从mTOR信号旁路研究慢性肾衰肾脏微徵积形成病机及抗纤灵对基因敲除小鼠抗肾纤维化作用

How to suppress the renal fibrosis and delaying progression of chronic renal failure is a difficult and key points in the research field of nephropathy. The theory of Traditional Chinese medicine considers there are multiple stasis in chronic disease, kidney fibrosis and accumulation of blood stasis in the kidney and cause of formation of micro-mass, promoting blood circulation and removing blood stasis has shown broad application prospects. Study found that in addition to the classical Smad, noncanonical PI3K/Akt/mTOR signal pathway also plays an important role in renal fibrosis. Select the Smad2( +/- ) gene knockout mice 5/6 nephrectomized kidney fibrosis model, dynamic observation of renal fibroblast proliferation and secretion of collagen and protein, study on chronic renal failure in the formation and development of micro-mass and different doses of Kangxianling at different time points against renal fibrosis, its mechanism is through inhibition of PI3K/Akt/mTOR and inhibition of TGF-β1, PDGF and VEGF,MMP2/TIMP and NF-κB、Th2 and IL10 changes were observed that this signaling pathway in supress of ECM proliferation and plays an important role in the maintenance of immune inflammatory balance，for chronic renal failure pathogenesis theory of micro-mass product in kidney and promoting blood circulation to remove blood stasis Kangxianling in addition to confirmed that inhibition of Smad,P38MAPK there are any other way of inhibiting renal fibrosis and provide new experimental evidence.

如何抑制肾纤维化延缓慢性肾衰进展一直是肾病研究领域中的难点和关键点。中医认为久病多瘀，肾纤维化与瘀血内结在肾脏进而导致微癥积形成理论相符，活血化瘀已显示广阔应用前景。最新研究发现除经典Smad，非经典PI3K/Akt/mTOR信号旁路在致肾纤维化中也有重要作用。选择Smad2（+/-）基因敲除小鼠制作5/6肾切除慢性肾衰肾纤维化模型，动态观察肾脏肌成纤维细胞增生和分泌胶原和蛋白，研究慢性肾衰微癥积形成和进展过程和不同剂量抗纤灵不同时间点的抗肾纤维化作用，其机制是否通过抑制PI3K/Akt/mTOR进而抑制TGF-β1、PDGF和VEGF等，观察MMP2/TIMP和NF-κB、Th2与IL10变化证明此信号旁路在抑制ECM增生及维持免疫炎症平衡的重要作用，为慢性肾衰肾脏微癥积病机理论和活血化瘀抗纤灵方除了已证实有抑制Smad、P38MAPK作用外是否还有其他途径抑制肾纤维化提供新的实验依据。

肾脏纤维化是慢性肾衰的主要病理基础之一，对其机制和治疗方法的研究具有重要的科学价值。肾纤维化的病理学形态改变符合中医有关“癥积”的认识，血瘀是形成肾脏微徵积的重要病理机制和中心环节，治疗以活血化瘀为主，抗纤灵是抗纤维化的经验方。.目前研究参与肾纤维的的主要信号通路有TGF-β/Smad信号转导通路及磷酯酰肌醇3激酶(PI3K)-丝氨酸/苏氨酸蛋白激(Akt)信号通路。通过现代基因技术，分别敲除小鼠肾脏特定的基因Smad2,Smad3，目的是阻断TGF-β1/Smad这一大家认可的致使纤维化发生的信号通路，从导致纤维化的另外一条通路，即PI3K-Akt-mTOR信号旁路途径入手，并使用mTOR的抑制剂作为对照药物，观察中药抗纤灵对模型小鼠肾组织病理形态学、细胞外基质及炎性细胞因子等的影响，观察中药抗纤灵是否通过PI3K-Akt-mTOR信号通路而达到抑制肾脏纤维化的发生。结果发现：抗纤灵能够改善慢性肾衰模型小鼠的血清肌酐、尿素氮及24小时尿蛋白定量水平，改善脂质代谢，减轻Smad2/3-KO小鼠肾功能的损害，减少纤维化面积，同时能够下调肾组织中的TGF-β1、α-SMA 、CollageI、FN、p-PI3K、p-Akt、p-mTOR、p38MAPK、NF-κB蛋白和mRNA表达，降低模型小鼠血清中IL-6、TNF-α的含量，减少F480细胞的阳性表达，其机制可能与抑制PI3K-Akt-mTOR信号通路有关。丰富了抗纤灵治疗慢性肾纤维化的作用机制，为其临床应用及新药开发奠定了基础。