铁死亡诱导剂抑制增生性瘢痕成纤维细胞的增殖

Erastin inhibits proliferation of hypertrophic scar fibroblasts

背景:增生性瘢痕是各种原因导致的皮肤创伤后愈合过程中出现的一种病理性瘢痕,目前缺乏特效治疗方法。目的:探讨铁死亡诱导剂Erastin对人增生性瘢痕成纤维细胞增殖的影响。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和同一个体正常皮肤,提取人增生性瘢痕成纤维细胞进行后续实验。将细胞分为对照组(不做处理)和铁死亡诱导剂组(用20μmol/L的Erastin干预细胞24 h),Western blot检测铁蛋白(Ferritin)的表达,铁离子检测试剂盒测定细胞铁离子浓度,丙二醛检测试剂盒检测细胞丙二醛水平;将细胞分为对照组(不做处理)、铁死亡诱导剂组(用20μmol/L的Erastin干预细胞24 h)和铁死亡诱导剂+铁死亡抑制剂组(用20μmol/L的Erastin和20μmol/L的Ferrostatin-1同时干预细胞24 h),qRT-PCR检测PCNA及p27的mRNA表达,Western blot检测PCNA及p27的蛋白表达,CCK-8、EdU检测细胞增殖活力和增殖水平。结果与结论:①与对照组相比,铁死亡诱导剂组铁蛋白减少(P<0.01),铁离子增多(P<0.05),丙二醛增多(P<0.01);②与对照组相比,铁死亡诱导剂组PCNA的表达降低(P<0.01),p27的表达增加(P<0.05),细胞增殖能力减弱(P<0.01),EdU阳性细胞数减少(P<0.01);③与铁死亡诱导剂组相比,铁死亡诱导剂+铁死亡抑制剂组PCNA的表达增加(P<0.05),p27的表达降低(P<0.05),细胞增殖能力增强(P<0.01),EdU阳性细胞数增多(P<0.05);④结果表明,铁死亡诱导剂Erastin通过诱导增生性瘢痕成纤维细胞发生铁死亡进而抑制其增殖。

BACKGROUND:Hypertrophic scar is a kind of pathological scar that appears in the healing process after skin trauma caused by various reasons and there is no effective treatment.OBJECTIVE:To investigate the effect of ferroptosis inducer(Erastin)on the proliferation of human hypertrophic scar fibroblasts.METHODS:Hypertrophic scar samples provided by the Burn Plastic Surgery Department of the General Hospital of Ningxia Medical University and normal skin samples of the same individual were collected.Human hypertrophic scar fibroblasts were then extracted for subsequent experiments.(1)The cells were divided into control group(without treatment)and ferroptosis inducer group(treated with 20μmol/L Erastin for 24 hours).The expression of Ferritin was detected by western blot.Iron ion detection kit was used to measure cellular iron ion concentration.Malondialdehyde detection kit was used to detect cellular malondialdehyde content.(2)The cells were divided into control group(without treatment),ferroptosis inducer group(treated with 20μmol/L Erastin for 24 hours)and ferroptosis inducer+ferroptosis inhibitor group(co-treated with 20μmol/L Erastin and 20μmol/L Ferrostatin-1 for 24 hours).The mRNA and protein expressions of proliferating cell nuclear antigen(PCNA)and cyclin-dependent kinase inhibitor(p27)were detected using qRT-PCR and western blot.Cell counting kit-8 and EdU were used to detect cell proliferation viability and levels.RESULTS AND CONCLUSION:Compared with the control group,Erastin decreased the ferritin expression(P<0.01),increased the content of iron ions(P<0.05),and elevated the malondialdehyde content in the cells(P<0.01).Compared with the control group,Erastin decreased the expression of PCNA(P<0.01),increased the expression of p27(P<0.05),weakened the cell proliferation ability(P<0.01),and reduced the number of EdU-positive cells(P<0.01).Compared with the ferroptosis inducer group,the ferroptosis inducer+ferroptosis inhibitor group had increased expression of PCNA(P<0.05),decreased p27 expression(P<0.05),enhanced cell proliferation(P<0.01),and increased number of EdU-positive cells(P<0.05).To conclude,the ferroptosis inducer(Erastin)induces ferroptosis in hypertrophic scar fibroblasts and then inhibits their proliferation.