益气活血化瘀汤对慢性肾衰竭大鼠钙磷代谢紊乱及血管钙化的影响及其机制研究

Effect and mechanism of Yiqi Huoxue Huayu Decoction on the disorder of calcium and phosphorus metabolism and vascular calcification in rats with chronic renal failure

摘要:

目的 探究益气活血化瘀汤对慢性肾衰竭（CRF）大鼠钙磷代谢紊乱及血管钙化的影响及其相关机制。方法 将50只大鼠随机分为5组：假手术组、肾衰组、低剂量组、高剂量组和骨化三醇组，每组10只。采用5/6肾切除法复制CRF大鼠，假手术组不切除肾脏。模型复制成功2周后开始灌胃益气活血化瘀汤：低剂量组8.8 g/kg，高剂量组35.2 g/kg；骨化三醇组灌胃骨化三醇25 ng/kg；假手术组灌胃等体积生理盐水，1次/d，连续8周。肾衰组未做任何处理。给药结束后，代谢笼收集大鼠24 h尿液，采用酶联免疫吸附试验（ELISA）检测尿蛋白（UP）、甲状旁腺激素（PTH）、血清肌酐（Scr）、成纤维细胞生长因子23（FGF-23）与25-羟维生素D₃ 25（OH）D₃；全自动生化仪检测血钙、血磷；化学显色法检测血尿素氮（BUN）。采用茜素红对大鼠主动脉进行大体染色和切片染色，观察血管钙化结节。采用实时荧光定量聚合酶链反应（qRT-PCR）和Western blotting检测结肠腺瘤样息肉蛋白（APC）、T细胞因子4（TCF4）、β-catenin mRNA和蛋白相对表达量。结果 与肾衰组比较，低剂量组、高剂量组和骨化三醇组血钙、25（OH）D₃升高（P <0.05）；高剂量组和骨化三醇组血钙、25（OH）D₃高于低剂量组（P <0.05）；骨化三醇组血钙、25（OH）D₃高于高剂量组（P <0.05）；与肾衰组比较，低剂量组、高剂量组和骨化三醇组血磷、PTH、BUN、Scr、24 h UP、FGF-23降低（P <0.05）；高剂量组和骨化三醇组血磷、PTH、BUN、Scr、24 h UP、FGF-23低于低剂量组（P <0.05）；骨化三醇组血磷、PTH、BUN、Scr、24 h UP、FGF-23低于高剂量组（P <0.05）。假手术组均未见红色钙化结节；肾衰组可见大面积红色钙化结节，呈弥散式分布；相比于肾衰组，低剂量组、高剂量组和骨化三醇组的钙化出现不同程度减轻。与肾衰组比较，低剂量组、高剂量组和骨化三醇组APC mRNA和蛋白相对表达量升高（P <0.05）；高剂量组和骨化三醇组APC mRNA和蛋白相对表达量高于低剂量组（P <0.05）；骨化三醇组APC mRNA和蛋白相对表达量高于高剂量组（P <0.05）；与肾衰组比较，低剂量组、高剂量组和骨化三醇组TCF4、β-catenin mRNA和蛋白相对表达量降低（P <0.05）。高剂量组和骨化三醇组TCF4、β-catenin mRNA和蛋白相对表达量低于低剂量组（P <0.05）；骨化三醇组TCF4、β-catenin mRNA和蛋白相对表达量低于高剂量组（P <0.05）。结论 益气活血化瘀汤可以改善CRF模型大鼠的钙磷代谢紊乱，缓解血管钙化程度，这可能与其调节Wnt/β-catenin通路有关。

Abstract:

Objective To explore the effect and related mechanism of Yiqi Huoxue Huayu Decoction on the disorder of calcium and phosphorus metabolism and vascular calcification in rats with chronic renal failure.Methods Fifty rats were randomly divided into sham operation group, renal failure group, low-dose group, high-dose group and calcitriol group. The rat models of chronic renal failure were established via 5/6 nephrectomy, while the rats in the sham operation group were not nephroectomized. Two weeks after the model establishment, the rats were given Yiqi Huoxue Huayu Decoction by gavage at a dose of 8.8 g/kg for the low-dose group and 35.2 g/kg for the high-dose group. The rats in the calcitriol group were given calcitriol at a dose of 25 ng/kg by gavage, while those in the sham operation group were administered with an equal volume of normal saline, once a day for consecutive eight weeks. The rats in the renal failure group were left untreated. After the administration, the 24-hour urine of rats was collected and the levels of urine protein (UP), parathyroid hormone (PTH), serum creatine (Scr), fibroblast growth factor-23 (FGF-23) and 25-hydroxyvitamin D₃ (25(OH)D₃) were measured via enzyme-linked immunosorbent assay (ELISA). The blood calcium and phosphorus were measured via automatic biochemical analyzer. The content of blood urea nitrogen (BUN) was detected via the chromogenic method. The aorta of rats was stained by alizarin red, and the calcified nodules within the aorta were observed. The mRNA and protein expressions of adenomatous polyposis coli (APC), T-cell factor 4 (TCF4) and β-catenin were detected via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB), respectively.Results Compared with renal failure group, the concentrations of blood calcium and 25(OH)D₃ were increased in low-dose group, high-dose group and calcitriol group (P < 0.05). The concentrations of blood calcium and 25(OH)D₃ were higher in the high-dose group and calcitriol group compared with those in the low-dose group (P < 0.05), while those in the calcitriol group were even higher than those in the high-dose group (P < 0.05). Compared with renal failure group, the concentrations of blood phosphorus, PTH, BUN, Scr, 24-hour UP and FGF-23 were decreased in the low-dose group, high-dose group and calcitriol group (P < 0.05). The concentrations of blood phosphorus, PTH, BUN, Scr, 24-hour UP and FGF-23 in the high-dose group and the calcitriol group were lower than those in the low-dose group (P < 0.05), while those in the calcitriol group were even lower than those in the high-dose group (P < 0.05). No calcified nodules were found in the sham operation group, whereas a large area of calcified nodules were seen and diffusely distributed in renal failure group. Compared with renal failure group, the calcification in low-dose group, high-dose group and calcitriol group decreased by varying degrees. The mRNA and protein expressions of APC in low-dose group, high-dose group and calcitriol group were increased compared with those in the renal failure group (P < 0.05), and they were higher in the high-dose group and calcitriol group relative to those in the low-dose group (P < 0.05). Besides, the mRNA and protein expressions of APC were even higher in the calcitriol group than those in the high-dose group (P < 0.05). Compared with the renal failure group, the mRNA and protein expressions of TCF4 and β-catenin were lower in the low-dose group, high-dose group and calcitriol group (P < 0.05). The mRNA and protein expressions of TCF4 and β-catenin were lower in the high-dose group and the calcitriol group (P < 0.05), while they were even lower in the calcitriol group relative to those in the high-dose group (P < 0.05).Conclusions Yiqi Huoxue Huayu Decoction can improve the disorder of calcium and phosphorus metabolism and alleviate the degree of vascular calcification in rats with chronic renal failure, which may be related to the regulation of Wnt/β-catenin pathway.