| Hepatic fibrosis is a reversible wound repair response and is also a necessary pathway for all chronic liver injury.A growing number of young patients have sequela of portal hypertension,liver failure and liver cirrhosis related to hepatocellular carcinoma.The global burden of disease predict liver cirrhosis and hepatocellular carcinoma accounted for 2.5%of all deaths in the world,the only effective treatment for decompensated liver disease is liver transplantation.However,a shortage of donor organs in North America and Europe has resulted in increased mortality of patients waiting for liver transplantation to about 20%.Not only adults but also infants and young children have hepatic fibrosis.1/5000～1/19000 live birth babies suffer from biliary atresia,the incidence in Asian countries is more common than in European countries.Hepatic fibrosis in biliary atresia is a common and prominet features,the progress of hepatic fibrosis is significantly faster than other hepatobiliary diseases.More than 90 days children are not suitable for Kasai procedure and need to undergo transplatation to long-term survival.Infant hepatitis syndrome occurs in the infant period(including neonatal period)and is a series of clinical syndrome of jaundice,liver pathological signs(hepatomegaly,abnormal texture)and liver function injury.Due to the continuous effect of pathogenic factors and immune function disorders liver cells are damaged directly or by immunity,fibrous tissue proliferates continuously,then the liver goes into the stage of fibrosis.The main pathological changes of hepatic fibrosis is collagen,proteoglycan and laminin and other extracellular matrix excessive deposition in the liver.At present the key signal pathway of hepatic fibrosis is TGF-β1/Smads signal pathway,drugs to block or intervene in TGF-β1/Smads signal pathway can effectively influence or inhibit the occurrence and development of hepatic fibrosis.Most drugs are used to treat adult fibrosis,pediatric drugs are less and less.Taking the early and effective intervention and less side effect as the goal,people turn their attention to traditional Chinese medicine,hoping to find effective drugs for prevention and treatment of hepatic fibrosis.The previous clinical research had shown Jia Wei Yin Chen Si Ni Decoction in the treatment of children with biliary atresia after Kasai procedure could improve the clinical symptoms,delay the onset of hepatic fibrosis and enhance the quality of survival,the previous experimental research proved that a certain effect of Jia Wei Yin Chen Si Ni Decoction on hepatic stellate cells of TGF-β1/Smads signal pathway,but the JWYCSND efficacy and mechanism in the animal experiment are still not reported.At the same time,this study is a part of National Natural Science Fund Project "Mechanism Study of Jia Wei Yin Chen Si Ni Decoction on TGF-β1/Smads Signal Pathway and MMPs/TIMPs in Billiary Atresia Model of Liver Fibrosis"(Project number:81373686).ObjectiveObserve preventive intervention effect of Jia Wei Yin Chen Si Ni Decoction on carbon tetrachloride induced hepatic fibrosis in unmatured rats,study its effect on organ index,hepatic pathological histology and serological indicators,and explore its possible mechanism about TGF-β1/Smads Signal Pathway.Methods42 male Wistar unmatured rats were randomly divided into normal group,oliva oil group,model group,colchicine group,JWYCSN high dose group,middle dose group and low dose group,6 rats in each group.Expect for the normal group and the oliva oil group every group was established by intraperitoneal injection of 0.15ml/kg 50%CC14,the oliva oil group was intraperitoneal injection of 0.15ml/kg oliva oil,2 times a week.The oliva oil group and model group were treated with intragastric administration of 10ml/kg sterile water per day,the colchicine group were treated with colchicine 0.lmg/kg,JWYCSN high dose group,middle dose group and low dose group were treated with Jia Wei Yin Chen Si Ni Decoction 16.46g/kg,8.23g/kg,4.12g/kg.The intragastric adminstration was once a day for 21 consecutive days.At the twenty-second days blood samples were taken from abdominal aorta and liver and spleen were collected.The live index and spleen index were calculated after weighing the liver and spleen.The pathological liver change was observed by performing hematoxylin-eosin staining and the fibrosis degree was understood by Inshak score system.The levels of serum ALT and AST were detected by micro plate method and the levels of serum HA,LN,COL-IV and PCOL-III were tested by competitive inhibition enzyme-linked immunosorbent assay.mRNA expression and protein expression of TGF-β receptor I,Smad3,Smad7,Collagen I and Collagen III were detected by RT-qPCR and western blot.Results1.The effect of Jia Wei Yin Chen Si Ni Decoction on liver index and spleen indexLiver index and spleen index in model group were higher than normal group(P<0.05),liver index and spleen index in oliva oil group were not different from normal group(P>0.05).Liver index and spleen index in colchicine group and all the JWYCSN group were lower than model group(P<0.05).2.The effect of Jia Wei Yin Chen Si Ni Decoction on liver tissue(1)In normal group and oliva oil group hepatic lobule structure was complete,the edge is shape,small bile duct and fiber hyperplasia were not found in portal area and its surrounding.In model group hepatic lobule structure was damaged or even disappear,hepatic cords arranged mussily,portal area of fiber hyperplasia were cord-like structure and formed fiber bridges to connect the portal area to adjacent portal area and set the liver into some parts,the liver was infiltrated by lots of inflammatory cells.In colchicine group and all the JWYCSN group hepatic lobule structure was not clear,liver cells were visible swelling and obvious fatty degeneration,liver mesenchyma was infiltrated by inflammatory cells,portal hepatic fibrosis were different,but their hepatic fibrosis were obviously relief compared with model group.(2)There was no fibrosis in normal group and oliva oil group(P>0.05).The pathological score in model group was higher than normal group(P<0.05),but the scores in colchicine group and all the JWYCSN group were lower than model group(P<0.05).3.The effect of Jia Wei Yin Chen Si Ni Decoction on serum ALT and ASTCompared with the normal group,ALT and AST in model group significantly increased(P<0.05),ALT and AST in oliva oil group had no difference(P>0.05).Compared with model group,ALT and AST in colchicine group and all the JWYCSN group decreased significantly(P<0.05),but each group of AST had no difference(P>0.05).4.The effect of Jia Wei Yin Chen Si Ni Decoction on serum HA,LN,COL-IV and PCOL-IIIHA,LN,COL-IV and PCOL-III in model group were higher than normal group(P<0.05),they were not different between oliva oil group and normal group(P>0.05).Expect for HA and LN in JWYCSN low dose group,all of indicators in all the treatment groups were obviously lower than model group(P<0.05).5.The effect of Jia Wei Yin Chen Si Ni Decoction on the expression of TβRI mRNA、Smad3 mRNA、Smad7 mRNA、Collagen I mRNA、Collagen III mRNA(1)The expression of TβRI mRNA in model group was obviously higher than normal group(P<0.05),but it had no different between oliva oli group and normal group(P>0.05).Except for JWYCSN low dose group,the expression of TβRI mRNA in all the treatment groups were remarkably lower than model group(P<0.05).(2)The expression of Smad3 mRNA in model group was observably higher than normal group(P<0.05),but it had no difference between oliva oil group and normal group(P>0.05).The expression of Smad3 mRNA in colchicine group and JWYCSN middle dose group were lower than model group(P<0.05),but the expression of Smad3 mRNA in JWYCSN high dose group and low dose group were not different from model group(P>0.05).(3)The expression of Smad7 mRNA in model group and oliva oil group were not significant different from normal group(P>0.05).The expression of Smad7 mRNA in JWYCSN middle dose group were lower than model group(P<0.05),but the expression of Smad7 mRNA in colchicine group,JWYCSN high dose group and low dose group had no different from model group(P>0.05).(4)The expression of Collagen I mRNA in model group was obviously higher than normal group(P<0.05),but it had no difference between oliva oil group and normal group(P>0.05).The expression of Collagen I mRNA in JWYCSN middle dose group were lower than model group(P<0.05),but the expression in colchicine group,JWYCSN high dose group and low dose group were not different from model group(P>0.05).(5)The expression of Collagen III mRNA in model group was remarkably higher than normal group(P<0.05),but the expression in oliva oil was the same as normal group(P>0.05).The expression of Collagen III mRNA in colchicine group,JWYCSN high dose group and middle dose group were lower than model group(P<0.05),but the expression in JWYCSN low dose group were not different from model group(P>0.05).6.The effect of Jia Wei Yin Chen Si Ni Decoction on the protein expression of TGF-β receptor I,Smad3,Smad7,Collagen I and Collagen III(1)The protein expression of TβRI in model group was obviously higher than normal group(P<0.05),but it had no different between oliva oli group and normal group(P>0.05).The protein expression of TβRI in all the treatment groups were the same as model group(P>0.05).(2)The protein expression of Smad3 in model group was evidently higher than normal group(P<0.05),but the expression in oliva oil group and normal group had no difference(P>0.05).The protein expression of Smad3 in JWYCSN middle dose group and low dose group were evidently lower than model group(P<0.05).(3)The protein expression of Smad7 in model group were remarkably higher than normal group(P>0.05),but the expression in oliva oil group was the same as normal group(P>0.05).The protein expression of Smad7 in all the treatment groups were not differnet from model group(P>0.05).(4)The protein expression of Collagen I in model group was obviously higher than normal group(P<0.05),it had no different between oliva oli group and normal group(P>0.05).The protein expression of Collagen I in all the treatment groups and model group had no significant difference(P>0.05).(5)The protein expression of Collagen III in model group was observably higher than normal group(P<0.05),but the expression in oliva oil was the same as normal group(P>0.05).The protein expression of Collagen III in JWYCSN middle dose group were lower than model group(P<0.05).ConclusionHepatic fibrosis rats induced by CCl4 are prophylactically treated with all of doses JWYCSN,which reduced liver inflammation,hepatic fibrosis and spleen symptoms.Jia Wei Yin Chen Si Ni Decoction could decrease the level of ALT,HA,LN,IV-C and PCIII and the changes of HA and LN are related to drug concentration.Jia Wei Yin Chen Si Ni Decoction has the preventive effect on hepatic fibrosis rats in some extent.Jia Wei Yin Chen Si Ni Decoction could suppress the expression of TβRI.Smad3 which lead to down-regulating the expression of Collagen I and Collagen III,thus attenuate the development of hepatic fibrosis.Inhibition effect of this drug on TβRI、Smad3 is not in proportion to drug concentration.The protein expression of Smad7 in all JWYCSN group are not higher than model group but the numbers have the rising trend,which are different from the expression of Smad7 mRNA.It conclude that other signal pathways may participate or other factors may affect the level of protein transcription. |
PDF Full Text Request