| Objective To study the pharmacodynamics and mechanisms of Yiqi Zhuyu Jiedu Formula on hepatic fibrosis in vivo and in vitro experiments.Method 1.Vivo experiments of Yiqi Zhuyu Jiedu Formula: 60 male Wistar rats were randomly divided into model group and blank control group.Except for blank group,CCl4 and olive oil mixture(CCl4 40%)was injected subcutaneously for 7 weeks.Then the rats were randomly divided into 6 groups and numbered,high dose group,middle dose group,low dose group,the positive group,model group and blank control group.The treatment group were given drug intervention: high,middle and low dose group each gave Yiqi Zhuyu Jiedu extract by gavage,the positive group gave Fuzheng Huayu,the rest gave distilled water to the same volume,and last for 5 weeks,and then sacrificed for blood and liver tissue.HA?LN in the blood were detected by Elisa,biochemical monitoringtest ALT?AST??-GT in the blood,and the liver tissue for HE,MASSON staining,the levels of MMP-1,MMP-2,MMP-13 and TIMP-1 were detected by q-PCR,the expression of ?-SMA,MMP-13 and TGF-?1 in liver tissue were detected by immunohistochemistry.2.Vitro experiments of Yiqi Zhuyu Jiedu Formula: All wistar rats were randomly divided into five groups,except the blank control group,the other groups were gavaged Yiqi Zhuyu Jiedu extract and Fuzheng Huayu by equal volume but different concentrations,and lasted for 1 week.Then all the rats were killed and their blood took for serum;The serum were used to cultivate HSC-T6 cells for 48 hours,and the plates were labeled.After 48 h,total RNA was isolated from HSC-T6,The expressions of ?-SMA,TIMP-1,MMP-1,MMP-2,MMP-9 and MMP-13 m RNA were detected by q-PCR.Results1.Pharmacodynamics Results of Yiqi Zhuyu Jiedu formula1.1 The result of Hepatic Fibrosis Rat Model by CCl4(1)General situation: except the blank group,the rest rats' mental state and appetite was obviously poor,and the gait was slow,with abnormal stools and urine,hair rough,yellow and dull,the injection site significant ulceration and local hair off.The modeling groups had death.liver tissue appearance: at the end of the seventh week,the liver specimens were atrophic,the color was dark and dull,there were obvious particles like bulge,with hard material and blunt edges.(2)pathological changes of the liver: At the end of modeling,hepatic fibrosis is not only limited to the central vein and portal area,fibrosis was seen extend around from the central vein and the harbor area,and there was a tendency to form false leaflets,the classification of the hepatic fibrosis had reached grade 3,and hepatic fibrosis model was successfully formed.1.2 Treatment Results(1)the general situation: rats in the treating were better in the spirit,appetite,fur and color than before.All the rats had faster weight gain.(2)Liver appearance: After the whole treatment,the livers in the treated groups showed no obvious particles like convex.In Yiqi Zhuyu Jiedu Formula groups showed smooth surface,dark red color,with soft quality and slightly blunt edge;More than half of the liver specimen of the positive control group showed the quality was right,the edge was not blunt,a few liver was atrophy,with no obvious granulelike bulging,dull surface,hard quality,blunt edge;The model group showed significantly atrophy and hard in the liver,dark red color,dull;Relatively speaking,the liver of the blank group had bright red color,soft and smooth surface.(3)Pathological results: The high,middle and low dose groups of collagen fibers were mainly distributed in the portal area,the central vein and its surroundings.The structure of the hepatic lobules was normal and the low dose group had collagen fibers extending to the surroundings.The hepatic lobule structure was divided into the annular structure by the thicker fibrous septum.The model group had thicker fibrous septum to segment the hepatic lobes.The blank control group had only a small amount of collagen fibers distributed in the portal vein and the central vein.Semiquantitative score of MASSON staining: The middle dose group was significantly lower than the model group(p <0.05).(4)Biochemical results: The levels of ALT,AST and ?-GT in the blank group were higher than normal.The levels of AST in model group were significantly higher than those in the blank group(P <0.01),and the levels of ALT and ?-GT were not significantly changed(p> 0.05).Compared with the model group,the levels of AST and ?-GT in the low dose group were significantly lower(P <0.05);Compared with the positive control group,the ALT in the low and middle dose group was significantly lower(p < 0.01),and the levels of AST and ?-GT in low dose group were significantly decreased(p <0.05).Compared with other groups,the levels of ALT and AST in high dose group were significantly increased(p <0.05).(5)Elisa results: Compared with the blank group,the level of LN in the model group was significantly higher(p <0.01),but there was no significant change on the level of HA in the model group(p> 0.05).Compared with the model group,the level of HA in high dose group and the level of LN in the high and low dose group were significantly lower(P <0.01).Compared with the positive control group,the level of HA in the high dose group and the level of LN in the high and low dose groups were significantly decreased(p <0.01).(6)The expression of Col1A1?Col1A2?MMP-1?MMP-2?MMP-9?MMP-13?TIMP-1??-SMA and TGF-?1 in liver tissue :Except for TIMP-1,the other indicators were significantly increased in model group.(1)Coll A1?Col1A2: The expression of Col1A1 in the treated groups had decreased(p <0.05),and the positive control group were more significant.Col1A2 in middle and low dose group of Chinese medicine had decreased(p <0.05),especially in middle dose group(p <0.01);(2)MMPs: Compared with the model group,the levels of MMP-2 in the high and middle dose groups had significantly increased(p <0.05),especially in the high dose group(p <0.01).The levels of MMP-13 in Chinese medicine groups had increased(p <0.05),especially in the high dose group(p <0.01).And compared with the control group,the levels of MMP-13 had significantly increased(p <0.01);(3)TIMP-1: Compared with the model group,the high and low dose groups of Chinese medicine had decreased(p<0.05);(4)?-SMA: The treated groups had decreased(p <0.05),especially in the high dose group of Chinese medicine and the positive control group(p <0.01);(5)TGF-?1: Compared with the model group,the high,middle dose group and the positive control group had significantly decreased(p <0.05),especially in the high dose group(p <0.01).2.Effect of Serum Containing Yiqi Zhuyu Jiedu Formula on HSC-T6 CellsThe relative gene expression in HSC-T6 of each group: Compared with the blank control group,?-SMA: the expression in the high and middle dose groups was higher,while the low dose group decreased a little;TIMP-1: the expression in the medication groups decreased significantly,especially in the Chinese medicine groups;MMP-1: the expression had decreased a little(or had no change);MMP-2: the expression in the Chinese medicine groups increased,while the positive control group changed little;MMP-9: the expression were significantly lower in the medication groups;MMP-13: the expression in the high dose group was significantly higher.Conclusions1.Yiqi Zhuyu Jiedu formule could improve the liver function and the fibrosis.2.Yiqi Zhuyu Jiedu formule could imblance the MMPs?TIMPs and induce the expression of TGF-?1.3.Yiqi Zhuyu formula can inhibit the activation of HSC. |
PDF Full Text Request