黄芩苷对糖尿病肾病小鼠肾小管上皮细胞凋亡的影响*

Inhibition of Baicalin on apoptosis of renal tubular epithelial cells induced by high glucose through Nrf2-ARE pathway*

摘要:

目的 研究黄芩苷对核因子E2相关因子2-抗氧化反应元件（Nrf2-ARE）信号通路的影响，探讨黄芩苷对糖尿病肾病（DN）的治疗机制。方法 30只C57BL/6雄性小鼠随机分成空白对照组（对照组）、DN模型组（DN组）和黄芩苷干预组（干预组），每组10只。通过腹腔注射链脲佐菌素复制DN模型并给予黄芩苷药物干预。ELISA法检测小鼠尿白蛋白。对肾小管上皮细胞进行分组：①高糖组：细胞在含25?mmol/L 葡萄糖的高糖培养基中培养。②激活组：细胞在高糖组处理的基础上，用Nrf2激活剂NK-252（20?μmol/ml） 预处理肾小管上皮细胞24?h。③黄芩苷组：细胞在高糖组处理的基础上，用黄芩苷（100?μmol/L）预处理肾小管上皮细胞24?h。④干扰对照组：高糖培养基中培养的细胞在加入黄芩苷的同时，通过Lipofectamine 2000对肾小管上皮细胞转染si-control（si-Nrf2的阴性对照），24?h后收集细胞。⑤Nrf2干扰组：高糖培养基中培养的细胞在加入黄芩苷的同时，通过Lipofectamine 2000对肾小管上皮细胞转染si-Nrf2（Nrf2的小干扰RNA），24?h后收集细胞。Western blotting检测Nrf2的表达水平，流式细胞仪检测肾小管上皮细胞凋亡状况。结果 与DN组比较，干预组小鼠的尿白蛋白水平降低、Nrf2的表达水平升高、ARE的活性增强，差异均有统计学意义（P?<0.05），且干预组小鼠的肾小管扩张变形程度减轻。细胞实验中发现，与高糖组比较，加入Nrf2激活剂或黄芩苷后，均能激活Nrf2，上调ARE活性，降低丙二醛（MDA）表达水平，并抑制肾小管上皮细胞凋亡（P?<0.05）。同时，干扰Nrf2能逆转黄芩苷的作用，促进肾小管上皮细胞凋亡（P?<0.05）。结论 黄芩苷可能通过上调Nrf2-ARE信号通路抑制高糖诱导的肾小管上皮细胞凋亡，发挥治疗DN的作用。

Abstract:

Objective To investigate the mechanism of Baicalin on diabetic nephropathy (DN) by studying the effects of baicalin on Nrf2-ARE signaling pathway. Methods The DN mouse model was constructed by intraperitoneal injection of streptozotocin, and baicalin was used to treat the mice. The urinary albumin in each mouse was detected by ELISA. The renal tubular epithelial cells were divided into two groups: ① high glucose group: the cells were treated with high glucose (25?mmol/L glucose); ② activation group: the cells were pretreated with Nrf2 activator NK-252 (20?μmol/ml) on the basis of high glucose group for 24 hours. ③ Baicalin group: cells were pretreated with baicalin (100?μmol/L) on the basis of high glucose group for 24 hours. ④ Interference control group: cells cultured in high glucose medium were transfected with Si-control (negative control of Si-Nrf2) by Lipofectamine 2000 while baicalin was added. Cells were collected 24 hours later. ⑤ Nrf2 interference group: cells treated with high glucose were transfected with Si-Nrf2 (small interfering RNA of Nrf2) by Lipofectamine 2000 while baicalin was added. Cells were collected 24 hours later. The expression level of Nrf2 was detected by Western blotting, and the apoptosis of renal tubular epithelial cells was measured by flow cytometry. Results Compared with the DN group, the level of urinary albumin in DN mice of the baicalin group was decreased, the expression of Nrf2 was increased and the activity of ARE was enhanced, and the difference was statistically significant (all P?