Phytochemical profile and biological activities of Bromelia antiacantha extracts / Perfil fitoquímico e atividades biológicas de extratos de Bromelia antiacantha

Reports from popular medicine usually act as a basis for the development of new drugs from natural compounds with therapeutic actions for serious diseases and prevalence such as cancer. Bromelia antiacantha Bertol. is a species of the Bromeliaceae family, considered an unconventional food plant, found in the south and midwest regions of Brazil. Despite the high nutritional content and pharmacological potential of its fruits, few scientific studies report its biological actions. Thus, this study evaluates the phytochemical profile of aqueous and ethanol extracts obtained from B. antiacantha fruits, as well as their possible antioxidant, antitumor, and cytotoxic activities. The aqueous extract exhibited phenolic compounds and flavonoids, while ethanol extracts indicated the presence of flavonoids and coumarin in their composition, regardless of the region of collection. The ethanolic extract demonstrated a more promising antioxidant effect than the aqueous extract and also induced a significant inhibition in the viability of human cervical cancer cells of the SiHa strain. In addition, treatment with both extracts did not alter the viability of non-tumor cells of the immortalized human keratinocyte lineage (HaCaT). These results bring new data about extracts obtained from a native plant, edible and traditionally used in popular medicine, opening new perspectives for its possible therapeutic application.

Relatos da medicina popular costumam atuar como referencial para o desenvolvimento de novos fármacos a partir de moléculas naturais com ações terapêuticas para doenças de alta gravidade e prevalência como o câncer. Bromelia antiacantha Bertol. é uma espécie da família Bromeliaceae, considerada uma planta alimentícia não convencional (PANC), encontrada nas regiões sul e centro-oeste do Brasil. Apesar do alto teor nutritivo e potencial farmacológico de seus frutos, poucos estudos científicos relatam suas ações biológicas. Desta forma, este estudo avalia o perfil fitoquímico de extratos aquoso e etanólico obtidos de frutos de B. antiacantha, bem como a sua possível ação antioxidante, antitumoral e citotóxica. O extrato aquoso apresentou compostos fenólicos e flavonoides, enquanto os extratos etanólicos apontam a presença de flavonóides e cumarina em sua composição, independente da região de coleta. O extrato etanólico demonstrou efeito antioxidante mais promissor do que o extrato aquoso e também induziu uma inibição significativa na viabilidade de células humanas de câncer cervical da linhagem SiHa. Além disso, o tratamento com ambos extratos não alterou a viabilidade de células não tumorais da linhagem de queratinócitos humanos imortalizados (HaCaT). Estes dados trazem novas informações sobre extratos obtidos de uma espécie vegetal nativa, comestível e já utilizada tradicionalmente, mas abrindo novas perspectivas quanto a possíveis aplicações terapêuticas.

Phytochemical profile and biological activities of Bromelia antiacantha extracts

Abstract Reports from popular medicine usually act as a basis for the development of new drugs from natural compounds with therapeutic actions for serious diseases and prevalence such as cancer. Bromelia antiacantha Bertol. is a species of the Bromeliaceae family, considered an unconventional food plant, found in the south and midwest regions of Brazil. Despite the high nutritional content and pharmacological potential of its fruits, few scientific studies report its biological actions. Thus, this study evaluates the phytochemical profile of aqueous and ethanol extracts obtained from B. antiacantha fruits, as well as their possible antioxidant, antitumor, and cytotoxic activities. The aqueous extract exhibited phenolic compounds and flavonoids, while ethanol extracts indicated the presence of flavonoids and coumarin in their composition, regardless of the region of collection. The ethanolic extract demonstrated a more promising antioxidant effect than the aqueous extract and also induced a significant inhibition in the viability of human cervical cancer cells of the SiHa strain. In addition, treatment with both extracts did not alter the viability of non-tumor cells of the immortalized human keratinocyte lineage (HaCaT). These results bring new data about extracts obtained from a native plant, edible and traditionally used in popular medicine, opening new perspectives for its possible therapeutic application.

Resumo Relatos da medicina popular costumam atuar como referencial para o desenvolvimento de novos fármacos a partir de moléculas naturais com ações terapêuticas para doenças de alta gravidade e prevalência como o câncer. Bromelia antiacantha Bertol. é uma espécie da família Bromeliaceae, considerada uma planta alimentícia não convencional (PANC), encontrada nas regiões sul e centro-oeste do Brasil. Apesar do alto teor nutritivo e potencial farmacológico de seus frutos, poucos estudos científicos relatam suas ações biológicas. Desta forma, este estudo avalia o perfil fitoquímico de extratos aquoso e etanólico obtidos de frutos de B. antiacantha, bem como a sua possível ação antioxidante, antitumoral e citotóxica. O extrato aquoso apresentou compostos fenólicos e flavonoides, enquanto os extratos etanólicos apontam a presença de flavonóides e cumarina em sua composição, independente da região de coleta. O extrato etanólico demonstrou efeito antioxidante mais promissor do que o extrato aquoso e também induziu uma inibição significativa na viabilidade de células humanas de câncer cervical da linhagem SiHa. Além disso, o tratamento com ambos extratos não alterou a viabilidade de células não tumorais da linhagem de queratinócitos humanos imortalizados (HaCaT). Estes dados trazem novas informações sobre extratos obtidos de uma espécie vegetal nativa, comestível e já utilizada tradicionalmente, mas abrindo novas perspectivas quanto a possíveis aplicações terapêuticas.

Punica granatum L. Extract Antibiofilm Action against Acinetobacter baumannii Carbapenem-Resistant and Biocompatibility over Human Keratinocytes / Ação Antibiofime de Extrato dePunica granatum L Contra Acinetobacter baumannii Resistente a Carbapnêmicos e Biocompatibilidade em Queratinócitos Humanos

Acinetobacter baumannii is a multi-drug resistant microorganism. The objective of this study was to evaluate the antimicrobial and antibiofilm action of the pomegranate natural extract against eight strains of multidrug resistant Acinetobacter baumanii and to assess the extract cytotoxicity in a culture of Human Keratinocytes (HaCat). Broth microdilution method was used to determine the minimum inhibitory and minimum microbicidal concentrations of the extracts. The extract antibiofilm action was analysed by the MTT colorimetric test. The cytotoxicity evaluation was performed by the MTT colorimetric test, which analysed the mitochondrial cellular action, after contact of the extract for 5 min. The results were statistically analysed by ANOVA and Tukey test with a significance level α≤ 0.05. Punica granatum L. (pomegranate) extract had antimicrobial action on all the 8 clinical strains of Acinetobacter baumannii evaluated. The extract showed a significant reduction in metabolic action in biofilm for all the strains, with results statistically different from growth control (p≤0.05%). P. granatum extract applied for 5 min on human keratinocytes (HaCat) promoted cell viability in all the tested concentrations. The pomegranate extract is effective in reducing the multidrug-resistant clinical strains of Acinetobacter baumanni and is biocompatible. (AU)

Acinetobacter baumannii é um microrganismo multirresistente. O objetivo deste estudo foi avaliar a ação antimicrobiana e antibiofilme do extrato natural de romã contra oito cepas de A. baumanii multirresistente e avaliar a citotoxicidade do extrato em uma cultura de queratinócitos humanos (HaCat). O método de microdiluição em caldo foi utilizado para determinar as concentrações inibitórias mínimas e microbicidas mínimas dos extratos. A ação antibiofilme do extrato foi analisada pelo teste colorimétrico MTT. A avaliação da citotoxicidade foi realizada pelo teste colorimétrico MTT, que analisou a ação celular mitocondrial, após contato do extrato por 5 min. Os resultados foram analisados ​​estatisticamente por ANOVA e teste de Tukey com nível de significância α≤ 0,05. O extrato de Punica granatum L. (romã) apresentou ação antimicrobiana em todas as 8 cepas clínicas avaliadas de A. baumannii. O extrato apresentou redução significativa na ação metabólica no biofilme para todas as linhagens, com resultados estatisticamente diferentes do controle de crescimento (p≤0,05%). O extrato de P. granatum aplicado por 5 min em queratinócitos humanos (HaCat) promoveu viabilidade celular em todas as concentrações testadas. O extrato de romã é eficaz na redução das cepas clínicas multirresistentes de Acinetobacter baumanni e é biocompatível. (AU)

Expression of miR-20b in plasma of patients with psoriasis vulgaris and its effect on keratinocytes / 中国医师杂志

Objective:To investigate the expression of microRNA-20b (miR-20b) in peripheral blood plasma of patients with psoriasis vulgis, and to further analyze the effect and potential mechanism of miR-20b on proliferation and apoptosis of human immortals keratinocytes (HaCaT cell line).Methods:The peripheral blood of 36 patients with psoriasis vulgaris and 36 healthy volunteers (as control group) were collected. The relative expression level of miR-20b in plasma was detected by real-time quantitative polymerase chain reaction (qRT-PCR). HaCaT cells were cultured in vitro and randomly divided into control group (untreated), interleukin-22 (IL-22)treatment group (stimulated with 100 ng/ml IL-22 for 24 h), IL-22+ inhibitor control group (after transfection of inhibitor negative control, stimulated with 100 ng/ml IL-22 for 24 h) and IL-22+ miR-20b inhibitor group (treated with 100 ng/ml IL-22 for 24 h after transfection with miR-20b inhibitor). The relative expression of miR-20b in HaCaT cells was detected by qRT-PCR. The proliferation and apoptosis of HaCaT cells were detected by cell counting kit-8 (CCK-8) and flow cytometry, respectively. Bioinformatics software was used to predict the downstream targets of miR-20b. The targeted binding relationship between miR-20b and killin, p53-regulated DNA replication inhibitor (KLLN) 3′UTR was verified by dual luciferase reporter gene assay. The expression level of KLLN protein in HaCaT cells was detected by Western blot. Results:The plasma level of miR-20b in psoriasis patients was significantly higher than that in healthy controls [(1.62±0.53) vs (1.00±0.42), P<0.001]. The results of qRT-PCR showed that the expression of miR-20b in IL-22 treatment group was higher than that in control group ( P<0.05). The expression of miR-20b in IL-22+ miR-20b inhibitor group was lower than that in IL-22+ inhibitor control group ( P<0.05). CCK-8 results showed that the absorbance values of HaCaT cells in IL-22 treatment group were significantly higher than those in control group at 24, 48, 72 and 96 h (all P<0.05). The absorbance values of HaCaT cells in IL-22+ miR-20b inhibitor group at 48, 72 and 96 h were significantly lower than those in IL-22+ inhibitor control group (all P<0.05). The apoptosis rate of IL-22 treatment group was significantly lower than that of control group [(4.12±0.37)% vs (7.06±0.58)%], with statistically significant difference ( P<0.05). The apoptosis rate of HaCaT cells in IL-22+ miR-20b inhibitor group was significantly higher than that in IL-22+ inhibitor control group [(6.59±0.53)% vs (3.94±0.46)%], with statistically significant difference ( P<0.05). Dual luciferase reporter assay was used to verify the interaction between miR-20b and KLLN, and the results showed that the luciferase activity of KLLN wild-type was significantly inhibited by miR-20b mimics. Western blot results showed that the protein expression of KLLN in the IL-22 treatment group was lower than that in the control group ( P<0.05); the protein expression of KLLN in IL-22+ miR-20b inhibitor group was higher than that in IL-22+ inhibitor control group, with statistically significant difference ( P<0.05). Conclusions:MiR-20b is highly expressed in the plasma of patients with psoriasis vulgaris, and miR-20b may promote the proliferation and anti-apoptotic ability of keratinocytes by targeting KLLN.

Expressions and roles of key enzymes in serine synthesis pathway in NaAsO2-treated HaCaT cells / 环境与职业医学

Background The key enzymes of serine synthesis pathway (SSP) play an important role in tumor growth, proliferation, and invasion, but their roles in arsenic carcinogenesis are unclear. Objective To observe the effects of NaAsO2 treatment on the expressions of key enzymes [such as phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH)] of SSP and on the ability to proliferate and migrate in human immortalized skin keratinocytes (HaCaT) and NaAsO2-induced malignantly transformed HaCaT (T-HaCaT), and to explore the roles of SSP key enzymes in arsenic carcinogenesis. Methods (1) The T-HaCaT cells constructed earlier by our research team were divided into a passage control (0 μmol·L−1 NaAsO2) group, a T-HaCaT (0.5 μmol·L−1 NaAsO2) group, a NCT503 (PHGDH inhibitor, 25 μmol·L−1) group, and a NCT503 (25 μmol·L−1) + T-HaCaT (0.5 μmol·L−1 NaAsO2) group. Western blotting was used to detect the protein expression levels of SSP key enzymes in the passage control group and the T-HaCaT group. CCK8 assay and cell scratch test were used to detect the proliferation and migration rates of cells in each group respectively. (2) Well-grown logarithmic-phase HaCaT cells were treated with 0, 0.625, 1.25, and 2.5 μmol·L−1 NaAsO2 for 0, 24, 48, and 72 h to detect cell proliferation rate and protein expression levels of SSP key enzymes. In the subsequent experiment, HaCaT cells were pretreated with 25 μmol·L−1 NCT503 for 6 h, and then treated with 2.5 μmol·L−1 NaAsO2 for 72 h continuously. The experimental groups included a control (0 μmol·L−1 NaAsO2) group, an exposure (2.5 μmol·L−1 NaAsO2) group, a pretreatment (25 μmol·L−1 NCT503) group, and a pretreatment (25 μmol·L−1 NCT503) + exposure (2.5 μmol·L−1 NaAsO2) group, to detect the proliferation rate of cells in each group. Results The protein expression level of PHGDH in the T-HaCaT group were 1.60 times higher than that in the passage control group (P<0.05), and its proliferation rate (177.51%±14.69%) and migration rate (53.85%±0.94%) were also higher than the passage control group’s (100.00%±0.00% and 24.30%±2.26%) (both Ps<0.05), respectively. After the NCT503 intervention, the proliferation rate (144.97%±8.08%) and migration rate (35.80%±0.99%) of cells in the NCT503 + T-HaCaT group were lower than those in the T-HaCaT group (both P<0.05). The proliferation rate of HaCaT cells after NaAsO2 exposure for 72 h increased with the increase of exposure concentration (r=0.862, P<0.05), and consistently, the protein levels of SSP key enzymes in HaCaT cells in each exposure group were higher than those in the control group (all P<0.05). The proliferation rate of HaCaT cells treated with 2.5 μmol·L−1 NaAsO2 increased with the extension of exposure time (r=0.775, P<0.05), which was consistent with the changes of PHGDH levels in cells. After the NCT503 intervention, the proliferation rate of the pretreatment + exposure group was significantly lower than that of the exposure group (P<0.05). Conclusion The key enzymes of SSP may play an important role in the proliferation of T-HaCaT cells induced by NaAsO2.

Toxicity of Carnoy's solution toward human keratinocytes: an in vitro study

Abstract: The present study aimed to characterize the chemical elements and cytotoxicity of Carnoy's solution (CS) by comparing two different trademarked products (one Brazilian [NCS] and another imported [ICS]) using inductively coupled plasma mass spectrometry (ICP-MS) and human keratinocyte (HaCaT) cultures. For performing ICP-MS, the solutions were diluted according to calibration curves, and the chemical elements were analyzed with a spectrometer. HaCaT cells were exposed to CS concentrations ranging from 0.10% to 20% for 3 or 5 min. Cell viability was evaluated immediately (T0), 24 h (T1), and 7 days (T2) after exposure to CS using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) reduction assay. Data were analyzed using a t-test for ICP-MS and analysis of variance followed by Tukey's post-hoc test for MTT assay, both considering statistical significance at p<0.05. ICP-MS results revealed that ICS presented significantly lower concentrations of 12 chemical elements than NCS. The results of MTT assay revealed that at T0, ICS was more cytotoxic than NCS regardless of the time of exposure (p < 0.05). At T1, the only difference between the groups was at a concentration of 0.10% after 5 min of exposure. At T2, at a concentration of 0.5%, ICS resulted in a significant reduction in cell viability compared to NCS (p < 0.05). Thus, the results showed that ICS was more cytotoxic than NCS. Collectively, our findings suggest that the individual compositions of different CS formulations should be investigated.

Lentinula edodes extract inhibits matrix metalloproteinase expression and increases typeⅠprocollagen expression via the p38 MAPK/c-Fos signaling pathway in ultraviolet A and B-irradiated HaCaT keratinocytes

Objective:To determine the effect of Lentinula edodes extract on ultraviolet (UV) A and UVB-induced changes in matrix metalloproteinase (MMP) and typeⅠprocollagen expression using human immortalized HaCaT keratinocytes. Methods:Lentinula edodes ethanol extract (LEE) was obtained by extraction with 80％ ethanol for 4 h at 80 ℃. Effect of LEE on UV-induced alteration on the expression and production of MMPs and typeⅠprocollagen in keratinocytes was investigated using ELISA, RT-PCR, and Western blotting assay. To determine the underlying mechanism of LEE-mediated effects, mitogen-activated protein kinase (MAPK) and activator protein 1 signaling pathways were analysed by Western blotting assay. Results:LEE significantly inhibited the expression of MMP-1 and MMP-9 and increased the expression of typeⅠprocollagen in UVA and UVB-irradiated HaCaT keratinocytes. The phosphorylation levels of p38 were significantly inhibited by LEE whereas it did not affect c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation. Suppression of p38 phosphorylation was also accompanied by downregulation of UVA and UVB-induced increase in c-Fos. Conclusions:LEE effectively inhibits the expression of MMP-1 and MMP-9 and increases typeⅠprocollagen production through the p38 MAPK/c-Fos signaling pathway in UVA and UVB-irradiated HaCaT keratinocytes. This findings suggest that Lentinula edodes may be developed as a cosmetic material to suppress UV exposure-mediated skin aging.

Standardized extract of Centella asiatica ECa 233 inhibits lipopolysaccharide-induced cytokine release in skin keratinocytes by suppressing ERK1/2 pathways

Objective: To evaluate the effect of standardized extract of Centella asiatica ECa 233 on inflammatory mediator production through cyclooxygenase-2 (COX-2), extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) pathway in keratinocyte HaCaT cells. Methods: HaCaT cells were treated with 0.1, 1, 10 and 100 μg/mL ECa 233 in the presence of lipopolysaccharide (LPS). Proinflammatory cytokines and prostaglandin E

Regulatory Effect of Yangxue Jiedu Decoction on Tight Junction of Keratinocytes in Psoriasis / 中国实验方剂学杂志

Objective::To observe the expressions of tight junction proteins (claduin-1, claudin-7 occludin)of psoriasis-like lesions in mice, and clarify the effect of Yangxue Jiedu decoction on the epidermal barrier of psoriasis, so as to provide scientific basis for the treatment of psoriasis with Yangxue Jiedu decoction. Method::C57BL/6J mice were randomly divided into control group, model group, methotrexate group and Yangxue Jiedu decoction.Methotrexate solution and water decoction of Yangxue Jiedu decoction were prepared, and the mice were given imiquimot to induce psoriasis-skin lesions after the hair was shaved.Daily photos were taken to record the forms of skin lesions and psoriasis area and severity index(PASI) scores.Water and oil test pens were used to detect skin moisture content. The pathological changes were observed by htoxylin eosin (HE) staining, and the epidermal thickness was measured.Ki67 was detected by immunofluorescence. Immunohistochemistry was used to detect the expressions of loricrin, CD3+ T lymphocyte infiltration and claduin-1, claudin-7, occludin.In addition, the expressions of claudin-7 and occludin in skin lesions were detected by Western blot.Meanwhile, interleukin-17(IL-17) (1 mg·L-1) was used to simulate the microenvironment of psoriasis skin lesions, and the intervention was conducted by making Yangxue component, Jiedu component and Yangxue Jiedu dry powder.The toxicity of the drug on Hacat cells was detected by cell counting kit-8(CCK-8)method.The effect of the drugs on the expressions of claudin-1, claudin-7, occludin in Hacat was detected by immunofluorescence assay. Result::Yangxue Jiedu decoction could significantly reduce the psoriasis skin lesions in mice, and reduce the PASI score and skin thickness (P<0.01). Compared with the model group, the skin moisture content in the lesion was increased (P<0.01). Abnormal expressions of ki67 and loricrin in epidermis and infiltration of CD3+ T cells were reduced (P<0.01). In addition, the expressions of claudin-1, claudin-7, occludin proteins (P<0.05), and the integrity of the tight junction structure were increased.In vitro studies, compared with the model group, the expressions of claudin-1, claudin-7, and occludin in the Yangxue Jiedu group and Yangxue group were increased (P<0.05). Compared with the model group, there was no statistically significant difference in protein expression in the Jiedu group. Conclusion::By regulating the expressions of tight junction proteins between keratinocytes, Yangxue Jiedu decoction can inhibit the abnormal proliferation and differentiation, and further restore the broken epidermal barrier.Yangxue Jiedu decoction plays a role in regulating tight junction mainly through Yangxue component.

Protective effect of Nrf2 protein against ultraviolet B-induced photodamage to HaCaT cells / 中华皮肤科杂志

Objective To evaluate the protective effect of nuclear factor E2-related factor 2(Nrf2) protein against ultraviolet B (UVB)-induced photodamage to HaCaT cells,and to explore its mechanisms.Methods Cultured HaCaT cells were divided into 4 groups:control group receiving no treatment,UVB group irradiated with 30 mJ/cm2 UVB for 30 s,Nrf2 group transfected with a lentiviral vector overexpressing the Nrf2 gene,and Nrf2 + UVB group transfected with a lentiviral vector overexpressing the Nrf2 gene followed by radiation with 30 mJ/cm2 UVB for 30 s.After the treatment,HaCaT cells in the above 4 groups were cultured for another 24 hours.Then,changes in the morphology of HaCaT cells were observed after UVB radiation,Western blot analysis was performed to determine Nrf2 protein expression,cell counting kit-8 (CCKS) assay to detect survival rates of HaCaT cells,flow cytometry to detect levels of reactive oxygen species (ROS),and a biochemical method to detect superoxide dismutase (SOD) levels in cells,and enzyme-linked immunosorbent assay to detect levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the culture supematant of HaCaT cells.One-way analysis of variance was used for comparing means in several groups,and least significant difference (LSD)-t test for multiple comparisons.Results Polygonal and clustered HaCaT cells were observed in the control group.After UVB radiation,HaCaT cells became shrunken and round,the number of floating cells increased,and the number of adherent cells markedly decreased.There was a significant difference in Nrf2 protein expression among the control group,UVB group,Nrf2 group and Nrf2 + UVB group (1.84 ± 0.047,0.63 ± 0.082,2.19 ± 0.168 and 1.43 ± 0.069 respectively;F =64.81,P ＜ 0.05),and the Nrf2 protein expression was significantly higher in the Nrf2 group than in the control group (t =14.82,P ＜ 0.05);the survival rates of HaCaT cells also significantly differed among the above 4 groups (98.00％ ± 2.39％,24.40％ ± 2.98％,71.63％ ± 3.39％and 43.38％ ± 3.39％ respectively;F =236.66,P ＜ 0.05),and the UVB group showed significantly decreased cell viability compared with the control group (t =33.34,P ＜ 0.05)and Nrf2 + UVB group (t=10.07,P ＜ 0.05);a significant difference in the ROS level in HaCaT cells was observed among the above 4 groups (1.27 ± 0.10,5.65 ± 0.19,2.10 ± 0.73 and 3.67 ± 0.19 respectively;F =481.39,P ＜ 0.05),and the UVB group showed a significantly increased ROS level compared with the control group (t =33.68,P ＜0.05) and Nrf2 + UVB group (t =12.47,P ＜ 0.05).The SOD level in HaCaT cells significantly differed among the above 4 groups (F =170.76,P ＜ 0.05),and was significantly lower in the UVB group than in the control group (t =11.25,P ＜ 0.05) and Nrf2 + UVB group (t =17.52,P ＜ 0.05).The IL-6 level also significantly differed among the above 4 groups (F =532.34,P ＜ 0.05),and was significantly higher in the UVB group than in the control group (t =28.48,P ＜ 0.05) and Nrf2 + UVB group (t =27.82,P ＜ 0.05).There was no significant difference in the TNF-α level among the above 4 groups (F =2.02,P =0.19).Conclusion Nrf2 can protect HaCaT cells from UVB-induced oxidative damage,by reducing intracellular ROS levels and increasing the activity of the endogenous antioxidant enzyme SOD.

DZ2002 improves psoriasis-like lesions of guinea pig via inhibiting secretion of chemokines in keratinocytes / 中国药理学通报

Aim To explore the therapeutic effect of DZ2002, a reversible S-adenosyl-L-homocysteine hydrolase inhibitor, on psoriasis-like skin lesions of guinea pig and its mechanism. Methods The guinea pig model of psoriasis was established with 50 g • L-1 propranolol hydrochloride liniment. The pathological changes of the skin were determined by hematoxylin and eosin (HE). Then the Baker score and epidermal thickness were measured based on HE. The infiltration of neutrophils was marked by immunohistochemical staining. The expression of chemokines in TNF-a/IFN-7-treated HaCa T cells in the present of DZ2002 or not were determined by real-time polymerase chain reaction (RT-PCR), and the production of chemokines from HaCa T cells were quantified by ELISA and Luminex x-MAP technology. In the same condition, supernatants were used to test the Chemotaxis effect on Jurkat and THP1 cells via Chemotaxis assays. Results Pathological features such as acanthosis, inflammatory cell infiltration, Munro microabscess, hyperkeratosis and parakeratasis appeared in the psoriasis-like skin lesions of guinea pigs. The Baker score and epidermal thickness of psoriasis-like guinea pig ear both increased significantly. Compared with vehicle group, DZ2002 cream not only significantly improved the pathological manifestations of guinea pig ear skin, but also reduced the skin Baker score and epidermal thickness. DZ2002 significantly down-regulated the expression of chemokines including IL-8 and CXCL9 in TNF-0/IFN-7treated HaCaT cells, and inhibited the Chemotaxis of THP1 and Jurkat cells. Conclusions DZ2002 cream can significantly improve the psoriasis symptoms in guinea pig model of psoriasis via inhibiting the secretion of chemokines by keratinocytes and reducing the infiltration of inflammatory cells.

Standardized extract of Centella asiatica ECa 233 inhibits lipopolysaccharide-induced cytokine release in skin keratinocytes by suppressing ERK1/2 pathways

Objective: To evaluate the effect of standardized extract of Centella asiatica ECa 233 on inflammatory mediator production through cyclooxygenase-2 (COX-2), extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) pathway in keratinocyte HaCaT cells.Methods: HaCaT cells were treated with 0.1, 1, 10 and 100 μg/mL ECa 233 in the presence of lipopolysaccharide (LPS). Proinflammatory cytokines and prostaglandin E2 were assessed with ELISA. Western blotting was used to determine the inhibition of COX-2, ERK1/2 and NF-κB protein expression. Results: ECa 233 suppressed LPS-induced release of interleukin-1β, tumor necrosis factor-α, and prostaglandin E2. ECa 233 also inhibited COX-2, phosphorylation of ERK1/2 and the activation of NF-κB. Moreover, the formation of reactive oxygen species (ROS) was decreased in response to LPS-inflamed keratinocytes. Conclusions: ECa 233 inhibits LPS-stimulated production of inflammatory mediators in keratinocytes via suppressing ERK1/2 and NF-κB pathways. The suppressive effect of ECa 233 may be related to an inhibition of ROS production.

Study on the effects of RCCS simulated microgravity on the metabolism of human keratinocyte / 解放军医学杂志

Objective: To investigate the effects of RCCS simulated microgravity on the metabolism of the human keratinocyte cell line HaCaT. Methods: The rotary cell culture system (RCCS) was used to simulate the microgravity environment, and HaCaT cells were cultured in vitro and divided randomly into simulated microgravity group (SMG) and normal gravity group (NG). The two group HaCaT cells were collected respectively after 1 d, 2 d and 3 d culture, and the samples were analyzed by LC/MS metabolomics. The differential metabolites between the SMG and NG cells were identified with partial least squares discriminant analysis ((O)PLS-DA), and the data were input into the KEGG database for the construction and functional analysis of metabolic pathways. Results: Comparing to NG cells, after 1 d culture, there were 74 different metabolites in SMG cells, among which 16 were up-regulated and 58 were down-regulated; after 2 d culture, there were 89 different metabolites, among which 15 were up-regulated and 74 down-regulated; after 3d culture, there were 100 different metabolites, of which 23 were up-regulated and 77 were down-regulated. The differentially expressed 49 metabolites (VIP>1 and P<0.05) after 3 d were set as target metabolites, within which the sphingosine, glutamate, and docosapentaenoic acid were down-regulated, and dehydrated sorbitol was up-regulated. KEGG analysis indicated that the metabolic pathways involved were amino acid metabolism, lipid metabolism, cell proliferation and apoptosis, substance transport, catabolism, and signal transduction. Conclusion: RCCS simulated microgravity may have significant impacts on keratinocyte metabolism mainly involving metabolites such as sphingolipids and glutamate as well as the related signaling pathways.

Effect of metformin on proliferation and differentiation of HaCaT cells and secretion of inflammatory factors by HaCaT cells / 中华皮肤科杂志

Objective To evaluate the effect of mefformin on the human keratinocyte line HaCaT,and to explore its molecular mechanism.Methods HaCaT cells were divided into several groups to be treated with mefformin at different concentrations of 1,2,5,10,20,50 mmol/L for 24,48 and 72 hours.Cell counting kit-8 (CCK-8) assay was performed to evaluate the effect of metformin on the survival rate of HaCaT cells.After 48-hour treatment with metformin at concentrations of 0 (control group),0.5,1,2,5,10 mmol/L,flow cytometry was conducted to evaluate the effect of metformin on cell cycle and apoptosis.Western blot analysis was performed to determine the expression of cell proliferation-and differentiation-related proteins (keratin-16 [K16],K17,K1,involucrin),apoptosis-related proteins (Bax,Bcl-2) and AKT/mTOR/STAT3 pathway proteins.Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the levels of interleukin (IL)-8,tumor necrosis factor (TNF)-α and IL-23 (inflammatory factors) in the culture supernatant of HaCaT cells.Statistical analysis was carried out with SPSS19.0 software using one-way analysis of variance for comparison of the above indices among the 0.5-,1-,2-,5-,10-mmol/L metformin groups and control group,repeated measures analysis of variance for comparisons among different time points or different metformin groups,least significant difference (LSD)-t test for multiple comparisons.Results CCK-8 assay showed that mefformin had inhibitory effects on the proliferation of HaCaT cells (F =116.87,P ＜ 0.05),and the cell survival rates gradually decreased along with the increase in the concentrations of mefformin.After 48-hour treatment with mefformin at concentrations of 0,0.5,1,2,5,10mmol/L,the proportion of HaCaT cells in G2/M phase gradually increased (5.55％ ± 1.03％,6.37％ ±0.93％,8.57％ ± 1.18％,10.05％ ± 0.60％,10.76％ ± 0.87％,13.63％ ± 1.41％,respectively,F =24.98,P ＜0.05),and the early apoptosis rate also gradually increased (0.78％ ± 0.71％,19.18％ ± 1.41％,25.67％ ±1.34％,28.45％ ± 0.92％,34.97％ ± 2.12％,40.41％ ± 1.49％,respectively,F =296.08,P ＜ 0.05).Along with the increase in the concentrations of metformin,there were increasing trends in the expression of K1 and the pro-apoptotic protein Bax (F =8.86,5.38 respectively,both P ＜ 0.05),while there were decreasing trends in the expression of K16,K17 and the apoptotic protein Bcl-2 (F =8.02,4.82,12.10 respectively,all P ＜ 0.05).There was no significant difference in the expression of involucrin among different metformin groups (F =0.57,P ＞ 0.05).After 48-hour treatment with mefformin at different concentrations,the levels of IL-8 and TNF-α in HaCaT cells significantly decreased (F =33.89,14.99 respectively,both P ＜0.05),while there was no significant change in the IL-23 level (F =2.12,P ＞ 0.05).Along with the increase in the concentrations of metformin,the expression of p-AKT,p-mTOR and p-STAT3 significantly decreased (F =11.38,0.35,4.38 respectively,all P ＜ 0.05),but there were no significant changes in the protein expression of AKT,mTOR and STAT3 (F =0.66,0.35,4.24 respectively,all P ＞ 0.05).Conclusion Metformin can inhibit the proliferation,promote the differentiation and apoptosis of HaCaT cells,and inhibit the secretion of inflammatory factors by regulating the AKT/mTOR/STAT3 signaling pathway.

Niacinamide Protects Skin Cells from Oxidative Stress Induced by Particulate Matter

Niacinamide (NIA) is a water-soluble vitamin that is widely used in the treatment of skin diseases. Moreover, NIA displays antioxidant effects and helps repair damaged DNA. Recent studies showed that particulate matter 2.5 (PM(2.5)) induced reactive oxygen species (ROS), causing disruption of DNA, lipids, and protein, mitochondrial depolarization, and apoptosis of skin keratinocytes. Here, we investigated the protective effects of NIA on PM(2.5)-induced oxidative stress in human HaCaT keratinocytes. We found that NIA could inhibit the ROS generation induced by PM(2.5), as well block the PM(2.5)-induced oxidation of molecules, such as lipids, proteins, and DNA. Furthermore, NIA alleviated PM(2.5)-induced accumulation of cellular Ca²⁺, which caused cell membrane depolarization and apoptosis, and reduced the number of apoptotic cells. Collectively, the findings show that NIA can protect keratinocytes from PM(2.5)-induced oxidative stress and cell damage.

Purpurogallin Protects Keratinocytes from Damage and Apoptosis Induced by Ultraviolet B Radiation and Particulate Matter 2.5

Purpurogallin, a natural phenol obtained from oak nutgalls, has been shown to possess antioxidant, anticancer, and anti-inflammatory effects. Recently, in addition to ultraviolet B (UVB) radiation that induces cell apoptosis via oxidative stress, particulate matter 2.5 (PM(2.5)) was shown to trigger excessive production of reactive oxygen species. In this study, we observed that UVB radiation and PM(2.5) severely damaged human HaCaT keratinocytes, disrupting cellular DNA, lipids, and proteins and causing mitochondrial depolarization. Purpurogallin protected HaCaT cells from apoptosis induced by UVB radiation and/or PM(2.5). Furthermore, purpurogallin effectively modulates the pro-apoptotic and anti-apoptotic proteins under UVB irradiation via caspase signaling pathways. Additionally, purpurogallin reduced apoptosis via MAPK signaling pathways, as demonstrated using MAPK-p38, ERK, and JNK inhibitors. These results indicate that purpurogallin possesses antioxidant effects and protects cells from damage and apoptosis induced by UVB radiation and PM(2.5).

Influence of traditional medicines on the activity of keratinocytes in wound healing: an in-vitro study / 대한해부학회지

Natural medicinal systems such as Ayurveda and folk medicine has remedies for wound management. However, the exact cellular and extracellular mechanisms involved in the healing process and its influence on keratinocytes is less discussed. Therefore, the present study was designed to evaluate the effect of certain natural wound healing medicines on the biology of the keratinocytes/HaCaT cells. Test materials such as honey (H), ghee (G), aqueous extracts of roots of Glycyrrhiza glabra (GG) and leaves of Nerium indicum (NI) were considered. The HaCaT cells were treated with the test materials singly and in combinations (H+G, all combined [Tot]) for a specific period (24, 48, and 72 hours). The cells were then subjected to cytotoxicity/proliferation and migration/scratch assays. All the test materials, except NI, were non-cytotoxic and showed increased cell proliferation at variable concentrations. Significant observations were made in the groups treated with honey (100 µg/ml at 48 hours, P<0.05; 1,000 µg/ml at 72 hours, P<0.05), GG (all concentrations at 48 hours, P<0.05; 750 µg/ml at 72 hours, P<0.05), H+G (250 µg/ml at 24 hours, P<0.001; 500 µg/ml at 48 and 72 hours, P<0.05), and Tot (50 µg/ml at 24, 48 and 72 hours, P<0.01). In the in-vitro wound healing assay, all the treated groups showed significant migration and narrowing of the scratch area by 24 and 48 hours (P<0.001) compared to control. The results obtained from the present study signifies the positive influence of these natural wound healing compounds on keratinocytes/HaCaT cells.

Effects of tacrolimus in treating vitiligo on the secretion of local chemokine / 中华医学美学美容杂志

Objective To analyze the abnormalities of local chemokines in patients with vitiligo and to explore the effect of tacrolimus on the secretion of chemokines in keratinocytes.Methods Blister fluids of 50 patients with vitiligo were collected,including lesion areas and normal areas.Luminex was used to analyze the concentration of local chemokines in patients with vitiligo to determine whether the chemokines were closely related to vitiligo.The effect of tacrolimus on chemokine secretion of was analyzed by Western blot in HaCaT cells.Results By Luminex analysis of blister fluid,it was found that CXCL9 and CXCL10 were significantly higher in the leukoplakia of vitiligo,and there was a significant difference,compared with the blister fluid in the normal site (P＜0.01).IFN-γ significantly stimulated the keratinocyte cell line HaCat to express CXCL9 and CXCL10.After pretreatment of HaCaT cells with 20 mg tacrolimus,the expression of CXCL9 and CXCL10 was significantly decreased,compared with the blank control (P＜0.01).Conclusions The leukoplakia chemokines CXCL9 and CXCL10 are highly expressed in vitiligo patients.The tacrolimus can significantly reduce the expression of CXCL9 and CXCL10 in keratinocytes under stress,and it therefore plays a therapeutic role in vitiligo.

Antineoplastic potential of the aqueous crude extract of Eugenia uniflora L. in human cervical cancer

Abstract Considering the high prevalence of human cervical cancer and the adverse effects of the available treatments, it is important to develop studies involving plants. Eugenia uniflora L. is a Brazilian native plant widely used in folk medicine and some biological effects have already been described. In this study, we investigated the biologicals effects of the aqueous crude extract of E. uniflora leaves in relation to the viability of human cervical cancer cells (SiHa), non-tumorigenic cells HaCaT and human lymphocytes. Our results demonstrated that different concentrations of E. uniflora's extract significantly inhibited the viability of the Siha cell line at 24, 48 and 72 hours of treatment, but did not induce significant changes in the HaCat cell line and human lymphocytes. Tumor cells had adhesion capacity, migration processes, ability of colony forming and the potential to recover its viability after treatment. withdrawal, significantly reduced. The nuclear morphology revealed chromatin condensation, and the flow cytometry showed predominantly cell death by apoptosis in the treated tumor cells. Therefore, the E. uniflora's extract may contribute for future studies aiming at new therapeutic perspectives for human cervical cancer.

Photosensitization of Lipofuscin in Skin Keratinocytes: Effect of Visible Light on Human Skin / Fotossensibilização de lipofuscina em queratinócitos da pele humana: Efeitos da luz visível na pele

Lipofuscin is an autofluorescent pigment progressively accumulated during cellular aging, in several tissues, such as heart, muscle and retina, especially in the postmitotic period. That phenomenon may result from oxidative stress, when biomolecules and organelles (mainly mitochondria) are damaged, generating non-degradable products inside lysosomes. Lipofuscin can be photosensitized, promoting photoxidative processes in cellular components. Many studies on lipofuscin were made using the human retinal pigment epithelial cells, but very little is known about lipofuscin from human skin. In this work we investigated the photoinduced formation (UVA and visible light) of lipofuscin and the consequence of its photosensitization by visible light. We also established an efficient protocol for the induction of lipofuscinogenesis, through specific damage in mitochondria and lysosomes. Cells that accumulated lipofuscin, after exposure to UVA and blue light, became sensitive to visible light (400-750 nm). We characterized the absorption and fluorescence emission of lipofuscin, as well as its fluorescence lifetime through the time resolved fluorescence microscopy (FLIM). We observed that lipofuscin in keratinocytes has absorption maximum in the blue region of light spectrum (420-450 nm), and maximum emission in the red. When photosensitized at 466 nm, lipofuscinloaded HaCaT cells had reduced cell viability, which was related with singlet oxygen generation, accumulated 8-oxo-dG premutagenic lesions and breaks in the DNA strand. Besides, we investigated the efficiency of different wavelengthsin visible light spectrum (408, 466, 522 and 650 nm) to promote lipofuscin formation due to damages in both mitochondria and lysosomes. Blue (408 and 466 nm) and green light (522 nm), but not red light (650 nm), promoted damage in mitochondria (membrane and DNA integrity) and lysosomes (membrane integrity and autophagic activity), effectively inducing lipofuscinogenesis. Thus, in addition to UVA, visible spectrum itself increases the sensitivity of keratinocytes to the visible light, through the generation of lipofuscin. Finally, we tested the carcinogenic potential of high-energy blue light (408 nm), by chronically irradiating HaCaT cells. For the first time in the literature, the formation of pyrimidine cyclobutane (CPD) dimers in the nuclear DNA of HaCaT cells was observed immediately or after several cycles of irradiation at 408 nm. We identified four major changes involved with the process of malignant transformation: genomic instability, decrease in the expression of tumor suppressor protein p16INK4a, increase in the proliferation rate and resistance to UVA-induced apoptosis

A lipofuscina é um pigmento autofluorescente acumulado progressivamente durante o envelhecimento celular em diversos tecidos, como o músculo cardíaco e retina, principalmente no período pós-mitótico. Esse fenômeno pode ocorrer em decorrência do estresse oxidativo, quando biomoléculas e organelas (principalmente mitocôndrias) sofrem danos, gerando produtos não degradáveis no interior dos lisossomos. A lipofuscina pode ser fotossensibilizada promovendo processos fotoxidativos nos componentes celulares. Muitos estudos de lipofuscina foram feitos em células do epitélio pigmentar da retina de olho humano, mas conhece-se muito pouco sobre a lipofuscina de pele humana. Neste trabalho nós investigamos a formação fotoinduzida (UVA e luz visível) de lipofuscina e as consequências da sua fotossensibilização pela luz visível. Nós também estabelecemos protocolos eficazes na indução de lipofuscinogênese, por meio de dano específico em mitocôndrias e lisossomos. Células que acumularam lipofuscina, após exposição à UVA ou luz azul, tornaram-se sensíveis à luz visível (400-750 nm). Caracterizamos as propriedades de absorção e de emissão da lipofuscina e seu tempo de vida de fluorescência, utilizando a microscopia de fluorescência resolvida no tempo (FLIM). Observamos que lipofuscina em queratinócitos tem máximo de absorção na região do azul (420-450 nm), com emissão máxima de fluorescência no vermelho. As células HaCaT carregadas com lipofuscina efotossensibilizadas no visível, tiveram redução da viabilidade celular, que foi relacionada com a geração de oxigênio singlete, bem como acumularam lesões pré-mutagênicas 8-oxo-dG e quebras na fita de DNA. Também, investigamos a eficiência de diferentes comprimentos de onda da luz visível (408, 466, 522 e 650 nm) em promover a formação de lipofuscina em consequência de lesões em mitocôndrias e lisossomos. Tanto a luz azul (408 e 466 nm) quanto a luz verde (522 nm), mas não vermelha (650 nm) promoveram dano em mitocôndrias (integridade de membrana e DNA) e lisossomos (integridade de membrana e atividade autofágica), induzindo eficientemente lipofuscinogênese. Logo, além de UVA, o próprio espectro do visível aumenta a sensibilidade de queratinócitos à luz visível, através da geração de lipofuscina. Por fim, testamos o potencial carcinogênico da luz azul de alta energia (408 nm), irradiando células HaCaT cronicamente. Identificamos quatro mudanças principais envolvidas com o processo de transformação maligna: instabilidade genômica, redução da expressão de proteína supressora de tumor p16INK4a, aumento da taxa de proliferação, e resistência à apoptose. Além disso, a formação de dímeros de pirimidina ciclobutano (CPD) no DNA nuclear de células HaCaT logo após ou depois de vários ciclos de irradiação com 408 nm foi observada pela primeira vez na literatura