五倍子酸通过TGF-β/Smads信号通路对人瘢痕成纤维细胞生长抑制效应的初步研究

A preliminary study on the inhibitory effect of gallic acid on the growth of human keloid fibroblasts by the transforming growth factor-β/Sma-and Mad-related proteins signaling pathway

目的研究五倍子酸对瘢痕疙瘩成纤维细胞的形态、增殖、细胞周期、胶原收缩程度及对转化生长因子β(TGF-β)/Sma和Mad相关蛋白(Smads)信号通路的影响,探讨五倍子酸治疗瘢痕疙瘩的作用及机制。方法2022年8-12月在武汉市第一医院皮肤外科获取术中切除的经临床及病理确诊的瘢痕疙瘩组织标本3份,采用组织块培养法进行原代成纤维细胞培养,取第3~8代细胞进行实验。设置低、中、高剂量五倍子酸组(分别用0.025、0.05、0.1 mg/ml五倍子酸干预细胞)及对照组(仅用含10%胎牛血清的DMEM培养细胞)。培养24、48、72 h后,细胞计数(CCK8)法检测各组细胞增殖活性,三维培养观察胶原收缩情况。按上述分组培养细胞24 h后,在微分干涉倒置荧光显微镜下拍照并使用流式细胞仪分析各组细胞周期变化。使用0.1 mg/ml五倍子酸干预细胞作为实验组(高剂量五倍子酸组),与对照组细胞分别培养24 h后,酶联免疫吸附试验(ELISA)测定两组TGF-β1、2、3浓度变化,实时荧光定量PCR检测TGF-β1、2、3,Smad2、3、4及α平滑肌肌动蛋白(SMA)mRNA相对表达水平。统计学分析采用t检验、单因素方差分析及两因素方差分析,两两多重比较采用LSD-t检验。结果与对照组相比,随五倍子酸剂量增加,镜下瘢痕疙瘩成纤维细胞形态发生改变,逐渐出现胞体缩小、细胞间距变大、萎缩、凋亡细胞增多现象。CCK8结果显示,随五倍子酸剂量增加及作用时间延长,细胞增殖活力变化显著(F组别=78.31,P<0.001;F时间=4.17,P=0.037),其中高剂量五倍子酸组24、48、72 h时瘢痕疙瘩成纤维细胞增殖活力均显著低于对照组(均P<0.05)。三维培养显示,随培养时间延长,各组胶原均发生收缩,但收缩程度不同,对照组胶原明显收缩,各浓度五倍子酸组胶原收缩程度较低;作用24、48、72 h,中、高剂量五倍子酸组胶原收缩指数均显著低于对照组(均P<0.05)。流式细胞仪检测显示,处理24 h后,低、中、高剂量五倍子酸组细胞凋亡率(38.68%±3.05%、41.82%±2.19%、43.56%±3.58%)均显著高于对照组(12.58%±1.56%,均P<0.001);与对照组相比,中、高剂量五倍子酸组G0/G1期细胞比例显著较低(均P<0.01),而S期和G2/M期细胞比例则显著较高(均P<0.05)。ELISA结果显示,高剂量五倍子酸组TGF-β1浓度[(758.58±31.42)pg/ml]显著低于对照组[(1081.30±44.72)pg/ml,t=11.81,P<0.001],TGF-β2浓度[(71.05±7.40)pg/ml]与对照组[(76.43±6.51)pg/ml]相比差异无统计学意义(t=1.09,P=0.317),而TGF-β3浓度[(5.70±3.87)pg/ml]高于对照组[(0.00±0.00)pg/ml,t=2.94,P=0.026]。实时荧光定量PCR显示,与对照组相比,高剂量五倍子酸组TGF-β1、Smad2、Smad3、Smad4、α-SMA mRNA表达量均较低(均P<0.05),TGF-β3 mRNA表达量较高(t=6.78,P=0.002),而TGF-β2 mRNA表达量无明显变化(t=0.05,P=0.962)。结论五倍子酸可通过改变瘢痕疙瘩成纤维细胞周期,抑制其增殖,促进其凋亡,改变细胞形态,从而减少瘢痕形成及挛缩,其机制可能与抑制TGF-β/Smads信号通路有关。

Objective To investigate the effect of gallic acid on the morphology,proliferation and cell cycle of keloid fibroblasts,as well as on collagen contraction and the transforming growth factor-β(TGF-β)/Sma-and Mad-related proteins(Smads)signaling pathway,and to explore the role and mechanisms of action of gallic acid in the treatment of keloids.Methods From August to December 2022,3 keloid tissue samples were collected from 3 patients with clinically and pathologically confirmed keloids after surgery in the Department of Dermatologic Surgery,Wuhan No.1 Hospital.Primary fibroblasts were isolated and cultured by using the tissue culture method,and 3-to 8-passage fibroblasts were used for subsequent experiments.Cultured keloid fibroblasts were divided into 4 groups:low-,medium-and high-dose gallic acid groups treated with 0.025,0.05 and 0.1 mg/ml gallic acid respectively,and a control group cultured with Dulbecco′s modified Eagle′s medium(DMEM)containing 10%fetal calf serum.After 24-,48-,and 72-hour treatment,cellular proliferative activity was evaluated by cell counting kit 8(CCK8)assay,and collagen contraction by using a three-dimensional culture method.After 24-hour treatment in the above groups,pictures were taken using a differential interference inverted fluorescence microscope,and changes in the cell cycle were analyzed by flow cytometry.Some keloid fibroblasts were divided into 2 groups:an experimental group(high-dose gallic acid group)treated with 0.1 mg/ml gallic acid,and a control group cultured with DMEM containing 10%fetal calf serum.After 24-hour treatment,enzyme-linked immunosorbent assay(ELISA)was performed to determine the changes in supernatant concentrations of TGF-β1,TGF-β2,and TGF-β3 in the two groups,real-time fluorescence-based quantitative PCR to detect the relative mRNA expression levels of TGF-β1,TGF-β2,TGF-β3,Smad2,Smad3,Smad4,andα-smooth muscle actin(α-SMA).Statistical analysis was carried out using t test,one-way analysis of variance and two-way analysis of variance,and least significant difference(LSD)-t test was used for multiple comparisons.Results Compared with the control group,the gallic acid groups showed gradual changes in the shape of keloid fibroblasts under the microscope as the dose of gallic acid increased,including gradually shrinking cell bodies,enlarged intercellular spaces,cell atrophy,increased number of apoptotic cells,etc.CCK8 assay showed that the cellular proliferative activity changed significantly as the dose of gallic acid increased and the treatment time was prolonged(Fgroup=78.31,P<0.001;Ftime=4.17,P=0.037),and the proliferative activity of keloid fibroblasts was significantly lower in the high-dose gallic acid group than in the control group at 24,48,and 72 hours(all P<0.05).The three-dimensional culture showed that different degrees of collagen contraction occurred in all groups over time,marked collagen contraction was observed in the control group,and a lower degree of collagen contraction in the gallic acid groups;the collagen contraction indices were significantly lower in the medium-and high-dose gallic acid groups than in the control group at 24,48,and 72 hours(all P<0.05).Flow cytometry showed that the cell apoptosis rates were significantly higher in the low-,medium-and high-dose gallic acid groups(38.68%±3.05%,41.82%±2.19%,43.56%±3.58%,respectively)than in the control group(12.58%±1.56%,all P<0.001)after 24-hour treatment;compared with the control group,the medium-and high-dose gallic acid groups showed significantly decreased proportions of cells in the G0/G1 phase(both P<0.01),but significantly increased proportions of cells in the S phase and G2/M phase(all P<0.05).ELISA revealed that the TGF-β1 concentration was significantly lower in the high-dose gallic acid group(758.58±31.42 pg/ml)than in the control group(1081.30±44.72 pg/ml,t=11.81,P<0.001),there was no significant difference in the TGF-β2 concentration between the high-dose gallic acid group(71.05±7.40 pg/ml)and the control group(76.43±6.51 pg/ml,t=1.09,P=0.317),while the TGF-β3 concentration was significantly higher in the high-dose gallic acid group(5.70±3.87 pg/ml)than in the control group(0.00±0.00 pg/ml,t=2.94,P=0.026).As real-time fluorescence-based quantitative PCR revealed,the high-dose gallic acid group showed significantly decreased mRNA expression levels of TGF-β1,Smad2,Smad3,Smad4,andα-SMA(all P<0.05),but significantly increased mRNA expression level of TGF-β3(t=6.78,P=0.002)compared with the control group;however,there was no significant difference in the TGF-β2 mRNA expression level between the above two groups(t=0.05,P=0.962).Conclusion Gallic acid could change the cell cycle,inhibit the proliferative activity,promote apoptosis and change the shape of keloid fibroblasts,and thus inhibit scar formation and contraction,which may be related to the inhibition of TGF-β/Smads signaling pathway.