铁死亡相关基因在瘢痕疙瘩中的表达及意义

The expression and significance of ferroptosis-related genes in human keloid tissue

目的:探究铁死亡相关基因在人瘢痕疙瘩组织中的表达及意义。方法:收集新疆医科大学第一附属医院整形科2022年1月至2022年6月诊治的35例瘢痕疙瘩及因其他手术切除的20例正常皮肤组织标本,应用免疫组织化学染色法检测瘢痕疙瘩和正常皮肤组织中铁死亡标志物谷胱甘肽过氧化物酶4(GPX4)的表达情况,实时定量聚合酶链反应技术(RT-qPCR)检测各组GPX4、溶质载体家族7成员11(SLC7A11,又称xCT)、转铁蛋白受体(TFRC)及核转录因子红系2相关因子2(Nrf2)mRNA的表达水平,蛋白质印迹法检测各组GPX4、xCT、TFRC及α-平滑肌肌动蛋白(α-SMA)的蛋白表达水平,比色法检测各组组织铁含量。计量资料符合正态分布时,组间比较采用t检验,若不符合正态分布,组间比较采用Mann-Whitney-U检验。结果:免疫组织化学染色结果显示,瘢痕疙瘩组铁死亡标志物GPX4的蛋白表达水平低于正常皮肤组(0.77±0.08比0.92±0.09,t=-3.54,P<0.01)。RT-qPCR结果显示,瘢痕疙瘩组GPX4、xCT及Nrf2 mRNA的表达水平低于正常皮肤组[GPX4:0.74±0.29比1.14±0.49,t=-3.22,P<0.01;xCT:0.81(0.71,1.25)比1.52(0.86,4.64),Z=-2.679,P<0.01;Nrf2:0.49(0.38,1.04)比1.25(0.49,1.73),Z=-2.234,P<0.05],TFRC mRNA的表达水平高于正常皮肤组[1.68(1.13,2.36)比0.87(0.48,1.35),Z=-3.886,P<0.01]。蛋白质印迹法结果显示,瘢痕疙瘩组GPX4、xCT的蛋白表达水平低于正常皮肤组[GPX4:0.75(0.42,0.91)比1.04(0.92,1.96),Z=-3.731;xCT:0.71(0.55,0.86)比0.98(0.95,1.14),Z=-4.941,P均<0.001],TFRC及α-SMA的蛋白表达水平高于正常皮肤组(TFRC:1.13±0.22比0.62±0.16,t=6.342;α-SMA:1.33±0.12比0.51±0.21,t=9.669,P均<0.001)。瘢痕疙瘩组铁含量高于正常皮肤组[(7.59±1.60)μg/g比(3.05±1.28)μg/g,t=9.086,P<0.01]。结论:瘢痕疙瘩中存在铁死亡现象。

Objective To explore the expression and significance of ferroptosis-related genes in human keloid tissue.Methods A total of 35 tissue specimens of keloid(13 males and 22 females,aged 10-65 years old)and 20 tissue specimens of normal skin(8 males and 12 females,aged 20-50 years old)excised by other operations were collected from the Department of Plastic Surgery,First Affiliated Hospital of Xinjiang Medical University from January 2022 to June 2022.The expression of ferroptosis marker glutathione peroxidase 4(GPX4)in keloid and normal skin tissues was detected by immunohistochemical staining,and the mRNA expression levels of GPX4,solute carrier family 7,member 11(SLC7A11,also known as xCT),transferrin receptor(TFRC)and nuclear factor erythroid 2-related factor 2(Nrf2)in each group were detected by real-time quantitative polymerase chain reaction(RT-qPCR).The protein expression levels of GPX4,xCT,TFRC andα-smooth muscle actin(α-SMA)in each group were detected by Western blotting,and the iron content of the two groups was detected by colorimetric method.When measurement data were in accordance with normal distribution,t-test was used for comparison between two groups.Otherwise,Mann-Whitney-U test was used for comparison between two groups.Results Immunohistochemical staining showed that the protein expression level of GPX4 in keloid group was lower than that in normal skin group(0.77±0.08 vs.0.92±0.09,t=-3.54,P<0.01).RT-qPCR showed that the mRNA expression levels of GPX4,xCT and Nrf2 in keloid group were lower than those in normal skin group[GPX4:0.74±0.29 vs.1.14±0.49,t=-3.22,P<0.01;xCT:0.81(0.71,1.25)vs.1.52(0.86,4.64),Z=-2.679,P<0.01;Nrf2:0.49(0.38,1.04)vs.1.25(0.49,1.73),Z=-2.234,P<0.05],and the mRNA expression level of TFRC was higher than that in normal skin group[1.68(1.13,2.36)vs.0.87(0.48,1.35),Z=-3.886,P<0.01].Western blotting showed that the protein expression levels of GPX4 and xCT in keloid group were lower than those in normal skin group[GPX4:0.75(0.42,0.91)vs.1.04(0.92,1.96),Z=-3.731;xCT:0.71(0.55,0.86)vs.0.98(0.95,1.14),Z=-4.941,all P<0.001],and the protein expression levels of TFRC andα-SMA were higher than those in normal skin group(TFRC:1.13±0.22 vs.0.62±0.16,t=6.342;α-SMA:1.33±0.12 vs.0.51±0.21,t=9.669,all P<0.001).The iron content in keloid group was higher than that in normal skin group(7.59±1.60)μg/g vs.(3.05±1.28)μg/g,t=9.086,P<0.01).Conclusion The phenomenon of ferroptosis exists in keloid tissue.