活性维生素D通过髓样细胞触发受体1降低糖尿病肾病大鼠肾组织巨噬细胞浸润

Active vitamin D reduces macrophage infiltration by TREM- 1 in renal tissue of diabetic nephropathy rats

目的探讨活性维生素D（vD）对糖尿病肾病（DN）大鼠肾组织中髓样细胞触发受体1（TREM-1）表达的影响及TREM-1对巨噬细胞迁移和黏附功能的作用。方法建立DN大鼠模型，随机分为正常对照（NC）组、VD组（骨化三醇0．1μg·kg-1·d-1灌胃）、DN组、DN＋VD组（DN大鼠以0．1μg·kg-1·d-1骨化三醇进行灌胃），分别于8周末、12周末处死，PAS和MASSON染色观察肾脏病理改变，Western印迹检测。肾组织CD68及TREM-1表达。体外培养RAW264．7细胞，分为NC组、高糖组、高糖＋VD组、VD组、乙醇组、甘露醇组。高糖＋VD组以10μmol／L1，25（OH）2D3干预高糖培养的巨噬细胞。免疫荧光和Western印迹检测TREM-1蛋白的表达，Transwell细胞迁移实验和黏附实验评估巨噬细胞的迁移和黏附能力。转染TREM-1 siRNA干扰及TREM-1质粒过表达，观察巨噬细胞黏附和迁移情况及1,25（OH）2D3对高糖诱导的巨噬细胞黏附和迁移的影响。结果（1）与对照组比较，DN组大鼠组肾组织CD68及TREM-1表达均增加（均P〈0．05），而DN＋VD组CD68及TREM-1表达均低于DN组（均P〈0．05）。（2）与正常对照组比较，高糖组巨噬细胞黏附、迁移数量和TREM．1的表达均增加（均P〈0．05），高糖＋VD组巨噬细胞黏附、迁移数量和TREM-1的表达均低于高糖组（均P〈0．05）。（3）TREM-1 siRNA干预下，巨噬细胞黏附和迁移数量均低于阴性对照组（均P〈0．05），VD能降低高糖诱导的巨噬细胞黏附和迁移（均P〈0．05）。（4）在TREM—1过表达巨噬细胞中，细胞黏附和迁移数量与阴性对照组比较均升高（均P〈0．05），VD对高糖诱导巨噬细胞黏附和迁移的抑制作用消失。结论活性维生素D通过降低TREM-1的表达抑制高糖诱导的巨噬细胞的黏附和迁移，从而降低巨噬细胞在糖尿病肾组织的浸润。

Objective To investigate the effects of active vitamin D （VD） on the expression of triggering receptor expressed on mycloid cells- 1 （TREM- 1） in renal tissue of diabetic ncphropathies （DN） rats and to explore the impact of TREM- 1 on adhesion and migration capacity of macrophage. Methods DN rat models were established by streptozotocin. Rats were randomly distributed into four groups： control （NC） group, VD group, DN group and DN＋VD group （DN rats with 0.1μg·kg-1·d-1 calcitriol by gavages）. Rats were sacrificed respectively at 8 weeks and 12 weeks after treatment. Pathological changes in kidney tissue were detected and the expressions of CD68 and TREM-1 were acquired by immunohistochemistry stain and Western blotting. In vitro, RAW264.7 cells were divided into NC group, VD group, high glucose （HG） group and HG＋VD group. In HG＋VD group rats were treated by high glucose with 10-8 mol/L 1,25（OH）2D3. TREM- 1 expression was measured by immunohistochemistry stain and Western blotting, and the ability of macrophage in migration and adhesion was evaluated by Transwell migration assay and adhesion assay. TREM- 1 siRNA was transferred to silence TREM - 1 expression, while plasmid of TREM - 1 was transferred for high expression. Their ability of adhesion and migration in macrophage and the effect of 1,25（OH）2D3 were examined. Results （1） Compared with the NC group, the expressions of CD68 and TREM-1 were increased in DN group （P 〈 0.05）, whereas markedly decreased in DN＋VD group （P 〈 0.05）. （2） The number of adhesion and migration cells, and the expression of TREM- 1 protein in macrophage were obviously increased in HG group as compared with those in NC group （all P 〈 0.05）; whereas above changes were markedly decreased in HG＋VD group than those in HG group （P 〈 0.05）. （3） The number of adhesion and migrated macrophage was reduced after TREM- 1 siRNA intervention （all P 〈 0.05）. VD could significantly decrease the effect of high glucose on adhesion and migrated macrophages after TREM- 1 siRNA （all P 〈 0.05）. （4） Adhesion and migration of macrophage were increased via TREM- 1 overexpression （all P 〈 0.05）, but the effects of VD on high glucose-induced adhesion and migration of macrophage were disappeared. Conclusions VD can suppress the adhesion and migration of macrophage via reducing the expression of TREM- 1, and inhibit infiltration of macrophage in renal tissue of DN rats.