Table5_Molecular mechanism of Cuscutae semen–radix rehmanniae praeparata in relieving reproductive injury of male rats induced with tripterygium wilfordii multiglycosides: A tandem mass tag-based proteomics analysis.docx

Background: We determined the effects of Cuscutae semen (Cuscuta chinensis Lam. or Cuscuta australis R. Br.)–Radix rehmanniae praeparata (Rehjnannia glutinosa Libosch.) on the protein levels in testicular tissues of rats gavaged with tripterygium wilfordii multiglycosides (GTW) and elucidated the molecular mechanism underlying Cuscutae semen–Radix rehmanniae praeparata for relieving GTW-induced reproductive injury.
Methods: A total of 21 male Sprague–Dawley rats were randomly divided into the control group, model group, and Cuscutae semen–Radix rehmanniae praeparata group based on their body weights. The control group was given 10 mLkg−1 of 0.9% normal saline by gavage daily. The model group (GTW group) was administered with 12 mg kg-1 GTW by gavage daily. Cuscutae semen–Radix rehmanniae praeparata group (the TSZSDH group) was administered with 1.56 gkg−1 of Cuscutae semen–Radix rehmanniae praeparata granules daily according to their model group dosing. The serum levels of luteinizing hormone, follicle-stimulating hormone, estradiol, and testosterone were measured after 12 weeks of continuous gavage, and the pathological lesion of testicular tissues was observed. Differentially expressed proteins were evaluated by quantitative proteomics and verified by western blotting (WB) and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR).
Results: Cuscutae semen–Radix rehmanniae praeparata can effectively relieve pathological lesions of GTW-induced testicular tissues. A total of 216 differentially expressed proteins were identified in the TSZSDH group and model group. High-throughput proteomics revealed that differentially expressed proteins are closely associated with the peroxisome proliferator-activated receptor (PPAR) signaling pathway, protein digestion and absorption, and protein glycan pathway in cancer. Cuscutae semen–Radix rehmanniae praeparata can significantly upregulate the protein expressions of Acsl1, Plin1, Dbil5, Plin4, Col12a1, Col1a1, Col5a3, Col1a2, Dcn, so as to play a protective role on testicular tissues. Acsl1, Plin1, and PPARγ on the PPAR signaling pathway were verified by WB and RT-qPCR experiments, which were found to be consistent with the results of proteomics analysis.
Conclusion: Cuscutae semen and Radix rehmanniae praeparata may regulate the PPAR signaling pathway mediated Acsl1, Plin1 and PPARγ to reduce the testicular tissue damage of male rats caused by GTW.

背景：本研究旨在探讨菟丝子（Cuscuta chinensis Lam.或Cuscuta australis R. Br.）合熟地黄（Rehmannia glutinosa Libosch.）对灌胃雷公藤多苷（tripterygium wilfordii multiglycosides, GTW）雄性大鼠睾丸组织蛋白表达水平的影响，并阐明该药对缓解GTW诱导生殖损伤的分子机制。
方法：将21只雄性Sprague-Dawley（SD）大鼠按体重随机分为对照组、模型组及菟丝子合熟地黄组。对照组每日灌胃10 mL·kg⁻¹ 0.9%生理盐水；模型组（GTW组）每日灌胃12 mg·kg⁻¹雷公藤多苷；菟丝子合熟地黄组（TSZSDH组）每日参照模型组给药剂量灌胃1.56 g·kg⁻¹菟丝子合熟地黄颗粒。连续灌胃12周后，检测各组大鼠血清黄体生成素、卵泡刺激素、雌二醇及睾酮水平，观察睾丸组织病理损伤情况。采用定量蛋白质组学技术筛选差异表达蛋白，并通过蛋白质印迹（western blotting, WB）及实时荧光定量聚合酶链反应（Real-Time Quantitative Polymerase Chain Reaction, RT-qPCR）对差异蛋白进行验证。
结果：菟丝子合熟地黄可有效缓解GTW诱导的大鼠睾丸组织病理损伤。TSZSDH组与模型组共鉴定出216个差异表达蛋白。高通量蛋白质组学分析显示，差异表达蛋白主要富集于过氧化物酶体增殖物激活受体（peroxisome proliferator-activated receptor, PPAR）信号通路、蛋白质消化与吸收及肿瘤蛋白聚糖通路。菟丝子合熟地黄可显著上调Acsl1、Plin1、Dbil5、Plin4、Col12a1、Col1a1、Col5a3、Col1a2及Dcn的蛋白表达水平，从而对睾丸组织发挥保护作用。通过WB及RT-qPCR实验验证了PPAR信号通路中的Acsl1、Plin1及PPARγ的表达，结果与蛋白质组学分析一致。
结论：菟丝子与熟地黄可能通过调控PPAR信号通路介导的Acsl1、Plin1及PPARγ表达，减轻GTW诱导的雄性大鼠睾丸组织损伤。