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目的:从天然药物中筛选出抑制紫外线照射诱导人黑色素瘤细胞A375-S2凋亡的有效单体。方 法:MTT法测定细胞生长抑制率;形态学观察,DNA凝胶电泳及LDH法;用半胱天冬酶活力检测试剂盒测定半胱天 冬酶活力;用免疫印记法检测Bcl-2家族成员(Bcl-2,Bcl-xL和Bax)的表达。结果:紫外线照射(2．4 J／cm2,5 min)能显著诱导A375-S2细胞发生凋亡,其作用呈明显时间依赖性。形态学观察可见凋亡小体的形成,琼脂糖凝 胶电泳可见凋亡典型的DNA梯带;水飞蓟素具有抑制紫外线照射(2．4 J／cm2,5 min)诱导A375-S2细胞凋亡的作 用,水飞蓟素作用于紫外线照射(2．4 J／cm2,5 min)的A375-S2细胞,培养12 h,使紫外线照射诱导的半胱天冬酶 -9、半胱天冬酶-3的活力降低;免疫印记法检测发现水飞蓟素作用的A375-S2细胞(紫外线照射)中Bcl-2蛋 白和Bcl-xL蛋白的表达增加。结论:水飞蓟素明显抑制紫外线照射诱导的A375-S2细胞的凋亡,其抑制凋亡作 用与半胱天冬酶途径和线粒体途径相关。
OBJECTIVE: To screen effective monomers that inhibit apoptosis of human melanoma cells A375-S2 induced by ultraviolet irradiation from natural drugs. METHODS: MTT assay was used to determine the cell growth inhibition rate; morphological observation, DNA gel electrophoresis and LDH assay; Caspase activity assay kit was used to determine caspase activity; immunoblotting was used to detect Bcl-2 family members. Expression of (Bcl-2, Bcl-xL and Bax). RESULTS: Ultraviolet radiation (2.4 J/cm2, 5 min) significantly induced apoptosis in A375-S2 cells, which was time-dependent. Morphological observation showed the formation of apoptotic bodies, apoptotic DNA ladders were observed by agarose gel electrophoresis, and silymarin inhibited apoptosis induced by ultraviolet irradiation (2.4 J/cm2, 5 min) in A375-S2 cells. Silymarin acts on A375-S2 cells exposed to ultraviolet light (2.4 J/cm2, 5 min) and cultured for 12 h to reduce the activity of caspase-9 and caspase-3 induced by ultraviolet irradiation. Immunoblotting revealed increased expression of Bcl-2 protein and Bcl-xL protein in A375-S2 cells exposed to silybin (UV irradiation). CONCLUSIONS: Silymarin significantly inhibited the apoptosis of A375-S2 cells induced by ultraviolet irradiation, and its inhibition of apoptosis was related to the caspase pathway and the mitochondrial pathway.