Arjunolic acid modulate pancreatic dysfunction by ameliorating pattern recognition receptor and canonical Wnt pathway activation in type 2 diabetic rats.

BACKGROUND: Arjunolic acid (AA) is a potent phytochemical with multiple therapeutics effects. In this study, AA is evaluated on type 2 diabetic (T2DM) rats to understand the mechanism of ß-cell linkage with Toll-like receptor 4 (TLR-4) and canonical Wnt signaling. However, its role in modulating TLR-4 and canonical Wnt/ß-catenin crosstalk on insulin signaling remains unclear during T2DM. Aim The current study is aimed to examine the potential role of AA on insulin signaling and TLR-4-Wnt crosstalk in the pancreas of type 2 diabetic rats. METHOD: Multiple methods were used to determine molecular cognizance of AA in T2DM rats, when treated with different dosage levels. Histopathological and histomorphometry analysis was conducted using masson trichrome and H&E stains. While, protein and mRNA expressions of TLR-4/Wnt and insulin signaling were assessed using automated Western blotting (jess), immunohistochemistry, and RT-PCR. RESULTS: Histopathological findings revealed that AA had reversed back the T2DM-induced apoptosis and necrosis caused to rats pancreas. Molecular findings exhibited prominent effects of AA in downregulating the elevated level of TLR-4, MyD88, NF-κB, p-JNK, and Wnt/ß-catenin by blocking TLR-4/MyD88 and canonical Wnt signaling in diabetic pancreas, while IRS-1, PI3K, and pAkt were all upregulated by altering the NF-κB and ß-catenin crosstalk during T2DM. CONCLUSION: Overall results, indicate that AA has potential to develop as an effective therapeutic in the treatment of T2DM associated meta-inflammation. However, future preclinical research at multiple dose level in a long-term chronic T2DM disease model is warranted to understand its clinical relevance in cardiometabolic disease.

Polyphenolics with Strong Antioxidant Activity from Acacia nilotica Ameliorate Some Biochemical Signs of Arsenic-Induced Neurotoxicity and Oxidative Stress in Mice.

Neurotoxicity is a serious health problem of patients chronically exposed to arsenic. There is no specific treatment of this problem. Oxidative stress has been implicated in the pathological process of neurotoxicity. Polyphenolics have proven antioxidant activity, thereby offering protection against oxidative stress. In this study, we have isolated the polyphenolics from Acacia nilotica and investigated its effect against arsenic-induced neurotoxicity and oxidative stress in mice. Acacia nilotica polyphenolics prepared from column chromatography of the crude methanol extract using diaion resin contained a phenolic content of 452.185 ± 7.879 mg gallic acid equivalent/gm of sample and flavonoid content of 200.075 ± 0.755 mg catechin equivalent/gm of sample. The polyphenolics exhibited potent antioxidant activity with respect to free radical scavenging ability, total antioxidant activity and inhibition of lipid peroxidation. Administration of arsenic in mice showed a reduction of acetylcholinesterase activity in the brain which was counteracted by Acacia nilotica polyphenolics. Similarly, elevation of lipid peroxidation and depletion of glutathione in the brain of mice was effectively restored to normal level by Acacia nilotica polyphenolics. Gallic acid methyl ester, catechin and catechin-7-gallate were identified in the polyphenolics as the major active compounds. These results suggest that Acacia nilotica polyphenolics due to its strong antioxidant potential might be effective in the management of arsenic induced neurotoxicity.

Arjunolic acid downregulates elevated blood sugar and pro-inflammatory cytokines in streptozotocin (STZ)-nicotinamide induced type 2 diabetic rats.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a worldwide health issue primarily due to failure of pancreatic ß-cells to release sufficient insulin. PURPOSE: The present work aimed to assess the antidiabetic potential of arjunolic acid (AA) isolated from Terminalia arjuna in type 2 diabetic rats. STUDY DESIGN: After extraction, isolation and purification, AA was orally administered to type 2 diabetic Sprague Dawley rats to investigate antidiabetic effect of AA. METHOD: T2DM was induced via single intraperitoneal injection of streptozotocin-nicotinamide (STZ-NIC) in adult male rats. After 10 days, fasting and random blood glucose (FBG and RBG), body weight (BW), food and water intake, serum C-peptide, insulin and glycated hemoglobin (HbA1c) was measured to confirm T2DM development. Dose dependent effects of orally administered AA (25 and 50 mg/kg/day) for 4 weeks was investigated by measuring BW variation, fasting and postprandial hyperglycemia, oral glucose tolerance test (OGTT), and levels of serum HbA1c, serum total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), high density lipoprotein (HDL), serum and pancreatic C-peptide, insulin, growth differentiation factor 15 (GDF-15), serum and pancreatic inflammatory cytokines. RESULTS: The oral administration of AA in preclinical model of T2DM significantly normalized FBG and RBG, restored BW, controlled polyphagia, polydipsia and glucose tolerance. In addition, AA notably reduced serum HbA1c, TC, TG, LDL with non-significant increase in HDL. On the other hand, significant increase in serum and pancreatic C-peptide and insulin was observed with AA treatment, while serum and pancreatic GDF-15 were non-significantly altered in AA treated diabetic rats. Moreover, AA showed dose dependent reduction in serum and pancreatic proinflammatory cytokines including TNF-α, IL-1ß and IL-6. CONCLUSION: For the first time our findings highlighted AA as a potential candidate in type 2 diabetic conditions.

Le Carbone prevents liver damage in non-alcoholic steatohepatitis-hepatocellular carcinoma mouse model via AMPKα-SIRT1 signaling pathway activation.

Le Carbone (LC), a fiber-enriched activated charcoal dietary supplement, claimed to be effective against inflammation associated with colitis, trimethylaminuria, and sclerosis. The study aimed to investigate the underlying mechanisms of LC to protect liver damage and its progression in non-alcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC) mice. To induce this model, C57BL/6J male baby mice were injected with a low-dose of streptozotocin and fed with a high-fat diet (HFD) 32 during 4 weeks-16 weeks of age. The LC suspension was administered orally at a dose of 5 mg/mouse/day started at the age of 6 weeks and continued until 16 weeks of age along with HFD32 feeding. At the end of the experiment, serum and liver tissues were collected for the biochemical, histological, and molecular analysis. We found that LC suspension improved the histopathological changes, serum aminotransferases in NASH mice. The hepatic expression of metabolic proteins, p-AMPKα and sirtuin 1, and proteins responsible for ß-oxidation of fatty acids, peroxisome proliferator-activated receptor (PPAR) γ coactivator-α, PPARα were significantly repressed in NASH mice. LC treatment markedly restored these expressions. LC treatment significantly reduced the hepatic proteins expressions of PPARγ, tissue inhibitor of metalloproteinases 4, p47phox, p-JNK, p-ERK1/2, glypican-3, and prothrombin in NASH mice. Our findings demonstrate that LC prevents the liver damage and progression of NASH, possibly by enhancing the AMPK-SIRT1 signaling pathway.

Brain adaptations of insulin signaling kinases, GLUT 3, p-BADser155 and nitrotyrosine expression in various hypoglycemic models of mice.

AIM AND OBJECTIVE: Insulin-induced moderate or severe hypoglycemia (MH or SH) impairs cognition and SH causes neuronal death. On the contrary, alternate day fasting (ADF) protects the brain during excitotoxic stress and improves cognitive function. Unlike the scenario in the periphery, insulin and its relationship towards brain glucose uptake and metabolism are considered to be less significant. Yet, the hypoglycemia associated brain metabolism is not clearly understood. The authors broadly investigated the brain metabolism in various hypoglycemic models such as insulin-induced MH, SH, SH with glucose reperfusion, 24 h fasting and ADF in the cortex or hippocampus of C57BL6/J mice. The authors analyzed the protein expression of insulin signaling kinases (plays a key role in neuronal survival and memory), Bcl-2 associated death promoter (p-BADser155) (dephosphorylation inhibits glucokinase activity and reduces glucose or increases ketone body metabolism in the brain), neuronal-specific glucose transporter 3 (GLUT 3) and nitrotyrosine (marker of nitric oxide which is involved in neuronal glucose uptake via GLUT 3) using western blotting analysis. RESULTS: Insulin-induced MH or SH differentially regulated the brain insulin signaling kinases. The expression of p-BADser155 decreased in all hypoglycemic models except the insulin-induced MH in hippocampus. The trended higher GLUT 3 and increased nitrotyrosine expression of insulin-induced SH were restored after glucose reperfusion. The trended higher or increased GLUT 3 and nitrotyrosine expression of ADF were positively correlated with serum beta-hydroxybutyrate levels. CONCLUSION: During hypoglycemia, it can be suggested that the brain might decrease glucose metabolism via glycolysis or prefer ketone body metabolism (except the insulin-induced MH in hippocampus) by modifying the p-BADser155 expression. In addition to the ketone body metabolism, the brain might adapt to uptake glucose in insulin-induced SH or ADF by modifying the GLUT 3 or nitrotyrosine expression.

Oral toxicity of arjunolic acid on hematological, biochemical and histopathological investigations in female Sprague Dawley rats.

BACKGROUND: Arjunolic acid (AA) is a potent phytochemical with wider pharmacological activities. Despite potential medicinal properties on various in vitro and in vivo studies, there is still a dearth of scientific data related to its safety profile and toxicological parameters. The current study aimed to investigate acute toxicity of AA in normal female Sprague Dawley rats. METHODS: In this study, AA was administered orally at an individual dose of 300 and 2000 mg/kg body weight to group 1 and 2 respectively, while group 3 served as normal control. All the animals were observed for 2 weeks to determine any behavioral and physical changes. On day 15, blood was collected for hematological and biochemical investigation, later animals from all the three groups were euthanized to harvest and store essential organs for histopathological analysis. Four different staining techniques; hematoxylin and eosin, Masson trichrome, Periodic acid Schiff and Oil O Red were used to investigate any alterations in different tissues through microscopical observation. RESULTS: The results of the study showed no morbidity and mortality at two different dosage of AA treatment. Daily food & water intake, body weight, relative organ weight, hematological and biochemical parameters were detected to be normal with no severe alteration seen through microscopical investigation in the structure of harvested tissues. Our findings support the safety profile of AA, which was well tolerated at higher dose. Thus, an in-detail study on the subacute disease model is warranted.

Curcumin reduces the risk of chronic kidney damage in mice with nonalcoholic steatohepatitis by modulating endoplasmic reticulum stress and MAPK signaling.

Developing confirmation recommends that in patients with dynamic type of NAFLD, particularly nonalcoholic steatohepatitis (NASH) may have the pathogenic parts in the advancement of kidney damage. In this study we have examined the impact of curcumin on NASH instigated chronic kidney damage (CKD) and the putative mechanisms. To prepare this NASH model, neonatal C57BL/6J male mice were exposed to low-dose streptozotocin (STZ) and were fed high-fat diet (HFD) at the age of 4weeks and continued up to 14weeks, curcumin was given at 100mg/kg dose by oral gavage daily after 10weeks of STZ injection and continued for 4weeks along with HFD feeding. NASH incited mice demonstrated nephrotoxicity as proved by declining renal capacity, which was evaluated by measuring blood urea nitrogen and creatinine in serum and histopathological variations from the norm. These progressions were switched by curcumin treatment, which brought about huge change in renal capacity. Furthermore, curcumin markedly decreased NAD(P)H oxidase subunits (p67phox, p47phox, p22phox), nitrotyrosine and CYP2E1 renal protein expression as well as reduced pro-inflammatory cytokine expression (TNFα, IL-1ß, IFNγ). Renal protein expression of mitogen activated protein kinases (MAPKs) (p-JNK, p-ERK1/2) and glucose regulated protein 78, CHOP were increased in NASH induced mice and curcumin treatment attenuated these increased expressions. In addition, curcumin treatment also decreased the apoptosis signaling proteins (cleaved caspase-3, cleaved caspase-12) in the NASH kidney. Taken together, our results suggest that curcumin preserves the renal function, probably by attenuating the ER stress mediated MAPK signaling.

Le Carbone, a charcoal supplement, modulates DSS-induced acute colitis in mice through activation of AMPKα and downregulation of STAT3 and caspase 3 dependent apoptotic pathways.

Le Carbone (LC) is a charcoal supplement, which contains a large amount of dietary fibers. Several studies suggested that charcoal supplement may be beneficial for stomach disorders, diarrhea, gas and indigestion. But no studies address whether LC intake would suppress inflammation, cell proliferation or disease progression in colitis. In the present study, the effect of LC on experimental colitis induced by dextran sulfate sodium (DSS) in mice and its possible mechanism of action were examined. A study was designed for 8days, using C57BL/6 female mice that were administered with 3% DSS in drinking water for 7days followed by another 1day consumption of normal water with or without treatment. LC suspension was administered daily for 7days via oral gavage using 5mg/mouse in treatment group and normal group was supplied with drinking water. LC suspension significantly attenuated the loss of body weight and shortening of colon length induced by DSS. The disease activity index, histopathologic changes were significantly reduced by LC treatment. The inflammatory mediators TNFα, IL-1ß, p-STAT3 and p-NF-κB induced in the colon by DSS were markedly suppressed by LC. The increased activation of AMPKα in the colon was also detected in LC group. Furthermore, the apoptotic marker protein cleaved caspase 3 was down-regulated and anti-apoptotic proteins Bcl2 and Bcl-xL were significantly up-regulated by LC treatment. Taken together, our results demonstrate the ability of LC to inhibit inflammation, apoptosis and give some evidence for its potential use as adjuvant treatment of inflammatory bowel disease.

Fasting time duration modulates the onset of insulin-induced hypoglycemic seizures in mice.

OBJECTIVE: Fasting (48h) in mice causes resistance to insulin-induced hypoglycemic seizures (IIHS) but in rats fasting (14-16h) predisposes IIHS. So we suspect the duration of fasting may possibly affect the onset of seizures and in this study, we investigated the IIHS by administering 8 Units (U) insulin (INS)/k.g., intraperitoneally to 8 weeks old male C57BL6/J mice. METHODS: The mice were divided into group 1 (non-fasted), group 2 (6h fasted) and group 3 (24h fasted) and we administered the 8U INS. The first behavioral hypoglycemic seizure symptoms such as jump, clonus or barrel rotations considered as seizure onset and we analyzed the blood glucose level (BGL) and serum beta-hydroxybutyrate (BHB) level. RESULTS: The time of first seizure onset in group 1 was 109.7±4.3min, group 2 was 46.50±3.9min and group 3 was 165.4±13.26min. The seizure onset time in group 2 was significantly decreased compared to group 1. The seizure onset time in group 3 was significantly increased compared to group 1 and group 2. The decreased BGL after INS administration was correlated with the seizure onset time in group 1 and group 2 but not in group 3. The BHB level in group 3 was significantly higher compared to group 1 and 2. CONCLUSION: Our data show that the fasting time duration significantly modulates the onset of hypoglycemic seizures. The opposite effect of 6h or 24h fasting time duration is likely caused by different BHB levels.

Hypothalamic glucagon signaling in fasting hypoglycemia.

AIMS: Sustained glucagon infusion increases hepatic glucose production, but this effect is transient due to hypothalamic glucagon signaling. In hypoglycemia, glucagon acts as a major defense to sustain the blood glucose level and this raises the question regarding glucagon signaling associated glucose production in prolonged fasting hypoglycemia. In this study, we investigated the proteins associated with hypothalamic glucagon signaling and liver gluconeogenesis during fasting hypoglycemia. MAIN METHODS: 8-9week old, male C57BL6/J mice were fasted for 4, 8, 12, 18, 24, 30, 36 or 42h. In the hypothalamus, we investigated glucagon signaling by analyzing the glucagon receptor and its downstream protein, peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC-1) expression. In the liver, we investigated gluconeogenesis by analyzing p-protein kinase A (PKA)(Ser/Thr) substrate and phosphoenolpyruvate carboxykinase - cytosolic (PEPCK-C) expression using the western blotting technique. KEY FINDINGS: The elevated or trended higher hypothalamic glucagon receptor and PGC-1 expressions at 18 and 42h were correlated with the attenuated liver p-PKA(Ser/Thr) substrate expression. The attenuated hypothalamic glucagon receptor and PGC-1 expressions at 12, 24, 30 and 36h were correlated with the elevated or trended higher liver p-PKA(Ser/Thr) substrate expression. SIGNIFICANCE: The hypothalamic glucagon signaling during fasting hypoglycemia might have been modulated by circadian rhythm and this possibly attenuates the liver p-PKA(Ser/Thr) substrate to modify the gluconeogenesis pathway. This mechanism will help to understand the hyperglucagonemia associated complications in diabetes.

Jumihaidokuto effectively inhibits colon inflammation and apoptosis in mice with acute colitis.

Jumihaidokuto, a Japanese kampo medicine, is prescribed in Japan for its anti-inflammatory activity. Here we have examined its beneficial effects against acute colitis induced by dextran sulfate sodium (DSS) in mice. We have used C57BL/6 female mice, divided into two groups and received 3% DSS in drinking water during the experimental period (8days). Treatment group mice received 1g/kg/day dose of Jumihaidokuto orally whereas DSS control group received equal volume of distilled water. Normal control group mice received plain drinking water. Jumihaidokuto treatment attenuated the colitis symptoms along with suppression of various inflammatory marker proteins such as IL-1ß, IL-2Rα, IL-4, CTGF and RAGE. It has also down-regulated the oxidative stress and apoptotic signaling in the colons of mice with colitis. The present study has confirmed the beneficial effects of Jumihaidokuto on DSS induced acute colitis in mice and suggests that it can be a potential agent for the treatment of colitis.

Telmisartan treatment targets inflammatory cytokines to suppress the pathogenesis of acute colitis induced by dextran sulphate sodium.

The renin angiotensin system (RAS) is essential for the regulation of cardiovascular and renal functions to maintain the fluid and electrolyte homeostasis. Recent studies have demonstrated a locally expressed RAS in various tissues of mammals, which is having pathophysiological roles in those organ system. Interestingly, local RAS has important role during the inflammatory bowel disease pathogenesis. Further to delineate its role and also to identify the potential effects of telmisartan, an angiotensin receptor blocker, we have used a mouse model of acute colitis induced by dextran sulphate sodium. We have used 0.01 and 5mg/kg body weight doses of telmisartan and administered as enema to facilitate the on-site action and to reduce the systemic adverse effects. Telmisartan high dose treatment significantly reduced the disease activity index score when compared with the colitis control mice. In addition, oxidative stress and endoplasmic reticulum stress markers expression were also significantly reduced when compared with the colitis control mice. Subsequent experiments were carried out to investigate some of the mechanisms underlying its anti-inflammatory effects and identified that the mRNA levels of pro-inflammatory cytokines such as tumour necrosis factor α, interleukin 1ß, interleukin 6 and monocyte chemoattractant protein 1 as well as cellular DNA damage were significantly suppressed when compared with the colitis control mice. Similarly the apoptosis marker proteins such as cleaved caspase 3 and 7 levels were down-regulated and anti-apoptotic protein Bcl2 level was significantly upregulated by telmisartan treatment. These results indicate that blockade of RAS by telmisartan can be an effective therapeutic option against acute colitis.

Effect of carvedilol against myocardial injury due to ischemia-reperfusion of the brain in rats.

We have previously reported the mechanism behind the myocardial injury and the activation of autonomic nervous system during the ischemia-reperfusion (IR) of the rat brain. This study was planned to investigate the effect of carvedilol, a ß-blocker, in improving the myocardial injury caused by IR of the rat brain. We have used a whole cerebral IR model in rats by clamping both the right and left common carotid arteries. Rats were divided into five groups; Sham surgery group (Group-Sham), carvedilol treatment before ischemia group (Group-Is+C), vehicle control group (Group-Is+V), carvedilol treatment before reperfusion group (Group-Re+C) and the vehicle control group (Group-Re+V). We have measured the blood pressure and heart rate via a catheter, myocardial tissue ß1-adrenaline receptor (ß1-AR) levels, phosphor-p38 mitogen-activated protein kinase (p-p38 MAPK) signaling factor, malondialdehyde (MDA), and apoptosis (TUNEL assay and expression of caspase-7 protein). The results indicated that the increased expressions of ß1-AR, p-p38 MAPK, caspase-7, apoptotic cells and MDA level in the myocardial tissue due to brain ischemia-reperfusion were significantly reduced by carvedilol treatment. From these observations we can suggest that, with the advantage of its antioxidant and ß blocking action, carvedilol had played the improvement of myocardial injury in ischemia-reperfusion of the brain.