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Glycine N-methyltransferase (GNMT) is a tumor suppressor for HCC. It is down-regulated in HCC, but the mechanism is not fully understood. MicroRNA-224 (miR-224) acts as an onco-miR in HCC. This study is the first to investigate miR-224 targeting the coding region of GNMT transcript. The GNMT-MT plasmid containing a miR-224 binding site silent mutation of the GNMT coding sequence can escape the suppression of miR-224 in HEK293T cells. Expression of both exogenous and endogenous GNMT was suppressed by miR-224, while miR-224 inhibitor enhanced GNMT expression. miR-224 counteracts the effects of GNMT on the reduction of cell proliferation and tumor growth. The levels of miR-224 and GNMT mRNA showed a significant inverse relationship in tumor specimens from HCC patients. Utilizing CCl4-treated hepatoma cells and mice as a cell damage of inflammatory or liver injury model, we observed that the decreased expression levels of GNMT were accompanied with the elevated expression levels of miR-224 in hepatoma cells and mouse liver. Finally, hepatic AAV-mediated GNMT also reduced CCl4-induced miR-224 expression and liver fibrosis. These results indicated that AAV-mediated GNMT has potential liver protection activity. miR-224 can target the GNMT mRNA coding sequence and plays an important role in GNMT suppression during liver tumorigenesis.

Excessive production of cytokines by microglia may cause cognitive dysfunction and long-lasting behavioral changes. Activating the peripheral innate immune system stimulates cytokine secretion in the central nervous system, which modulates cognitive function. Histone deacetylases (HDACs) modulate cytokine synthesis and release. Trichostatin A (TSA), an HDAC inhibitor, is documented to be anti-inflammatory and neuroprotective. We investigated whether TSA reduces lipopolysaccharide- (LPS-) induced neuroinflammation and cognitive dysfunction. ICR mice were first intraperitoneally (i.p.) injected with vehicle or TSA (0.3 mg/kg). One hour later, they were injected (i.p.) with saline orEscherichia coliLPS (1 mg/kg). We analyzed the food and water intake, body weight loss, and sucrose preference of the injected mice and then determined the microglia activation and inflammatory cytokine expression in the brains of LPS-treated mice and LPS-treated BV-2 microglial cells. In the TSA-pretreated mice, microglial activation was lower, anhedonia did not occur, and LPS-induced cognitive dysfunction (anorexia, weight loss, and social withdrawal) was attenuated. Moreover, mRNA expression of HDAC2, HDAC5, indoleamine 2,3-dioxygenase (IDO), TNF-α, MCP-1, and IL-1βin the brain of LPS-challenged mice and in the LPS-treated BV-2 microglial cells was lower. TSA diminished LPS-induced inflammatory responses in the mouse brain and modulated the cytokine-associated changes in cognitive function, which might be specifically related to reducing HDAC2 and HDAC5 expression.

PURPOSE: Intervertebral disc degeneration is a major cause of back pain. Novel therapies for prevention or reversal of disc degeneration are needed. It is desirable for potential therapies to target both inflammation and matrix degeneration. MATERIALS AND METHODS: The combined regenerative potential of link protein N-terminal peptide (LN) and fullerol on annulus fibrosus (AF) cells was evaluated in a 3D culture model. RESULTS: Interleukin-1α (IL-1α)-induced AF cell degeneration was counteracted by fullerol, LN, and fullerol + LN, with the latter having the greatest effect on matrix production as evaluated by real-time polymerase chain reaction and glycosaminoglycan assay. IL-1α-induced increases in pro-inflammatory mediators (interleukin-6 and cyclooxygenase-2) and matrix metalloproteinases (MMP-1, -2, -9, and -13) were also counteracted by fullerol and LN. CONCLUSION: Our data demonstrate that LN and fullerol individually, and in combination, promote matrix production and have anti-inflammatory and anti-catabolic effects on AF cells.

Background Interleukin (IL)-19, a member of the IL-10 cytokine family, is involved in keratinocyte proliferation in psoriasis. Objectives We investigated the role of IL-19 in the wound-healing process in vivo and in vitro . Methods Two full-thickness circular wounds (4 mm in diameter) were punched into the skin of BALB/C mice. IL-19 and keratinocyte growth factor (KGF) mRNA in wounded skin were determined using real-time PCR. The wounds were treated with PBS, vehicle, IL-19 (400 ng/mL), or IL-20 (400 ng/mL) ( n = 6 in each group) twice daily and the percentage of wound healing was measured daily for 7 days. In vitro , human skin fibroblast CCD966-SK cells and keratinocyte HaCaT cells were treated with IL-19 or KGF. Cell proliferation and migration were determined using bromodeoxyuridine (BrdU) and transwell assays, respectively. The expression of IL-19 and KGF mRNA was also analyzed. Results In wounded mouse skin, IL-19 mRNA was upregulated at 12 h, and KGF at 24 h after the injury. Both increases in gene expression declined 72 h after the skin had been wounded. The percentage of wound healing in IL-19-treated mice was higher than in control mice. In vitro , IL-19 upregulated KGF expression in the CCD966-SK cells; IL-19 was upregulated in KGF-treated HaCaT cells. KGF but not IL-19 promoted HaCaT cell proliferation. However, IL-19 significantly increased the migration of HaCaT cells. HaCaT cells treated with the cultured supernatants of IL-19-stimulated CCD966-SK cells showed significantly more proliferation than in controls. Conclusions IL-19 is important for cutaneous wound healing because it upregulates KGF expression.

Bone morphogenetic protein (BMP)-7 protects sepsis-induced acute kidney injury (AKI). Dexmedetomidine (DEX), an α2-adrenoceptor (α2-AR) agonist, has anti-inflammatory effects. We investigated the protective effects of DEX on sepsis-induced AKI and the expression of BMP-7 and histone deacetylases (HDACs). In vitro , the effects of DEX or trichostatin A (TSA, an HDAC inhibitor) on TNF-α, monocyte chemotactic protein (MCP-1), BMP-7, and HDAC mRNA expression in LPS-stimulated rat renal tubular epithelial NRK52E cells, was determined using real-time PCR. In vivo, mice were intraperitoneally injected with DEX (25 μg/kg) or saline immediately and 12 h after cecal ligation and puncture (CLP) surgery. Twenty-four hours after CLP, we examined kidney injury and renal TNF-α, MCP-1, BMP-7, and HDAC expression. Survival was monitored for 120 h. LPS increased HDAC2, HDAC5, TNF-α, and MCP-1 expression, but decreased BMP-7 expression in NRK52E cells. DEX treatment decreased the HDAC2, HDAC5, TNF-α, and MCP-1 expression, but increased BMP-7 and acetyl histone H3 expression, whose effects were blocked by yohimbine, an α2-AR antagonist. With DEX treatment, the LPS-induced TNF-α expression and cell death were attenuated in scRNAi-NRK52E but not BMP-7 RNAi-NRK52E cells. In CLP mice, DEX treatment increased survival and attenuated AKI. The expression of HDAC2, HDAC5, TNF-α, and MCP-1 mRNA in the kidneys of CLP mice was increased, but BMP-7 was decreased. However, DEX treatment reduced those changes. DEX reduces sepsis-induced AKI by decreasing TNF-α and MCP-1 and increasing BMP-7, which is associated with decreasing HDAC2 and HDAC5, as well as increasing acetyl histone H3.

Nonalcoholic fatty liver disease is frequently associated with type 2 diabetes; however, this idea is challenged by recent studies because hepatic steatosis is not always associated with insulin resistance (IR). Oleic acid (OA) is known to induce hepatic steatosis with normal insulin sensitivity; however, the mechanism is still unknown. Previous studies depict that activation of peroxisome proliferator-activated receptor δ (PPARδ) improves hepatic steatosis and IR, whereas the role of PPARδ in the improvement of insulin sensitivity by OA is unknown. Here we induced steatosis in HepG2 cells by incubation with OA and OA significantly increased the expression of PPARδ through a calcium-dependent pathway. OA also induced the expression of G protein-coupled receptor 40 (GPR40), and deletion of GPR40 by small interfering ribonucleic acid transfection partially reversed the effect of OA on PPARδ. Inhibition of phospholipase C (PLC) by U73122 also reversed OA-induced PPARδ expression. Otherwise, deletion of PPARδ augmented the OA-induced steatosis in HepG2 cells. Furthermore, IR was developed in OA-treated HepG2 cells with PPARδ deletion, while insulin-related signals and insulin-stimulated glycogen synthesis were reduced through increase of phosphatase and tensin homolog (PTEN) expression. In conclusion, OA activates GPR40-PLC-calcium pathway to increase the expression of PPARδ and PPARδ further decreased the expression of PTEN to regulate insulin sensitivity in hepatic steatosis.

The nuclear receptor farnesoid X receptor (FXR) regulates pathways in lipid, glucose, and energy metabolism. Activation of FXR in mice significantly improved high-fat diet-induced hepatic steatosis. It has been reported that activation of imidazoline I-1 receptor by rilmenidine increases the expression of FXR in human hepatoma cell line, Hep G2 cell, to regulate the target genes relating to lipid metabolism; activation of FXR by rilmenidine exerts an antihyperlipidemic action. However, signals for this action of rilmenidine are still unknown. In the present study, hepatic steatosis induced in mice by high-fat diet was improved by rilmenidine after intraperitoneal injection at 1 mg/kg daily for 12 weeks. Also, mediation of I-1 receptors was identified using the specific antagonist efaroxan. Moreover, rilmenidine decreased the oleic acid-induced lipid accumulation in Hep G2 cells. Otherwise, rilmenidine increased the phosphorylation of p38 to increase the expression of FXR. Deletion of calcium ions by BAPTA-AM reversed the rilmenidine-induced p38 phosphorylation. In conclusion, we suggest that rilmenidine activates I-1 receptor to increase intracellular calcium ions that may enhance the phosphorylation of p38 to higher the expression of FXR for improvement of hepatic steatosis in both animals and cells.

Metformin (dimethylbiguanide) belongs to guanidinium-derivative and is widely used for treatment of diabetic disorders in clinic. Metformin lowers blood glucose in diabetic animals through increase of glucose uptake into skeletal muscle. Recent evidence indicates that activation of imidazoline I2B receptor (I2BR) by guanidinium-derivatives also increased glucose uptake; however, the effect of metformin on I2BR is still unknown. The blood glucose levels were determined by a glucose kit. The ability of glucose uptake into isolated skeletal muscle or cultured C2C12 cells was determined using 2-[14C]-deoxyglucose as tracer. The expressions of 5' AMP-activated protein kinase (AMPK) and glucose transporter 4 (GLUT-4) were identified by Western blotting analysis. The metformin-induced blood glucose-lowering action was dose-dependently blocked by BU224, a specific I2R antagonist, in Wistar rats. Also, similar reversion by BU224 was observed in isolated skeletal muscle regarding the metformin-induced glucose uptake. Moreover, AMPK phosphorylation by metformin was concentration-dependently reduced by BU224 in isolated skeletal muscle. In addition, signals for metformin increased glucose uptake were identified via I2R/PI3K/PKC/AMPK dependent pathway in C2C12 cells. Thus, we suggest that metformin can activate I2BR to increase glucose uptake and I2BR will be a new target for diabetic therapy.

Background. Pro-inflammatory cytokines and free radicals damage renal tissue leading to acute kidney injury (AKI) during sepsis. Bone morphogenetic protein-7 (BMP-7) represses tumour necrosis factor (TNF)-α-induced inflammatory responses and protects kidney from injury. The sedative agent, propofol, has immunomodulatory and antioxidative properties. The present study investigated whether propofol could reduce AKI in caecal ligation and puncture (CLP) mice and the possible mechanism behind this.Methods. Mice were treated with propofol or saline immediately and 12 h after CLP surgery. Kidney injury, survival and cytokine expressions of CLP mice were observed 24 h after CLP surgery. In vitro, lipopolysaccharide (LPS)-stimulated rat mesangial cells (RMCs) or hydrogen peroxide (H 2 O 2 )-exposed murine kidney epithelial cells (M1) were treated with propofol. The expression of BMP-7, TNF-α and monocyte chemotactic protein (MCP)-1 in CLP mice kidney, RMCs or M1 cells was determined by RT–PCR. Free radical generation and cell death of RMCs and M1 cells were analysed. Nuclear factor (NF)-κB and peroxisome proliferator-activated receptor (PPAR)-γ expressions in LPS-stimulated RMCs were determined by western blotting.Results. Propofol increased survival and ameliorated AKI in CLP mice. Propofol increased BMP-7 expression but decreased TNF-α and MCP-1 expressions in the kidney of CLP mice and LPS-stimulated RMCs. Propofol also inhibited free radical generation and cell death in LPS-stimulated RMCs and decreased the TNF-α expression and cell death in H 2 O 2 -exposed M1 cells. Moreover, propofol decreased NF-κB but increased PPAR-γ expression in LPS-stimulated RMCs.Conclusions. Propofol treatment could protect kidney from sepsis-induced AKI by increasing BMP-7 expression, decreasing inflammatory cytokines and inhibiting oxidative stress.

Racecadotril is known as an inhibitor of enkephalinase. Increase of plasma insulin by racecadotril has been observed in rats while the mechanism of the action remains obscure. In the present study, intravenous injection of male Wistar rats with racecadotril significantly decreased blood glucose levels. However, this effect of racecadotril was not modified by naloxone at the dose sufficient to block opioid receptors. Thus, the blood glucose-lowering action of racecadotril might be through an endogenous opioid independent mechanism. Otherwise, we found that C-peptide content was also raised by racecadotril in parallel with the increase of insulin in Wistar rats. Thus, the blood glucose-lowering action of racecadotril was related to insulin secretion, but not through the inhibition of plasma insulin degradation. In addition, racecadotril showed no direct effect on insulin secretion in isolated islets or cultured HIT-T15 beta cells. The increase of plasma insulin and blood glucose-lowering action induced by racecadotril were reduced by pretreatment with atropine and enhanced by physotigmine. Direct inhibition of cholinesterase was not observed in brain homogenates treated with racecadotril. Moreover, actions of racecadotril were significantly reduced in rats receiving hemicholinium-3 at a sufficient dose to decrease endogenous acetylcholine. Activation of cholinergic tone is possibly involved in the blood glucose-lowering effect of racecadotril. Our results suggested that racecadotril increased insulin secretion to lower blood glucose mainly via regulation of parasympathetic tone in Wistar rats.

Background Peripheral innate immune response may induce sickness behavior through activating microglia, excessive cytokines production, and neuroinflammation. Dexmedetomidine (Dex) has anti-inflammatory effect. We investigated the effects of Dex on lipopolysaccharide (LPS)-induced neuroinflammation and sickness behavior in mice. Materials and methods BALB/c mice were intraperitoneally (i.p.) injected with Dex (50 ug/kg) or vehicle. One hour later, the mice were injected (i.p.) with Escherichia coli LPS (0.33 mg/kg) or saline (n = 6 in each group). We analyzed the food and water intake, body weight loss, and sucrose preference of the mice for 24h. We also determined microglia activation and cytokines expression in the brains of the mice. In vitro, we determine cytokines expression in LPS-treated BV-2 microglial cells with or without Dex treatment. Results In the Dex-pretreated mice, LPS-induced sickness behavior (anorexia, weight loss, and social withdrawal) were attenuated and microglial activation was lower than vehicle control. The mRNA expression of TNF-α, MCP-1, indoleamine 2, 3 dioxygenase (IDO), caspase-3, and iNOS were increased in the brain of LPS-challenged mice, which were reduced by Dex but not vehicle. Conclusion Dexmedetomidine diminished LPS-induced neuroinflammation in the mouse brain and modulated the cytokine-associated changes in sickness behavior.

Bone morphogenetic protein-7 (BMP-7) expression is known to be protective for renal damage during diabetic nephropathy and disappears early during the progression of diabetic nephropathy. However, changes in expression of BMP-7 and BMP-7 type II receptor (BMP-RII) during kidney nephropathy response to high glucose-induced oxidative stress remain unclear. In this study, we used streptozotocin-induced diabetic rats with diabetic nephropathy and treated them with insulin, phloridzin, or antioxidant tiron. The insulin, phloridzin, or tiron treatment improved the renal function and decreased fibronectin expression in the streptozotocin-induced diabetic rats. Both insulin and phloridzin could reverse the attenuation effects of hyperglycemia on BMP-7 and BMP-RII expressions in the kidneys of streptozotocin-induced diabetic rats through the correction of hyperglycemia. However, the decrease of BMP-7 and BMP-RII expressions in kidney of streptozotocin-induced diabetic rats could be reversed by tiron through decreasing the high glucose-induced oxidative stress but not through changing the levels of glucose. We further confirmed the effect on reversing the BMP-7 and BMP-RII expressions through decreasing oxidative stress by tiron treatment in high glucose exposed mesangial cells. Thus, we suggest that a decrease in oxidative stress is responsible for the improvement of renal function and recovery of renal BMP-7 and BMP-RII expression in streptozotocin-induced diabetic rats.

Bone morphogenetic protein-7 (BMP-7) protects kidneys from diabetic nephropathy (DN), and high glucose (HG)-induced oxidative stress is involved in DN. We investigated the antioxidative ability of BMP-7 using HG-treated mesangial cells. We treated rat mesangial cells (RMCs) with recombinant human BMP-7 (rhBMP-7) and examined changes in reactive oxygen species (ROS) levels and intracellular signals in response to HG-induced oxidative stress. rhBMP-7 decreased the level of ROS in HG-treated RMCs. In contrast, lowering endogenous BMP-7 by siRNA or BMP receptor II (BMP-RII) by anti-BMP-RII antibodies increased the level of ROS in HG-treated RMCs. rhBMP-7 increased Smad-1,5,8 phosphorylation, decreased PKCzeta and c-Jun N-terminal kinase (JNK) phosphorylation, and decreased fibronectin and collagen IV synthesis in HG-treated RMCs. In conclusion, we found that BMP-7 could protect mesangial cells from HG-induced oxidative stress by activating BMP-RII. The antioxidative activity of BMP-7 was primarily due to inhibition of PKCzeta, JNK phosphorylation, and c-jun activation.

Adipocyte plays an important role in lipid regulation in mammals. Understanding of adipocyte differentiation becomes a key issue for the development of anti-obesity agent. Glucocorticoids (GCs) regulate lipid metabolism through promoting lipogenesis in adipose tissue. Ginsenoside Rh2, with a similar chemical structure as GCs, shows antidiabetic, anti-inflammatory, and anticancer actions both in vivo and in vitro. However, effect of Rh2 on glucocorticoid receptor (GR) for an increase of adipogenesis like GCs remains unclear. In the present study, we employed ginsenoside Rh2 to investigate the changes in adipogenetic process of 3T3-L1, one of the widely used preadipocytes, through activating GR or not. In leuciferase assay, we found that ginsenoside Rh2 induced GRs transitivity in a way as dexamethasone, which was deleted by RU486 at concentrations sufficient to block GR. Moreover, 3T3-L1 preadipocytes were differentiated into adipocytes by adipogenic induction medium containing 0.01 to 1 microM of ginsenoside Rh2. Also, RU486 blocked this adipogenesis induced by ginsenoside Rh2 or dexamethasone. The obtained results suggest that ginsenoside Rh2 can promote preadipocytes differentiation through activating GR. This finding seems helpful for the understanding of ginsenosides in the regulation of lipid metabolism.

Various factors involved in the development of liver fibrosis, including hepatic stellate cells (HSCs), cholinergic nervous activity and fibrogenetic cytokines. The present study aims to investigate the role of cholinergic regulation in the promoting of liver fibrogenesis relating to bone morphogenetic protein-6 (BMP-6) and/or transforming growth factor-beta1 (TGFbeta1). We treated carbon tetrachloride (CCl(4)) into rats for eight weeks to induce liver fibrosis and arranged these rats for cholinergic denervation, hepatic branch vagotomy or atropine administration. Acetylcholinesterase (AChE) staining showed the distribution of cholinergic nerve around fibrosis scaring septa. The immunohistochemical staining for alpha smooth muscle actin (alphaSMA) indicated the less HSCs in CCl(4) treated rat liver with cholinergic denervation as compared to the sham-operated CCl(4) treated rats. It seems that cholinergic nerve not only innervates around the fibrosis area but also promotes HSCs. We also detected TGFbeta1 and BMP-6 expressions using RT-PCR and immunohistochemistry. The obtained results show that cholinergic denerveration decreases BMP-6 and TGF-beta1 expressions in CCl(4) induced liver fibrosis of rats. In conclusion, cholinergic nerve may influence HSCs in addition to the lowering of BMP-6 and TGF-beta1 gene expressions to modify liver fibrosis.

Osteoblasts and adipocytes share a common progenitor in bone marrow. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a critical role in adipogenesis. Using a mouse pluripotent mesenchymal cell, D1, as a model, several reports have demonstrated that dexamethasone, a glucocorticoid, can induce adipogenesis. We first examined whether adipogenesis induction in D1 cells is initiated by activation of PPAR-gamma. The results revealed that pioglitazone induces adipogenesis in D1 cells in a dose-dependent manner and decreases alkaline phosphatase activity in D1 cells. Interestingly, this adipogenesis was not blocked by bisphenol A diglycidyl ether, a PPAR-gamma antagonist. A PPAR-gamma-mediated reporter gene assay showed no response to pioglitazone. We then asked whether dexamethasone-induced adipogenesis can be repressed by mifepristone (RU486), an antagonist of glucocorticoid receptor. The results disclosed that mifepristone cannot counteract dexamethasone-induced adipogenesis, and mifepristone itself induced adipogenesis in D1 cells. Moreover, glucocorticoid receptor-mediated reporter gene assay was not responsive to dexamethasone or mifepristone. We concluded that the adipogenesis induced by pioglitazone and dexamethasone in D1 cells may not occur via a PPAR-gamma and glucocorticoid receptor pathway. Finally, we analyzed the gene expression profile of D1 by cDNA microarray after treatment with dexamethasone. We found that the expression of several adipogenesis-related genes is highly provoked by this agent.

Nonsteroidal anti-inflammatory drugs (NSAIDs) were found to suppress proliferation and induce cell death in cultured osteoblasts, and steroids were found to decrease the osteogenesis potential of mesenchymal stem cells. In this study, we further tested the effects of anti-inflammatory drugs (AIDs) on the functions of bone marrow mesenchymal stem cells (BMSCs). The BMSCs from mice (D1-cells) and humans (hBMSCs) were treated with dexamethasone (10(-7) to 10(-6) M), cyclooxygenase-2 (COX-2) selective NSAIDs (10(-6) to 10(-5) M) and non-selective NSAIDs (10(-5) to 10(-4) M). Drug effects on proliferation, cell cycle kinetics, cytotoxicity and mRNA and protein expressions of cell cycle regulators were tested. The osteogenesis potential of D1-cells were evaluated by testing mRNA expressions of type Ialpha collagen and osteocalcin 2-8 days after treatments, and testing mineralization 1-3 weeks after treatments. The results showed that all the tested drugs suppressed proliferation and arrested cell cycle of D1-cells, but no significant cytotoxic effects was found. Prostaglandin E1, E2 and F2alpha couldn't rescue the effects of AIDs on proliferation. The p27kip1 expression was up-regulated by indomethacin, celecoxib and dexamethasone in both D1-cells and hBMSCs. Higher concentrations of indomethacin and dexamethasone also up-regulated p21Cip1/Waf1 expression in hBMSCs, and so did celecoxib on D1-cells. Expressions of cyclin E1 and E2 were down-regulated by these AIDs in D-cells, while only cyclin E2 was down-regulated by dexamethasone in hBMSCs. All the tested NSAIDs revealed no obvious detrimental effects on osteogenic differentiation of D1-cells. These results suggest that the proliferation suppression of AIDs on BMSCs may act via affecting expressions of cell cycle regulators, but not prostaglandin-related mechanisms.

Runx2 is one of the most important transcription factors directing the osteogenesis of mesenchymal stem cells and osteoblastic functions. It is likely that the factors controlling Runx2 expression would trigger the early steps of osteoblast differentiation. By using a reporter gene assay for 4.5 kb Runx2 promoter, it was found that the first 305 bp of Runx2 promoter are active in D1 cells. Within this region, electromobility shift assays (EMSAs) delineated a 6 bp of CACATG bound specifically by the proteins from D1 cell nuclear extract. Antibody super-shift and DNA-coupling magnetic bead pull-down assay indicated that the protein bound to this sequence is USF2. Site-specific mutagenesis revealed that this sequence contributed to the activity of 305 bp Runx2 promoter. Thus, we suggest that USF2 might be one of the regulators for the expression of the Runx2 gene in D1 cells.

Background. Ischemia/reperfusion (I/R) injury is a major cause of acute renal failure and allograft dysfunction in kidney transplantation. ROS/inflammatory cytokines are involved in I/R injury. 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, inhibits inflammatory cytokine expression and is an antiangiogenic and antitumor agent. We investigated the inhibitory effect of 2ME2 on renal I/R injury and possible molecular actions.Methods. BALB/c mice were intraperitoneally injected with 2ME2 (10 or 20 mg/kg) or vehicle 12 h before and immediately after renal I/R experiments. The kidney weight, renal function, tubular damages, and apoptotic response were examined 24 h after I/R injury. The expression of mRNA of interleukin-1β, tumor necrosis factor- (TNF)α, caspase-3, hypoxia inducible factor- (HIF) 1α, and proapoptotic Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) in kidney tissue was determined using RT-PCR, while the expression of nuclear factorκB (NF-κB), BCL-2, and BCL-xL, activated caspase-9, and HIF-1αwas determined using immunoblotting.In vitro, we determined the effect of 2ME2 on reactive oxygen species (ROS) production and cell viability in antimycin-A-treated renal mesangial (RMC) and tubular (NRK52E) cells.Results. Serum creatinine and blood urea nitrogen were significantly higher in mice with renal I/R injury than in sham control and in I/R+2ME2-treated mice. Survival in I/R+2ME2-treated mice was higher than in I/R mice. Histological examination showed that 2ME2 attenuated tubular damage in I/R mice, which was associated with lower expression TNF-α, IL-1β, caspase-9, HIF-1α, and BNIP3 mRNA in kidney tissue. Western blotting showed that 2ME2 treatment substantially decreased the expression of activated caspase-9, NF-κB, and HIF-1αbut increased the antiapoptotic proteins BCL-2 and BCL-xL in kidney of I/R injury.In vitro, 2MR2 decreased ROS production and increased cell viability in antimycin-A-treated RMC and NRK52E cells.Conclusions. 2ME2 reduces renal I/R injury in mice because it inhibits the expression of ROS and proinflammatory cytokines and induces antiapoptotic proteins.

Purpose: Interleukin (IL)-19 was expressed in invasive ductal carcinoma (IDC) of the breast tissue but not in healthy breast tissue. We explored the effects of IL-19 on the pathogenesis of breast cancer and its clinical outcome. Experimental Design: Tumor expression of IL-19 was assessed by immunohistochemistry and/or real-time quantitative PCR between two groups of patients with breast IDC (n = 60 and 143, respectively) with available clinical and survival data. We examined the effects of IL-19 on cytokine and chemokine production as well as proliferation and migration in breast cancer cells. Mice were injected with IL-19–overexpressing or vector control 67NR cells and the tumor growth and lung metastatic micronodules were measured. Results: Of the IDC specimens, high IL-19 expression was associated with advanced tumor stage, high tumor metastasis, and worse survival. In vitro, IL-19 induced transcripts of IL-1β, IL-6, TGF-β, matrix metalloproteinase (MMP)2, MMP9, and CXCR4 in 4T1 breast cancer cells; induced fibronectin expression and assembly; and promoted cancer cell proliferation and migration, which were inhibited by anti-IL-19 monoclonal antibody (mAb). Endogenous fibronectin expression and cancer cell migration were lower in IL-19 knockdown 4T1 cells. In 4T1 cells, hypoxia induced IL-19 and CXCR4 expression, which was inhibited by anti-IL-19 mAb. IL-19 overexpression in noninvasive 67NR cancer cells increased cell proliferation and migration. In vivo, mice injected with IL-19–overexpressing 67NR cell clones showed larger tumors and more metastatic micronodules in the lung. Conclusions: High IL-19 expression in breast cancer tissue is associated with a poor clinical outcome. IL-19 is pivotal in the pathogenesis of breast cancer. Clin Cancer Res; 18(3); 713–25. ©2011 AACR.

Hyperlipidemia is an important risk factor for cardiovascular diseases. Agents for the treatment of hyperlipidemia are well-developed in the clinic while PPARα is a target for lipid-lowering agents. Shan-Zha (Crataegus pinnatifida) is a traditional Chinese medicine used to increase digestion. Also, Shan-Zha fruit extract showed merit to improve obesity and hyperlipidemia in hamsters; however, the mechanism remained obscure. In the present study, hypertriglycemia and hypercholesterolemia were induced by high fat diet in C57BL/6 J male mice. Then, they were orally administered with Shan-Zha fruit extract at an effective dose of 250 mg/kg for 7 days. The liver was removed to estimate the expressions of PPARα and β-oxidation-related enzyme. Oral intake of Shan-Zha extract significantly improved hyperlipidemia in high fat diet-fed mice with an increase of PPARα expression in liver. Also, expression of PPARα-regulated β-oxidation-related enzymes was raised in liver by Shan-Zha extract. However, adipose tissue and others were not modified by this treatment of Shan-Zha fruit extract. Thus, Shan-Zha can increase the expression of PPARα to facilitate β-oxidation-related enzymes in liver for lipid degradation and blood lipid decrement. Also, this is the first report showing Shan-Zha fruit extract can influence liver to lower hyperlipidemia prior to the action in adipose tissue.

The mechanisms regarding hepatic steatosis related to hepatic insulin resistance have been well documented. However, the agents for treatment of hepatic steatosis and insulin resistance remain poorly developed. Peroxisome proliferator-activated receptors (PPARs) are transcription factors that are responsible for the regulation of glucose and/or lipid metabolism. There are 3 distinct isoforms of PPARs family: PPARα, PPARγ, and PPARδ. Both PPARα and PPARγ agonists are widely used in clinic for the treatment of hyperlipidemia and hyperglycemia. However, the therapeutic efficacy of PPARδ agonists for diabetic disorders remains obscure. In the present study, we used L-165041 as PPARδ agonist to treat the high fat diet (HFD) fed mice. Administration of L-165041 improved the hepatic steatosis and increased the insulin sensitivity in HFD-mice. In addition to the histological identification of hepatic steatosis, the improvement of insulin sensitivity was characterized by the enhanced insulin signals and the increase of hepatic glycogen content. This is the first report showing that pharmacological activation of PPARδ improves insulin resistance in diet-induced diabetic mice. Thus, we suggest that pharmacological activation of PPARδ may be a new strategy for the treatment of diabetic patients with hepatic steatosis.

Catalpol is one of the active principles from roots of Rehmannia glutinosa Steud (Scrophulariaceae) that is widely used to treat diabetic disorders in Chinese traditional medicine using the name of Di-Huang, which is used to investigate the mechanisms for lowering of plasma glucose in streptozotocin-induced diabetic rats (STZ-diabetic rats). Catalpol decreased plasma glucose in a dose-related manner, and this action was reduced by pretreatment with naloxone or naloxonazine. An increase of plasma β-endorphin by catalpol was also observed in parallel. The plasma glucose lowering action of catalpol was deleted in bilateral adrenalectomized rats. Moreover, catalpol enhanced β-endorphin release from the isolated adrenal medulla of STZ-diabetic rats. Otherwise, plasma glucose lowering action of catalpol failed to produce in opioid μ-receptor knockout mice. Also, repeated administration of catalpol for 3 days in STZ-diabetic rats resulted in a marked reduction of phosphoenolpyruvate carboxykinase (PEPCK) expression in liver and an increased expression of glucose transporter subtype 4 (GLUT 4) in skeletal muscle. These effects were also reversed by blockade of opioid μ-receptors. Our results suggested that catalpol increased glucose utilization through increase of β-endorphin secretion from adrenal gland in STZ-diabetic rats.

Inflammation is involved in the development of insulin resistance and diabetes. However, the effectiveness of anti-inflammatory drugs in diabetic therapy remains obscure. In the present study, the possible mechanisms of indomethacin, one of the nonsteroidal anti-inflammatory drugs, in the improvement of insulin resistance were investigated. Indomethacin treatment significantly decreased cotton pellet implantation induced white blood cell count elevation and immune cells infiltration in epididymal white adipose tissue. Also, cotton pellet implantation induced impaired glucose utilization and insulin resistance were improved by indomethacin. The decrement in phosphoinsulin receptor and phospho-Akt levels induced by cotton pellet implantation was improved by indomethacin as well. Moreover, indomethacin decreased cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in epididymal white adipose tissue with a marked reduction of prostaglandin 2 (PGE2) and nitrite/nitrate (NOx) levels in cotton pellet-implanted mice. Furthermore, pretreatment of peroxisome proliferator-activated receptor γ (PPARγ) antagonist, GW9662 not only reversed indomethacin-modified COX-2 and iNOS levels but also reversed indomethacin-improved insulin sensitivity determined by homeostasis model assessment-insulin resistance (HOMA-IR). Taken together, indomethacin might elevate the expression of PPARγ to decrease serum NOx and PGE2 to result in the improvement of insulin resistance.

Chlorella is a type of unicellular fresh water algae. In an attempt to develop new agents for handling insulin resistance, Chlorella was employed to screen the effect on insulin resistance in rats induced by fructose-rich chow. A single oral administration of Chlorella for 90 min decreased the plasma glucose in a dose-dependent manner in rats receiving 4-week fructose-rich chow. In addition, chronic treatment with Chlorella for 15 days also lowered plasma glucose in the same manner. Then, the insulin action on glucose disposal rate was measured using the glucose-insulin index, values of the areas under the curves of glucose and insulin during the intraperitoneal glucose tolerance test (IPGTT). Oral administration (three times daily for 5 days) of Chlorella to rats receiving 4 weeks of fructose-rich chow abolished the elevated value of the glucose-insulin index, indicating that Chlorella has an ability to improve insulin resistance. An increase of insulin sensitivity by Chlorella was further evaluated using the plasma glucose lowering action of exogenous insulin in streptozotocin-induced diabetic rats (STZ-diabetic rats). Oral administration of Chlorella three times daily to STZ-diabetic rats increased the response to exogenous insulin 15 days later. The obtained results suggest that oral administration of Chlorella has the ability to improve insulin sensitivity, which may be used as an adjuvant therapy for patients with insulin resistance.

Aim The present study is designed to investigate the effect of ShanZha ( Crataegus pinnatifida ) on obesity or dyslipidemia induced by high-fat diet in hamsters and characterize the role of PPARα in this action of ShanZha. Materials and methods We induced dyslipidemia and obesity in hamsters using high-fat diet and treated hamsters with ShanZha or vehicle for 7 days. We measured the body weight, adipose tissue weights, and food intake of hamsters. Plasma total cholesterol (TC), triglyceride (TG), LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) were determined at the beginning and end of this treatment. Effect of ShanZha on adipogenesis was examined in vitro and change of PPARα was analyzed using Western blot. Results The food intake, body weights, and weights of both brown and white adipose tissues were markedly reduced in hamsters receiving ShanZha as compared with the vehicle-treated control. Plasma levels of TC, TG and LDL-C were decreased by this ShanZha treatment while HDL-C was elevated. The effects of ShanZha were reversed by the combined treatment with PPARα antagonist, MK886. ShanZha inhibited the fat droplet accumulation in 3T3-L1 adipocytes in a dose-dependent manner and this effect was abolished by MK886. Western blot results showed activation of PPARα by ShanZha in hamster adipose tissue. Conclusion We suggest that ShanZha could activate PPARα to improve dyslipidemia or obesity.