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.
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.
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。