Effects and mechanism of metformin on the proliferation and expression of fibrotic proteins of human hypertrophic scar fibroblasts
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摘要:
目的 探讨二甲双胍对人增生性瘢痕（HS）成纤维细胞（Fb）增殖及纤维化蛋白表达的影响及其机制。 方法 该研究为实验研究。收集2021年6月—2022年6月于空军军医大学第一附属医院烧伤与皮肤外科行HS切除术的5例HS患者（男3例、女2例,年龄21~36岁）的HS组织,分离、培养Fb并取第5~7代Fb进行实验。取Fb,分别在其培养基中加入磷酸盐缓冲液（PBS）或终物质的量浓度为5、10、20、40 mmol/L的二甲双胍培养,培养48 h,采用细胞计数试剂盒-8（CCK-8）检测细胞增殖活性并计算细胞增殖抑制率,采用羟脯氨酸测定试剂盒检测细胞培养上清液中羟脯氨酸含量,采用蛋白质印迹法检测细胞中蛋白激酶B（Akt）、哺乳动物雷帕霉素靶蛋白（mTOR）的磷酸化水平并计算磷酸化Akt（p-Akt）与Akt比值、磷酸化mTOR（p-mTOR）与mTOR比值；培养24 h,采用实时荧光定量反转录PCR法检测细胞中Ⅰ型胶原、Ⅲ型胶原和α-平滑肌肌动蛋白（α-SMA）的mRNA表达。另取Fb分为对照组（常规培养）和LY294002组、二甲双胍组、LY294002+二甲双胍组,后3组细胞培养基中分别加入LY294002、二甲双胍、LY294002+二甲双胍培养,LY294002与二甲双胍的终物质的量浓度分别为20 μmol/L、10 mmol/L,于培养0（即刻）、24、48 h采用CCK-8检测细胞增殖活性,培养48 h采用蛋白质印迹法检测细胞中Akt、mTOR的磷酸化水平并计算p-Akt与Akt比值、p-mTOR与mTOR比值。细胞增殖抑制率实验样本数为4,其余实验样本数为3。 结果 培养48 h,与经PBS处理比较,经5、10、20、40 mmol/L二甲双胍处理细胞的增殖抑制率均显著升高（t值分别为10.69、14.20、19.73、52.54,P<0.05）,经10、20、40 mmol/L二甲双胍处理细胞培养上清液中羟脯氨酸含量均显著降低（t值分别为8.06、7.86、10.25,P<0.05）,经10、20、40 mmol/L二甲双胍处理细胞中p-Akt与Akt比值及经20、40 mmol/L二甲双胍处理细胞中p-mTOR与mTOR比值均显著降低（t值分别为2.82、4.28、9.88及5.66、9.08,P<0.05）；培养24 h,与经PBS处理比较,经5、10、20、40 mmol/L二甲双胍处理细胞中Ⅰ型胶原及α-SMA的mRNA表达及经10、20、40 mmol/L二甲双胍处理细胞中Ⅲ型胶原的mRNA表达均显著降低（t值分别为4.35、8.53、9.57、14.77与4.14、5.58、7.89、9.37及5.18、6.85、9.15,P<0.05）。培养24、48 h,LY294002组（t值分别为6.30、13.60）、二甲双胍组（t值分别为6.47、10.69）细胞增殖活性均较对照组显著下降（P<0.05）。培养48 h,LY294002组、二甲双胍组细胞中p-Akt与Akt比值分别为0.554±0.027、0.681±0.029,均显著低于对照组的1.053±0.193（t值分别为4.45、3.31,P<0.05）；LY294002+二甲双胍组细胞中p-Akt与Akt比值为0.387±0.023,显著低于二甲双胍组（t=5.95,P<0.05）。培养48 h,LY294002组细胞中p-mTOR与mTOR比值显著低于对照组（t=4.01,P<0.05）,LY294002+二甲双胍组细胞中p-mTOR与mTOR比值显著低于二甲双胍组（t=6.05,P<0.05）。 结论 二甲双胍可以通过磷脂酰肌醇3-激酶/Akt/mTOR信号通路抑制人HS中Fb增殖及Ⅰ型胶原、Ⅲ型胶原、α-SMA等纤维化蛋白的表达。 Abstract:Objective To investigate the effects and mechanism of metformin on the proliferation and expression of fibrotic proteins of human hypertrophic scar (HS) fibroblasts (Fbs). Methods The study was an experimental study. From June 2021 to June 2022, 5 patients with HS were admitted to the Department of Burns and Cutaneous Surgery of the First Affiliated Hospital of Air Force Medical University, including 3 males and 2 females, aged from 21 to 36 years. HS tissue was collected, Fbs were isolated and cultured, and Fbs of passage 5 to 7 were used for experiment. Fbs were taken and cultured in their respective media supplemented with phosphate buffered solution (PBS) or metformin at final molarities of 5, 10, 20, and 40 mmol/L for 48 hours. The cell proliferation activity was detected using the cell counting kit-8 (CCK-8), and the proliferation inhibition rate of cells was calculated. The content of hydroxyproline in the cell culture supernatant was measured using a hydroxyproline assay kit. The phosphorylation levels of protein kinase B (Akt) and mammalian target of rapamycin (mTOR) in the cells were detected by Western blotting, and the ratios of phosphorylated Akt (p-Akt) to Akt and phosphorylated mTOR (p-mTOR) to mTOR were calculated. After 24 hours of culture, the mRNA expressions of type Ⅰ collagen, type Ⅲ collagen, and α-smooth muscle actin (α-SMA) in the cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Another batch of Fbs were divided into control group (with conventional culture), LY294002 group, metformin group, and LY294002+metformin group. LY294002, metformin, and LY294002+metformin were added to the culture media of the last three groups, respectively, with the final molarities of LY294002 and metformin being 20 μmol/L and 10 mmol/L, respectively. CCK-8 was used to detect the cell proliferation activity at 0 (immediately), 24, and 48 hours of culture. After 48 hours of culture, Western blotting was used to detect the phosphorylation levels of Akt and mTOR in the cells, and the ratios of p-Akt to Akt and p-mTOR to mTOR were calculated. The sample size for the cell proliferation inhibition rate experiment was 4, and the sample size for the other experiments was 3. Results After 48 hours of culture, compared with the cells treated with PBS, the proliferation inhibition rates of the cells treated with 5, 10, 20, and 40 mmol/L metformin were significantly increased (with t values of 10.69, 14.20, 19.73, and 52.54, respectively, P<0.05), the content of hydroxyproline in the culture supernatants of the cells treated with 10, 20, and 40 mmol/L metformin was significantly decreased (with t values of 8.06, 7.86, and 10.25, respectively, P<0.05), and the ratios of p-Akt to Akt in the cells treated with 10, 20, and 40 mmol/L metformin and the ratios of p-mTOR to mTOR in the cells treated with 20 and 40 mmol/L metformin were significantly decreased (with t values of 2.82, 4.28, 9.88, 5.66, and 9.08, respectively, P<0.05). After 24 hours of culture, compared with those treated with PBS, the mRNA expressions of type Ⅰ collagen and α-SMA in the cells treated with 5, 10, 20, and 40 mmol/L metformin and the mRNA expressions of type Ⅲ collagen in the cells treated with 10, 20, and 40 mmol/L metformin were significantly decreased (with t values of 4.35, 8.53, 9.57, 14.77, 4.14, 5.58, 7.89, 9.37, 5.18, 6.85, and 9.15, respectively, P<0.05). At 24 and 48 hours of culture, the proliferation activities of the cells in LY294002 group (with t values of 6.30 and 13.60, respectively) and metformin group (with t values of 6.47 and 10.69, respectively) were significantly lower than those in control group (P<0.05). After 48 hours of culture, the ratios of p-Akt to Akt in the cells of LY294002 group and metformin group were 0.554±0.027 and 0.681±0.029, respectively, which were significantly lower than 1.053±0.193 in control group (with t values of 4.45 and 3.31, respectively, P<0.05). The ratio of p-Akt to Akt in the cells of LY294002+metformin group was 0.387±0.023, which was significantly lower than that in metformin group (t=5.95, P<0.05). After 48 hours of culture, the ratio of p-mTOR to mTOR in the cells of LY294002 group was significantly lower than that in control group (t=4.01, P<0.05), and the ratio of p-mTOR to mTOR in the cells of LY294002+metformin group was significantly lower than that in metformin group (t=6.05, P<0.05). Conclusions Metformin can inhibit the proliferation and expression of fibrotic proteins type Ⅰ collagen, type Ⅲ collagen, and α-SMA of human HS Fbs through phosphatidylinositol 3-kinase/Akt/mTOR signaling pathway. -
参考文献
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图 1 蛋白质印迹法检测的人增生性瘢痕成纤维细胞经加入PBS或各种终物质的量浓度二甲双胍培养48 h后Akt与mTOR磷酸化水平
注：Akt为蛋白激酶B,p‑Akt为磷酸化Akt,mTOR为哺乳动物雷帕霉素靶蛋白,p‑mTOR为磷酸化mTOR,PBS为磷酸盐缓冲液；条带上方1、2、3、4、5分别指示经PBS或5、10、20、40 mmol/L二甲双胍处理细胞
图 2 蛋白质印迹法检测的4组人增生性瘢痕成纤维细胞培养48 h细胞中Akt和mTOR磷酸化水平（
注：Akt为蛋白激酶B,p-Akt为磷酸化Akt,mTOR为哺乳动物雷帕霉素靶蛋白,p-mTOR为磷酸化mTOR；条带上方1、2、3、4分别指示细胞常规培养的对照组和细胞培养基中加入LY294002、二甲双胍、LY294002+二甲双胍培养的LY294002组、二甲双胍组、LY294002+二甲双胍组,LY294002与二甲双胍的终物质的量浓度分别为20 μmol/L、10 mmol/L
Table 1. 人增生性瘢痕成纤维细胞经加入PBS或各种终物质的量浓度二甲双胍培养24 h后细胞中纤维化蛋白的mRNA表达比较（
处理因素 样本数 Ⅰ型胶原 Ⅲ型胶原 α-平滑肌肌动蛋白 PBS 3 0.98±0.07 1.01±0.09 1.003±0.078 5 mmol/L二甲双胍 3 0.76±0.05 0.82±0.05 0.750±0.020 10 mmol/L二甲双胍 3 0.56±0.05 0.59±0.07 0.627±0.046 20 mmol/L二甲双胍 3 0.46±0.07 0.46±0.07 0.457±0.054 40 mmol/L二甲双胍 3 0.32±0.04 0.39±0.03 0.427±0.032 F值 64.26 30.58 41.72 P值 <0.001 <0.001 <0.001 t1值 4.35 2.57 4.14 P1值 0.012 0.062 0.014 t2值 8.53 5.18 5.58 P2值 0.001 0.007 0.005 t3值 9.57 6.85 7.89 P3值 0.001 0.002 0.001 t4值 14.77 9.15 9.37 P4值 0.010 0.001 0.001 注：F值、P值为加入磷酸盐缓冲液（PBS）或各种终物质的量浓度二甲双胍培养后各指标总体比较所得；t1值与P1值、t2值与P2值、t3值与P3值、t4值与P4值分别为加入5、10、20、40 mmol/L二甲双胍培养与加入PBS培养后各指标比较所得 
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Table 2. 人增生性瘢痕成纤维细胞经加入PBS或各种终物质的量浓度二甲双胍培养48 h后Akt与mTOR磷酸化水平比较（
处理因素 样本数 p-Akt与Akt比值 p-mTOR与mTOR比值 PBS 3 1.101±0.101 0.875±0.087 5 mmol/L二甲双胍 3 1.064±0.065 0.843±0.052 10 mmol/L二甲双胍 3 0.915±0.053 0.806±0.058 20 mmol/L二甲双胍 3 0.799±0.069 0.584±0.018 40 mmol/L二甲双胍 3 0.509±0.025 0.396±0.028 F值 37.68 42.81 P值 <0.001 <0.001 t1值 0.53 0.54 P1值 0.623 0.617 t2值 2.82 1.13 P2值 0.048 0.321 t3值 4.28 5.66 P3值 0.013 0.005 t4值 9.88 9.08 P4值 <0.001 <0.001 注：Akt为蛋白激酶B,mTOR为哺乳动物雷帕霉素靶蛋白,p-Akt为磷酸化Akt,p-mTOR为磷酸化mTOR；Akt、mTOR磷酸化水平分别以p-Akt与Akt比值、p-mTOR与mTOR比值表示；F值、P值为加入磷酸盐缓冲液（PBS）或各种终物质的量浓度二甲双胍培养后各指标总体比较所得；t1值与P1值、t2值与P2值、t3值与P3值、t4值与P4值分别为加入5、10、20、40 mmol/L二甲双胍培养与加入PBS培养后各指标比较所得
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Table 3. 4组人增生性瘢痕成纤维细胞培养各时间点增殖活性比较（
组别 样本数 0 h（即刻） 24 h 48 h 对照组 3 0.350±0.021 0.907±0.071 1.377±0.051 LY294002组 3 0.347±0.031 0.557±0.065 0.807±0.051 二甲双胍组 3 0.361±0.040 0.621±0.029 0.763±0.085 LY294002+二甲双胍组 3 0.372±0.013 0.760±0.086 0.811±0.020 t1值 0.16 6.30 13.60 P1值 0.882 0.003 <0.001 t2值 0.39 6.47 10.69 P2值 0.718 0.003 <0.001 t3值 1.59 1.57 0.96 P3值 0.190 0.192 0.393 注：对照组细胞常规培养,后3组细胞培养基中分别加入LY294002、二甲双胍、LY294002+二甲双胍培养,LY294002与二甲双胍的终物质的量浓度分别为20 μmol/L、10 mmol/L；处理因素主效应,F=411.00,P<0.001；时间因素主效应,F=78.10,P<0.001；两者交互作用,F=25.79,P<0.001；t1值与P1值、t2值与P2值分别为LY294002组、二甲双胍组与对照组各时间点比较所得,t3值与P3值为LY294002+二甲双胍组与二甲双胍组各时间点比较所得
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Table 4. 4组人增生性瘢痕成纤维细胞培养48 h细胞中Akt和mTOR磷酸化水平比较（
组别 样本数 p-Akt与Akt比值 p-mTOR与mTOR比值 对照组 3 1.053±0.193 0.958±0.181 LY294002组 3 0.554±0.027 0.531±0.025 二甲双胍组 3 0.681±0.029 0.787±0.057 LY294002+二甲双胍组 3 0.387±0.023 0.321±0.089 F值 24.57 25.32 P值 <0.001 <0.001 t1值 4.45 4.01 P1值 0.011 0.016 t2值 3.31 1.55 P2值 0.030 0.195 t3值 5.95 6.05 P3值 0.004 0.004 注：Akt为蛋白激酶B,mTOR为哺乳动物雷帕霉素靶蛋白,p-Akt为磷酸化Akt,p-mTOR为磷酸化mTOR；Akt、mTOR磷酸化水平分别以p-Akt与Akt比值、p-mTOR与mTOR比值表示；对照组细胞常规培养,后3组细胞培养基中分别加入LY294002、二甲双胍、LY294002+二甲双胍培养,LY294002与二甲双胍的终物质的量浓度分别为20 μmol/L、10 mmol/L；F值、P值为4组间各指标总体比较所得；t1值与P1值、t2值与P2值分别为LY294002组、二甲双胍组与对照组各指标比较所得,t3值与P3值为LY294002+二甲双胍组与二甲双胍组各指标比较所得
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