桃红四物汤影响ERK磷酸化表达延缓小鼠胚胎成纤维细胞生长的机理研究

Mechanism Study of Taohong Siwu Decoction on Delaying Growth of BALB/c 3T3 Cells by Affecting ERK Phosphorylation

目的探讨活血化瘀名方桃红四物汤(THSWD)减缓小鼠胚胎成纤维细胞(BALB/c 3T3,3T3)生长和增殖可能的分子靶点及信号通路。方法在3T3细胞内分别过表达碱性成纤维细胞生长因子(bFGF)、沉默bFGF以及用PD98059阻断细胞内ERK上游信号通路;然后观察桃红四物汤对细胞活性,细胞周期,bFGF、ERK1/2及p-ERK1/2基因或蛋白表达的影响。桃红四物汤含药血清培养基浓度为20%,作用时间48 h。结果与正常对照组比较,桃红四物汤组的3T3细胞活性降低(P<0.01);G0/G1期占比升高(P<0.05,P<0.01),S期占比降低(P<0.05,P<0.01);bFGF和ERK1/2基因表达降低(P<0.01);bFGF和p-ERK1/2蛋白表达降低(P<0.05,P<0.01),ERK1/2蛋白表达的差异无统计学意义(P>0.05)。与bFGF过表达组比较,bFGF过表达+桃红四物汤组的3T3细胞活性降低(P<0.05);G0/G1期占比升高(P<0.01),S期占比的差异无统计学意义(P>0.05);bFGF和ERK1/2基因表达降低(P<0.01);bFGF和p-ERK1/2蛋白表达降低(P<0.01),ERK1/2蛋白表达的差异无统计学意义(P>0.05)。与PD98059组比较,PD98059+桃红四物汤组的3T3细胞活性降低(P<0.05);G0/G1期占比升高(P<0.01),S期占比降低(P<0.01);ERK1/2基因表达的差异无统计学意义(P>0.05);ERK1/2和p-ERK1/2蛋白表达的差异无统计学意义(P>0.05)。由于bFGF基因的结构独特性及沉默引物设计的方法学缺陷,3T3细胞内未能沉默掉bFGF。结论桃红四物汤在3T3细胞中首先降低bFGF,进而降低ERK信号通路的标志性蛋白p-ERK的表达,减缓3T3细胞的增殖速度。

Objective To explore the possible molecular targets and signaling pathways of THSWD on delaying growth and proliferation of BALB/c 3 T3 Cells.Methods 3 T3 cells were transfected with bFGF overexpression lentivirus,bFGF silencing lentivirus and were either treated with PD98059 to block ERK upstream pathway.Then we investigated the effects of THSWD on cell activity,cell cycle,and the expressions of bFGF,ERK1/2 and p-ERK1/2 in 3 T3 cells.The concenteation of THSWD drug-containing serum in culture medium was 20%.The cells were cultured for 48 h.Results Compared with the normal control group,the activity of 3 T3 cells in THSWD group was decreased(P<0.01),the percentage of cells in G0/G1 phase was increased(P<0.05,P<0.01),and the percentage of cells in S phase was decreased(P<0.05,P<0.01).THSWD downregulated the gene expression of bFGF and ERK1/2(P<0.01)and reduced the protein expression of bFGF and p-ERK1/2(P<0.05,P<0.01).The difference in protein expression of ERK1/2 was not statistically significant(P>0.05).Compared with the bFGF overexpression group,the activity of 3 T3 cells in bFGF overexpression+THSWD group was decreased(P<0.05),the percentage of cells in the G0/G1 phase was increased(P<0.01).The difference of cell percentage in S phase was not statistically significant(P>0.05).THSWD downregulated the gene expression of bFGF and ERK1/2(P<0.01)and reduced the protein expression of bFGF and p-ERK1/2(P<0.01).The difference in protein expression of ERK1/2 was not statistically significant(P>0.05).Compared with the PD98059 group,the activity of 3 T3 cells in PD98059+THSWD group was decreased(P<0.05),the percentage of cells in the G0/G1 phase was increased(P<0.01),and the percentage of cells in S phase was decreased(P<0.01).The difference in gene expression of ERK1/2 was not statistically significant(P>0.05).In addition,the difference in protein expression of ERK1/2 and p-ERK1/2 was not statistically significant(P>0.05).Silencing bFGF lentivirus showed no affects on 3 T3 cells maybe because the special sequence of bFGF or the shortage of silence techniques.Conclusion THSWD could reduce the expression of bFGF.Subsequently,the expression of p-ERK protein of ERK signaling pathway was decreased.Finally,the proliferation of 3 T3 cells was retarded.