摘要
目的 探讨红景天苷对瘢痕疙瘩(KD)成纤维细胞异常增殖及细胞外基质沉积的作用机制.方法 将原代KD成纤维细胞随机分为模型组(正常培养)、低剂量组(50 μmol·L-1红景天苷)、高剂量组(100 µmol·L-1红景天苷)、Vector组(100 μmol·L-1红景天苷+转染Vector)、泛素羧基末端水解酶L1(UCHL1)组(100 µmol·L-1红景天苷+过表达UCHL1).用实时荧光定量聚合酶链反应(RT-qPCR)实验检测UCHL1 mRNA表达水平,用流式细胞术检测细胞凋亡,用蛋白质印迹(Western blot)法实验检测各组细胞Ⅰ型胶原蛋白(Collagen Ⅰ)、谷胱甘肽过氧化物酶4(GPX4)、α平滑肌肌动蛋白(α-SMA)蛋白相对表达水平,用DCFH-DA法检测细胞活性氧(ROS)水平.结果 模型组、低剂量组、高剂量组、Vector组和UCHL1组的UCHL1 mRNA相对表达水平分别为 1.01±0.11、0.71±0.07、0.49±0.05、0.50±0.05 和 2.36±0.13,细胞凋亡率分别为(4.59±0.56)％、(12.11±0.80)％、(22.16±3.21)％、(23.54±2.08)％和(9.04±0.72)％,Collagen Ⅰ蛋白相对表达水平分别为0.97±0.06、0.82±0.07、0.53±0.04、0.54±0.07 和 0.91±0.11,α-SMA 蛋白相对表达水平分别为 1.11±0.07、0.79±0.06、0.61±0.08、0.60±0.04 和 0.96±0.11,GPX4 蛋白相对表达水平分别为 0.93±0.06、0.66±0.04、0.41±0.03、0.43±0.03和0.78±0.04,ROS 水平分别为(100.00±5.03)％、(127.18±10.15)％、(146.09±10.15)％、(144.75±10.95)％和(106.57±11.28)％.低、高剂量组的上述指标与模型组比较,在统计学上差异均有统计学意义(均P＜0.05),UCHL1组的上述指标与Vector组比较,在统计学上差异均有统计学意义(均P＜0.05).结论 红景天苷可诱导铁死亡,促进成纤维细胞凋亡,并抑制细胞外基质过度沉积,进而减缓KD进展,这可能与抑制UCHL1表达有关.
Abstract
Objective To investigate the mechanism of salidroside on abnormal proliferation and extracellular matrix deposition of keloid(KD)fibroblasts.Methods The primary KD fibroblasts were randomly divided into model group(normal culture),experimental-L group(50 μmol·L-1 salidroside),experimental-H group(100 μmol·L-1 salidroside),Vector group(100 μmol·L-1 salidroside+Vector),ubiquitin C-terminal hydrolase L1(UCHL1)group(100 μmol·L-1 salidroside+overexpression UCHL1);The mRNA expression level of UCHL1 was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR);flow cytometry was used to detect apoptosis;Western blot assay was used to detect the expression levels of Collagen Ⅰ,glutathione peroxidase 4(GPX4)and α-smooth muscle actin(α-SMA);the reactive oxygen species(ROS)levels was detected by DCFH-DA method.Results The relative expression levels of UCHL1 mRNA in model group,experimental-L,experimental-H,vector group and UCHL1 group were 1.01±0.11,0.71±0.07,0.49±0.05,0.50±0.05 and 2.36±0.13,respectively;the apoptosis rates were(4.59±0.56)％,(12.11±0.80)％,(22.16±3.21)％,(23.54±2.08)％,(9.04±0.72)％,respectively;Collagen Ⅰ protein expression levels were 0.97±0.06,0.82±0.07,0.53±0.04,0.54±0.07 and 0.91±0.11,respectively;α-SMA protein expression levels were 1.11±0.07,0.79±0.06,0.61±0.08,0.60±0.04,0.96±0.11,respectively;GPX4 protein expression levels were 0.93±0.06,0.66±0.04,0.41±0.03,0.43±0.03,0.78±0.04,respectively;ROS levels were(100.00±5.03)％,(127.18±10.15)％,(146.09±10.15)％,(144.75±10.95)％,(106.57±11.28)％,respectively.Compared with the model group,the above indexes in the experimental-L,-H groups were statistically significant(all P＜0.05).Compared with the Vector group,the above indexes in the UCHL1 group were statistically significant(all P＜0.05).Conclusion Salidroside can induce iron death,promote fibroblast apoptosis,inhibit excessive extracellular matrix deposition,and slow down KD progression,which may be related to the inhibition of UCHL1 expression.
维普
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