从肌动蛋白骨架重排探讨益气清热解毒法对肾小球足细胞损伤的影响

Proteinuria plays a major role in the progression of chronic nephritis. we have realized that the pathogenesis of chronic nephritis is deficiency of the lung qi to protect the surface, heat and toxin invasion, and violation of the kidney to store the essence of the body. Based on this theory, we established the treatment notion that "Treat the kidney by treating the lung.", and Yiqiqingregao was created according to this theory. Based on the previous principles, we also explored the mechanisms of Yiqiqingregao in treatment of proteinuria in animal experiments. In the previous project of National Natural Science Fund of China (NSFC, 2011/01－2013/12，No.81072971), we demonstrated that, in rat Puromycin aminonucleoside model, Yiqiqingregao could decrease proteinuria, elevate serum albumin, attenuate kidney pathological lesions, ameliorate podocyte and glomerular filtration barrier (GFB) injury, restore slit diaphragm, upregulate the protein expression levels of nephrin, podocin and CD2 associated protein (CD2AP), and decrease the mRAN expression levels of those proteins probably by negative feedback mechanism. In another project of NSFC (2011/01－2014/12，No.81072971) of our group, we explored the mechanism of Yiqiqingregao in regulation of mesangial homeostasis and wnt/β-catenin pathway by in vitro studies in rat mesangial cell line HBZY-1. We found that Yiqiqingregao contained serum could inhibit mesangial cell proliferation stimulated by lipopolysaccharide(LPS), inhibit wnt4, TGF-β1 mRNA expression, and inhibit wnt4, β-catenin, and TGF-β1protein levels, a effect equivalent to that of the fosinopril. Podocyte injury is critical for the formation of proteinuria. The actin cytoskeletal structure dis-arrangement is the direct mechanism of increased podocyte activity. In this study, we will conduct experiments to study the mechanism of Yiqiqingregao on regulatory proteins of podocyte cytoskeleton (e.c.α-actinin-4, synaptopodin, vimentin, Desmin, nestin), and key proteins of RhoA/ROCK signaling pathway (e.c. RhoA, Rac1, Cdc42) in vivo experiment of PAN rat model and in vitro experiment of podocyte. We are aimed to explore the objectivity of the hypothesis that "increased podocyte activity as a result of re-arrangement of actin cytoskeleton is the pathogenesis of podocyte injury, and the treatment method of reinforcing Qi and clearing heat and toxin can attenuate proteinuria by affecting actin cytoskeleton re-arrangement." This study will further provide molecular mechanism for the treatment method of reinforcing Qi and clearing heat and toxin, the notion of "treating the kidney by treating the lung", and the theory of "The kidney governs the storage of essence, and proteinuria is the result of deficency of the kidney to store the essence".

蛋白尿属中医肾精的范畴，郁于中医"肾主藏精、封藏失职"理论，补肾固摄法成为慢性肾炎主要治法，但疗效甚微。我们根据患者容易"外感"并因此加重蛋白尿的特征，以益气清热解毒法治疗取得较好疗效。以往研究从肾小球系膜细胞和基质、系膜稳态及肾小球滤过屏障等证实了益气清热解毒法降低蛋白尿的机制。本研究通过整体研究：益气清热膏对嘌呤霉素肾病大鼠足细胞的干预研究；离体研究：从actin骨架相关蛋白探讨益气清热膏对体外培养足细胞干预作用并探索其作用的信号通路。从不同角度探讨益气清热膏对足细胞骨架相关调控蛋白如α-辅肌动蛋白-4等及足细胞RhoA/ROCK信号通路关键蛋白如RhoA等分子表达的影响，阐述"actin骨架重排引起足细胞活动度增加导致足细胞损伤的机制及益气清热解毒法通过影响actin骨架重排减轻蛋白尿"假说的客观性，为慢性肾炎从"肺"论治提供客观依据，更加丰富中医"肾主藏精、封藏失职"理论内涵。

肾小球足细胞损伤是慢性肾小球进展肾炎蛋白尿形成的关键环节，肌动蛋白骨架重排是导致足细胞损伤的重要分子机制，因此通过干预肌动蛋白调节蛋白可稳定细胞骨架，减少足细胞的活动性，降低蛋白尿。课题组根据患者容易“外感”并因此加重蛋白尿的特点，提出从“肺”论治慢性肾炎，以益气清热解毒法获得良好疗效。据此提出益气清热解毒法通过影响肌动蛋白细胞骨架重排减轻蛋白尿的假说，本研究包括体内研究和体外研究两部分。体内研究通过建立嘌呤霉素氨基核苷（PAN）肾病大鼠模型，采用光镜、透射电镜、ELISA、RT-PCR、Western Blot等方法，观察益气清热膏对肾小球足细胞的保护作用。体外研究通过离体培养条件永生性小鼠足细胞系，采用G蛋白活化检测法（G-LISA）、流式细胞术、免疫荧光染色、划痕实验等方法，观察益气清热膏对PAN损伤致足细胞骨架重排的影响；采用RhoA/ROCK信号通路选择性抑制剂和激动剂预干预，检测该通路关键分子的表达情况及其下游重要靶点的磷酸化水平，进一步探讨益气清热膏对RhoA/ROCK信号通路的影响，为益气清热膏治疗慢性肾小球肾炎蛋白尿提供客观依据。体内研究结果表明，益气清热膏能有效降低蛋白尿，减轻肾脏病理损伤，减少足突宽度，降低肾小球内足细胞肌动蛋白细胞骨架相关蛋白mRNA与蛋白的异常表达。体外研究结果提示，益气清热膏能减轻PAN引起的离体培养足细胞损伤，促进足细胞增殖并抑制其凋亡；还可降低足细胞的运动能力，增加其黏附能力；抑制肌动蛋白微丝重排，稳定细胞骨架；同时也能下调肌动蛋白细胞骨架相关蛋白的异常升高，结果与整体研究一致。体外研究还说明，益气清热膏可激活RhoA/ROCK信号通路，增加下游MLC和MYPT1的磷酸化水平以保持应力纤维的形成，同时增加另一靶点LIMK和Cofilin的磷酸化水平以抑制肌动蛋白解聚，促进肌动蛋白微丝形成，恢复肌动蛋白微丝在细胞内的网状排列结构，以此稳定细胞骨架，发挥足细胞保护作用。本研究是既往研究的有力深化和补充，为益气清热解毒法治疗慢性肾小球蛋白尿提供客观依据。.

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