紫草素调控MicroRNA-382-5p抑制人增生性瘢痕成纤维细胞纤维化

Shikonin inhibits fibrosis of human hypertrophic scar fibroblasts via MicroRNA-382-5p

背景:目前已有研究表明紫草素具有治疗增生性瘢痕的潜力,且miRNA参与增生性瘢痕的病理机制调控,猜测紫草素有可能通过影响miRNA调控增生性瘢痕的发生发展。目的:探讨紫草素通过影响MicroRNA-382-5p对人增生性瘢痕成纤维细胞纤维化的作用机制。方法:收集宁夏医科大学总医院烧伤整形外科提供的增生性瘢痕组织及瘢痕旁正常皮肤组织(瘢痕旁3 cm以内),并分别分离出人增生性瘢痕成纤维细胞和正常成纤维细胞用于后续实验。苏木精-伊红染色鉴定正常皮肤和增生性瘢痕;免疫荧光鉴定成纤维细胞;采用实时荧光定量PCR从组织水平检测MicroRNA-382-5p相对表达水平。将增生性瘢痕成纤维细胞随机分为紫草素组(紫草素溶于二甲基亚砜配成浓度为13.46μmol/L的药物干预细胞24 h)、二甲基亚砜组、MicroRNA-382-5p阴性对照组、MicroRNA-382-5p抑制组、紫草素+MicroRNA-382-5p阴性对照组及紫草素+MicroRNA-382-5p过表达组。实时荧光定量PCR检测Ⅰ型胶原蛋白α1、Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白mRNA表达;Western blot检测Ⅰ型胶原蛋白α1、Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的蛋白表达;CCK-8法检测细胞活性;划痕实验检测细胞迁移能力。结果与结论:①与正常皮肤成纤维细胞相比,增生性瘢痕成纤维细胞增殖活性更强(P<0.01);②与二甲基亚砜组相比,紫草素组可以下调增生性瘢痕成纤维细胞中Ⅰ型胶原蛋白α1、Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的表达水平(mRNA:P<0.01,蛋白:P<0.01),并抑制增生性瘢痕成纤维细胞迁移(P<0.05);③与正常皮肤相比,MicroRNA-382-5p在增生性瘢痕中呈高表达(P<0.01),且紫草素能下调增生性瘢痕成纤维细胞中MicroRNA-382-5p的表达(P<0.01);④敲低MicroRNA-382-5p能下调增生性瘢痕成纤维细胞中Ⅰ型胶原蛋白α1、Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的表达水平(mRNA:P<0.01,蛋白:P<0.01),并抑制增生性瘢痕成纤维细胞迁移(P<0.05);⑤过表达MicroRNA-382-5p能上调在紫草素影响下增生性瘢痕成纤维细胞中Ⅰ型胶原蛋白α1、Ⅲ型胶原蛋白α1和α-平滑肌肌动蛋白的表达水平(mRNA:P<0.01,蛋白:P<0.01),并促进增生性瘢痕成纤维细胞迁移(P<0.01);⑥提示紫草素可通过下调MicroRNA-382-5p的表达抑制增生性瘢痕成纤维细胞的纤维化和迁移。

BACKGROUND:Previous studies have shown that shikonin has the potential to treat hypertrophic scar and that microRNAs are involved in the regulation of the pathological mechanism of hypertrophic scar.It is speculated that shikonin may regulate the occurrence and development of hypertrophic scar through microRNAs regulation.OBJECTIVE:To investigate the mechanism of shikonin on the fibrosis of human hypertrophic scar fibroblasts via MicroRNA-382-5p.METHODS:Hypertrophic scar tissue and normal skin tissue adjacent to the scar(within 3 cm around the scar)were provided by the Department of Burn and Plastic Surgery,General Hospital of Ningxia Medical University to extract human hypertrophic scar fibroblasts and human normal skin fibroblasts,respectively.Hematoxylin-eosin staining was used to identify normal skin and hypertrophic scar and immunofluorescence was used to identify fibroblasts.The relative expression level of MicroRNA-382-5p was detected by quantitative real-time PCR at the tissue level.Hypertrophic scar fibroblasts were randomly divided into shikonin group(shikonin was dissolved in dimethyl sulfone to make drug solution at a concentration of 13.46μmol/L,which was used for cell culture for 24 hours),dimethyl sulfone group,MicroRNA-382-5p negative control group,MicroRNA-382-5p inhibitor group,shikonin+MicroRNA-382-5p negative control group and shikonin+MicroRNA-382-5p overexpression group.Real-time fluorescence quantitative PCR and western blot were used to detect the expression of Collagen Type Iα1,Collagen Type IIIα1 andα-smooth muscle actin at mRNA and protein levels,respectively.Cell counting kit-8 was used to detect cell viability.Cell scratch test was used to detect the migration ability of cells.RESULTS AND CONCLUSION:Compared with normal skin fibroblasts,hypertrophic scar fibroblasts had stronger proliferative activity(P<0.01).Compared with the dimethyl sulfone group,shikonin down-regulated the expression levels of Collagen Type Iα1,Collagen Type IIIα1 andα-smooth muscle actin in human hypertrophic scar fibroblasts(mRNA:P<0.01,protein:P<0.01),and inhibited the migration of hypertrophic scar fibroblasts(P<0.05).Compared with normal skin,MicroRNA-382-5p was highly expressed in hypertrophic scar(P<0.01),and shikonin could down-regulate the expression of MicroRNA-382-5p(P<0.01).Inhibition of MicroRNA-382-5p down-regulated the expression level of Collagen Type Iα1,Collagen Type IIIα1 andα-smooth muscle actin in hypertrophic scar fibroblasts(mRNA:P<0.01,protein:P<0.01),and inhibited the migration of hypertrophic scar fibroblasts(P<0.05).Under the influence of shikonin,overexpression of MicroRNA-382-5p upregulated the expression levels of Collagen Type Iα1,Collagen Type IIIα1 andα-smooth muscle actin in hypertrophic scar fibroblasts(mRNA:P<0.01,protein:P<0.01),and promoted the migration of hypertrophic scar fibroblasts(P<0.01).To conclude,shikonin can inhibit the fibrosis and migration of hypertrophic scar fibroblasts by down-regulating the expression of MicroRNA-382-5p.