Pharmacological Study On The Effects And It's Mechanism Of Ganbao Capsule On The Liver Fibrosis Induced By BDL In Rats

Objective:To evaluate the therapeutic effect and mechanism of Ganbao Capsule(GBC)on the liver fibrosis induced by bile duct ligation(BDL)in rats,and it's General pharmacological effects on beagle dogs or mice.Methods:(1)Female SD rats were commited bile duct ligation to develop cholestatic liver fibrosis(BDL),while the sham group rats only free hepatic portal vein.The BDL rats were randomly divided into two groups after 5 days:bicyclol 0.1 g·kg-1,silymarin 0.1 g·kg-1,Ganbao 1.0 g·kg-1,Ganbao2.0 g·kg-1 or Ganbao4.0 g·kg-1 and BDL model goup.The animals were ig once a day in the following 4 weeks,and the model and conrol groups were given the same volume of blank solvent.24 h after the last administration,the liver and spleen were weighted to calculate the organ index.Meanwhile,the biochemical index of serum were measured:the activities of Alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP)and gamma glutamyl transpeptidase(?-GT),the levels of total bilirubin(TBIL),direct bilirubin(DBIL),albumin(ALB)and total protein(TP).Enzyme linked immunosorbent assay were used to measure the levels of hyaluronic acid(HA),rat procollagen peptide(?PC)and laminin(LN)in serum.The extent of liver fibrosis was determined by the content of hydroxyproline(HyP)in liver,the degree of liver fibrosis under the microscope(HE and Masson staining)and the expression of a-SMA by immunohistochemistry.The expression of Smad4,Smad7,PDGF-BB,IFN-?,TGF?1,IL-10,IL-13,IL-12p70,IL-4,IL-6,IL-12,IL-18,IL-1?,IL-1?,MCP-1?,MCP-1,MIP-1?,MIP-2 and TNF-?,the extent of phosphorylation of P-CREB,P-JNK,P-NF-?B,P-P38,P-ERK1/2,P-Akt,P-p70S6K,P-STAT3 and P-STAT5 in liver were examined by multi-functional liquid protein chip or ELISA.(2)Healthy beagle dogs,ip pentobarbital sodium 35mg·kg-1.Ganbao were given by duodenum.the blood pressure,ECG and respiration wave curve of dogs were monitored via sixteen channel physiological recorder.Meanwhile the spontaneous activity,rotating rod movement,the effect induced sleep time with pentobarbital sodium threshold dose and hypnotic synergy with pentobarbital sodium subthreshold dose were carried out in mice.Results:(1)Bile duct ligation significantly increased the serum activities of ALT,AST,TBIL,DBIL,y-GT and ALP,decreased the serum level of ALB and ratio of A/G,increased the content of Hyp and collagen,and the expression of ?-SMA,IL-18,MIP-1?,TGF-?1,p70S6K,but markedly decreased the expression of the IL-1?,IL-4,IL-10,TNF-?,and total phosphorylation of Smad4,NF-?B,P38,Akt and STAT5 in liver of rat.Ganbao capsule 1.0 g·kg-1,2.0 g·kg-1 and 4.0 g·kg-1 reduced the activity of AST,ALT,TBIL,DBIL,?-GT and ALP,elevated the level of ALB and ratio of A/G in serum.Ganbao capsule 1.0 g·kg-1 also decreased the content of Hyp,IL-18 and TGF-?1,decreased the expression of IL-1? in the liver in BDL rats.Ganbao capsule 2.0 g·kg-1 significantly reduced the level of Hyp,TGF-?1 and p70S6K,and increased the expression of IL-4,TNF-?,and total phosphorylation of NF-?B,P38,Akt and STAT5 in the liver in BDL rats.Meanwhile Ganbao capsule 4.0 g·kg-1 significantly decreased the expression of Hyp,MIP-1?,IL-18,IL-6 and TGF-?1,increased the contented of IL-1?,TNF-?,IL-13 and PDGF-BB,and total phosphorylation of P38 and STAT5 in the liver in BDL rats.The results of microscopic examination showed that Ganbao 4.0 g·kg-1 and 2.0 g·kg-1 can decrease expression of a-SMA and improve the liver fibrosis in BDL rats.(2)Ganbao capsule 9.0 g·kg-1,5.0 g·kg-1and 1.0 g·kg-1don't induce the beagle dogs salivation,muscle tremors or pupil change.It has no effect on the blood pressure,electrocardiogram,heart rate,the depth or frequency or rhythm of breathing in beagle dogs.Meanwhile,there have some effect on spontaneous activity and the synergic effect inducig sleep time with pentobarbital sodium in mice.But there were no effect on rotating rod movement and sleeping time with a threshold dose of sodium pentobarbital in mice.Conclusion:The effects of Ganbao capsule on liver fibrosis may be related to suppression of expression of IL-18,TGF-?1,MIP-1? and p70S6K,decreased the activation and proliferation of HSC,inhabited the generation of ECM induced by BDL.Ganbao 9.0 g·kg-1 had no adverse effect on the cardiovascular and respiratory system in beagle dogs.But Ganbao 26.0 g·kg-1 have some effect on the central nervous system in mice.