SMAD2调控ERK通路干预M2巨噬细胞活化在糖尿病肾病小鼠肾脏纤维化中的作用及机制

Macrophages are associated with the process of diabetic nephropathy.M2 macrophages are involved in the kidney fibrosis of diabetic mice in our previous research. ERK and SMAD2 are all activated in M2 macrophages. But the process of M2 macrophage can not be fully blocked by the use of ERK inhibitor, and the activation of SMAD2 can not also be inhibited with it. The effect of SMAD2 pathway in the process of M2 macrophage of diabetic mice, and the relationship between the ERK and SMAD2 in M2 macrophage are not clear. In this research, the plasmid of SMAD7 ,shSMAD7,SMAD2 and shSMAD2 would be constructed and transfected to the diabetic mice with the use of ultrasound microbubble, then the renal M2 macrophages and the renal fibrosis would be measured ,which will confirm the effect of SMAD2 in the process of M2 macrophages.Furthermore,RAW264.7 macrophages would be transfected with these plasmids in vitro, then the macrophages will be induced to M2 phenotype with the high ambient glucose and TGF-β1,and the activation of ERK and the ratio of M2 phenotype in these macrophages will be investigated, which will confirm the relationship between ERK and SMAD2.From this research, a new pathogenesis of diabetic nephropathy will be found ,as a result, a new target to treat the diabetic nephropathy will also be discovered.

巨噬细胞与糖尿病肾病(DN)发病密切相关，我们前期研究发现:DN小鼠肾脏M2型巨噬细胞渗出增多，并参与了肾脏纤维化进程，且巨噬细胞向M2分化过程中伴有ERK1/2及SMAD2活化，但阻断ERK仅可部分阻断巨噬细胞向M2分化，且阻断ERK对SMAD2活化无影响。SMAD2是否为DN肾脏巨噬细胞向M2分化的关键通路，且ERK是否受控于SMAD2活化目前尚不清楚。本研究中，我们将构建SMAD2、 SMAD7正义及反义质粒：①通过超声微泡技术转染DN小鼠，观察其肾脏巨噬细胞向M2分化是否受SMAD2调控，且是否伴随M2分化的减弱存在肾脏纤维化程度减低。②体外转染RAW264.7巨噬细胞株，并以高糖及TGF-β1诱导其向M2分化，观察ERK1/2活化是否受控于SMAD2的活化。通过该研究提出DN新的发病机制，为临床通过干预SMAD2及ERK1/2活化治疗DN提供新的靶点。

研究背景：糖尿病肾病是终末期肾病的主要病因之一，巨噬细胞参与了糖尿病肾病的发病过程，但M2巨噬细胞在其中的作用及机制尚不清楚。.主要研究内容：通过STZ诱导C57BL/6j小鼠造糖尿病肾病模型，通过共聚焦显微镜观察肾小球及肾间质巨噬细胞型别变化,通过尾静脉注射脂质体包裹的氯磷酸钠清除糖尿病小鼠肾脏巨噬细胞，观察糖尿病小鼠肾小球硬化及肾间质纤维化缓解情况。体外通过高糖及TGF-β1诱导巨噬细胞向M2分化，观察分化过程中ERK通路及SMAD2通路活化情况，并分别利用SMAD2通路特异性抑制剂SB431542，及ERK通路抑制剂U0126分别抑制上述两条通路，观察巨噬细胞向M2分化是否受到抑制及SMAD2通路与ERK通路间是否存在交叉对话。.重要结果：糖尿病小鼠成模后8周，肾小球渗出巨噬细胞增多，且以表达CD206的M2巨噬细胞为主。通过脂质体包裹的氯磷酸钠静脉注射清除巨噬细胞后，糖尿病肾病小鼠肾脏纤维化指标胶原-4及纤连蛋白表达减低。高糖及TGF-beta1共同刺激可诱导RAW264.7细胞向M2分化，表现为高表达Arginase-1及CD206。SMAD2通路及ERK通路在M2分化过程中活化，且两条通路间相互独立。.关键数据及其科学意义：M2巨噬细胞参与了糖尿病肾病小鼠肾脏纤维化过程，且SMAD2通路及ERK通路参与了上述过程，阻断巨噬细胞渗出或者阻断SMAD2通路或ERK通路的活化在预防糖尿病肾病患者迅速走向终末期肾病可能有较大临床应用前景。