CN1994325B - Dog gallbladder powder and its application in medicine preparation - Google Patents
Dog gallbladder powder and its application in medicine preparation Download PDFInfo
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- CN1994325B CN1994325B CN2006100165052A CN200610016505A CN1994325B CN 1994325 B CN1994325 B CN 1994325B CN 2006100165052 A CN2006100165052 A CN 2006100165052A CN 200610016505 A CN200610016505 A CN 200610016505A CN 1994325 B CN1994325 B CN 1994325B
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Abstract
The invention relates to a dog biliary powder, wherein its production comprises that: providing fresh dog biliary and taking out bile; screening with 100 deals; drying at 60Deg. C; grinding and screening at 100deals; disinfecting. The invention also provides application to prepare liver fibrosis resistant drug, caused by dimethyl nitrosamine and caused by carbon tetrachloride.
Description
Technical field
The present invention relates to dog gallbladder powder and the new purposes in pharmacy thereof, the particularly application of dog gallbladder powder in the medicine of preparation control hepatic fibrosis.
Background technology
Fel Canitis is the bile of Canis animals Canis familiaris L., and to early on the books in the application Compendium of Material Medica of Fel Canitis, " congestion to the greatest extent down for all QI and blood pains and trauma person, hot wine clothes half.Control cutter arrow skin ulcer, remove pus in the intestinal, the Ding ear comes to a head, and it is black that dawn is changed Tang Buhuan, and vim and vigour are taken the photograph pain, and food is told in regurgitation, mass in the abdomen infantile malnutrition, red white dysentery ".As medical material application among the people for a long time, has liver heat removing and eyesight improving, the effect of hemostasia and detumescence.In modern age, Fel Canitis is incorporated into own forces in " Chinese medicine dictionary " as a kind of Chinese medicine material, but very few to research, the utilization of Fel Canitis.Consult document, do not see the research report of Fel Canitis aspect anti-hepatic fibrosis up to now both at home and abroad as yet, more do not find to utilize the application of Fel Canitis in the medicine of preparation control hepatic fibrosis.
Summary of the invention
The object of the present invention is to provide a kind of dog gallbladder powder and new purposes thereof, especially the application in the medicine of preparation control hepatic fibrosis.
In fact, a kind of dog gallbladder powder that the present invention relates to, step makes by the following method: fresh Fel Canitis capsule is provided and gets bile, filter through 100 mesh sieves, 60 ℃ dry down, after grinding after 100 mesh sieves, terminal disinfection.
Dog gallbladder powder of the present invention relates to the application in the medicine of preparation control hepatic fibrosis.
In order to understand essence of the present invention better, pharmacological evaluation and the result with dog gallbladder powder illustrates its new purposes in pharmaceutical field below.
One, (Dimethylnitrosamine DMN) brings out the preventive and therapeutic effect of rat liver fibrosis to dog gallbladder powder to N-nitrosodimethylamine
(1) dog gallbladder powder brings out the inhibitory action of rat liver fibrosis to N-nitrosodimethylamine
This experiment prepares the rat liver fibrosis model with DMN, is the medicine contrast with similar product-Fel Ursi, proves that further Fel Canitis suppresses the effect of hepatic fibrosis.
1 material and method
1. laboratory animal: S-D male white rat, body weight 160-180g.
2. reagent and medicine:
N-nitrosodimethylamine (DMN); Directly red (Direct Red 80); SABC monoclonal antibody ED1 (Serotec.co.uk) and α SMA (Dako, Denmak).
Dog gallbladder powder of the present invention; Fel Ursi powder (Jilin Province prolongs the bright moon mountain board that limit Xiong Chang provides).
3. animal model is made:
60 animals are divided into 6 groups at random, poisoning group (10): 1%DMN (normal saline dilution) 1ml/kg for three days on end/week, intraperitoneal injection totally 4 weeks, irritate stomach totally 4 weeks with normal saline 2cc/d simultaneously.1,2,3 groups of Fel Canitis (10 every group): the 1%DMN intraperitoneal injection, dosage, time are the same, use Fel Canitis low dose (400mg/kg), middle dosage (600mg/kg), heavy dose of (800mg/kg) simultaneously, irritate stomach totally 4 weeks every day.Fel Ursi group (10): the 1%DMN intraperitoneal injection, dosage, time are the same, irritate stomach totally 4 weeks with Fel Ursi 400mg/kg/d simultaneously.Matched group (10): injection normal saline 1ml/kg for three days on end/week, intraperitoneal injection totally 4 weeks, irritate stomach totally 4 weeks with normal saline 2cc/d simultaneously.Test the 4th weekend anesthetized rat, heart blood sampling centrifugal treating is got liver after putting to death animal immediately.
4. the mensuration of serum AST, ALT, total protein, T-CHOL: the mensuration of serum AST, ALT adopts the ReimanShi method, the mensuration of total serum protein adopts the Biuret method, the mensuration of serum total cholesterol adopts enzyme process, all Kit is available from Eiken Chemical (Tyoko, Japan) company, strictness is operated by the Kit description, (Ultraspec 4050 to utilize spectrophotometer, LKB, Switzerland) measure absorbance, reference standard curve calculation AST, ALT value, total protein, total cholesterol level.
5. pathologic finding:
The detection of rats'liver/body weight: measure the body weight of animal before putting to death, get liver after the blood sampling immediately and weigh, calculate liver/body weight percentage ratio.Get two in tissue in the leftlobe of liver same area after weighing, neutral formalin through 10% is fixed, routine paraffin wax embedding, slice row H-E, direct red colouring (0.1% direct red picric acid saturated solution), light microscopic is observed the pathological change and the proliferation of fibrous tissue degree of hepatic tissue down.Directly the section of red colouring utilizes CMIAS true color pathology image analysis system (BJ University of Aeronautics ﹠ Astronautics) to carry out the quantitative analysis of collagen fiber.Observation condition: 10 times on object lens, 4 visuals field are selected in every section at random, image acquisition, dividing processing, the parametric statistics analysis draws the ratio of a target gross area/statistics gross area.Immunohistochemical staining: slice thick 4～5 μ m, routine dewaxes to water, carries out immunohistochemical staining with the ABC method, and the ED1 working concentration is 1: 500, and α-SMA working concentration is 1: 50.
6. statistical procedures: data are got average row t check.
2 results
1. serum AST, ALT, total protein, T-CHOL testing result see the following form 1.
Table 1 is respectively organized the testing results (x ± s) of laboratory animal 4 week back serum biochemical indicators
* * and poisoning group be p＜0.001 relatively, and * and poisoning group be p＜0.05 relatively
2. liver/weight ratio testing result sees the following form 2.
Table 2 is respectively organized laboratory animal 4 week back liver/body weight percentage ratios (x ± s)
* *Compare p＜0.001 with the poisoning group
3. hepatic tissue proliferation of fibrous tissue image analysis the results are shown in following table 3.
Table 3 is respectively organized laboratory animal 4 week back hepatic tissue collagen fiber area density % (x ± s)
▲ ▲ compare P＜0.01 with the poisoning group,
*Compare P＜0.01 with the Fel Ursi group.
4. the pathological observation result is as follows:
Matched group: the lobules of liver structure is normal, does not have obvious degeneration, necrosis, sees a small amount of cell infiltration in the portal area in indivedual specimen.
Poisoning group: lobules of liver structure disturbance, swelling of liver cell, degeneration, the part of hepatocytes endochylema concentrates, nuclear hyperchromatism, as seen more regenerated hepatocyte, hepatocyte kitchen range shape or lamellar necrosis, a large amount of proliferations of fibrous tissue in the portal area, form thicker fibrous septum and go deep in the hepatic tissue, form 7/9 of diffusivity pseudolobuli.
The Fel Ursi group: the lobules of liver structure disturbance, hepatocellular degeneration, and visible more point-like, the necrosis of kitchen range shape, more proliferation of fibrous tissue in the portal area forms thinner fibrous septum and gos deep in the hepatic tissue, forms 4/9 of diffusivity pseudolobuli.
Each group of Fel Canitis: the lobules of liver structure is normal substantially, accidental spotty necrosis in slight hepatic cell degeneration, the indivedual specimen, and proliferation of fibrous tissue in the portal area forms very thin fibrous septum and gos deep in the hepatic tissue, only has individual animal to form pseudolobuli.Pathological change and proliferation of fibrous tissue degree no significant difference between 3 kinds of various dose groups of Fel Canitis.
The immunohistochemical staining result:
Matched group: the ED1+ cell is being dispersed in distribution around portal area, the central veins of hepatic lobules and in the liver parenchyma on a small quantity.α-SMA+ cell distributes on the various blood vessel wall of portal area, and the central veins of hepatic lobules wall has a small amount of positive expression, and no positive cell distributes between hepatocyte.Poisoning group: ED1+, α-SMA+ cell fills the air distribution in a large number between outgrowth fibrous septum, be dispersed in distribution between liver parenchyma.Fel Ursi group: ED1+, α-SMA+ cell distribution is the same, but quantity reduces than poisoning group.Each group of Fel Canitis: ED1+, α-SMA+ cell distribution is similar to the Fel Ursi group, but quantity lacks than the Fel Ursi group, and the quantity of two kinds of positive cells of three various dose groups of Fel Canitis is similar.
Conclusion
DMN is a kind of chemical substance with liver toxicity, genotoxicity and immunotoxicity, and DMN forms acetaldehyde after the microsome metabolism, thus the damage hepatocyte, and nucleic acid and protein are methylated, cause hepatic necrosis.DMN can make hepatocellular degeneration, necrosis, and 4 week backs form the pathology finding similar to human alcoholic cirrhosis, and the characteristics of still keeping the liver cirrhosis some months after the drug withdrawal are arranged.
The content detection result of the serum AST of this experiment, ALT value and total protein sees, Fel Canitis and Fel Ursi all have and fall enzyme preferably and increase the total serum protein effect, and the effect of Fel Canitis is better than Fel Ursi, but the Fel Ursi group has the effect that reduces serum cholesterol preferably.The Image Detection result of this experiment liver/weight ratio, liver fibrous tissue, relatively there were significant differences for Fel Canitis group and Fel Ursi group, proliferation of fibrous tissue degree no significant difference between 3 groups of Fel Canitis; Hepatic tissue pathology is observed and is shown that poisoning group hepatocyte arrangement disorder loses normal configuration, the degeneration of hepatocyte diffusivity, visible more kitchen range shape, lamellar necrosis.Fel Ursi group pathological changes is lighter than poisoning group, but still visible more hepatocellular degeneration, visible more point-like, the necrosis of kitchen range shape.The degeneration of Fel Canitis group slight hepatic cell, accidental spotty necrosis.The above results has confirmed that further Fel Canitis is to hepatocellular protective effect on pathologic angle.According to the pathological examination results of detection, liver/weight ratio, liver fibrous tissue image analysis result and the hepatic tissue of serum AST, ALT in this experiment, dog gallbladder powder has the effect that suppresses the hepatic fibrosis that DMN brings out preferably, and its effect is better than Fel Ursi.
(2) dog gallbladder powder is at the therapeutical effect that N-nitrosodimethylamine is brought out rat liver fibrosis
Treat bring out the model of hepatic fibrosis with 4 weeks of 1% N-nitrosodimethylamine lumbar injection after.60 rats are divided into Fel Canitis group (3 kinds of various dose), Fel Ursi group, poisoning group and matched group at random.Observe and respectively organize rats'liver/weight ratio, serum AST, ALT value, total protein, total cholesterol level.The direct red colouring of hepatic tissue specimen row is measured the fibriilar area of hepatic tissue hose lining through the pathology image analysis system.SABC adopts the ABC method, observes the quantity and the distribution of ED1, α in the hepatic tissue-SMA positive cell.
1 material and method
1. laboratory animal: S-D male white rat, body weight 160-180g.
2. reagent and medicine:
(1) N-nitrosodimethylamine (DMN); Directly red (Direct Red 80); SABC monoclonal antibody ED1 (Serotec.co.uk) and α SMA (Dako, Denmak).
(2) dog gallbladder powder of the present invention; Fel Ursi powder (Jilin Province prolongs the bright moon mountain board that limit Xiong Chang provides).
3. animal model is made:
60 animals are divided into 6 groups at random, poisoning group (10): 1%DMN (normal saline dilution) 1ml/kg for three days on end/week, intraperitoneal injection totally 4 weeks, begin in the 5th week to irritate stomach totally 3 weeks with normal saline 2cc/d.1,2,3 groups of Fel Canitis (10 every group): the 1%DMN intraperitoneal injection, dosage, time are the same, begin in the 5th week with Fel Canitis low dose (400mg/kg), middle dosage (600mg/kg), heavy dose of (800mg/kg), irritate stomach totally 3 weeks every day.Fel Ursi group (10): the 1%DMN intraperitoneal injection, dosage, time are the same, begin in the 5th week to irritate stomach totally 3 weeks with Fel Ursi 400mg/kg/d.Matched group (10): injection normal saline 1ml/kg for three days on end/week, intraperitoneal injection totally 4 weeks, begin in the 5th week to irritate stomach totally 3 weeks with normal saline 2cc/d.Test the 7th weekend anesthetized rat, heart blood sampling centrifugal treating is got liver after putting to death animal immediately.
4. the mensuration of serum AST, ALT, total protein, T-CHOL: the mensuration of serum AST, ALT adopts the ReimanShi method, the mensuration of total serum protein adopts the Biuret method, the mensuration of serum total cholesterol adopts enzyme process, all Kit is available from Eiken Chemical (Tyoko, Japan) company, strictness is operated by the Kit description, (Ultraspec 4050 to utilize spectrophotometer, LKB, Switzerland) measure absorbance, reference standard curve calculation AST, ALT value, the content of total protein, T-CHOL.
5. pathologic finding:
The detection of rats'liver/body weight: measure the body weight of animal before putting to death, get liver after the blood sampling immediately and weigh, calculate liver/body weight percentage ratio.Get two in tissue in the leftlobe of liver same area after weighing, neutral formalin through 10% is fixed, the routine paraffin wax embedding, slice row H-E, direct red colouring (0.1% direct red picric acid saturated solution), light microscopic is observed the pathological change and the proliferation of fibrous tissue degree of hepatic tissue down, liver fibrosis classification is divided into 4 grades: do not see that obvious fibrous septum former is "-", there is very thin fibrous septum to form, but do not form pseudolobuli "+" as yet, the hepatic tissue subregion forms pseudolobuli (below 50%) " ++ ", formation diffusivity pseudolobuli in the hepatic tissue " +++".
Directly the section of red colouring utilizes CMIAS true color pathology image analysis system (BJ University of Aeronautics ﹠ Astronautics) to carry out the quantitative analysis of collagen fiber.Observation condition: 10 times on object lens, 4 visuals field are selected in every section at random, image acquisition, dividing processing, the parametric statistics analysis draws the ratio of a target gross area/statistics gross area.Immunohistochemical staining: slice thick 4～5 μ m, routine dewaxes to water, carries out immunohistochemical staining with the ABC method, and the ED1 working concentration is 1: 500, and α-SMA working concentration is 1: 50.
6. statistical procedures: data are got average row t check.
2 results
1. serum AST, ALT, total protein, T-CHOL testing result see the following form 4.
Table 4 is respectively organized the testing result of 7 week of laboratory animal back serum biochemical indicator
2. the classification of hepatic fibrosis sees Table 5
Table 5 is respectively organized the classification of laboratory animal 7 all heptic fibrosis
3. liver/weight ratio testing result sees the following form 6.
Table 6 is respectively organized 7 week of laboratory animal back liver/body weight percentage ratio
4. hepatic tissue proliferation of fibrous tissue image analysis the results are shown in following table 7.
The effect of table 7DMN7 week Fel Canitis anti-hepatic fibrosis
5. the pathological observation result is as follows:
Matched group: the lobules of liver structure is normal, does not have obvious degeneration, necrosis, sees a small amount of cell infiltration in the portal area in indivedual specimen.
Poisoning group: lobules of liver structure disturbance, swelling of liver cell, degeneration, the part of hepatocytes endochylema concentrates, nuclear hyperchromatism, as seen more regenerated hepatocyte, hepatocyte kitchen range shape or lamellar necrosis, a large amount of proliferations of fibrous tissue in the portal area, form thicker fibrous septum and go deep in the hepatic tissue, form 6 of diffusivity pseudolobuli.
The Fel Ursi group: the lobules of liver structure disturbance, hepatocellular degeneration, and visible more point-like, the necrosis of kitchen range shape, more proliferation of fibrous tissue in the portal area forms thinner fibrous septum and gos deep in the hepatic tissue, forms 4/8 of diffusivity pseudolobuli.
Each group of Fel Canitis: the lobules of liver structure is normal substantially, accidental spotty necrosis in slight hepatic cell degeneration, the indivedual specimen, and proliferation of fibrous tissue in the portal area forms very thin fibrous septum and gos deep in the hepatic tissue, only has individual animal to form pseudolobuli.Along with the increase pathological change and the proliferation of fibrous tissue degree of Fel Canitis dosage alleviates.
The immunohistochemical staining result:
Matched group: the ED1+ cell is being dispersed in distribution around portal area, the central veins of hepatic lobules and in the liver parenchyma on a small quantity.α-SMA+ cell distributes on the various blood vessel wall of portal area, and the central veins of hepatic lobules wall has a small amount of positive expression, and no positive cell distributes between hepatocyte.Poisoning group: ED1+, α-SMA+ cell fills the air distribution in a large number between outgrowth fibrous septum, be dispersed in distribution between liver parenchyma.Fel Ursi group: ED1+, α-SMA+ cell distribution is the same, but quantity reduces than poisoning group.Each group of Fel Canitis: ED1+, α-SMA+ cell distribution is similar to the Fel Ursi group, but quantity lacks than the Fel Ursi group, along with the quantity minimizing of two kinds of positive cells of increase of Fel Canitis dosage.
Conclusion
The content detection result of the serum AST of this experiment, ALT value and total protein sees that Fel Canitis and Fel Ursi all have the enzyme of falling and increase the total serum protein effect, but the statistics there was no significant difference, and the Fel Ursi group has the effect that reduces serum cholesterol preferably.The Image Detection result of this experiment liver/weight ratio, liver fibrous tissue, relatively there were significant differences for Fel Canitis group and Fel Ursi group, along with the increase difference of Fel Canitis dosage is more remarkable; Hepatic tissue pathology is observed and is shown that poisoning group hepatocyte arrangement disorder loses normal configuration, the degeneration of hepatocyte diffusivity, visible more kitchen range shape, lamellar necrosis.Fel Ursi group pathological changes is lighter than poisoning group, but still visible more hepatocellular degeneration, visible more point-like, the necrosis of kitchen range shape.The degeneration of Fel Canitis group slight hepatic cell, accidental spotty necrosis.The above results has confirmed that further Fel Canitis is to hepatocellular protective effect on pathologic angle.Pathological examination results according to detection, liver/weight ratio, liver fibrous tissue image analysis result and the hepatic tissue of serum AST, ALT in this experiment, Fel Canitis has the effect for the treatment of the hepatic fibrosis that DMN brings out preferably, effect is better than Fel Ursi, along with its therapeutic effect of increase of dosage is better.
Two, Fel Canitis brings out the rat liver fibrosis preventive and therapeutic effect to carbon tetrachloride
This experiment carbon tetrachloride (CCl
4) preparation rat liver fibrosis model, be the medicine contrast with similar product Fel Ursi, further confirm the effect of Fel Canitis anti-hepatic fibrosis
1 material and method
1. the male too white mouse of laboratory animal: S-D (cleaning level), body weight 160-180g.
2. reagent and medicine:
CCl
4Directly red (Direct Red 80); SABC monoclonal antibody ED1, monoclonal antibody α-SMA; Serum ALT, AST, ALP, TP (total protein), TB (total bilirubin) detectable; SABC monoclonal antibody ED1, monoclonal antibody α-SMA.
Medicine: dog gallbladder powder of the present invention; Fel Ursi powder (Jilin Province prolongs the bright moon mountain board that limit Xiong Chang provides).
3. animal model is made:
Experiment 1: 46 animals are divided into 5 groups at random, and matched group (6): in 1ml/kg/2 time/week of injection normal saline, intraperitoneal injection is totally 12 weeks, and the stomach of usefulness normal saline 2cc/d filling simultaneously is totally 12 weeks.Poisoning group (10): 50%CCl
4In (olive oil dilution) 2 times/week of 1ml/kg, intraperitoneal injection is totally 12 weeks, and the stomach of usefulness normal saline 2cc/d filling simultaneously is totally 12 weeks.Fel Canitis group low dose (400mg/kg), middle dosage (600mg/kg), heavy dose of (800mg/kg), 10 every group; 50%CCl
41ml/kg2 time/all intraperitoneal injections, dosage, time are the same, simultaneously with irritating Fel Canitis low dose, heavy dose of every day stomach totally 12 weeks.Fel Ursi group (10): 50%CCl
42 times/all intraperitoneal injections of 1ml/kg, dosage, time are the same, irritate stomach totally 12 weeks with Fel Ursi 400mg/kg/d simultaneously.Test and use the etherization rat the 12nd weekend, heart blood sampling centrifugal treating is got liver behind the execution animal immediately.
Experiment 2: 46 animals are divided into 5 groups at random, and matched group (6): in 1ml/kg/2 time/week of injection normal saline, intraperitoneal injection is totally 8 weeks, begins in the 9th week with normal saline 2cc/d filling stomach totally 4 weeks.Poisoning group (10): 50%CCl
4In (olive oil dilution) 2 times/week of 1ml/kg, intraperitoneal injection is totally 8 weeks, begins in the 9th week to irritate stomach totally 4 weeks with normal saline 2cc/d.Fel Canitis group low dose (400mg/kg), middle dosage (600mg/kg), heavy dose of (800mg/kg), 10 every group; 50%CCl
42 times/all intraperitoneal injections of 1ml/kg, dosage, time are the same, begin in the 9th week with irritating Fel Canitis low dose, heavy dose of every day stomach totally 4 weeks.Fel Ursi group (10): 50%CCl
41ml/kg2 time/all intraperitoneal injections, dosage, time are the same, begin in the 9th week to irritate stomach totally 4 weeks with Fel Ursi 400mg/kg/d.Test and use the etherization rat the 12nd weekend, heart blood sampling centrifugal treating is got liver behind the execution animal immediately.
4. the detection of serum biochemistry index:
The mensuration of serum AST, ALT adopts the Reiman-RankelShi method, ALP adopts kind-King method (the K-K method of improvement), total serum protein adopts the Biuret method, serum total bilirubin adopts the Evelyn-Malloy method, and strictness is operated by the Kit description, and (Ultraspec 4050 to utilize spectrophotometer, LKB, Switzerland) measure absorbance, reference standard curve calculation AST, ALT, ALP value, the content of total protein, total bilirubin at 490nm.
5. pathological examination:
(1) detection of rats'liver/weight ratio: measure the body weight of animal before putting to death, get liver after the blood sampling immediately and weigh, calculate liver/body weight percentage ratio.
(2) liver histopathology is observed: two block organizations are got in the leftlobe of liver same area in the back of weighing, neutral formalin through 10% is fixed, conventional dehydration, paraffin embedding, section is done H-E dyeing and is observed the hepatic tissue pathology morphological change, directly red colouring (0.1% direct red picric acid saturated solution) is observed proliferation of fibrous tissue degree in the hepatic tissue.
(3) classification of hepatic fibrosis: 0 grade: no fibrosis; The I level: the portal area proliferation of fibrous tissue has very thin fibrous septum to enter in the hepatic tissue, bridge between the fiber of formation portal area-portal area.II level: the visible indivedual pseudolobulis of hepatic tissue on the basis of I level.The III level: the obvious hypertrophy of portal area fibrous tissue, the fibrous septum is thicker, and hepatic tissue subregion (50%) forms big pseudolobuli.The IV level: a large amount of hypertrophy of portal area fibrous tissue, the fibrous septum is thick, the pseudolobuli that form diffusivity in the hepatic tissue, differs in size.
(4) quantitative analysis of collagen fiber: directly the section of red colouring utilizes CMIAS true color pathology image analysis system (BJ University of Aeronautics ﹠ Astronautics) to carry out the quantitative analysis of collagen fiber.Observation condition: 4 times on object lens, 2 visuals field are selected in every section at random, image acquisition, dividing processing, the parametric statistics analysis draws the ratio of a target gross area/statistics gross area.
(5) immunohistochemical staining: slice thick 4～5 μ m, routine dewaxes to water, carries out immunohistochemical staining with the ABC method, and the ED1 working concentration is 1: 100, and α-SMA working concentration is 1: 50.
6. statistical procedures: data are got average row t check.
The result
1. the testing result of serum biochemistry index sees the following form.
Test 1 rat blood serum biochemical indicator testing result
| ALT(U/L) | AST(U/L) | ALP(U/L) | TP(g/dl) | TB(mg/dl) | |
| Matched group | 28.33±10.04 | 101.89±39.79 | 37.53±7.45 | 6.53±0.56 | 0.30±0.23 |
| The poisoning group | 154.06±37.82 | 350.68±81.18 | 138.68±51.03 | 6.49±0.51 | 2.08±1.78 |
| The Fel Ursi group | 108.33±33.84 | 113.29±23.58 | 120.39±49.90 | 7.44±0.45 | 0.80±0.58 |
| 1 group of Fel Canitis | 63.48±18.05 | 143.92±26.37 | 90.85±20.30 | 7.22±0.86 | 0.25±0.12 |
| 2 groups of Fel Canitis | 87.71±20.33 | 174.40±25.56 | 111.28±34.61 | 7.63±0.50 | 0.33±0.26 |
ALT: Fel Ursi group, 1 group of Fel Canitis and poisoning group be P＜0.01 relatively, and 2 groups of Fel Canitis and poisoning group be P＜0.05 relatively.AST: Fel Ursi group, each group of Fel Canitis compare P＜0.01 with the poisoning group.ALP: Fel Ursi group, each group of Fel Canitis and the relatively ALP value decline of poisoning group, but not statistically significant.TP: Fel Ursi group, 2 groups of Fel Canitis and poisoning group be P＜0.01 relatively, TB: Fel Ursi group, each group of Fel Canitis compare the TB value with the poisoning group and descend, but not statistically significant.
Test 2 rat blood serum biochemical indicator testing results
| ALT(U/L) | AST(U/L) | ALP(U/L) | TP(g/dl) | TB(mg/dl) | |
| Matched group | 22.60±18.24 | 170.53±72.29 | 39.02±7.70 | 8.21±1.19 | 0.27±0.19 |
| The poisoning group | 61.23±18.40 | 173.38±26.05 | 45.03±6.36 | 6.34±0.42 | 0.32±0.22 |
| The Fel Ursi group | 12.90±14.60 | 126.55±30.68 | 34.80±6.20 | 6.74±0.64 | 0.12±0.05 |
| 1 group of Fel Canitis | 26.23±15.28 | 89.06±57.15 | 35.92±6.81 | 7.01±0.40 | 0.22±0.10 |
| 2 groups of Fel Canitis | 36.55±23.25 | 125.85±35.82 | 48.10±6.71 | 7.44±0.74 | 0.30±0.19 |
ALT: Fel Ursi group and poisoning group be P＜0.05 relatively.AST: 2 groups of Fel Canitis and poisoning group be P＜0.05 relatively.ALP: Fel Ursi group, each group of Fel Canitis compare ALP value not statistically significant with the poisoning group.TP: each group of Fel Canitis compares P＜0.01, TB with the poisoning group: Fel Ursi group and poisoning group be P＜0.05 relatively.
2. pathological examination result
(1) rats'liver/weight ratio
Experiment 1: because CCl
4Cause hepatocyte diffusivity steatosis, injection CCl
4The rat liver volume increase, the weight ratio matched group obviously increases, the most of diffusivity liver cirrhosis that forms of poisoning group, a large amount of proliferations of fibrous tissue, liver volume is dwindled relatively, weight saving, and the Fel Ursi group is similar to the poisoning group, Fel Canitis group liver/weight ratio and poisoning group and Fel Ursi group relatively have significant difference, see the following form.
Test 1 rats'liver/weight ratio (X ± SD)
| Grouping | n | Dead | Body weight (g) | Liver/weight ratio |
| Matched group | 6 | 0 | 386.00±43.94 | 2.55983±0.24027 |
| The poisoning group | 6 | 4 | 329.33±23.97 | 3.89950±0.84987 |
| The Fel Ursi group | 6 | 4 | 289.33±38.85 | 3.80317±0.69710 |
| 1 group of Fel Canitis | 10 | 0 | 316.30±21.74 | 4.36270±0.41689 * |
| 2 groups of Fel Canitis | 8 | 2 | 314.00±33.38 | 4.34712±0.86259 * |
*: there was no significant difference between P＜0.001 Fel Canitis group
Experiment 2: because CCl
4At 8 weekends of lumbar injection to the, begin to stop to inject CCl the 9th week
4, steatosis disappears in poisoning at the 12nd weekend group hepatic tissue, and hepatic fibrosis alleviates, and liver weight is near recovering normal, and Fel Canitis group liver/weight ratio is a little more than poisoning group and Fel Ursi group, but there was no significant difference on the statistics.
Test 2 rats'liver/weight ratio X ± SD
| Grouping | n | Dead | Body weight (g) | Liver/weight ratio S ± X |
| Matched group | 6 | 0 | 413.67±37.74 | 2.57717±0.20747 |
| The poisoning group | 8 | 2 | 357.63±19.29 | 2.67711±0.65582 |
| The Fel Ursi group | 7 | 3 | 373.57±14.71 | 2.58114±0.17604 |
| 1 group of Fel Canitis | 6 | 4 | 380.17±15.90 | 2.97217±0.54168 |
| 2 groups of Fel Canitis | 6 | 4 | 358.67±33.95 | 2.93617±0.29108 |
(2). the pathology of hepatic tissue change
Experiment 1:
Matched group: the lobules of liver structure is normal, is the center with the central vein, and hepatic cords is radial arrangement, and hepatocyte does not have degeneration, necrosis, in the portal area a small amount of cell infiltration is arranged in indivedual specimen.
Poisoning group: lobules of liver structural deterioration, the obvious swelling of hepatocyte, the interior visible diffusivity of endochylema, the fat vacuole that differs in size, as seen point-like, the necrosis of kitchen range shape, a large amount of proliferations of fibrous tissue in the portal area, thick fibrous septum is goed deep in the hepatic tissue, most of pseudolobuli (5/6) that forms diffusivity, differs in size.
The Fel Ursi group: the lobules of liver structure disturbance, hepatic cell fattydegeneration is light than the poisoning group, and more proliferation of fibrous tissue in the portal area forms thicker fibrous septum and gos deep in the hepatic tissue, forms big pseudolobuli (4/6).
Each group of Fel Canitis: the lobules of liver structure is slightly disorderly, the slight hepatic cell steatosis, and proliferation of fibrous tissue in the portal area forms very thin fibrous septum and gos deep in the hepatic tissue, forms pseudolobuli individually.
Experiment 2:
Matched group: the lobules of liver structure is normal, is the center with the central vein, and hepatic cords is radial arrangement, and hepatocyte does not have degeneration, necrosis, in the portal area a small amount of cell infiltration is arranged in indivedual specimen.
The poisoning group: the slight degeneration of rarely seen mistake that disappears of lobules of liver structure disturbance, hepatic cell fattydegeneration, accidental spotty necrosis, proliferation of fibrous tissue in the portal area, the fibrous septum is very thin, it is light to dye, cell component is few, with the poisoning group of 1 group of experiment relatively, hepatic fibrosis begins to recover.
The Fel Ursi group: the lobules of liver structure disturbance, the slight hepatic cell degeneration, fibrous tissue is few than the poisoning group in the portal area, and the fibrous septum is very thin, and it is light to dye, and cell component is few.
Each group of Fel Canitis: the slight structure disturbance of lobules of liver, the slight hepatic cell degeneration, fibrous tissue is few than the Fel Ursi group in the portal area, and the fibrous septum is very thin, and it is light to dye, and cell component is few.
(3). the classification of hepatic fibrosis:
Experiment 1:
Poisoning group fibrous septum is thick, gos deep in the hepatic tissue, most of pseudolobuli that forms diffusivity, differs in size.The Fel Ursi group forms thicker fibrous septum and gos deep in the hepatic tissue, the big pseudolobuli of most of formation.Fel Canitis is respectively organized very thin fibrous septum and is goed deep in the hepatic tissue, forms the diffusivity pseudolobuli individually, sees the following form.
Test the classification of 1 hepatic fibrosis
Experiment 2:
The poisoning group alleviates with experiment 1 poisoning group comparison hepatic fibrosis, and the portal area fibrous tissue reduces, light dying, and the fibrous septum attenuates in the liver, gos deep in the hepatic tissue, and half forms big pseudolobuli.Each group of Fel Ursi group and Fel Canitis forms very thin fibrous septum and gos deep in the hepatic tissue, forms big pseudolobuli individually, sees the following form.
Test the classification of 2 hepatic fibrosis
(4). the collagen fiber surface density:
Experiment 1:
A large amount of proliferations of fibrous tissue in the poisoning group hepatic tissue, the collagen fiber surface density obviously increases, the proliferation of fibrous tissue of Fel Ursi group reduces, collagen fiber surface density and poisoning group relatively have significant difference, the fibroplasia of Fel Canitis group obviously reduces, with the Fel Ursi group significant difference is arranged relatively, there was no significant difference between the Fel Canitis group sees the following form.
Test 1 rat collagen fiber surface density X ± SD
| Grouping | n | Dead | The collagen fiber surface density |
| Matched group | 6 | 0 | 0.005184±0.002995 |
| The poisoning group | 6 | 4 | 0.05867±0.01127 ** |
| The Fel Ursi group | 6 | 4 | 0.03484±0.008761 ** |
| 1 group of Fel Canitis | 10 | 0 | 0.01680±0.005401 ** |
| 2 groups of Fel Canitis | 8 | 2 | 0.01401±0.003914 ** |
*: there was no significant difference between P＜0.001 Fel Canitis group
Experiment 2:
Still have more proliferation of fibrous tissue in the poisoning group hepatic tissue, but the poisoning group hepatic fibrosis than experiment 1 alleviates, Fel Ursi group and poisoning group relatively fibrous tissue reduce, the collagen fiber surface density has significant difference, Fel Canitis group and Fel Ursi group more also have significant difference, there was no significant difference between the Fel Canitis group sees the following form.
Test 2. rats'liver collagen fiber surface density X ± SD
| Grouping | n | Dead | The collagen fiber surface density |
| 2 groups of 1 group of Fel Canitis of matched group poisoning group Fel Ursi group Fel Canitis | 6 8 7 6 6 | 0 2 3 4 4 | 0.006531±0.003363 0.03579±0.006850 ** 0.02624±0.004338 ** 0.01729±0.003770 ** 0.01871±0.003177 ** |
*: there was no significant difference between P＜0.001 Fel Canitis group
(5). the immunohistochemical staining result:
Experiment 1:
ED1: matched group: the ED1+ cell is being dispersed in distribution around portal area, the central veins of hepatic lobules and in the liver parenchyma.
The poisoning group: the ED1+ cell distributes in outgrowth fibrous tissue in portal area and fibrous septum in a large number, showed increased in the liver parenchyma.
The Fel Ursi group: the ED1+ cell distributes in the outgrowth fibrous tissue in portal area, fibrous septum and liver parenchyma.But its quantity is few than the poisoning group.
Each group of Fel Canitis: the ED1+ cell distribution is the same, but quantity lacks than the Fel Ursi group, along with the quantity of the increase ED1+ cell of Fel Canitis dosage reduces.
α-SMA: matched group: α-SMA+ cell distributes on the various blood vessel wall of portal area, and the central veins of hepatic lobules wall has a small amount of positive expression, and no positive cell distributes between hepatocyte.Poisoning group: α-SMA+ cell distributes in the outgrowth fibrous tissue in portal area, fibrous septum in a large number, and distributes near hepatocyte.Fel Ursi group: α-SMA+ cell distribution is the same, but quantity reduces than poisoning group.Each group of Fel Canitis: α-SMA+ cell distribution is similar to the Fel Ursi group, but quantity reduces.Except matched group, between poisoning group, Fel Ursi group and Fel Canitis group rat in outgrowth fibrous tissue in portal area and the fibrous septum α-SMA positive expression be uneven.
Experiment 2:
ED1: matched group: the ED1+ cell is being dispersed in distribution around portal area, the central veins of hepatic lobules and in the liver parenchyma.The poisoning group: the ED1+ cell distributes in outgrowth fibrous tissue in portal area and fibrous septum in a large number, showed increased in the liver parenchyma.The Fel Ursi group: the ED1+ cell distributes in the outgrowth fibrous tissue in portal area, fibrous septum and liver parenchyma.But its quantity is few than the poisoning group.Each group of Fel Canitis: the ED1+ cell distribution is the same, but quantity is lacked than the Fel Ursi group.
α-SMA: matched group: α-SMA+ cell distributes on the various blood vessel wall of portal area, and the central veins of hepatic lobules wall has a small amount of positive expression, and no positive cell distributes between hepatocyte.
Poisoning group: α-SMA+ cell distribution is the same, and no positive cell is expressed in outgrowth fibrous tissue in portal area and the fibrous septum.
Each group of Fel Ursi group and Fel Canitis: α-SMA+ cell distribution is the same, and no positive cell is expressed in outgrowth fibrous tissue in portal area and the fibrous septum.
Conclusion
CCl
4Be classical Hepatoxic substance, form Free radical in vivo, produce lipid peroxidation, and influence intracellular calcium concentration, thus the damage hepatocyte, CCl
4But the coup injury liver causes hepatic cell fattydegeneration, central vein hepatic necrosis on every side, and long-term chronic poisoning can cause the liver proliferation of fibrous tissue, forms hepatic fibrosis and liver cirrhosis.Single-throw CCl in zoopery
46 weeks can form hepatic fibrosis, and 12-15 week, liver cirrhosis can take place, at present CCl
4The hepatic fibrosis of bringing out, liver cirrhosis animal model still are widely used, but in a single day this model halts attacks and can recover.
The content detection result of the serum AST of this experiment, ALT value and total protein sees, Fel Canitis and Fel Ursi all have and fall enzyme preferably and increase the total serum protein effect, and the effect of Fel Canitis is better than Fel Ursi, but Fel Ursi has the effect that reduces serum cholesterol preferably.The Image Detection result of liver/weight ratio, liver fibrous tissue, relatively there were significant differences for the Fel Canitis group of experiment 1 and Fel Ursi group, no significant difference between the Fel Canitis group; Hepatic tissue pathology is observed and is shown, the normal configuration of poisoning group hepatic tissue is destroyed, hepatocyte diffusivity steatosis, and fat vacuole is not of uniform size, the kitchen range shape that as seen is dispersed in, lamellar necrosis.Fel Ursi group pathological changes is lighter than poisoning group, but still visible more hepatic cell fattydegeneration, visible point-like, the necrosis of kitchen range shape.Fel Canitis group slight hepatic cell steatosis, accidental spotty necrosis.The classification results of hepatic fibrosis is seen a large amount of proliferations of fibrous tissue in poisoning group portal area, and form thick fibrous septum, most diffusivity liver cirrhosis (5/6) that form, hepatic fibrosis of Fel Ursi group and poisoning group relatively alleviate, and the Fel Canitis group only has indivedual formation diffusivity liver cirrhosis.The experiment 2 fat-free degeneration in the 4 week back hepatic tissues that halts attacks, liver weight is recovered near normal, and Fel Canitis group liver/weight ratio is organized a little more than poisoning, but there was no significant difference on the statistics.Except matched group, the hepatic fibrosis of poisoning group alleviates, but still the recovery fully as yet of visible fibrous septum, the Image Detection poisoning group as a result of fibrous tissue still has more fibrous tissue, Fel Ursi group and poisoning group comparison group of fibers are woven with significant difference, and Fel Canitis group and Fel Ursi group more also have significant difference.Hepatic tissue pathology is observed and is shown, poisoning group hepatic tissue structure disturbance, the slight hepatic cell degeneration, collagen fiber are light to be dyed, and absorbs a part naturally, and the fibrous septum attenuates, fibrosis alleviates, but still have half to form big pseudolobuli, and Fel Ursi group and the hepatic fibrosis of Fel Canitis group are lighter than poisoning group, and the big pseudolobuli of indivedual formation is only arranged.The above results has further confirmed the effect of Fel Canitis anti-hepatic fibrosis from pathologic angle.According to pathological change, the classification of hepatic fibrosis, the liver fibrous tissue quantitative analysis results of serum AST, ALT detection, liver/weight ratio, hepatic tissue in this experiment, Fel Canitis has good preventing and treatment CCl
4Bring out the effect of rat liver fibrosis, its effect is better than Fel Ursi.
The specific embodiment
Embodiment 1
Dog gallbladder powder step by the following method makes: fresh Fel Canitis capsule is provided and gets bile, filter through 100 mesh sieves, and dry under 60 ℃, after grinding,, used disinfection by ultraviolet light at last 30 minutes after 100 mesh sieves.Under aseptic condition, dog gallbladder powder is incapsulated, every capsules 0.25g, packing: 10/plate, a box 4 plates.Usage and consumption: 1-2 grain, 2 times on the one, oral.
Embodiment 2
The preparation method of dog gallbladder powder is with embodiment 1, and weight such as the dog gallbladder powder of preparation and Cordyceps mycelium, Radix Salviae Miltiorrhizae are prepared capsule, every capsules 0.25g, and packing: 10/plate, a box 4 plates.Usage and consumption: 1-2 grain, 2 times on the one, oral.In addition, dog gallbladder powder also can have the Chinese crude drug equivalent preparation compound preparation capsule of effect of anti hepatic fibrosis, the effect of raising control hepatic fibrosis with other.
Claims (1)
1. the application of dog gallbladder powder in the medicine of preparation control hepatic fibrosis.
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