CN1827132A - Medicine for treating cholestasis and hepatic fibrosis and method for preparing the same - Google Patents
Medicine for treating cholestasis and hepatic fibrosis and method for preparing the same Download PDFInfo
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Abstract
The invention relates to a medicine for treating cholestasis and hepatic fibrosis and method for preparation, which is preparing from traditional Chinese medicinal herbs including (by weight ratio): astragalus root 300-900, licorice root 100-300, and Chinese date 100-300.
Description
Technical field
The present invention relates to a kind of medicine for the treatment of cholestasis, hepatic fibrosis; It specifically is the Chinese patent medicine of feedstock production with the Chinese herbal medicine; The invention still further relates to the preparation method of this medicine in addition.
Background technology
Cholestasis is bile dyspoiesis and flow stagnation or is subjected to press down that increasing with jaundice, pruritus and serum alkaline phosphatase clinically is feature; In the relevant human liver disease of many cholestasis, the bile duct epithelial cell hypertrophy with mode is arranged in various degree.As 1. primary biliary cirrhosiss (primary biliary cirrhosis, PBC) be to cause one of modal disease of the chronic silt gallbladder of adult, its, pathological characters was in mid-term: bile duct infringement in portal area causes biliary ductuli reactive hyperplasia around it, and can extend to liver parenchyma, outgrowth biliary ductuli crosses one another identical, the many hypoplasia of tube chamber, flat or columnar epithelium (the Desmet VJ of the rarely seen one deck that has, Roskams T, Van Eyken P.Ductular reaction in the liver.Pathol ResPract, 1995,191:513-24.).2. primary sclerosing cholangitis (Primary sclerosing Cholangitis) also is a kind of chronic cholestasis disease, its pathological changes can be involved the outer and/or stones in intrahepatic bile duct of liver, show as thickening and stenosis of bile duct of bile duct wall, liver organization pathology characteristics are the fibrosis of bile duct and the hypertrophy of inflammation and little bile duct.Chronic hepatitis, liver cirrhosis and patients with biliary hepatic tissue biopsy specimen, the microhepatia cell and the hepatocyte of find around the lobules of liver, fiber being arranged every, the common outgrowth biliary ductuli of liver cirrhosis trifle joint edge part, little bile duct sample epithelial cell and bile duct sample, these cell peripherals always follow basement membrane thickened, collagen fiber to increase, even fibrosis is (big vast sensible fully, Huang Xianghong, Li Yunsheng, Deng. the experiment biliary cirrhosis forms the pathological study of mechanism. Capital University of Medical Sciences's journal, 1996,17 (1): 36-39.).The medicine cholestasis mainly influences the epithelial cell of little bile duct.Hepatic fibrosis is the total pathological change of multiple chronic hepatic diseases, be development of chronic hepatitis liver cirrhosis must be through pathological process.
Ursodesoxycholic acid is for treatment cholestasis, especially primary biliary cirrhosis early stage active drug, and is limited to the effect of middle and late stage pathological changes, and the cholestasis of other chronic hepatitiss, liver cirrhosis be there is no clear and definite therapeutic effect.Chinese medicine preparation based on Herba Artemisiae Scopariae, Fructus Gardeniae, Radix Et Rhizoma Rhei etc. is traditional medicine, but certain limitation is all arranged.Still the anti-hepatic fibrosis medicine that does not have clear and definite curative effect in chemistry and the bio-pharmaceutical.
Summary of the invention
The objective of the invention is to be to propose a kind of medicine that can effectively improve liver cholestasis, anti-liver injury, reverse hepatic fibrosis.
Another object of the present invention provides the preparation method of this medicine.
Medicine of the present invention (to call " stilbene is sweet logical " in the following text) is made by following component: (consumption is a weight portion)
Radix Astragali 300-900 Radix Glycyrrhizae 100-300 Fructus Jujubae 100-300
The preferential weight of prescription (part) ratio range of preparation medicine of the present invention is:
Radix Astragali 400-800 Radix Glycyrrhizae 150-250 Fructus Jujubae 150-250
The optimum weight of medicine of the present invention (part) proportioning is:
The Radix Astragali 600 Radix Glycyrrhizaes 200 Fructus Jujubaes 200
The method that above-mentioned each component is made medicine of the present invention is:
The Radix Astragali, Radix Glycyrrhizae is in small, broken bits, add 10 times of amounts of water with Fructus Jujubae, soaked 2 hours, decocted 30 minutes, filter extracting juice; Medicinal residues add 8 times of amounts of water, decoct 1 hour, filter extracting juice, and concentrate drying becomes dried cream, mix and pulverize, tabletting, get final product the medical ointment of treatment cholestasis, hepatic fibrosis.
Above-mentioned unguentum also can be reprocessed into the solid block dosage form of similar cookies size, and need add an amount of binding agent this moment again, and compression moulding under negative pressure is dry and with the vacuum mode packing, can obtain bulk block dosage form.
Find in our the effect research of overall merit at the hepatic fibrosis of different method of treatment example side intervention rat bile alluvial property, carbon tetrachloride rat liver fibrosis and the N-nitrosodimethylamine rat liver fibrosis of liver cirrhosis basic pathogenesis, " stilbene sweet logical " all demonstrates the effect that improves cholestasis and alleviate liver tissue fibrosis to above-mentioned 3 kinds of animal models, to bile duct epithelial cell breed this-the chronic hepatopathy common pathological changes and has remarkable inhibitory action.
Description of drawings
Fig. 1 be hepatic tissue HE dyeing (on) and collagen staining (descending) photo;
Fig. 2 is a hepatic tissue LM immunohistochemistry photo.
Fig. 3 is a hepatic tissue FN immunohistochemistry photo.
Fig. 4 is a hepatic tissue Col I immunohistochemistry photo.
Fig. 5 is a hepatic tissue Col VI immunohistochemistry photo.
Fig. 6 is hepatic tissue α-SMA immunohistochemistry photo.
Fig. 7 is the Ultrastructural variation photo of liver tissues of rats under the transmission electron microscope.
Fig. 8 is a hepatic tissue PCNA immunohistochemistry photo.
Fig. 9 is the scarlet collagen staining photo of hepatic tissue sky wolf.
The specific embodiment
Below in conjunction with drawings and Examples the present invention is described in further detail.
Embodiment 1
The Radix Astragali 600 gram, Radix Glycyrrhizae 200 grams is in small, broken bits, add 10 kilograms in water with Fructus Jujubae 200 grams, soaked 2 hours, decocted the filtration extracting juice 30 minutes; Medicinal residues add 8 kilograms in water, decoct 1 hour, filter extracting juice, and concentrate drying becomes dried cream, mix and pulverize, and tabletting promptly makes " stilbene is sweet logical " dry extract 309 grams.
Embodiment 2
The Radix Astragali 300 grams, Radix Glycyrrhizae 300 grams, Fructus Jujubae 300 grams make " stilbene is sweet logical " dry extract 280 grams according to embodiment 1 described method.
Embodiment 3
The Radix Astragali 500 grams, Radix Glycyrrhizae 300 restrain, restrain with Fructus Jujubae 200, make " stilbene is sweet logical " dry extract 313 grams according to embodiment 1 described method.
Embodiment 4
The Radix Astragali 500 grams, Radix Glycyrrhizae 100 grams, Fructus Jujubae 300 grams make " stilbene is sweet logical " dry extract 274 grams according to embodiment 1 described method.
Embodiment 5
With the Radix Astragali 400 grams, Radix Glycyrrhizae 150 grams, Fructus Jujubae 250 grams, make " stilbene is sweet logical " dry extract 240 grams according to embodiment 1 described method.
Embodiment 6
The Radix Astragali 800 grams, Radix Glycyrrhizae 150 grams, Fructus Jujubae 250 grams make " stilbene is sweet logical " dry extract 371 grams according to embodiment 1 described method.
Embodiment 7
The Radix Astragali 600 grams, Radix Glycyrrhizae 300 grams, Fructus Jujubae 100 grams make " stilbene is sweet logical " dry extract 301 grams according to embodiment 1 described method.
Embodiment 8
The Radix Astragali 700 grams, Radix Glycyrrhizae 200 grams, Fructus Jujubae 300 grams make " stilbene is sweet logical " dry extract 352 grams according to embodiment 1 described method.
Example I (" stilbene is sweet logical " control bile rat liver fibrosis)
1 experiment medicine: each prepares " stilbene is sweet logical " dry extract embodiment 1-8.
2 laboratory animals: the SD rat, male, the cleaning level, body weight 200 ± 20g is available from Chinese Academy of Sciences's Shanghai Experimental Animal Center.
3 experimental techniques
3.1 model preparation: (200 ± 20g), along the incision of abdomen median line, the exposure common bile duct drives in the wrong direction and injects sclerosing agent to hepatic portal portion, and distance is held the ligation common bile duct, and cut off the centre, the pass abdomen to select cleaning grade male SD rat for use.Sham-operation is closed abdomen after only opening abdomen and free common bile duct.
3.2 medication and dosage: be divided into model control group and pharmaceutical intervention group after one week of modeling at random, the pharmaceutical intervention per os is irritated stomach (dry extract adding dissolved in distilled water), and model control group gives the equivalent normal saline.Irritate stomach dosage 1ml/100g (be equivalent to 65kg body weight adult per kilogram dosage 8 times).The intervention medication is killed Mus after 4 weeks and is drawn materials.
3.3 sample collecting and processing: after medication 4 week finishes, rat with 2% pentobarbital sodium with 2ml/kg body weight dosage intraperitoneal injection of anesthesia after, dorsal position is fixed, and opens the abdominal cavity, observes the cardinal principle situation, the color of liver spleen, matter, form, volume and ascites situation.Through the postcava blood sampling, win liver, spleen, after weighing, obtain the thick hepatic tissue of 0.3-0.4cm, the embedding of OTC liquid, liquid nitrogen is freezing rapidly, and-70 ℃ of preservations are used to prepare frozen section; Cut 2 of 1.0 * 0.8 * 0.3cm size hepatic tissues in the thick leaf in liver right side, 10% neutral formalin is fixed, automatic dehydration machine dehydration of alcohol, dimethylbenzene step by step is transparent, 4um is thick for embedding, section, be used for HE and collagen staining, the pathological change of tissues observed, the liver tissue fibrosis degree, and formulate the sxemiquantitative criterion of hepatic tissue collagen fiber hyperplasia degree and the image semi-quantitative analysis of collagen deposition.
3.4 liver function detects
Serum alanine aminotransferase (Alanine aminotransferase, ALT) activity, serum Aspartic Acid aminotransferase (Aspartateaminotransferase, AST), reitman-frankel method (Rriman-Frankel); Serum total bilirubin (Totalbilirubin, T-BIL), the J-P method; Serum albumin (Albumin, Alb) content bromocresol green method (BBG); Total serum protein (Total protein, TP) content biuret colorimetry; Adopt Shanghai Biological Products Inst., Ministry of Public Health's test kit description to measure; Serum alkaline phosphatase (alkaline phosphatase, ALP), (γ-glutamyltranspetidase, γ GT GGT) build up bio-engineering research institute test kit description according to Nanjing and measure gamma glutamyl transpeptidase; TOTAL BILE ACID TBA (TBA U.S. AEROSET company, Abbott full automatic biochemical apparatus).
3.5 hepatic tissue hydroxyproline (Hydroxyproline, Hyp) assay: JamallShi method (Jamall IS, Finelli VN, Que Hee SS.A simple method to determine nanogram levels of 4-hydroxyproline in biological tissues.AnalBiochem 1981; 112:70-75.).
3.6 hepatic tissue dyeing: conventional H E dyeing; The scarlet collagen fiber dyeing of it wolf
3.7 immunohistochemical staining (two-step method): PBS (pH7.4) does blank dyeing.
SABC image semi-quantitative analysis: adopt HPIAS-1000 high-definition color pathology picture and text analytical system, immunohistochemical staining is cut into slices every group and is got 3, select 5 different visuals field at random, under 20 * 10 amplifications, measure to calculate the area ratio of positive region and canonical measure window.
3.8 it is positive that PCNA positive staining cell counting: PCNA judgement is well-defined brown reaction with nucleus.Get 3 SABC sheets for every group, observe and define the representative PCNA stained positive visual field under the low power lens (* 100), the back is counted positive bile duct epithelial cell of PCNA and hepatocyte respectively in each visual field of high power lens (* 400), every tissue is observed 5 visuals field, gets its positive cell counting mean and carries out statistical analysis.
3.9 the detection of bile duct epithelial cell Procoll α 1 (IV), PDGF-B, CTGF, TGF-β 1mRNA
Frozen section is carried out HE dyeing back adopt LCM microdissection instrument cutting bile duct epithelial cell.Every group of hepatic tissue sheet of getting 3 rats, 1,000 of cutting bile duct epithelial cell, computer is counted automatically, adds the RLT solution of 5-7 μ l on the medicated cap of eppendorf, and bile duct epithelial cell is collected on the medicated cap, adds RLT100 μ l mixing, low-temperature centrifugation.
The real-time fluorescence quantitative PCR method that bile duct epithelial cell mRNA expresses detects: extract total RNA of bile duct epithelial cell, and reverse transcription is cDNA, it is quantitative to carry out real-time fluorescence PCR then.
4 experimental results
4.1 the variation of respectively organizing general situation such as survival of rats rate, ascites occurrence rate, body weight, liver and spleen weight, liver/body weight ratio, spleen/body weight ratio and portal vein diameter is shown in table 1.1, table 1.2.
Begin to observe to 5 weekends from modeling, the model control group mortality rate is 41%, and " stilbene is sweet logical " group is 56.3%; 5 weekend the Sham-operated control group rat there is no obvious ascites, the AS occurrence model control group is 90%, " stilbene is sweet logical " group is 29% (2 are a small amount of ascites), significantly is lower than model control group (P＜0.05); The portal vein diameter of model control group rat significantly increases (P＜0.01) than Sham-operated control group, and " stilbene is sweet logical " group reduces to some extent, but compares there was no significant difference (P＞0.05) with model control group.The results are shown in Table shown in 1.1.
Table 1.1 is respectively organized rats death rate, AS occurrence and portal vein diameter variation (x ± s)
| Group (n) | Survival number/sum (mortality rate %) | Ascites number/survival number (ascites rate %) | Portal vein diameter (mm) |
| Sham-operated control group (12) model control group (10) " stilbene is sweet logical " group (7) | 12/13(0) 10/17(41.2) 7/16(56.3) | 0/12(0) 9/10(90.0) 2/7(28.6) * | 2.15±0.54 * 3.64±0.81 3.40±0.68 |
Annotate: compare with model control group,
*, P＜0.05
Compare with Sham-operated control group, the model control group rat body weight significantly alleviates, the remarkable increase of liver, spleen weight and liver/body weight ratio and spleen/body weight ratio (P＜0.01).With model control group relatively, " stilbene is sweet logical " liver of group is heavy, spleen is heavy, liver/body weight ratio and spleen/body weight ratio all significantly reduce (P＜0.05).The results are shown in Table 1.2.
Table 1.2. organizes respectively that rat body weight, liver are heavy, spleen is heavy, liver/body weight variation of spleen/weight ratio when (x ± s)
| Group (n) | Body weight (g) | Liver heavy (g) | Spleen heavy (g) | Liver/body is than (%) | Spleen/body is than (‰) |
| Sham-operated control group (12) model control group (10) " stilbene is sweet logical " group (7) | 354.6±20.4 ** 302.5±31.1 298.6±43.6 | 10.0±1.1 ** 18.7±6.3 13.4±2.0 * | 0.91±0.20 ** 2.35±0.57 1.87±0.94 * | 2.82±2.15 ** 6.11±1.60 4.62±1.14 * | 2.57±0.55 ** 7.73±1.72 5.57±1.66 * |
Annotate: compare with model control group,
*, P＜0.05;
*, P＜0.01
4.2 respectively organize the variation of rats'liver function
Compare with Sham-operated control group, the serum T BiL content of model control rats (average is 22 times of Sham-operated control group rat), TBA content, ALP, GGT, AST, ALT activity all significantly increase (P＜0.01); Alb content significantly reduces (P＜0.01).Compare with the model control rats, the serum T BiL of " stilbene is sweet logical " intervention group rat, TBA content, ALP, GGT, AST, ALT activity all significantly reduce (P＜0.05), and Alb content, A/G ratio significantly raise; The results are shown in Table 1.3, shown in the table 1.4.
Table 1.3. respectively organizes rat blood serum TBil, TBA content and the active variation of ALP, GGT (x ± s)
| Group (n) | TBiL(mg/dl) | TBA(umol/l) | ALP(u/l) | GGT(u/l) |
| Sham-operated control group (12) model control group (10) " stilbene is sweet logical " group (7) | 0.26±0.14 ** 5.78±3.00 2.03±1.35 * | 26.40±10.79 ** 76.33±24.93 51.00±25.63 * | 326.58±94.48 ** 595.90±144.11 434.43±126.46 * | 3.01±1.82 ** 115.94±63.15 58.95±27.34 * |
Annotate: compare with model control group,
*, P＜0.01;
*, P＜0.05
Table 1.4 is respectively organized the variation (x ± s) of rat blood serum ALT, AST activity and Alb content, A/G ratio
| Group (n) | AST(u/l) | ALT(u/l) | Alb(g/l) | A/G |
| Sham-operated control group (12) model control group (10) " stilbene is sweet logical " group (7) | 128.24±14.08 ** 276.77±53.60 213.36±67.48 * | 38.55±4.26 ** 106.49±31.69 72.05±30.84 * | 26.68±2.61 ** 14.21±3.02 20.65±7.35 * | 0.88±0.11 ** 0.31±0.07 0.56±0.29 * |
Annotate: compare with model control group,
*, P＜0.01;
*, P＜0.05
4.3 liver histopathology changes
4.3.1 HE dyeing: Sham-operated control group rats'liver leaflet structure is clear.The a large amount of hypertrophy of little bile duct in the model control group rats'liver, outgrowth bile duct is the garland sample, and more extrtacellular matrix deposition is arranged around the bile duct epithelial cell; Inflammation and necrosis are all not obvious, and the hepatocyte ratio obviously reduces in the hepatic tissue, and hepatocellular morphosis is still normal.Compare with model control group, " stilbene is sweet logical " group biliary ductuli hypertrophy significantly reduces hepatocyte quantity more (Fig. 1)." stilbene is sweet logical " group bile duct proliferation degree significantly is lower than model control group (P＜0.05), the results are shown in Table shown in 1.5.
Table 1.5 is respectively organized liver tissues of rats bile duct proliferation degree and is changed
| Group (n) | - | + | ++ | The bile duct proliferation classification +++ | The P value |
| Model control group (10) " stilbene is sweet logical " group (7) | 0 3 | 1 1 | 1 2 | 8 1 | p＜0.01 |
The scarlet dyeing of it wolf: sham operated rats is only seen a small amount of collagen fiber in portal area and central vein wall.Intensive collagen fiber deposition is arranged around a large amount of hypertrophy bile duct epithelial cells of model control group rat; A spot of extrtacellular matrix deposition is arranged in the liver parenchyma.Have around the outgrowth bile duct intensive collagen fiber to form at interval, with close on outgrowth interval interconnection, hold, cut apart hepatic tissue and form pseudolobuli.Compare the significantly minimizing of collagen fiber deposition of " stilbene is sweet logical " group rat with model control group; Liver tissue fibrosis degree score evaluation result shows that " stilbene is sweet logical " group liver tissue fibrosis degree significantly is lower than model control group relatively (P＜0.05), the result as shown in Figure 1, shown in the table 1.6.
Table 1.6. respectively organizes rat bile degree of hepatic fibrosis scoring and changes (x ± s)
| Group | n | Integration |
| Sham-operated control group model control group " stilbene is sweet logical " group | 12 10 7 | 0# 9.10±2.69 6.71±3.45 * |
Annotate: compare #, P＜0.001 with model control group;
*, P＜0.05
4.4 the variation of liver tissues of rats Hyp content
Hepatic tissue Hyp content significantly increases (P＜0.01) the model control group rat, be 4 times of Sham-operated control group rat; " stilbene is sweet logical " group significantly is lower than model control group (P＜0.05), the results are shown in Table shown in 1.7.
Table 1.7. respectively organizes the variation (x ± s) of liver tissues of rats Hyp content
| Group | n | Hyp (ug/g wet liver) |
| Sham-operated control group model control group " stilbene is sweet logical " group | 11 10 7 | 130.7±25.5 ** 538.2±105.6 394.1±135.2 * |
Annotate: compare with model control group,
*, P＜0.01;
*, P＜0.05
4.5 immunohistochemistry is observed
4.5.1 liver tissues of rats LM change of Expression
LM is detected in around blood vessel and the large bile duct in the Sham-operated control group liver tissues of rats, and is not obvious in the sinus hepaticus.Model control group rat LM around outgrowth bile duct expresses significantly and increases.LM positive staining of " stilbene sweet logical " group significantly is lower than model control group (P＜0.05), the result as shown in Figure 2, shown in the table 1.8.
Table 1.8 is respectively organized the variation (x ± s) of liver tissues of rats LM SABC graphical analysis
| Group | n | LM (positive area ratio, %) |
| Sham-operated control group model control group " stilbene is sweet logical " group | 3 3 3 | 0.60±0.12# 7.63±1.36 5.83±1.20 * |
Annotate: compare with model control group,
*, P＜0.05; #, P＜0.001
4.5.2 liver tissues of rats FN change of Expression
FN is continuous expression in the Sham-operated control group liver tissues of rats in sinus hepaticus, and positive staining is also arranged around blood vessel.Model control group FN is expressed in around the outgrowth bile duct consumingly except that having in sinus hepaticus the positive staining.With model control group relatively, " stilbene is sweet logical " group positive staining area significantly is lower than model control group (P＜0.05), the result as shown in Figure 3, shown in the table 1.9.
Table 1-9 respectively organizes the variation (x ± s) of liver tissues of rats FN SABC graphical analysis
| Group | n | FN (positive area ratio, %) |
| Sham-operated control group model control group " stilbene is sweet logical " group | 3 3 3 | 5±0.82# 15.5±1.62 10.98±1.37 * |
Annotate: compare with model control group, #, p＜0.001,
*, P＜0.05;
4.5.3 liver tissues of rats type i collagen change of Expression
Sham-operated control group Col I has slight positive staining in blood vessel wall.The intraparenchymatous Col I of model control group liver tissues of rats positive staining strengthens, and does not see obvious positive staining around the bile duct epithelial cell of propagation." stilbene is sweet logical " group dyeing area significantly is lower than model control group (P＜0.05).The result is shown in Fig. 4, table 1.10.
Table 1-10. respectively organizes the variation (x ± s) of liver tissues of rats Col I SABC image analysis
| Group | n | Col I (positive area ratio, %) |
| Sham-operated control group model control group " stilbene is sweet logical " group | 3 3 3 | 0.23±0.04# 6.05±0.96 4.58±0.74 * |
Annotate: compare #, P＜0.001 with model control group;
*, P＜0.05
4.5.4 liver tissues of rats IV Collagen Type VI change of Expression
Sham-operated control group rat liver Col IV mainly is present in trunk wall and bile duct wall, and the sinus hepaticus wall is continuously weak positive expression.Compare with the Sham-operated control group rat, model control group liver tissues of rats sinus hepaticus wall Col IV expresses and does not see significant change, but strong expression is around the bile duct epithelial cell of propagation.Col IV dyeing area significantly is lower than model control group (P＜0.05) in " stilbene sweet logical " group liver tissues of rats, the result as shown in Figure 5, shown in the table 1.11.
Table 1.11 is respectively organized the variation (x ± s) of liver tissues of rats Col IV SABC image analysis
| Group | n | Col IV (positive area ratio, %) |
| Sham-operated control group model control group " stilbene is sweet logical " group | 3 3 3 | 0.42±0.07# 8.41±0.69 5.13±0.68 * |
Annotate: compare #, P＜0.001 with model control group;
*, P＜0.05
4.5.5 liver tissues of rats α-SMA change of Expression
Sham-operated control group rat liver α-SMA positive staining is detected in blood vessel wall, does not see positive kitchen range in the lobules of liver.Model control group rat α-SMA positive staining sees on the myofibroblast on every side of portal area, and these myofibroblasts are centered around around the outgrowth bile duct epithelial cell." stilbene sweet logical " group rat liver α-SMA stained positive kitchen range significantly is less than model control group (P＜0.05), the result as shown in Figure 6, table 1.12.
Table 1.12 is respectively organized the variation (x ± s) of liver tissues of rats α-SMA SABC image analysis
| Group | n | α-SMA (positive area ratio, %) |
| Sham-operated control group model control group " stilbene is sweet logical " group | 3 3 3 | 0.58±0.11# 8.71±1.21 6.3±0.70 * |
Annotate: compare #, P＜0.001 with model control group;
*, P＜0.05
4.6 the Ultrastructural variation of liver tissues of rats under the transmission electron microscope
Rare fibroblast around the no silt gallbladder in Sham-operated control group rat hepatocytes and the bile duct epithelial cell, bile duct epithelial cell, bile duct intracavity microvillus is more, and a little collagen fiber is arranged around the no gallbladder bolt, bile duct epithelial cell.Silt gallbladder in model control group rat hepatocytes and the outgrowth bile duct epithelial cell endochylema has more gallbladder bolt to form in the tube chamber, microvillus reduces, and has a large amount of collagen depositions and fibroblast to assemble around the bile duct epithelial cell.Fibroblastic gathering is less around " stilbene is sweet logical " group rat bile duct epithelial cell, the silt gallbladder is light in hepatocyte and the bile duct epithelial cell, the collagen fiber deposition is less (as shown in Figure 7, among the figure: a small amount of collagen deposition is arranged around the normal bile duct epithelial cell of A around the bile duct epithelial cell; B. a large amount of ECM deposit around the model control group bile duct; C. the ECM deposition is less around the sweet logical group of bile duct of stilbene).
4.7 liver tissues of rats PCNA change of Expression
Proliferative cell nuclear antigen (PCNA) is expressed in the newly-increased celliferous karyon, is newly-increased celliferous specific marker, with positive outgrowth BEC of counting of PCNA and hepatocyte.
4.7.1 the variation of the positive hepatocyte number of PCNA in the liver tissues of rats
Compare with the Sham-operated control group rat, the significantly minimizing (P＜0.001) of the positive hepatocyte counting of PCNA is 11.2% of Sham-operated control group in the model control group liver tissues of rats; Compare with the model control rats, the positive hepatocyte counting of " stilbene is sweet logical " group P of Rats CNA significantly increases (P＜0.05); As shown in Figure 8, shown in the table 1.13.
4.7.2 respectively organize the variation of the positive bile duct epithelial cell counting of liver tissues of rats PCNA
Compare with the Sham-operated control group rat, the positive bile duct epithelial cell digital display of model control group liver tissues of rats PCNA work increases (photo P＜0.001); The positive bile duct epithelial cell number of " stilbene is sweet logical " group P of Rats CNA significantly reduces (P＜0.05) than the model control group rat; The results are shown in Table 1.13.
The positive hepatocyte change in count analysis of table 1.13 PCNA (x ± s)
| Group | n | The positive hepatocyte number (times visual field, individual/400) of PCNA | The positive bile duct epithelial cell number (times visual field, individual/400) of PCNA |
| Sham-operated control group model control group " stilbene is sweet logical " group | 3 3 3 | 19.6±2.06 * 2.20±0.86 7.93±1.53 ** | 0 10.87±1.64 4.13±1.19 ** |
Annotate: compare with model control group,
*, P＜0.001;
*, P＜0.05
4.8 the variation of bile duct epithelial cell Procollagen α 1 (IV), PDGF-B, CTGF and TGF-β 1mRNA expression
4.8.1 the variation of bile duct epithelial cell Procollagen α 1 (IV) mRNA expression
Procollagen α 1 (IV) mRNA has trace expression at Sham-operated control group rat bile duct epithelial cell, and the expression of rat model bile duct epithelial cell significantly increases (P＜0.001), is 16 times of sham-operation control rats; The expression of " stilbene is sweet logical " group rat bile duct epithelial cell significantly is lower than model control rats (P＜0.01), reduces to 31% of model control rats.Each is organized the rat bile duct epithelial cell and expresses the results are shown in Table shown in 1.14 of Procollagen α 1 (IV) mRNA amount.
Table 1.14 is respectively organized the variation (x ± s) of rat bile duct epithelial cell Procollagen α 1 (IV) mRNA expression
| Group | n | Procollagen α1(IV)mRNA (copies×10 4/10 6GAPDH) |
| Sham-operated control group model control group " stilbene is sweet logical " group | 3 3 3 | 0.40±0.11 * 6.54±0.27 2.03±0.14 ** |
Annotate: compare with model control group,
*, P＜0.001;
*, P＜0.01
4.8.2 respectively organize the variation that the rat bile duct epithelial cell is expressed the CTGFmRNA amount
Sham-operated control group rat bile duct epithelial cell has micro-CTGF mRNA to express, and the amount that model control group rat bile duct epithelial cell is expressed CTGF mRNA significantly increases (P＜0.001), is 9 times of sham-operation control rats; Compare with the model control group rat, the amount that " stilbene is sweet logical " group rat bile duct epithelial cell is expressed CTGF mRNA significantly reduces (P＜0.01), is 26% of model control rats.Each is organized the rat bile duct epithelial cell and expresses the results are shown in Table shown in 1.15 of CTGF mRNA amount.
Table 1.15. respectively organizes the rat bile duct epithelial cell and expresses the variation of CTGF mRNA amount (x ± s)
| Group | n | CTGFmRNA (copies×10 4/10 6GAPDH) |
| Sham-operated control group model control group " stilbene is sweet logical " group | 3 3 3 | 0.27±0.12 * 2.44±0.11 0.63±0.07 ** |
Annotate: compare with model control group,
*, P＜0.001;
*, P＜0.01;
4.8.3 respectively organize the variation that the rat bile duct epithelial cell is expressed TGF-β 1mRNA amount
Sham-operated control group rat bile duct epithelial cell has a small amount of TGF-β 1mRNA to express, and the amount that model control group rat bile duct epithelial cell is expressed TGF-β 1mRNA significantly increases (P＜0.001), is 4.3 times of sham-operation control rats; With the model control rats relatively, the amount that " stilbene is sweet logical " group rat bile duct epithelial cell is expressed TGF-β 1mRNA significantly reduces (P＜0.01), each is organized the rat bile duct epithelial cell and expresses the results are shown in Table shown in 1.16 of TGF-β 1mRNA amount.
Table 1.16. respectively organizes the rat bile duct epithelial cell and expresses the variation of TGF-β 1mRNA amount (x ± s)
| Group | n | TGF-β1mRNA (copies×10 4/10 6GAPDH) |
| Sham-operated control group model control group " stilbene is sweet logical " group | 3 3 3 | 0.42±0.12 * 1.83±0.12 0.99±0.14 * |
Annotate: compare with model control group,
*, P＜0.001;
*, P＜0.01;
5 conclusions:
" stilbene is sweet logical " can suppress the pathological change of the bile duct proliferation and the hypertrophy bile duct epithelial cell of bile duct obstruction hepatic fibrosis rats effectively, suppresses the development of hepatic fibrosis.
Example II (" stilbene is sweet logical " suppresses the effect of carbon tetrachloride rat liver fibrosis progress)
1 experiment medicine: each prepares " stilbene is sweet logical " dry extract embodiment 1-8.
2 laboratory animals and reagent: the Wistar rat, male, 44, the cleaning level, body weight 170-230g is available from Chinese Academy of Sciences's Shanghai Experimental Animal Center.Shanghai Univ. of Traditional Chinese Medicine's Experimental Animal Center is raised.Carbon tetrachloride (CCl
4, lot number: 20021219), olive oil is all available from Shanghai chemical reagents corporation of Chinese Medicine group.
3 experimental techniques
3.1 Preparation of model: with olive oil dilution CCl
4Be 50% concentration, with 2ml.kg
-1The dosage abdominal part hypodermic 2 times weekly, is total to 12w.
3.2 the grouping of model and administration: behind the modeling 8w, get 6 model group rats at random and do to observe before the medication.And all the other rats are divided into 14 of model control group, 12 of " stilbene is sweet logical " groups by the method for completely random.In the 9th the 1st day week, " stilbene is sweet logical " group awards " stilbene is sweet logical " extractum solution per os and irritates stomach, and drug dose amounted to for 4 weeks with experiment one.Normal control group, model control group award commensurability normal saline per os and irritate stomach.Finish in modeling the 12nd week, with 2% pentobarbital sodium with 2ml/kg body weight dosage intraperitoneal injection of anesthesia, the postcava blood sampling, the observation general form is won liver, spleen and is weighed.
3.3 liver function, hepatic tissue Hyp assay and liver histological are observed:
The same example I of method.
4 experimental results
4.1 respectively organize the variation of rats'liver function
With the rats in normal control group comparison same period, the rats'liver function of modeling 8w, 12w is significantly unusual, shows as Serum ALT, AST, GGT, ALP activity and serum T bil content and significantly increases; Compare with 12w model control group rat, Serum ALT, AST, ALP activity of " stilbene is sweet logical " medication group decrease, but there was no significant difference; Serum GGT activity and Tbil content significantly reduce (P＜0.05), the results are shown in Table shown in 2.1.
Table 2.1 CCl
4Variation (the x ± s) of rats'liver function is respectively organized in hepatic fibrosis
| Group (n) | ALT(U) | AST(U) | GGT(U) | ALP(U) | Tbil(mg/dl) |
| Normal control group (8) CCl 4-8w model group (6) CCl 4-12w model group (14) " stilbene is sweet logical " group (10) | 30.65±12.96 * 237.44±41.60 230.36±24.73 202.77±48.66 | 156.69±211 * 292.60±25.6 305.65±24.5 281.49±29.0 | 3.30±0.79 * 6.60±3.04 * 14.49±6.65 10.74±2.84 ** | 15.97±5.25 * 20.26±030 * 40.46±9.0 36.57±8.10 | 0.20±0.04 * 0.28±0.03 ** 0.37±0.12 0.29±0.08 ** |
Annotate: compare with the 12w model control group,
*, p＜0.01;
*, p＜0.05
4.2 respectively organize the variation of liver tissues of rats Hyp content
Compare with the normal control rat, 12w model control rats hepatic tissue Hyp content significantly increases (P＜0.01), and " stilbene is sweet logical " therapeutic intervention group rat not only significantly is lower than 12w model control group (P＜0.05), and the trend of reduction is also arranged than the 8w model control group before the medication therapeutic intervention.The results are shown in Table shown in 2.2.
Table 2.2 is respectively organized the variation (x ± s) of liver tissues of rats Hyp content
| Group (n) | Hepatic tissue Hyp |
| Normal rat matched group (12) 8w-CCl 4Model control group (6) 12w-CCl 4Model control group (12) " stilbene is sweet logical " group (9) | 180.17±27.90 * 243.52±73.39 262.08±68.57 217.58±32.72 ** |
Annotate: compare with the 12w model control group,
*, p＜0.01;
*, p＜0.05
4.3 liver histological changes
Model group mainly shows the hepatocellular large tracts of land balloon sample change in central vein week district during 8w, fat becomes and spotty necrosis, and with massive inflammatory cells infiltrated; Have thinner collagen fiber to stretch in lobule from the portal area, there is tangible little bile duct proliferation the portal area.Model group further increases the weight of except that above-mentioned pathological manifestations during 12 weeks, and sedimentary collagen fiber further stretch in lobule, form incomplete pseudolobuli; The collagenous fibrosis deposition is light than the 12w model group in " stilbene is sweet logical " liver, rests on the level of 8 all model group basically, and the result as shown in Figure 9.
5 conclusions:
Under the situation of modeling stimulating factor continuous action, " stilbene is sweet logical " can improve cholestasis effectively, stop the progress of hepatic fibrosis.
EXAMPLE III (effect of " stilbene is sweet logical " treatment N-nitrosodimethylamine rat liver fibrosis)
1 experiment medicine: each prepares " stilbene is sweet logical " dry extract embodiment 1-8.
2 animals and reagent: the Wistar rat, male, 44, the cleaning level, body weight 160 ± 15g is available from Chinese Academy of Sciences's Shanghai Experimental Animal Center.N-nitrosodimethylamine (dimethylinittrosamine, DMN), Tokyo HuaCheng Industry Co., Ltd's product, lot number: MAL05.
3 experimental techniques
3.1 model preparation: with reference to Jenkins method (Jenkins SA, Grandison A, Baxter JN, et al.Adimethylnitrosamine-induced model of cirrhosis and portal hypertension in the rat.J Hepatol 1985; 1:489-99.), normal saline dilution DMN is 0.5% (1:199 normal saline) concentration, with 2ml.kg
-1Dosage is made lumbar injection to rat, continuous weekly 3d, every day 1 time, 4w altogether.
3.2 the grouping of model and administration: after modeling finishes, get 6 model group rats execution and draw materials, do to observe before the medication.And all the other rat completely randoms are divided into 16 of model control group, 12 of " stilbene sweet logical " treatment groups; " stilbene is sweet logical " treatment group is pressed 8 times of amounts that 65kg becomes kg body weight dosage such as clinical 1 consumption per day of body weight for humans, and the dosage of 10ml/kg rat body weight is irritated stomach, amounts to 2w, free of discontinuities.Normal control group and model control group rat are with the capable respective handling of volume normal saline.Behind the medication 2w, with 2ml/kg body weight dosage intraperitoneal injection of anesthesia, general form is observed in postcava blood sampling with 2% pentobarbital sodium, wins liver, spleen and weighs.
3.3 liver function, hepatic tissue Hyp assay and liver histological are observed: the same example I of method.
4 experimental results
4.1 DMN rats with liver cirrhosis body weight, liver spleen, ascites and death condition
With the normal rat comparison same period, modeling the 2nd all body weight begin to descend, and each organizes the same model group of body weight change.Each organizes body weight, liver body, spleen body index referring to shown in the table 3.1.
Table 3.1 is respectively organized rat body weight, liver/body than the variation of, spleen/body ratio (x ± s)
| Group (n) | Body weight (g) | Liver/body (%) | Spleen/body (%) |
| 4w normal rat matched group (6) DMN+4w model control group (8) 6w normal rat matched group (12) DMN-6w model control group (16) astragali seu Hedysari Decoction treatment group (12) | 269.5±19.4 209.9±23.2 * 296.8±20.3 212.0±28.9 * 240.8±23.3 | 4.07±0.007 2.80±0.002 * 3.02±0.007 2.85±0.006 2.92±0.003 | 0.307±0.001 0.716±0.001 * 0.25±0.0003 0.563±0.001 * 0.541±0.001 |
Annotate: with the normal rat matched group comparison same period,
*, p＜0.01
Behind the modeling 4w, 4 rats appearance ascites is in various degree arranged in 8 rat models; 17 rats of 6w model control group have 9 a large amount of ascites to occur.In 12 rats of astragali seu Hedysari Decoction group P＜0.05 4 of ascites (33.3%) appears.
4.2 respectively organize the variation of rats'liver function
With normal rat relatively, rat blood serum AST, ALT activity and Tbil content all significantly increase behind the DMN modeling 4w, albumin content significantly reduces (P＜0.01); When stopping 6w behind the modeling 2w improvement is arranged all than 4w, but still significantly unusual; In the 6w model control group relatively, " stilbene is sweet logical " treatment group serum AST, ALT activity descend to some extent, but there was no significant difference (P＞0.05); Serum T bil content then significantly reduces, albumin content significantly increases (P＜0.05).Detailed results sees Table shown in 3.2.
Variation (the x ± s) of rat blood serum liver function is respectively organized in table 3.2 DMN hepatic fibrosis
| Group (n) | AST(u/l) | ALT(u/l) | Tbil(mg/dl) | Alb(g/l) |
| Normal rat matched group (12) DMN+4w model control group (8) DMN-6w model control group (16) " stilbene is sweet logical " treatment group (12) | 64.52±9.42 * 143.9±28.0 115.6±26.7 97.6±6.5 | 32.86±8.70 * 86.14±13.39 * 61.89±19.98 59.96±9.79 | 0.25±0.05 * 0.53±0.20 0.46±0.21 0.33±0.08 ** | 27.4±1.5 * 21.8±2.0 23.2±3.7 26.5±1.5 ** |
Annotate: compare with the DMN-6w model control group,
*, P＜0.01;
*, P＜0.05
4.3 liver histopathology changes
Normal control group lobules of liver clear in structure.Modeling is as seen hemorrhage during 4 weeks, and see a large amount of swelling hepatocyte, show as turbid expanding, endochylema is loose, the obvious broadening in portal area, extensive outgrowth fibrous tissue is cut apart original lobules of liver and is held the tuberosity that becomes to differ in size, cell infiltration such as lymphocyte, mononuclear cell, sinus hepaticus parietal cell hypertrophy is obvious, a large amount of spindle shape cell attachment is arranged at Dou Bi, hole wall thickening, even sinus hepaticus distortion.During 6w, the fibrous septum attenuates during than 4w and fine and close, swelling of liver cell, and cell infiltration reduces, and sees in the sinus hepaticus that fibroblast adheres to." stilbene is sweet logical " treatment group all significantly alleviates (photo) to hepatocellular degeneration, fibroplasia.
Normal group is only seen a small amount of collagen fiber in portal area and central vein wall.The collagen hypertrophy is obvious during modeling 4w, and fibrous tissue diffusivity hypertrophy also stretches in the lobules of liver tissue, cuts apart to hold hepatic tissue, and great majority form thicker complete interval, the disorderly pseudolobuli that forms of normal hepatocytes leaflet structure.Rat proliferation of fibrous tissue degree alleviates during 6w, and the fibrous septum narrows down." stilbene is sweet logical " treatment group rat proliferation of fibrous tissue degree alleviates, and the fibrous septum narrows down, and is painted shallow, mostly is incomplete interval, and seriality ring-type collagen reduces or interrupted (photo) in the sinus hepaticus.Each is organized rat collagen fiber hyperplasia degree and sees Table shown in 3.3.
4 weeks of table 3.2 DMN modeling, 6 all rats and each medication group liver collagen deposition sxemiquantitative Riddit analyze
| Group (n) | The classification of collagen hyperplasia degree | The Riddit value | ||||
| 0 | I | II | III | IV | ||
| Normal control rat (12) DMN+4w model control group (8) DMN-6w model control group (16) " stilbene is sweet logical " treatment group (12) | 12 0 0 0 | 0 0 0 0 | 0 1 6 9 | 0 4 4 3 | 0 3 6 0 | 0.9118 * 0.23 0.3071 0.5184 * |
Annotate: compare with the DMN-6w model control group,
*, P＜0.05
4.4 the variation of hepatic tissue Hyp content
Compare with the normal control rat, the rat model hepatic tissue Hyp content of DMN modeling 4w significantly raises (p＜0.01), and the 6w rat model hepatic tissue Hyp content behind the termination modeling 2w still is significantly higher than normal group (p＜0.01)." stilbene is sweet logical " treatment group liver tissues of rats Hyp content significantly is lower than the model control group same period (p＜0.05).See Table shown in 3.3.
Liver tissues of rats Hyp changes of contents (x ± s) is respectively organized in table 3.3 DMN hepatic fibrosis
| Group (n) | Hepatic tissue Hyp |
| Normal rat matched group (12) DMN-4w model control group (8) DMN-6w model control group (16) " stilbene is sweet logical " treatment group (12) | 143.92±28.04 * 341.61±46.30 288.55±41.58 252.22±30.95 ** |
Annotate: compare with the DMN-6w model control group,
*, P＜0.01;
*, P＜0.05
5 conclusions:
" stilbene is sweet logical " can reverse the hepatic fibrosis that has formed effectively, promotes the removing of serum bilirubin, improves intrahepatic cholestasis.
Claims (6)
1, a kind of medicine for the treatment of cholestasis, hepatic fibrosis is characterized in that it is the medicament of being made by the following weight proportion raw material:
Radix Astragali 300-900;
Radix Glycyrrhizae 100-300;
Fructus Jujubae 100-300.
2, the medicine of treatment cholestasis according to claim 1, hepatic fibrosis, wherein the weight proportion of each raw material is:
Radix Astragali 400-800;
Radix Glycyrrhizae 150-250;
Fructus Jujubae 150-250.
3, the medicine of treatment cholestasis according to claim 1, hepatic fibrosis, wherein the weight proportion of each raw material is:
The Radix Astragali 600;
Radix Glycyrrhizae 200;
Fructus Jujubae 200.
4, according to the medicine of claim 1 or 2 or 3 described treatment cholestasis, hepatic fibrosis, it is characterized in that said medicament is a said dosage form on any pharmaceutics.
5, the medicine of treatment cholestasis according to claim 4, hepatic fibrosis is characterized in that said medicament is unguentum or piece agent.
6, the method for the medicine of any one described treatment cholestasis of preparation claim 1-3, hepatic fibrosis is characterized in that elder generation is in small, broken bits with the Radix Astragali, Radix Glycyrrhizae, adds 10 times of amounts of water with Fructus Jujubae, soaks 2 hours, decocts 30 minutes, filters extracting juice; Medicinal residues add 8 times of amounts of water, decoct 1 hour, filter extracting juice, and concentrate drying becomes dried cream, mix and pulverize, and tabletting makes unguentum.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102370686A (en) * | 2010-08-26 | 2012-03-14 | 上海中医药大学附属曙光医院 | Medicinal composition for treating chronic liver disease and application thereof |
| CN104873665A (en) * | 2015-05-28 | 2015-09-02 | 陈爱华 | Medicine for treating hepatic fibrosis |
| CN110141610A (en) * | 2019-05-28 | 2019-08-20 | 山西大学 | A kind of Chinese herbal ointment formula is preparing the application in hepatic |
| CN110585262A (en) * | 2019-09-24 | 2019-12-20 | 上海中医药大学 | Traditional Chinese medicine composition for treating and/or preventing liver and gall diseases and application thereof |
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2005
- 2005-03-02 CN CNB2005100241649A patent/CN100408061C/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102370686A (en) * | 2010-08-26 | 2012-03-14 | 上海中医药大学附属曙光医院 | Medicinal composition for treating chronic liver disease and application thereof |
| CN102370686B (en) * | 2010-08-26 | 2013-09-18 | 上海中医药大学附属曙光医院 | Medicinal composition for treating chronic liver disease and application thereof |
| CN104873665A (en) * | 2015-05-28 | 2015-09-02 | 陈爱华 | Medicine for treating hepatic fibrosis |
| CN110141610A (en) * | 2019-05-28 | 2019-08-20 | 山西大学 | A kind of Chinese herbal ointment formula is preparing the application in hepatic |
| CN110141610B (en) * | 2019-05-28 | 2021-07-27 | 山西大学 | Application of traditional Chinese medicine ointment formula in preparation of liver-protecting medicine |
| CN110585262A (en) * | 2019-09-24 | 2019-12-20 | 上海中医药大学 | Traditional Chinese medicine composition for treating and/or preventing liver and gall diseases and application thereof |
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