Rossicaside B Protects against Carbon Tetrachloride‐induced Hepatotoxicity in Mice

Abstract: The protective effect of rossicaside B, the major phenylpropanoid glycoside from Boschniakia rossica, on CCl₄‐induced hepatotoxicity and the mechanisms underlying its protective effect were investigated. The mice were administered orally with rossicaside B (100 or 200 mg/kg of body weight) 48, 24 and 1 hr before CCl₄ (0.5 ml/kg of body weight) administration. The CCl₄ challenge caused a marked increase in the levels of serum aspartate aminotransferase, alanine aminotransferase and of tumour necrosis factor‐α, and propagated lipid peroxidation with a concomitant reduction in reduced glutathione (GSH) and antioxidative enzyme activities in the liver. The administration of rossicaside B to CCl₄‐treated mice not only decreased the serum toxicity marker enzymes and TNF‐α but also reduced hepatic oxidative stress, as demonstrated by decreased lipid hydroperoxide and thiobarbituric acid‐reactive substance concentrations, combined with elevated GSH content and antioxidative enzyme activities in the liver tissues. Furthermore, the contents of hepatic nitrite, inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2) and haem oxygenase‐1 (HO‐1) were elevated after CCl₄ treatment while the cytochrome P450 2E1 (CYP2E1)‐specific monooxygenase activity was suppressed. Rossicaside B treatment inhibited the formation of liver nitrite, reduced the over‐expression of iNOS and COX‐2 proteins, but increased the CYP2E1 function compared with the CCl₄‐treated mice. However, the protein expression of HO‐1 was further elevated by rossicaside B treatment. The results demonstrate that rossicaside B provides a protective action on CCl₄‐induced acute hepatic injury, which may be related to its antioxidative activity, suppressed inflammatory responses, induced HO‐1 expression and improved CYP2E1 function in the liver.     

Zinc Protects Human Kidney Cells from Depleted Uranium‐induced Apoptosis

Depleted uranium (DU) is a weak radioactive heavy metal, and zinc (Zn) is an effective antidote to heavy metal poisoning. However, the effect of Zn on DU‐induced cytotoxicity and apoptosis is not completely understood. The purpose of this study was to evaluate the effect of Zn on DU‐induced cell apoptosis in human kidney cells (HK‐2) and explore its molecular mechanism. Pre‐treatment with Zn significantly inhibited DU‐induced apoptosis. It reduced the formation of reactive oxygen species in the cells, increased the catalase (CAT) and glutathione (GSH) concentrations, suppressed the DU‐induced soluble Fas receptor (sFasR) and soluble Fas ligand (sFasL) overexpression, suppressed the release of cytochrome c and apoptosis inhibitor factor (AIF) from mitochondria to cytoplasm, inhibited the activation of caspase‐9, caspase‐8 and caspase‐3, and induced metallothionein (MT) expression. Furthermore, exogenous MT effectively inhibited DU‐induced cell apoptosis. In conclusion, mitochondrial and FasR‐mediated apoptosis pathways contribute to DU‐induced apoptosis in HK‐2 cells. Through independent mechanisms, such as indirect antioxidant effects, inhibition of the activation of caspase‐9, caspase‐8 and caspase‐3, and induction of MT expression, Zn inhibits DU‐induced apoptosis.     

Clonidine Attenuates Naloxone‐Induced Opioid‐Withdrawal Syndrome in Cholestatic Mice*

Abstract: Cholestasis is associated with elevated plasma level of endogenous opioid peptides. Naloxone‐precipitated withdrawal syndrome has been described in a mouse model of acute cholestasis. Thus we aimed at determining whether central noradrenergic hyperactivity is involved in manifestation of naloxone‐precipitated withdrawal syndrome in mice with obstructive cholestasis. Acute cholestasis was induced by bile duct resection in mice and physical dependence was observed by precipitating a withdrawal syndrome with naloxone (2 mg/kg, intraperitoneally) 5 days after induction of cholestasis. Administration of clonidine (0.1 mg/kg, intraperitoneally), an α₂‐adrenoceptor agonist, 15 min. before naloxone injection significantly alleviates withdrawal severity in cholestatic mice. However, pretreatment of animals with yohimbine (3 mg/kg, intraperitoneally), an α₂‐adrenoceptor antagonist, 15 min. before clonidine blocked this ameliorative effect of clonidine. The results of this study support the evidence for involvement of the α₂‐adrenoceptors in the withdrawal syndrome of cholestasis in a mouse model.     

A Novel CC Chemokine Receptor 4 Antagonist RS‐1269 Inhibits Ovalbumin‐Induced Ear Swelling and Lipopolysaccharide‐Induced Endotoxic Shock in Mice

Abstract: There is growing evidence that chemokines recruit leukocytes in allergic, inflammatory and immune responses. CC chemokine receptor 4 (CCR4) is implicated as a preferential marker for T helper 2 cells, and the cells selectively respond to CC chemokine ligand 17 (CCL17) and CCL22. We searched for compounds having a profile as a CCR4 antagonist from an in‐house library and have previously reported that 3‐{2‐[(2R)‐2‐phenyl‐4‐(4‐pyridin‐4‐ylbenzyl)morpholin‐2‐yl]ethyl}quinazoline‐2,4(1H,3H)‐dione (named RS‐1154) was capable of significantly inhibiting the binding of [¹²⁵I]CCL17 to human CCR4‐expressing CHO cells. From further synthesis of its derivatives, we newly focused on 3‐(isobutyrylamino)‐N‐{2‐[(2R)‐2‐phenyl‐4‐(4‐pyridin‐4‐ylbenzyl)morpholin‐2‐yl]ethyl}benzamide (RS‐1269), which showed potency comparable to RS‐1154 in inhibiting CCL17‐induced migration of DO11.10 mice‐derived T helper 2 cells with an IC₅₀ value of 5.5 nM in vitro. We then investigated the pharmacological effects of RS‐1269 on ovalbumin‐induced ear swelling and lipopolysaccharide‐induced endotoxic shock in mice. The ear thickness was significantly decreased by oral administration of RS‐1269 at the dose of 30 mg/kg. Treatment with lipopolysaccharide significantly increased the serum level of tumour necrosis factor‐α. Compared with an anti‐CCL17 antibody, RS‐1269 significantly inhibited the production at the dose of 100 mg/kg. These results raise the possibility that RS‐1269 or one of its derivatives has potential to serve as a prototype compound to develop therapeutic agents for atopic dermatitis and inflammatory diseases.     

Calcitriol Attenuates Weight‐Related Systemic Inflammation and Ultrastructural Changes in the Liver in a Rodent Model

The role of vitamin D in maintaining calcium homoeostasis and bone mineralization is well‐established. The aim of the current investigation was to evaluate the effect of calcitriol treatment on inflammation, insulin resistance and liver changes induced by increased body‐weight. Four groups of mice (n = 11 each) were maintained on either low‐fat diet (LFD) or high‐fat diet (HFD) with and without 1α, 25‐dihydroxyvitamin D3 (calcitriol) for 16 weeks. Body‐weight of animals was recorded at the start of the study and every 4 weeks thereafter. At the end of the experiment, blood samples were collected for the determination of biochemical parameters, and liver tissues were harvested for the histopathological evaluation. A significant gradual decrease in weight was observed in HFD‐fed mice treated with calcitriol compared with a steady increase in controls (p < 0.01). Furthermore, calcitriol treatment reduced concentrations of various inflammatory markers including TNF‐α, CRP and IL‐6 (p < 0.05). Treated animals also exhibited lower levels of C‐peptide and insulin (539.4 ng/ml versus 718.9 ng/ml and 0.77 ng/ml versus 1.7 ng/ml, respectively; p < 0.05), which are consistent with improved insulin resistance. Liver histology and ultrastructural studies showed a marked accumulation of fat droplets in approximately 60–70% of hepatocytes of mice fed on HFD, while calcitriol administration rendered the whole structure more normal. Overall, our data signify an important effect of calcitriol on inflammation under HFD conditions and a protective effect on the liver structure.     

Up‐regulated GLT‐1 Resists Glutamate Toxicity and Attenuates Glutamate‐induced Calcium Loading in Cultured Neurocytes

Glutamate transporter‐1 (GLT‐1) plays a dual role in glutamate transportation: both normally devotion to the clearance of glutamate and during some pathological conditions extruding glutamate to the extracellular space. Therefore, it is uncertain whether increased expression of GLT‐1 will actually be helpful against glutamate excitotoxicity. In this study, GLT‐1 up‐regulation was induced by ceftriaxone, and L‐glutamate was added to induce glutamate toxicity in primary cultured rat cortical cells. The results showed that up‐regulated GLT‐1 induced by 1 μM ceftriaxone for 2 days markedly increased cell viability, decreased apoptotic cell death and alleviated ultrastructural damage induced by 50 μM glutamate 15 min. as well as promoted L‐[³H]‐glutamate uptake in cultured cells. GLT‐1 up‐regulation had no effect on the intracellular free calcium concentration ([Ca²⁺]_i) in the resting situation, while relieved intracellular calcium overloading by reducing the elevation and promoting the recovery of [Ca²⁺]_i following stimulation of 50 μM glutamate for 2 min. Applying 100 μM dihydrokainic acid (GLT‐1 antagonist) 30 sec. before glutamate eliminated the above effect of GLT‐1 up‐regulation on [Ca²⁺]_i. In conclusion, GLT‐1 up‐regulation induced by ceftriaxone plays a positive glutamate transporting role against glutamate toxicity in primary cultured rat cortical cells.     

Myrica nagi Attenuates Cumene Hydroperoxide‐Induced Cutaneous Oxidative Stress and Toxicity in Swiss Albino Mice

In recent years, considerable efforts have been made to identify new chemopreventive agents which could be useful for man. Myrica nagi, a subtropical shrub, has been shown to possess significant activity against hepatotoxicity and other pharmacological and physiological disorders. We have shown a chemopreventive effect of Myrica nagi on cumene hydroperoxide‐induced cutaneous oxidative stress and toxicity in mice. Cumene hydroperoxide treatment at a dose level of 30 mg/animal/0.2 ml acetone enhances susceptibility of cutaneous microsomal membrane for iron‐ascorbate‐induced lipid peroxidation and induction of xanthine oxidase activity which are accompanied by decrease in the activities of cutaneous antioxidant enzymes such as catalase, glutathione peroxidase, glutathione reductase, glucose‐6‐phosphate dehydrogenase and depletion in the level of cutaneous glutathione. Parallel to these changes a sharp decrease in the activities of phase II metabolizing enzymes such as glutathione S‐transferase and quinone reductase has been observed. Application of Myrica nagi at doses of 2.0 mg and 4.0 mg/kg body weight in acetone prior to that of cumene hydroperoxide (30 mg/animal/0.2 ml acetone) treatment resulted in significant inhibition of cumene hydroperoxide‐induced cutaneous oxidative stress and toxicity in a dose‐dependent manner. Enhanced susceptibility of cutaneous microsomal membrane for lipid peroxidation induced by iron ascorbate and xanthine oxidase activities were significantly reduced (P<0.05). In addition the depleted level of glutathione, the inhibited activities of antioxidants, and phase II metabolizing enzymes were recovered to a significant level (P<0.05). The protective effect of Myrica nagi was dose‐dependent. In summary our data suggest that Myrica nagi is an effective chemopreventive agent in skin and capable of ameliorating cumene hydroperoxide‐induced cutaneous oxidative stress and toxicity.     

Rho Kinase Inhibitor,Fasudil, Attenuates Contrast‐induced Acute Kidney Injury

In this study, we tested the hypothesis that fasudil, a Rho kinase inhibitor, would protect against contrast‐induced acute kidney injury (CI‐AKI) in a mouse model and attempted to elucidate the mechanism involved. Mice subjected to unilateral ligation of the left anterior renal pedicle were divided into four groups: (1) control group, (2) CI‐AKI induced by contrast media (CM group), (3) CI‐AKI plus low‐dose fasudil (LD group) and (4) CI‐AKI plus high‐dose fasudil (HD group). Animals from groups 2–4 received iodixanol (4.0 g iodine/kg), and the control group received saline. At 12, 2 hr before iodixanol injection and 4 hr after iodixanol administration, the animals in groups 3–4 received 3 or 10 mg/kg fasudil, respectively. Renal blood flow, renal function parameters, kidney histology and the expression of proteins that regulates apoptosis and inflammation were determined 24 hr later. Fasudil treatment notably ameliorated contrast medium‐induced medullary damage, restored renal function, suppressed renal tubular apoptosis, ameliorated redox imbalance and DNA damage. Fasudil had a nephroprotective effect that was partially attributed to its anti‐inflammatory, anti‐apoptotic and antioxidant effects of inhibiting the Rho/ROCK pathway.     

Epigallocatechin‐3‐Gallate Attenuates Oxidative Stress and Inflammation in Obstructive Nephropathy via NF‐κB and Nrf2/HO‐1 Signalling Pathway Regulation

Oxidative stress and inflammation contribute importantly to the pathogenesis of chronic kidney disease (CKD). Epigallocatechin‐3‐gallate (EGCG), which is the most abundant and most active catechin polyphenol extracted from green tea, has been proved to have many bioactivities. In this study, the renoprotective effect of EGCG was evaluated in a widely used kidney disease model, the unilateral ureteral obstruction (UUO) mice model. After 14 days of EGCG administration, mean arterial blood pressure, body‐weight and obstructed kidney weight were measured. Levels of blood urea nitrogen (BUN) and creatinine (CR) and activities of glutamic–pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) in serum were estimated as indicators of renal function. Periodic acid–Schiff (PAS) staining was performed to observe the pathological changes of the obstructed kidney. Antioxidant enzymes and pro‐inflammatory cytokine production were estimated to reflect the oxidative stress and inflammatory state in the obstructed kidney. Finally, the main proteins in the NF‐κB and Nrf2 signalling pathway and DNA binding activity of NF‐κB and Nrf2 were measured to investigate the effect of EGCG on these two pathways. The results demonstrated that EGCG could restore UUO‐induced kidney weight loss and renal dysfunction. In addition, UUO‐induced oxidative stress and inflammatory responses in the obstructed kidney were also prevented by EGCG. Furthermore, EGCG could induce both NF‐κB and Nrf2 nuclear translocation in the UUO kidney and promote heme oxygenase‐1 (HO‐1) production. These results indicated that the renoprotective effect of EGCG might be through its NF‐κB and Nrf2 signalling pathway regulations.     

Design,Synthesis, and Biological Evaluation of Novel Quinazoline Derivatives as Anti‐inflammatory Agents against Lipopolysaccharide‐induced Acute Lung Injury in Rats

Quinazoline has been reported to exhibit multiple bioactivities. The aim of this study was to discover new quinazoline derivatives with preventive effect on lipopolysaccharide‐induced acute lung injury via anti‐inflammatory actions. Thirty‐three 4‐amino quinazolin derivatives were synthesized and screened for anti‐inflammatory activities in lipopolysaccharide‐induced macrophages. The most potent four compounds, 6h, 6m, 6p , and 6q , were shown dose‐dependent inhibition against lipopolysaccharide‐induced TNF‐α and IL‐6 release. Then, the preliminary structure–activity relationship and quantitative structure–activity relationship analyses were conducted. To further determine the effects of quinazolines on acute lung injury treatment, lipopolysaccharide‐induced acute lung injury model was employed. Male Sprague Dawley rats were pretreated with 6m or 6q before instillation of lipopolysaccharide. The results showed that 6m and 6q, especially 6q , obviously alleviated lung histopathological changes, inflammatory cells infiltration, and cytokines mRNA expression initiated by lipopolysaccharide. Taken together, this work suggests that 6m and 6q suppressed the lipopolysaccharide‐induced acute lung injury through inhibition of the inflammatory response in vivo and in vitro, indicating that quinazolines might serve as potential agents for the treatment of acute lung injury and deserve the continuing drug development and research.     

Oral Administration of Polaprezinc Attenuates Fluorouracil‐induced Intestinal Mucositis in a Mouse Model

5‐Fluorouracil (5‐FU) has broadly been applied to treat colorectal cancer as one of the most effective chemotherapeutic agents. However, it frequently causes intestinal mucosal injury and related side effects, such as abdominal pain and diarrhoea, which limit the use of 5‐FU in a clinic setting. Polaprezinc has gradually become known as a mucosal protective agent for the management of gastric ulcer. This study aimed to investigate the prophylactic efficacy of Polaprezinc administered orally against intestinal mucositis induced by 5‐FU in mice on the condition that the antitumour effect could not be compromised. We induced intestinal mucositis in SPF‐grade ICR mice with 5‐FU, and evaluated intestinal damage in the absence or presence of Polaprezinc. We examined the score of diarrhoea and the loss of weight after the 5‐FU treatment and assessed the integrity of villus and the proliferation of small intestine crypt cells by haematoxylin and eosin staining and PCNA immunohistochemical detection. The antitumour effect of 5‐FU on colorectal cancer was assessed with or without Polaprezinc in a xenograft model. The result showed that Polaprezinc significantly reduced the elevated diarrhoea score and the body‐weight loss caused by 5‐FU abolished histological abnormality and crypt cell hypoproliferation in a dose‐dependent manner, without affecting 5‐FU efficacy on colon xenograft tumour in mice. We conclude that Polaprezinc could inhibit 5‐FU‐induced diarrhoea and alleviate the weight loss during 5‐FU chemotherapy, as a possible candidate for treatment and prevention of intestinal mucositis, through protecting intestinal mucosa and improving the quality of life after chemotherapy.     

Metalloproteinase Inhibition Protects against Reductions in Circulating Adrenomedullin during Lead‐induced Acute Hypertension

Intoxication with lead (Pb) results in increased blood pressure by mechanisms involving matrix metalloproteinases (MMPs). Recent findings have revealed that MMP type two (MMP‐2) seems to cleave vasoactive peptides. This study examined whether MMP‐2 and MMP‐9 levels/activities increase after acute intoxication with low lead concentrations and whether these changes were associated with increases in blood pressure and circulating endothelin‐1 or with reductions in circulating adrenomedullin and calcitonin gene‐related peptide (CGRP). Here, we expand previous findings and examine whether doxycycline (a MMPs inhibitor) affects these alterations. Wistar rats received intraperitoneally (i.p.) 1st dose 8 μg/100 g of lead (or sodium) acetate, a subsequent dose of 0.1 μg/100 g to cover daily loss and treatment with doxycycline (30 mg/kg/day) or water by gavage for 7 days. Similar whole‐blood lead levels (9 μg/dL) were found in lead‐exposed rats treated with either doxycycline or water. Lead‐induced increases in systolic blood pressure (from 143 ± 2 to 167 ± 3 mmHg) and gelatin zymography of plasma samples showed that lead increased MMP‐9 (but not MMP‐2) levels. Both lead‐induced increased MMP‐9 activity and hypertension were blunted by doxycycline. Doxycycline also prevented lead‐induced reductions in circulating adrenomedullin. No significant changes in plasma levels of endothelin‐1 or CGRP were found. Lead‐induced decreases in nitric oxide markers and antioxidant status were not prevented by doxycycline. In conclusion, acute lead exposure increases blood pressure and MMP‐9 activity, which were blunted by doxycycline. These findings suggest that MMP‐9 may contribute with lead‐induced hypertension by cleaving the vasodilatory peptide adrenomedullin, thereby inhibiting adrenomedullin‐dependent lowering of blood pressure.     

Crocin Attenuates Kindling Development and Associated Cognitive Impairments in Mice via Inhibiting Reactive Oxygen Species‐Mediated NF‐κB Activation

Crocin is a pharmacologically active carotenoid pigment mainly present in the stigmas of Crocus sativus L. (Iridaceae). It has been well explored in experimental animal models of cognitive impairments, depression, anxiety and epilepsy. This study was designed to understand the effect of crocin on pentylenetetrazol (PTZ)‐induced kindling development and its associated cognitive deficit in mouse. Crocin treatment at 5, 10 and 20 mg/kg p.o. doses showed a marked reduction in severity of PTZ‐induced seizures. There was an increase in novel object preference index and discrimination ratio in the crocin‐treated groups in the novel object recognition test. Its treatment also increased percentage spontaneous alternations in T‐maze test at all the tested doses. Histopathological examination by Nissl staining showed a reduction in dark neurons in the hippocampal pyramidal layer of crocin‐treated animals in contrast to vehicle control, indicating a decrease in neuronal damage. Biochemical estimations showed a significant increase in superoxide dismutase activity and reduced reactive oxygen species (ROS) in the hippocampus of crocin‐treated animals. Immunohistochemistry results revealed attenuation in the levels of nuclear factor‐κB (NF‐κB) and phosphorylated NF‐κB in the hippocampal sections of crocin‐treated animals. The results of this study concluded that crocin treatment increased seizure threshold, thus inhibiting PTZ‐induced kindling development and improving cognitive functions. The effect was found to be due to suppression of seizure‐induced ROS generation and its linked NF‐κB pathway‐associated neuronal damage.     

Chitosan Oligosaccharides Inhibit the Expression of Interleukin‐6 in Lipopolysaccharide‐induced Human Umbilical Vein Endothelial Cells Through p38 and ERK1/2 Protein Kinases

Abstract: Chitosan oligosaccharides (COS) have been reported to exert anti‐fungal activities, antitumour activities and immuno‐enhancing effects. However, the potential roles of COS in the treatment of vascular inflammations remain unknown. In the present study, we examined the effects of COS on interleukin‐6 (IL‐6) production in human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS). Induction of HUVECs with LPS (100 ng/ml) increased the mRNA expression and protein secretion of IL‐6 (versus the vehicle‐treated group, p < 0.01), which were significantly reverted by the pre‐treatment with COS (50–200 μg/ml) for 24 hr before LPS exposure (versus the LPS‐treated group, p < 0.05 or 0.01). Signal transduction studies showed that the pre‐treatment of HUVECs with COS (50–200 μg/ml) for 24 hr markedly inhibited the LPS‐induced over‐expression of phosphorylated p38 mitogen‐activated protein kinase (MAPK), phosphorylated ERK1/2 and nuclear factor κB (NF‐κB). Moreover, the LPS‐induced NF‐κB activation was suppressed by the specific ERK1/2 inhibitor PD98059 (30 μM) (versus the LPS‐treated group, p < 0.01), but not by the specific p38 MAPK inhibitor SB203580 (25 μM). Additionally, both MAPK inhibitors markedly suppressed LPS‐induced IL‐6 mRNA expression in HUVECs (versus the LPS‐treated group, p < 0.01). In conclusion, our results suggest that COS inhibit LPS‐induced up‐regulation of IL‐6 in HUVECs, and this can be regulated by at least two parallel signalling pathways: one via p38 MAPK pathway independent of NF‐κB activation and one via ERK1/2 pathway dependent on NF‐κB activation.     

Organ differences in the impact of p27kip1 deficiency on carcinogenesis induced by N‐methyl‐N‐nitrosourea

To evaluate the impact of p27 on carcinogenesis in various organs, N‐methyl‐N‐nitrosourea (MNU), a direct‐acting alkylating agent, was given to p27 knock‐out mice. Groups of 20–40 male and female mice with null, hetero‐ or wild‐type p27 alleles were given drinking water containing 240 ppm MNU or distilled water every other week for five cycles. The incidence and multiplicity of the induced proliferative lesions were then histologically evaluated at weeks 14 and 20. MNU treatment induced various lesions including squamous hyperplasia and squamous cell carcinoma in the forestomach, atypical hyperplasia and adenocarcinomas in the fundic and pyloric glands, adenomas and adenocarcinomas in the duodenum, malignant lymphomas in the thymus, liver, kidney and spleen and alveolar hyperplasia, adenomas, adenocarcinomas and malignant lymphomas in the lung. Although the incidences of the lesions in the forestomach, fundic and pyloric glands did not differ among the p27 genotypes, those of alveolar hyperplasia of the lung and malignant lymphoma of the thymus were significantly increased in p27‐null males as compared with both wild‐ and hetero‐type animals. Moreover, in both p27^+/+ and p27^+/? cases, the rates for p27‐positive cells were obviously increased in proliferative lesions of the pyloric gland and the lung. However, an increased rate of p27‐positive cells was not observed in malignant lymphoma of the thymus. These findings suggest that p27 does not control the cell cycle equally in all organs affected by MNU‐induced carcinogenesis. Copyright © 2011 John Wiley & Sons, Ltd.