基于TGF-βl和NOX的五酯片抵抗他克莫司慢性肾毒性的作用及新机制研究

Tacrolimus (FK506) is a well-known potent immunosuppressant agent for the prevention and/or treatment of graft rejection in solid organ transplantation patients. However, its chronic nephrotoxicity is one of the main limiting factors in the long-term outcome of organ transplants, leading to tissue fibrosis and ultimate organ failure. The cytokines TGF-βl and NADPH oxidases (NOX) were considered key factors in this process. Wuzhi Tablet (WZ) is a preparation of ethanolic herb extract of Wuweizi (Schisandra sphenanthera), which has been widely used to treat viral and drug-induced hepatitis in China. It is also popularly prescribed for Chinese renal or liver transplant patients with FK506-induced hepatitis. Our previous study showed that WZ could regulate the NRF2-ARE signaling pathway to prevent oxidative stress and prevent liver fibrosis. Meanwhile, combination of WZ and FK506 not only reduced rats transaminase (AST and ALT), but also significantly reduced rats usea nitrogen(BUN) and creatinine (Cr), suggesting WZ could inhibit FK506-induced nephrotoxicity. However, whether long-term treatment with WZ had protective and therapeutic effects against FK506-induced chronic nephrotoxicity and the involved molecular mechanism is still unclear. Therefore, the current study was conducted to investigate the protective and therapeutic effects of WZ on FK506-induced chronic nephrotoxicity in vitro and in vivo Furthermore, based on the characteristics and mechanism of FK506-induced chronic nephrotoxicity , the effect of WZ on TGF-βl pathway and NOX pathway and their mediated molecular events were investigated. Thus, the molecular mechanism of WZ in preventing FK506-induced chronic nephrotoxicity was well clarified. The current study would provide a theoretical basis for the new application of WZ in preventing FK506-induced chronic nephrotoxicity.

他克莫司(FK506)是临床上重要的一线免疫抑制剂, 但其高发的慢性肾毒性严重影响移植患者及移植物的远期存活率。如何减轻或避免FK506慢性肾毒性一直备受医学界关注。目前认为转化生长因子(TGF-βl)及NADPH氧化酶系(NOX)在FK506慢性肾毒性中起重要作用。华中五味子的乙醇提取物制剂五酯片(WZ）作为护肝药在临床上常与FK506合用，我们前期发现WZ通过抗氧化应激及抗纤维化起护肝作用，且WZ与FK506合用可显著降低大鼠尿素氮及肌酐，提示WZ可抵抗FK506所致肾功能损害。但是，长期合用WZ能否抵抗FK506慢性肾毒性及相关机制尚未见报道。因此，本课题拟通过体内体外实验考察WZ对FK506慢性肾毒性的保护和治疗作用，从影响TGF-βl通路介导的纤维化及NOX通路介导的氧化应激等方面阐释WZ防治FK506慢性肾毒性的分子机制，为WZ抵抗FK506慢性肾毒性的新应用提供理论依据。

他克莫司（FK506）是临床上器官移植患者长期使用的一线免疫抑制剂，但其所致慢性肾损伤严重地影响着移植物的远期存活率及移植受者的生活质量。五酯片（WZ）是华中五味子的乙醇提取物，是常用的护肝中成药。我们前期的研究表明合用WZ可显著降低其大鼠的尿素氮（BUN）及肌酐（Cr），提示WZ可能具有抵抗FK506所致肾功能损伤的潜在作用。基于此，本研究采用钠耗竭法建立FK506慢性肾损伤大鼠模型，考察WZ对FK506所致慢性肾损伤的抵抗作用及相关机制，为WZ改善FK506所致慢性肾损伤提供理论依据。 结果表明：（1）在低钠饮食的基础上，连续灌胃给予FK506 28天可使大鼠的血清中Cr、BUN显著升高，促进大鼠肾组织中肾小囊腔萎缩、肾小管上皮细胞脱落和坏死钙化、胶原纤维增生、肾小球基底膜增厚，说明长期灌胃给予FK506可致大鼠肾功能及肾组织病理结构损伤。表明采用钠耗竭法成功建立了FK506慢性肾损伤大鼠模型。（2）与FK506组相比，合用低剂量（62.5 mg/kg）的WZ显著减少大鼠血清中Cr（P<0.001）和BUN（P<0.05）的含量，改善肾组织中胶原纤维增生，说明低剂量的WZ对FK506所致慢性肾功能及肾组织病理损伤具有保护作用。但中剂量（125 mg/kg）、高剂量（250 mg/kg）的WZ未显示出对FK506慢性大鼠肾功能及肾组织病理损伤的保护作用。（3）合用低、中、高剂量的WZ使大鼠肾组织的还原型谷胱甘肽（GSH）显著性升高，而使丙二醛（MDA）呈剂量依赖性降低，表明合用不同剂量的WZ均可不同程度地增加大鼠的抗氧化能力、抑制脂质过氧化，从而减少FK506慢性肾损伤大鼠肾脏的氧化应激损伤。（4）不同剂量的WZ对FK506慢性肾损伤大鼠肾组织中TGF-β/Smad通路和NOX通路相关因子的mRNA及蛋白表达无显著影响，提示WZ改善FK506所致大鼠慢性肾损伤并非通过影响TGF-β/Smad通路和NOX通路相关因子的基因和蛋白表达。总之，低剂量WZ可以改善FK506所致慢性肾功能及肾组织损伤，但是并非通过影响响TGF-β/Smad通路和NOX通路相关因子的基因和蛋白表达。本研究初步为WZ抵抗FK506所致慢性肾损伤提供实验基础及理论依据，为临床上防治FK506所致慢性肾损伤提供新的治疗方法。