Poria cocos Extract from Mushrooms Stimulates Aquaporin-3 via the PI3K/Akt/mTOR Signaling Pathway.

Background: Poria cocos (PC), a fungus, has been used for more than 2000 years as a food and medicine in China. PC and its components have various pharmacological effects on the skin, including immunomodulatory activities, barrier function improvement, and anti-tumor effects. However, the effect of PC in aquaporin-3 (AQP3) expression, which is essential for epidermal water permeability barrier maintenance, was not reported. Methods: This study examined the mechanism through which the ethanol extract of the sclerotium of PC (EPC) promoted the expression of AQP3 in cultured human keratinocytes. Western blotting was used to investigate the expression of AQPs and the activation of phosphoinositide 3-kinase (PI3K)/Akt-related signaling molecules in HaCaT cells. Cells were treated with inhibitors of PI3K/Akt and mechanistic target of rapamycin (mTOR) prior to EPC treatment. Results: EPC promoted the expression of AQP3 in HaCaT cells without affecting AQP1 and AQP2 expression. Phosphorylated Akt levels were increased by EPC treatment, and the inhibition of PI3K by LY2940002 resulted in a reduction in EPC-induced AQP3 expression. Furthermore, EPC stimulated the phosphorylation of p70S6K and AktSer473, which are downstream targets of mTORC1 and mTORC2, respectively. The mTOR complex inhibitors, rapamycin and Torin 1, partially reduced EPC-induced AQP3 expression. Conclusion: These results suggest that EPC increased expression of AQP3, which is important for skin moisturization, by activating the PI3K/Akt/mTOR signaling pathway in human keratinocytes.

Arecae pericarpium water extract alleviates chronic pancreatitis by deactivating pancreatic stellate cells.

Chronic pancreatitis (CP) is a chronic inflammatory disease of the pancreas with irreversible morphological changes. Arecae pericarpium (ARP), known to improve gastrointestinal disorders, has not yet been reported to inhibit fibrosis in CP. Therefore, we investigated the beneficial effects of ARP on cerulein-induced CP. Cerulein (50 µg/kg) was administered intraperitoneally to mice every hour, six times a day, four times a week for a total of 3 weeks to induce CP. To ascertain the prophylactic effects of ARP, ARP water extract (50, 100, or 200 mg/kg) or saline was administered intraperitoneally 1 h before the onset of CP. To determine the therapeutic effects of ARP, ARP water extract (200 mg/kg) or saline was administered for a total of 1 week or 2 weeks, starting 2 weeks or 1 week after the onset of CP. The pancreas was collected immediately for histological analysis. Additionally, to determine the effectiveness and mechanism of ARP in alleviating pancreatic fibrosis, pancreatic stellate cells (PSCs) were isolated. ARP treatment considerably improved glandular atrophy and inflammation and repressed collagen deposition in the pancreas. Furthermore, ARP water extract inhibited extracellular matrix (ECM) constituents such as alpha-smooth muscle actin (α-SMA), collagen I, and fibronectin 1 (FN1) in pancreatic tissue and PSCs. ARP also suppressed transforming growth factor-ß (TGF-ß) signaling by inhibiting Smad2 phosphorylation. Our study suggests that ARP exhibits anti-fibrotic effects in cerulein-induced CP by inhibiting TGF-ß/Smad signaling.

The Modulation of Nrf-2/HO-1 Signaling Axis by Carthamus tinctorius L. Alleviates Vascular Inflammation in Human Umbilical Vein Endothelial Cells.

Carthamus tinctorius L., known as safflower, has been used in traditional treatment for cardiovascular, cerebrovascular, and diabetic vascular complications. We proposed to investigate how the ethanol extract of Carthamus tinctorius L. (ECT) can be used ethnopharmacologically and alleviate vascular inflammatory processes under cytokine stimulation in human vascular endothelial cells. Using the optimized HPLC method, six markers were simultaneously analyzed for quality control of ECT. Pretreatment with ECT (10-100 µg/mL) significantly reduced the increase of leukocyte adhesion to HUVEC by TNF-α in a dose-dependent manner. Cell adhesion molecules (CAMs) such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial cell selectin (E-selectin) are decreased by ECT. In addition, ECT significantly suppressed TNF-α-induced oxidative stress referring to reactive oxygen species (ROS) production. p65 NF-κB nuclear translocation and its activation were inhibited by ECT. Furthermore, pretreatment of ECT increased the HO-1 expression, and nuclear translocation of Nrf-2. These data suggest the potential role of ECT as a beneficial therapeutic herb in vascular inflammation via ROS/NF-kB pathway and the regulation of Nrf-2/HO-1 signaling axis is involved in its vascular protection. Thus, further study will be needed to clarify which compound is dominant for protection of vascular diseases.

Gardeniae Fructus Attenuates Thioacetamide-Induced Liver Fibrosis in Mice via Both AMPK/SIRT1/NF-κB Pathway and Nrf2 Signaling.

Liver fibrosis, which means a sort of the excessive accumulation of extracellular matrices (ECMs) components through the liver tissue, is considered as tissue repair or wound-healing status. This pathological stage potentially leads to cirrhosis, if not controlled, it progressively results in hepatocellular carcinoma. Herein, we investigated the pharmacological properties and underlying mechanisms of Gardeniae Fructus (GF) against thioacetamide (TAA)-induced liver fibrosis of mice model. GF not only attenuated hepatic tissue oxidation but also improved hepatic inflammation. We further confirmed that GF led to ameliorating liver fibrosis by ECMs degradations. Regarding the possible underlying mechanism of GF, we observed GF regulated epigenetic regulator, Sirtuin 1 (SIRT1), in TAA-injected liver tissue. These alterations were well supported by SIRT1 related signaling pathways through regulations of its downstream proteins including, AMP-activated protein kinase (AMPK), p47phox, NADPH oxidase 2, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1, respectively. To validate the possible mechanism of GF, we used HepG2 cells with hydrogen peroxide treated oxidative stress and chronic exposure conditions via deteriorations of cellular SIRT1. Moreover, GF remarkably attenuated ECMs accumulations in transforming growth factor-ß1-induced LX-2 cells relying on the SIRT1 existence. Taken together, GF attenuated liver fibrosis through AMPK/SIRT1 pathway as well as Nrf2 signaling cascades. Therefore, GF could be a clinical remedy for liver fibrosis patients in the future.

Scopoletin Stimulates Melanogenesis via cAMP/PKA Pathway and Partially p38 Activation.

Scopoletin was recently shown to stimulate melanogenesis through cAMP-response element-binding protein (CREB) phosphorylation. In this study, we investigated the molecular events of melanogenesis-induced by scopoletin. After exposure to scopoletin, the protein levels of tyrosinase and tyrosianse related protein-1 (TRP-1) were significantly increased in B16F10 cells. The mRNA levels of tyrosinase and microphthalmia-associated transcription factor (MITF) were also enhanced by scopoletin. cAMP production and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were increased by scopoletin treatment. Scopoletin-mediated increase of intracellular melanin and tyrosinase expression were significantly attenuated by protein kinase A (PKA) inhibitors (H-89 and KT5720), while a protein kinase C (PKC) inhibitor (Ro-32-0432) had no effect and a p38 MAPK inhibitor (SB203580) partially blocked the scopoletin-induced intracellular melanin and tyrosinase expression. Moreover, scopoletin synergistically with cell-permeable cAMP analog (dibutyryl cAMP) significantly induced tyrosinase activity and melanin content in B16F10 cells. The silencing of p38 MAPK by small interfering RNA (siRNA) decreased the scopoletin-induced tyrosinase expression in B16F10 cells. These results suggest that scopoletin could induce melanin synthesis through the cAMP/PKA pathway and partially p38 MAPK activation in B16F10 cells.

EPA attenuates ultraviolet radiation-induced downregulation of aquaporin-3 in human keratinocytes.

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid (ω-3 PUFA) that protects against photodamage and photocarcinogenesis in mammals. Aquaporin-3 (AQP3) is a water/glycerol transport protein that is found in basal layer keratinocytes. In this study, we have investigated the protective effect of EPA against ultraviolet B (UVB)-induced AQP3 downregulation in human keratinocytes. EPA treatment was found to increase AQP3 gene and protein expression in human epidermal keratinocytes (HaCaT). Using a specific inhibitor, we observed that the effect of EPA on AQP3 expression was mediated by extracellular signal-regulated kinase (ERK) activation. UVB radiation induced AQP3 downregulation in HaCaT cells, and it was found that EPA treatment attenuated UVB-induced AQP3 reduction and the associated cell death. UVB-induced downregulation of AQP3 was blocked by EPA and p38 inhibitor SB203580. Collectively, the present results show that EPA increased AQP3 expression and that this led to a reduction UVB-induced photodamage.

Diosgenin Induces Apoptosis in HepG2 Cells through Generation of Reactive Oxygen Species and Mitochondrial Pathway.

Diosgenin, a naturally occurring steroid saponin found abundantly in legumes and yams, is a precursor of various synthetic steroidal drugs. Diosgenin is studied for the mechanism of its action in apoptotic pathway in human hepatocellular carcinoma cells. Based on DAPI staining, diosgenin-treated cells manifested nuclear shrinkage, condensation, and fragmentation. Treatment of HepG2 cells with 40 µM diosgenin resulted in activation of the caspase-3, -8, -9 and cleavage of poly-ADP-ribose polymerase (PARP) and the release of cytochrome c. In the upstream, diosgenin increased the expression of Bax, decreased the expression of Bid and Bcl-2, and augmented the Bax/Bcl-2 ratio. Diosgenin-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK, p38 MAPK and apoptosis signal-regulating kinase (ASK)-1, as well as generation of the ROS. NAC administration, a scavenger of ROS, reversed diosgene-induced cell death. These results suggest that diosgenin-induced apoptosis in HepG2 cells through Bcl-2 protein family-mediated mitochndria/caspase-3-dependent pathway. Also, diosgenin strongly generated ROS and this oxidative stress might induce apoptosis through activation of ASK1, which are critical upstream signals for JNK/p38 MAPK activation in HepG2 cancer cells.

BBR induces apoptosis in HepG2 cell through an Akt-ASK1-ROS-p38MAPKs-linked cascade.

Berberine (BBR) has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, and fungi. The mechanism by which BBR initiates apoptosis remains poorly understood. In the present study, we demonstrated that BBR exhibited significant cytotoxicity in human hepatoma HepG2 cells. Herein, we investigated cytotoxicity mechanism of BBR in HepG2 cells. The results showed that the induction of apoptosis in HepG2 cells by BBR was characterized by DNA fragmentation, an increased percentage of annexin V, and the activation of caspase-3. The expressions of Bcl-2 protein and pro-caspase-3 were reduced by BBR in HepG2 cells. However, Bax protein was increased in the cells. BBR-induced apoptosis was preceded by increased generation of reactive oxygen species (ROS). NAC treatment, a scavenger of ROS, reversed BBR-induced apoptosis effects via inhibition of Bax activation and Bcl-2 inactivation. BBR-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of MAP Kinases (JNK and p38 MAPK), ASK1, Akt, and p53. Furthermore, SB203580, p38 inhibitor, reduced the apoptotic effect of BBR, and blocks the generation of ROS and NO as well as activation of Bax. We found that the treatment of HepG2 cells with BBR triggers generation of ROS through Akt phosphorylation, resulting in dissociation of the ASK1-mediated activation of JNK and p38 pathways.

Selaginella tamariscina induces apoptosis via a caspase-3-mediated mechanism in human promyelocytic leukemia cells.

Cell apoptosis is now known to play an important role in the maintenance of cellular homeostasis and anticarcinogenesis. Selaginella tamariscina (ST) is a traditional medicinal plant for treatment of advanced cancer in the Orient. In the present study, the anticancer effect of ST was investigated by analyzing its potential to induce apoptosis in human leukemia HL-60 cells. ST-induced cytotoxicity of HL-60 cells was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The apoptosis was determined by microscopic examination of apoptotic morphology, determination of DNA fragmentation by electrophoresis, activation of caspase-3, and protein expression of procaspase-3, poly(ADP-ribose) polymerase (PARP) cleavage, Bcl-2, and Bax. ST was cytotoxic to HL-60 cells in a dose-dependent manner. However, ST-induced cytotoxicity was suppressed by reactive oxygen species scavengers, including superoxide dismutase (SOD) and catalase. ST caused DNA fragmentation and nuclear condensation, all characteristics of apoptosis. ST-induced apoptosis is accompanied by the activation of caspase-3 and the specific proteolytic cleavage of PARP. Concomitantly, ST treatments led to an increase in the proapoptotic Bax levels, while Bcl-2 expression was decreased. Moreover, this effect was attenuated by SOD and catalase. These results suggest that oxidative stress may be involved in the cytotoxicity of ST, and that ST-induced apoptosis of HL-60 cells is primarily mediated by the caspase activation pathway.

Yukmijihwang-tang ameliorates ischemia/reperfusion-induced renal injury in rats.

The present study was designed to examine whether Yukmijihwang-tang (YJT), which is a Korean decoction for the treatment of renal disease, has an effect on renal functional parameters in association with the expression of aquaporin 2 (AQP 2), Na,K-ATPase, heme oxygenase-1 (HO-1) in rats with ischemia/reperfusion-induced acute renal failure (ARF). Polyuria caused by down-regulation of renal AQP 2 in the ischemia/reperfusion-induced ARF rats was markedly restored by administration of YJT (100 or 200 mg/kg, p.o.) with restoring expression of AQP 2 in the kidney. The expressions of Na,K-ATPase alpha1 and beta1 subunits in the renal medulla and cortex of the ARF rats were also restored in them by the administration of YJT. Administration of YJT lowered the expression of renal HO-1, which was up-regulated in rats with ischemia/reperfusion-induced ARF. The renal functional parameters including creatinine clearance, urinary sodium excretion, urinary osmolality, and solute-free reabsorption were also markedly restored in ischemia-ARF rats by administration of YJT. Histological study also showed that renal damages in the ARF rats were abrogated by administration of YJT. Taken together, these data indicate that YJT ameliorates renal defects in rats with ischemia/reperfusion-induced ARF.

Anti-atherogenic effects of the methanol extract of Sorbus cortex in atherogenic-diet rats.

The present study was designed to examine whether the methanol extract of Sorbus commixta cortex (MSC) could prevent the development of atherosclerosis through regulating the vascular nitric oxide (NO) and endothelin-1 (ET-1) systems in atherogenic-diet rats. Our findings show that aortic NO production as well as endothelial nitric oxide synthase (ecNOS) expression was significantly decreased in atherogenic-diet rats compared with those in the control group. Aortic ET-1 expression was augmented in rats fed an atherogenic-diet while NF-kappaB p65 was upregulated. Treatment of atherogenic-diet rats with either low (100 mg/kg/d) or high (200 mg/kg/d) doses of MSC led not only to significant increases in the aortic NOS/NO system, but also to decreases in aortic ET-1 expression. The aortic expression level of NF-kappaB p65 was also attenuated in atherogenic-diet rats by chronic treatment with low or high doses of MSC. Atherogenic-diet induced increases in the expression of adhesion molecules including intercellular adhesion molecules-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin were markedly decreased by treatment with MSC. From the histopathological examination, MSC treatment was shown to lessen the thickening noted in the aortic intima and media of the atherogenic-diet rats. These results suggest that MSC affects the atherogenic process via the suppression of proinflammatory and adhesion molecules in atherogenic-diet rats, which may be, at least in part, causally related with the regulation of vasoactive systems such as the NO and ET-1 systems.

Inhibitory effect of miconazole on melanogenesis.

Miconazole (MIC), a regional antifungal agent, has been used worldwide in the treatment of superficial mycosis. However, the effect of MIC on skin pigmentation is not known. In this study, we investigated the inhibitory effect of MIC on melanogenesis in B16 melanoma cells. Tyrosinase activity and melanin content were dose dependently decreased by MIC as compared with untreated cells. The level of tyrosinase protein expression was reduced with treatment MIC. A decrease in cell proliferation was observed in B16 cells treated with MIC 30 microM, indicating that the MIC-induced depigmenting effect was caused by inhibition of melanin synthesis and not by destruction of B16 cells. Furthermore, MIC markedly suppressed alpha-melanocyte stimulating hormone or forskolin-induced tyrosinase activity in B16 cells. Therefore the depigmenting effect of MIC might be due to the inhibition of tyrosinase activity and tyrosinase expression, which eventually slows melanin biosynthesis. These results indicate that MIC may be a useful inhibitor of melanogenesis in B16 cells and suggest that it may have beneficial effects in the treatment of hyperpigmentation disorders such as ephelis and melasma.

Butein ameliorates renal concentrating ability in cisplatin-induced acute renal failure in rats.

The present study examined whether the cisplatin-induced nephropathy could be ameliorated by administration of butein isolated from the stems of Rhus verniciflua STOCKS. The present study showed that polyuric profile was revealed in cisplatin-induced acute renal failure (ARF) rats associated with decreases in urinary sodium, potassium, chloride, and creatinine excretion, and osmolality. Among these renal functional parameters, urinary volume and osmolality were partially restored by administration of butein (10 mg/kg, i.p.), but electrolytes and creatinine excretion were not restored. Both solute-free water reabsorption and creatinine clearance were also significantly decreased in rats subjected to cisplatin. When butein was administered in rats with cisplatin-induced ARF for 4 d, solute-free water reabsorption was improved by 91% compared with that of cisplatin-induced ARF rats, but creatinine clearance was not restored. The expression levels of aquaporin 2 (AQP 2) in the inner, outer medulla, and cortex were significantly decreased in the kidney of ARF, which were partially reverted by administration of butein. In histological examination of the kidney, butein treatment partially prevented the lesions at tubules of renal cortex in cisplatin-induced ARF rats, while the lesions at glomeruli were not ameliorated. Taken together, butein ameliorates renal concentrating ability via up-regulation of renal AQP 2 water channel in rats with cisplatin-induced ARF without ameliorating effect on renal filtration defect.

Glycyrrhizin ameliorates renal function defects in the early-phase of ischemia-induced acute renal failure.

The aim of this study was to investigate the effect of glycyrrhizin administration (200 mg/kg/day) on renal function parameters in the early-phase of ischaemia-reperfusion induced acute renal failure (ARF) in rats. The present study showed that the urinary fl ow rate in ischaemia-ARF was significantly increased in association with decreases in water balance, urinary sodium excretion and urine osomolality, which were partially restored by administration of glycyrrhizin. Both solute-free water reabsorption (T(c)H2O) and creatinine clearance (Ccr) were significantly decreased in rats subjected to ischaemia-reperfusion for 72 h compared with the control. Histopathological examination of the kidneys from ARF rats at 72 h after release of the bilateral renal artery clamping, showed that the glomerulus, proximal tubules and distal tubules were severely disrupted and left a denuded basement membrane. When glycyrrhizin was administered in rat ARF for 72 h, Ccr reached almost 96% compared with that of the sham-operated control rats and T(c)H2O was improved by 47% compared with that of the ischaemia-ARF rats. The lesions in the glomerulus, proximal and distal tubule of the renal cortex were also restored by the administration of glycyrrhizin. Taken together, glycyrrhizin administration ameliorates both renal function defects, especially the renal concentrating ability, and structural lesions in renal tissues in rats in the early-phase of ischaemia-ARF.

Effect of Radix Ginseng and Radix Trichosanthis on the melanogenesis.

Melanogenesis is a well known physiological response of human skin exposed to ultraviolet light, genetic reasons and other sources. In this study, we conducted to evaluate the effects of Radix Ginseng (RG) and Radix Trichosanthis (RT) on the melanogenesis in the B16 melanoma cells. The cells were treated for 48 h with RT at concentrations ranging from 1 to 50 microg/ml, RG at concentration of 10-1000 microg/ml, or RG at various doses (10-1000 microg/ml) with 25 microg/ml RT. Treatment with RT alone dose-dependently suppressed tyrosinase activity and melanin content compared with untreated control, and significantly inhibited cell proliferation. However, RG at various concentrations did not exhibit any significant change of them. Treatment with RT in the presence of various concentrations of RG suppressed tyrosinase activity and melanin content, similar to treatment with RT alone, but slightly increased cell proliferation. Furthermore, tyrosinase protein level was significantly decreased in treatment with 25 microg/ml RT alone and with a combination of 100 microg/ml RG. These results indicate that treatment with RG and RT significantly inhibits the melanogenesis in B16 cells, and raise the possibility that this combination may be effective in the whitening agent for the skin.

Cucurbitacins from Trichosanthes kirilowii as the inhibitory components on tyrosinase activity and melanin synthesis of B16/F10 melanoma cells.

Cucurbitacins 1 and 2 were isolated from the root of Trichosanthes kirilowii by tyrosinase inhibitory activity-guided fractionation. Spectroscopic analysis revealed that compounds 1 and 2 were cucurbitacin D and 23,24-dihydro-cucurbitacin D, respectively. Compounds 1 and 2 effectively inhibited the activity of tyrosinase (IC(50) = 0.18 microM and 6.7 microM, respectively), and the synthesis of melanin (IC(50) = 0.16 microM and 7.5 microM, respectively) in B16/F10 melanoma cells.

Effects of the aqueous extract of Epimedii Herba on the induction of oral tolerance in mice.

We investigated the effects of the aqueous extract of Epimedii Herba (AEEH) on the induction of oral tolerance. Oral tolerance was induced in mice by giving an oral administration of 20 mg ovalbumin (OVA) 7 d before immunization with the antigen. AEEH at 40 mg/kg was given orally daily for 6 d from 24 h after the feeding of OVA. The results showed that oral administration of OVA greatly suppressed total serum and antigen-specific immunoglobulin (Ig) levels, phagocytic activity and delayed-type hypersensitivity (DTH) reaction to the antigen. The suppression of these immune responses to OVA by the oral antigen was associated with a marked reduction of the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) from spleen cells. However, AEEH treatment significantly blocked the suppression of total serum and antigen-specific IgG2a antibodies, phagocytic activity and DTH response by the oral OVA. The suppression of IFN-y production by the oral antigen was also greatly decreased by AEEH treatment. Therefore, AEEH appears to be effective in preventing the induction of oral tolerance to OVA.

Effects of the ethanol extract of Cichorium intybus on the immunotoxicity by ethanol in mice.

Effects of the ethanol extract of Cichorium intybus (CIEE) on the immunotoxicity of ethanol (EtOH) were investigated in ICR mice. Mice were divided into four groups, and CIEE at dose of 300 mg/kg was orally administered to mice daily for 28 consecutive days, and normal mice were given vehicle. Mice treated with EtOH were given freely with 20% w/v EtOH solution. The results of this study are summarized as follows: The combination of CIEE and EtOH showed significant increases in the circulating leukocytes and the relative weights of liver, spleen and thymus, as compared with those in mice treated with EtOH alone. However, the body weight gain was not affected. Splenic plaque forming cells (PFC) and hemagglutination (HA) titers to sheep red blood cells (SRBC), and the secondary IgG antibody response to bovine serum albumin (BSA) were markedly enhanced by CIEE plus EtOH treatment as compared with the treatment of EtOH alone. In mice receiving the combination of CIEE and EtOH when compared with EtOH alone-treated mice, there were also significant increases in delayed-type hypersensitivity (DTH) reaction, phagocytic activity, natural killer (NK) cell activity and cell proliferation as well as interferony (IFN-gamma) secretion. In the case of interleukin-4 (IL-4) content, however, an insignificant induction observed by CIEE plus EtOH treatment. These findings indicate that the immunotoxicity induced by EtOH is significantly restored or prevented by CIEE treatment.