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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.
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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.
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Objective To investigate the effects of simulated microgravity by RCCS on proliferation and cell cytoskeleton of human HaCaT keratinocyte. Methods The rotary cell culture system (RCCS) was used to simulate the microgravity environment, and human HaCaT keratinocytes were divided randomly(random number) into the simulated microgravity group (SMG) and normal gravity group (NG). HaCaT cells in the two groups were harvested respectively after 32, 36 and 42 h culture. The HaCaT cells proliferation and cycles were detected by flow cytometry, the concentration of hb-EGF in supernatant was detected by ELISA, and the cell cytoskeleton was observed after 42 hours' culture under laser confocal microscope with FITC-labeled technique. SPSS 23.0 statistical software was used for statistical analysis, and P <0.05 was considered statistically significant. Results The flow cytometry showed that the proportions of human HaCaT keratinocytes in G1 and G2/M phases were increased while the proportion of HaCaT cells in S stage was decreased significantly after 32, 36 and 42 h RCCSculture compared with those in the normal gravity group. The HaCaT cells in G1 stage were declined along with incubation time. ELISA results showed that the hb-EGF concentration in HaCaT supernatant under simulated microgravity culture for 24 and 36 h was lower than that in the normal control group (P<0.01). The laser confocal microscope revealed that the HaCaT fluorescence intensity was decreased,and there were disordered microfilaments, structural ambiguity, pseudopodia reduction and irregularshape among FITC-labeled HaCaT cells cultured 42 h in RSSC compared with the normal gravity group.Conclusions RCCS simulated microgravity environment could inhibit the cell cycle transformation and proliferation of human HaCaT keratinocyte, affect the keratinocyte-secreting function, and induce alterations of the cell cytoskeleton.
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Objective:To explore the expression level of serum interleukin-17 (IL-17) in the patients with psoriasis vulgaris and the changes of interleukin-6 (IL-6) and interleukin-23 (IL-23) levels secrered by the HaCaT cells after stimulated with IL-17,and to clarify its clinical significance and the intervention effect of shikonin.Methods:Twenty-five normal controls,29 patients with psoriasis vulgaris and different groups of HaCaT cells (blank control group,IL-17-24 h group,IL-17-36 h group,IL-17-48 h group,shikonin+-IL-17 group,CsA+-IL-17 group and IL-17 group) were used as the subjects.The levels of serum IL-17 in the patients with psoriasis vulgaris and the levels of IL-6 and IL-23 in supernatant of HaCaT cells in various groups were measured by double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction RT-PCR was used to detect the expression levels of IL-6 and IL-23 p19 mRNA in HaCaT cells in various groups.At the same time,Cell Counting Kit-8 (CCK-8) method was used to detect the viabilities of HaCaT cells in various groups.Results:The level of serum IL-17 of the patients in psoriasis vulgaris group was increased compared with nomral control group,especially in the severe skin lesion group (P＜0.05).The levels of IL-6 and IL-23 in HaCaT cells and supernatant and their mRNA expression levels in IL-17-24 h,IL-17-36 h and IL-17-48 h groups were significantly higher than those in blank control group (P＜0.01).The expression levels of IL-6 and IL-23 in HaCaT cells and supernatant and their mRNA expression levels in shikonin+IL-17 and CsA+IL-17 groups were lower than those in IL-17 groups (P＜0.05).No statistical differences were found in the cell viabilities between each drug treatment group and blank control group (P＞0.05).Conclusion:The expression level of IL-17 in the patients with psoriasis vulgaris is significantly increased,especially in the patients with severe psoriasis vulgaris.IL-17 can promote the secretion of IL-6 and IL-23 in HaCaT cells in a time-dependent manner.Shikonin can inhibit the proinflammatory effect of IL-17.
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Objective:To explore the expression level of serum interleukin-17 (IL-17) in the patients with psoriasis vulgaris and the changes of interleukin-6 (IL-6) and interleukin-23 (IL-23) levels secrered by the HaCaT cells after stimulated with IL-17,and to clarify its clinical significance and the intervention effect of shikonin.Methods:Twenty-five normal controls,29 patients with psoriasis vulgaris and different groups of HaCaT cells (blank control group,IL-17-24 h group,IL-17-36 h group,IL-17-48 h group,shikonin+-IL-17 group,CsA+-IL-17 group and IL-17 group) were used as the subjects.The levels of serum IL-17 in the patients with psoriasis vulgaris and the levels of IL-6 and IL-23 in supernatant of HaCaT cells in various groups were measured by double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction RT-PCR was used to detect the expression levels of IL-6 and IL-23 p19 mRNA in HaCaT cells in various groups.At the same time,Cell Counting Kit-8 (CCK-8) method was used to detect the viabilities of HaCaT cells in various groups.Results:The level of serum IL-17 of the patients in psoriasis vulgaris group was increased compared with nomral control group,especially in the severe skin lesion group (P＜0.05).The levels of IL-6 and IL-23 in HaCaT cells and supernatant and their mRNA expression levels in IL-17-24 h,IL-17-36 h and IL-17-48 h groups were significantly higher than those in blank control group (P＜0.01).The expression levels of IL-6 and IL-23 in HaCaT cells and supernatant and their mRNA expression levels in shikonin+IL-17 and CsA+IL-17 groups were lower than those in IL-17 groups (P＜0.05).No statistical differences were found in the cell viabilities between each drug treatment group and blank control group (P＞0.05).Conclusion:The expression level of IL-17 in the patients with psoriasis vulgaris is significantly increased,especially in the patients with severe psoriasis vulgaris.IL-17 can promote the secretion of IL-6 and IL-23 in HaCaT cells in a time-dependent manner.Shikonin can inhibit the proinflammatory effect of IL-17.
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Objective To study the rhubarb phenol and P38 inhibitor suppression on light aging of human skin HaCaT induced by UVB. Methods The potential of cell proliferation of the different concentrations of rhubarb phenol and P38 inhibitor (SB203580) on human skin HaCaT was detected by MTT method. The cells was divided into the control group, the model group, the rhubarb phenol group, the SB203580 group by random number table method after the 24 h incubation. The 10-6 mol/L rhubarb phenol and 10-7 mol/L SB203580 were added to the rhubarb phenol group and SB203580 group for 24h, The competence of cultured cell proliferation which was irradiated with UVB of intensity of 0.61mW/cm2, mutiply time of 7 min and distance of 10 cm for 24 h except the control group; Western Blot method detected rhubarb phenol and P38 inhibitor of the influence of P38, P-P38, TNF-α, IL-6 protein. Results Compared with control group, the cell proliferation in UVB group significantly reduced (P<0.01); Compared with UVB group, the expression of the P-P38 (0.419 ± 0.029, 0.398 ± 0.015 vs. 0.497 ± 0.051), TNF-α (0.435 ± 0.025, 0.411 ± 0.021 vs. 0.509 ± 0.040) and IL-6 (0.457 ± 0.027, 0.432 ± 0.018 vs. 0.478 ± 0.036) in rhubarb phenol and P38 inhibitor group significantly reduced (P<0.01). Conclusions The rhubarb phenol and P38 inhibitor could significantly suppress HaCaT cells light aging, and its mechanism may be related with inhibiting P38 signaling pathways, and inhibiting the secretion of inflammatory cytokines.
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Objective To explore the mechanism of cell apoptosis of immortalized human keratinocytes (HaCaT cells) and protein expression related to this process after long term exposure to sodium arsenite (NaAsO2,1.0 μmol/L).Methods Malignant transformation model was set up through long-term exposure of HaCaT cells to 1.0 μmol/L NaAsO2.Cell passage for 0,1,7,14,21,28 and 35 generations in the process of malignant transformation were collected for measurement of cell apoptosis rate by flow cytometry,and apoptosis related proteins by Western blotting,including activation of cysteine protease 3,8 (cleaved-caspase-3,8),C/EBP homologous protein (CHOP),B-cell leukemia/lymphoma 2 (Bcl-2),and Bcl-2 associated X protein (Bax).Results Along with the arsenite treatment,the apoptosis levels were significantly decreased (F =26.770,all P ＜ 0.05),the apoptosis levels (0.307 ± 0.049,0.213 ± 0.055,0.163 ± 0.057,0.147 ± 0.035,0.053 ± 0.012) of the 7th,14th,21st,28th and 35thgenerations of cells after arsenite treatment were lower than that of control group of the 0th generation (0.393 ±0.021,all P ＜ 0.05).Compared between generations,there were statistical differences of the protein expression levels of cleaved-caspase-3,Chop,Bax and Bcl-2 in arsenite group (cleaved-caspase-3:1.000 ± 0.000,1.030 ± 0.027,1.104 ± 0.069,1.016 ± 0.087,0.838 ± 0.075,0.753 ± 0.082,0.677 ± 0.073;Chop:1.000 ± 0.000,1.059 ± 0.018,0.934 ± 0.095,0.976 ± 0.216,0.793 ± 0.136,0.651 ± 0.042,0.564 ± 0.056;Bax:1.000 ± 0.000,1.069 ± 0.037,1.028 ± 0.042,0.954 ± 0.118,0.641 ± 0.135,0.531 ± 0.132,0.429 ± 0.085;Bcl-2:1.000 ± 0.000,1.072 ± 0.023,1.249 ± 0.134,1.334 ± 0.143,1.633 ± 0.221,1.507 ± 0.152,1.461 ± 0.145,F =7.730,7.355,27.802,12.438,all P ＜ 0.05),compared with control group of the 0th generation (1.000 ± 0.000) and the same generation control group (1.000 ± 0.000),after the 21st generation,the differences were statistically significant (all P ＜ 0.05),while there was no difference of the protein expression levels of cleaved-caspase-8 (F =0.832,P ＞ 0.05).Conclusion In the process of malignant transformation,the apoptosis levels of HaCaT cells are inhibited after long term sodium arsenite exposure through mitochondria and endoplasmic reticulum (ER) stress signaling pathways.
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Objective To explore the effect of tert?butylhydroquinone(tBHQ)on ultraviolet B(UVB)?induced oxidative damages in human im?mortalized keratinocytes(HaCaT),and discuss its mechanism. Methods The cultured HaCaT cells were randomly divided into 4 groups:control group(G1),ultraviolet irradiation group(G2),25μmol/L tBHQ pretreatment before ultraviolet irradiation group(G3),and 50μmol/L tBHQ pre?treatment before ultraviolet irradiation group(G4). The content of reactive oxygen species was detected by DCFH?DA method,and the cell prolifera?tion was evaluated by MTT. Western blot was used to measure the protein expression of nuclear factor E2?related factor 2(Nrf2)in both nuclear fac?tions and whole?cell of HaCaT. The mRNA expressions of CAT and SRX were determined by real?time RT?PCR. Results The content of reactive oxygen species in HaCaT cells was increased,and the cell proliferation rate was decreased significantly after ultraviolet irradiation. The pretreatment of 25 and 50μmol/L tBHQ can inhibit the UVB?induced oxidative damage in a dose?dependent manner in HaCaT cells. Compared with G2 group, tBHQ pretreatment could dose?dependently increase the level of Nrf2 protein in nuclear factions and whole?cell of HaCaT,and also the mRNA ex?pressions of CAT and SRX. Conclusion UVB irradiation can induce oxidative stress damages of HaCaT cells. tBHQ may inhibit the UVB?induced oxidative damages through enhancing Nrf2 expressions and nuclear translocation,then activating the transcription of the downstream antioxidant en?zymes CAT and SRX.
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BACKGROUND/OBJECTIVES: Chronic ultraviolet (UV) exposure-induced reactive oxygen species (ROS) are commonly involved in the pathogenesis of skin damage by activating the metalloproteinases (MMP) that break down type I collagen. Adenophora remotiflora (AR) is a perennial wild plant that inhabits Korea, China, and Japan. The present study investigated the protective effects of AR against UVB-induced photo-damage in keratinocytes. MATERIALS/METHODS: An in vitro cell-free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and nitric oxide (NO). The effect of AR on ROS formation, antioxidant enzymes, elastase, MMP-1 level, and mRNA expression of MMP-1 were determined in UVB-irradiated human keratinocyte HaCaT cells. RESULTS: AR demonstrated strong DPPH free radical and NO scavenging activity in a cell-free system exhibiting IC50 values of 1.88 mg/mL and 6.77 mg/mL, respectively. AR pretreatment dose-dependently attenuated the production of UVB-induced intracellular ROS, and antioxidant enzymes (catalase and superoxide dismutase) were enhanced in HaCaT cells. Furthermore, pretreatment of AR prevented UVB-induced elastase and collagen degradation by inhibiting the MMP-1 protein level and mRNA expression. Accordingly, AR treatment elevated collagen content in UVB-irradiated HaCaT cells. CONCLUSION: The present study provides the first evidence of AR inhibiting UVB-induced ROS production and induction of MMP-1 as a result of augmentation of antioxidative activity in HaCaT human keratinocytes. These results suggest that AR might act as an effective inhibitor of UVB-modulated signaling pathways and might serve as a photo-protective agent.
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Humans , Campanulaceae , Cell-Free System , China , Collagen , Collagen Type I , In Vitro Techniques , Inhibitory Concentration 50 , Japan , Keratinocytes , Korea , Metalloproteases , Nitric Oxide , Pancreatic Elastase , Plants , Reactive Oxygen Species , RNA, Messenger , Skin , SuperoxidesABSTRACT
Objective To observe the effect of glycyrrhizin (CG) on the proliferation and the expressions of interleukin (IL)-2, IL-4 in human keratinocytes cell line HaCaT cells, and to investigate the therapeutic mechanism of CG in the pa?tients with psoriasis. Methods The HaCaT cells were divided into four groups:blank group (without CG), low concentration group (50μL CG/mL cell supernatant), medium concentration group (100μL CG/mL cell supernatant) and high concentra?tion group (200μL CG/mL cell supernatant). Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the prolifer?ation of HaCaT cells that were treated with CG at different concentrations respectively for 24 hours. The contents of IL-2 and IL-4 in HaCaT cells were examined by enzyme linked immunosorbent assay (ELISA). Results Compared with the blank group, there were obviously inhibitory effects in the proliferation of HaCaT cells treated by low,medium and high concentra?tions of CG for 24 hours. There was no significant difference in IL-2 level between low concentration CG group and blank group (ng/L:234.51±17.98 vs 225.31±16.23, P>0.05). After 24 hours of intervention with CG of medium and high concen?trations, the expression levels of IL-2 was significantly lower in HaCaT cells than that in blank group and low concentration group. The IL-2 level was significantly lower in high concentration group than that in medium concentration group (ng/L:188.99±19.22 vs 208.49±18.40, P<0.05). At the same condition, the secretion of IL-4 in HaCaT cells was significantly up-regulated in high concentration group than that in other three groups (ng/L:45.67±10.29 vs 37.62±3.90, 39.68±6.08, 43.85± 10.26, P<0.05). Conclusion Glycyrrhizin may suppress the proliferation of human skin keratinocytes by regulating secre?tion of cytokines IL-2 and IL-4.
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Objective To investigate the action mode of borneol on activity of epidermal skin;To investigate action mode of borneol as penetration enhancer. Methods The well-established and standard penetration enhancer Azone was employed as a positive control in this study. The cytotoxicities of borneol and Azone on HaCaT cells were detected by CCK-8 assay, and their half 50% inhibitory concentrations (IC50) were calculated. The fluorescence recovery after photo bleaching was employed to investigate the effect of borneol and Azone on membrane fluidity, and the flow cytometer was used to monitor the changes of membrane potential of HaCaT cell after treated with these penetration enhancers. Results The IC50 values of borneol and Azone were 2.826 , 0.172 mmol/L, respectively. Borneol could significantly improve the membrane fluidity in a concentration-dependent manner, and effectively decrease the membrane potential of HaCaT cell, which exhibited the performances similar to those of Azone. Conclusion The penetration enhancement mechanism of borneol was associated with the concentrations of Ca2+ in keratinocytes, which changes the membrane fluidity and membrane potential of HaCaT cell.
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Objective To discuss effects of Wugong Baidu Drink on expressions of Bcl-2, NF-κB mRNA genes related to the apoptosis of HaCaT in psoriasis. Methods Wistar rats were randomly divided into 6 groups:blank group, Western medicine control group, TCM control group, and Wugong Baidu Drink high, medium, and low dose groups. They were given normal saline, methotrexate suspension, compound natural indigo capsule suspension, and the different doses of Wugong Baidu Drink for gavage, respectively. Drug-containing serum of each group was prepared, and was put in the HaCaT respectively to subculture. The intervention effect of different Drug-containing serums on expressions of objective genes were detected through PR-PCR method. Results Drug-containing serum of each group could decrease the expressions of Bcl-2 mRNA and NF-κB mRNA. The most obvious effects were shown in the groups of Western medicine control group and Wugong Baidu Drink high dose group. All Wugong Baidu Drink groups showed a dose-response relationship. Conclusion Wugong Baidu Drink probably can promote the apoptosis of HaCaT by reducing the gene expressions of Bcl-2 and NF-κB. It can also decrease immune response by regulating serum factors related to T cells to play the role of anti-psoriasis.
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Objective To investigate the influence of low-dose,long-term arsenite exposure on keratinocyte (HaCat) cell apoptosis and the expression of Bcl-2 and Bax proteins.Methods HaCat cells were exposed to different concentrations of NaAsO2[0(control group),0.05,0.10 μmol/L] for 15 weeks.A total of 10 000 cells in each group were measured to detect the level of apoptosis by flow cytometry.The protein expressions of Bcl-2 and Bax were determined by Western blotting method.Results The cell apoptosis rate was significantly different between groups (F =17.19,P ＜ 0.05).Compared with that of the control [(1.42 ± 0.13)％],the apoptosis rates of the 0.05 and 0.10 μmol/L groups [(1.23 ± 0.08)％ and (1.04 ± 0.06)％] decreased significantly (all P ＜ 0.05); the 0.10 μmol/L group was significantly lower than that of the 0.05 μmol/L group(P ＜ 0.05).The protein expressions of Bcl-2 and Bax were significantly different between groups (F=107.38,346.45,all P＜ 0.05).The protein expression of Bcl-2 of the 0.05 and 0.10 μmol/L groups were (143.89 ± 7.74)％ and (199.96 ± 12.18)％,respectively,which were significantly higher than that of the control group[(100.00 ± 1.45)％,all P ＜ 0.05] ; the 0.10 μmol/L group was significantly higher than that of the 0.05 μmol/L group (P ＜ 0.05).The protein expression of Bax of the 0.05 and 0.10 μmol/L groups were (70.78 ± 1.53)％ and (54.80 ± 1.34)％,respectively,which were markedly lower than that of the control group[(100.00 ± 3.09)％,all P ＜ 0.05]; the 0.10 μmoL/L group was significantly lower than that of the 0.05 μmol/L group(P ＜ 0.05).Conclusion Decreased level of apoptosis induced by low level and long-term arsenic exposure may be associated with increased expression of Bcl-2 protein and decreased expression of Bax protein.
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Sargassum fusiforme has traditionally been widely consumed in Asia as a food, and it has gained much attention due to its high nutritional, pharmaceutical, and industrial value. This study aimed to examine the promotional effects of ethanol extract (ET) and fraction obtained from ethyl acetate (FR) of S. fusiforme on hair growth in C57BL/6 mice and HaCaT cells. Five-week-old mice were used to compare hair regrowth during application of ET and FR for 21 days. Hair regrowth was evaluated by macroscopic observation and verified by hematoxylin-eosin tissue staining. Levels of mRNA expression of factors relevant to the hair growth cycle such as keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1) were examined by quantitative polymerase chain reaction (qPCR). Our results showed that ET and FR successfully promoted hair regrowth in shaved C57BL/6 mice at a dose >20 mg/kg. Moreover, ET and FR were effective in stimulating expression of KGF and VEGF mRNAs in a dose-dependent manner, whereas TGF-beta1 was not activated. These results indicate that ET and FR of S. fusiforme effectively promoted hair growth and gene expression relevant to hair growth cycles in both in vitro and in vivo models.
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Animals , Mice , Alopecia , Asia , Ethanol , Fibroblast Growth Factor 7 , Gene Expression , Hair , Polymerase Chain Reaction , RNA, Messenger , Sargassum , Transforming Growth Factor beta1 , Vascular Endothelial Growth Factor AABSTRACT
BACKGROUND: UVB irradiation induces apoptosis or/and autophagy through several molecular pathways in keratinocytes. However, the precise molecular mechanism of UVB-induced autophagy is largely unknown in keratinocytes. OBJECTIVE: The purpose of this study was to investigate the molecular mechanisms of UVB-induced apoptosis and autophagy in HaCaT cell lines. METHODS: Cells were irradiated by UVB (Westinghouse FS-40 sunlamps) with various doses (0, 30, 60, 120, 240 mJ/cm2). The expression levels of caspase-3, Bax, Bcl2, Bcl-X(L) and LC3 were confirmed by Western blot analysis in UVB-irradiated HaCaT cell lines. Apoptotic cells were analyzed by PI staining, and autophagy cells were analyzed by immunofluorescent staining. RESULTS: The expression of Bcl-X(L) decreased from UVB 60 mJ/cm2 and Bcl2 decreased from UVB 240 mJ/cm2. The expression of caspase-3 was increased from UVB 120 mJ/cm2. These data showed that UVB-induced apoptosis is mediated by up-regulation of caspase-3 and down-regulation of Bcl2 and Bcl-X(L). Furthermore, the expression of LC3 increased from UVB 120 mJ/cm2. In addition, autophagy formation was observed in few fractions of apoptotic HaCaT cells in immunofluorescent staining; most apoptotic cells did not show autophagy formation. Moreover, autophagy formation inhibitor treatment induced a slight increment of apoptotic cell population under UVB irradiation. CONCLUSION: UVB irradiation induces not only apoptotic cell death but also autophagy formations; these events may create a defense mechanism for the prevention of apoptosis in UVB-treated HaCaT cells.
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Apoptosis , Autophagy , Blotting, Western , Caspase 3 , Cell Death , Cell Line , Down-Regulation , Keratinocytes , Up-RegulationABSTRACT
Objective To investigate the influence of Fe3 04 magnetic nanoparticles on human keratinocyte cells (HaCaT cells). Methods HaCaT cells were incubated with different concentrations of Fe304magnetic nanoparticles for 4 h at 37°C with 5% C02. Then transmission electron microscopy (TEM) was used to observe the way nanoparticles entering HaCaT cells and the ultrastructure of HaCaT cells. Results The mean diameter of Fe3 04 magnetic nanoparticles was 12 nm. The nanoparticles of different concentrations could enter the HaCaT cells by phagocytosis. After entering the cells the particles were released from phagocytic vesicles and exerted influence on the nearby mitochondria, leading to mitochondria swelling and cristae dissolving. Conclusion Fe3 04 magnetic nanoparticles can damage the ultrastructure of mitochondria near the particles in HaCaT cells, and the effect is in a concentration-dependent manner.
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Objective To investigate whether IL-17 could stimulate the vascular endothelial growth factor (VEGF) production on HaCaT cells alone. We also investigated whether shikonin could inhibited the proinflamation effects of interleukin-17(IL-17) acting on HaCaT cells. MethodsWe examined the expression of VEGF by double antibody sandwich enzyme-linked immunosorbent assay ( ELISA ) and realtime polymerase chain reaction(RT-PCR) in HaCaT cells and the cell supernatant. The viability of HaCaT cells in the drug group was detected by the Cell Counting Kit-8 (CCK-8). ResultsThe expression of VEGF in different time IL-17-stimulated groups on HaCaT cells and the cell supernatant were higher than the control group( P＜0.001 ). The expression of VEGF in different drug treatment groups on HaCaT cells and the cell supematant were lower than the stimulated group by IL-17 ( P＜0. 001 ). The cell viability of different drug treatment groups have no significant difference( P＞0.05 ). ConclusionWe show that IL-17 specifically and time-dependently augmented and induced VEGF expression on HaCaT cells and the cell supernatantThen shikonin markedly inhibited the increase tengency of IL-17 effection on HaCaT cells and the cell supematant level.
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BACKGROUND: Acne is a multifactorial disease. Particularly, Propionibacterium acne acts on triglycerides and releases cytokines, which trigger inflammatory reactions and alter the infundibular keratinization status. Recently, visible light, especially blue light, has been shown to activate the endogenous porphyrins of P. acne, which results in a photodynamic reaction that destroys the bacteria. However, the effect of blue light on inflamed lesion, inflammatory papules, pustules, are largely unknown. OBJECTIVE: We investigated the bactericidal effect of blue light on P. acnes as well as the inhibitory effect on the expression of inflammatory cytokines in HaCaT cells. METHODS: We performed the colony count assay in blue light irradiated-culture media of P. acnes and RT-PCR analysis in blue light irradiated-HaCaT immortalized keratinocytes. RESULTS: A 7 minute irradiation with blue light was bactericidal to P. acnes. It decreased the expression of tumor necrosis factor (TNF)-alphaand toll-like receptor (TLR) 2 in HaCaT cells. Expression of TNF-alpha-induced TNF-alpha and TLR 2 were down-regulated by blue light in HaCaT cells. CONCLUSION: Blue light is a potential anti-acne treatment.
Subject(s)
Acne Vulgaris , Bacteria , Cytokines , Keratins , Light , Polyenes , Porphyrins , Propionibacterium , Propionibacterium acnes , Toll-Like Receptors , Triglycerides , Tumor Necrosis Factor-alphaABSTRACT
BACKGROUND: Ultraviolet B (UVB) radiation is a major harmful environmental factor causing cutaneous changes such as sunburn, skin aging and skin cancer. Excessive UVB irradiation induces apoptosis of keratinocytes through several molecular pathways. However, the precise molecular mechanisms have been underinvestigated. OBJECTIVE: The purpose of this study was to investigate expression levels of apoptosis-related genes in UVB- irradiated HaCaT ketratinocyte cell lines. METHODS: Cells were irradiated by UVB at various doses (0, 100, 200 and 400 mJ/cm2). Expression levels of caspases, Bax, Bcl2, and Bcl-XL were confirmed by RT-PCR analysis and western blotting. RESULTS: Expression of cytochrome C was increased followed by activation of caspase-3, 8 and 9 in UVB-irradiated HaCaT cells. Furthermore, the expression of Bcl-XL was decreased from UVB 200 mJ/cm2 and Bcl2 was decreased weakly from UVB 400 mJ/cm2, implying that the expression of Bcl-XL is more sensitive to UVB. Interestingly, the down-regulation of Bcl-XL may be mediated by proteasome dependent pathways. CONCLUSION: Excessive UVB-irradiated HaCaT cells undergo apoptotic cell death following activation of caspases; degradation of Bcl-XL is particularly sensitive to UVB.
Subject(s)
Apoptosis , Caspase 3 , Caspases , Cell Death , Cytochromes c , Down-Regulation , Keratinocytes , Proteasome Endopeptidase Complex , Skin Aging , Skin Neoplasms , SunburnABSTRACT
BACKGROUND: Ultraviolet B (UVB) radiation is a major harmful environmental factor causing cutaneous changes such as sunburn, skin aging and skin cancer. Excessive UVB irradiation induces apoptosis of keratinocytes through several molecular pathways. However, the precise molecular mechanisms have been underinvestigated. OBJECTIVE: The purpose of this study was to investigate expression levels of apoptosis-related genes in UVB- irradiated HaCaT ketratinocyte cell lines. METHODS: Cells were irradiated by UVB at various doses (0, 100, 200 and 400 mJ/cm2). Expression levels of caspases, Bax, Bcl2, and Bcl-XL were confirmed by RT-PCR analysis and western blotting. RESULTS: Expression of cytochrome C was increased followed by activation of caspase-3, 8 and 9 in UVB-irradiated HaCaT cells. Furthermore, the expression of Bcl-XL was decreased from UVB 200 mJ/cm2 and Bcl2 was decreased weakly from UVB 400 mJ/cm2, implying that the expression of Bcl-XL is more sensitive to UVB. Interestingly, the down-regulation of Bcl-XL may be mediated by proteasome dependent pathways. CONCLUSION: Excessive UVB-irradiated HaCaT cells undergo apoptotic cell death following activation of caspases; degradation of Bcl-XL is particularly sensitive to UVB.