双参芎连颗粒对高同型半胱氨酸血症慢性肾病大鼠血管舒张及PI3K/Akt/eNOS通路的影响

Effect of Shuangshen Xionglian Granule on Vasodilation and PI3K/Akt/e NOS Pathway in Hyperhomocysteinemia Chronic Kidney Disease Rats

目的:研究双参芎连颗粒对高同型半胱氨酸血症慢性肾病大鼠血管病变及磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)/内皮型一氧化氮合酶(eNOS)信号通路影响。方法:大鼠随机分为5组,分别为假手术组、模型组、双参芎连颗粒高、中、低剂量(8,4,2 g·kg^(-1))组。采用高蛋氨酸饲料喂养结合5/6肾摘除法建立大鼠高同型半胱氨酸血症慢性肾病模型,5/6肾摘除术后4周后开始连续灌胃给药4周。术后4,8周测定大鼠动脉血压;术后8周实验结束,取血测定血清肌酐、尿素氮、同型半胱氨酸、蛋氨酸及血脂水平;蛋白免疫印迹法(Western blot)检测胸主动脉磷酸化(p-)p85,p-Akt和p-Ser177等PI3K/Akt/eNOS信号通路相关蛋白表达,测定血清一氧化氮(NO),eNOS;采用离体血管环灌流方法,测定内皮依赖性舒张和非内皮依赖性舒张变化;苏木素-伊红(HE)染色观察肾脏组织及胸主动脉血管改变。结果:实验8周时,与假手术组比较,模型组动脉收缩压、血清尿素氮、肌酐、高同型半胱氨酸、蛋氨酸、总胆固醇及低密度脂蛋白明显增高;给予双参芎连颗粒4周后,各剂量组动脉收缩压、血清尿素氮、高同型半胱氨酸、蛋氨酸、血清总胆固醇及血清低密度脂蛋白均明显降低(P<0.05,P<0.01),双参芎连颗粒8,4 g·kg^(-1)组肌酐明显降低(P<0.05)。双参芎连颗粒各剂量组NO含量较模型组明细升高(P<0.05,P<0.01),双参芎连颗粒8 g·kg^(-1)组血清eNOS活性较模型组明显升高(P<0.05)。模型组动物离体胸主动脉环的内皮依赖性及非内皮依赖性血管舒张均受到明显损伤,双参芎连颗粒8 g·kg^(-1)组在乙酰胆碱累积浓度1×10^(-5).5~1×10^(-4)mmo1·L^(-1)舒张程度较模型组明显改善(P<0.05,P<0.01);双参芎连颗粒8 g·kg^(-1)组血管环在硝普钠积浓度1×10-10~1×10^(-7)mmo1·L^(-1)舒张程度较模型组明显,差异具有明显统计学意义(P<0.05)。Western blot结果显示,与假手术组比较,模型组血管p-85,p-Akt及p-Ser177的表达均显著升高(P<0.01);与模型组比较,双参芎连颗粒各剂量组该通路磷酸化水平较模型组呈升高趋势,其中双参芎连颗粒8 g·kg^(-1)组p-Akt及p-Ser177的表达升高更明显,差异具有明显统计学意义(P<0.05)。双参芎连颗粒各剂量组的胸主动脉和肾脏组织病理变化与模型组比较有不同程度的减轻。结论:双参芎连颗粒可以改善高同型半胱氨酸血症慢性肾病大鼠高同型半胱氨酸及血脂代谢异常,降低血清肌酐及尿素氮水平,降低动脉收缩压,改善血管形态及舒张功能,其作用可能与调控PI3K/Akt/eNOS信号通路有关。

Objective:To study the effect of Shuangshen Xionglian(SSXL)granules on vasculopathy and phosphatidylinositol 3 kinase(PI3 K)/serine threonine kinase(Akt)/nitrogen oxide synthase(eNOS)signal in hyperhomocysteinemia chronic kidney disease rats. Method: Rats were randomly divided into 5 groups:sham operation group,model group,and high,medium and low-dose(8,4,2 g·kg^(-1))SSXL groups. The model of hyperhomocysteinemia chronic kidney disease in rats was established with high methionine feed combined with 5/6 nephrectomy. After 5/6 nephrectomy,continuous intragastric administration lasted for four weeks. Arterial blood pressure was measured at the 4 thand 8 thweeks after operation. At the end of the 8 thweek after the operation,blood was collected to determine serum creatinine,urea nitrogen,homocysteine(Hcy),methionine and blood lipid. Western blot was used to detect the expressions of PI3 K/Akt/eNOS pathway-related proteins,such as p-p85,p-Akt and p-Ser177 in thoracic aorta,and serum NO and eNOS were measured. The changes of endothelium-dependent relaxation and non-endothelium-dependent relaxation were measured by the method of isolated thoracic aorta ring. Pathological htoxylin eosin(HE)staining was used to observe the changes of renal tissue and thoracic aorta. Result:At the 8 thweek of the experiment,compared with the sham operation group,arterial systolic blood pressure,serum urea nitrogen,creatinine,Hcy,methionine,total cholesterol and low-density lipoprotein of the model group were significantly increased. Four weeks later after administration,arterial systolic blood pressure,serum urea nitrogen,Hcy,methionine,serum total cholesterol and serum lowdensity lipoprotein were significantly reduced in each dose group(P<0.05,P<0.01). The creatinine in the SSXL8,4 g·kg^(-1) group was significantly reduced(P<0.05). The nitric oxide content of SSXL in each dose groups were increased compared with that in the model group(P<0.05,P<0.01),and the serum eNOS activity of the SSXL in the SSXL 8 g·kg^(-1) group was significantly increased compared with that in the model group(P<0.05).The endothelium dependent and non-endothelium dependent vasodilation of thoracic aortic rings in the model group were significantly damaged. The cumulative concentration of acetylcholine(1×10^(-5).5~1×10^(-4) mmo1·L^(-1))in the SSXL 8 g·kg^(-1) group was significantly improved(P<0.05,P<0.01). The diastolic degree of the vascular ring in the SSXL 8 g·kg^(-1) group was significantly higher than that in the model group(P<0.05). Western blot results showed that the expressions of p-85,p-Akt and p-Ser177 in blood vessels increased in the sham group compared with those in the model group(P<0.01). Compared with the model group,the phosphorylation level of this pathway was increased in the SSXL groups,and the expressions of p-Akt and p-Ser177 in the SSXL 8 g·kg^(-1) group were significantly increased(P<0.05). The pathological results showed that the pathological changes of thoracic aorta and renal tissue in the dosages of SSXL were significantly reduced compared with those in the model group. Conclusion: SSXL granules can improve hyperhomocysteine and dyslipidemia in rats of chronic kidney disease with hyperhomocysteine,reduce serum creatinine,urea nitrogen levels and arterial systolic blood pressure,and improve vascular morphology and diastolic function,which may be related to the regulation of the PI3 K/Akt/eNOS signaling pathway.