Influence and mechanisms of metformin on the proliferation and apoptosis of human keloid fibroblasts
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摘要:
目的 探讨二甲双胍对人瘢痕疙瘩成纤维细胞（Fb）增殖及凋亡的影响及其机制。 方法 该研究为实验研究。收集于2020年9月—2023年9月在西安交通大学第一附属医院整形美容·颌面外科接受瘢痕疙瘩切除术治疗的7例瘢痕疙瘩患者（男2例、女5例,年龄20~65岁,病程均>1年）的瘢痕疙瘩组织,分离、培养原代Fb并选取第3~6代细胞进行实验。将细胞分为对照组和二甲双胍组,均采用完全培养基培养,二甲双胍组培养基中另加入终物质的量浓度为60 mmol/L的二甲双胍。采用细胞计数试剂盒-8检测2组细胞培养12、24 h后增殖活性并计算二甲双胍组细胞培养12、24 h后增殖抑制率（样本数为6）,采用细胞凋亡检测试剂盒检测对照组细胞培养0 h（即刻）后和二甲双胍组细胞培养12、24 h后凋亡细胞分布情况（样本数为3）,采用细胞周期检测试剂盒检测2组细胞培养12、24 h后周期分布（样本数为3）。将2组适量细胞培养24 h后进行真核mRNA测序,筛选2组间差异表达显著的差异表达基因（DEG）后,进行京都基因和基因组百科全书功能注释分析与功能富集分析。采用蛋白质印迹法检测2组细胞培养24 h后磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路中PI3K、磷酸化Akt（p-Akt）、磷酸化mTOR（p-mTOR）蛋白表达（样本数为3）。 结果 培养12、24 h后,二甲双胍组细胞增殖活性均显著低于对照组（t值分别为4.70、24.02,P<0.05）；二甲双胍组细胞培养24 h后增殖活性显著高于组内培养12 h后（t=4.73,P<0.05）。与组内培养12 h后比较,二甲双胍组细胞培养24 h后增殖抑制率显著升高（t=5.29,P<0.05）。与对照组培养0 h后比较,二甲双胍组细胞培养12、24 h后早期凋亡细胞比例均显著升高（t值分别为6.62、4.58,P<0.05）,早期凋亡+晚期凋亡细胞比例均显著升高（t值分别为4.84、3.75,P<0.05）；二甲双胍组细胞培养24 h后晚期凋亡细胞比例显著高于组内培养12 h后（t=4.55,P<0.05）。培养12 h后,二甲双胍组S期细胞比例显著低于对照组（t=5.90,P<0.05）；培养24 h后,与对照组比较,二甲双胍组G0/G1期细胞比例显著升高（t=5.36,P<0.05）,G2/M期细胞比例显著降低（t=17.63,P<0.05）；二甲双胍组培养24 h后S期细胞比例显著高于组内培养12 h后（t=7.60,P<0.05）。培养24 h后,与对照组相比,从二甲双胍组细胞中检测到4 814个差异表达显著的DEG,显著上调和显著下调的DEG主要参与了信号转导、细胞生长与死亡、运输与分解代谢、内分泌系统、免疫系统以及癌症相关的生物学功能；差异表达显著的DEG显著富集的通路包括细胞周期、DNA复制等,且在PI3K/Akt信号通路方面基因数量最多。培养24 h后,二甲双胍组细胞的PI3K、p-Akt与p-mTOR蛋白表达分别为0.190±0.017、0.170±0.017、0.247±0.005,显著低于对照组的0.440±0.026、0.300±0.060、0.547±0.025（t值分别为13.69、3.61、20.12,P值均<0.05）。 结论 二甲双胍可通过PI3K/Akt/mTOR信号通路显著抑制人瘢痕疙瘩Fb增殖,同时有效诱导其凋亡过程,从而发挥抗纤维化作用。 Abstract:Objective To investigate the influence and mechanisms of metformin on the proliferation and apoptosis of human keloid fibroblasts (Fbs). Methods This study was an experimental research. The keloid tissue was collected from 7 keloid patients (2 males and 5 females, aged 20-65 years, with a disease course of more than 1 year) who underwent keloid excision surgery at the Department of Plastic, Cosmetic and Maxillofacial Surgery of the First Affiliated Hospital of Xi'an Jiaotong University from September 2020 to September 2023. The primary Fbs were isolated and cultured, and cells from passages 3 to 6 were used for experiments. The cells were divided into control group and metformin group, and were cultured in complete medium. The medium for metformin group was supplemented with metformin at a final molarity of 60 mmol/L. The cell counting kit-8 was used to assess the proliferation activity of cells in two groups after 12 and 24 hours of culture, and the proliferation inhibition rate of cells in metformin group after 12 and 24 hours of culture was calculated, with a sample size of 6. The apoptosis detection kit was used to detect the apoptotic distribution of cells in control group after 0 hour (immediately) of culture and in metformin group after 12 and 24 hours of culture, with a sample size of 3. The cell cycle detection kit was used to detect the cycle distribution of cells in two groups after 12 and 24 hours of culture, with a sample size of 3. The eukaryotic mRNA sequencing was performed on suitable number of cells of two groups after 24 hours of culture, and the Kyoto encyclopedia of genes and genomes functional annotation analysis and functional enrichment analysis were performed after screening for differentially expressed genes (DEGs) with significantly differential expression between two groups. Western blotting was conducted to detect the protein expressions of phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt), and phosphorylated mammalian target of rapamycin (p-mTOR) in the PI3K/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway of cells in two groups after 24 hours of culture, with a sample size of 3. Results After 12 and 24 hours of culture, the proliferation activity of cells in metformin group was significantly lower than that in control group (with t values of 4.70 and 24.02, respectively, P<0.05); the proliferation activity of cells in metformin group after 24 hours of culture was significantly lower than that after 12 hours of culture within the group (t=4.73, P<0.05). Compared with that after 12 hours of culture within the group, the proliferation inhibition rate of cells in metformin group was significantly increased after 24 hours of culture (t=5.29, P<0.05). Compared with that in control group after 0 hour of culture, the proportion of early apoptotic cells in metformin group was significantly increased (with t values of 6.62 and 4.58, respectively, P<0.05), and the proportion of early and late apoptotic cells was significantly increased after 12 and 24 hours of culture (with t values of 4.84 and 3.75, respectively, P<0.05). After 24 hours of culture, the proportion of late apoptotic cells in metformin group was significantly higher than that after 12 hours of culture within the group (t=4.55, P<0.05). After 12 hours of culture, the proportion of S-phase cells in metformin group was significantly lower than that in control group (t=5.90, P<0.05). After 24 hours of culture, compared with that in control group, the proportion of G0/G1-phase cells in metformin group was significantly increased (t=5.36, P<0.05), while the proportion of G2/M-phase cells was significantly decreased (t=17.63, P<0.05). The proportion of S-phase cells in metformin group after 24 hours of culture was significantly higher than that after 12 hours of culture within the group (t=7.60, P<0.05). After 24 hours of culture, 4 814 DEGs with significantly differential expression were detected in the cells of metformin group compared with control group. The significantly upregulated and downregulated DEGs were mainly involved in biological functions related to signal transduction, cell growth and death, transport and catabolism, the endocrine system, the immune system, and cancer. The pathways that were significantly enriched with DEGs with significantly differential expression included the cell cycle and DNA replication, with the highest number of genes in the PI3K/Akt signaling pathway. After 24 hours of culture, the protein expressions of PI3K, p-Akt, and p-mTOR of cells in metformin group were 0.190±0.017, 0.170±0.017, and 0.247±0.005, respectively, which were significantly lower than 0.440±0.026, 0.300±0.060, and 0.547±0.025 in control group (with t values of 13.69, 3.61, and 20.12, respectively, P values all <0.05). Conclusions Metformin can significantly inhibit the proliferation of human keloid Fbs through the PI3K/Akt/mTOR signaling pathway and effectively induce its apoptotic process, thereby exerting antifibrotic effects. -
参考文献
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图 1 2组人瘢痕疙瘩成纤维细胞培养不同时间点形态及数量 倒置荧光显微镜（非荧光通道）×400。1A.对照组细胞培养0 h（即刻）后呈长梭形,数量较多；1B.二甲双胍组细胞培养12 h后数量较图1A减少,出现棒状形态；1C.二甲双胍组培养24 h后数量较图1B减少,伴细胞碎片增多
注：对照组、二甲双胍组细胞均采用完全培养基培养,二甲双胍组培养基中另加入终物质的量浓度为60 mmol/L的二甲双胍
图 2 火山图展示2组人瘢痕疙瘩成纤维细胞培养24 h后差异表达基因
注：对照组、二甲双胍组细胞均采用完全培养基培养,二甲双胍组培养基中另加入终物质的量浓度为60 mmol/L的二甲双胍；图中红色点表示二甲双胍组较对照组显著上调基因,蓝色点表示二甲双胍组较对照组显著下调基因,灰色点表示二甲双胍组较对照组无显著变化基因
图 3 2组人瘢痕疙瘩成纤维细胞培养24 h后差异表达显著的DEG的京都基因与基因组百科全书功能注释分析结果。3A.二甲双胍组较对照组显著上调DEG中基因数排前20的条目；3B.二甲双胍组较对照组显著下调DEG中基因数排前20的条目
注：对照组、二甲双胍组细胞均采用完全培养基培养,二甲双胍组培养基中另加入终物质的量浓度为60 mmol/L的二甲双胍；图3A中横坐标从左至右对应富集条目为氨基酸代谢、脂质代谢、蛋白质折叠-分选-降解、信号分子及其相互作用、信号转导、细胞群落-真核、运输与分解代谢、细胞生长与死亡、神经系统、消化系统、内分泌系统、免疫系统、免疫疾病、心血管疾病、内分泌和代谢疾病、神经退行性疾病、特定类型癌症、细菌传染病、病毒传染病、癌症概览；图3B中横坐标从左至右对应富集条目为氨基酸代谢、脂质代谢、碳水化合物代谢、翻译、蛋白质折叠-分选-降解、复制和修复、信号分子及其相互作用、信号转导、细胞群落-真核、细胞生长与死亡、运输与分解代谢、免疫系统、内分泌系统、特定类型癌症、心血管疾病、内分泌和代谢疾病、神经退行性疾病、细菌传染病、病毒传染病、癌症概览；DEG为差异表达基因
图 4 气泡图展示2组人瘢痕疙瘩成纤维细胞培养24 h后差异表达显著的差异表达基因的京都基因与基因组百科全书功能富集分析富集分数排名前20的通路
注：对照组、二甲双胍组细胞均采用完全培养基培养,二甲双胍组培养基中另加入终物质的量浓度为60 mmol/L的二甲双胍；mTOR为哺乳动物雷帕霉素靶蛋白,PI3K/Akt为磷脂酰肌醇3-激酶/蛋白激酶B,JAK/STAT为Janus激酶/信号转导及转录激活因子
图 5 蛋白质印迹法检测的2组人瘢痕疙瘩成纤维细胞培养24 h后PI3K与p-Akt和p-mTOR蛋白表达
注：对照组、二甲双胍组细胞均采用完全培养基培养,二甲双胍组培养基中另加入终物质的量浓度为60 mmol/L的二甲双胍；p-mTOR为磷酸化哺乳动物雷帕霉素靶蛋白,p-Akt为磷酸化蛋白激酶B,PI3K为磷脂酰肌醇3-激酶,GAPDH为3-磷酸甘油醛脱氢酶
Table 1. 2组人瘢痕疙瘩成纤维细胞培养不同时间点各期凋亡细胞比例比较（%,
组别与时间点 样本数 早期凋亡 晚期凋亡 早期凋亡+晚期凋亡 对照组 培养0 h（即刻）后 3 6.25±0.82 0.59±0.21 7.24±1.58 二甲双胍组 培养12 h后 3 11.67±1.16 0.47±0.10 12.58±1.08 培养24 h后 3 13.51±2.62 0.98±0.16 13.98±2.68 F值 14.41 7.82 10.49 P值 0.005 0.021 0.011 t1值 6.62 0.89 4.84 P1值 0.004 0.426 0.008 t2值 4.58 2.54 3.75 P2值 0.010 0.064 0.020 t3值 1.11 4.55 0.84 P3值 0.328 0.010 0.449 注：对照组、二甲双胍组细胞均采用完全培养基培养,二甲双胍组培养基中另加入终物质的量浓度为60 mmol/L的二甲双胍；F值、P值为组间各指标总体比较所得；t1值与P1值、t2值与P2值分别为对照组培养0 h后与二甲双胍组培养12、24 h后各指标比较所得,t3值与P3值为二甲双胍组培养12 h后与组内培养24 h后各指标比较所得 
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Table 2. 2组人瘢痕疙瘩成纤维细胞培养不同时间点各期细胞比例比较（%,
组别与时间点 样本数 G0/G1期 S期 G2/M期 对照组 培养12 h后 3 88.50±4.21 19.07±2.94 3.81±1.29 培养24 h后 3 46.03±0.18 39.20±6.71 22.37±1.05 二甲双胍组 培养12 h后 3 82.49±10.38 7.18±1.88 10.33±8.57 培养24 h后 3 64.32±5.91 29.28±4.68 5.74±1.25 t1值 0.93 5.90 1.30 P1值 0.405 0.004 0.262 t2值 5.36 2.10 17.63 P2值 0.006 0.104 <0.001 t3值 2.64 7.60 0.92 P3值 0.058 0.002 0.411 注：对照组、二甲双胍组细胞均采用完全培养基培养,二甲双胍组培养基中另加入终物质的量浓度为60 mmol/L的二甲双胍；G0/G1期中,处理因素主效应,F=2.82,P=0.132；时间因素主效应,F=68.77,P<0.001；二者交互作用,F=11.04,P=0.011；S期中,处理因素主效应,F=18.05,P=0.003；时间因素主效应,F=67.70,P<0.001；二者交互作用,F=0.15,P=0.711；G2/M期中,处理因素主效应,F=3.93,P=0.083；时间因素主效应,F=7.52,P=0.025；二者交互作用,F=20.60,P=0.002；t1值与P1值为对照组与二甲双胍组培养12 h后各指标比较所得,t2值与P2值为对照组与二甲双胍组培养24 h后各指标比较所得,t3值与P3值为二甲双胍组培养12 h后与组内培养24 h后各指标比较所得
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