The protective effects of ß-sitosterol and vermicularin from Thamnolia vermicularis (Sw.) Ach. against skin aging in vitro.

Aged skin, featured with dryness and wrinkles, has received mounting attention due to its adverse influences on beauty. ß-Sitosterol and vermicularin are two common active ingredients of Thamnolia vermicularis (Sw.) Ach., a traditional Chinese medicine, of which the anti-aging effect has been discovered. Their protective performance against skin aging was assayed by co-culturing with skin cells in this work. Results showed that ß-sitosterol promoted the biosynthesis of hyaluronic acid by increasing the expression of hyaluronic acid synthases in fibroblasts and enhanced the expression of skin barrier functional proteins including aquaporin 3, loricrin, filaggrin and involucrin in keratinocytes, which conduced to the moisture retention within skin. Moreover, vermicularin might function as an anti-wrinkle agent by preventing the loss of collagen type I. Specifically, vermicularin reduced the amount of reactive oxygen species within hydrogen-peroxide-induced fibroblasts; together with suppressing the activation of mitogen-activated protein kinases, it could inhibit the production of matrix metalloproteinases-1. The present research will contribute to the development of the compounds as anti-aging ingredients for future applications in cosmetic formulations and functional food as well as promote further studies of raw materials containing alike compounds.

The proteomic study of serially passaged human skin fibroblast cells uncovers down-regulation of the chromosome condensin complex proteins involved in replicative senescence.

Dermal fibroblast is one of the major constitutive cells of skin and plays a central role in skin senescence. The replicative senescence of fibroblasts may cause skin aging, bad wound healing, skin diseases and even cancer. In this study, a label-free quantitative proteomic approach was employed to analyzing the serial passaged human skin fibroblast (CCD-1079Sk) cells, resulting in 3371 proteins identified. Of which, 280 proteins were significantly changed in early passage (6 passages, P6), middle passage (12 passages, P12) and late passage (21 passages, P21), with a time-dependent decrease or increase tendency. Bioinformatic analysis demonstrated that the chromosome condensin complex, including structural maintenance of chromosomes protein 2 (SMC2) and structural maintenance of chromosomes protein 4 (SMC4), were down-regulated in the serially passaged fibroblast cells. The qRT-PCR and Western Blot experiments confirmed that the expression of these two proteins were significantly down-regulated in a time-dependent manner in the subculture of human skin fibroblasts (HSFb cells). In summary, we used serially passaged human skin fibroblast cells coupled with quantitative proteomic approach to profile the protein expression pattern in the temporal progress of replicative senescence in HSFb cells and revealed that the down-regulation of the chromosome condensin complex subunits, such as SMC2 and SMC4, may play an important role in the fibroblast senescence.

Synergy of GHK-Cu and hyaluronic acid on collagen IV upregulation via fibroblast and ex-vivo skin tests.

INTRODUCTION: GHK-Cu and HA are two commonly used skin care ingredients, both of which were reported to enhance collagen synthesis. This work aims to investigate their co-effect on collagen regulation. MATERIALS AND METHODS: In cell experiments, human dermal fibroblasts were treated by a series of GHK-Cu and HA combinations, and the expressions of collagen I, IV, and VII were measured by qRT-PCR. The best formula screened out from cell experiments were further studied by ex-vivo skin model, and the content of collagen IV was quantified by immunofluorescence method. RESULTS: The combination GHK-Cu and HA was found to promote the generation of collagen I, IV, and VII. Especially, they form a synergy on collagen IV. At the ratio of 1:9, GHK-Cu and LMW HA deliver the strongest effect to elevate collagen IV synthesis by 25.4 times in cell test and 2.03 times in ex-vivo skin test. CONCLUSION: The co-effect of GHK-Cu and HA was revealed. Their synergy brings an insight to anti-aging technology: choosing proper molecular weight of HA and managing its ratio with GHK-Cu could enhance DEJ health via stimulating collagen IV synthesis.

Serine 363 of the {delta}-opioid receptor is crucial for adopting distinct pathways to activate ERK1/2 in response to stimulation with different ligands.

Distinct opioid receptor agonists have been proved to induce differential patterns of ERK activation, but the underlying mechanisms remain unclear. Here, we report that Ser363 in the δ-opioid receptor (δOR) determines the different abilities of the δOR agonists DPDPE and TIPP to activate ERK by G-protein- or ß-arrestin-dependent pathways. Although both DPDPE and TIPP activated ERK1/2, they showed different temporal, spatial and desensitization patterns of ERK activation. We show that that DPDPE employed G protein as the primary mediator to activate the ERK cascade in an Src-dependent manner, whereas TIPP mainly adopted a ß-arrestin1/2-mediated pathway. Moreover, we found that DPDPE gained the capacity to adopt the ß-arrestin1/2-mediated pathway upon Ser363 mutation, accompanied by the same pattern of ERK activation as that induced by TIPP. Additionally, we found that TIPP- but not DPDPE-activated ERK could phosphorylate G-protein-coupled receptor kinase-2 and ß-arrestin1. However, such functional differences of ERK disappeared with the mutation of Ser363. Therefore, the present study reveals a crucial role for Ser363 in agonist-specific regulation of ERK activation patterns and functions.

Quantitative proteomic analysis uncovers inhibition of melanin synthesis by silk fibroin via MITF/tyrosinase axis in B16 melanoma cells.

AIMS: Silk fibroin (SF), a natural product from silkworms, has been used to promote anti-inflammation, induce wound healing, and reduce melanin production. However, the underlying regulatory mechanism of SF on melanin production remains unknown. The aim of this study was to investigate the distinct regulatory mechanism of SF in B16 melanoma cells by applying quantitative proteomic approach. MATERIALS AND METHODS: B16 melanoma cells were treated with PBS, KA or SF for 48 h, respectively. Cell viability, melanin content, and tyrosinase activity were examined. A label-free quantitative proteomic approach was utilized to investigate the regulatory mechanism of SF. The differentially expressed proteins and their related biological processes were subsequently identified by bioinformatics methods. Furthermore, the identified differentially expressed proteins were validated by western blot. KEY FINDINGS: Both SF and KA were able to suppress the melanin synthesis of B16 melanoma cells without appreciable toxicity; yet, SF had a distinct effect on mushroom tyrosinase activity in vitro. Moreover, quantitative proteomic approach identified 141 proteins differentially expressed only in SF/Con group. Bioinformatic analysis of these proteins revealed that oxidation-reduction process, RNA processing, fatty acid degradation, as well as melanin biosynthetic process were enriched with SF treatment. The proteins associated with melanin biosynthetic process, including microphthalmia-associated transcription factor (MITF) and tyrosinase, were down-regulated in SF group, which was confirmed by western blot. SIGNIFICANCE: SF inhibited melanin synthesis in B16 melanoma cells via down-regulation of MITF and tyrosinase expression, which provides a rationale for future utilization of SF.

Enhancement of anti-acne effect of Scutellaria baicalensis extract by fermentation with symbiotic fungus Penicillium decumbens.

Inflammatory responses stimulated by Propionibacterium acnes have been shown to be major etiological factors in the pathogenesis of acne. Scutellaria baicalensis, a popular traditional Chinese medicine, has been widely shown to have anti-inflammatory effects. In this study, primary component analysis and primary effective component analysis were conducted. The results showed that wogonin (1.15 mg/g S. baicalensis extract) possessed better anti-acne effects than wogonoside (8.71 mg/g S. baicalensis extract) in inhibiting the up-regulation of IL-1ß and IL-8 level caused by P. acnes via inactivation of the MAPK and NF-κB signaling pathways. To enhance the anti-acne effects of S. baicalensis extract, an environmentally friendly and healthy plant fermentation strategy was used to efficiently convert glycoside-type constituents into bioactive aglycone. S. baicalensis extract was fermented by symbiotic fungus Penicillium decumbens f3-1 to transform wogonoside into wogonin with a conversion rate of 91.0% after 4 days. Fermented S. baicalensis extract (FSE) showed higher potential anti-acne effects than non-fermented S. baicalensis extract (NSE) by inhibiting the up-regulation of IL-1ß and IL-8. Thus, P. decumbens-fermented S. baicalensis Extract may be used for developing new anti-acne cosmetic ingredients.

Role of Src in ligand-specific regulation of delta-opioid receptor desensitization and internalization.

The opioid receptors are a member of G protein-coupled receptors that mediate physiological effects of endogenous opioid peptides and structurally distinct opioid alkaloids. Although it is well characterized that there is differential receptor desensitization and internalization properties following activation by distinct agonists, the underlying mechanisms remain elusive. We investigated the signaling events of delta-opioid receptor (deltaOR) initiated by two ligands, DPDPE and TIPP. We found that although both ligands inhibited adenylyl cyclase (AC) and activated ERK1/2, only DPDPE induced desensitization and internalization of the deltaOR. We further found that DPDPE, instead of TIPP, could activate GRK2 by phosphorylating the non-receptor tyrosine kinase Src and translocating it to membrane receptors. Activation of GRK2 led to the phosphorylation of serine residues in the C-terminal tail, which facilitates beta-arrestin1/2 membrane translocation. Meanwhile, we also found that DPDPE promoted beta-arrestin1 dephosphorylation in a Src-dependent manner. Thus, DPDPE appears to strengthen beta-arrestin function by dual regulations: promoting beta-arrestin recruitment and increasing beta-arrestin dephosphorylation at the plasma membrane in a Src-dependent manner. All effects initiated by DPDPE could be abolished or suppressed by PP2, an inhibitor of Src. Morphine, which has been previously shown to be unable to desensitize or internalize deltaOR, also behaved as TIPP in failure to utilize Src to regulate deltaOR signaling. These findings point to the existence of agonist-specific utilization of Src to regulate deltaOR signaling and reveal the molecular events by which Src modulates deltaOR responsiveness.

Comparative effects of schisandrin A, B, and C on Propionibacterium acnes-induced, NLRP3 inflammasome activation-mediated IL-1ß secretion and pyroptosis.

Propionibacterium acnes, a common pathogen associated with acne, is also responsible for various surgical infections. Schisandrin A, schisandrin B and schisandrin C, the representative lignans of Schisandra chinensis (Turcz.) Baill. extract, inhibit P. acnes-induced inflammation. However, their effects on P. acnes-induced IL-1ß secretion and pyroptosis mediated by NLRP3 inflammasome activation remain unknown. In this study, we compared the effects of schisandrin A, B, and C (Sch A, B, and C) on IL-1ß secretion and pyroptosis in P. acnes-infected THP-1 cells. As NLRP3 plays important roles in P. acnes-mediated inflammation and pyroptosis, we also investigated the effects of Schs on P. acnes-induced NLRP3 inflammasome activation by measuring the levels of NLRP3, active caspase-1, and mature IL-1ß, and activity of caspase-1. Our results showed that Sch A, B, and C suppressed P. acnes-induced pyroptosis. Further, the three lignans significantly suppressed NLRP3 inflammasome activation, with the following potency: Sch C > Sch B > Sch A. Three lignans also inhibited the production of mitochondrial ROS and ATP release. Additionally, Sch B and C almost completely prevented the efflux of K+., whereas Sch A had a relatively weak effect. Collectively, our novel findings showed that Sch A, B, and C effectively suppressed IL-1ß secretion and pyroptosis by inhibiting NLRP3 inflammasome activation in P. acnes-infected THP-1 cells. Thus, Schs may be promising agents for the treatment of P. acnes-related infections.

Comparative Effects of Schisandrin A, B, and C on Acne-Related Inflammation.

Inflammatory responses induced by Propionibacterium acnes are a major etiological factor in the pathogenesis of acne vulgaris. Schisandrin A, schisandrin B, and schisandrin C are the representative lignans of Schisandra chinensis (Turcz.) Baill. extract. Although anti-inflammatory effects of the lignans have been shown, their effects on acne-related inflammation caused by P. acnes have not been investigated and compared. We pretreated THP-1 human monocytic cells with 5, 10, and 20 µM schisandrin A, B, and C, and stimulated the cells with P. acnes. Schisandrin B and C inhibited the release of inflammatory cytokines at a concentration of 5 µM, while schisandrin A required a concentration of 10 µM to exert the effects. All of the schisandrins decreased the levels of toll-like receptor 2, and schisandrin B and C reduced the intracellular mRNA expression of the receptor gene. We also studied the influence of schisandrins on the MAPK signaling pathway. Schisandrin A suppressed the P. acnes-induced activation of JNK, while exerting only a weak effect on ERK and p38. Schisandrin B exerted a strong effect on p38, a lesser effect on ERK, and almost no effect on JNK. Schisandrin C inhibited the phosphorylation of all three proteins, especially ERK. Furthermore, the three lignans also prevented the nuclear translocation of NF-κB. These results contribute to our understanding of the mechanisms underlying the effects of the three lignans on P. acnes-induced inflammation and suggest that schisandrins might be developed as pharmacological agents for acne therapy.

Role for engagement of ß-arrestin2 by the transactivated EGFR in agonist-specific regulation of δ receptor activation of ERK1/2.

BACKGROUND AND PURPOSE: ß-Arrestins function as signal transducers linking GPCRs to ERK1/2 signalling either by scaffolding members of ERK1/2s cascades or by transactivating receptor tyrosine kinases through Src-mediated release of transactivating factor. Recruitment of ß-arrestins to the activated GPCRs is required for ERK1/2 activation. Our previous studies showed that δ receptors activate ERK1/2 through a ß-arrestin-dependent mechanism without inducing ß-arrestin binding to the δ receptors. However, the precise mechanisms involved remain to be established. EXPERIMENTAL APPROACH: ERK1/2 activation by δ receptor ligands was assessed using HEK293 cells in vitro and male Sprague Dawley rats in vivo. Immunoprecipitation, immunoblotting, siRNA transfection, intracerebroventricular injection and immunohistochemistry were used to elucidate the underlying mechanism. KEY RESULTS: We identified a new signalling pathway in which recruitment of ß-arrestin2 to the EGFR rather than δ receptor was required for its role in δ receptor-mediated ERK1/2 activation in response to H-Tyr-Tic-Phe-Phe-OH (TIPP) or morphine stimulation. Stimulation of the δ receptor with ligands leads to the phosphorylation of PKCδ, which acts upstream of EGFR transactivation and is needed for the release of the EGFR-activating factor, whereas ß-arrestin2 was found to act downstream of the EGFR transactivation. Moreover, we demonstrated that coupling of the PKCδ/EGFR/ß-arrestin2 transactivation pathway to δ receptor-mediated ERK1/2 activation was ligand-specific and the Ser(363) of δ receptors was crucial for ligand-specific implementation of this ERK1/2 activation pathway. CONCLUSIONS AND IMPLICATIONS: The δ receptor-mediated activation of ERK1/2 is via ligand-specific transactivation of EGFR. This study adds new insights into the mechanism by which δ receptors activate ERK1/2.

Discovery of fruquintinib, a potent and highly selective small molecule inhibitor of VEGFR 1, 2, 3 tyrosine kinases for cancer therapy.

VEGF/VEGFR signal axis has been proven to be an important target for development of novel cancer therapies. One challenging aspect in small molecular VEGFR inhibitors is to achieve sustained target inhibition at tolerable doses previously seen only with the long-acting biologics. It would require high potency (low effective drug concentrations) and sufficient drug exposures at tolerated doses so that the drug concentration can be maintained above effective drug concentration for target inhibition within the dosing period. Fruquintinib (HMPL-013) is a small molecule inhibitor with strong potency and high selectivity against VEGFR family currently in Phase II clinical studies. Analysis of Phase I pharmacokinetic data revealed that at the maximum tolerated dose of once daily oral administration fruquintinib achieved complete VEGFR2 suppression (drug concentrations were maintained above that required to produce >85% inhibition of VEGFR2 phosphorylation in mouse) for 24 hours/day. In this article, the preclinical data for fruquintinib will be described, including kinase enzyme activity and selectivity, cellular VEGFR inhibition and VEGFR-driven functional activity, in vivo VEGFR phosphorylation inhibition in the lung tissue in mouse and tumor growth inhibition in a panel of tumor xenograft and patient derive xenograft models in mouse. Pharmacokinetic and target inhibition data are also presented to provide a correlation between target inhibition and tumor growth inhibition.

Chronic high-dose morphine treatment promotes SH-SY5Y cell apoptosis via c-Jun N-terminal kinase-mediated activation of mitochondria-dependent pathway.

Chronic high doses of morphine inhibit the growth of various human cancer cell lines. However, the mechanisms by which such high-dose morphine inhibits cell proliferation and induces cell death are not fully understood. Here we show that c-Jun N-terminal kinase (JNK) plays a pivotal role in high-dose morphine-induced apoptosis of SH-SY5Y cells in a mitochondria-dependent manner. Activation of JNK by morphine led to reactive oxygen species (ROS) generation via the mitochondrial permeability transition pore, because the mPTP inhibitor cyclosporin A significantly inhibited ROS generation. ROS in turn exerted feedback regulation on JNK activation, as shown by the observations that cyclosporin A and the antioxidant N-acetylcysteine significantly inhibited the phosphorylation of JNK induced by morphine. ROS-amplified JNK induced cytochrome c release and caspase-9/3 activation through enhancement of expression of the proapoptotic protein Bim and reduction of expression of the antiapoptotic protein Bcl-2. All of these effects of morphine could be suppressed by the JNK inhibitor SP600125 and N-acetylcysteine. The key role of the JNK pathway in morphine-induced apoptosis was further confirmed by the observation that decreased levels of JNK in cells transfected with specific small interfering RNA resulted in resistance to the proapoptotic effect of morphine. Thus, the present study clearly shows that morphine-induced apoptosis in SH-SY5Y cells involves JNK-dependent activation of the mitochondrial death pathway, and that ROS signaling exerts positive feedback regulation of JNK activity.