Attenuation of in vitro and in vivo melanin synthesis using a Chinese herbal medicine through the inhibition of tyrosinase activity.

BACKGROUND: In traditional Chinese medicine, the skin reflects the health of body organs. A skin whitening agent, named seven whitening creams (also called Chi-Bai-San), has been used since ancient times in China. Chi-Bai-San reduces melanin and helps to reduce wrinkles. PURPOSE: We aimed to determine the skin-whitening ability and safe dose of the seven compounds in Chi-Bai-San. STUDY DESIGN: A common use for Chinese medicine is decocted in water. To mimic the function of Chi-Bai-San apply in clinical, we boiled all seven compound in water, respectively. These single recipe extractions and a mixture of these seven items were used in zebrafish embryo and B16F10 melanoma cell to identify the anti-melanogenesis function. METHODS: Chi-Bai-San comprises Bai-Lian (Ampelopsis japonica), Bai-Ji (Bletilla striata), Bai-Zhi (Angelica dahurica), Bai-Zhu (Atractylodes macrocephala), Bai-Shau (Paeonia lactiflora), Fu-Ling (Wolfiporia cocos), and Jen-Ju-Fen (Pearl powder). All components were extracted by heating in distilled water. The supernatant was collected after centrifugation. The extracted components were introduced into zebrafish embryos at different doses to determine the safe dose. B16F10 melanoma cells were treated with the final dose of each component and the component mixture. Melanin content and tyrosinase activity were assessed in zebrafish and B16F10 cells. Chi-Bai-San and its components were exposed to α MSH-induced B16F10 cells, and detected for mechanism of anti-melanogenesis pathway. RESULTS: Most compounds were not toxic at a low dose (0.1 mg/ml), except A. macrocephala, which resulted in a survival rate of only 30% at 72 hpf. The final dose of A. dahurica, P. lactiflora, W. cocos, and pearl was 1 mg/ml; that of A. japonica was 0.5 mg/ml; and that of A. macrocephala and B. striata was 0.1 mg/ml. Chi-Bai-San markedly decreased melanin content 37.47% in zebrafish embryos. Further, Chi-Bai-San abolished tyrosinase activity and MITF-mediated tyrosinase expression by down regulating the upstream transcription factors ZEB2, ß-catenin, and CREB2 in α MSH-induced B16F10 cells. Additionally, Chi-Bai-San might reduce melanosome secretion from melanocytes. CONCLUSION: Our findings indicate that safety and efficacy of heat-extracted Chi-Bai-San, which can reduce αMSH-induced melanin production by inhibiting the key role of melogenic-related transcription factor and promote the synergic effect of seven types of traditional Chinese herbal medicines.

Down-Regulated Expression of Magnesium Transporter Genes Following a High Magnesium Diet Attenuates Sciatic Nerve Crush Injury.

BACKGROUND: Magnesium supplementation has potential for use in nerve regeneration. The expression of some magnesium transporter genes is reflective of the intracellular magnesium levels. OBJECTIVE: To assess the expression of various magnesium transporter genes as they relate to neurological alterations in a sciatic nerve injury model. METHODS: Sciatic nerve injury was induced in rats, which were then fed either basal or high magnesium diets. Magnesium concentrations and 5 magnesium transporter genes (SLC41A1, MAGT1, CNNM2, TRPM6, and TRPM7) were measured in the tissue samples. RESULTS: The high magnesium diet attenuated cytoskeletal loss in a dose-dependent manner in isolated nerve explants. The high magnesium diet augmented nerve regeneration and led to the restoration of nerve structure, increased S-100, and neurofilaments. This increased regeneration was consistent with the improvement of neurobehavioral and electrophysiological assessment. The denervated muscle morphology was restored with the high magnesium diet, and that was also highly correlated with the increased expression of desmin and acetylcholine receptors in denervated muscle. The plasma magnesium levels were significantly elevated after the animals consumed a high magnesium diet and were reciprocally related to the down-regulation of CNNM2, MagT1, and SCL41A1 in the blood monocytes, nerves, and muscle tissues of the nerve crush injury model. CONCLUSION: The increased plasma magnesium levels after consuming a high magnesium diet were highly correlated with the down-regulation of magnesium transporter genes in monocytes, nerves, and muscle tissues after sciatic nerve crush injury. The study findings suggest that there are beneficial effects of administering magnesium after a nerve injury.

Novel Combination of Arsenic Trioxide (As2O3) Plus Resveratrol in Inducing Programmed Cell Death of Human Neuroblastoma SK-N-SH Cells.

AIM: Arsenic trioxide (As2O3), known as pi-shuang and the most toxic compound in traditional Chinese medicine, has been used as an antitumor agent for thousands of years. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural phenol that has significant anti-bacterial, anti-fungaI and antiaging activities. Our study aimed to examine the combined anticancer effects of As2O3 and resveratrol against human neuroblastoma SK-N-SH cells, and elucidate the underlying intracellular signaling. MATERIALS AND METHODS: SK-N-SH cells were treated with an extremely low-dose (2-4 µM) of As2O3 alone or combined with 75 µg/ml resveratrol for further comparisons. Cell viability, apoptotic signaling as well as synergistic cytotoxic effects were estimated using the MTT assay, microscopy observation, flow cytometric analysis for loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), and typical quantitative western blotting analysis. Student's t-test, and one- and two-way analysis of variance (ANOVA) were used for examination of significant differences. RESULTS: The combined treatment was more effective than single treatment of As2O3 or resveratrol alone in suppressing cell viability, which correlated with the elevation of ROS levels. The intracellular mechanisms of cytotoxicity of As2O3 plus resveratrol were revealed as ROS accumulation and relative decrease of MMP, leading to activation of caspase-3 and -9, but not of caspase-1, -7 and-8. Combination treatment reduced the expression of B-cell lymphoma 2 (BCL2), BH3 interacting domain death agonist (BID), and BCL-x/L. CONCLUSION: Combined treatment at extremely low concentration of two agents from natural products, As2O3 and resveratrol, has high potential as a cocktail of anticancer drugs for neuroblastoma.

Late administration of high-frequency electrical stimulation increases nerve regeneration without aggravating neuropathic pain in a nerve crush injury.

BACKGROUND: High-frequency transcutaneous neuromuscular electrical nerve stimulation (TENS) is currently used for the administration of electrical current in denervated muscle to alleviate muscle atrophy and enhance motor function; however, the time window (i.e. either immediate or delayed) for achieving benefit is still undetermined. In this study, we conducted an intervention of sciatic nerve crush injury using high-frequency TENS at different time points to assess the effect of motor and sensory functional recovery. RESULTS: Animals with left sciatic nerve crush injury received TENS treatment starting immediately after injury or 1 week later at a high frequency(100 Hz) or at a low frequency (2 Hz) as a control. In SFI gait analysis, either immediate or late admission of high-frequency electrical stimulation exerted significant improvement compared to either immediate or late administration of low-frequency electrical stimulation. In an assessment of allodynia, immediate high frequency electrical stimulation caused a significantly decreased pain threshold compared to late high-frequency or low-frequency stimulation at immediate or late time points. Immunohistochemistry staining and western blot analysis of S-100 and NF-200 demonstrated that both immediate and late high frequency electrical stimulation showed a similar effect; however the effect was superior to that achieved with low frequency stimulation. Immediate high frequency electrical stimulation resulted in significant expression of TNF-α and synaptophysin in the dorsal root ganglion, somatosensory cortex, and hippocampus compared to late electrical stimulation, and this trend paralleled the observed effect on somatosensory evoked potential. The CatWalk gait analysis also showed that immediate electrical stimulation led to a significantly high regularity index. In primary dorsal root ganglion cells culture, high-frequency electrical stimulation also exerted a significant increase in expression of TNF-α, synaptophysin, and NGF in accordance with the in vivo results. CONCLUSION: Immediate or late transcutaneous high-frequency electrical stimulation exhibited the potential to stimulate the motor nerve regeneration. However, immediate electrical stimulation had a predilection to develop neuropathic pain. A delay in TENS initiation appears to be a reasonable approach for nerve repair and provides the appropriate time profile for its clinical application.

Green Tea Catechin Prevents Hypoxia/Reperfusion-Evoked Oxidative Stress-Regulated Autophagy-Activated Apoptosis and Cell Death in Microglial Cells.

Defective activation and proliferation in microglial cells has been suggested to be associated with the increase of cerebral ischemia/reperfusion injury. We investigated the protection and molecular mechanism of green tea catechin on hypoxia/reperfusion-induced microglial cell injury in vitro. Microglial cells were cultured in hypoxia condition (O2 < 1%) and then re-incubated to the complete normal culture medium (reperfusion). Hypoxia/reperfusion obviously decreased cell viability and induced apoptosis in microglial cells, but not in neuronal cells. Catechin significantly inhibited the hypoxia/reperfusion-induced decreased cell viability and increased reactive oxygen species (ROS) and apoptosis in microglia. The administration of both PI3K/Akt inhibitor LY294002 and mTOR inhibitor rapamycin demonstrated that Akt/mTOR-regulated autophagy was involved in the hypoxia/reperfusion-induced microglia apoptosis/death. Catechin up-regulated the Akt and mTOR phosphorylation and inhibited the hypoxia/reperfusion-induced autophagy in microglia. These results suggest that hypoxia/reperfusion can evoke autophagy-activated microglia apoptosis/death via an ROS-regulated Akt/mTOR signaling pathway, which can be reversed by catechin.

Honokiol thwarts gastric tumor growth and peritoneal dissemination by inhibiting Tpl2 in an orthotopic model.

Honokiol is known to suppress the growth of cancer cells; however, to date, its antiperitoneal dissemination effects have not been studied in an orthotopic mouse model. In the present study, we evaluated the antiperitoneal dissemination potential of Honokiol in an orthotopic mouse model and assessed associations with tumor growth factor-ß1 (TGFß1) and cells stimulated by a carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Our results demonstrate that tumor growth, peritoneal dissemination and peritoneum or organ metastasis of orthotopically implanted MKN45 cells were significantly decreased in Honokiol-treated mice and that endoplasmic reticulum (ER) stress was induced. Honokiol-treated tumors showed increased epithelial signatures such as E-cadherin, cytokeratin-18 and ER stress marker. In contrast, decreased expression of vimentin, Snail and tumor progression locus 2 (Tpl2) was also noted. TGFß1 and MNNG-induced downregulation of E-cadherin and upregulation of Tpl2 were abrogated by Honokiol treatment. The effect of Tpl2 inhibition in cancer cells or endothelial cells was associated with inactivation of CCAAT/enhancer binding protein B, nuclear factor kappa-light-chain-enhancer of activated B cell and activator protein-1 and suppression of vascular endothelial growth factor. Inhibition of Tpl2 in gastric cancer cells by small interfering RNA or pharmacological inhibitor was found to effectively reduce growth ability and vessel density in vivo. Honokiol-induced reversal of epithelial-to-mesenchymal transition (EMT) and ER stress-induced apoptosis via Tp12 may involve the paralleling processes. Taken together, our results suggest that the therapeutic inhibition of Tpl2 by Honokiol thwarts both gastric tumor growth and peritoneal dissemination by inducing ER stress and inhibiting EMT.

Magnesium supplement promotes sciatic nerve regeneration and down-regulates inflammatory response.

Magnesium (Mg) supplements have been shown to significantly improve functional recovery in various neurological disorders. The essential benefits of Mg supplementation in peripheral nerve disorders have not been elucidated yet. The effect and mechanism of Mg supplementation on a sciatic nerve crush injury model was investigated. Sciatic nerve injury was induced in mice by crushing the left sciatic nerve. Mice were randomly divided into three groups with low-, basal- or high-Mg diets (corresponding to 10, 100 or 200% Mg of the basal diet). Neurobehavioral, electrophysiological and regeneration marker studies were conducted to explore nerve regeneration. First, a high Mg diet significantly increased plasma and nerve tissue Mg concentrations. In addition, Mg supplementation improved neurobehavioral, electrophysiological functions, enhanced regeneration marker, and reduced deposits of inflammatory cells as well as expression of inflammatory cytokines. Furthermore, reduced Schwann cell apoptosis was in line with the significant expression of bcl-2, bcl-X(L) and down-regulated expression of active caspase-3 and cytochrome C. In summary, improved neurological function recovery and enhanced nerve regeneration were found in mice with a sciatic nerve injury that were fed a high- Mg diet, and Schwann cells may have been rescued from apoptosis by the suppression of inflammatory responses.

Honokiol ameliorates renal fibrosis by inhibiting extracellular matrix and pro-inflammatory factors in vivo and in vitro.

BACKGROUND AND PURPOSE: Renal fibrosis acts as the common pathway leading to the development of end-stage renal disease. The present study investigated, in vivo and in vitro, the anti-fibrotic and anti-inflammatory effects, particularly on the epithelial to mesenchymal transition of renal tubular cells, exerted by honokiol, a phytochemical used in traditional medicine, and mechanisms underlying these effects. EXPERIMENTAL APPROACH: Anti-fibrotic effects in vivo were assayed in a rat model of renal fibrosis [the unilateral ureteral obstruction (UUO) model]. A rat tubular epithelial cell line (NRK-52E) was stimulated by transforming growth factor-ß1 (TGF-ß1) and treated with honokiol to explore possible mechanisms of these anti-fibrotic effects. Gene or protein expression was analysed by Northern or Western blotting. Transcriptional regulation was investigated using luciferase activity driven by a connective tissue growth factor (CTGF) promoter. KEY RESULTS: Honokiol slowed development of renal fibrosis both in vivo and in vitro. Honokiol treatment attenuated tubulointerstitial fibrosis and expression of pro-fibrotic factors in the UUO model. Honokiol also decreased expression of the mRNA for the chemokine CCL2 and for the intracellular adhesion molecule-1, as well as accumulation of type I (α1) collagen and fibronectin in UUO kidneys. Phosphorylation of Smad-2/3 induced by TGF-ß1 and CTGF luciferase activity in renal tubular cells were also inhibited by honokiol. CONCLUSIONS AND IMPLICATIONS: Honokiol suppressed expression of pro-fibrotic and pro-inflammatory factors and of extracellular matrix proteins. Honokiol may become a therapeutic agent to prevent renal fibrosis.

Escalated regeneration in sciatic nerve crush injury by the combined therapy of human amniotic fluid mesenchymal stem cells and fermented soybean extracts, Natto.

Attenuation of inflammatory cell deposits and associated cytokines prevented the apoptosis of transplanted stem cells in a sciatic nerve crush injury model. Suppression of inflammatory cytokines by fermented soybean extracts (Natto) was also beneficial to nerve regeneration. In this study, the effect of Natto on transplanted human amniotic fluid mesenchymal stem cells (AFS) was evaluated. Peripheral nerve injury was induced in SD rats by crushing a sciatic nerve using a vessel clamp. Animals were categorized into four groups: Group I: no treatment; Group II: fed with Natto (16 mg/day for 7 consecutive days); Group III: AFS embedded in fibrin glue; Group IV: Combination of group II and III therapy. Transplanted AFS and Schwann cell apoptosis, inflammatory cell deposits and associated cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. The deterioration of neurological function was attenuated by AFS, Natto, or the combined therapy. The combined therapy caused the most significantly beneficial effects. Administration of Natto suppressed the inflammatory responses and correlated with decreased AFS and Schwann cell apoptosis. The decreased AFS apoptosis was in line with neurological improvement such as expression of early regeneration marker of neurofilament and late markers of S-100 and decreased vacuole formation. Administration of either AFS, or Natto, or combined therapy augmented the nerve regeneration. In conclusion, administration of Natto may rescue the AFS and Schwann cells from apoptosis by suppressing the macrophage deposits, associated inflammatory cytokines, and fibrin deposits.