基于Wnt/β-catenin信号通路探讨黄芪甲苷对慢性肾衰竭大鼠钙磷代谢紊乱的改善作用及机制

Research of Effect and Mechanism of Astragaloside-Ⅳon Calcium and Phosphorus Metabolism Disorder in Rats with Chronic Renal Failure Based on Wnt/β-catenin Signaling Pathway

[目的]分析黄芪甲苷(astragaloside-Ⅳ,AS-Ⅳ)对慢性肾衰竭(chronic renal failure,CRF)大鼠钙磷代谢紊乱的改善作用,以及对Wnt/β-连环蛋白(β-catenin)信号通路的调控机制。[方法]将无特定病原体(specific pathogen free,SPF)级雄性Wistar大鼠60只随机分为假手术组、模型组、西药组、AS-Ⅳ低剂量组和AS-Ⅳ高剂量组。各组干预6周后检测各组大鼠血清肌酐(serum creatinine,Scr)、尿素氮(blood urea nitrogen,BUN)、钙(calcium,Ca)、磷(phosphorus,P)、甲状旁腺激素(parathyroid hormone,PTH)、碱性磷酸酶(alkaline phosphatase,ALP)水平;检测24h尿蛋白定量;采用苏木精-伊红(hematoxylin-eosin,HE)染色观察肾组织病理变化;以免疫组化及实时荧光定量聚合酶链式反应(quantificational Real-time reverse transcription polymerase chain reaction,qRT-PCR)检测Wnt4、β-catenin蛋白及mRNA表达。[结果]与假手术组比较,模型组血清Ca水平降低,P、ALP、PTH、Scr、BUN及24h尿蛋白水平升高(P<0.01)。与模型组比较,西药组及AS-Ⅳ各剂量血清Ca水平升高,P、ALP、PTH、Scr、BUN及24h尿蛋白水平降低(P<0.05,P<0.01)。与西药组比较,AS-Ⅳ低剂量组血清Ca水平更低,ALP、PTH水平更高,AS-Ⅳ高剂量组P、Scr、BUN及24h尿蛋白水平更低(P<0.05,P<0.01)。与AS-Ⅳ低剂量组比较,AS-Ⅳ高剂量组血清Ca水平更高,P、ALP、PTH、Scr、BUN、24h尿蛋白水平更低(P<0.01)。HE染色提示,西药组病理改变较模型组轻,与AS-Ⅳ低剂量组相当;AS-Ⅳ高剂量组病理改变较西药组轻。与假手术组比较,模型组Wnt4、β-catenin蛋白平均光密度值(integral optical density,IOD)及mRNA相对表达量升高(P<0.01);与模型组比较,西药组与AS-Ⅳ各剂量组的Wnt4、β-catenin蛋白平均IOD及mRNA相对表达量更低(P<0.05,P<0.01);而AS-Ⅳ高剂量组Wnt4、β-catenin蛋白IOD及mRNA相对表达量低于西药组(P<0.05,P<0.01)。[结论]AS-Ⅳ能改善CRF大鼠的肾功能及钙磷代谢紊乱,抑制肾脏损伤,其机制可能与抑制Wnt/β-catenin信号通路的激活有关。

[Objective]To analyze the improvement effect of astragaloside-Ⅳ(AS-Ⅳ) on calcium and phosphorus metabolism disorders in chronic renal failure(CRF) rats, and the effect on regulatory mechanism of Wnt/β-catenin signaling pathway. [Methods] Sixty male specific pathogen free(SPF) Wistar rats were randomly divided into sham operation group, model group, western medicine group, AS-Ⅳ low-dose group and AS-Ⅳ high-dose group. After 6 weeks of intervention, the levels of serum creatinine(Scr), blood urea nitrogen(BUN), calcium(Ca), phosphorus(P), parathyroid hormone(PTH), alkaline phosphatase(ALP) were detected in each group, as well as 24h urine protein. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of renal tissue. The expressions of Wnt4 and β-catenin protein and mRNA were detected by immunohistochemistry and quantificational Real-time reverse transcription polymerase chain reaction(qRT-PCR).[Results] Compared with sham operation group, the serum Ca level of model group decreased, and the P, ALP, PTH, Scr, BUN and 24h urine protein levels increased(P<0.01). Compared with model group, the serum a level of western medicine group and AS-Ⅳ each dose group was higher, the levels of P, ALP, PTH, Scr, BUN and 24h urine protein were lower(P<0.05,P<0.01). Compared with western medicine group, the serum Ca level in AS-Ⅳ low-dose group was lower, and the ALP and PTH levels were higher, and AS-Ⅳ high-dose group had lower levels of P, Scr, BUN and 24h urine protein(P<0.05,P<0.01). Compared with AS-Ⅳ low-dose group, AS-Ⅳ high-dose group had higher serum levels of Ca and lower levels of P, ALP, PTH, Scr, BUN, and 24h urine protein(P<0.01). HE staining observation indicated that, the western medicine group had less pathological changes than model group, but equal to AS-Ⅳ low-dose group;the pathological changes of AS-Ⅳ high-dose group were lighter than those of western medicine group. Compared with sham operation group, the average integral optical density (IOD) value and relative expression of mRNA of Wnt4, β-catenin increased in model group(P<0.01).Compared with model group, the IOD of Wnt4 and β-catenin protein and the relative expression of mRNA were lower in western medicine group and AS-Ⅳ each dose group(P<0.05,P<0.01). The IOD of Wnt4 and β-catenin protein and the relative expression of mRNA in AS-Ⅳ high-dose group were lower than those in western medicine group(P<0.05,P<0.01). [Conclusion] AS-Ⅳ can improve the renal function, relieve calcium and phosphorus metabolism disorder and inhibit renal injury in CRF rats, and the mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway activation.