| Objective: Hepatic fibrosis is a pathology process that liver diffuseextracellular matrix excessively deposit because of various pathogenic factorscausing abnormal intrahepatic connective tissue hyperplasia,it is also themajor aspects of the development of chronic liver disease tocirrhosis．Therefore,timely blocking and reverse liver fibrosis is the key toprevent cirrhosis and improve the prognosis of liver disease,A variety ofcytokines and signal transduction pathways involved in liver fibrosisdevelopment process,transforming growth factor beta （transforming growthfactor beta, TGF-beta） is a kind of polypeptide can regulate cell growth andcell differentiation,it is the most critical cytokine mediated liver fibrosis.It canactivate hepatic stellate cells,activated HSC further transformed intomyofibroblasts,expressing α-smooth muscle actin （alpha-SMA）,ensynthesizeand secrete ECM.TGF-β1/Smads signal pathway is one of the mostimportant regulatory mechanism of liver fibrosis,it is of great significance tothe formation of liver fibrosis.The researchers conducted a mass of studies forthe liver fibrosis,but blocking of liver fibrosis is still difficult inclinical,recently,theory of traditional Chinese medicine research and clinicalpractices have proved that,using the Chinese traditional medicine to preventthe liver fibrosis indicates the certain advantage via muti-pathway andmuti-target spots to integrate,Chinese traditional medicine Biejiajianwan（BJJW） which is the main medicine for treating mother-of-malaria comes fromJinguiyaolue wrote by Zhang zhongjing,it promotes the blood circulate，removing the blood stasis, using the gentle method and force,replenishing qi toinvigorate the spleen.Demonstrated by a great number of experiment,Biejiajianwan （BJJW） has the function for liver fibrosis and liver cirrhosis,This research will take the CCL4method for SD mouse to establishthe liver fibrosis mouse model, Biejiajianwan（BJJW）,which promote the bloodcirculate and remove the blood stasis is put in use to interpose for the model.Observing the general situation of experiment animal, inspect the content ofLN, Col-IV, ALT, AST, ALB. Observing the liver nosology shape, detectingthe expressionof TGF-β1mRNA, Smad3mRNA, and α-SMA protein.Wewant to explore the curative effect and possible antifibrotic mechanism ofChinese traditional medicine.Methods:56healthy male SD rats, weighted360-410g, after raising oneweek,were randomly divided into6groups: control group（A）, model group（B）,colchicina-group（C）, low-dose [0.55g/（kg.d）] BJJW group （D）, medium-dose[1.1g/（kg.d）] BJJW group （E）, and high-dose[2.2g/（kg.d）] BJJW group （F）.Excepting the control group, which were injected olive oil only, the othergroups of rats were induced hepatic fibrosis by subcutaneous injections of40%CCl4olive oil solution.The injected dose was5ml/kg for the first time,and then3ml/kg, twice a week. At the same time, drugs were intragastricallyadministrated at a volume of10mL/（kg.d） in groups C-E, whereas the rats ofgroups A and B were given the same volume of distilled water.At week6,hepatic fibrosis was found, so stop the injections of CCl4or olive oil.But, eachgroup continues to be gaven drugs or water accordingly for5weeks. At theend of11thweek, abrosia12hours, we took blood from heart to measure theserum ALT,AST,ALB level, detect the serum Col-Iv and LN level byradioimmunoassay,take the liver,put the left liver tissue in the4%paraformaldehyde,dyed by HE and Masson, observe liver nosology change ofmouse in each group,the expression of alpha SMA protein in liver tissue wasdetected by Immunohistochemical method,then,preserve the right liver tissuein the refrigerator with-80degree after quick-freezing by the liquid nitrogen,The content of TGF-β1mRNA and Smad3mRNA level in the liver tissuewere detected by reverse transcription-polymerase chain reaction RT-PCR.Results:1General condition of experimental rats Normal group of rats whose weight increased，were well, normal size,sensitive reaction, lustrous hair.The rats in other groups were poor eating, darkhair, slim down，poor reaction，bad temper.During the experiment, some ratswere dead:2rats dead in model group,1rat dead in colchicina-group andBJJW dose groups respectively.2The pathology changes of hepatic tissue in each groupOptical microscope observation: in the normal group, structure of hepaticlobule were complete and clear.There was no connective tissue proliferation incentral vein and portal area.The liver cell was more than a single nucleus andnucleolus was clearly visible, there was no fatty degeneration andnecrosis.The liver cells arranged cord-like radiation to the surroundings.Inmodel group, normal hepatic lobule structure was destroyed. Liver tissueshowed extensive steatosis and necrosis, seems like the empty mesh, the portalarea expanded, inflammatory cells infiltrated.The proliferation of fibrousconnective tissue formed varied thickness of fiber connected interval, andhepatic fibrosis was significant.Compared with modle group, in the colchicinagroup and BJJW dose groups, the damage of hepatic lobule significantlyreduced, so does the hepatic steatosis necrosis and inflammatory cellinfiltration.There was mild fibrous tissue proliferation,which formed a thinfiber bundles.The pseudolobule significantly reduced. The degree of liverfibrosis in High-dose BJJW group significantly reduced than in the low-dosegroup and the colchicine group,There was no significant difference in liverpathology between colchicine group and low-dose BJJW group.3The levels of ALT, AST and ALB in the serum of different groupsThe level of ALT and AST in the serum of different groups display:compared with normal group, the content of ALT, AST in the rats serumincreased obviously （P<0.05） in model group, colchicina group and BJJWdose groups. Compared with model group,the serum ALT, AST contentdecreased obviously （P<0.05） in colchicina group and BJJW dosegroups.Compared with colchicina group, the serum ALT, AST contentdecreased obviously （P<0.05） in high-dose BJJW group, the level of serum ALT, AST in the low-dose BJJW group was more higher than in colchicinagroup （P<0.05）, and there were no significant differences between the BJJWmedium-dose group and colchicina group （P>0.05）.Among the BJJW dosegroups, there were significant differences between them （P<0.05）, high-dosegroup was the lowest, low-dose group was the highest, and medium-dosegroup was middle.The level of ALB in the serum of different groups display: compared withnormal group,the content of ALB in the rats serum decreased obviously（P<0.05） in model group, colchicina group and BJJW dose groups.Comparedwith model group, the serum ALB content increased obviously （P<0.05） incolchicina group, BJJW medium-dose group and BJJW high-dose group, therewere no significant differences between the model group and BJJW low-dosegroup （P>0.05）.Compared with colchicina group, the serum ALB contentincreased obviously （P<0.05） in high-dose BJJW group,the level of serumALB in the low-dose BJJW group was more lower than in colchicina group（P<0.05）,and there were no significant differences between the BJJWmedium-dose group and colchicina group （P>0.05）.Among the BJJW dosegroups,there were significant differences between them （P<0.05）, high-dosegroup was the highest,low-dose group was the lowest,and medium-dose groupwas middle.4The levels of Col-IV and LN in the serum of different groupsThe level of Col-IV in the serum of different groups display: comparedwith normal group, the content of Col-IV in the rats serum increasedobviously （P<0.05） in model group, colchicina group and BJJW dose groups.Compared with model group, the serum Col-IV content decreased obviously（P<0.05） in colchicina group, BJJW medium-dose group and BJJW high-dosegroup, there were no significant differences between the model group andBJJW low-dose group （P>0.05）.Compared with colchicina group, the serumCol-IV content decreased obviously （P<0.05） in high-dose BJJW group, thelevel of serum Col-IV in the low-dose BJJW group was more higher than incolchicina group （P<0.05）, and there were no significant differences between the BJJW medium-dose group and colchicina group （P>0.05）. Among theBJJW dose groups, there were significant differences between them （P<0.05）,high-dose group was the lowest, low-dose group was the highest, andmedium-dose group was middle.The level of LN in the serum of different groups display: compared withnormal group, the content of LN in the rats serum increased obviously（P<0.05） in model group,colchicina group and BJJW dose groups.Comparedwith model group,the serum LN content decreased obviously （P<0.05） incolchicina group and BJJW dose groups. Compared with colchicina group, theserum LN content decreased obviously （P<0.05） in high-dose BJJW group,the level of serum LN in the low-dose BJJW group was more higher than incolchicina group （P<0.05）, and there were no significant differences betweenthe BJJW medium-dose group and colchicina group （P>0.05）.Among theBJJW dose groups,there were significant differences between them（P<0.05）,high-dose group was the lowest,low-dose group was the highest, andmedium-dose group was middle.5The expression levels of TGF-β1mRNA and Smad3mRNA in the hepatictissue of different groupsThe expression levels of TGF-β1mRNA in different groups: comparedwith normal group, the expression levels of TGF-β1mRNA increasedsignificantly （P<0.05） in model group, colchicina group and BJJW dosegroups. Compared with model group, the expression levels of TGF-β1mRNAdecreased significantly （P<0.05） in colchicina group and BJJW dose groups.Compared with colchicina group,The expression levels of TGF-β1mRNAdecreased significantly （P<0.05） in high-dose BJJW group,the level ofTGF-β1mRNA in the low-dose BJJW group was more higher than incolchicina group （P<0.05）, and there were no significant differences betweenthe BJJW medium-dose group and colchicina group （P>0.05）.Among theBJJW dose groups,there were obvious differences between them （P<0.05）,high-dose group was the lowest, low-dose group was the highest,andmedium-dose group was middle. The expression levels of Smad3mRNA in different groups: comparedwith normal group,the expression levels of Smad3mRNA increasedsignificantly （P<0.05） in model group, colchicina group and BJJW dosegroups.Compared with model group, the expression levels of Smad3mRNAdecreased significantly （P<0.05） in colchicina group and BJJW dose groups.Compared with colchicina group,the expression levels of Smad3mRNAdecreased significantly （P<0.05） in high-dose BJJW group, the level of Smad3mRNA in the low-dose BJJW group was more higher than in colchicina group（P<0.05）,and there were no significant differences between the BJJWmedium-dose group and colchicina group （P>0.05）.Among the BJJW dosegroups, there were obvious differences between them （P<0.05）, high-dosegroup was the lowest, low-dose group was the highest, and medium-dosegroup was middle.6The expression levels of α-SMA protein in the hepatic tissue of differentgroupsIn the normal group, α-SMA protein of liver tissue expressed only in thesmall arteries and small veins, and there was no expression in the bile duct. Inmodel group, the expression of α-SMA protein in the liver tissue markedlyincreased, enhanced, and the degree of positive staining also increased. Itexpressed mainly in portal area and fiber separation, as oblong or spindleshape.Compared with the modle group, the positive staining ofα-SMA proteinin the colchicine group and BJJW dose groups significantly reduced（P<0.05）,and the positive staining of interstitial cells and inflammatory cellsin fibrous septa reduced（P<0.05）too. The positive staining of BJJW high-dosegroup, was significantly lower than the low-dose group, medium-dose groupand the colchicine group（P<0.05）. There were no significant differencesbetween the colchicina group and BJJW medium-dose group （P>0.05）.Conclusions:1Rats with hepatic fibrosis are liver damage, proliferation of fibrousconnective tissue and excessive deposition of extracellular matrix.TheChinese traditional medicine which promote blood circulate and remove the stasis can significantly improve liver function and reduce the degree ofhepatic fibrosis.2The Chinese traditional medicine which promote the blood circulateand remove the stasis can significantly lower the expression of TGF-β1mRNAand Smad3mRNA,its antifibrotic mechanism may be through regulating theTGF-β1/Smad3signaling pathways.3The Chinese traditional medicine which promote the blood circulateand remove the stasis can inhibit α-SMA protein,so inhibiting HSCs activationand proliferation may be one of the mechanisms of anti-hepatic fibrosis. |
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