| Objective:Hepatic fibrosis is a pathologic course,which induced by all kinds of chronic hepatic injury.Hepatic fibrosis can result in hepatic cirrhosis,which is very harmful to human health.The formation of hepatic cirrhosis is a complicated process in which multi-factor and multi-cell involved.TGFβ1 is the most effective cytokine we have found which can result in hepatic fibrosis.Smad protein is the important substrate of TGFβ1.The information that HSCs are actived by TGFβ1 only can be delivered by Smad,only that the information can be transmited into the cell nucleus,and expression of collagen gene was induced,all that result in hepatic fibrosis.The activation and corresponding pathological changes of TGFβ1/Smads play an important role in hepatic fibrosis.TGFβ1, TGFβRII,Smad3,Smad7 have different roles in the development of hepatic fibrosis.Curcumin is active component of traditional Chinese medicine turmeric yellow,which was used to treat hepatic fibrosis depending on its functions of anti-oxygen free radical,adjusting cell cycle.But what happened in signalling pathways were not clear.In this study, experimental models of hepatic fibrosis were established by given C57BL6/J mice the mixture of 10%CCL4 and olive oil by intraperitoneal injecting.Then curcumin was used to prevent and treat hepatic fibrosis.Our objective was to investigate the expression and possible roles of TGFβ1/Smads signalling pathway in liver of mice with hepatic fibrosis,and the role of curcumin in prevention and treatment of hepatic fibrosis. Our objective also was to find the new mechanism that how curcumin can depress the development of hepatic fibrosis.Methods:40 healthy female C57BL6/J mice which were 8 weeks old were divided into 4 groups randomly:control group,model group,prevention group and treatment group. There were 10 mice in each group.The mixture of 10%CCL4 and olive oil(5ul/g) was given by intraperitoneal injection to all mice except control group two times a week for 8 weeks. 0.9%Sodium Chloride(5ul/g) was given by intraperitoneal injection to mice in control group two times a week for 8 weeks. Curcumine(50 ug/g)was given by intragastric administration to mice in prevention group and treatment group three times a week for 8 weeks at the beginning of 1st and 7th week.All mice were killed after 8th week.Serum alanine aminotransferase (ALT) was tested by enzymic method with automatic biochemistry analyzer.Hyaluronic acid(HA) was tested by radio-immunity.Liver inflammation was graded under HE staining,and liver fibrosis by Masson staining.The expression of protein ofα-SMA,TGFβ1,TGFβRII,Smad3 and Smad7 of liver organization were examined by immunohistochemistry. The expression of TGF-β1,Smad3 and Smad7 mRNA were analyzed by RT-PCR.Results:1 The common behavior of mice:control mice were active, their hair was bright and weight increased gradually.Body weight of mice in model group decreased remarkably compareing with control group.Body weight of mice in control, model,prevention and treatment group were 22.82±0.84g, 18.07±0.68g,20.07±0.86g,19.46±0.59g.Compareing with control group,body weight of mice in the other three groups decreased(P＜0.05).Compareing with model group,Body weight of mice in prevention and treatment groups increased (P＜0.05),there was no difference between prevention and treatment groups(P＞0.05 )2 The examination of ALT and HA:The levels of ALT of each group:model group＞treatment group＞prevention group＞control group(168.62±13.75 U/L,104.86±11.68 U/L, 82.98±9.57U/L,47.91±8.08 U/L),the difference of the levels of ALT among above groups was significant(P＜0.05).The levels of HA of mice:model group＞treatment group＞prevention group＞control group(1609.95±125.91 ng/ml,1290.26±127.59ng/ml,953.39±98.48ng/ml,759.10±77.31 ng/ml),the difference of the levels of HA among above groups was significant(P＜0.05).3 Routine pathologic examinations:The liver histology is normal in control group,and there were no cellular necrosis and fibrosis in liver.However,severe cellular necrosis and fibrosis were found in model group.In model group mice hepatic lobe obvious punctual or stove shape liver cell necrosis can be seen. Large areas mixed inflammatory infiltration can be seen throughout the hepatic lobule.Massive fiber structure can be seen in portal canal area and hepatic sinusoid obviously (G2～3S3～4).The extent of inflammation,cellular necrosis and fibrosis in liver of curcumine treatment group is dramatically decreased,and more dramatically in prevention group.4α-SMA expresses mostly in vessel wall in the liver of control group.However,expression ofα-SMA increased in model group.Overexpression ofα-SMA in vessel wall,portal canal area and fiber structure in the liver of model group can be found.Protein expression ofα-SMA of each group:model group＞treatment group＞prevention group＞control group (8.21±1.48%,6.25±1.69%,4.78±1.32%,1.51±0.95%),the difference among above groups was significant(P＜0.05).5 TGFβ1 expresses mostly in groundplasm of portal canal area and hepatic sinusoid,central veins wall,and expresses mostly in endochylema in the liver of control group.However, compared with control group,TGFβ1 masculine cell number increases obviously in model group.TGFβ1 expresses in endochylema of the cell around portal canal area,central veins wall,fibrosis district.Protein expression of TGFβ1 of each group:model group＞treatment group＞prevention group＞ control group(5.52±0.89%,4.05±0.69%,2.70±0.82%, 1.31±0.81%),the difference among above groups was significant.(P＜0.05).6 The expression of TGFβ1 mRNA of each group:model group＞treatment group＞prevention group＞control group (1.00±0.05,0.68±0.04,0.58±0.04,0.31±0.03).the difference among above groups was significant(P＜0.05).7 TGFβRII expresses mostly in cell membrane of liver cell in control group.However,compared with control group, TGFβRII masculine cell number increased obviously. Expression of TGFβRII of control,model,prevention and treatment group:0.37±0.04%,0.52±0.04%,0.44±0.03%, 0.46±0.05%.Compareing with control group,the expression of TGFβRII of the other three groups increased(P＜0.05); compareing with model group,the expression of TGFβRII of prevention and treatment groups decreased(P＜0.05),there was no difference between prevention and treatment groups(P＞0.05 )8 Smad3 expresses mostly in groundplasm of portal canal area,less in endochylema of interstitial cell.Positive expression can be seen in portal canal area,endochylema of interstitial cell in fibrosis district in the liver of model group.Protein expression of Smad3 of each group:model group＞treatment group＞prevention group＞control group(5.86±1.49%, 4.25±1.32%,3.10±0.99%,1.41±0.95%),the difference among above groups was significant(P＜0.05). 9 The expression of Smad3 mRNA of control,model, prevention and treatment group:0.23±0.02,0.46±0.03, 0.34±0.02,0.36±0.02.Compareing with control group,the expression of Smad3 mRNA of the other three groups increased (P＜0.05),compareing with model group,the expression of Smad3 mRNA of prevention and treatment groups decreased (P＜0.05),there was no difference between prevention and treatment groups(P＞0.05)10 Smad7 expresses mostly in endochylema of hepatic cell and HSCs in the liver of control group.Smad7 masculine cell number decreased obviously in model group,and positive expression was only seen in hepatic cell and fusiform cell in fabric compartment.The expression of Smad7 of control,model, prevention and treatment group:6.32±1.18%,1.83±0.62%, 4.45±1.07%,4.17±0.99%.Compareing with control group,the expression of Smad7 of the other three groups decreased (P＜0.05);compareing with model group,the expression of Smad7 of prevention and treatment groups increased(P＜0.05). there was no difference between prevention and treatment groups(P＞0.05).11 The expression of Smad7 mRNA of each group:control group＞prevention group＞treatment group＞model group (0.69±0.03,0.63±0.04,0.51±0.04,0.40±0.03 )the difference among above groups was significant(P＜0.05).Conclusion:1 The hepatic fibrosis models could be established successfully by intraperitoneal injection with the mixture of 10%CCL4 and olive oil for 8 weeks.This model is characteristed by rapidly formation,easily performed and consistent with the pathological features of patients of hepatic fibrosis.2 TGFβ1/Smads signalling pathway play an important role in hepatic fibrosis.The high expression of TGFβ1,TGFβRII,Smad3 accelerates the development of hepatic fibrosis.The high expression of Smad7 depress the development of hepatic fibrosis.3 Curcumine has the function of depressing the development of hepatic fibrosis.4 We found that expression of TGFβ1,TGFβRII,Smad3 decreased and Smad7 increased in the liver of mice with hepatic fibrosis after treatment with curcumine.It provided the theory that curcumine might be used to treat hepatic fibrosis by affecting TGFβ1/Smads signalling pathway.
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