低聚原花青素通过介导Wnt/β-catenin信号通路对糖尿病肾病大鼠肾功能的保护作用研究

Protective effects of oligomeric proanthocyanidins on renal function in rat models of diabetic nephropathy via Wnt/β-catenin signaling pathway

摘要:

目的 研究低聚原花青素通过介导Wnt/β-catenin信号通路对糖尿病肾病（DN）大鼠肾功能的保护作用。方法 选择60只SD雄性大鼠，随机分为正常对照组（NC组）、DN模型组（DN组）、DN模型+不同剂量低聚原花青素组（低、中、高剂量组），采用单次腹腔注射链脲佐菌素复制DN模型，低、中、高剂量组分别给予50 mg/kg、100 mg/kg、150 mg/kg低聚原花青素灌胃，NC组和DN组给予等体积蒸馏水灌胃。分别于第4周、8周、12周、16周，检测各组大鼠24 h尿蛋白定量；16周末检测大鼠血糖、血肌酐（Scr）、尿素氮（BUN）水平；处死大鼠，取肾脏组织行苏木精-伊红及过碘酸希夫染色，观察肾组织形态学变化；检测肾组织匀浆上清液中超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量；实时荧光定量聚合酶链反应和Western blotting检测肾组织Wnt4、β-catenin mRNA和蛋白的表达。结果 DN组血糖、Scr、BUN水平较NC组升高（P <0.05）；低、中、高剂量组血糖、Scr、BUN水平较DN组降低（P <0.05）。各组大鼠灌胃4周、8周、12周、16周的24 h尿蛋白定量比较，采用重复测量设计的方差分析，结果 ①不同时间点24 h尿蛋白定量有差异（F =102.38，P =0.000）；②各组24 h尿蛋白定量有差异（F =86.58，P =0.000）。③各组24 h尿蛋白定量变化趋势有差异（F =174.64，P =0.000）。DN组大鼠肾组织活检可见肾小管萎缩、小管间质区基质增厚、纤维化等病理改变。DN组左肾指数较NC组升高（P <0.05）；低、中、高剂量组左肾指数较DN组下降（P <0.05）。各组大鼠SOD活性比较，差异无统计学意义（P >0.05）；与NC组比较，DN组MDA含量升高（P <0.05），与DN组比较，不同剂量组MDA含量降低（P <0.05）。DN组大鼠肾组织Wnt4、β-catenin mRNA和蛋白相对表达量高于NC组（P <0.05），不同剂量组Wnt4、β-catenin mRNA和蛋白相对表达量低于DN组（P <0.05）。结论 低聚原花青素能有效降低DN大鼠24 h尿蛋白定量，减轻肾脏病理损害。低聚原花青素可能通过抗氧化作用及抑制Wnt/β-catenin信号通路，发挥肾脏保护作用。

Abstract:

Objective To investigate the protective effects of oligomeric proanthocyanidins on renal function in rat models with diabetic nephropathy (DN) via Wnt/β-catenin signaling pathway.Methods Sixty male SD rats were randomly divided into normal control group (NC group), DN model group (DN group), and DN model + oligomeric proanthocyanidins groups (low-, medium- and high-dose groups). DN models were established via intraperitoneal injection of streptozotocin. After model establishment, low-, medium- and high-dose groups were given 50 mg/kg, 100 mg/kg, and 150 mg/kg of oligomeric proanthocyanidins by gavage, while NC group and DN group were given equal volumes of distilled water. Quantitative test of the 24-hour urine protein was performed at the 4th, 8th, 12th and 16th weeks. Levels of blood glucose (Glu), serum creatinine (Scr) and blood urine nitrogen (BUN) were detected at the end of the 16th week. The histomorphometric changes in renal tissues were observed under hematoxylin and eosin staining and periodic acid-Schiff staining. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in the supernatants of renal tissue homogenates were detected. Western blotting and quantitative real-time polymerase chain reaction were used to assess the protein and mRNA expressions of Wnt4 and β-catenin in the renal interstitium, respectively.Results Compared with NC group, the levels of Glu, Scr and BUN in DN group were all significantly increased (P < 0.05). In contrast, the levels of Glu, Scr and BUN in low-, medium- and high-dose groups were significantly lower than those in DN group (P < 0.05). The 24-hour urine protein in each group at week 4, 8, 12 and 16 was compared via repeated measures analysis of variance, and the results revealed that the 24-hour urine protein was different among the time points (F = 102.38, P < 0.001) and the groups (F = 86.58, P < 0.001), and that the change trend of the 24-hour urine protein was also different among the groups (F = 174.64, P < 0.001). Renal biopsies of rats in the DN group showed tubular atrophy, tubulointerstitial thickening and fibrosis, and other pathological changes. The left renal index was higher in the DN group relative to NC group (P < 0.05), while that in low-, medium- and high-dose groups was lower compared with the DN group (P < 0.05). The SOD activity was not different among groups (P > 0.05). The MDA content in the DN group was increased compared with the NC group (P < 0.05), while that in low-, medium- and high-dose groups was decreased compared with the DN group (P < 0.05). The mRNA and protein expressions of Wnt4 and β-catenin in the renal interstitium of the DN group were significantly higher than those of the NC group (P < 0.05), while they were significantly lower in the low-, medium- and high-dose groups compared with the DN group (P < 0.05).Conclusions Oligomeric proanthocyanidins can effectively reduce the 24-hour urine protein and attenuate the renal damage in DN rats. Moreover, the renal protective effect of oligomeric proanthocyanidins may be related to its antioxidant activity and the inhibition of Wnt/β-catenin signaling pathway.