Associated Evaluation Of Efficacy And Toxicity Of Bupleurum Chinense On Rat Of Hepatic Fibrosis With Syndrome Of Liver Depression And Spleen Deficiency

Objective: Under the guidance of Chinese medicine theory and the background of efficacy and syndromes,the rat model of liver depression and spleen deficiency type hepatic fibrosis is established by means of the traditional Chinese medicine literature mining combined with modern pharmacology and toxicology research,with the basis of the Bupleurum chinense's effect of soothing the liver and relieving depression and resisting liver fibrosis.the use of research methods in vivo and external were carried out in the whole,cell level to study the Pharmacodynamic activity of different Bupleurum samples.To affirm the effect of Bupleurum on Hepatic Fibrosis of Liver Depression and Spleen Deficiency,and to explore its preliminary pharmacological mechanism.The pharmacological effects associated with side effects were studied to reveal the characteristics of target organ and toxic and side effects.Verify the activity of different samples of disease model,which will help to clear material basis for efficacy and toxicity of Bupleurumand carry out "effect and toxicity" relational evaluation under the background of syndrome,to provide a new strategy for the study of traditional Chinese medicine and drugs for liver fibrosis.Methods: Liver fibrosis rat model were fed with edible alcohol of different proportionand high-lipid and low-protein fodder,induced by intrapulmonary injection with pig serum,To establish an animal model of liver fibrosis suitable for the evaluation of liver depression and spleen deficiency,To optimize and establish a rat model of liver stagnation and spleen deficiency and liver fibrosis,observe the general condition of the rats during the experimental period,and evaluate the use of spleen deficiency syndrome integral liver,to observe the inhibitory effect of total saponins of Bupleurum water extract and Radix Bupleurum on Model Rats.The indexes change of ALT,AST,HA,LN,IV-C,PCIII,TGF-?1,BUN and Cr in serum were tested while analyze the degree of liver fibrosis by the method of misdiagnosis of liver tissue.Clarify the toxicity of different components of Bupleurum on model rats.Culture the HSC-T6 cells and L-02 cellsin vitro and performe MTT assay,observe morphological changes to investigate the infiunce of different components of Bupleurum chinense on human liver cell L-02 and HSC-T6.And the contents of LDH,GSH and GSH-Px in supernatant were measured to further explore the effect of different components of Bupleurum chinense in vitro on the effect of "effect-toxicity",and clarified the safe dose range.Results: Obvious hepatic fibrosis occurred to all rats in model groups at 3 week following model induction.Both main symptoms and secondary symptoms were consistent with the syndrome manifestation of liver depression and spleen deficiency.Compared with normal control group,the activity of ALT,AST,TGF-?1 and four parameters of hepatic fibrosis of LN,HA,PC-III,IV-C in serum all increased significantly.Liver tissue pathological morphology showed the formation of liver cell fibers in hepatocytes of rats in model groups.The results showed that in the syndrome of liver depression and spleen deficiency group,the rat no deaths occurred.Giving water extracts of Bupleurum chinense ranged from 0.90 g·kg^-1 to 3.60 g·kg^-1 and 7.04mg·kg^-1 to13.90mg·kg^-1 Bupleurum total saponins can obviously improve the diet,drinking and physical activities of rats.The content of HA,LN,IV-C and PCIII in the blood and liver coefficient of rats with hepatic fibrosis were significantly decreased,histopathological changes were reduced.But the levels of ALT,AST,BUN and Cr in the high dose group were higher.In vitro administration of hepatic stellate HSC-T6 cells and normal liver L-02 cells of different doses of Bupleurum chinense water extract and Bupleurum total saponins.The IC₅₀ of the water extract of Bupleurum chinense was 6.23 mg·mL^-1 for HSC-T6 cells and 10.70 mg·mL^-1 for L-02 cells.The IC₅₀ of Bupleurum total saponins was 3.52 mg·mL^-1 for HSC-T6 cells and 6.68 mg·mL^-1 for L-02 cells.Detected the supernatant of the cells,the activity of LDH increased with the increase of drug concentration after 24 hours of administration,which was positively correlated with the dose.The activities of GSH and GSH-Px in the supernatant of the cells decreased with the increase of the concentration,and negatively correlated with the dose.Conclusions:By the improved high fat diet and alcohol concentration,rat models of hepatic fibrosis with syndrome of liver depression and spleen deficiency can be successfully induced.The model has high agreement with clinical syndrome.Both water extracts of Bupleurum chinense and total saponins have an obviously effective for liver fibrosis with syndrome of liver depression and spleen deficiency rats and shows a good dose-effect relationship.At the same time,3.60 g·kg^-1 the water extract of Bupleurum and 13.90mg·kg^-1 total saponins of Radix Bupleuri have side effects and liver,kidney toxicity.HSC-T6 cells and L-02 cells showed a positive correlation with the increase in the dose of water extract of Bupleurum,suggesting that the dose safety window range of "efficacy toxicity" of Bupleurum water extract was 6.23 mg.ml-1 10.70mg·ml-1,TI is 1.72;the inhibitory rate of HSC-T6 cells and L-02 cells increased with the dose of total saponins of Bupleurum,which showed a positive correlation,suggesting that the dose safety window range of "total efficacy and toxicity" of the total saponin of Bupleurum was 3.52mg·ml^-16.68mg·ml^-1,TI is 1.90.After administration,the GSH,GSH-Px activity in HSC-T6 and L-02 supernatant were detected to decrease,and with the increase of concentration of LDH,presumably due to water extract and saikosaponins inhibit HSC-T6 and L-02 cell growth and the effects of free radicals in the cells.