Conclusion
SFHXF can regulate melanin metabolism and enhance tyrosinase activity and melanogenesis through the autophagy pathway to inhibit the proliferation of B16 cells in vitro. 目的: 观察自噬在疏风活血方对体外培养的鼠B16黑素瘤细胞增殖及黑素合成中的作用。 方法: 体外培养鼠B16细胞，设立空白对照组（C组），0.12、0.25、0.49、0.98、1.96 mg/mL疏风活血方组（S组），自噬诱导剂雷帕霉素组（R组），自噬诱导+疏风活血方组（RS组），采用MTT法测定各组对B16细胞增殖的影响，酶标仪检测法测定各组酪氨酸酶活性及黑素含量，Western blot法测定自噬相关蛋白P62、p-mTOR、LC3B、Beclin1含量，比较分析结果。 结果: 与C组相比，S组及RS组对B16细胞增殖均起浓度依赖的抑制作用（P < 0.05）；对酪氨酸酶活性及黑素合成有促进作用（P < 0.05）；0.98 mg/mL疏风活血方组（R0.98）、自噬诱导+ 0.98 mg/mL疏风活血方组（RS0.98）、50 nmol/L自噬诱导剂雷帕霉素组（Rap50）均能上调自噬相关蛋白LC3B-Ⅱ、Beclin1，下调P62、p-mTOR含量。 结论: 疏风活血方能通过激活自噬途径对体外培养的鼠B16黑素瘤细胞黑素代谢起调节作用，对其细胞增殖有抑制作用；对酪氨酸酶活性及黑素合成起促进作用。
Methods
B16 cells were treated with solutions containing 0.12, 0.25, 0.49, 0.98, or 1.96 mg/mL SFHXF preparations, rapamycin (an autophagy inducer), or rapamycin+SFHXF. The changes in the proliferation of B16 cells were assessed using MTT assay, and tyrosinase activity and melanin content in the cells were determined. The expressions of autophagy-related proteins P62, p-mTOR, LC3B, and beclin 1 in the cells were detected using Western blotting. Result: Compared with the blank control cells, treatments with SFHXF both in the presence and in the absence of rapamycin concentration-dependently inhibited the cell proliferation (P<0.05) and obviously increased tyrosinase activity and melanogenesis in B16 cells (P<0.05); 0.98 mg/mL SFHLF, rapamycin+0.98 mg/mL SFHXF, and 50 nmol/L rapamycin all significantly up-regulated the expressions of LC3B-II and beclin 1 and down-regulated the expressions of P62 and p-mTOR in the cells.
Objective
To investigate the role of autophagy in the regulatory effect of Shufeng Huoxue Fumula (SFHXF) on the proliferation and melanin metabolism in cultured murine B16 melanoma cells.