小檗碱通过调节蛋白激酶B缓解对比剂肾病的作用及机制研究  

Effect and mechanism of berberine in alleviating contrast-induced nephropathy through regulating protein kinase B

作　　者：于然 肖瑶 王万鹏 李庆菊[1,2] 陈皓瑜 宋坚 陈佳佳 Yu Ran;Xiao Yao;Wang Wan-peng;Li Qing-ju;Chen Hao-yu;Song Jian;Chen Jia-jia(Department of Central Laboratory,Lianshui People’s Hospital,Affiliated Kangda college of Nanjing Medical University,Huai’an 223400,China;School of Clinical Medicine,Medical College of Yangzhou University,Jiangsu Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Treatment of Senile Diseases,Yangzhou 225009,China)

机构地区：[1]南京医科大学康达学院附属涟水人民医院中心实验室,淮安223400 [2]扬州大学医学院临床学院,江苏省中西医结合老年病防治重点实验室,扬州225009

出　　处：《临床肾脏病杂志》2024年第10期847-854,共8页Journal Of Clinical Nephrology

基　　金：国家自然科学基金资助项目(81600549);江苏省卫生健康委科研项目(Z2021059);淮安市自然科学研究计划(HABL202260);南京医科大学康达学院科研发展基金项目(KD2023KYJJ169,KD2023KYJJ172,KD2023KYJJ175)。

摘　　要：目的探究小檗碱(berberine,BBR)对对比剂肾病(contrast-induced nephropathy,CIN)的作用及潜在机制。方法SD大鼠用于构建大鼠CIN模型,BBR治疗组(model+BBR,M+B)大鼠给予200 mg/kg BBR灌胃治疗,对照组(control,Con)大鼠和模型组(model,Mod)大鼠给予等量生理盐水灌胃。人肾皮质近曲小管上皮细胞(human kidney cortex proximal tubule epithelial cells,HK-2)用于构建CIN体外模型,Con组细胞不进行特殊处理,Mod组细胞使用50 g/L碘佛醇作用24 h,M+B组细胞使用50 g/L碘佛醇和30μmol/L BBR处理24 h。蛋白激酶B(protein kinase B,Akt)激动剂(SC79)和Akt抑制剂(MK2206)用于检测BBR对蛋白激酶B-叉头盒O3-核因子E2相关因子2(protein kinase B-forkhead box O3-nuclear factor erythroid 2-related factor 2,Akt-Foxo3a-Nrf2)调控轴的调节作用。血肌酐(serum creatinine,Scr)检测和HE染色用于评估大鼠肾脏损伤情况。膜联蛋白V-碘化丙啶(annexin V-propidium iodide,Annexin V-PI)染色用于检测各组HK-2细胞凋亡情况。蛋白质印迹法用于检测Akt-Foxo3a-Nrf2调控轴蛋白的表达水平。结果Scr检测结果显示,Mod组大鼠的Scr水平[(58.83±7.96)μmol/L比(23.52±0.78)μmol/L]高于Con组,M+B组Scr水平[(39.64±2.52)μmol/L比(58.83±7.96)μmol/L]低于Mod组(P均<0.05)。HE染色结果显示,Con组大鼠肾脏形态正常,肾小管上皮细胞完好,Mod组大鼠肾脏中肾小管结构受损,大量肾小管上皮细胞死亡,肾小管上皮细胞空泡样变明显,M+B组大鼠肾脏中部分肾小管形态恢复,肾小管上皮细胞空泡样变减少,细胞死亡减少。蛋白质印迹法表明,与Con组相比,在Mod组大鼠和HK-2细胞中磷酸化蛋白激酶B(Phospho-protein kinase B,p-Akt)表达增加,差异均具有统计学意义(P均<0.05);M+B组p-Akt表达较Mod组下降(P<0.05);Annexin V-PI凋亡检测显示,与Con组相比,Mod组中HK-2细胞凋亡[(12.65±1.25)%比(27.44±0.73)%]增加,与Mod组相比,M+B组中HK-2细胞凋亡[(27.44±0.73)%比(24.81±0.49)%]下降(P均<0.05)�Objective To explore the inhibitory effect of berberine(BBR)on contrast-induced nephropathy(CIN)and clarify the underlying mechanism.Methods Sprague-Dawley(SD)rats were utilized for constructing a rat CIN model.BBR treatment group(M+B)received a gavage of 200 mg/kg BBR while control group(Con)and model group(Mod)had an oral intake of the same volume of normal saline.Human kidney cortex proximal tubule epithelial cells(HK-2)were utilized for constructing a CIN cell model in vitro.No special treatment was offered to HK-2 cells in Con group.HK-2 cells in Mod group were treated with 50 g/L ioversol for 24 h and HK-2 cells in M+B group 50 g/L ioversol and 30μmol/L BBR for 24 h.Akt agonist(SC79)and Akt inhibitor(MK2206)were utilized for confirming the effect of BBR on protein kinase B-forkhead box O3-nuclear factor erythroid 2-related factor 2(Akt-Foxo3a-Nrf2)regulatory axis.Serum creatinine(Scr)detection and hematoxylin-eosin(HE)stain were employed for evaluating renal injury in rats.Annexin V-propidium iodide(annexin V-PI)stain was used for detecting cell apoptosis.And Western blot was utilized for detecting the protein expression of Akt-Foxo3a-Nrf2 axis.Results The level of Scr was higher in Mod group than that in Con group[(58.83±7.96)μmol/L vs(23.52±0.78)μmol/L,P<0.05].And it was lower in M+B group than that in Mod group[(39.64±2.52)μmol/L vs(58.83±7.96)μmol/L,P<0.05].HE stain indicated that kidney morphology was normal and renal tubular epithelial cells remained intact in Con group.In Mod group,structure of renal tubules was disarrayed with diffuse death of epithelial cells and obvious vacuolar degeneration of renal tubular epithelial cells.In M+B group,morphology of renal tubules was partially restored and vacuolar degeneration of renal tubule epithelial cells and cell death lessened.Western blot results indicated that,as compared with Con group,the expression of phospho-protein kinase B(p-Akt)was up-regulated in CIN rats and ioversol treated HK-2 cells.And the difference was statistically significant(P

关 键 词：对比剂肾病 小檗碱 蛋白激酶B 凋亡 

分 类 号：R692[医药卫生—泌尿科学]