雷公藤甲素影响线粒体脂肪酸β-氧化致睾丸支持细胞毒性的机制研究

Triptolide (TP), a major active ingredient of the Chinese herb Tripterygium wilfordii Hook f. (TWHF), which is widely used in Traditional Chinese Medicine for centuries, has been shown multiple biological activities. However, the clinical applications of TP are limited by its narrow therapeutic window and severe toxicity on male fertility . Our previous studies demonstrated that TP exerted arrenotokous toxic action on rat and Beagle dog, and recent results also indicated that TP interfered with the function of mitochondia in TM4 sertoli cell which could generate ATP by promoting fatty acid β-oxidation . Whether TP interfered with fatty acid β-oxidation in TM4 sertoli cell or not and the relative mechanism should be elucidated. In this study, activiate SIRT1 which PGC-1α can be deacetylated and then, PGC-1α combines with PPARα, or AMPK which ACC can be phosphorylated by agnoist in TM4 cell and Wistar rat will be used to activiate CPT1 in mitochondrion fatty acid β-oxidation and determine the arrenotokous toxicity of TP. Levels of mitochondrial membrane potential depolarization, ATP, LDH, CPT-1 enyzme activity and the key transcription factors in mitochondrial electron transport chain expression were assessed as the toxic indices for TP. This research would suggest that TP modulates the CPT-1 enyzme activity by inhibiting SIRT1 and AMPK, and then interfered with fatty acid β-oxidation in sertoli cell. The results could clarify the mechanisms of TP-induced toxicity in arrenotokous toxicity, and provide a rational basis for seeking method to attenuate TP-induced male unfertility in clinical application, further can be applied to the similar Traditional Chinese Medicine.

中药单体雷公藤甲素（Triptolide,TP）具明确的生殖毒性，是限制其临床应用的主要原因之一。我们前期研究观察到TP能引起动物睾丸组织出现病理改变及近期预实验发现TP对睾丸支持细胞的线粒体有明显毒性。睾丸支持细胞主要是通过促进脂肪酸β-氧化产生能量，那么TP会否对线粒体能量代谢、脂肪酸β-氧化过程产生影响？机制如何？本课题拟在细胞和动物水平上，干扰与线粒体脂肪酸β-氧化相关的限速酶CPT-1相互作用的基因PPARα和ACC及调控β-氧化的基因SIRT1与AMPK, 观察激活SIRT1与AMPK前后， TP对睾丸支持细胞的线粒体脂肪酸β-氧化过程及动物睾丸组织和精子的影响，以论证线粒体脂肪酸β-氧化中的CPT-Ⅰ酶是TP致睾丸支持细胞毒性的关键，SIRT1／AMPK的扰动可能参与了TP对细胞线粒体能量代谢的影响之假说，详析TP致雄性生殖毒性作用机制，为其临床应用的减毒策略提供理论依据。

雷公藤甲素（Triptolide，TP）是中药雷公藤的主要活性成分，具抗炎、抗免疫等多种药理活性，有良好的临床应用前景；但治疗窗口窄、有严重毒副作用的特点限制了其临床应用，其中雄性生殖毒性是TP的明确毒性。本研究应用体内外模型研究了TP影响CPT-1酶的因子过氧化物酶增殖受体α(PPARα)与乙酰辅酶A 羧化酶(ACC), 并调控脂肪酸β-氧化的关键因子SIRT1与AMPK，扰乱支持细胞线粒体能量代谢的毒性机理。同时，对SIRT1与AMPK活性进行干预，以论证线粒体脂肪酸β-氧化的CPT-1酶是TP致睾丸支持细胞毒性的关键蛋白。研究结果显示TP诱导小鼠睾丸支持细胞系TM4细胞的线粒体氧化损伤，影响线粒体ATP合成功能，导致细胞凋亡；TP显著抑制SIRT1的蛋白水平，影响SIRT1-PGC-1α通路导致支持细胞线粒体损伤；TP引起小鼠雄性生殖毒性机制与影响SIRT1和AMPK对PGC-1α的调控相关，并通过PGC-1α影响下游PPARα与ACC，增加CPT-1表达，诱导脂质过氧化损伤； TP通过GATA4/Sp1/PFKP通路抑制支持细胞糖酵解，减少乳酸生成，导致细胞能量代谢紊乱。RSV可促进SIRT1的表达及活性，减少TP对支持细胞的毒性。本研究阐明了TP致支持细胞毒性的机制，为临床寻求减轻TP致男性不育症的方法提供了合理依据，可进一步应用于类似的中药。