基于内质网应激-自噬途径探讨益气养阴活血法防治糖尿病肾病的分子机制

Diabetic nephropathy has become a serious public health problem. In our previous studies, we put forward the Yiqi Yangyin Huoxue therapy for treating diabetic nephropathy by comprehensive literature search and evidence-based medicine analysis. Our previous work found that Yiqi Yangyin Huoxue therapy can effectively reduce proteinuria in diabetic nephropathy, as well as decrease the expression of endoplasmic reticulum stress related protein ATF4 and improve the activity of podocyte autophagy. Therefore, we speculate that Yiqi Yangyin Huoxue therapy could regulate the endoplasmic reticulum stress-autophagy way to delay the development of diabetic nephropathy. This project intends to use normal mice and ATF4 knockout mice given with streptozotocin (STZ) to establish diabetic nephropathy model and observe the expression of ATF4 in kidney and the activity of podocyte autophagy. We plan to over expression and inhibit ATF4 in podocyte cultured under the condition of high sugar using the technology of gene transfection and siRNA. Combined with studies in vivo and vitro, the role of ATF4 in the regulation of podocyte autophagy in diabetic nephropathy will be confirmed. On this foundation, we apply the method of administration in vivo and serum pharmacology to clarify the effects of Yiqi Yangyin Huoxue therapy in the regulation of ATF4 and the activity of podocyte autophagy. It is of great significance for prevention and control of diabetic nephropathy.

糖尿病肾病已成为严重的公共卫生问题。本课题组前期通过文献调研、循证分析，提出益气养阴活血法治疗糖尿病肾病，前期研究发现益气养阴活血法可有效减少糖尿病肾病蛋白尿的同时，能够降低内质网应激相关蛋白ATF4的表达，提高足细胞自噬活性。因此我们推测，益气养阴活血法可能通过调节内质网应激-自噬途径延缓糖尿病肾病发生发展。本项目拟利用正常小鼠和ATF4基因敲除小鼠给予链脲佐菌素(STZ)诱导，建立糖尿病肾病小鼠模型，观察模型小鼠肾脏ATF4表达水平及足细胞自噬活性；应用RNA干扰技术及基因转染方法对高糖条件下体外培养足细胞进行ATF4抑制和过表达，结合体内外实验以明确ATF4对糖尿病肾病足细胞自噬的调控作用。在此基础上，采用体内给药及血清药理学方法，阐明益气养阴活血法对糖尿病肾病ATF4及其调控的足细胞自噬活性的干预效应。这对于糖尿病肾病的防治具有重要意义。

糖尿病肾病已成为严重的公共卫生问题。本课题组前期研究发现益气养阴活血法可有效减少糖尿病肾病蛋白尿，但机制尚不明确。本研究首先利用正常C57BL/6J小鼠和ATF4KO小鼠给予链脲佐菌素(STZ)诱导，成功建立糖尿病肾病小鼠模型，WB及免疫荧光结果显示糖尿病肾病小鼠自噬活性降低。与糖尿病肾病模型小鼠相比，给予STZ腹腔注射的ATF4KO小鼠细胞自噬活性得到一定的恢复。其次，我们应用RNA干扰技术及基因转染方法对高糖条件下体外培养NRK-52E细胞进行ATF4抑制和过表达，结果表明过表达ATF4后NRK-52E细胞p62蛋白表达升高，LC3-Ⅱ蛋白表达降低；抑制ATF4后NRK-52E细胞p62蛋白表达降低，LC3-Ⅱ蛋白表达升高，透射电镜显示自噬体数量增加，自噬双标腺病毒法显示自噬流增强。以上体内外实验明确了ATF4对糖尿病肾病细胞自噬的调控作用。再次，我们采用体内给药的方法，给予糖尿病肾病小鼠益气养阴活血中药干预，结果显示益气养阴活血方可以显著改善糖尿病肾病小鼠空腹血糖、尿白蛋白含量、血肌酐、尿素氮水平。最后，我们采用益气养阴活血方干预体外高糖损伤的NRK-52E细胞，WB结果显示益气养阴活血方可以显著降低ATF4的表达水平、降低p62表达水平、提高LC3-Ⅱ表达水平，透射电镜结果显示给予益气养阴活血方干预可以增加高糖损伤的NRK-52E细胞自噬体的形成，提示益气养阴活血方可在一定程度上恢复高糖诱导的NRK-52E细胞自噬抑制。本研究阐明了益气养阴活血法对糖尿病肾病ATF4介导的内质网应激-自噬失衡的调节作用，这为益气养阴活血法治疗糖尿病肾病的临床应用提供了科学依据，对于糖尿病肾病的防治具有重要意义。