三七总皂苷对大鼠血管平滑肌细胞增殖及JNK磷酸化的影响

Effects of Total Panax Notoginseng Saponin on Proliferation and JNK Phosphorylation of Rat Vascular Smooth Muscle Cells

【目的】研究发现,三七总皂苷(TPNS)具有抑制和促进细胞增殖的双向调节作用,但是具体的作用机制尚不明确。本研究旨在观察TPNS对大鼠血管平滑肌细胞(VSMC)增殖的影响及经由何种信号通路介导。【方法】采用四甲基偶氮唑盐比色法(MTT法)和蛋白印迹(Western blot)法观察了体外培养的大鼠VSMC在不同浓度及一定浓度不同作用时间TPNS作用下,VSMC增殖活性的变化及c-Jun氨基末端激酶(JNK)磷酸化的变化;并用DAPI染细胞核,观察TPNS作用后核形态的变化。【结果】MTT法观察到:TPNS的浓度为0.5g/L,1.0g/L,1.5g/L时,无论是处于静息状态或50mL/L血清刺激下,VSMC的代谢活性均增高;TPNS的浓度为3.0g/L时,静息培养的VSMC的代谢活性没有明显的变化,而50mL/L血清刺激的VSMC的代谢活性仍增高;TPNS的浓度为6.0g/L,12.0g/L时,静息培养或50mL/L血清刺激下的VSMC的代谢活性均降低,并随着浓度的增大,代谢活性降低越明显。同时,Westernblot结果显示:TPNS能快速的激活JNK1/2,并呈时间和浓度依赖性。当TPNS的浓度为0.5g/L时,即可引起JNK的磷酸化,并随着浓度的增大,磷酸化作用增强;当TPNS的浓度为1.5g/L时,作用5min,JNK呈现最大程度的激活,随后逐渐减弱。DAPI染核观察到,6.0g/LTPNS作用48h后,VSMC出现了核固缩、核碎片等细胞凋亡的特征。【结论】较低浓度的TPNS可促进血管平滑肌细胞的增殖,而较高浓度的TPNS可抑制其增殖,JNK1/2磷酸化可能是引起细胞增殖受到抑制的机制之一。

[Objective] Studies have been showed that total Panax Notoginseng Saponin （TPNS） have ability to both inhibit and/or prompt cell proliferation, but its mechanisms are largely unknown. To explore the effects of TPNS on proliferation of vascular smooth muscle cells （VSMC） and by which signal pathway mediates. [Methods] The cultured VSMC treated by TPNS with different concentrations for different durations were detected for changed proliferative viability via MTr and phosphorylation of c-Jun N-terminal kinase（JNK） of VSMC by Western blot. The morphological changes of VSMC nuclei were observed by DAPI staining. [ Results] The metabolic viabilities of quiescent VSMC in the absence or presence of 50 mL/L fetal calf serum（FCS） were increased after treatment with 0.5 g/L, 1.0 g/L, andl.5 g/L of TPNS. When treated by 3.0 g/L of TPNS, the metabolic viability of quiescent VSMC was no obvious changes, while the metabolic viability of 50 mL/L serum-stimulated VSMC was increased. When treated by 6.0 g/L, 12.0 g/L of TPNS, the metabolic viabilities of quiescent VSMC in the absence or presence of 50 mL/L FCS were decreased and become more declined with increased TPNS concentrations.Meanwhile, TPNS could rapidly induce the activation of JNK1/2 of VSMC in dose- and time-dependent manners by Western blot analysis. When the VSMC were treated by 0.5 g/L of TPNS, phosphorylation of JNK1/2 was induced and more obvious with increased TPNS concentrations. When the VSMC were treated by 1.5 g/L of TPNS, phosphorylation of JNK1/2 peaked at 5 min and declined thereafter. Furthermore, the VSMC with condensed and/or fragmented nuclei were observed via DAPI staining after 6.0 g/L TPNS treatment. [Conclusion] The lower concentration of TPNS can induce VSMC proliferation, whereas the higher concentration of TPNS can inhibit VSMC proliferation, and the activation of JNK1/2 signal pathway may be involved in the inhibitory mechanisms of TPNS on rat VSMC proliferation.