Screening Effective Herbal Monomer"IR"Formula Based on Uniform Design and Its Effect on Renal Fibrosis in Rats with Chronic Renal Failure
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 mod-eling,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 Mas-son staining,and the expressions of fibrosis marker proteins[fibronectin(FN),collagen Ⅰ(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 formu-la 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-Iproteins.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 CT-GF proteins were detected by immunoblotting.Results The results of screening experiment suggested that groups 1～8 could de-crease the Scr level of 5/6(A/I)rats in different degrees,and combined with pathological staining and Western-blot analysis,the effect of group 8 was optimal.The equation obtained by regression analysis was:(y)=88.381-0.539X1-0.473X2+0.01X1X2(X1 icariin,X2 rosmarinic acid),indicating that the combination of icariin and rosmarinic acid was optimal with a ratio of 9.6∶1.The results of validation experiment showed that the IR formula could significantly improve kidney function,attenuate tubu-lointerstitial damage and fibrosis,and inhibit the expressions of FN and Col-Ⅰ proteins in 5/6(A/I)rats,and its renoprotective effect was comparable to that of SSR and superior to that of the western drug control.In vitro,the results revealed that IR formula could significantly inhibit expressions of FN,Col-1 and CTGF proteins in TGF-β1-stimulated NRK-52E cells and NRK-49F cells,and was superior to icariin or rosmarinic acid alone intervention.Conclusion IR formula of effective monomer screened by the uniform design method improved renal function and exerted anti-fibrotic effects in rats with chronic renal failure.
renal fibrosisuniform design methodeffective monomersShenshuai Ⅱ Recipe(肾衰Ⅱ号方)IR formula
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