印度块菌提取物对长波紫外线诱导人皮肤成纤维细胞光损伤的保护作用研究

Protective effect of Tuber indicum extract on UVA-induced photodamage in HSF cells

目的:探讨印度块菌提取物对长波紫外线(UVA)诱导的人皮肤成纤维细胞(HSF)光损伤的保护作用及其相关机制。方法:通过UVA辐射建立细胞光损伤模型。不同剂量印度块菌提取物预处理HSF细胞后,采用四甲基偶氮唑蓝(MTT)法检测HSF细胞活力,于倒置显微镜观察HSF细胞形态变化;采用Hoechst 33342染色观察细胞凋亡形态;应用荧光探针法分别检测活性氧(ROS)及羰基化蛋白的表达水平;应用酶联吸附免疫(ELISA)法检测Ⅰ型胶原蛋白(ColⅠ)和透明质酸(HA)含量。结果:应用10 J/cm^2 UVA辐射后,HSF细胞数量减少,融合度下降,细胞形态皱缩,细胞活力降低,细胞凋亡增加,ROS和羰基蛋白表达水平升高,ColⅠ和HA含量均降低。与模型组相比,100 mg/L和200 mg/L印度块菌提取物预处理后,细胞形态趋于正常,细胞活力分别由61.97%提高为72.27%和73.94%(P<0.05),且细胞凋亡减少,ROS表达水平分别由156.57%降低为133.70%和118.45%(P<0.05),羰基蛋白荧光比值显著降低,同时ColⅠ和HA含量均显著升高。结论:印度块菌提取物能减轻UVA诱导的皮肤光损伤效应,其机制可能与抑制细胞的氧化损伤,减少ColⅠ和HA的降解有关。

Objective:To determine the protective effect of Tuber indicum extract on UVA-induced photodamage in human skin fibroblasts(HSF)and to explore the underlying mechanism.Methods:Cultured normal skin fibroblasts were pretreated with different doses of Tuber indicum extract and irradiated with UVA to develop photodamage.Cell viability was detected by MTT method.The damage of fibroblasts was observed with inverted microscope.Apoptosis morphology was observed by Hoechst 33342 staining.The level of reactive oxygen species(ROS)was observed by H2DCFDA detection.The level of carbonyl protein(CP)was observed by FTSC detection.ColⅠand hyaluronic acid(HA)were detected by ELISA.Results:Sustained exposure to 10 J/cm^2 UVA induced photodamage in HSF cells.Compared with the control group,the cell number and HSF fusion were decreased.Cell morphology shrinkage was also observed.Cell viability of HSF cells was decreased.The apoptosis of HSF was promoted.The levels of ROS and CP were evidently increased.The contents of ColⅠand HA were significantly decreased.Compared with the model group,Tuber indicum extract(100 mg/L and 200 mg/L)restored the cell morphology with the survival rate of HSF cells increasing from 61.97%to 72.27%and 73.94%(P<0.05).The apoptosis was diminished and the ROS expression level decreased from 156.57%to 133.70%and 118.45%(P<0.05).Furthermore,CP expression level obviously decreased,while the contents of ColⅠand HA significantly increased after the treatment of Tuber indicum extracts at the concentrations of 100 mg/L and 200 mg/L.Conclusion:Tuber indicum extract can protect HSF from UVA-induced injury,which may be attributed to the inhibition of cell oxidative damage and the reduction of ColⅠand HA degradation.