, stability, suitable dose, route of administration, diet regimen, animal species with respect to age, body weight, gender and the target blood glucose level used to represent hyperglycemia. Therefore, protocol for STZ-induced diabetes in experimental animals must be meticulously planned. This review highlights specific skills and strategies involved in the execution of STZ-induced diabetes model. The present review aims to provide insight into diabetogenic mechanisms of STZ, specific toxicity of STZ with its significance and factors responsible for variations in diabetogenic effects of STZ. Further this review also addresses ways to minimize STZ-induced mortality, suggests methods to improve STZ-based experimental models and best utilize them for experimental studies purported to understand diabetes pathogenesis and preclinical evaluation of drugs.

Neuroprotective potential of ferulic acid in the rotenone model of Parkinson’s disease

Parkinson’s disease (PD) is a chronic, progressive, and the second most common form of neurodegenerative disorders. In order to explore novel agents for the treatment of PD, in the current study, we have evaluated the neuroprotective efficacy of ferulic acid (FA) using rotenone (ROT)-induced rat model of PD. ROT was administered 2.5 mg/kg body weight to male Wistar rats for 4 weeks to induce the PD. Since PD is progressive and chronic in nature, the paradigm for evaluating FA was based on chronic administration for 4 weeks at the dose of 50 mg/kg, 30 minutes prior to ROT administration. ROT administration caused significant reduction in endogenous antioxidants such as superoxide dismutase, catalase, and glutathione. ROT challenge-induced lipid peroxidation evidenced by increased malondialdehyde following perturbation of antioxidant defense. Apart from oxidative stress, ROT also activated proinflammatory cytokines and enhanced inflammatory mediators such as cyclooxygenase-2 and inducible nitric oxide synthase. The immunofluorescence analysis revealed a significant increase in the number of activated microglia and astrocytes accompanied by a significant loss of dopamine (DA) neurons in the substantia nigra pars compacta area upon ROT injection. However, treatment with FA rescued DA neurons in substantia nigra pars compacta area and nerve terminals in the striatum from the ROT insult. FA treatment also restored antioxidant enzymes, prevented depletion of glutathione, and inhibited lipid peroxidation. Following treatment with FA, the inflammatory mediators such as cyclooxygenase-2 and inducible nitric oxide synthase and proinflammatory cytokines were also reduced. Further, the results were supported by a remarkable reduction of Iba-1 and GFAP hyperactivity clearly suggests attenuation of microglial and astrocytic activation. Results of our study suggest that FA has promising neuroprotective effect against degenerative changes in PD, and the protective effects are mediated through its antioxidant and anti-inflammatory properties.

Seabuckthorn Pulp Oil Protects against Myocardial Ischemia–Reperfusion Injury in Rats through Activation of Akt/eNOS

Seabuckthorn (SBT) pulp oil obtained from the fruits of seabuckthorn [Hippophae rhamnoides L. (Elaeagnaceae)] has been used traditionally for its medicinal and nutritional properties. However, its role in ischemia–reperfusion (IR) injury of myocardium in rats has not been elucidated so far. The present study reports the cardioprotective effect of SBT pulp oil in IR-induced model of myocardial infarction in rats and underlying mechanism mediating activation of Akt/eNOS signaling pathway. Male albino Wistar rats were orally administered SBT pulp oil (5, 10, and 20 ml/kg/day) or saline for 30 days. On the day 31, ischemia was induced by one-stage ligation of left anterior descending coronary artery for 45 min followed by reperfusion for 60 min. SBT pulp oil pretreatment at the dose of 20 ml/kg observed to stabilize cardiac function and myocardial antioxidants such as glutathione, superoxide dismutase, catalase, and inhibited lipid peroxidation evidenced by reduced malondialdehyde levels as compared to IR-control group. SBT pulp oil also improved hemodynamic and contractile function and decreased tumor necrosis factor and activities of myocyte injury marker enzymes; lactate dehydrogenase and creatine kinase-MB. Additionally, a remarkable rise in expression of pAkt–eNOS, Bcl-2 and decline in expression of IKKβ/NF-κB and Bax was observed in the myocardium. The histopathological and ultrastructural salvage of cardiomyocytes further supports the cardioprotective effect of SBT pulp oil. Based on findings, it can be concluded that SBT pulp oil protects against myocardial IR injury mediating favorable modulation of Akt-eNOS and IKKβ/NF-κB expression.

Cannabinoid Type 2 (CB2) Receptors Activation Protects against Oxidative Stress and Neuroinflammation Associated Dopaminergic Neurodegeneration in Rotenone Model of Parkinson's Disease

The cannabinoid type two receptors (CB2), an important component of the endocannabinoid system, have recently emerged as neuromodulators and therapeutic targets for neurodegenerative diseases including Parkinsons disease (PD). The downregulation of CB2 receptors has been reported in the brains of PD patients. Therefore, both the activation and the upregulation of the CB2 receptors are believed to protect against the neurodegenerative changes in PD. In the present study, we investigated the CB2 receptor-mediated neuroprotective effect of β-caryophyllene (BCP), a naturally occurring CB2 receptor agonist, in, a clinically relevant, rotenone (ROT)-induced animal model of PD. ROT (2.5 mg/kg BW) was injected intraperitoneally (i.p.) once daily for 4 weeks to induce PD in male Wistar rats. ROT injections induced a significant loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and DA striatal fibers, following activation of glial cells (astrocytes and microglia). ROT also caused oxidative injury evidenced by the loss of antioxidant enzymes and increased nitrite levels, and induction of proinflammatory cytokines: IL-1β, IL-6 and TNF-α, as well as inflammatory mediators: NF-κB, COX-2, and iNOS. However, treatment with BCP attenuated induction of proinflammatory cytokines and inflammatory mediators in ROT-challenged rats. BCP supplementation also prevented depletion of glutathione concomitant to reduced lipid peroxidation and augmentation of antioxidant enzymes: SOD and catalase. The results were further supported by tyrosine hydroxylase immunohistochemistry, which illustrated the rescue of the DA neurons and fibers subsequent to reduced activation of glial cells. Interestingly, BCP supplementation demonstrated the potent therapeutic effects against ROT-induced neurodegeneration, which was evidenced by BCP-mediated CB2 receptor activation and the fact that, prior administration of the CB2 receptor antagonist AM630 diminished the beneficial effects of BCP. The present study suggests that BCP has the potential therapeutic efficacy to elicit significant neuroprotection by its anti-inflammatory and antioxidant activities mediated by activation of the CB2 receptors.

Kaempferol Attenuates Myocardial Ischemic Injury via Inhibition of MAPK Signaling Pathway in Experimental Model of Myocardial Ischemia-Reperfusion Injury.

Kaempferol (KMP), a dietary flavonoid, has antioxidant, anti-inflammatory, and antiapoptotic effects. Hence, we investigated the effect of KMP in ischemia-reperfusion (IR) model of myocardial injury in rats. We studied male albino Wistar rats that were divided into sham, IR-control, KMP-20 + IR, and KMP 20 per se groups. KMP (20 mg/kg; i.p.) was administered daily to rats for the period of 15 days, and, on the 15th day, ischemia was produced by one-stage ligation of left anterior descending coronary artery for 45 min followed by reperfusion for 60 min. After completion of surgery, rats were sacrificed; heart was removed and processed for biochemical, morphological, and molecular studies. KMP pretreatment significantly ameliorated IR injury by maintaining cardiac function, normalizing oxidative stress, and preserving morphological alterations. Furthermore, there was a decrease in the level of inflammatory markers (TNF-α, IL-6, and NFκB), inhibition of active JNK and p38 proteins, and activation of ERK1/ERK2, a prosurvival kinase. Additionally, it also attenuated apoptosis by reducing the expression of proapoptotic proteins (Bax and Caspase-3), TUNEL positive cells, and increased level of antiapoptotic proteins (Bcl-2). In conclusion, KMP protected against IR injury by attenuating inflammation and apoptosis through the modulation of MAPK pathway.