摘要
目的:初步探讨藏红花素(Crocin)调节Hippo信号通路对病理性瘢痕成纤维细胞增殖和凋亡的影响。方法:体外分离培养人瘢痕疙瘩成纤维细胞(HKF),免疫荧光鉴定成纤维细胞;MTT法计算细胞增殖抑制率,筛选Crocin最佳干预浓度,将HKF细胞分为control组和Crocin低、中、高剂量组(Crocin浓度分别为20、50、100μmol/L);细胞转染实验中将HKF细胞分为control组、Crocin组(100μmol/L)、pcDNA3.1组(转染pcDNA3.1)、pcDNA3.1+Crocin组(转染pcDNA3.1+100μmol/L Crocin干预)、pcDNA3.1-YAP/TAZ组(转染pcDNA3.1-YAP/TAZ)、pcDNA3.1-YAP/TAZ+Crocin组(转染pcDNA3.1-YAP/TAZ+100μmol/L Crocin干预)。qRT-PCR法检测YAPmRNA、TAZ mRNA表达;EdU染色、流式细胞仪分别检测细胞增殖、凋亡情况;Western blot检测细胞中增殖细胞核抗原(PCNA)、Bcl-2相关X蛋白(Bax)、细胞抗凋亡因子B淋巴细胞瘤-2(Bcl-2)及Hippo通路蛋白表达。结果:Crocin以浓度依赖性的方式抑制HKF细胞增殖,IC50=111.56μmol/L;与control组比较,Crocin低、中、高剂量组HKF细胞中YAP、TAZ mRNA表达水平EdU阳性细胞率、PCNA、Bcl-2、p-YAP/YAP、TAZ蛋白表达水平显著降低,凋亡率及Bax蛋白表达水平升高(P<0.05);转染YAP/TAZ后,显著减弱了藏红花素对HKF细胞增殖的抑制作用及对HKF细胞凋亡的促进作用。结论:藏红花素可能通过抑制YAP/TAZ信号通路,抑制HKF细胞增殖,促进其凋亡。
Objective:To preliminary investigate the impacts of Crocin on the proliferation and apoptosis of fibroblasts from pathological scars by regulating Hippo signaling pathway.Method:Human keloid fibroblasts(HKF)were isolated and cultured in vitro,and identified by immunofluorescence assay.The inhibition rate of cell proliferation was calculated by MTT method,and the optimal intervention concentration of Crocin was screened.The expression of YAP and TAZ mRNA was detected by qRT-PCR method,cell proliferation and apoptosis were detected by EdU staining,and flow cytometry,respectively.Western blot was applied to detect the expression of proliferating cell nuclear antigen(PCNA),Bcl-2 associated X protein(Bax),cell anti apoptotic factor B cell lymphocyte tumor 2(Bcl-2),and Hippo pathway protein.Results:Crocin inhibited the proliferation of HKF cells in a concentration-dependent manner,IC50=111.56μmol/L.The positive rates of EdU in control group,Crocin low,medium and high dose groups were(39.56±2.45)%,(31.42±2.17)%,(23.81±1.79)%,(14.37±1.12)%,respectively.The apoptosis rates were(8.67±1.06)%,(14.85±1.12)%,(23.72±1.35)%,(31.47±2.64)%,respectively.Compared with the control group,the expression levels of YAP,TAZ mRNA,EdU positive cell rate,the expression levels of PCNA,Bcl-2,p-YAP/YAP,and TAZ proteins in HKF cells in Crocin low,medium,and high dose groups were obviously decreased,the apoptosis rate and the expression level of Bax protein were increased(P<0.05).After overexpression of YAP/TAZ,the inhibitory effect of crocin on the proliferation of HKF cells and the promoting effect on the apoptosis of HKF cells were significantly weakened(P<0.05).Conclusion:Crocin may inhibit the proliferation of HKF cells and promote their apoptosis by inhibiting the YAP/TAZ signaling pathway.
作者
王献珍
崔强
吴晓伟
张科伟
WANG Xian-zhen;CUI Qiang;WU Xiao-wei;ZHANG Ke-wei(Department of Burn Plastic Surgery,Qinghai University Affiliated Hospital,Xining 810001,China)
出处
《微循环学杂志》
2024年第1期14-22,共9页
Chinese Journal of Microcirculation
基金
青海省2023年中央引导地方科技发展资金计划项目(20232Y021)。
