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目的观察六味地黄汤对慢性肾衰模型大鼠肾组织中脯氨酸羟化酶2(PHD2)、缺氧诱导因子-1α(HIF-1α)表达的影响,探讨慢性肾衰的发病机制以及六味地黄汤防治慢性肾脏病的分子机制。方法 60只SD大鼠随机分为假手术组,模型组,六味地黄汤低剂量、中剂量和高剂量组。5/6肾切除法复制慢性肾衰模型。六味地黄汤低剂量、中剂量和高剂量组分别给予3.38 g/(kg·d)、6.75 g/(kg·d)、13.5 g/(kg·d)六味地黄汤灌胃。假手术组和模型组给予10 mL/(kg·d)生理盐水灌胃,每天1次,连续灌胃12周。造模12周后,检测各组大鼠24 h尿蛋白、血尿素氮和血肌酐,HE染色观察各组大鼠肾组织形态学变化,免疫组化法和RT-PCR法分别检测各组大鼠肾组织中PHD2、HIF-1α蛋白水平和基因表达。结果模型组大鼠24 h尿蛋白、血尿素氮和血肌酐较假手术组明显升高(P<0.05),六味地黄汤低剂量、中剂量和高剂量组较模型组明显降低(P<0.05)。HE染色假手术组大鼠肾组织肾间质纤维化不明显,模型组肾间质纤维化明显,六味地黄汤低剂量、中剂量和高剂量组较模型组肾间质纤维化程度明显减轻。与假手术组比较,模型组大鼠肾组织PHD2蛋白水平和mRNA表达均明显降低(P<0.05),HIF-1α蛋白水平和mRNA表达均明显升高(P<0.05)。与模型组比较,六味地黄汤低剂量、中剂量和高剂量组大鼠肾组织中PHD2蛋白水平和mRNA表达均明显升高(P<0.05),HIF-1α蛋白水平和mRNA表达均明显降低(P<0.05),且PHD2与HIF-1αmRNA的表达呈负相关(r=-0.653,P=0.00)。结论 PHD2/HIF-1α信号途径参与肾间质纤维化,六味地黄汤可有效防治慢性肾脏病,其作用机制可能与上调PHD2、降低HIF-1α表达有关。
Objective To observe the effect of Liuwei Dihuang decoction on the expression of proline hydroxylase 2 (PHD2) and hypoxia inducible factor-1α (HIF-1α) in the rat model of chronic renal failure and to explore the pathogenesis of chronic renal failure The Molecular Mechanism of Dihuang Decoction in Preventing and Treating Chronic Kidney Disease. Methods Sixty SD rats were randomly divided into sham operation group, model group and Liuweidihuang Decoction low dose, middle dose and high dose group. 5/6 nephrectomy replicates chronic renal failure model. Liuwei Dihuang Decoction low dose, middle dose and high dose group were given 3.38 g / (kg · d), 6.75 g / (kg · d), 13.5 g / (kg · d) Liuweidihuangtang orally. Sham operation group and model group were given 10 mL / (kg · d) saline, gavage once daily for 12 weeks. Twelve weeks after model establishment, 24-hour urinary protein, blood urea nitrogen and serum creatinine were detected in each group. The morphological changes of renal tissue in each group were observed by HE staining. Immunohistochemistry and RT-PCR were used to detect PHD2, HIF-1α protein levels and gene expression in rat kidney. Results The 24 h urinary protein, blood urea nitrogen and serum creatinine in model group were significantly higher than those in sham-operation group (P <0.05), while those in low dosage, middle dosage and high dosage group of Liuweidihuang Decoction were significantly lower than those in model group ). The renal interstitial fibrosis was not obvious in the model group and the renal interstitial fibrosis was obvious in the model group. The level of renal interstitial fibrosis in the low dose, middle dose and high dose group of the Liuweidihuang decoction group was significantly reduced. Compared with the sham-operation group, the protein and mRNA expression of PHD2 in the kidney of the model group were significantly decreased (P <0.05), and the protein and mRNA expression of HIF-1α in the model group were significantly increased (P <0.05). Compared with the model group, the protein and mRNA expression of PHD2 in the kidney of the low dose, middle dose and high dose groups of Liuweidihuang Decoction group were significantly increased (P <0.05), HIF-1α protein level and mRNA expression were significantly decreased P <0.05). There was a negative correlation between PHD2 and HIF-1αmRNA expression (r = -0.653, P = 0.00). Conclusion PHD2 / HIF-1α signaling pathway is involved in renal interstitial fibrosis. Liuweidihuang Decoction can effectively prevent and treat chronic kidney disease. Its mechanism may be related to up-regulating PHD2 and decreasing HIF-1α expression.