阿魏酸钠对人皮肤增生性瘢痕成纤维细胞增殖和凋亡的调控作用及其信号机制

Regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts

目的探讨阿魏酸钠对人皮肤增生性瘢痕成纤维细胞(HSFb)增殖、凋亡的调控作用和信号机制。方法采用实验研究方法。取第4~6代人皮肤HSFb用于后续实验。将HSFb分别与终质量浓度1、1×10^(-1)、1×10^(-2)、1×10^(-3)、1×10-4、1×10^(-5)、1×10^(-6) mg/mL阿魏酸钠共培养48 h,采用噻唑蓝法测定细胞吸光度值并采用线性回归法分析阿魏酸钠的半数致死浓度(LC50),样本数为6。将HSFb分别与终质量浓度0.1、0.2、0.3、0.4 mg/mL的阿魏酸钠共培养24、48、72、96 h后,采用噻唑蓝法测定细胞吸光度值并计算细胞增殖抑制率(样本数为3)。取细胞,按随机数字表法分为采用相应终质量浓度阿魏酸钠处理的0.300 mg/mL阿魏酸钠组、0.030 mg/mL阿魏酸钠组、0.003 mg/mL阿魏酸钠组和不进行任何处理的阴性对照组。培养72 h后,采用噻唑蓝法测定细胞吸光度值(样本数为5),采用透射电子显微镜观察细胞微观形态(样本数为3),采用原位末端标记(TUNEL)法检测细胞凋亡情况并计算凋亡指数(样本数为4),采用免疫组织化学法检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(caspase-3)蛋白表达(样本数为4),采用蛋白质印迹法检测转化生长因子β1(TGF-β1)、磷酸化Smad2/3、磷酸化Smad4、磷酸化Smad7的蛋白表达(样本数为4)。对数据行单因素方差分析、Dunnett检验。结果阿魏酸钠的LC50为0.3075 mg/mL。培养24~96 h,4个质量浓度阿魏酸钠处理的细胞增殖抑制率均呈先升高后降低的趋势,于培养72 h达到最高,选择72 h作为后续实验的处理时间。培养72 h后,0.003 mg/mL阿魏酸钠组、0.030 mg/mL阿魏酸钠组、0.300 mg/mL阿魏酸钠组细胞吸光度值分别为0.57±0.06、0.53±0.04、0.45±0.05,均明显低于阴性对照组的0.69±0.06(P<0.01)。培养72 h后,与阴性对照组比较,用阿魏酸钠处理的3组细胞出现不同程度的核固缩或断裂、裂解,染色质丢失,胞质中可见线粒体肿胀、粗面内质网扩张,局部逐渐空泡化。培养72 h后,与阴性对照组比较,0.003 mg/mL阿魏酸钠组、0.030 mg/mL阿魏酸钠组、0.300 mg/mL阿魏酸钠组细胞凋亡指数均显著升高(P<0.05或P<0.01)。培养72 h后,与阴性对照组比较,0.300 mg/mL阿魏酸钠组细胞Bcl-2蛋白表达量明显减少(P<0.01),0.030 mg/mL阿魏酸钠组、0.300 mg/mL阿魏酸钠组细胞Bax蛋白表达量均明显增加(P<0.05),0.300 mg/mL阿魏酸钠组细胞caspase-3蛋白表达量显著增加(P<0.01)。培养72 h后,与阴性对照组比较,0.030 mg/mL阿魏酸钠组、0.300 mg/mL阿魏酸钠组细胞TGF-β1、磷酸化Smad2/3、磷酸化Smad4蛋白表达量均明显减少(P<0.05或P<0.01),0.003 mg/mL阿魏酸钠组、0.030 mg/mL阿魏酸钠组、0.300 mg/mL阿魏酸钠组细胞磷酸化Smad7蛋白表达量均显著增加(P<0.01)。结论阿魏酸钠可通过阻断TGF-β/Smad信号通路上的关键蛋白的表达,协同激活线粒体凋亡途径共同发挥抑制人皮肤HSFb增殖和促进人皮肤HSFb凋亡的作用。

Objective To investigate the regulatory effects and signaling mechanism of sodium ferulate on the proliferation and apoptosis of human skin hypertrophic scar fibroblasts(HSFbs).Methods The experimental research methods were used.The 4th-6th passage of HSFbs from human skin were used for the following experiments.HSFbs were co-cultured with sodium ferulate at final mass concentrations of 1,1×10^(-1),1×10^(-2),1×10^(-3),1×10-4,1×10^(-5),and 1×10^(-6) mg/mL for 48 hours,and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and linear regression was used to analyze the half lethal concentration(LC50)of sodium ferulate(n=6).HSFbs were co-cultured with sodium ferulate at final mass concentrations of 0.1,0.2,0.3,and 0.4 mg/mL for 24,48,72,and 96 hours,and methyl thiazolyl tetrazolium method was used to determine the cell absorbance values and the cell proliferation inhibition rate was calculated(n=3).According to the random number table,the cells were divided into 0.300 mg/mL sodium ferulate group,0.030 mg/mL sodium ferulate group,0.003 mg/mL sodium ferulate group treated with sodium ferulate at corresponding final mass concentrations,and negative control group without any treatment.After 72 hours of culture,the cell absorbance values were determined by methyl thiazolyl tetrazolium method(n=5),the microscopic morphology of cells was observed by transmission electron microscope(n=3),the cell apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling(TUNEL)assay and the apoptosis index was calculated(n=4),the protein expressions of B lymphocystoma-2(Bcl-2),Bcl-2-associated X protein(Bax),and cysteine aspartic acid specific protease-3(caspase-3)were determined by immunohistochemistry(n=4),and the protein expressions of transformed growth factorβ1(TGF-β1),phosphorylated Smad2/3,phosphorylated Smad4,and phosphorylated Smad7 were detected by Western blotting(n=4).Data were statistically analyzed with one-way analysis of variance and Dunnett test.Results The LC50 of sodium ferulate was 0.3075 mg/mL.After being cultured for 24-96 hours,the cell proliferation inhibition rates of cells treated with sodium ferulate at four different mass concentrations tended to increase at first but decrease later,which reached the highest after 72 hours of culture,so 72 hours was chosen as the processing time for the subsequent experiments.After 72 hours of culture,the cell absorbance values in 0.003 mg/mL sodium ferulate group,0.030 mg/mL sodium ferulate group,and 0.300 mg/mL sodium ferulate group were 0.57±0.06,0.53±0.04,0.45±0.05,respectively,which were significantly lower than 0.69±0.06 in negative control group(P<0.01).After 72 hours of culture,compared with those in negative control group,the cells in the three groups treated with sodium ferulate showed varying degrees of nuclear pyknosis,fracture,or lysis,and chromatin loss.In the cytoplasm,mitochondria were swollen,the rough endoplasmic reticulum was expanded,and local vacuolation gradually appeared.After 72 hours of culture,compared with that in negative control group,the apoptosis indexes of cells were increased significantly in 0.003 mg/mL sodium ferulate group,0.030 mg/mL sodium ferulate group,and 0.300 mg/mL sodium ferulate group(P<0.05 or P<0.01).After 72 hours of culture,compared with those in negative control group,the protein expressions of Bcl-2 of cells in 0.300 mg/mL sodium ferulate group was significantly decreased(P<0.01),the protein expressions of Bax of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly increased(P<0.05),and the protein expression of caspase-3 of cells in 0.300 mg/mL sodium ferulate group was significantly increased(P<0.01).After 72 hours of culture,compared with those in negative control group,the protein expression levels of TGF-β1,phosphorylated Smad2/3,and phosphorylated Smad4 of cells in 0.030 mg/mL sodium ferulate group and 0.300 mg/mL sodium ferulate group were significantly decreased(P<0.05 or P<0.01),and the protein expression levels of phosphorylated Smad7 of cells in 0.003 mg/mL sodium ferulate group,0.030 mg/mL sodium ferulate group,and 0.300 mg/mL sodium ferulate group were significantly increased(P<0.01).Conclusions Sodium ferulate can inhibit the proliferation of HSFbs of human skin and promote the apoptosis of HSFbs of human skin by blocking the expression of key proteins on the TGF-β/Smad signaling pathway and synergistically activating the mitochon-drial apoptosis pathway.