【摘要】 目的 研究活性维生素(AV)D3对阿霉素肾病(AN)大鼠肾组织蛋白激酶B(Akt)磷酸化(Ser473)的影响。方法 将90只雄性SD大鼠均分为正常对照(NC)组、AN组及AVD3干预(AN+AVD3)组，每组各30只。除NC组外，其余均一次性尾静脉注射阿霉素7.5 mg/kg造模，造模成功后，AN+AVD3组0.25μg/(kg·d)AVD3灌胃治疗，其余两组给予等量的花生油灌胃，持续治疗10 w。采用免疫荧光(IF)观察p-Akt蛋白在肾小球中的定位；免疫组织化学染色(IHC)检测p-Akt蛋白的平均光密度；实时荧光定量聚合酶链反应(PCR)检测Akt mRNA水平；Western印迹检测Akt及p-Akt蛋白表达。结果 造模大鼠精神、饮食、排泄都出现异常(大量蛋白尿),组织化学染色呈现严重的病理学损伤；IF结果显示：p-Akt蛋白定位于肾小球系膜基质区，连续样分布，AN组较NC组阳性信号明显增强，AN+AVD3组较AN组阳性信号减弱；IHC结果显示：实验后，AN组p-Akt蛋白的平均吸光度(第6周起)显著高于NC组，AN+AVD3组p-Akt蛋白的平均吸光度显著低于AN组(P<0.001);实时荧光定量PCR和Western印迹实验显示：实验后，各组Akt mRNA和蛋白水平未见明显差异(P>0.05),但AN组p-Akt/Akt较NC组显著升高，AN+AVD3组p-Akt/Akt较AN组显著降低(P<0.01,P<0.001)。结论 AVD3显著抑制AN肾小球内Akt的磷酸化，提示AVD3可能通过抑制磷脂酰肌醇3-激酶(PI3K)/Akt通路发挥肾保护作用。更多还原

【Abstract】 Objective To investigate the effect of active vitamin(AV)D3 on protein kinase B(Akt) phosphorylation(Ser473) in renal tissue of adriamycin nephropathy(AN) rats. Methods A total of 90 male SD rats were divided into normal control(NC) group, AN group, and AVD3 intervention(AN+AVD3) group, with 30 rats in each group. Except for the NC group, the others were given a one-time tail vein injection of adriamycin 7.5 mg/kg to establish the model. After successful modeling, the AN+AVD3 group was given 0.25 μg/(kg·d) AVD3 by gavage, and the other two groups were given the same amount of peanut oil by gavage, for a continuous treatment of 10 w. Immunofluorescence(IF) was used to observe the localization of p-Akt protein in the glomerulus; immunohistochemical staining(IHC) was used to detect the average optical density of p-Akt protein; real-time fluorescence quantitative polymerase chain reaction(PCR) was used to detect the Akt mRNA level; Western blot was used to detect the expressions of Akt and p-Akt protein.Results The model rats showed abnormalities in mental, dietary and excretion(massive proteinuria),and histochemical staining showed severe pathological damage; IF results showed that p-Akt protein was localized in the glomerular mesangial matrix area, with a continuous pattern distribution. The positive signal of the AN group was significantly stronger than that of the NC group, and the positive signal of the AN+AVD3 group was significantly weaker than that of the AN group; IHC results showed that after the experiment, the average absorbance of p-Akt protein(starting from the sixth week) in the AN group was significantly higher than that of the NC group, and the average absorbance of p-Akt protein in the AN+AVD3 group was significantly lower than that of the AN group(P<0.001); real-time fluorescence quantitative PCR and Western blot experiments showed that after the experiment, there was no significant difference in Akt mRNA and protein levels among each group(P>0.05), but the p-Akt/Akt in AN group was significantly higher than that in the NC group, and p-Akt/Akt in the AN+AVD3 group was significantly lower than that in the AN group(P<0.01,P<0.001). Conclusions AVD3 could significantly inhibit the phosphorylation of Akt in the glomerulus of AN, suggesting that AVD3 might exert a renal protective effect by inhibiting the phosphatidyl kinase 3-kinase(PI3K)/Akt pathway.更多还原