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Maslinic acid (MA), also named crategolic acid, is a pentacyclic triterpene extracted from fruits and vegetables. Although various beneficial pharmacological effects of MA have been revealed, its effect on renal fibrosis remains unclear. This study was designed to clarify whether MA could attenuate renal fibrosis and determine the putative underlying molecular mechanisms. We demonstrated that MA-treated mice with unilateral ureteral obstruction (UUO) developed a histological injury of low severity and exhibited downregulated expression of fibrotic markers, including α-smooth muscle actin (α-SMA), vimentin, and fibronectin by 38, 44 and 40%, and upregulated expression of E-cadherin by 70% as compared with untreated UUO mice. Moreover, MA treatment restored the expression levels of α-SMA, connective tissue growth factor, and vimentin to 10, 7.8 and 38% of those induced by transforming growth factor (TGF)-β in NRK49F cells. MA decreased expression of Smad2/3 phosphorylation and Smad4 in UUO kidneys and TGF-β treated NRK49F cells (p < 0.05, respectively). Notably, MA specifically interferes with MyD88, an adaptor protein, thereby mitigating Smad4 nuclear expression (p < 0.01 compared to TGF-β treated group) and ameliorating renal fibrotic changes (p < 0.01 for each fibrotic markers compared to TGF-β induced cells). In addition, in the UUO model and lipopolysaccharide-induced NRK49F cells, MA treatment decreased the expression of IL-1β, TGF-α and MCP-1, ICAM-1, associated with the suppression of NF-κB signaling. These findings suggest that MA is a potential agent that can reduce renal interstitial fibrosis, to some extent, via targeting TGF-β/Smad and MyD88 signaling.

G-protein-coupled receptor 40 (GPR40) has an anti-apoptotic effect in pancreatic &beta, cells. However, its role in renal tubular cell apoptosis remains unclear. To explore the role of GPR40 in renal tubular apoptosis, a two-week unilateral ureteral obstruction (UUO) mouse model was used. The protein expression of GPR40 was decreased, while the Bax/Bcl-2 protein expression ratio, the expression of tumor necrosis factor (TNF)-&alpha, mRNA, and angiotensin II type 1 receptor (AT1R) protein were increased in mice with UUO. In vitro, pretreatment of rat proximal tubular (NRK52E) cells with GW9508, a GPR40 agonist, attenuated the decreased cell viability, increased the Bax/Bcl-2 protein expression ratio, increased protein expression of cleaved caspase-3 and activated the nuclear translocation of nuclear factor-&kappa, B (NF-&kappa, B) p65 subunit induced by TNF-&alpha, treatment. TNF-&alpha, treatment significantly increased the expression of AT1R protein and the generation of reactive oxygen species (ROS), whereas GW9508 treatment markedly reversed these effects. Pretreatment with GW1100, a GPR40 antagonist, or silencing of GPR40 in NRK52E cells promoted the increased expression of the cleaved caspase-3 protein by TNF-&alpha, treatment. Our results demonstrate that decreased expression of GPR40 is associated with apoptosis via TNF-&alpha, and AT1R in the ureteral obstructed kidney. The activation of GPR40 attenuates TNF-&alpha, induced apoptosis by inhibiting AT1R expression and ROS generation through regulation of the NF-&kappa, B signaling pathway.

We present electrophysiological (EP) signals correlated with cellular cell activities in the adrenal cortex and medulla using an adrenal gland implantable flexible EP probe. With such a probe, we could observe the EP signals from the adrenal cortex and medulla in response to various stress stimuli, such as enhanced hormone activity with adrenocorticotropic hormone, a biomarker for chronic stress response, and an actual stress environment, like a forced swimming test. This technique could be useful to continuously monitor the elevation of cortisol level, a useful indicator of chronic stress that potentially causes various diseases.

Tubulointerstitial fibrosis is a common feature of kidney disease. Histone deacetylase (HDAC) inhibitors have been reported to attenuate renal fibrosis progression. Here, we investigated the effect of CG200745, a novel HDAC inhibitor, on renal fibrosis development in a mouse model of unilateral ureteral obstruction (UUO). To examine the effects of CG200745 on renal fibrosis in UUO, C57BL/6 J male mice were divided into three groups: control, UUO, and CG200745 (30 mg/kg/day)-treated UUO groups. CG 200745 was administered through drinking water for 1 week. Human proximal tubular epithelial (HK-2) cells were also treated with CG200745 (10 µM) with or without TGF-β (2 ng/mL). Seven days after UUO, plasma creatinine did not differ among the groups. However, plasma neutrophil gelatinase-associated lipocalin (NGAL) levels were markedly increased in the UUO group, which were attenuated by CG200745 treatment. UUO kidneys developed marked fibrosis as indicated by collagen deposition and increased α-smooth muscle actin (SMA) and fibronectin expression. CG200745 treatment attenuated these fibrotic responses and suppressed UUO-induced production of transforming growth factor-beta1 (TGF-β) and phosphorylation of Smad-2/3. CG200745 treatment also attenuated UUO-induced inflammation as indicated by the expression of inflammatory markers. Furthermore, CG200745 attenuated phosphorylation of p38 mitogen-activated protein kinase in UUO kidneys. In HK-2 cells, TGF-β induced the expression of α-SMA and fibronectin, which were attenuated by CG200745 cotreatment. These results demonstrate that CG200745, a novel HDAC inhibitor, has a renoprotective effect by suppressing renal fibrosis and inflammation in a UUO mouse model.

G-protein-coupled receptor 40 (GPR40) is known to play a role in the regulation of fatty acids, insulin secretion and inflammation. However, the pathophysiological roles of GPR40 in kidney disease have not yet been identified. In the present study, we investigated the expression of GPR40 during cisplatin-induced kidney injury using male Sprague-Dawley rats that were treated with 8 mg/kg cisplatin. Control rats were treated with saline. Following treatment with cisplatin, the protein expression of GPR40 in the kidneys was decreased in association with an increase in serum creatinine levels and the Bax/Bcl-2 expression ratio. To further investigate the function of GPR40, the human renal proximal tubule epithelial cell line (HK-2) was cultured with cisplatin in the absence or presence of GW9508, a selective GPR40 agonist. Pre-treatment of the HK-2 cells with GW9508 attenuated the decrease in cell viability induced by treatment with cisplatin. Treatment with cisplatin increased the number of cells with condensed nuclei, which was ameliorated by GW9508 pre-treatment. TUNEL assay also revealed that pre-treatment with GW9508 ameliorated cisplatin-induced apoptosis. Treatment with cisplatin increased the Bax/Bcl-2 expression ratio and cleaved caspase-3 expression, and promoted the activation of nuclear factor-κB (NF-κB). These changes were attenuated by pre-treatment with GW9508. The cisplatin-induced generation of reactive oxygen species (ROS) and the activation of the Src/epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK) pathway were also counteracted by pre-treatment with GW9508. Thus, the activation of GPR40 attenuates cisplatin-induced apoptosis by inhibiting the generation of ROS, the activation of the Src/EGFR/ERK signaling pathway and the nuclear activation of NF-κB and pro-apoptotic factors.

This study aimed to examine the protective effects of aliskiren on gentamicin-induced nephropathy. Rats were injected with gentamicin (100 mg/kg per day) for 14 days. Aliskiren was infused for two weeks. Human proximal tubular epithelial cell lines (HK-2) were cultured with gentamicin in the absence or presence of aliskiren. Inflammatory profibrotic and apoptotic markers were evaluated in vivo and in vitro. Aliskiren treatment attenuated the decreased creatinine clearance, increased fractional sodium excretion, glomerulosclerosis and tubulointerstitial fibrosis and counteracted the increased ED-1 expression in gentamicin-treated rats. The levels of inflammatory cytokines (TNF-α, IL-1β and IFN-γ) and adhesion molecules (MCP-1, ICAM-1 and VCAM-1) increased in the gentamicin-treated kidneys. These changes were restored by aliskiren co-treatment. Aliskiren effectively reversed transforming growth factor-β-induced fibrotic responses such as induction of α-smooth muscle actin in gentamicin-treated rat kidneys. Along with these changes, aliskiren also attenuated the increase in nuclear factor κB and phosphorylated extracellular signal-regulated kinase (pERK 1/2) levels in HK-2 cells cultured with gentamicin. In addition, aliskiren decreased the number of TUNEL-positive nuclei and reduced the expression of proapoptotic markers in gentamicin-treated HK-2 cells. These findings suggest that aliskiren attenuates gentamicin-induced nephropathy by suppression of inflammatory, profibrotic and apoptotic factors through inhibition of the nuclear factor κB, Smads and mitogen-activated protein kinase signaling pathways.

Purpose: The purpose of this study was to determine whether periurethral injection of allogenic mesenchymal stem cells (MSCs) could increase the leak point pressure (LPP) in a rat model of stress urinary incontinence. Materials and Methods: Female Sprague-Dawley rats (230–240 g, n = 30) were divided into 3 groups: sham operation (group C), saline-treated (group S) and MSC-treated (group M). Bilateral pudendal nerve dissection followed by normal saline or MSC injection on both sides of the urethra was done. LPP and closing pressure (CP) testing was performed after the treatment. The specific markers for smooth muscle cells in the transplantation sites of the urethra were determined. Results: Both the LPP and CP were significantly lower in group S than controls. However, these were restored to the control values in group M (p < 0.05). The LPPs of groups C, S and M were 29.1 ± 2.1, 22.0 ± 2.2 and 43.1 ± 3.2 cm H2O, respectively. The CPs of groups C, S and M were 27.1 ± 3.1, 21.1 ± 3.2, and 32.1 ± 2.1 cm H2O, respectively. The injected MSCs stained positive for muscle-specific markers. Conclusion: This study suggests that MSCs might differentiate into muscle lineage cells and may contribute to the repair of damaged muscle tissue.

Background Nicotine is, to a large extent, responsible for smoking-mediated renal dysfunction. This study investigated nicotine's effects on renal tubular epithelial cell apoptosis in vitro and it explored the mechanisms underlying its effects. Methods Human proximal tubular epithelial (HK-2) cells were treated with nicotine. Cell viability was examined by using the WST-1 assay. Intracellular levels of reactive oxygen species (ROS) and the expression of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-kappa B) proteins were determined. The messenger ribonucleic acid and the protein expression associated with the nicotine acetylcholine receptors (nAChRs) in HK-2 cells was examined, and apoptosis was detected using flow cytometry, cell cycle analysis, and immunoblot analysis. Results The HK-2 cells were endowed with nAChRs. Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression. Nicotine increased NF-kappa B activation, which was attenuated by N-acetyl-L-cysteine, and ERK and JNK inhibitors, but was not affected by a p38 MAPK inhibitor. Nicotine increased the Bax/Bcl-2 ratio, which was attenuated by N-acetyl-L-cysteine, the NF-kappa B inhibitor, Bay 11-7082, and hexamethonium, a non-specific nAChR blocker. Flow cytometry revealed nicotine-induced G2/M phase arrest. While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11-7082 pretreatment reduced their expression. Conclusions Nicotine caused apoptosis in HK-2 cells by inducing ROS generation that activated the NF-kappa B signaling pathway via the MAPK pathway and it arrested the cell cycle at the G2/M phase. Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.

Curcumin, a yellow pigment of turmeric in curry, is reported to interfere with nuclear factor (NF)-kappaB. This study was designed to investigate the underlying pathway of antiinflammation of curcumin on endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with 10 ng/mL tumor necrosis factor (TNF)-alpha. Curcumin blocked the activation of NF-kappaB by TNF-alpha. Curcumin also reduced the intracellular reactive oxygen species (ROS), monocyte adhesion, phosphorylation of c-Jun N-terminal kinase (JNK), p38, and signal transducer and activator of transcription (STAT)-3 in TNF-alpha-stimulated HUVECs. The expression of intracellular cell adhesion molecule (ICAM)-1, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-8 were attenuated by curcumin at both mRNA and protein level. Curcumin, however, did not affect the expression of TNF receptor I and II in TNF-alpha-stimulated HUVECs. We suggest that curcumin could contribute to protection against the adverse vascular effect of the proinflammatory response through the modulation of p38 and STAT-3 in addition to NF-kappaB and JNK in endothelial cells.

Transferrin (Tf) has been used as a targeting ligand for delivering liposome/interleukin-12 (IL-12) pDNA complexes to cancer cells mostly due to the greater number of transferrin receptors (TfR) found on tumor cells than on normal cells. Tf was conjugated to liposomes via the reaction of MPB-PE with thiol groups of Tf introduced by a heterobifunctional cross-linking agent,N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Four days after C-26 inoculation when the tumor volume reached ∼100 mm3, tumor-bearing Balb/c mice were injected intravenously with Tf -liposome/IL-12 pDNA complexes twice a week for 3 weeks. Significant suppression of tumor growth was achieved in the group treated with the Tf -liposome/IL-12 pDNA complexes, with a dose of 10 μg of IL-12 pDNA showing the highest suppression effect among the tested doses. Similar results were obtained when the therapy was initiated one day after tumor inoculation, although in this case 30 μg IL-12 pDNA/Tf -liposome complexes showed a significant suppression of tumor growth between 19 and 23 days after tumor inoculation. This result indicates that the transferrin receptor-targeted liposomal system is an efficient delivery agent of therapeutic genes, such as IL-12, in mice and that its potential clinical use warrants further research investigation.

The farnesoid X receptor (FXR) is mainly expressed in liver, intestine and kidney. We investigated whether 6-ethyl chenodeoxycholic acid (6ECDCA), a semisynthetic derivative of chenodeoxycholic aicd (CDCA, an FXR ligand), protects against kidney injury and modulates small heterodimer partner (SHP) in cisplatin-induced kidney injury. Cisplatin inhibited SHP protein expression in the kidney of cisplatin-treated mice and human proximal tubular (HK2) cells; this effect was counteracted by FXR ligand. Hematoxylin and eosin staining revealed the presence of tubular casts, obstructions and dilatations in cisplatin-induced kidney injury, which was attenuated by FXR ligand. FXR ligand also attenuated protein expression of transforming growth factor-beta 1 (TGF-beta 1), Smad signaling, and the epithelial-to-mesenchymal transition process, inflammatory markers and cytokines, and apoptotic markers in cisplatin-treated mice. Cisplatin induced NF-kappa B activation in HK2 cell; this effect was attenuated by pretreatment with FXR ligand. In SHP knockdown by small interfering RNA, cisplatin-induced activation of TGF-beta 1, p-JNK and Bax/Bcl-2 ratio was not attenuated, while SHP overexpression and FXR ligand inhibited expression of these proteins in cisplatin-pretreated HK2 cells. In conclusion, FXR ligand, 6ECDCA prevents cisplatin-induced kidney injury, the underlying mechanism of which may be associated with anti-fibrotic, anti-inflammatory, and anti-apoptotic effects through SHP induction.

4-Hydroxy-2-hexenal (HHE), the aldehyde product of lipid peroxidation, may be responsible for the pathogenesis of progressive renal disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to be renoprotective through its anti-inflammatory and antifibrotic effects in various experimental nephropathy models. In this study, we investigated the effects of paricalcitol on inflammation and epithelial-mesenchymal transition (EMT) after HHE-induced renal tubular epithelial cell injury. To investigate the molecular mechanisms underlying HHE-induced renal tubular cell injury, the human proximal tubular epithelial (HK-2) cells cultured with 10 µM HHE in the presence or absence of paricalcitol. In HK-2 cells, paricalcitol attenuated the HHE-induced expression of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, and prevented nuclear factor-κB (NF-κB) activation. The expression of the inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2 was attenuated by paricalcitol pretreatment. In addition, HHE increased the expression of the transforming growth factor (TGF)-β/Smad signaling proteins and fibrotic proteins, such as α-smooth muscle actin and connective tissue growth factor; this inducible expression was suppressed by pretreatment with paricalcitol. Treatment with HHE resulted in the activation of the β-catenin signaling pathway, and paricalcitol pretreatment reduced the expression of β-catenin in HHE-treated HK-2 cells. Coimmunoprecipitation shows that paricalcitol induced vitamin D receptor (VDR)/β-catenin complex formation in HK-2 cells. Also immunofluorescence staining revealed that co-localization of VDR and β-catenin in the nuclei. ICG-001, an inhibitor of β-catenin, decreased the expression of TGF-β1 and attenuated HHE-induced tubular EMT. These results show that paricalcitol attenuated HHE-induced renal tubular cell injury by suppressing inflammation and EMT process through inhibition of the NF-κB, TGF-β/Smad, and β-catenin signaling pathways.

The present study investigated the changes that occurred in the mammalian target of rapamycin (mTOR) signaling pathway in the kidney as a result of deoxycorticosterone acetate (DOCA)-salt hypertension. Rats were implanted with DOCA strips (200 mg/kg) 1 week after unilateral nephrectomy and were then supplied with 0.9% saline to drink. Four weeks after DOCA implantation, systolic blood pressure (SBP) was measured by use of the tail-cuff method. The expression levels of phosphorylated phosphatidylinositol-3-kinase (PI3K), Akt, and mTOR, as well as the protein expression levels of ED-1 and cyclooxygenase-2 (COX-2), transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (SMA), caspase-3, Bax, and Bcl-2, were then examined in the kidney by semiquantitative immunoblotting. DOCA-salt hypertensive rats were found to have significantly increased SBP as well as an increased kidney weight-to-body weight ratio. Moreover, the phosphorylation of PI3K, Akt, and mTOR was increased in the kidney of DOCA-salt hypertensive rats compared with the control, as was the protein expression of ED-1, COX-2, TGF-β1, and α-SMA. The expression levels of caspase-3 and Bax were increased significantly, whereas Bcl-2 expression was decreased. In conclusion, the phosphorylation of PI3K/Akt/mTOR was increased in the kidney of DOCA-salt hypertensive rats.

Background/Aims: Sildenafil, the first selective phosphodiesterase-5 (PDE5) inhibitor to be widely used for treating erectile dysfunction, has been investigated with regard to its cardioand renoprotective effects in animal models. This study further investigated the renoprotective effects of sildenafil and their molecular mechanisms in deoxycorticosterone acetate (DOCA)-salt hypertensive (DSH) rats. Methods: DOCA strips (200 mg/kg) were implanted in rats 1 week after unilateral nephrectomy. These rats were fed on a control diet, with or without sildenafil (50 mg·kg–1day–1), for 2 weeks. Systolic blood pressure (SBP) was measured by the tail cuff method, and the urinary albumin-to-creatinine ratio (ACR) was calculated. The extent of glomerulosclerosis and tubulointerstitial fibrosis was determined by Masson’s trichrome stain. Renal expression of ED-1, transforming growth factor-β1 (TGF-β1), Bax, and Bcl-2 were determined by semiquantitative immunoblotting, polymerase chain reaction (PCR), and immunohistochemistry. TUNEL staining was used for detecting apoptotic cells. Results: The increased SBP in DSH rats was not attenuated by sildenafil treatment. The decreased creatinine clearance and increased ACR in DSH rats, compared with control animals, were attenuated by sildenafil treatment. Further, sildenafil treatment attenuated glomerulosclerosis and tubulointerstitial fibrosis in DSH rats and counteracted the increased expression of ED-1, TGF-β1, and Bax and the decreased expression of Bcl-2 in the kidneys of these rats. The increase in the number of apoptotic cells in DSH rats was attenuated by sildenafil treatment. Conclusion: Sildenafil effectively prevented the progression of renal injury in DSH rats via its anti-inflammatory, antifibrotic, and antiapoptotic effects.

The present study was performed to examine whether paricalcitol may prevent the cisplatin-induced kidney injury. Furthermore, potential molecular mechanisms underlying the protective effect of paricalcitol were explored. Male Sprague-Dawley rats were treated with vehicle (n=12), cisplatin (n=12, 6 mg/kg/day, i.p.), or cisplatin+paricalcitol (n=12, 0.2 μg/kg/day, s.c.) for 4 days. In another series of experiment, HK-2 cells were treated with cisplatin (50 μM), with or without paricalcitol (0.2 ng/ml). Paricalcitol counteracted the cisplatin-induced decline in renal function. Paricalcitol also suppressed the expression of TGF-β1, Smad signaling, and the subsequent epithelial-to-mesenchymal process in cisplatin-treated rats. The expression of P-p53 and p21 was increased in cisplatin-induced nephropathy. These changes were completely prevented or significantly attenuated with paricalcitol co-treatment. The expression of p27(kip1) was increased in cisplatin-treated rats, which was, however, further augmented by the paricalcitol co-treatment. In HK-2 cells, cisplatin increased the expression of p-ERK1/2 and P-p38. Cisplatin also increased the expression of fibronectin and CTGF. Cisplatin increased the expression of pro-apoptotic markers. The expression of CDK2 and Cyclin E as well as that of PCNA was increased. These changes were completely prevented or significantly attenuated by the paricalcitol pretreatment. In contrast, cisplatin increased the expression of p27(kip1), which was further augmented by the paricalcitol-pretreatment. These results suggest that paricalcitol may ameliorate cisplatin-induced renal injury by suppressing the fibrotic, apoptotic and proliferative factors. Its underlying mechanisms may include inhibition of TGF-β1, mitogen-activated protein kinase signaling, p53-induced apoptosis, and augmentation of p27(kip1).

Background. The aldehyde products of lipid peroxidation such as 4-hydroxy-2-hexenal (HHE) might be responsible for the pathogenesis of kidney injury. The present study was aimed to investigate the effects of HHE on renal tubular epithelial cells and its signaling mechanisms. Methods. Human proximal tubular epithelial (HK-2) cells were treated with 10 lM of HHE. Cell viability was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The fluorescent probe 2#,7#-dichlorofluorescein diacetate was used to measure intracellular levels of reactive oxygen species (ROS). The protein expression of NF-jB, mitogen-activated protein kinase (MAPK), pro-apoptoic Bax and anti-apoptotic protein Bcl-2 was determined by semiquantitative immunoblotting. Apoptosis was assessed by flow cytometry analysis after the cells were stained by fluorescein isothiocyanateconjugated annexin V protein and propidium iodine. Results. Treatment with various doses of HHE resulted in dose-dependent decreases of cell viability and increases of ROS. HHE increased the expression of p38 MAPK, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). HHE induced NF-jB activation and IjB-a degradation. Increased nuclear NF-jB activation was blocked by inhibitors of ERK (PD98059) or JNK (SP600125), but not affected by p38 MAPK inhibitor (SB203580). Flow cytometry analysis revealed HHE-induced apoptosis. HHE decreased the expression of Bcl-2, while it increased that of Bax, which was attenuated by the treatment of NF-jB inhibitor (Bay 11-7082) or N-acetyl-L-cysteine (NAC). An inhibition of NF-jB prevented HHE-induced apoptosis. Conclusions. HHE-induced tubular cell apoptosis is mediated by modulation of Bax and Bcl-2 via ROS generation. HHE-mediated accumulation of ROS may induce redoxsensitive transcription factor, NF-jB, through activation of ERK and JNK, resulting in cellular apoptosis in HK-2 cells.

Resveratrol (RSV) may provide numerous protective eff ects against chronic inflammatory diseases. Due to local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress, and aldehyde products formed during lipid peroxidation, such as 4-hydroxy-2-hexenal (HHE), might be responsible for tubular injury. This study aimed at investigating the eff ects of RSV on renal and its signaling mechanisms. While HHE treatment resulted in decreased expression of Sirt1, AQP2, and nuclear factor erythroid 2-related factor 2 (Nrf2), mouse cortical collecting duct cells (M1) cells treated with HHE exhibited increased activation of p38 MAPK, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and increased expression of NOX4, p47(phox), Kelch ECH associating protein 1 (Keap1) and COX2. HHE treatment also induced NF-κB activation by promoting IκB-α degradation. Meanwhile, the observed increases in nuclear NF-κB, NOX4, p47(phox), and COX2 expression were attenuated by treatment with Bay 117082, N-acetyl-l-cysteine (NAC), or RSV. Our findings indicate that RSV inhibits the expression of inflammatory proteins and the production of reactive oxygen species in M1 cells by inhibiting NF-κB activation.

The conventional method for the analysis of myocardial cell transplantation depends on postmortem histology. Here, we have sought to demonstrate the feasibility of a longitudinal monitoring of transplanted cell survival in living animals, accomplished with optical imaging techniques and pharmacological interventions.Human cord blood (50 ml) was donated with parental consent. After getting cord blood derived mesenchymal stem cells (CBMSCs), cells were transfected (MOI = 100) overnight with adenovirus encoding firefly luciferase gene (Ad-CMV-Fluc). Our experimental Sprague-Dawley rats (n = 12) were given intramyocardial injections containing 1 x 10(6) CBMSCs, which had been made to express the firefly luciferase (Fluc) reporter gene. Optical bioluminescence imaging was then conducted using a cooled charged-coupled device (CCD) camera (Xenogen), beginning on the day after the transplantation (day 1). Groups of mice were intraperitoneally injected with cyclosporine (5 mg/kg) or tacrolimus (1 mg/kg), in an attempt to determine the degree to which cell survival had been prolonged, and these values were then compared with the cell survival values of the negative control group. The presence of transplanted CBMSCs on in vivo images confirmed by in situ hybridization for human specific Alu in the myocardium.Cardiac bioluminescence signals were determined to be present for 6 days after transplantation: day 1 (97000 +/- 9100 x 10(5) p/s/cm2/sr), day 3 (9600 +/- 1110 p/s/cm2/sr), and day 5 (3200 +/- 550 p/s/cm2/sr). The six mice that received either cyclosporine or tacrolimus displayed cardiac bioluminescence signals for a period of 8 days after transplantation. We observed significant differences between the treated group and the non-treated group, beginning on day 3 (tacrolimus; 26500 +/- 4340 p/s/cm2/sr, cyclosporine; 27200 +/- 3340 p/s/cm2/sr, non-treated; 9630 +/- 1180 p/s/cm2/sr, p0.01), and persisting until day 7 (tacrolimus; 12500 +/- 2946 p/s/cm2/sr, cyclosporine; 7310 +/- 1258 p/s/cm2/sr, non-treated; 2460 +/- 160 p/s/cm2/sr, p0.01). The human-derived CBMSCs were detected in the myocardium 3 days after transplantation by in situ hybridization.The locations, magnitude, and survival duration of the CBMSCs were noninvasively monitored with a bioluminescence optical imaging system. We determined that optical molecular imaging expedites the fast throughput screening of pharmaceutical agents, allowing for the noninvasive tracking of cell survival within animals. In rat cardiac CBMSC transplant models, transient immunosuppressive treatment with tacrolimus or cyclosporine was shown to improve donor cell survival.

Objective This study was designed to examine whether mesenchymal stem cells (MSCs) transduced with Akt enhance cardiac repair after transplantation into the ischemic porcine heart. Methods MSCs isolated from porcine bone marrow and transduced with myr-Akt were transplanted into porcine hearts after experimental myocardial infarction (MI) using intracoronary injection [Group I, vehicle; Group II, MSCs; Group III, Akt-MSCs]. Myocardial single photon emission tomography (M-SPECT) was performed to assess myocardial function and the infarcted area. Pigs were also sacrificed for immunohistochemical characterization and histologic analysis. In addition, in vitro assays were performed to examine the resistance of Akt-MSCs to H2O2 stimulation. Results Transplantation of MSCs into the ischemic porcine myocardium (Group II) increased the left ventricular ejection fraction (δLV EF; − 6.3±15.1% versus 0.5±6.4%, P

Transferrin-conjugated cationic liposome (Tf-DDAB liposome) was developed as a targeted gene delivery system by using heterobifunctional cross-linking agent, N-succimidyl-3-(2-pyridyldithio) propionate (SPDP), and gradient metrizamide ultracentrifugation method. Physico-chemical properties of Tf-liposome were determined by scanning/transmission electron microscopy (SEM/TEM) and dynamic laser-light scattering method (DLS) with a mean diameter of 584 +/- 15 nm. Gel retardation assay was performed using various DDAB:DNA ratios, and proved that the 6:1 weight ratio formulation gave the most neutralized complex. In vitro transfection was done in human cervical cancer cells, HeLa, and the transfection efficiency of Tf-liposome was found to be fivefold higher than that of unconjugated (plain) DDAB liposome and twofold higher than that of Lipofectin. In conclusion, a target-oriented Tf-DDAB liposome was made successfully and proved to be very efficient in DNA delivery into the cervical cancer cells in culture.

The present study aimed to investigate changes in the mammalian target of rapamycin (mTOR) signaling pathway in the obstructed kidney of rats with unilateral ureteral obstruction (UUO). Male Sprague-Dawley rats were unilaterally obstructed by ligation of the left proximal ureter for 7 days. Control rats were treated in the same way except that no ligature was made. The expression levels of phosphorylated phosphatidylinositol 3-kinase (PI3K), Akt, and mTOR were determined in the kidney by semiquantitative immunoblotting. The protein expression levels of transforming growth factor (TGF)-β1, Bax, and Bcl-2 were also determined in the kidney. The phosphorylation of PI3K, Akt, and mTOR was increased in the kidney of ureteral obstruction rats compared with the control. In the obstructed kidney, the protein expression of TGF-β1 and Bax was also increased, whereas Bcl-2 expression was decreased. In conclusion, the phosphorylation of PI3K/Akt/mTOR was increased in the obstructed kidney of rats with UUO.

배경: 일상 식이 생활에서 흔히 쓰이는 생강(ginger)의 한 성분인 6-gingerol은 질병 치료 과정 중에 사용하는 약물과 상호 작용을 일으킬 수 있다. 그러나 인체 간 대사 효소인 시토크롬 P450 활성도에 대한 6-gingerol의 효과는 아직 알려져 있지 않다. 본 연구에서는 인체 간 미세소체에서 6-gingerol이 시토크롬 P450대사 효소에 미치는 억제 효과를 연구하고자 하였다. 방법: 인체 간 미세소체 모델을 이용하여 다양한 농도의 6-gingerol과 각각의 CYP450에 특이적인 기질을 함께 배양하였으며, 각각의 대사물을 HPLC로 측정하였다. 본 연구에서 사용된 CYP효소에 의해 촉매 되는 반응에는 CYP1A2에 의한 phenacetin O-deethylation, CYP2C9에 의한 tolbutamide 4-hydroxyltion, CYP2C19에 의한 S-mephenytoin 4'-hydroxylation, CYP2D6에 의한 dextromethorphan O-demethylation, CYP2E1에 의한 chlorzoxazone 6-hydroxylation, 그리고 CYP3A4에 의한 midazolam 1-hydroxylation이었다. 결과: 6-gingerol은 $IC_{50}$ 가 $100{\mu}M$ 을 상회할 정도로 CYP2C9, CYP2D6, CYP2E1 그리고 CYP3A4의 활성도에 큰 영향을 미치지 않았다. 그러나 6-gingerol은 CYP 2C19과 CYP 1A2에 대하여 Ki 수치가 각각 $13{\mu}M$ 과 $29{\mu}M$ 이 될 정도로 강한 억제 효과를 나타내었다. CYP1A2에 대하여 6-gingerol은 상경적인 형태의 억제를 초래하였다. 결론: 이러한 결과는 6-gingerol이 CYP1A2과 CYP2C19의 활성도를 잠재적으로 억제할 수 있음을 시사하였다.

Background and Objectives: Myocardial ischemia-reperfusion (I/R) injury is one of the major causes of cardiac mortality. Curcumin, an active component extracted from turmeric in curry, inhibits inflammatory responses. This study was designed to investigate whether curcumin can exert beneficial effects on myocardial I/R injury. Materials and Methods: Sprague-Dawley male rats received a normal diet or a curcumin diet (80 mg/kg/d) for one week, and I/R injury was induced by ligating the left anterior descending artery (LAD) for 30 min followed by release. After 24 hours, the myocardium was extracted to evaluate the myeloperoxidase (MPO) activity and the vascular cellular adhesion molecule (VCAM)-1 protein level. The apoptotic cardiomyocytes and neutrophils were counted and quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining at 14 days after I/R. Results: In the infarcted myocardium of the curcumin-fed rats, the MPO activity (32.9±2.2% of the control, p=0.001) and the VCAM-1 protein (28.7±2.9% of control, p=0.001) level were significantly attenuated. The number of neutrophils was lower in the curcumin-fed rats (57±12% of the control, p=0.024). A reduction of the apoptotic cardiomyocytes was also observed in the curcumin-fed I/R rats (36± 9.2% of the control, p=0.032). Conclusion: The cardioprotective effects of curcumin on an I/R injury rat model could include anti-inflammation activities and inhibition of apoptosis that occurred in the cardiomyocytes. Our findings suggest that curcumin has a positive contribution as a dietary supplement for the prevention of heart disease. (Korean Circ J 2008;38:353-359)

Purpose: Several study trials have used stem cells to treat stress incontinence in an animal model. In this study, we compared injecting either periurethral mesenchymal stem cells (MSC) or normal saline (C) to increase the leak point pressure (LPP) and closing pressure (CP) in a rat model of stress urinary incontinence. Materials and Methods: Sprague Dawley rats (250g each, 12 weeks old) were divided into the MSC group (n=5) and group C (n=5). They were anesthetized and the pudendal nerve was transected bilaterally via a ventral incision in order to denervate the external urethral sphincter. The MSCs were obtained from both femurs of Sprague Dawley rats (150g each, 6 weeks, n=10). After 1 week, the MSCs were stained by 4'-6-diamidino2-phenylindole (DAPI), which was injected into both sites of the proximal external urethra (n=1.5x10). At 3 weeks after injection, cystometry was performed and this was followed by cord transection at the T9-10 level with the rat under anesthesia. Visually identified LPP and CP measurements were evaluated with using a vertical tilt/intravesical pressure clamp. The urethral tissues of the rats were harvested for histology. Results: Both the LPP and CP measurements were significantly higher in the MSC group when compared with that of the C group (p

Background and Objectives：Curcumin, a yellow pigment of turmeric in curry, has been reported to interfere with nuclear factor (NF)-κB. This study was designed to investigate the underlying pathway of the anti-inflammation effect of curcumin on endothelial cells. Materials and Methods：Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor (TNF)-α (10 ng/mL). The levels of intracellular reactive oxygen species (ROS) were examined using a fluorescent dye DCFH-DA, and the adhesion of U-937 monocytes to the HUVECs was then examined. Nuclear factor kappa B (NF-B) activation was determined by the NF-κB p65 translocation to the nucleus via immunocytochemistry. The expression of the NF-κB dependent pro-inflammatory molecules were measured by RT-PCR and ELISA. The phosphorylations of c-Jun N-terminal protein kinase (JNK), p38 and STAT-3 (signal transducer and activator of transcription-3) were measured by Western blotting. Results：Curcumin blocked the activation of NF-κB by TNF-α, and it also reduced the ROS, monocyte adhesion and the phosphorylation of JNK, p38 and STAT-3 in the TNF-α-stimulated HUVECs. The expression of intracellular cell adhesion molecule (ICAM)-1, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-8 were attenuated by curcumin at both the transcription and translation levels. Conclusion：We suggest that curcumin could contribute to protection against the adverse vascular effects of the pro-inflammatory response through the modulation of NF-κB, JNK, p38 and STAT-3, and this is in addition to its antioxidant effect in endothelial cells. (Korean Circulation J 2006;36:482-489)

Background and Objectives：Carvedilol is an anti-oxidative, the cardioprotective effects of which are mediated by the inhibition of NF-κB activation. The present study was designed to examine the effects of carvedilol, an α1- and β-blocker, on tumor necrosis factor (TNF)-α stimulated human umbilical vein endothelial cells (HUVEC). Materials and Methods：HUVEC were treated with TNF-α (10 ng/mL) in either the absence or presence of carvedilol. The levels of intracellular reactive oxygen species (ROS) were examined using a fluorescent dye DCFH-DA, with the adhesion of U-937 monocyte to the HUVEC. Nuclear factor kappa B (NF-κB) activation was determined by NF-κB p65 translocation to the nucleus using Western blotting and immunocytochemistry. The expressions of NF-κB dependent pro-inflammatory molecules, i.e., vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8, were measured by RT-PCR and ELISA. Bcl-2 and phosphorylation of c-Jun N-terminal protein kinase (JNK) were measured using Western blotting. Results：TNF-α treatment increased the activation of NFκB, suppressed Bcl-2, and increased the phosphorylation of JNK, the ROS level and the adhesion of U-937. The levels of mRNA and protein expressions of VCAM-1, ICAM-1, MCP-1 and IL-8 were up-regulated by TNFα. Carvedilol inhibited the phosphorylation of JNK, ROS formation and the adhesion of U-937 monocyte. In addition, carvedilol reduced the production of VCAM-1, ICAM-1, MCP-1 and IL-8 at the mRNA and protein levels, via the suppression of NF-B activation. Conclusion：These results suggested that the anti-inflammatory effects of carvedilol on TNF-α stimulated endothelial cells could be explained by its ROS-scavenging and NF-κ B inactivation properties. (Korean Circulation J 2005;35:576-582)