基于SIRT1对P-gp表达的调控作用研究活血化瘀法逆转狼疮肾炎激素耐药机制

Lupus nephritis (LN) is a severe complication of systemic lupus erythematosus (SLE). When we refer to its treatment, steroid resistance is always the main factor of treatment failure and poor therapeutic effect. Also, we find that promoting blood circulation therapy can improve the therapeutic effect in different degrees. Our previous study demonstrates that panax notoginseng saponin, the active component of panax notoginseng, can inhibit the expression of P-gp in peripheral lymphocytes of SLE patients, thus it can reverse the steroid resistance. But the mechanism of its down-regulating the expression of P-gp is keep unknown. Recent studies indicate that SIRT1 is the key factor of regulating P-gp. SIRT1 enters the nucleus by the deacetylation of FoxO1, then promotes the transcription of MDR1, and finally up-regulates the expression of P-gp. But there is few reports mentioned about this pathway of reversing the steroid resistance of LN. We infer that the effect of traditional Chinese drug for promoting blood circulation on reversing the P-gp mediated steroid resistance may associate with its interfering this pathway. In our study, we will establish the NZB/WF1 lupus model, and form the control groups of SIRT1 low expression and high expression by the transfection of siRNA and lentiviral vector plasmid. Then, we will observe the effect of panax notoginseng saponin on the expression of SIRT1, intranuclear FoxO1,MDR1,P-gp gene and protein and intracellular corticosteroids in splenic lymphocytes of lupus mice, also the improvement of renal impairment of lupus mice and the local expression of P-gp. All of these observations will be finished on both the cellular level and the general level. Finally, we will discuss the pathway, target site and effect of panax notoginseng saponin on reversing steroid resistance, illustrate the mechanism of enhancing therapeutic effect and reducing side-effect which is reached by Promoting Blood Circulation Therapy on LN treatment, and afford a novel sight to improve the therapeutic effect of traditional Chinese drug on the treatment of LN.

狼疮肾炎（LN）是系统性红斑狼疮的严重并发症，激素耐药是导致治疗失败、影响疗效的主要因素。采用活血化瘀法治疗能不同程度提高临床疗效。我们前期研究证实活血化瘀中药三七的有效成分三七皂苷能抑制狼疮患者外周血淋巴细胞P-gp表达，具有逆转激素耐药作用，但其下调P-gp表达的机制尚不明确。新近研究表明SIRT1能使FoxO1脱乙酰后进入细胞核、启动MDR1转录并上调P-gp表达，但基于这一通路研究逆转LN激素耐药鲜有报道。我们推测活血化瘀中药逆转P-gp介导的激素耐药可能与这一途径有关。本研究拟通过NZB/WF1狼疮肾炎鼠，构建siRNA及慢病毒质粒转染形成SIRT1低表达及高表达作为对照，分别从细胞和整体水平研究三七皂苷逆转LN激素耐药作用途径、靶位和效应，阐明活血化瘀法治疗LN增效减毒机制，为提高临床疗效提供新的研究思路和科学依据。

狼疮肾炎(LN)是系统性红斑狼疮最常见和严重的脏器损害，也是终末期肾衰最重要的原发病之一。激素是迄今国际公认的一线治疗药，但激素耐药成为了导致治疗失败、影响预后的主要因素。故阐明激素耐药机制及关键靶点，探索运用活血化瘀中药逆转耐药，对提高临床疗效具有重要的理论和现实意义。课题组利用自发LN小鼠构建激素耐药细胞和动物模型，运用siRNA和慢病毒质粒转染形成SIRT1低表达及高表达作为对照，分别从细胞和整体水平研究三七皂苷（PNS）逆转LN激素耐药作用途径、靶位和效应。取得以下主要结果：1.通过小剂量甲强龙（2µl/ml）体外刺激小鼠脾淋巴细胞，及小剂量甲强龙（0.8mg/kg.d）连续灌胃28天，使小鼠脾淋巴细胞P-gp高表达和罗丹明-123水平显著下降，成功构建激素耐药细胞和动物模型，弥补了狼疮激素耐药研究领域此项技术的缺陷，对研究激素耐药机制和关键靶点，进一步探寻逆转耐药方法奠定了研究基础。2.阐明了SIRT1对P-gp介导激素耐药调控作用及信号通路。体外研究证实SIRT1通过干预淋巴细胞核内转录因子Fox01，调控MDR1mRNA表达，影响P-gp水平；体内研究表明下调SIRT1后，激素耐药鼠脾淋巴细胞和肾组织P-gp表达明显降低。证明激素耐药淋巴细胞中存在SIRT1- Fox01-MDR1-P-gp信号通路，SIRT1是调控P-gp关键因子，这为进一步研究逆转激素耐药提供了新的干预靶点和信号通路。3.证实了PNS基于SIRT1对P-gp的调控在逆转LN激素耐药中发挥了重要作用。PNS通过干预SIRT1，下调MDR1基因表达，降低了淋巴细胞P-gp水平，使细胞内激素水平明显升高；并协同增强大剂量激素诱导淋巴细胞凋亡和抑制其活化。动物实验进一步证实PNS能协同大剂量激素下调激素耐药鼠脾淋巴细胞SIRT1基因和蛋白表达，降低脾和肾组织P-gp水平；改善小鼠尿蛋白、血清白蛋白和肾病理损伤。PNS为上市药，安全性已为长期临床实践所证实。本研究结果丰富了PNS的作用内涵，为其在治疗LN中增效减毒作用提供了科学依据。本项目为进一步研究活血化瘀法提高LN临床疗效，及开发逆转激素耐药的中药新药提供了新的研究思路和方向。