三七蛋白对小鼠黑色素瘤B16细胞黑色素的抑制作用及机制研究

Inhibitory effect and mechanism of Panax notoginseng protein on melanin in murine melanoma B16 cell

目的:探讨三七蛋白对小鼠黑色素瘤B16细胞黑色素的抑制作用及机制。方法:采用硫酸铵沉淀法提取三七蛋白,氨基酸自动分析仪测定三七蛋白氨基酸组成。采用NaOH裂解法测定三七蛋白不同质量浓度(0.125、0.25、0.5 mg·mL–1)作用下B16细胞内黑色素含量,CCK8法检测细胞增殖率,流式细胞仪检测线粒体膜电位及细胞凋亡,多巴氧化法测定细胞内酪氨酸酶(tyrosinase,Tyr)活性,同时采用体外抑制Tyr活性试验观察对Tyr活性的影响,Western blot检测B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、小眼畸形相关转录因子(microphthalmia-associated transcription factor,MITF)、Tyr以及Tyr相关蛋白1(Tyr-related protein 1,TRP-1)表达变化。结果:与对照组相比,三七蛋白显著降低B16细胞内黑色素含量,抑制细胞增殖,提高细胞凋亡率,降低线粒体膜电位,上调Bax蛋白表达,下调Bcl-2蛋白表达及Bcl-2/Bax表达比值;此外,三七蛋白显著抑制B16细胞内及体外Tyr活性,显著下调p-MIFP/MITF、Tyr及TRP-1蛋白表达。结论:三七蛋白对B16细胞黑色素生成具有抑制作用,且可能与诱导细胞凋亡、降低Tyr活性相关。

OBJECTIVE To explore the inhibitory effect and mechanism of Panax notoginseng protein on melanin in murine melanoma B16 cell.METHODS Panax notoginseng protein was extracted by ammonium sulfate precipitation.The amino acid contents of P.notoginseng protein were determined by an automatic amino acid analyzer.The intracellular melanin content under different concentrations(0.125,0.25,0.5 mg·mL–1) of P.notoginseng protein was determined by NaOH lysis.CCK8 method was employed for detecting cell proliferation rate.Flow cytometry was utilized for detecting mitochondrial membrane potential and cell apoptosis.Intracellular and in vitro tyrosinase(Tyr) activity were measured by dopamine oxidation and in vitro Tyr inhibition test.The protein expression changes of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),microphthalmiaassociated transcription factor(MITF),Tyr and Tyr-related protein 1(TRP-1) were measured by Western blot.RESULTS As compared with control group,P.notoginseng protein significantly decreased melanin content,arrested the proliferation of B16cells,reduced mitochondrial membrane potential level,accelerated cell apoptosis rate,boosted Bax protein expression,downregulated the expression of Bcl-2 protein and lowered Bcl-2/Bax expression ratio.Moreover,P.notoginseng protein significantly reduced Tyr activity in B16 cells and in vitro and significantly down-regulated the protein expressions of p-MIFP/MITF,Tyr and TRP-1.CONCLUSION P.notoginseng protein has an inhibitory effect on melanin production in B16 cells.It may be correlated with an induction of apoptosis and a reduction of Tyr activity.