Raspberry ketone promotes FNDC5 protein expression via HO-1 upregulation in 3T3-L1 adipocytes.

Obesity is a global health problem and a risk factor for cardiovascular diseases and cancers. Exercise is an effective intervention to combat obesity. Fibronectin type III domain containing protein 5 (FNDC5)/irisin, a myokine, can stimulate the browning of white adipose tissue by increasing uncoupling protein 1 (UCP1) expression, and therefore may represent a link between the beneficial effects of exercise and improvement in metabolic diseases. Thus, upregulating the endogenous expression of FNDC5/irisin by administering medication would be a good approach for treating obesity. Herein, we evaluated the efficacy of raspberry ketone (RK) in inducing FNDC5/irisin expression and the underlying mechanisms. The expression of brown fat-specific proteins (PR domain containing 16 (PRDM16), CD137, and UCP1), heme oxygenase-1 (HO-1), FNDC5, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) in differentiated 3T3-L1 adipocyte was analyzed by western blotting or immunofluorescence. The level of irisin in the culture medium was also assayed using an enzyme-linked immunosorbent assay kit. Results showed that RK (50 µM) significantly induced the upregulation of FNDC5 protein in differentiated 3T3-L1 adipocytes; however, the irisin level in the culture media was unaffected. Moreover, RK significantly increased the levels of PGC1α, brown adipocyte markers (PRDM16, CD137, and UCP1), and HO-1. Furthermore, the upregulation of PGC1α and FNDC5 and the browning effect induced by RK were significantly reduced by SnPP or FNDC5 siRNA, respectively. In conclusion, RK can induce FNDC5 protein expression via the HO-1 signaling pathway, and this study provides new evidence for the potential use of RK in the treatment of obesity.

Hyperbaric oxygen suppressed tumor progression through the improvement of tumor hypoxia and induction of tumor apoptosis in A549-cell-transferred lung cancer.

Tumor cells have long been recognized as a relative contraindication to hyperbaric oxygen treatment (HBOT) since HBOT might enhance progressive cancer growth. However, in an oxygen deficit condition, tumor cells are more progressive and can be metastatic. HBOT increasing in oxygen partial pressure may benefit tumor suppression. In this study, we investigated the effects of HBOT on solid tumors, such as lung cancer. Non-small cell human lung carcinoma A549-cell-transferred severe combined immunodeficiency mice (SCID) mice were selected as an in vivo model to detect the potential mechanism of HBOT in lung tumors. HBOT not only improved tumor hypoxia but also suppressed tumor growth in murine xenograft tumor models. Platelet endothelial cell adhesion molecule (PECAM-1/CD31) was significantly increased after HBOT. Immunostaining of cleaved caspase-3 was demonstrated and apoptotic tumor cells with nuclear debris were aggregated starting on the 14th-day after HBOT. In vitro, HBOT suppressed the growth of A549 cells in a time-dependent manner and immediately downregulated the expression of p53 protein after HBOT in A549 cells. Furthermore, HBOT-reduced p53 protein could be rescued by a proteasome degradation inhibitor, but not an autophagy inhibitor in A549 cells. Our results demonstrated that HBOT improved tissue angiogenesis, tumor hypoxia and increased tumor apoptosis to lung cancer cells in murine xenograft tumor models, through modifying the tumor hypoxic microenvironment. HBOT will merit further cancer therapy as an adjuvant treatment for solid tumors, such as lung cancer.

Involvement of the p62/Nrf2/HO-1 pathway in the browning effect of irisin in 3T3-L1 adipocytes.

Irisin has gained attention because of its potential applications in the treatment of metabolic diseases. Accumulating evidence indicates that irisin attenuates obesity via the browning of white adipose tissue; however, the underlying mechanisms are unclear. Here, we evaluated the effects of irisin on adipocyte browning and the underlying mechanisms. The western blotting and immunofluorescence analyses demonstrated that irisin significantly induced the up-regulation of brown fat-specific proteins (PGC1α, PRDM16, and UCP-1) and HO-1 in 3T3-L1 adipocytes. Moreover, irisin significantly increased the levels of cytosolic p62 and nuclear Nrf2. These effects of irisin in the adipocytes were attenuated by treatment with SnPP or p62 siRNA. In addition, the browning effect of irisin was observed in BAT-WT-1 cells. These findings suggest that irisin induced browning effect via the p62/Nrf2/HO-1 signalling pathway and that it may be a potential candidate for preventing or treating obesity.

17-DMAG, an Hsp90 inhibitor, ameliorates ovariectomy-induced obesity in rats.

AIMS: Obesity is not only associated with metabolic diseases but is also a symptom of menopause in women. To date, there are no effective drugs for the management of obesity, and it is important to find new agents with fewer side effects, for the treatment of obesity. This study aimed to determine the anti-obesity effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein 90 (Hsp90) inhibitor, and its underlying mechanism in rats with ovariectomy-induced obesity. MAIN METHODS: Ovariectomy (Ovx) rats were treated with 17-DMAG (1 mg kg-1, intraperitoneally) for eight weeks from one week after surgery. The body weight, food intake, locomotor activity, adipogenic- and autophagy-related protein expression in white adipose tissue (WAT) and plasma triglyceride (TG) levels were measured in sham and Ovx rats. KEY FINDINGS: Compared with sham rats, Ovx rats showed increased weight gain, food intake, WAT mass, TG levels, adipogenic protein expression, and decreased locomotor activity. Furthermore, autophagy-related proteins and Foxo3a of WAT were significantly increased in Ovx rats. However, with the exclusion of increased food intake, the changes induced by Ovx were all reversed in 17-DMAG-treated Ovx rats. In addition, the expression of Hsp70 and phosphorylation of Akt increased in 17-DMAG-treated Ovx rats. SIGNIFICANCE: These results suggest that 17-DMAG significantly ameliorated obesity induced by Ovx, and this phenomenon is accompanied by the downregulation of adipogenic-related and autophagy-related proteins as well as the upregulation of Akt-phosphorylation and Hsp70 expression. Therefore, 17-DMAG may be a potential agent for preventing or treating obesity in postmenopausal women.

YC-1 alleviates bone loss in ovariectomized rats by inhibiting bone resorption and inducing extrinsic apoptosis in osteoclasts.

Osteoporosis is a major health problem in postmenopausal women and the elderly that leads to fractures associated with substantial morbidity and mortality. Current osteoporosis therapies have significant drawbacks, and the risk of fragility fractures has not yet been eliminated. There remains an unmet need for a broader range of therapeutics. Previous studies have shown that YC-1 has important regulatory functions in the cardiovascular and nervous systems. Many of the YC-1 effector molecules in platelets, smooth muscle cells and neurons, such as cGMP and µ-calpain, also have important functions in osteoclasts. In this study, we explored the effects of YC-1 on bone remodeling and determined the potential of YC-1 as a treatment for postmenopausal osteoporosis. Micro-computed tomography of lumbar vertebrae showed that YC-1 significantly improved trabecular bone microarchitecture in ovariectomized rats compared with sham-operated rats. YC-1 also significantly reversed the increases in serum bone resorption and formation in these rats, as measured by enzyme immunoassays for serum CTX-1 and P1NP, respectively. Actin ring and pit formation assays and TRAP staining analysis showed that YC-1 inhibited osteoclast activity and survival. YC-1 induced extrinsic apoptosis in osteoclasts by activating caspase-3 and caspase-8. In osteoclasts, YC-1 stimulated µ-calpain activity and inhibited Src activity. Our findings provide proof-of-concept for YC-1 as a novel antiresorptive treatment strategy for postmenopausal osteoporosis, confirming an important role of nitric oxide/cGMP/protein kinase G signaling in bone.

Heme oxygenase-1 mediates anti-adipogenesis effect of raspberry ketone in 3T3-L1 cells.

BACKGROUND: Obesity is caused by excessive accumulation of body fat and is closely related to complex metabolic diseases. Raspberry ketone (RK), a major aromatic compound in red raspberry, was recently reported to possess anti-obesity effects. However, its mechanisms are unclear. AIM: Adipogenesis plays a critical role in obesity and, therefore, this study aimed to investigate the effect and mechanisms of action of RK on adipogenesis in 3T3-L1 preadipocytes. MATERIALS AND METHODS: 3T3-L1 preadipocytes were differentiated in medium containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Adipocyte lipid contents were determined using oil-red O staining while adipogenic transcription factor and lipogenic protein expressions were determined using western blotting. RESULTS: RK (300-400µM) strongly inhibited lipid accumulation during 3T3-L1 preadipocyte differentiation into adipocytes. RK reduced the CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferation-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and fatty acid-binding protein 4 (FABP4) expressions and increased heme oxygenase-1 (HO-1), Wnt10b, and ß-catenin expressions in 3T3-L1 adipocytes. Additionally, RK inhibited lipid accumulation, and adipogenic transcription factor and lipogenic protein expressions were all decreased by inhibiting HO-1 or ß-catenin using tin protoporphyrin (SnPP) or ß-catenin short-interfering RNA (siRNA), respectively. Furthermore, Wnt10b and ß-catenin expressions were negatively regulation by SnPP. CONCLUSION: RK may exert anti-adipogenic effects through modulation of the HO-1/Wnt/beta-catenin signaling pathway.

Genistein suppresses leptin-induced proliferation and migration of vascular smooth muscle cells and neointima formation.

Obesity is a strong risk factor for the development of cardiovascular diseases and is associated with a marked increase in circulating leptin concentration. Leptin is a peptide hormone mainly produced by adipose tissue and is regulated by energy level, hormones and various inflammatory mediators. Genistein is an isoflavone that exhibits diverse health-promoting effects. Here, we investigated whether genistein suppressed the atherogenic effect induced by leptin. The A10 cells were treated with leptin and/or genistein, and then the cell proliferation and migration were analysed. The reactive oxygen species (ROS) and proteins levels were also measured, such as p44/42MAPK, cell cycle-related protein (cyclin D1 and p21) and matrix metalloproteinase-2 (MMP-2). Immunohistochemistry and morphometric analysis were used for the neointima formation in a rat carotid artery injury model. Genistein (5 µM) significantly inhibited both the proliferation and migration of leptin (10 ng/ml)-stimulated A10 cells. In accordance with these finding, genistein decreased the leptin-stimulated ROS production and phosphorylation of the p44/42MAPK signal transduction pathway. Meanwhile, genistein reversed the leptin-induced expression of cyclin D1, and cyclin-dependent kinase inhibitor, p21. Genistein attenuated leptin-induced A10 cell migration by inhibiting MMP-2 activity. Furthermore, the leptin (0.25 mg/kg)-augmented neointima formation in a rat carotid artery injury model was attenuated in the genistein (5 mg/kg body weight)-treated group when compared with the balloon injury plus leptin group. Genistein was capable of suppressing the atherogenic effects of leptin in vitro and in vivo, and may be a promising candidate drug in the clinical setting.

Heat shock protein 70 and AMP-activated protein kinase contribute to 17-DMAG-dependent protection against heat stroke.

Heat shock protein 70 (Hsp70) preconditioning induces thermotolerance, and adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a role in the process of autophagy. Here, we investigated whether 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) protected against heat stroke (HS) in rats by up-regulation of Hsp70 and phosphorylated AMPK (pAMPK). To produce HS, male Sprague-Dawley rats were placed in a chamber with an ambient temperature of 42°C. Physiological function (mean arterial pressure, heart rate and core temperature), hepatic and intestinal injury, inflammatory mediators and levels of Hsp70, pAMPK and light chain 3 (LC3B) in hepatic tissue were measured in HS rats or/and rats pre-treated with 17-DMAG. 17-DMAG pre-treatment significantly attenuated hypotension and organ dysfunction induced by HS in rats. The survival time during HS was also prolonged by 17-DMAG treatment. Hsp70 expression was increased, whereas pAMPK levels in the liver were significantly decreased in HS rats. Following pre-treatment with 17-DMAG, Hsp70 protein levels increased further, and pAMPK levels were enhanced. Treatment with an AMPK activator significantly increased the LC3BII/LC3BI ratio as a marker of autophagy in HS rats. Treatment with quercetin significantly suppressed Hsp70 and pAMPK levels and reduced the protective effects of 17-DMAG in HS rats. Both of Hsp70 and AMPK are involved in the 17-DMAG-mediated protection against HS. 17-DMAG may be a promising candidate drug in the clinical setting.

The effect of ferulic acid ethyl ester on leptin-induced proliferation and migration of aortic smooth muscle cells.

Leptin is a peptide hormone, which has a central role in the regulation of body weight; it also exerts many potentially atherogenic effects. Ferulic acid ethyl ester (FAEE) has been approved for antioxidant properties. The aim of this study was to investigate whether FAEE can inhibit the atherogenic effects of leptin and the possible molecular mechanism of its action. Both of cell proliferation and migration were measured when the aortic smooth muscle cell (A10 cell) treated with leptin and/or FAEE. Phosphorylated p44/42MAPK, cell cycle-regulatory protein (for example, cyclin D1, p21, p27), ß-catenin and matrix metalloproteinase-9 (MMP-9) proteins levels were also measured. Results demonstrated that leptin (10, 100 ng ml(-1)) significantly increased the proliferation of cells and the phosphorylation of p44/42MAPK in A10 cells. The proliferative effect of leptin was significantly reduced by the pretreatment of U0126 (0.5 µM), a MEK inhibitor, in A10 cells. Meanwhile, leptin significantly increased the protein expression of cyclin D1, p21, ß-catenin and decreased the expression of p27 in A10 cells. In addition, leptin (10 ng ml(-1)) significantly increased the migration of A10 cells and the expression of MMP-9 protein. Above effects of leptin were significantly reduced by the pretreatment of FAEE (1 and 10 µM) in A10 cells. In conclusion, FAEE exerts multiple effects on leptin-induced cell proliferation and migration, including the inhibition of p44/42MAPK phosphorylation, cell cycle-regulatory proteins and MMP-9, thereby suggesting that FAEE may be a possible therapeutic approach to the inhibition of obese vascular disease.

The role of heat shock protein 70 in the protective effect of YC-1 on ß-amyloid-induced toxicity in differentiated PC12 cells.

Neurodegenerative brain disorders such as Alzheimer's disease (AD) have been well investigated. However, significant methods for the treatment of the progression of AD are unavailable currently. Heat shock protein 70 (Hsp70) plays important roles in neural protection from stress by assisting cellular protein folding. In this study, we investigated the effect and the molecular mechanism of YC-1, an activator of guanylyl cyclase (GC), on Aß25-35-induced cytotoxicity in differentiated PC12 cells. The results of this study showed that Aß25-35 (10 µM) significantly increased p25 protein production in a pattern that was consistent with the increase in µ-calpain expression. Moreover, Aß25-35 significantly increased tau hyperphosphorylation and induced differentiated PC12 cell death. YC-1 (0.5-10 µM) prevented the cell death induced by Aß25-35. In addition, YC-1 (1, 10 µM) significantly blocked Aß25-35-induced µ-calpain expression and decreased the formation of p25 and tau hyperphosphorylation. Moreover, YC-1 (5-20 µM) alone or combined with Aß25-35 (10 µM) significantly increased the expression of Hsp70 in differentiated PC12 cells. The neuroprotective effect of YC-1 was significantly attenuated by an Hsp70 inhibitor (quercetin, 50 µM) or in PC12 cells transfected with an Hsp70 small interfering RNA. However, pretreatment of cells with the GC inhibitor ODQ (10 µM) did not affect the neuroprotective effect of YC-1 against Aß25-35 in differentiated PC12 cells. These results suggest that the neuroprotective effect of YC-1 against Aß25-35-induced toxicity is mainly mediated by the induction of Hsp70. Thus, YC-1 is a potential agent against AD.

α-Lipoic acid enhances endogenous peroxisome-proliferator-activated receptor-γ to ameliorate experimental autoimmune encephalomyelitis in mice.

ALA (α-lipoic acid) is a natural, endogenous antioxidant that acts as a PPAR-γ (peroxisome-proliferator-activated receptor-γ) agonist to counteract oxidative stress. Thus far, the antioxidative and immunomodulatory effects of ALA on EAE (experimental autoimmune encephalomyelitis) are not well understood. In this study, we found that ALA restricts the infiltration of inflammatory cells into the CNS (central nervous system) in MOG (myelin oligodendrocyte glycoprotein)-EAE mice, thus reducing the disease severity. In addition, we revealed that ALA significantly suppresses the number and percentage of encephalitogenic Th1 and Th17 cells and increases splenic Treg-cells (regulatory T-cells). Strikingly, we further demonstrated that ALA induces endogenous PPAR-γ centrally and peripherally but has no effect on HO-1 (haem oxygenase 1). Together, these data suggest that ALA can up-regulate endogenous systemic and central PPAR-γ and enhance systemic Treg-cells to inhibit the inflammatory response and ameliorate MOG-EAE. In conclusion, our data provide the first evidence that ALA can augment the production of PPAR-γ in vivo and modulate adaptive immunity both centrally and peripherally in EAE and may reveal further antioxidative and immunomodulatory mechanisms for the application of ALA in human MS (multiple sclerosis).

The role of heat shock protein 70 in the protective effect of YC-1 on heat stroke rats.

Heat stroke is a life-threatening illness characterized by an elevated core body temperature. Despite adequate lowering of the body temperature and support treatment of multiple organ-system function, heat stroke is often fatal. 3-(5'-Hydoxymethyl-2'-furyl)-1-benzyl-indazol (YC-1) been identified as an activator of soluble guanylate cyclase. To evaluate whether YC-1 protects multiple organ dysfunctions and improves survival during heat stroke and its mechanism. Male Sprague-Dawley rats untreated or treated with either YC-1 or quercetin (heat shock protein (Hsp) 70 inhibitor) were exposures to heat as a model of heat stroke. The mean arterial pressure (MAP), heart rate, rectal temperature (Tco), survival time, and plasma biochemical data, intracellular Hsp70 and heat shock factor-1 expression were measured. The value of MAP, heart rate and Tco of untreated heat stroke (HS) group were all significantly lower than that of normothermal (NT) group. Biochemical markers evidenced that liver and kidney injuries of HS group were significantly higher than that of NT groups. YC-1 (20mg/kg) pretreatment with heat stroke (YC-1+HS) group, the MAP and heart rate were return to normal, and the biochemical markers were all significantly recovered to normal. The survival time of HS group, NT group and YC-1+HS group were 21, 480, and 445 min, respectively. The expression of Hsp70 and HSF-1 in liver and renal of YC-1+HS group was significantly higher than that of HS group. All of the protective effects of YC-1 were all significantly suppressed when pretreated with quercetin (400mg/kg). Results indicate that YC-1 may improve survival due to induce Hsp70 overexpression.

Role of AMP-activated protein kinase in α-lipoic acid-induced vasodilatation in spontaneously hypertensive rats.

BACKGROUND: Adenosine monophosphate (AMP)-activated protein kinase (AMPK) has recently emerged as an attractive and novel target for the regulation of vascular smooth muscle contraction. The present study investigated the vasodilatory effects of α-lipoic acid (α-LA) and the possible mechanism of its action on aortic rings from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: Aortae were removed from WKY and SHR, and contractile responses to acetylcholine and α-LA studied in organ chamber. Phosphorylated AMPK (pAMPK), phosphorylated Peutz-Jeghers syndrome kinase LKB1 (pLKB1) and calcium/calmodulin-dependent protein kinase (CaMKK) protein level were measured in SHR, WKY, and aortic smooth muscle (A10) cells. RESULTS: α-LA (1-500 µmol/l) produced a concentration-dependent relaxation of precontracted aortic rings from 8- and 16-week-old SHR, but not in those from WKY rats. This vasodilatory effect of α-LA did not change after preincubation with N(G)-nitro-L-arginine methyl ester (100 µmol/l), but significantly suppressed by an AMPK inhibitor, compound C (40 µmol/l). The expression of pAMPKα, pLKB1, and CaMKK were also significantly reduced in endothelium-denuded arteries from 16-week-old SHR compared with those from younger SHR or age-matched WKY rats. After incubation with α-LA (100 µmol/l), the expression of pAMPKα and pLKB1 was significantly increased in the endothelium-denuded aortas from 16-week-old SHR, the expression of CaMKK was more reduced in the endothelium-denuded aortas of 8-week-old SHR, but this was not observed in WKY rats. α-LA also activated AMPKα phosphorylation in A10 cells. CONCLUSIONS: The effects of α-LA on vascular relaxation in SHR result from the enhanced phosphorylation of LKB1-AMPK in aortic smooth muscle.

Reciprocal effects of α-lipoic acid on adenosine monophosphate-activated protein kinase activity in obesity induced by ovariectomy in rats.

OBJECTIVE: Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays an important role in regulating whole-body energy homeostasis. The aim of this study was to investigate dietary α-lipoic acid (α-LA) supplementation on the activation of AMPK in both central and peripheral tissues in obese rats induced by ovariectomy. METHODS: Ovariectomized (Ovx) rats were treated with α-LA (200 mg/kg) 3 to 10 weeks after surgery. Body weight, food intake, fat mass, phosphorylated AMPKα (pAMPKα), and phosphorylated acetyl-CoA carboxylase (ACC) protein expression in both the hypothalamus and white adipose tissue (WAT) as well as plasma leptin and adiponectin levels were measured in rats after either Ovx or sham operations. RESULTS: Compared with control rats, ovariectomy led to increased body weight, food intake, and WAT mass 2 to 10 weeks after surgery. Furthermore, plasma leptin and adiponectin levels as well as hypothalamic pAMPKα expression were significantly increased after ovariectomy, accompanied by a reduction in pAMPKα expression in WAT after ovariectomy. However, after treatment with α-LA, the elevation of leptin and adiponectin levels and the activation of hypothalamic AMPKα and ACC, as induced by ovariectomy, were significantly suppressed. Meanwhile, decreased fat mass and increased pAMPKα and phosphorylated ACC expression in the WAT were observed in Ovx rats treated with α-LA. CONCLUSIONS: α-LA significantly decreased appetite and fat accumulation, possibly through the regulation of central and peripheral AMPK activities in rats. Therefore, this study provides a rationale for the therapeutic use of α-LA for obesity in postmenopausal women.

In vivo anticoagulant effect of ethyl pyruvate in endotoxemic rats.

INTRODUCTION: Disseminated intravascular coagulation (DIC), a disorder of the blood coagulation function, is a common complication of severe sepsis. Ethyl pyruvate (EP), a derivative of pyruvate, is known for its anti-inflammatory and antioxidant properties. Recently, EP showed anticoagulant effects in vitro. Therefore, the aim of study is to investigate the in vivo anticoagulant effect of EP on a severe DIC model induced by lipopolysaccharide (LPS) in anesthetized Wistar rats. MATERIALS AND METHODS: The animals were intravenously infused with LPS (30 mg/kg) over a period of 4h. One hour after LPS initiation, rats were treated with EP in three dosages (20, 40 and 60 mg/kg/4h, i.v.). RESULTS: The intermediate and high doses of EP (40 and 60 mg/kg) prevented circulatory failure, improved renal and hepatic function, and reduced the plasma levels of TNF-α and IL-6 after LPS administration. All used doses of EP significantly prevented prothrombin time prolongation, and reduced mRNA expression of tissue factor in lung tissue induced by LPS. The two higher doses of EP attenuated plasma concentrations of thrombin-antithrombin complex. The high dose of EP (60 mg/kg) significantly preserved platelet counts, and improved survival rate. However, EP did not reduce the elevation of plasma plasminogen activator inhibitor-1 during endotoxemia. CONCLUSION: EP demonstrates the in vivo anticoagulant effect and improved organ functions in a severe DIC model in rats, which is likely associated with the inhibitory effect on tissue factor mRNA expression and cytokines release. Its effectiveness in preventing DIC is not mediated by increase in fibrinolysis.

Opioid-like compound exerts anti-fibrotic activity via decreased hepatic stellate cell activation and inflammation.

Hepatic fibrosis is characterized by excess type I collagen deposition and exacerbated inflammatory response. Naltrexone, an opioid receptor antagonist used for treating alcohol abuse, attenuates hepatocellular injury in fibrotic animal models, which can be accompanied by deleterious side effects. Additionally, opioid neurotransmission is upregulated in patients with inflammatory liver disease. Several derivatives of Naltrexone, Nalmefene (Nal) and JKB-119, exert immunomodulatory activity; however, unlike Nal, JKB-119 does not show significant opioid receptor antagonism. To delineate the potential hepatoprotective effects of these compounds, we investigated if JKB-119 and Nal could modulate activation of hepatic stellate cells (HSCs), primary effector cells that secrete type I collagen and inflammatory mediators during liver injury. Our results demonstrated that Nal or JKB-119 treatment decreased smooth muscle α-actin, a marker of HSC activation, mRNA and protein expression. Despite decreased collagen mRNA expression, both compounds increased intracellular collagen protein expression; however, inhibition of collagen secretion was observed. To address a possible mechanism for suppressed collagen secretion or retention of intracellular collagen, endoplasmic (ER) protein expression and matrix metalloproteinase (MMP) activity were examined. While no change in ER protein expression (Grp78, PDI, Hsp47) was observed, MMP13 mRNA expression was dramatically increased. In an acute LPS inflammatory injury animal model, JKB-119 treatment decreased liver injury (ALT), plasma TNFα and PMN liver infiltration. Overall, these results suggest that JKB-119 can directly inhibit HSC activation attributed to anti-inflammatory activity and may, therefore, attenuate inflammation associated with HSC activation and liver disease.

Ethyl pyruvate reduces acute lung injury via regulation of iNOS and HO-1 expression in endotoxemic rats.

BACKGROUND: Ethyl pyruvate (EP) has been shown to attenuate lipopolysaccharide (LPS)-induced acute lung injury (ALI). Induction of heme oxygenase-1 (HO-1) and suppression of inducible nitric oxide synthase (iNOS) expression provide cytoprotection in lung and vascular injury. The aim of this study is to evaluate whether the beneficial effect of EP on lung inflammation is related to HO-1 induction in a rat model of LPS-induced ALI. MATERIALS AND METHODS: Rats were administered LPS (30 mg/kg) by intravenous infusion for 4 h to induce ALI. EP (20, 40, and 60 mg/kg/4 h i.v. infusion) or vehicle was given 1 h after LPS initiation. RESULTS: EP 40 and 60 mg/kg attenuated plasma levels of TNF-α and IL-6 caused by LPS, and further increased IL-10 levels compared with the LPS group. At 6 h after LPS initiation, iNOS protein expression in lungs and plasma NO metabolite levels were markedly increased, which were reduced by EP 60 mg/kg. LPS caused a significant HO-1 induction, whereas administration of EP 60 mg/kg significantly induced higher HO-1 expression compared with the LPS group. The beneficial effects of EP on cytokines and iNOS expression were reversed by HO-1 inhibitor SnPP. EP significantly suppressed phosphorylated p38 MAPK and increased phosphorylated ERK1/2 protein levels in the lung tissue. The edema and infiltration of neutrophils into lungs was reduced by EP. CONCLUSION: EP reduced LPS-induced ALI, which may be mediated by induction of HO-1. The underlying mechanisms are associated with suppression of p38 MAPK and increase of ERK1/2 signaling pathway activation.