茶黄素通过磷脂酰肌醇3-激酶/蛋白激酶B信号通路促进小鼠乳腺肿瘤细胞凋亡

Theaflavin Promoting Apoptosis of Mouse Mammary Tumor Cells through Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway

本试验旨在研究茶黄素促进小鼠乳腺肿瘤细胞凋亡的作用机制。采用不同浓度[0(A组)、48(B组)、88(C组)、128μmol/L(D组)]茶黄素处理小鼠乳腺肿瘤细胞(C-127细胞)24 h,采用CCK-8法检测细胞存活率,流式细胞术检测细胞凋亡情况,荧光显微镜检测细胞活性与坏死情况,透射电镜观察细胞形态,实时荧光定量PCR(qRT-PCR)检测半胱氨酸特异性天冬氨酸蛋白酶-3(Caspase-3)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、B淋巴细胞瘤-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的mRNA相对表达量,Western Blot法检测活化后的Caspase-3(Cleaved-Caspase-3)、PI3K、Akt、Bcl-2和Bax的蛋白表达水平。结果显示:1)与A组相比,B组细胞存活率显著降低(P<0.05),C组、D组细胞存活率极显著降低(P<0.01)。2)与A组相比,B组、C组、D组细胞凋亡率极显著升高(P<0.01)。3)与A组相比,B组Akt的mRNA相对表达量极显著降低(P<0.01),PI3K、Bcl-2的mRNA相对表达量显著降低(P<0.05),Caspase-3、Bax的mRNA相对表达量显著升高(P<0.05);C组和D组PI3K、Bcl-2和Akt的mRNA相对表达量极显著降低(P<0.01),Caspase-3和Bax的mRNA相对表达量极显著升高(P<0.01)。4)与A组相比,B组Bcl-2的蛋白表达水平极显著降低(P<0.01),Cleaved-Caspase-3的蛋白表达水平极显著升高(P<0.01);C组和D组Cleaved-Caspase-3和Bax的蛋白表达水平极显著升高(P<0.01),PI3K、Akt、Bcl-2的蛋白表达水平极显著降低(P<0.01)。5)荧光显微镜和透视电镜观察可见,随着茶黄素浓度的升高,细胞活性逐渐降低,坏死细胞逐渐增多,线粒体变大,线粒体嵴增多;染色质浓缩分布在核膜周围或一侧,呈眼球状;自噬体内吞噬线粒体、内质网和一些细胞内的胞质成分,再与细胞膜融合后,形成凋亡小体。综上所述,茶黄素可通过PI3K/Akt信号通路促进促凋亡基因的表达,降低抗凋亡基因的表达,从而促进小鼠乳腺肿瘤细胞凋亡,达到抗肿瘤的作用;并且,随着茶黄素浓度的增大,茶黄素促进小鼠乳腺肿瘤细胞凋亡的作用增强。

The purpose of this experiment was to study the mechanism of theaflavins(TF)in promoting apoptosis of mouse mammary tumor cells.Mouse mammary tumor cells(C-127 cells)were treated with different concentrations[0(group A),48(group B),88(group C)and 128μmol/L(group D)]of TF for 24 h.The CCK-8 method was used to detect the survival rate of C-127 cells,flow cytometry was used to detect the cell apoptosis,fluorescence microscopy was used to detect cell viability and necrosis,transmission electron microscopy was used to observe cell morphology,real-time quantitative PCR(qRT-PCR)was used to detect the mRNA relative expression levels of cysteine-containing aspartate-specific protease-3(Caspase-3),phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax),and Western Blot were used to detect the protein expression levels of cleaved cysteine-containing aspartate-specific protease-3(Cleaved-Caspase3),PI3K,Akt,Bcl-2 and Bax.The results showed as follows:1)compared with group A,the cell survival rate of group B was significantly decreased(P<0.05),and the cell survival rate of groups C and D were extremely significantly decreased(P<0.01).2)Compared with group A,the cell apoptosis rate of groups B,C and D was extremely significantly increased(P<0.01).3)Compared with group A,the mRNA relative expression levels of Akt of group B were extremely significantly decreased(P<0.05),the mRNA relative expression levels of PI3K and Bcl-2 were significantly decreased(P<0.05),and the mRNA relative expression levels of Caspase-3 and Bax were significantly increased(P<0.05).The mRNA relative expression levels of PI3K,Bcl-2 and Akt of groups C and D were extremely significantly decreased(P<0.01),and the mRNA relative expression levels of Caspase-3 and Bax were extremely significantly increased(P<0.01).4)Compared with group A,the protein expression level of Bcl-2 of group B was extremely significantly decreased(P<0.01),and the protein expression level of Cleaved-Caspase-3 was extremely significantly increased(P<0.01);the protein expression levels of Cleaved-Caspase-3 and Bax of groups C and D were extremely significantly increased(P<0.01),and the protein expression levels of PI3K,Akt and Bcl-2 were extremely significantly decreased(P<0.01).5)Observation by fluorescence microscope and fluoroscopic electron microscope,with the increase of TF concentration,the cell viability gradually decreased,the necrotic cells gradually increased,the mitochondria became larger,and the mitochondrial cristae increased;chromatin was concentrated and distributed in the nuclear membrane around or on one side,giving an eyeball-shaped;the autophagy body contained mitochondria,endoplasmic reticulum and some intracellular cytoplasmic components,and then fused with the cell membrane to form apoptotic bodies.In conclusion,TF can promote the expression of pro-apoptotic genes and reduce the expression of anti-apoptotic genes through the PI3K/Akt signaling pathway,thereby promote the apoptosis of mouse mammary tumor cells and achieve anti-tumor effects;with the increase of TF concentration,the promoting effect of TF on the apoptosis of mouse mammary tumor cells is enhanced.