基于"异类相制"的雷公藤复方对小鼠精母细胞凋亡及凋亡蛋白表达的影响

目的:探讨雷公藤复方单体成分对小鼠精母细胞凋亡及凋亡相关蛋白B细胞淋巴瘤/白血病-2(Bcl-2),Bcl-2相关X蛋白(Bax),半胱天冬蛋白酶-3(Caspase-3)表达的影响。方法:建立体外培养小鼠精母细胞体系,实验分为空白组,雷公藤甲素3.5 mg·L~(-1)组,雷公藤甲素3.5 mg·L~(-1)+梓醇3 mg·L~(-1)组,雷公藤甲素3.5 mg·L~(-1)+三七总皂苷12 mg·L~(-1)组,雷公藤甲素3.5 mg·L~(-1)+草酸铵1.5 mg·L~(-1)组,雷公藤甲素3.5 mg·L~(-1)+青藤碱0.75 mg·L~(-1)组。流式细胞仪测定药物作用24 h后精母细胞凋亡率,Western blot测定精母细胞Bax,Bcl-2,Caspase-3蛋白表达情况。结果:梓醇、三七总皂苷可减轻雷公藤甲素对小鼠精母细胞凋亡的诱导作用,与雷公藤甲素比较有明显差异(P0.05),草酸铵和青藤碱组与雷公藤甲素组相比无明显差异;雷公藤甲素促使Bax,Caspase-3蛋白表达上调,Bcl-2蛋白表达下调,与正常比较差异显著(P0.01),雷公藤甲素配伍梓醇、三七总皂苷后Bax,Caspase-3蛋白表达下调,Bcl-2蛋白表达上调,与雷公藤甲素比较较有明显差异(P0.05)。而配伍草酸铵及青藤碱则差异不明显。结论:雷公藤甲素可引起精母细胞凋亡,并通过Bax/Bcl-2,Caspase-3系统启动精母细胞凋亡。而雷公藤复方组成中其他药物组分可不同程度抑制雷公藤甲素引起的精母细胞凋亡,减轻其雄性生殖毒性。

Effects of Compound Tripterygii Radix et Rhizoma on Apoptosis Spermatocytes and Expression of Apoptotic Proteins Based on 'Heterogeneous Phase System'

Objective:To investigate the effect of compound Tripterygii Radix et Rhizoma on spermatocytes apoptosis in mice and their expression of apoptosis-related Bax, Bcl-2, and Caspase-3. Method:In vitro culture system for mice spermatocytes was established. The mice were divided into blank group, triptolide 3.5 mg·L^-1 group, triptolide 3.5 mg·L^-1+catalpol 3 mg·L^-1 group, triptolide 3.5 mg·L^-1+panax notoginseng saponins 12 mg·L^-1 group, triptolide 3.5 mg·L^-1+oxalate 1.5 mg·L^-1 group, triptolide 3.5 mg·L^-1+sinomenine 0.75 mg·L^-1 group. Flow cytometry was used to detect the spermatocytes apoptosis rate after 24 h drug treatment, Western blot assay was done to determine the protein expression of Bax, Bcl-2, and Caspase-3 in spermatocytes. Result:Catalpol and Panax notoginseng saponions could reduce the induction effect of triptolide on apoptosis in mouse spermatocytes, with significant differences(P<0.05) compared with triptolide group, but there was no significant difference between ammonium oxalate, Sinomenine group and triptolide group. Triptolide induced increase in Bax, Caspase-3 protein expression, decrease in Bcl-2 protein expression, with significant difference with blank group (P<0.01). After combination of triptolide with catalpol and panax notoginseng saponins, Bax, Caspase-3 protein expression was reduced, while Bcl-2 protein expression was increased, with significant difference compared with triptolide group (P<0.05). The combination of triptolide with ammonium oxalate and Sinomenine did not make significant difference. Conclusion:Triptolide can cause spermatocytes apoptosis through Bax/Bcl-2, and Caspase-3 system. The other drug components in compound in Tripterygii Radix et Rhizoma can inhibited apoptosis in spermatocytes to varying degrees, and reduce its reproductive toxicity.