ABSTRACT
Objective To investigate effects of polyphylin Ⅰ on the proliferation and apoptosis of human melanoma cell line A375,and to explore their mechanisms.Methods Normal human melanocytes isolated from healthy human foreskin were divided into 6 groups to be treated with 0,1.5,3.0,6.0,9.0,12.0 mg/L polyphyllin Ⅰ respectively.A375 melanoma cells were divided into 4 groups,i.e.,control group,1.5-,3.0-,6.0-mg/L polyphyllin Ⅰ groups,to be treated with 0,1.5,3.0,6.0 mg/L polyphyllin Ⅰ,respectively.Cell counting kit-8 (CCK8) assay was performed to evaluate the effect of polyphyllin Ⅰ on the proliferation of normal human melanocytes and A375 cells.Hoechst 33258 fluorescent staining was conducted to observe the morphology of apoptotic cells,flow cytometry to estimate cell cycle phase distribution and apoptosis rate,dichloro-dihydro-fluorescein diacetate (DCFH-DA) fluorescent probe assay to detect the level of reactive oxygen species (ROS),rhodamine-123 staining to evaluate changes of mitochondrial membrane potential,spectrophotography to detect the level of ATP in A375 cells,as well as levels of lactic acid and glucose in the culture supernatant of A375 cells,and Western blot analysis to determine the protein expression of Bcl-2,Bcl-2-related X protein (Bax),cleaved-caspase-3,cyclin D1 and pyruvate kinase isozyme type M2 (PKM2).Statistical analysis was carried out by using one-way analysis of variance (ANOVA) for comparisons among groups and Student-Newman-Keuls-q (SNK-q) test for multiple comparisons.Results CCK8 assay showed that the treatment with polyphyllin Ⅰ at concentrations of 1.5,3.0,6.0 mg/L for 48 hours had no effects on the proliferation of normal human melanocytes,but significantly inhibited the proliferation of A375 cells.The survival rate of A375 cells was significantly lower in the 1.5-,3.0-,6.0-mg/L polyphyllin Ⅰ groups than in the control group (P ＜ 0.01).After the treatment with polyphyllin Ⅰ,distinct apoptotic morphology of A375 cells was observed under fluorescence microscope.Additionally,along with the increase of polyphyllin Ⅰ concentrations (0,1.5,3.0,6.0 mg/L),there were gradual increasing trends in the apoptosis rate of A375 cells (4.25％ ± 1.27％,10.03％ ± 1.49％,36.62％ ± 1.97％,44.11％ ± 2.47％ respectively,F =665.7,P ＜ 0.01),the percentage of A375 cells at G0/G1 phase (54.13％ ± 2.57％,67.35％ ± 3.79％,74.39％ ± 3.29％,82.29％ ± 3.99％ respectively,F =71.81,P ＜ 0.01),the level of ROS in A375 cells (P ＜ 0.01),the level of glucose in the culture supernatant (P ＜ 0.01),and the protein expression of Bax and cleaved-caspase-3 (both P ＜ 0.01),while gradual decreasing trends were found in the levels of mitochondrial membrane potential and ATP in A375 cells (both P ＜ 0.01),the level of lactic acid in the culture supernatant (P ＜ 0.01),and the protein expression of Cyclin D1,Bcl-2 and PKM2 (all P ＜ 0.01).Conclusion Polyphyllin Ⅰ can effectively induce A375 cell apoptosis by promoting the production of ROS in A375 cells and decreasing the mitochondrial membrane potential,and arrest A375 cells at G0/G1 phase by inhibiting the expression of PKM2 and Cyclin D1.
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Objective Toexplore the expression and significance of microRNA-155 (miR-155) in psoriasis vulgaris. Methods Areal-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) method with TaqMan probe technology was performed to detect miR-155 expression in skin lesion area and nonskinlesionalarea of 35 patients with psoriasis vulgaris , compared with that of 30 normal controls. The correlations among miR-155 expression, psoriasis area and severity index (PASI) score and Interleukin-17A(IL-17A)expression were studied. Results The expressions of miR-155 and IL-17A in lesional and non-lesionalgroups were higher than that of control group (all P < 0.01). Also expressions in lesional skin were higher than non-lesional skin (both P < 0.01). In skin lesion group, significant positive correlations existed betweenmiR-155 or IL-17A expression and PASI score as well as miR-155 and IL-17A expression (all P < 0.05). Conclusions Up-expression of miR-155 was relevant to psoriasis development , which is related withthe hyperfunctionof Th17 cells in psoriasis.
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Objective To explore the expression alteration and significance of inteleukin (IL)-37 in pso-riasis valguris (PV) patients. Methods Patients with PV had been treated with oral acitretin for 8 weeks. PASI score, ELISA and qRT-PCR were used to exam the data of 38 patients (PV group) and 32 controls (control group). Results IL-37 in PV group was significantly higher than that in control group (P 0.05) but decreased obviously after 8 weeks(P < 0.001). Signif-icant correlations existed among PASI scores, IL-37, IFN-γ and IL-17, as well as among IL-37 and IFN-γ, IL-17 (P < 0.05). Conclusions The increase of IL-37 is relevant to PV development and is associated with pa-tients’ conditions, IFN-γ and IL-17 but the alteration of IL-37 is not related with IL-4.
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Objective To discuss the expressions and clinical significance of p53 and p33ING1b in human psoriasis and basal cell carcinoma (BCC). Methods Immunohistochemistry EnVision technique was used to detect the expressions of p53 and p33ING1b in samples of 36 psoriasis vulgaris, 28 BCC and 14 normal skins. Results The expression of p53 increased while p33ING1b had a degressive expression in the control group, the psoriasis group and the BCC group. It was found significant statistical difference between the two groups (all P < 0.05). Prominent positive correlation between p53 and p33ING1b were found in both psoriasis group and BCC group (all P<0.05). Conclusions p53 coacts with p33ING1b at local lesions of abnormal proliferative diseases . It′s one of the most prominent mechanisms contributing to deviant cell proliferation.