基于均匀设计的中药有效单体“IR”方筛选及其对慢性肾衰竭大鼠肾纤维化的作用  

Screening Effective Herbal Monomer“IR”Formula Based on Uniform Design and Its Effect on Renal Fibrosis in Rats with Chronic Renal Failure

作　　者：王蒙 王凌晨 王琛[1,2,3,4] WANG Meng;WANG Lingchen;WANG Chen(Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;TCM Institute of Kidney Disease,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Key Laboratory of Liver and Kidney Diseases,Ministry of Education,Shanghai University of Traditional Chinese Medicine;Shanghai Key Laboratory of Traditional Chinese Clinical Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院,上海201203 [2]上海中医药大学中医肾病研究所,上海201203 [3]上海中医药大学肝肾疾病病证教育部重点实验室,上海201203 [4]上海市中医临床重点实验室,上海201203

出　　处：《中华中医药学刊》2024年第2期63-68,I0015,I0016,共8页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82104769,81973770);上海市科技创新行动扬帆计划项目(21YF1447900)。

摘　　要：目的运用“均匀设计法”从“肾衰Ⅱ号方”中筛选抗肾纤维化中药有效单体组方(淫羊藿苷合迷迭香酸配伍,简称IR方),并进行验证。方法采用5/6(Ablation and infarction,A/I)慢性肾衰大鼠模型,将肾衰Ⅱ号方中已知的5种抗肾纤维化有效单体(淫羊藿苷、迷迭香酸、阿魏酸、苦杏仁苷、大黄素)作为研究对象,按照均匀设计法“5因素8水平”分组,共设8种配比组方。造模后4周,组1~组8给予相应配比的组方药液,假手术组和模型组给予生理盐水,连续干预8周。末次给药后,检测各组大鼠血清肌酐(Serum creatinine,Scr)水平,苏木素-伊红(Hematoxylin-eosin,HE)和马松(Masson)染色观察肾组织病理形态,Western Blot法检测纤维化标志蛋白Ⅰ型胶原(CollagenⅠ,Col-Ⅰ)、纤维连接蛋白(Fibronectin,FN)、结缔组织生长因子(Connective tissue growth factor,CTGF)表达。以各药物组大鼠Scr为筛选指标,通过多元逐步回归分析筛选中药单体成分组方并确定特定配比。再用5/6(A/I)大鼠模型,以氯沙坦钾、母方“肾衰Ⅱ号方”和淫羊藿苷(20 mg·kg^(-1)·d^(-1))作为对照,有效成分组方(淫羊藿苷20 mg·kg^(-1)·d^(-1)+迷迭香酸2.1 mg·kg^(-1)·d^(-1))作为实验药物,连续干预8周。通过各组大鼠Scr、血尿素氮(Blood urea nitrogen,BUN)、病理染色(HE和Masson)和FN、Col-Ⅰ蛋白表达比较各组大鼠肾功能和肾纤维化程度。体外构建转化生长因子-β1(Transforming growth factor-β1,TGF-β1)诱导肾小管细胞(NRK-52E)损伤和肾成纤维细胞(NRK-49F)活化模型,以有效成分组方和相应单一成分作为干预药物,Western Blot法检测FN、Col-Ⅰ、CTGF蛋白表达。结果筛选实验结果提示,组1~组8均可不同程度降低5/6(A/I)大鼠Scr水平,结合病理染色和Western Blot分析,组8效果最优。经过多元逐步回归分析,得到方程为:=88.381-0.539X1-0.473X2+0.01X1X2(X1淫羊藿苷,X2迷迭香酸),提示淫羊藿苷和迷迭香酸配伍为最佳Objective To screen and verify the effective formulae of herbal monomers(icariin combined with rosmarinic acid,abbreviated as IR formula)against renal fibrosis from Shenshuai Ⅱ Recipe(肾衰Ⅱ号方,SSR)using the uniform design method.Methods Using the 5/6(ablation and infarction,A/I)rat model with chronic renal failure,the five known effective monomers(icariin,rosmarinic acid,ferulic acid,amygdalin,emodin)against renal fibrosis in SSR were selected for the study subjects,and a total of eight formulae were designed according to the“five-factor,eight-level”uniform design method.Four weeks after modeling,groups 1~8 were given the corresponding proportion of the drug solution,and the sham-operated and model groups were given saline for 8 consecutive weeks of intervention.After the last administration,the serum creatinine(Scr)levels of rats in each group were measured,and the histopathological morphology of kidney tissues was observed by hematoxylin-eosin(HE)and Masson staining,and the expressions of fibrosis marker proteins[fibronectin(FN),collagenⅠ(Col-Ⅰ),connective tissue growth factor(CTGF)]were detected by Western blot.Using Scr of rats in each drug group as the index,it screened the effective formula of herbal monomers and determined the specific ratio by multiple stepwise regression analysis.The 5/6(A/I)rats were treated with losartan,SSR and icariin(20 mg·kg^(-1)·d^(-1))as controls and the effective ingredient formulation(icariin 20 mg·kg^(-1)·d^(-1)+rosmarinic acid 2.1 mg·kg^(-1)·d^(-1))as the experimental drug for 8 weeks.The degree of renal fibrosis in the rats of each group was compared by Scr,blood urea nitrogen(BUN),pathological staining(HE and Masson)and expressions of FN and Col-Ⅰ proteins.In vitro,the renal tubular cells(NRK-52E)and renal fibroblasts(NRK-49F)stimulated by transforming growth factor-β1(TGF-β1)were treated with effective ingredient formulation or single components,and the expressions of FN,Col-Ⅰand CTGF proteins were detected by immunoblotting.Results The results

关 键 词：肾纤维化 均匀设计法 有效单体 肾衰Ⅱ号方 IR方 

分 类 号：R285.5[医药卫生—中药学]