Effect Of Curcuma Kwangsiensis S . G . Lee Et C . F . Liang On TGF-β1/Smads Signaling Pathway In Mice With Hepatic Fibrosis Induced By Blood Stasis

Aims:This article mainly discusses the effect of Curcuma kwangsiensis S.G.Lee et C.F.Liang on TGF-β1/Smads signaling pathway in mice with hepatic fibrosis induced by blood stasis.Methods:（1）Extracting the volatile oil of Curcuma kwangsiensis S.G.Lee et C.F.Liang by distillation,and comparing with the curcumol standard sample,detecting the main active constituents in Curcuma kwangsiensis S.G.Lee et C.F.Liang by gas chromatography-mass spectrometry（GC-MS）and verify that this main active constituents contains Curcumol.（2）72 SPF Kunming healthy male mice were randomly divided into normal group（A）;blood stasis syndrome hepatic fibrosis mouse model group（B）;Curcuma kwangsiensis S.G.Lee et C.F.Liang treatment group（C）（Dividing into hight,medium and low dose groups,marked C1,C2,C3）;colchicine group（D）and Salvianolic acid B group（E）.There are 12 mice in group B and 10 in each group.Firstly,Group A was routinely fed with water and feed.Secondly the other mice in each group were subcutaneously injected with 40%concentration of CCl₄+peanut oil solvent at the dose of0.05 m L/100 g on the first experimental day.And the subsequent injection dose was changed to be 0.03 m L/100g,3 times a week.Thirdly from the second week,each mouse was injected intraperitoneally norepinephrine（concentration 0.01 mg/m L）at a dose of 0.5 m L/dose.Fourth,fetal bovine serum was intraperitoneally injected at a dose of 50 mg/m L on the 1st day and 10th day of the experiment.In addition,daily fed 20%alcoholic water,egg yolk and peanut kernels something with high-fat/low-protein+fodder.During the experiment（the 8th,10th,and 11th week）,randomly selected one mouse from each group for pathological observation.This model costed 12 weeks to be built.（3）After model successfully built,group C1-C3 were respe ctively induced by intragastric infusion once a day at high,medium and low doses of0.75mg/100g,0.5mg/100g,0.25mg/100g Curcuma kwangsiensis S.G.Lee et C.F.Liang.Group D was administered by intragastrically once a day at a dose of 0.025 mg/100g.Group E was administered by intragastrically once a day at a dose of 0.25 mg/100 g.Group A and Group B were administered by intragastrically once a day according to the dose of group C1 added 0.9%NaCl.Except group A,the other groups were given“40%CCl₄+alcohol drinking water+high fat low protein diet”to maintain hepatic fibrosis while intragastric administration.Intragastric administration continued total 5weeks.（4）Mice in each group was been weighed before observing and taking photos to record their appearance and morphological characteristics,then assessing the symptom of symptomatic blood stasis syndrome in mice.After taking the material,weigh the weight of each liver and spleen,and calculate the index of liver and spleen.Using the HE and Masson staining method to observe the pathological changes of hepatic fibrosis in each liver,then grade.Observing the expression of TGF-beta 1,Smad 2 and Smad 3 protein in hepatic tissue by immuno histo chemistry（IHC method）and Western blotting（Western blot method）.Observing the TGF-beta 1,Smad 2 and Smad 3’s mRNA expression in hepatic tissues by real-time fluorescent quantitative PCR（RT-PCR）.Results:（1）The results of Gas chromatography-mass spectrometry（GC-MS）:By the computer software MassHunter ChemStation,total of 133active ingredients of Curcuma kwangsiensis S.G.Lee et C.F.Liang were been detected,after carefully contrast,40 kinds of ingredients with a matching degree more than 80%were been screened out.The chromatograms show that the different peak times in the chromatogram of the Curcuma kwangsiensis S.G.Lee et C.F.Liang sample represent different active ingredients.The peak time of the curcumol standard sample was on 28.731min,the same as the peak time of the curcumol standard sample which found in the chromatogram of the Curcuma kwangsiensis S.G.Lee et C.F.Liang sample extracted by distillation,which indicated that the Curcuma kwangsiensis S.G.Lee et C.F.Liang contained curcumol.（2）The appearance of blood stasis syndrome in TCM symptoms is sluggishness of mind,unresponsive,difficult to move,rough hair,tongue appeared dark purple,the prominent of sublingual collaterals was over than the tongue’s 3/4.Compared with group A,the score of TCM syndrome of blood stasis syndrome in group B was significantly increased（P<0.01）;Respectively compared with group B,group C1-C3,group D and group E with different degrees decrease of TCM symptom scores in blood stasis syndrome（P<0.05 or P<0.01）,above all the decreased of C1 was noteworthy（P<0.01）.The results of the Curcuma kwangsiensis S.G.Lee et C.F.Liang treatment group was similar with the result of positive drug control group（group D and E）.（3）Before and after modeling and treatment,Compared with group A,the increase of weight in group B’s mice was significantly low（P<0.01）;Respectively compared with group B,group C1-C3,group D and group E with different degree increase in body weight（P<0.05 or P<0.01）,especially group C1（P<0.01）,the increase is the most significant.The results of Curcuma kwangsiensis S.G.Lee et C.F.Liang treatment group was similar with the result of the positive drug control group（group D and E）.（4）The gross morphology of the liver in each group’s mice was appeared:The size of liver in group A was more moderate than other groups,and liver with blood red color,smooth surface,without graininess and nodules,soft and elastic,edges complete and sharp,no greasy feeling.The liver of group B was significantly larger than normal group,and the color was dark and dull,the surface was rough and covered with small nodules,the graininess was obvious,hard and inelastic,breaking easily to touch,the edge became thickened and dull,also with a small amount of orange peel-like changes and became greasy.The appearance of the liver in the group C1 was closer to the normal group,the color was brighter than group B.There was no or only few particles on the surface of the liver,and the elasticity was acceptable.For the size of liver,color and texture of the group C2 were similar to those in group C1,but the surface was less glossy than group C1,and with few nodules,edge tenacious.The appearance of the liver in the group C3 was between the group C2 and the group B.The liver expanded or shrunk,surface was not smooth or with had nodules,when touched it,it felt hard and slightly greasy.Liver in group D and group E was the most similar with group C1,sometimes also close to group C2.（5）The liver index and spleen index of each group’s mice:Compared with group A,the liver index and spleen index of group B’s mice were significantly increased（P<0.01）;Respectively compared with group B,group C1-C3,group D and group E,the liver index and spleen index of each group were with different degrees drop（P<0.05 or P<0.01）,above all the reduce of group C1 was the most obvious（P<0.01）.And the results of Curcuma kwangsiensis S.G.Lee et C.F.Liang treatment group were similar to the result of the positive drug control group（group D and E）.（6）Results of hepatic tissue（HE staining and Masson staining）of each group’s mice:In group A,the hepatocytes and hepatic sinusoids were arranged radially around the central vein.The structure of the hepatic lobule was clear,and the portal area was rounded,without largen and deformed.There also was no cell inflammatory change,necrosis and abnormal fibrosis;Group B,the liver cells and hepatic sinusoids were disordered,and the hepatic lobule was destroyed by abnormally proliferating fibrous tissue into be pseudolobules.The management area with varying degrees largen and deformed.The pathological changes in group C1 was closer to group A,but somtimes still appearing a small amount of hepatocyte necrosis and fibrosi;The structure,size and necrosis of hepatocytes in group C2 were similar to those in group C1.The proliferation of fibrous tissue was much more obvious,and some of the portal area became larger and deformed.The pathological morphology of liver in group C3 was between group C2 and group B,the changes of hepatocyte necrosis,degeneration and fibrosis were more obvious and increased.The pathological changes of liver in group D and group E were the most similar to those in group C1,sometimes closed to group C2.（7）Percentage of collagen deposition area and pathological scores of hepatic tissue of each group’s mice:Compared with group A,the percentage of collagen deposition area and pathological score of group B were significantly increased（P<0.01）.Respectively compared with group B,the percentage of collagen deposition area and pathological score of groups C（1-3）,D and E were significantly decreased（P<0.05 or P<0.01）.The degrease of percentage of collagen deposition area and pathological score of hepatic tissue in group C1 were the most significant（P<0.01）.And the results of the Curcuma kwangsiensis S.G.Lee et C.F.Liang treatment group was similar to the result of the positive drug control group（group D and E）.（8）Immunohistochemistry（IHC）results of hepatic tissues of mice in each group:The positive cells that TGF-β1,Smad 2 and Smad 3’s IHC sections under microscope appeared yellow or brownish yellow,and mainly concentrated in the portal area,the fibrous septum and Portal area.The positive expression of TGF-β1 and Smad3 occurred in the cytoplasm among others,while the positive expression of Smad 2 occurred in the nucleus.Compared with group A,the positive rate of TGF-beta 1,Smad 2 and Smad3 in hepatic tissue and the semi-quantitative analysis of protein expression in group B increased significantly（P<0.01）.Group C1-C3,group D and group E respectively compared with group B,the positive rate score and semi-quantitative analysis of protein expression of all target proteins were significantly decreased in varying degrees（P<0.05 or P<0.01）,and the decrease in group C1 was the most obvious（P<0.01）.And the results the of Curcuma kwangsiensis S.G.Lee et C.F.Liang treatment group was similar to the positive drug control group（group D and E）.（9）The results of western blot:Compared with group A,the proteins’s relative expression of TGF-beta 1,Smad 2 and Smad 3 in group B was significantly increased（P<0.01）.Group C1-C3,group D and group E respectively compared with group B,the relative expression of all target proteins decreased in varying degrees（P<0.05 or P<0.01）.Among them,group C1 with the most significant decrease（P<0.01）.And the results of the Curcuma kwangsiensis S.G.Lee et C.F.Liang treatment group was similar to the positive drug control group（group D and E）.（10）The results of RT-PCR:Compared with group A,the mRNA’s relative expressions of TGF-beta 1,Smad 2 and Smad 3 in hepatic tissue of group B were significantly increased（P<0.01）.Group C1-C3,group D and group E respectively compared with group B,the relative expression of each target gene decreased in varying degrees（P<0.05 or P<0.01）.Among them,group C1 with the most significant decrease（P<0.01）.And the results of the Curcuma kwangsiensis S.G.Lee et C.F.Liang treatment group was similar to the positive drug control group（group D and E）.Conclusions:（1）The Curcuma kwangsiensis S.G.Lee et C.F.Liang is complex and diverse,including curcumol,curcumin,gamma ketone,elemene andβ-elemene.Its anti-liver fibrosis is the result of a combination of various active ingredients,mutual restraint and interaction;（2）Curcuma kwangsiensis S.G.Lee et C.F.Liang can effectively alleviate the symptoms of TCM syndrome in hepatic fibrosis mice,increase their body weight and reduce liver’s swelling and reduce liver and spleen index,improve the degree of fibrosis in the liver to some extent;（3）Through the regulatory effects of TGF-β1/Smads signalling pathway,Curcuma kwangsiensis S.G.Lee et C.F.Liang can decrease the expression of TGF-beta 1,Smad 2 and Smad 3 proteins and genes（mRNA）in hepatic tissue of mice with hepatic fibrosis of blood stasis syndrome,thereby reducing the degree of hepatic fibrosis of mice with hepatic fibrosis of blood stasis syndrome.