Human HaCaT Cells Research Articles

Comparison of the in vitro and in vivo toxic effects of thymoquinone using oral cancer HSC-3 and HSC-4 cell lines, oral fibroblasts, HACAT cell line, and Zebrafish embryos

Abstract The discovery and development of anticancer agents were made through long processes from screening new compounds in various cell lines, animal experimental studies, until clinical trials Majority of cancer chemotherapeutic agents have been initially screened in an in vitro cell-based system and then subsequently studied in an in vivo model using small animals destined for pharmacokinetic properties. Zebrafish (Danio rerio) has become one of the most popular animal model systems used for chemical toxicity tests and drug discovery. Thymoquinone (TQ) is the main constituent of black seed (Nigella sativa, spp) essential oil which shows anti-neoplastic activities in various tumor cells. However, to date, studies on the toxicity of TQ on normal cells or animal model are still insufficient. Here we report that TQ is selective toxic to different human cell lines and show negligible effect on the Zebrafish. The MTT assay demonstrated that TQ induced cytotoxicity on oral cancer cells (HSC-3 and HSC-4) but not on the normal cells (human oral fibroblast and HACAT cells). Moreover, no significant morphological change was observed in the Zebrafish model following TQ exposure at the concentration (0.003 mg/mL), similar to those in the cancer cells. The study suggests that TQ as a cancer chemotherapeutic candidate without apparent adverse effects.

Cytotoxic activity of Rauvolfia tetraphylla L. on human cervical cancer (HeLa) cells

Background: Although the plant Rauvolfia tetraphylla is used for the treatment of cancer in the traditional medicine of some regions of Mexico, its cytotoxic activity has not been subjected to rigorous investigation. Objective: The aim of the present study was to carry out a bioassay-guided fractionation of Rauvolfia tetraphylla leaves to evaluate the activity on cervical cancer (HeLa) and normal cells. Materials and Methods: Of hexane, dichloromethane and methanol extracts, hexane extract showed the most cytotoxic activity. Its most active fraction was characterized by mass spectrometry and assessed for cytotoxicity (by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assays and trypan blue staining) on cervical cancer (HeLa) cells, normal human (HaCat) cells, and other cancer cell lines: A549, Caco-2, MDA-MB-231, and MCF7. Cisplatin served as the reference drug. Apoptosis was explored as a possible mechanism of action by examining DNA fragmentation and phosphatidylserine translocation. Results: The active fraction, composed of at least 5 constituents, was cytotoxic to all cancer cell lines, especially HeLa cells (IC30= 20.10 ± 1.1 μg/mL). To a lesser extent, it was also cytotoxic to normal cells (HaCat; IC30= 40.04 ± 12.78 μg/mL), indicating certain selectivity. Its mechanism of action in HeLa cells involved apoptosis. Conclusion: The active fraction of the hexane extract of R. tetraphylla was cytotoxic to cancer cells and did not produce excessive damage to normal cells. Apoptosis was apparently part of the mechanism of cytotoxicity.

The Antioxidant and Anti-aging Effects of Treatment with Schisandra chinensis Seeds Fractions in UVB-irradiated Human HaCaT Cells

The Antioxidant and Anti-aging Effects of Treatment with Schisandra chinensis Seeds Fractions in UVB-irradiated Human HaCaT Cells

Chlorpyrifos stimulates expression of vitamin D3 receptor in skin cells irradiated with UVB

Skin, the organ responsible for vitamin D synthesis, is fully exposed to many xenobiotics, e.g. polycyclic aromatic hydrocarbons and pesticides. A broad spectrum organophosphorus insecticides (OP's), such as chlorpyrifos (CPS), are commonly used in agriculture and to control domestic insects. Thus, the aim of this study was to investigate the effect of chlorpyrifos, on the expression of vitamin D3 receptor (VDR) in human keratinocytes cell line HaCaT and fibroblasts cell line BJ. The impact of CPS and UVB radiation on cell viability were examined by Neutral Red assay. The effect of CPS on VDR expression was evaluated by RT-qPCR and flow cytometry (FC). The presented study demonstrated that exposure to CPS and UVB significantly affects the viability of HaCaT and BJ cells lines. Results also revealed that exposure to CPS induced the expression at mRNA and protein level of VDR nuclear receptor in both cell lines exposed to UVB. In HaCaT incubated with 250 μM CPS and 15 mJ/cm2 UVB, the relative VDR expression was ∼2-fold higher; whereas in BJ incubated with 250 μM CPS and 20 mJ/cm2, UVB was∼3-fold higher. Results from FC confirmed this result, as VDR expression increased by ~250% in HaCaT incubated with 250 μM CPS and 20 mJ/cm2 UVB, and in BJ incubated with 250 μM CPS, and 20 mJ/cm2 UVB cells VDR expression increased by ~190%, compared with control. It can therefore be concluded that OPs pesticide might interfere with vitamin D3 metabolism in skin cells.

Schisandrin B protects human keratinocyte-derived HaCaTcells from tert-butyl hydroperoxide-induced oxidative damage through activating the Nrf2 signaling pathway.

Schisandrin B (Sch B), an active extract of Schisandrachinensis, has demonstrated antioxidant activity in a number of invitro and invivo models. In the present study, the capacity of Sch B to protect against oxidative injury in keratinocytes using the human keratinocyte‑derived HaCaT cell line was investigated. To induce oxidative injury, tert‑Butyl hydroperoxide (tBHP) was employed. The results indicate that Sch B efficiently reduced tBHP‑induced cell death, reactive oxygen species (ROS) generation, protein oxidation, lipid peroxidation and DNA damage. Sch B also effectively attenuated the loss of mitochondrial membrane potential (MMP), and restored adenosine triphosphate (ATP) levels in tBHP‑injured HaCaT cells. Furthermore, Sch B enhanced the expression of key antioxidant enzymes, including catalase, heme oxygenase‑1, glutathione peroxidase, and superoxide dismutase, and further engaged the nuclear factor‑erythroid 2‑related factor 2 (Nrf2) signaling pathway by modulating its phosphorylation through activating multiple upstream kinases, including protein kinase B, adenosine monophosphate‑activated protein kinase and mitogen‑activated protein kinases (MAPKs). The present study suggests that Sch B provides a protective effect in keratinocytes in response to oxidative injury via reinforcing the endogenous antioxidant defense system. Therefore, it may be applied as an adjuvant therapy or in health foods to delay the skin aging process and the onset of skin diseases caused by oxidative stress.

BLIMP1 transcriptionally induced by EGFR activation and post-translationally regulated by proteasome and lysosome is involved in keratinocyte differentiation, migration and inflammation

BackgroundB lymphocyte-induced maturation protein-1 (BLIMP1) is a transcriptional repressor, and plays a crucial role in the regulation of development and functions of various immune cells. Currently, there is limited understanding about the regulation of BLIMP1 expression in keratinocytes and crosstalk between EGFR and BLIMP1 in skin homeostasis. ObjectiveThe aim of the study was to investigate the regulation and functional link between EGFR and BLIMP1 in human epidermal keratinocytes. MethodsImmunoblotting and Q-PCR were used to determine the molecular mechanism of BLIMP1 expression induced by EGFR in primary human epidermal keratinocytes (NHEK) and HaCaT cells. In functional assay, effects of BLIMP1 knockdown on EGFR-mediated cytokine production, differentiation, and migration in NHEK were evaluated by Q-PCR, ELISA, immunoblotting, and/or wound-healing assay. ResultsEGFR activation by EGFR ligands could upregulate the protein and mRNA levels of BLIMP1 in NHEK and HaCaT cells. This effect was dependent on PKC, p38, and ERK activation. Additionally, the stability of BLIMP1 protein was under the control of the proteasome and lysosome degradation systems. EGF could also upregulate BLIMP1 expression in skin squamous cell carcinomas. In addition, BLIMP1 knockdown enhanced the EGFR-mediated IL8, CXCL5 and IL6 gene expression and keratinocyte migration, but reduced the EGFR-mediated suppression of differentiation marker K10. ConclusionsOur findings shed new insights into the regulation of BLIMP1 expression by EGFR-mediated gene transcription and proteasome/lysosome-mediated degradation in keratinocytes. Functionally, BLIMP1 is a negative regulator of EGF-induced inflammation and migration in keratinocytes, and exerts a gene-specific regulation on keratinocyte differentiation.

Effects of a ceramide-containing water-in-oil ointment on skin barrier function and allergen penetration in an IL-31 treated 3D model of the disrupted skin barrier.

Atopic dermatitis (AD) is a chronically relapsing, pruritic inflammation of the skin with dryness and disturbed skin barrier function. Recently, we established that IL-31 treatment of human 3D skin models resulted in a disrupted skin barrier phenotype resembling AD. In this model, we found that IL-31 interferes with the differentiation of keratinocytes and inhibits the expression of terminal differentiation markers. In the present study, we investigated the effects of a ceramide-containing water-in-oil skin care ointment on the physical skin barrier structure and function in disrupted skin barrier models, generated either by using primary normal human epidermal keratinocytes (NHEK) or HaCaT cells. We observed that the physical skin barrier of the models recovered after daily topical treatment with the ceramide-containing ointment. Topical application of the ointment prevented downregulation of filaggrin and disorganization of other differentiation markers, such as keratin 10 and β4-integrin, as demonstrated by immunohistological analysis. The expression of Ki67 was also upregulated in response to the ointment. Furthermore, functional studies revealed that local application of the ointment diminished the increased uptake of fluorescently labelled recombinant allergens of timothy grass (phl p1) in our model. In conclusion, our data revealed that topical application of a ceramide-containing skin care ointment reduced IL-31 induced impairments of the physical skin barrier and skin barrier function in an in vitro model of the disrupted skin barrier. This standardized model can be utilized in the future to monitor ex vivo effects of various topical therapies on skin morphology, physiology, and gene expression.

Omega-7 inhibits inflammation and promotes collagen synthesis through SIRT1 activation

Excessive accumulation of reactive oxygen species (ROS) during oxidative stress accelerates the skin aging process. ROS stimulate inflammatory processes in the skin, leading to activation of matrix metalloprotease-1 (MMP-1). Silent information regulator 1 (SIRT1) controls a broad range of cellular functions including the expression of MMP-1. Omega-7 fatty acids such as palmitoleic acid have many beneficial effects on health, including improvement in cardiovascular risk factors and increased insulin sensitivity. However, the effectiveness of omega-7 fatty acids (herein referred to as omega-7) related to skin aging, characterized by the degradation of collagen and loss of elasticity, remains unclear. We here investigated the effects of palmitoleic acid, a representative omega-7, on collagen regeneration through its ability to activate SIRT1 and inhibit MMP-1 in the presence of hydrogen peroxide (H2O2)-induced oxidative stress in human HaCaT cells. SIRT1 activation by omega-7 decreased signaling levels of nuclear transcription factor kappa B (NF-κB) and inflammatory cytokines. However, inhibition of SIRT1 by sirtinol counteracted the advantage effects of omega-7 in H2O2-treated HaCaT cells. In addition, omega-7 significantly counteracted the decrease in collagen abundance and loss of elasticity induced by H2O2. Consistent with this observation, omega-7 significantly decreased H2O2-induced upregulation of MMP-1 in HaCaT cells. Together, these studies suggest the potential efficacy of SIRT1 in collagen regeneration and indicate that omega-7 is a possible functional food to improve skin health for the prevention of aging.

E3 Ligase Trim21 Ubiquitylates and Stabilizes Keratin 17 to Induce STAT3 Activation in Psoriasis

Keratin 17 (K17), a marker of keratinocyte hyperproliferation, is a type I intermediate filament that is overexpressed in psoriatic epidermis and plays a critical pathogenic role by stimulating T cells. However, the posttranslational modification of K17, which is reversible and targetable, has not been elucidated. Herein, we reported that K17 could be modified through ubiquitination that controlled its stability and led to the phosphorylation and nuclear translocation of its interactor signal transducers and activators of transcription 3 (STAT3), which is a key regulator of cell proliferation in psoriasis. First, we stimulated human keratinocyte cell line HaCaT cells with psoriasis (pso)-mix, which is a cytokine pool (IL-17, IL-22, tumor necrosis factor-α, and IFN-γ) mimicking the invitro "psoriasis-like" status and found that the ubiquitination of K17 was essential tostabilize its protein expression in pso-mix-treated HaCaT cells. Subsequently, tripartite motif-containing protein 21 was identified as the E3 ligase of K17, which ubiquitylated K17 via K63 linkage to maintain K17 stabilization. More importantly, we uncovered that K17 was a direct interactor of STAT3, and K17 ubiquitination could promote STAT3 activation in pso-mix-treated HaCaT cells. Our study demonstrated that targeting K17 ubiquitination may be a potential therapeutic approach by attenuating STAT3 signaling in psoriasis.

Bone Marrow-Derived Mesenchymal Stem Cells Promoted Cutaneous Wound Healing by Regulating Keratinocyte Migration via β2-Adrenergic Receptor Signaling.

Mesenchymal stem cells (MSCs) play an important role in cutaneous wound healing; however, the functional mechanisms involved in the healing process are poorly understood. A series of studies indicate that keratinocytes that migrate into the wound bed rely on an epithelial-mesenchymal transition (EMT)-like process to initiate re-epithelialization. We therefore examined whether bone marrow-derived MSCs (BMSCs) could affect biological behavior and induce EMT-like characteristics in the human epidermal keratinocytes (HEKs) and in the immortalized human keratinocyte cell line HaCaT cells, and we investigated the signaling pathways of BMSC-mediated phenotypic changes. By assessing the expression of EMT-related markers including E-cadherin, α-SMA, and Snail family transcription factors by β2-adrenergic receptor (β2-AR) blockage using ICI-118,551, a β2-AR selective antagonist, or β2-AR small interfering RNA (siRNA), we showed an involvement of β2-AR signaling in the induction of EMT-like alterations in human keratinocytes in vitro. β2-AR signaling also affected collective and individual cell migration in human keratinocyte cell lines, which was attenuated by administration of ICI-118,551. Treating the cells with BMSC-conditioned media (BMSC-CM) not only recapitulated the effect of isoproterenol (ISO) on cell migration but also induced the expression of β2-AR and a panel of proteins associated with mesenchymal phenotype in HEKs and HaCaT cells. Similarly, a blockade of the β2-AR by either ICI-118,551 or β2-AR siRNAs reversed both responses of the epidermal keratinocyte cell lines relative to BMSC-CM exposure. These results were further verified in our vivo findings and indicated that the exogenous application of MSCs promoted cutaneous wound healing and endowed the keratinocytes surrounding the wound area with an increased migratory phenotype through activation of β2-AR signaling. Our findings suggest a biochemical mechanism underlying the function of MSCs in wound re-epithelization, which provides a reliable theoretical basis for the wide application of MSCs in the treatment of chronic wounds.

PSORI-CM02 Formula Increases CD4+ Foxp3+ Regulatory T Cell Frequency and Ameliorates Imiquimod-Induced Psoriasis in Mice.

Psoriasis is an autoimmune and inflammatory disease, which is estimated to affect 2–3% of the population in the world. PSORI-CM02 is an empirical formula of Chinese medicine optimized from Yin Xie Ling, which is widely used to treat psoriasis in China for decades. However, its antipsoriatic mechanisms are still not well understood. Here, we explored the therapeutic effects of PSORI-CM02 on psoriasis and its mechanisms of action in imiquimod-induced psoriasis-like mouse models and human HaCaT cells. In experiments in vitro, PSORI-CM02 significantly inhibited HaCaT cell proliferation in dose-dependent and time-dependent manners. Furthermore, it hindered the progression of HaCaT cell cycle and arrested HaCaT cells at G1 phase. On the other hand, our in vivo studies demonstrated that PSORI-CM02 dramatically reduced psoriasis area and severity index scores and lesion temperature in imiquimod-induced psoriatic mice. The antioxidative activities of glutathione, catalase, and superoxide dismutase were increased while oxidative activity of malonaldehyde was markedly decreased after treatments with PSORI-CM02. PSORI-CM02 also suppressed the mRNA expression of proinflammatory cytokines, including TNF-α, IL-6, and IL-17, and lowered their protein levels in the serum as well. In addition, PSORI-CM02 could reduce the expression of IKKα and NF-κB in psoriatic skin tissue. It also upregulated the proportion of CD4+ Foxp3+ regulatory T cells (Tregs) in both lymph nodes and spleens and promoted CD4+ CD25+ Treg proliferation in vitro. Taken together, our research demonstrated that PSORI-CM02 inhibited HaCaT cell proliferation by arresting them at G1 phase and alleviated systemic inflammation and psoriasis in mice via altering the oxidative/anti-oxidative status, tipping the balance between Th17 responsiveness and CD4+ Foxp3+ Treg generation, and suppressing the expression of proinflammatory cytokines as well as NF-κB signaling.

MiR-124 inhibits proliferation, migration and invasion of malignant melanoma cells via targeting versican.

MicroRNA (miR)-124 has been implicated in malignant melanoma (MM). However, the detailed regulatory mechanism of miR-124 in the malignant phenotypes of MM cells has remained largely elusive. A total of 68 pairs of MM tissues and adjacent tissues were collected. Reverse-transcription quantitative polymerase chain reaction was used to examine the mRNA expression of versican as well as the expression of miR-124, and the protein expression of versican was assessed by western blot analysis. MTT, wound healing and Transwell assays were used to determine cell proliferation, migration and invasion, respectively. A bioinformatics analysis and a luciferase reporter assay were used to confirm the targeting association between miR-124 and versican. miR-124 was significantly downregulated in MM tissues compared with that in adjacent non-tumorous tissues, and decreased expression of miR-124 was associated with increased tumor thickness, advanced clinical stage and node metastasis of MM. Furthermore, the expression levels of miR-124 were also reduced in MM cell lines compared with normal human skin HACAT cells. Forced overexpression of miR-124 caused a significant reduction in the proliferation, migration and invasion of MM A375 cells. Versican was significantly upregulated in MM tissues and cell lines, and was identified as a novel target of miR-124 in A375 cells using a luciferase reporter gene assay, and miR-124 was revealed to negatively regulate the protein expression of versican in A375 cells. Overexpression of versican impaired the suppressive effects of miR-124 on the proliferation, migration and invasion of A375 cells. In conclusion, miR-124 inhibited the malignant phenotypes of MM cells at least partly via inhibition of versican. Therefore, the miR-124/versican axis may be used as a promising therapeutic target for inhibiting MM growth and metastasis.

Possible role of PAPR-1 in protecting human HaCaT cells against cytotoxicity of SiO2 nanoparticles

Nano-SiO2 materials play a significant role in the engineered nanomaterials (ENMs) field. The ease of their production as well as their relatively low cost has promoted the wide use of these products in many fields. Nano-SiO2 exposure is known to cause severe DNA damage; however, the underlying mechanisms remain poorly understood. In a previous study, we found that nano-SiO2 exposure regulate the expression of the poly(ADP-ribose) polymerases-1 (PARP-1), a pivotal DNA repair gene, in human HaCaT cells. Here, we employed lentivirus-mediated RNA interference (RNAi) to knock down PAPR-1 expression in HaCaT cells and explored the potential role of PARP-1 in nano-SiO2 induced cytotoxicity. We found that nano-SiO2 treatment of HaCaT cells causes decreased cell viability, increased apoptosis and DNA damage. Nano-SiO2-treated HaCaT cells were also found to have slightly changed cell cycle distribution. Lentivirus-mediated PAPR-1 knockdown partially aggravated cytotoxicity and increased apoptosis induced by nano-SiO2 treatment. Nano-SiO2 had significant toxicity to human HaCaT cells and causes DNA damage. PAPR-1 knock-down cell line appears more sensitive to nano-SiO2 than the control cells in DNA damage. The results suggest that PAPR-1 is involved in protecting cells from damage caused by nano-SiO2.

Effects of fermented black ginseng on wound healing mediated by angiogenesis through the mitogen-activated protein kinase pathway in human umbilical vein endothelial cells

BackgroundFermented black ginseng (FBG) is produced through several cycles of steam treatment of raw ginseng, at which point its color turns black. During this process, the original ginsenoside components of raw ginseng (e.g., Re, Rg1, Rb1, Rc, and Rb2) are altered, and less-polar ginsenosides are generated (e.g., Rg3, Rg5, Rk1, and Rh4). The aim of this study was to determine the effect of FBG on wound healing. MethodsThe effects of FBG on tube formation and on scratch wound healing were measured using human umbilical vein endothelial cells (HUVECs) and HaCaT cells, respectively. Protein phosphorylation of mitogen-activated protein kinase was evaluated via Western blotting. Finally, the wound-healing effects of FBG were assessed using an experimental cutaneous wounds model in mice. Results and ConclusionThe results showed that FBG enhanced the tube formation in HUVECs and migration in HaCaT cells. Western blot analysis revealed that FBG stimulated the phosphorylation of p38 and extracellular signal-regulated kinase in HaCaT cells. Moreover, mice treated with 25 μg/mL of FBG exhibited faster wound closure than the control mice did in the experimental cutaneous wounds model in mice.

House Dust Mite Increases pro-Th2 Cytokines IL-25 and IL-33 via the Activation of TLR1/6 Signaling

House dust mites have been implicated in the etiology and exacerbation of atopic dermatitis. Diverse factors contribute to house dust mite allergenicity through the activation of innate immunity. We investigated whether Dermatophagoides farinae extract (DFE) allergens mediate innate immune activation through specific toll-like receptors (TLRs) in epidermal keratinocytes, a DFE-induced murine atopic dermatitis model, and human atopic dermatitis lesions. DFE activated the expression of TLR1, TLR6, IL-25, and IL-33 in human primary keratinocytes and HaCaT cells. Knockdown of TLR6 inhibited DFE-induced upregulation of IL-25 or IL-33. In addition, the suppression of TLR1 inhibited the release of IL-33. DFE induced the expression of IL-25 and IL-33 by upregulation of IL-1 receptor-associated kinase 1, transforming growth factor-β activated kinase-1, IκB kinase, and NF-κB pathways. Tlr6-/- mice did not show DFE-induced upregulation of IL-25 and IL-33. Furthermore, DFE-induced upregulation of IL-25 was not induced in Tlr1-/- mice. We also identified upregulated mRNA and protein expression of TLR1, TLR6, IL-25, and IL-33 in human atopic dermatitis skin lesions with high house dust mite sensitization. We found that DFE-induced activation of TLR1 and TLR6 may cause polarization toward a T helper type 2 immune response via the release of IL-25 and IL-33.

MicroRNA-9 inhibits the proliferation and migration of malignant melanoma cells via targeting sirituin 1.

MicroRNA (miR) are a class of small non-coding RNA that are able to inhibit gene expression by directly binding to the 3′ untranslated region (UTR) of their target mRNA and thus promote translational repression or mRNA degradation. Recently, miR-9 was reported to have a suppressive role in malignant melanoma; however, the underlying mechanism remains largely unclear. In the present study, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to examine the mRNA and protein expression levels in malignant melanoma tissues and cell lines. The MTT assay and wound healing assay were used to examine the cell viability, proliferation and migratory capacities. Bioinformatics prediction and luciferase reporter assay were performed to investigate the relationship between miR-9 and its potential target gene. The present data revealed that miR-9 expression was significantly downregulated in malignant melanoma tissues when compared with their matched adjacent non-tumor tissues. Furthermore, the expression levels of miR-9 were reduced in malignant melanoma cell lines when compared with human normal skin HACAT cells. Moreover, the ectopic expression of miR-9 significantly suppressed the proliferation and migration of malignant melanoma cells, accompanied with a remarkable decrease in the protein expression levels of sirtuin 1 (SIRT1), which were markedly upregulated in malignant melanoma tissues and cell lines. Additionally, restoration of SIRT1 reversed the suppressive effects of miR-9 on the proliferation and migration of malignant melanoma cells. Luciferase reporter assay data further identified SIRT1 as a direct target gene of miR-9. To conclude, the present findings indicate that miR-9 has a suppressive role in malignant melanoma cell viability and migration, at least in part, via directly inhibiting the protein expression of its target gene, SIRT1. Therefore, miR-9 may serve as a potential candidate for the treatment of malignant melanoma.

Performance of a High-Pressure Liquid Chromatography-Ion Mobility-Mass Spectrometry System for Metabolic Profiling.

A commercial liquid chromatography/drift tube ion mobility-mass spectrometer (LC/IM-MS) was evaluated for its utility in global metabolomics analysis. Performance was assessed using 12 targeted metabolite standards where the limit of detection (LOD), linear dynamic range, resolving power, and collision cross section (Ω) are reported for each standard. Data were collected in three different instrument operation modes: flow injection analysis with IM-MS (FIA/IM-MS), LC/MS, and LC/IM-MS. Metabolomics analyses of human plasma and HaCaT cells were used to compare the above three operation modes. LC/MS provides linearity in response, data processing automation, improved limits of detection, and ease of use. Advantages of LC/IM-MS and FIA/IM-MS include the ability to develop mobility-mass trend lines for structurally similar biomolecules, increased peak capacity, reduction of chemical/matrix noise, improvement in signal-to-noise, and separations of isobar/isomer compounds that are not resolved by LC. We further tested the feasibility of incorporating IM-MS into conventional LC/MS metabolomics workflows. In general, the addition of ion mobility dimension has increased the separation of compounds in complex biological matrixes and has the potential to largely improve the throughput of metabolomics analysis.

Porphyra-334, a mycosporine-like amino acid, attenuates UV-induced apoptosis in HaCaT cells.

The main aim of the current research was to study the effect of porphyra-334, one of mycosporine-like amino acids (MAAs), well known as UV-absorbing compounds, on UVinduced apoptosis in human immortalized keratinocyte (HaCaT) cells. Due to their UV-screening capacity and ability to prevent UV-induced DNA damage, MAAs have recently attracted considerable attention in both industry and research in pharmacology. Herein, human HaCaT cells were used to determine the biological activities of porphyra- 334 by various in vitro assays, including proliferation, apoptosis and Western blot assays. The proliferation rate of UV-irradiated HaCaT cells was significantly decreased compared to the control group. Pretreatment with porphyra- 334 markedly attenuated the inhibitory effect of UV and induced a dramatic decrease in the apoptotic rate. Expression of active caspase-3 protein was increased in response to UV irradiation, while caspase-3 levels were similar between cells treated with porphyra-334 and the non-irradiated control group. Taken together, our data suggest that porphyra-334 inhibits UV-induced apoptosis in HaCaT cells through attenuation of the caspase pathway.

The effect of dehydroglyasperin C on UVB-mediated MMPs expression in human HaCaT cells.

The ultraviolet B (UVB) from solar radiation increases the generation of reactive oxygen species (ROS), which mediate the production of matrix metalloproteinases (MMPs), and acts mainly on the epidermis layer of the skin. This study was aimed at assessing the anti-photoaging effects of dehydroglyasperin C isolated from Glycyrrhiza uralensis Fisch on MMPs levels in HaCaT human keratinocytes and to elucidate the underlying mechanism. The cell viability was measured by MTT assay. Expression, phosphorylation and enzymatic activity of the protein were examined using ELISA, Western blot or gelatin zymography. Intracellular ROS measurement was evaluated by fluorescent ELISA and 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) assay. In the present study, we found that dehydroglyasperin C markedly inhibited UVB-mediated expression of collagenase (MMP-1) and gelatinase (MMP-9) by inhibiting ROS generation. Dehydroglyasperin C treatment also decreased the UVB irradiation-mediated activation of mitogen-activated protein kinase (MAPK), c-Jun phosphorylation, and c-Fos expression. In addition, the down-regulation of UVB-induced c-Jun phosphorylation caused by dehydroglyasperin C treatment was more than the down-regulation of c-Fos expression in the HaCaT human keratinocytes. Our results indicated that dehydroglyasperin C may function as a potential anti-photoaging agent by inhibiting UVB-mediated MMPs expression via suppression of MAPK and AP-1 signaling.

Analysis of PM2.5-induced cytotoxicity in human HaCaT cells based on a microfluidic system

Human exposure to PM2.5 causes several adverse health effects. Skin is the first barrier against harmful environmental substances and can directly contact with PM2.5, but there is no study about PM2.5-induced cytotoxicity in human skin cells on the molecular level partially due to the shortcomings of traditional research methods. In present study, we established a microfluidic system including a cell culture chip integrated with a high-throughput protein microarray chip to investigate the mechanism of PM2.5-mediated cytotoxicity in human HaCaT cells. We found that PM2.5 was lodged inside the cytoplasm, mitochondria and nucleus of HaCaT cells by TEM. Flow cytometry analysis indicated that the cell apoptosis rate increased from 0.49% to 53.4%. The results of protein microarray showed that NF-κB and NALP3 signal transductions were activated in HaCaT cells after PM2.5 stimulations, up-regulating the expression of IL-1β and IL-6, which resulted in inflammatory response in HaCaT cells. Our findings provide a molecular insight into PM2.5-induced skin injury.