基于均匀设计的抗肝纤维化中药单体成分组方筛选与验证

Screening and verification on prescription of Chinese herbal monomers for anti-liver fibrosis based on uniform design

目的:以丹参酚酸B、红景天苷和青蒿琥酯3种具有抗肝纤维化作用且机制明确的中药单体成分组方,基于均匀设计方法筛选最佳配伍剂量,并进行验证。方法:雄性C57BL/6J小鼠,腹腔注射15%四氯化碳(CCl4)6周,诱导肝纤维化模型。①筛选实验:采用均匀设计法"三因素八水平"分组,共设8种配比组方。造模第2周起,各组小鼠灌胃给予相应剂量配比的组方溶液,连续5周。末次给药后,检测血清丙氨酸氨基转移酶(ALT)水平和肝脏羟脯胺酸(Hyp)含量,天狼猩红染色后光镜下观察肝组织形态学变化;以肝脏Hyp含量为主要指标,采用NOMEN法分析最优组方配比;综合ALT、Hyp和病理学变化,筛选药效最佳的配比组方。②验证实验:以扶正化瘀方为阳性对照药,验证筛选的成分组方。组方1为均匀设计分析得到的最优配比组方;组方2为课题组前期研究的经验配比组方,即丹参酚酸B(17.5 mg/kg)+红景天苷(610 mg/kg)+青蒿琥酯(79 mg/kg);组方3为筛选实验得到的药效最佳的配比组方。造模和干预方法同前。末次给药后,检测血清ALT、天冬氨酸氨基转移酶(AST)和肝脏Hyp含量,HE染色和天狼猩红染色后光镜下观察肝组织形态学变化,比较各组肝纤维化程度。结果:①筛选实验:均匀设计筛选的最优组方配比为红景天苷(610 mg/kg)+青蒿琥酯(79 mg/kg);丹参酚酸B(14 mg/kg)+红景天苷(610 mg/kg)+青蒿琥酯(46 mg/kg)的配比组方抗肝纤维化效应最为显著,可显著降低血清ALT水平和肝脏Hyp含量(P<0.01),明显减轻胶原沉积。②验证实验:丹参酚酸B(14 mg/kg)+红景天苷(610 mg/kg)+青蒿琥酯(46 mg/kg)配比的组方3组血清ALT、AST水平及肝脏Hyp含量较模型组显著降低(P<0.05,P<0.01),肝细胞变性、坏死、出血、纤维增生、胶原沉积、炎症浸润等病理变化明显减轻,且胶原沉积程度分期显著改善,胶原阳性染色面积占比明显减少(P<0.01),其作用与扶正化瘀方相当。结论:丹参酚酸B(14 mg/kg)+红景天苷(610 mg/kg)+青蒿琥酯(46 mg/kg)的配比组方具有较好的抗肝纤维化作用,其药效与扶正化瘀方相当。

Objective:Three Chinese herbal monomers with clear anti-liver fibrosis effects and mechanisms, salvianolic acid B, salidroside and artesunate, are made as the formulation. The aim is to screen and verify the optimal compatible dose of this formulation based on uniform design method. Methods:Male C57 BL/6 J mice were intraperitoneally injected with 15% carbon tetrachloride(CCl4) for 6 weeks to induce liver fibrosis model. ①Screening experiment:The groups were divided based on "three factors and eight levels" in uniform design method, and a total of eight formulations were set up. From the second week of modeling, mice in each group were treated with these formulation solutions at the corresponding dose by intragastric administration for 5 weeks. After the last administration, the serum alanine aminotransferase(ALT) level and liver hydroxyproline acid(Hyp) content were detected, and the morphological changes of liver tissue were observed under light microscope after Sirius red staining. Taken the content of Hyp in liver as the main index, the optimal formulation ratio was analyzed by NOMEN method. Combined with ALT, Hyp and pathological changes, the formulation with the best efficacy was screened.②Verification experiment:Fuzheng Huayu Formula was used as the positive control drug to verify the screened formulations. Formulation 1 was the optimum formula obtained by uniform design analysis, Formulation 2 was the empirical formula from previous studies of the research group, namely salvianolic acid B(17.5 mg/kg)+salidroside(610 mg/kg)+artesunate(79 mg/kg), Formulation 3 was the formula with the best efficacy obtained from the screening experiment. Modeling and intervention were same as above. After the last administration, the serum ALT and aspartate aminotransferase(AST) levels and the liver Hyp content were detected. After HE staining and Sirius red staining, the morphological changes of liver tissue were observed under light microscope to compare the degree of liver fibrosis in each group. Results:①Screening test:Based on uniform design, the optimal formula ratio was salidroside(610 mg/kg)+artesunate(79 mg/kg).The formulation of salvianolic acid B(14 mg/kg)+salidroside(610 mg/kg)+artesunate(46 mg/kg) showed the most significant anti-liver fibrosis effects, which significantly reduced the serum ALT level and liver Hyp content(P<0.01), and obviously alleviated the collagen deposition. ②Verification experiment:The Formulation 3, with formula ratio of salvianolic acid B(14 mg/kg)+salidroside(610 mg/kg)+artesunate(46 mg/kg), significantly decreased the serum levels of ALT and AST and liver Hyp content compared with the model group(P<0.05, P<0.01), the pathological changes such as hepatocyte degeneration, necrosis, hemorrhage, fiber proliferation, collagen deposition and inflammatory infiltration were significantly alleviated, the stages of collagen deposition degree were improved obviously, and the proportion of collagen positive staining area was significantly reduced(P<0.01). Its effect was similar to Fuzheng Huayu Formula. Conclusion:The formulation of salvianolic acid B(14 mg/kg)+salidroside(610 mg/kg)+artesunate(46 mg/kg) shows good anti-liver fibrosis effects, and its effect is comparable to that of Fuzheng Huayu Formula.