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.

Curcumin ameliorates liver damage and progression of NASH in NASH-HCC mouse model possibly by modulating HMGB1-NF-κB translocation.

Curcumin, a phenolic compound, has a wide spectrum of therapeutic effects such as antitumor, anti-inflammatory, anti-cancer and so on. The study aimed to investigate the underlying mechanisms of curcumin to protect liver damage and progression of non-alcoholic steatohepatitis (NASH) in a novel NASH-hepatocellular carcinoma (HCC) mouse model. To induce this model neonatal C57BL/6J male mice were exposed to low-dose streptozotocin and were fed a high-fat diet (HFD) from the age of 4weeks to 14weeks. Curcumin was given at 100mg/kg dose daily by oral gavage started at the age of 10weeks and continued until 14weeks along with HFD feeding. We found that curcumin improved the histopathological changes of the NASH liver via reducing the level of steatosis, fibrosis associated with decreasing serum aminotransferases. In addition, curcumin treatment markedly reduced the hepatic protein expression of oxidative stress, pro-inflammatory cytokines, and chemokines including interferon (IFN) γ, interleukin-1ß and IFNγ-inducible protein 10, in NASH mice. Furthermore, curcumin treatment significantly reduced the cytoplasmic translocation of high mobility group box 1 (HMGB1) and the protein expression of toll like receptor 4. Nuclear translocation of nuclear factor kappa B (NF-κB) was also dramatically attenuated by the curcumin in NASH liver. Curcumin treatment effectively reduced the progression of NASH to HCC by suppressing the protein expression of glypican-3, vascular endothelial growth factor, and prothrombin in the NASH liver. Our data suggest that curcumin reduces the progression of NASH and liver damage, which may act via inhibiting HMGB1-NF-κB translocation.

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.

Attenuation of Endoplasmic Reticulum Stress-Mediated Liver Damage by Mulberry Leaf Diet in Streptozotocin-Induced Diabetic Rats.

Endoplasmic reticulum stress (ERS) plays a crucial role in the development of insulin resistance and diabetes mellitus. Although antidiabetic use of mulberry leaves (MLs) has been popular due to their many anti-oxidative flavonoid compounds and free radical scavenging effects, ML's effects on ERS in experimental diabetic hepatocyte injury remain unknown. To investigate how ML affect ERS in diabetic liver, Sprague-Dawley (SD) rats were assigned to induce diabetes by a single intraperitoneal injection of streptozocin (STZ; 55 mg/kg) and fed with either normal chow or a diet containing 25% mulberry leaf powder diet (MLD) and examined for 56 days. We observed that MLD improved the rats' morphological and histopathological changes. Levels of ERS markers such as phosphorylated double-stranded RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) and X-box binding protein 1 (XBP1) and the protein expression of glucose regulated protein 78 (GRP78) were significantly higher in the diabetic liver compared to normal liver. MLD for 8 weeks significantly reduced all of these markers. MLD also significantly decreased hepatocyte apoptosis, hepatic macrophage recruitment, cellular infiltration, and CCAAT/enhancer-binding protein homologous protein (CHOP), tumor necrosis factor receptor associated factor 2 (TRAF2), interleukin 1[Formula: see text] (IL-1[Formula: see text]) and sterol regulatory element binding protein isoform 1c (SREBP 1c) levels in diabetic liver. These results may suggest that MLs can preserve hepatic function in experimental diabetes by modulating ERS mediated apoptosis and liver damage.

Tannic acid modulates NFκB signaling pathway and skin inflammation in NC/Nga mice through PPARγ expression.

Polyphenolic compound tannic acid, which is mainly found in grapes and green tea, is a potent antioxidant with anticarcinogenic activities. In this present study, we hypothesized that tannic acid could inhibit nuclear factor (NF)κB signaling and inflammation in atopic dermatitis (AD) NC/Nga mice. We have analyzed the effects of tannic acid on dermatitis severity, histopathology and expression of inflammatory signaling proteins in house dust mite extract induced AD mouse skin. In addition, serum levels of T helper (Th) cytokines (interferon (IFN)γ, interleukin (IL)-4) were measured by enzyme-linked immunosorbent assay. Treatment with tannic acid ameliorated the development of AD-like clinical symptoms and effectively inhibited hyperkeratosis, parakeratosis, acanthosis, mast cells and infiltration of inflammatory cells in the AD mouse skin. Serum levels of IFNγ and IL-4 were significantly down-regulated by tannic acid. Furthermore, tannic acid treatment inhibited DfE induced tumor necrosis factor (TNF)α, high mobility group protein (HMG)B1, receptor for advanced glycation end products (RAGE), extracellular signal-regulated kinase (ERK)1/2, NFκB, cyclooxygenase (COX)2, IL-1ß and increased the protein expression of peroxisome proliferator-activated receptor (PPAR)γ. Taken together, our results demonstrate that, DfE induced skin inflammation might be mediated through NFκB signaling and tannic acid may be a potential therapeutic agent for AD, which may possibly act via induction of PPARγ protein.

Pruni cortex ameliorates skin inflammation possibly through HMGB1-NFκB pathway in house dust mite induced atopic dermatitis NC/Nga transgenic mice.

Pruni cortex, the bark of Prunus jamasakura Siebold ex Koidzumi, has been used in the Japanese systems of medicine for many years for its anti-inflammatory, antioxidant and antitussive properties. In this study, we investigated the effect of pruni cortex on atopic dermatitis NC/Nga mouse model. Atopic dermatitis-like lesion was induced by the application of house dust mite extract to the dorsal skin. After induction of atopic dermatitis, pruni cortex aqueous extract (1 g/kg, p.o.) was administered daily for 2 weeks. We evaluated dermatitis severity, histopathological changes and cellular protein expression by Western blotting for nuclear and cytoplasmic high mobility group box 1, receptor for advanced glycation end products, nuclear factor κB, apoptosis and inflammatory markers in the skin of atopic dermatitis mice. The clinical observation confirmed that the dermatitis score was significantly lower when treated with pruni cortex than in the atopic dermatitis group. Similarly pruni cortex inhibited hypertrophy and infiltration of inflammatory cells as identified by histopathology. In addition, pruni cortex significantly inhibited the protein expression of cytoplasmic high mobility group box 1, receptor for advanced glycation end products, nuclear p-nuclear factor kappa B, apoptosis and inflammatory markers. These results indicate that pruni cortex may have therapeutic potential in the treatment of atopic dermatitis by attenuating high mobility group box 1 and inflammation possibly through the nuclear factor κB pathway.

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.

Modulation of HMGB1 translocation and RAGE/NFκB cascade by quercetin treatment mitigates atopic dermatitis in NC/Nga transgenic mice.

Quercetin, glycosylated form of flavonoid compound, has potent antioxidant and anti-inflammatory properties. In this study, we have investigated the effects of quercetin on skin lesion, high-mobility group box (HMGB)1 cascade signalling and inflammation in atopic dermatitis (AD) mouse model. AD-like lesion was induced by the application of house dust mite extract to the dorsal skin of NC/Nga transgenic mouse. After AD induction, quercetin (50 mg/kg, p.o) was administered daily for 2 weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for HMGB1, receptor for advanced glycation end products (RAGE), toll-like receptor (TLR)4, nuclear factor (NF)κB, nuclear factor erythroid-2-related factor (Nrf)2, kelch-like ECH-associated protein (Keap)1, extracellular signal-regulated kinase (ERK)1/2, cyclooxygenase (COX)2, tumor necrosis factor (TNF)α, interleukin (IL)-1ß, IL-2Rα and other inflammatory markers in the skin of AD mice. In addition, serum levels of T helper (Th) cytokines (interferon (IFN)γ, IL-4) were measured by enzyme-linked immunosorbent assay. Quercetin treatment attenuated the development of AD-like skin lesions. Histological analysis showed that quercetin inhibited hyperkeratosis, parakeratosis, acanthosis, mast cells and infiltration of inflammatory cells. Furthermore, quercetin treatment downregulated cytoplasmic HMGB1, RAGE, nuclear p-NFκB, p-ERK1/2, COX2, TNFα, IL-1ß, IL-2Rα, IFNγ and IL-4 and upregulated nuclear Nrf2. Our data demonstrated that the HMGB1/RAGE/NFκB signalling might play an important role in skin inflammation, and quercetin treatment could be a promising agent for AD by modulating the HMGB1/RAGE/NFκB signalling and induction of Nrf2 protein.

Myocardial 14-3-3η protein protects against mitochondria mediated apoptosis.

There is a definite cardioprotective role for 14-3-3η protein against pressure overload induced cardiac hypertrophy and streptozotocin induced cardiac dysfunction in type 1 diabetes mellitus (DM). But it is not conclusive whether it has any influence on mitochondrial mediated cardiomyocyte apoptosis in type 2 DM. In order to test this hypothesis, we have used C57BL6/J (WT) mice with cardiac specific dominant negative mutation of 14-3-3η protein (DN 14-3-3η). Both WT and DN 14-3-3η mice were fed with high fat diet (HFD) for 12weeks. Their body weight and blood glucose levels were measured weekly and compared with standard diet (SD) fed mice. By the end of 12weeks, echocardiography was performed. Frozen myocardial sections were prepared to stain the apoptotic cardiomyocytes using TUNEL staining. DN 14-3-3η mice fed with HFD showed cardiac dysfunction as identified by the decreased fractional shortening and ejection fraction and increased cardiomyocyte apoptosis in TUNEL staining. Western blotting analysis using mitochondrial fraction of the ventricular tissue homogenates showed a significant reduction in the level of cytochrome c suggesting its translocation into cytoplasm, which may be crucial in inducing cardiomyocyte apoptosis. In addition, DN 14-3-3η mice depicted significantly increased levels of NADPH oxidase subunits suggesting oxidative stress, a significant reduction in phospho apoptosis signal-regulating kinase-1 (p-Ask-1) and increase in Ask-1 and phospho c-Jun N-terminal kinase (p-JNK) levels suggesting activation of Ask-1/JNK signaling. These results suggest that 14-3-3η has a protective role against mitochondria mediated cardiomyocyte apoptosis with the involvement of Ask-1/JNK signaling during HFD induced type 2 DM.

Resveratrol attenuates HMGB1 signaling and inflammation in house dust mite-induced atopic dermatitis in mice.

Resveratrol is a polyphenol abundantly found in red grape skin and is effective against antiaging and anti-inflammation associated with immune responses. In this study, we have investigated the effect of resveratrol on skin lesion, high mobility group box (HMGB)1 and inflammation pathway in an atopic dermatitis (AD) mouse model. AD-like lesion was induced by the application of house dust mite extract to the dorsal skin of NC/Nga mouse. After AD induction, resveratrol (20 mg/kg, p.o.) was administered daily for 2 weeks. We evaluated dermatitis severity, histopathological changes, serum levels of T helper (Th) cytokines (interferon (IFN)γ, interleukin (IL)-4) and changes in protein expression by Western blotting for HMGB1, receptor for advanced glycation end products (RAGE), toll like receptor (TLR)4, nuclear factor (NF)κB, phosphatidylinositide 3-kinase (PI3K), extracellular signal-regulated kinase (ERK)1/2, cyclooxygenase (COX)2, tumor necrosis factor (TNF)α, IL-1ß, IL-2Rα and other inflammatory markers in the skin of AD mice. Treatment of resveratrol inhibited the development of the AD-like skin lesions. Histological analysis showed that resveratrol inhibited hypertrophy, intracellular edema, mast cells and infiltration of inflammatory cells. Furthermore, resveratrol treatment down-regulated HMGB1, RAGE, p-NFκB, p-PI3K, p-ERK1/2, COX2, TNFα, IL-1ß, IL-2Rα, IFNγ and IL-4. Considering all these findings together, the HMGB1 pathway might be a potential therapeutic target in skin inflammation, and resveratrol treatment could have beneficial effects on AD by modulating the HMGB1 protein expression.

Fasting mediated increase in p-BAD(ser155) and p-AKT(ser473) in the prefrontal cortex of mice.

BAD-deficient mice and fasting have several common functional roles in seizures, beta-hydroxybutyrate (BHB) uptake in brain and alteration in counterregulatory hormonal regulation during hypoglycemia. Neuronal specific insulin receptor knockout (NIRKO) mice display impaired counterregulatory hormonal responses during hypoglycemia. In this study we investigated the fasting mediated expression of p-BAD(ser155) and p-AKT(ser473) in different regions of brain (prefrontal cortex, hippocampus, midbrain and hypothalamus). Fasting specifically increases p-BAD(ser155) and p-AKT(ser473) in prefrontal cortex and decreases in other regions of brain. Our results suggest that fasting may increase the uptake BHB by decreasing p-BAD(ser155) in the brain during hypoglycemia except prefrontal cortex and it uncovers specific functional area of p-BAD(ser155) and p-AKT(ser473) that may regulates counter regulatory hormonal response. Overall in support with previous findings, fasting mediated hypoglycemia activates prefrontal cortex insulin signaling which influences the hypothalamic paraventricular nucleus mediated activation of sympathoadrenal hormonal responses.

Comparative evaluation of torasemide and furosemide on rats with streptozotocin-induced diabetic nephropathy.

Nephropathy is one of the complications of diabetes mellitus in human and experimental animals. There are various pathological renal remodeling processes leading to diabetic nephropathy because of the chronic hyperglycemia during diabetes mellitus. Various reports suggest the involvement of oxidative stress, inflammation and fibrosis during this progression. As antihypertensive drugs are reported to provide renoprotection in various animal models and clinical studies, we have carried out this study to identify the effect of torasemide, a loop diuretic, against streptozotocin-induced diabetic nephropathy and compare with furosemide. Here we have performed the measurement of blood and urine parameters and renal protein expression levels for measuring the disease severity in streptozotocin-induced diabetic rats treated torasemide or furosemide and compared with the vehicle treated rats. Furosemide treatment significantly increased the urinary protein excretion when compared with the normal rats. Torasemide treatment has reduced the expression of mineralocorticoid receptor and oxidative stress marker p67phox levels with improved mRNA levels of heme oxygenase-1 in the kidneys. In addition, torasemide treated diabetic rats showed significantly reduced expression of renal fibrosis related proteins when compared with the vehicle treated diabetic rats. Although furosemide treatment has produced improvement, its effects are comparably less than that of torasemide. Thus with the present results, we can suggest that torasemide treatment can improve the diabetic kidney disease in this rat model and which is superior to furosemide against renal fibrotic remodeling.