| Objective:To study effects of Haobie Yangyin Ruanjian Decoction on hepatic fibrosis induced by carbon tetrachloride composite factor and their mechanism.Method:in vivo:Healthy Wistar rats were divided randomly into 7 groups.Negative control group(n=6);model group(n=14);small,middle and large dosage groups of Haobie Yangyinruanjian Decoction(n=12 respectively),positive control groups of Fufang Biejia Rungan tablets and colchicin(n=12 respectively).Except negative control group,solution of carbon tetrachloride combined with peanut oil(CCl4:peanut oil=4:6,V/V)were administered to each rat by subcutaneous injection,0.5ml/100g body weight for the first time,and then the same solution were administered to each rat by subcutaneous injection every 3 days,0.3ml/100g body weight,16 times in total.In the former 2 weeks,each rat was raised by feedstuff I(80%corn meal+20%grease+0.5%cholesterol).After 2 weeks they were raised by feedstuff II(corn meal+0.5%cholesterol).Except negative control group,30%alcohol solution were given to each rat by oral administration every other day from the beginning,1ml for each rat.in the rest of time,they drank the tap water.For the negative control group,the peanut oil were administered to each rat by subcutaneous injection.For the therapeutic groups,the rats were treated with Haobie Yangyin ruanjian Decoction suspension,0.82g/kg/d;2.5g/kg/d;8.2g/kg/d,lml/100g respectively.For the positive control groups of Fufang Biejia Rungan tablets and colchicin,the rats were treated with Fufang Biejia Rungan suspension,0.55g/kg/d,and colchicin solution,0.1 g/kg/d,lml/100g;For the negative control and model groups,the rats were given purified tap water,lml/100g.l.body weight,liver weight,liver weight/body weight ratio,spleen weight,and spleen weight/body weight ratio were detected by weighting method.MDA content,ALT and AST activity,total protein and albumin level were detected by chromatometry.2.CIV,PCIII,HA and LN level were detected by radio immunoassay,hydroxyproline content in the liver were detected by chromatometry.3.Matrix metalloproteinase MMP2 and MMP9 activity in the liver were detected by gelatin zymography.4.Pathological change of liver were observed by means of HE staining and Collagen staining,a-SMA expression in the liver was detected by immunohistochemical method.5.TGF-β1 expression were detected by Real time PCR,Smad 3 expression were detected by Western blotting.In vitro:1.HSC-LX02 cell strain were cultured.Sterile drug serum were collected from the rats with hepatic fibrosis induced by carbon tetrachloride composite factor after they are sacrificed and healthy rats.2.HSC cell proliferation were monitored by colorimetric MTT assay,hydroxyproline level in cell culture supernatant were measured by chromatometry.3.HSC cell cycle was observed by flow cytometry.4.Smad 3 expression in HSC cell was measured by Western blotting.Result:in vivo:1.Effects of Haobie Yangyin Ruanjian Decoction on physiology and biochemistry in the rats with hepatic fibrosis induced by carbon tetrachloride composite factor.Compared with those of negative control group(319.82±56.2g,14.95±10.4g,45.04±26.82),the body weight,liver weight and liver weight/body weight ratio were decreased in the model group(234.92±34.13 g;8.82±1.12g;37.87±4.37,P<0.01).Compared with those of model group,they were increased in small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction gradually(248.02±53.31g,9.14±2.89g,36.22±5.59;243.02±43.91g,9.25±1.76g,38.16±3.74;233.86±53.16g,8.98±1.91g,38.39±5.63),but less than the negative control group.They were also increased in Fufang Biejia Rungan tablets and colchicin groups(238.97±53.16g,9.46±2.16g,39.52±2.15;264.28±46.56g,9.13±1.44g,34.799±2.89).Compared with those of negative control group(0.64±0.10g,2.01±0.09),the spleen weight and spleen weight/body weight ratio were increased in the model group(1.15±0.34g;4.97±1.45,P<0.01).Compared with those of model group,they were decreased in small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction(1.18±0.39g,4.83±1.48;0.87±0.19g,’3.61±0.74,7<0.01;0.72±0.14g,3.07±0.37,7<0.01),but less than the control group.They were also decreased in Fufang Biejia Rungan tablets and colchicin groups(11.13±0.33g,4.97±2.20;0.99±0.23g,3.83±0.99).Compared with those of negative control group(7.0±1.0 nmol/ml),MDA content were increased(10.4±1.5nmol/ml,P<0.01)in the model group.Compared with those of model group,They were decreased in small,middle and large dosage groups of Haobie YangyinruanjianDecoctiongradually(9.1±1.7nmol/ml;8.6±1.6 nmol/ml,p<0.05;7.9±2.0 nmol/ml,P<0.01),but less than the negative control group.They were also decreased in Fufang Biejia Rungan tablets and colchicin groups(9.1±1.2 nmol/ml;9.1±1.7 nmol/ml).Compared with those of negative control group(23.8±8.5Carmen’s unit;30.0±11.4 Carmen’s unit),the ALT and AST activity in the model group were increased(62.0±23.7 Carmen’s unit,p<0.01;98.8±40.0 Carmen’s unit,P<0.01).Compared with those of model group,the ALT and AST activity in small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction were decreased gradually(48.8±18.8 Carmen’s unit,68.8±26.7 Carmen’s unit,p<0.05;45.3±19.5 Carmen’s unit,p<0.05,73.3±26.7 Carmen’s unit,P<0.05:36.3±9.8 Carmen’s unit,P<0.01,50.4±15.6 Carmen’s unit,P<0.01),but less than the control group.the ALT and AST activity in Fufang Biejia Rungan tablets and colchicin groups were also decreased(50.02±3.7 Carmen’s unit,57.2±30.0 Carmen’s unit,P<0.01;46.1±14.8 Carmen’s unit,66.0±33.2 Carmen’s unit,P<0.05).Compared with those of negative control group(76.9±13.1g/L,36.3±5.2g/L),the total protein and albumin level in the model group were decreased(45.8±14.8 g/L,p<0.05;24.3±5.3 g/L,P<0.01).Compared with those of model group,the total protein and albumin level in small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction were increased gradually(53.2±23.5 g/L,28.0±7.4 g/L;58.8±24.9g/L,30.6±7.5 g/L,P<0.05;67.1±20.2 g/L,p<0.05,33.0±5.6 g/L,p<0.01),but less than the control group.the total protein and albumin level in Fufang Biejia Rungan tablets and colchicin groups were also increased(60.4±20.2g/L,32.7±7.4g/L,P<0.01;42.7±26.9 g/L,31.9±8.3 g/L,P<0.05).2.Effects of Haobie Yangyin Ruanjian Decoction on extracellular matrix in the rats with hepatic fibrosis induced by carbon tetrachloride composite factor.Compared with those of negative control group(0.18±0.13mg/g dry liver),the hydroxyproline content in the model group were increased(2.39±0.28mg/g dry liver,P<0.01).Compared with those of model group,hydroxyproline content in small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction were decreased gradually(1.89±0.99 mg/g dry liver;1.29±0.56 mg/g dry liver,p<0.01;1.22±0.45mg/g dry liver,p<0.01),but less than the control group.the hydroxyproline content in Fufang Biejia Rungan tablets and colchicin groups were also decreased(1.77±0.72 mg/g dry liver,P<0.05;2.19±0.66 mg/g dry liver).Compared with those of negative control group(21.71±1.76ng/ml,15.16±15.12μg/ml,205.30±48.92ng/ml,82.0218.86ng/ml),the CIV,PCIII,HA and LN level in the model group were increased(29.20±6.17 ng/ml;35.73±17.90μg/ml,p<0.01;563.82±335.54 ng/ml,p<0.05;89.57±7.59 ng/ml).Compared with those of model group,They were decreased gradually in small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction(26.38±8.61 ng/ml,27.87±10.13μg/ml,464.19±283.41 ng/ml,79.86±9.52 ng/ml;24.41±4.46 ng/ml,24.72±10.87μg/ml,P<0.05,256.46±95.98 ng/ml,P<0.01,86.34±8.30 ng/ml;26.22±7.97 ng/ml,20.34±13.92μg/ml,P<0.01,161.51±48.29 ng/ml,P<0.01,83.26±13.20 ng/ml),but less than the control group.They were also decreased in Fufang Biejia Rungan tablets and colchicin groups(28.02±9.45 ng/ml,30.18±9.41 μg/ml,456.18±410.83 ng/m,85.46±7.51 ng/ml;29.22±7.9μg/ml,34.08±9.19 ng/ml,313.17±230.06 ng/ml,P<0.05,88.61±8.97 ng/ml).Compared with those of negative control group(0.46±0.46×108),the activity of MMP2 in the model group was increased(1.2±0.57×108,P<0.01).Compared with those of model group,the activity of MMP2 in small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction were decreased gradually((0.93±0.47×108;0.65±0.48×108,p<0.05;0.44±0.21×108,p<0.01).the activity of MMP2 in Fufang Biejia Rungan tablets and colchicin groups were also decreased(0.46±0.32x 10,p<0.01;0.86±0.58×l08).the activity of MMP9 were lower,and have no significant change in each group.3.Effects of Haobie Yangyin Ruanjian Decoction on hepatic pathomorphology in the rats with hepatic fibrosis induced by carbon tetrachloride composite factor.Compare with the negative control group,hepatic injury,collgen deposotion and a-SMA expression were increased in the model group.Compare with the model group,Haobie Yangyin Ruanjian Decoction can alliviate hepatic injury,inhibit collgen deposotion and down regulate a-SMA expression,Fufang Biejia Rungan tablets and colchicin can also produce the same effects,but compare with the model group,colchicin has no significant effects on the above indexes.4.Effects of Haobie Yangyin Ruanjian Decoction on TGF-β1/Smad3 signal transduction pathway in rats with hepatic fibrosis induced by carbon tetrachloride composite factor.Compared with those of negative control group,TGF-β1 expression was increased(3.29±2.08 folds,P<0.01)in the model group,TGF-β1 expression were also increased in the small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction(2.52±1.57 folds;2.14± 1.42 folds;1.68±0.51 folds,P<0.05),but less than the model group.TGF-β1 expression were also increased in Fufang Biejia Rungan tablets and colchicin groups(2.08±0.57folds,2.25±0.82folds),also less than the model group.Compared with those of negative control group(0.62±0.08),the Smad3 expression in the model group were increased(1.33±0.10,P<0.01).Compared with those of model group,the Smad3 expression in small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction were decreased gradually(1.20±0.07,p<0.01;1.16±0.05,P<0.01;0.79±0.06,P<0.01),but less than the model group.Smad 3 expression in Fufang Biejia Rungan tablets and colchicin groups were also decreased(0.95±0.12,P<0.01;1.15±0.06,P<0.01).In vitro:Serum pharmacology of Haobie Yangyin-ruanjian Decoction on HSC1.Effects of drug Serum of Haobie Yangyin Ruanjian Decoction from the rats with hepatic fibrosis induced by carbon tetrachloride composite factor on Hepatic Stellate Cells.Compared with the control group(100%),cell proliferation was increased in the model group(101.78%).Compared with the model group,cell proliferation was inhibited by small,middle and large dosage of Haobie Yangyin Ruanjian Decoction gradually(99.08%,90.29%,85.52%),cell proliferation can also be inhibited by Fufang Biejia Rungan tablets and colchicin groups(89.61%,94.52%).Compared with those of control group(2.77±0.20μg/ml),the hydroxyproline content in the model group were increased(7.91±0.15μg/ml,P<0.01).Compared with those of model group,they were decreased gradually in small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction(7.52±0.80μg/ml;7.02±0.44μg/ml,p<0.01;6.69±0.26μg/ml,P<0.01),but less than the control group.The hydroxyproline content in Fufang Biejia Rungan tablets and colchicin groups were decreased(6.84t0.47μg/ml,p<0.01;7.22±0.26μg/ml,P<0.01).Compared with those of control group(46.66%,41.67%),G0/G1 was decreased(34.59%)and S was increased significantly(50.93%)in the model group.Compared with those of model group,G0/G1 was increased slightly(35,89%,36.89%)and S was decreased slightly(50.39%,49.29%)in the small,middle dosage of Haobie Yangyin Ruanjian Decoction.Large dosage can cause HSC apoptosis(21%).G0/G1 was increased(35.03%)and S was decreased(48.14%)in the Fufang Biejia Rungan tablets group,colchicin groups can cause HSC apoptosis(3.91%).Compared with those of control group,smad 3 expression was increased in the model group.Compare with the model group,smad 3 expression were decreased gradually in the different dosage of Haobie Yangyin Ruanjian Decoction groups.2.Effects of drug Serum of Haobie Yangyin Ruanjian Decoction from healthy rats on Hepatic Stellate Cells.Compared with the control group(100%),cell proliferation was inhibited by small,middle and large dosage of Haobie Yangyin Ruanjian Decoction(89.46%,81.02%,71.68%),cell proliferation can also be inhibited by Fufang Biejia Rungan tablets and colchicin(69.14%,72.01%).Compared with those of control group(2.35t0,12μg/ml),hydroxyproline content in small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction were decreased gradually(2.14±0.08μg/ml,P<0.01;1.95±0.11μg/ml,P<0.01;1.77±0.11μg/ml,P<0.01).the hydroxyproline content in Fufang Biejia Rungan tablets and colchicin groups were also decreased(1.78±0.06μg/ml,P<0.01;1.91+0.14μg/ml,P<0.01).Compared with those of control group(46.7±0.0%,42.1 ±0.5%),G0/G1 was increased(45.6v2.8%,47.2± 1.7%,47.7±2.5%)and S was decreased(43.1 ± 10.4%,40.0±0.9%,39.4±4.2%)in the small,middle and large dosage groups of Haobie Yangyin Ruanjian Decoction.G0/G1 was increased(52.6±1.2%,45.2±3.6%)and S was decreased(34.99±7.9%,43.0±5.9%)in the Fufang Biejia Rungan tablets and colchicin groups.Conclusion:1.Haobie Yangyin Ruanjian Decoction can improve general state of rats.Body weight,liver weight and liver weight/body weight ratio can be increased.spleen weight and spleen weight/body weight ratio can be decreased.MDA content,ALT and AST activity can be decreased.total protein and albumin level can be increased,which can alleviate hepatic injury induced by carbon tetrachloride composite factor,and improve hepatic function2.Hydroxyproline content in the liver,CIV,PCⅢ,HA and LN le’vel in serum can be decreased,which can inhibit collagen deposition,and liver injury can be lessened.Matrix metalloproteinase MMP2 activity were decreased by Haobie Yangyin Ruanjian Decoction,which can keep basal membrane intact,inhibit HSC activation,hepatic fibrosis degree can be decreased.Haobie Yangyin Ruanjian Decoction has no significant effects on MMP9.3.Histomorphology show hepatic injury was relieved by Haobie Yangyin Ruanjian Decoction,and a-SMA expression was decreased,which means HSC activation was inhibited.4.TGF-βland Smad 3 expression can be decreased by Haobie Yangyin Ruanjian Decoction,TGF-β1/Smad3 signal transduction pathway activation can be repressed.hepatic fibrosis degree can be decreased.5.HSC cell proliferation and collogen production were inhibited by Haobie Yangyin Ruanjian Decoction.HSC cell cycle transformation can be blocked by Haobie Yangyin Ruanjian Decoction,cell division can be influenced.Haobie Yangyin Ruanjian Decoction can down regulate smad 3 expression in HSC. |
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