清热消癥法疏通糖尿病肾病肾脏固有细胞自噬不同节点的作用机制

Diabetic nephropathy (DN) is one of the diabetic microvascular complications. Obstruction of autophagic degradation pathway in the renal innate cells is the key site in the pathogenesis of DN. The specific blocked nodes were unclear. We proposed that “ShenLuoZhengJia” is the core pathogenesis of DN in Traditional Chinese Medicine. The therapy of “QingReXiaoZheng” guided by the theory of "ShenluoZhengjia" can regulate the protein expression in the process of autophagy-degradation related protein. We proposed a hypothesis that “QingReXiaoZheng can dredge the autophagy pathway in the renal innate cells of DN, promote autophagy induction and increasing the degradation of autophagic vesicles for alleviating the injury and apoptosis of renal innate cells, and then delay the progress of DN.” In this study, we want to explore the autophagy blocked nodes in the renal innate cells in vivo and in vitro. The diabetic animal and renal innate cells induced by Atg5 gene transfection are used as models. We well explore the medicine of “QingReXiaoZheng “and related serum can enhance autophagy induction for clarifing the correlation between the therapeutic effect of DN and the enhancement of the degradation of autophagic vesicles, so as to verify the scientificity and objectivity of the hypothesis. The specific targets and mechanisms of the therapeutic effect of “QingReXiaoZheng” method of DN also are studied. Therefore, we can provide the experimental evidences and theoretical basis for the treatment of DN in Traditional Chinese Medicine based on the theory of "ShenLuoZhengJia ".

糖尿病肾病(DN)是糖尿病微血管并发症之一，DN发病与肾脏固有细胞自噬降解通路受阻密切相关，但其具体受阻节点尚未阐明。我们认为“肾络癥瘕”是DN核心病机，在此理论指导下的清热消癥法可干预自噬降解相关蛋白的表达。我们提出“清热消㿂法可以通过促进自噬诱导及增加自噬泡降解疏通肾脏固有细胞的自噬通路，减轻肾脏固有细胞的损伤和凋亡，从而延缓DN进展”的科学假说。本研究拟从体内外筛查DN肾脏固有细胞自噬通路受阻的节点，以Atg5基因干预的糖尿病动物及肾脏细胞模型为研究对象，探讨清热消癥方及其含药血清是否通过增强自噬诱导治疗DN，阐明清热消癥方及其含药血清治疗DN与增强自噬泡降解的相关性，并验证假说的科学性及客观性；探讨清热消癥方治疗DN的确切作用靶点及效应机制，为以“肾络癥瘕”理论为指导的中医药防治糖尿病肾病研究提供理论基础和实验依据。

研究目的：研究表明调节自噬或疏通自噬通路可以减少与糖尿病肾病相关的肾损伤、蛋白尿和肾纤维化，并延缓糖尿病进展。清热消癥方（QRXZF）是我院肾病科医生多年来治疗DN的临床经验方，然而，目前尚不清楚QRXZF是否能调节自噬对DN的影响。故需要明确清热消癥法对肾脏固有细胞的保护作用，验证清热消癥法是否通过减少自噬应激，疏通自噬降解通路，恢复溶酶体功能，抑制DN肾损伤，从而阐释清热消癥法治疗DN的生物学内涵。.研究结果：.Ⅰ、体内实验:.1．各组大鼠24 h尿蛋白、血清肌酐及尿素氮、血脂和血清胱抑素C等水平显著高于假手术组；QRXZF和海藻糖治疗DN大鼠能够显著降低尿蛋白排泄量，改善肾脏功能。.2．HE、PAS、Masson等病理染色结果显示，DN组大鼠经QRXZF和海藻糖干预治疗后，能够改善DN大鼠肾脏的病理损伤。透射电镜下发现与DN大鼠相比，QRXZF组和海藻糖组足突病变程度相对较轻。雷帕霉素组大鼠肾脏病理损伤较DN组有加重趋势。.3．免疫组化结果：与DN大鼠组相比，QRXZF和海藻糖能下调III型、IV型胶原和纤维连接蛋白FN的表达。.4.透射电镜观察自小体和溶酶体：QRXZF组自噬体数量较DN组大鼠明显减少。海藻糖组大鼠肾脏有较小的自噬体和溶酶体分布。雷帕霉素组大鼠有大量溶酶体堆积。.5．免疫荧光结果：QRXZF可下调DN大鼠肾脏LC3、LAMP1和p62的表达。.6．免疫印迹法结果：QRXZF干预治疗16周后，可以下调LC3II/LC3I、p62的表达，上调TFEB的表达。.Ⅱ、体外实验:.1．免疫印迹法结果：.HK-2细胞：与模型组相比，QRXZF和海藻糖均可增加Beclin-1、Atg 7表达水平（*P＜0.05）。MPC-5细胞：海藻糖和QRXZF加海藻糖组均可增加ATG5、ATG7、Beclin1的表达水平。.2. 免疫荧光结果：HK-2细胞：与模型组相比，QRXZF和海藻糖可增加LC3B表达水平。MPC-5细胞：海藻糖联合QRXZF可降低P62表达，LC3B表达水平Lc3B各组无显著性差异。.研究结论：清热消癥法可能通过减轻自噬应激，疏通自噬降解，修复溶酶体功能，改善肾脏细胞衰老和凋亡，减少肾损伤，改善DKD肾脏纤维化，进而延缓DKD病情进展。