CN102614363B - Chinese medicine combination for preventing and curing lung injury and pulmonary fibrosis and preparation method thereof - Google Patents
Chinese medicine combination for preventing and curing lung injury and pulmonary fibrosis and preparation method thereof Download PDFInfo
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Abstract
The invention discloses Chinese medicine combination for preventing and curing lung injury and pulmonary fibrosis and a preparation method thereof. The Chinese medicine combination comprises the following components in percentage by weight: 28.6 percent of balloonflower root, 28.6 percent of Zhejiang fritillary bulb, 21.4 percent of flos lonicerae, and 21.4 percent of moutan bark; the method for preparing oral liquid comprises the following steps: weighing all the components in percentage by weight, mixing and smashing the components through a 100-mesh sieve, adding mixed powder into a container, adding 500 ml of water into the container, boiling the water and then keeping boiling for 45 minutes, precipitating, filtering, fetching filtrate to be conducted to vacuum concentration to 100 ml, filling concentrated solution into bottles, ensuring that each bottle is 100 milliliters, and then performing sterilizing sealing; the method for preparing capsules comprises the following steps: weighing all the components in percentage by weight, mixing and smashing the components through a 100-mesh sieve, boiling 3/4 of the medicine for 45 mutinies, precipitating, filtering, fetching filtrate to be conducted to vacuum concentration to 100 ml, smashing 1/4 of the medicine again through a 200-mesh sieve to be uniformly mixed with the liquid medicine, performing vacuum drying or low-temperature drying, sterilizing, encapsulating, and ensuring that each capsule is 0.25 g. The Chinese medicine combination ensures that lung is prevented from chemotherapeutics injury and radiation injury, is resistant to pulmonary fibrosis, and does not have toxic side effect and untoward effect.
Description
Technical field
The present invention relates to Chinese medicine, be specifically related to a kind of Chinese medicine composition of preventing and treating injury of lung and pulmonary fibrosis and preparation method thereof.
Background technology
As everyone knows, the injury of lung that chemotherapeutics and radiotherapy cause often shows as alveolitis in early days, a large amount of inflammatory cell infiltrations are arranged in alveolar, subsequently in the presence of inflammatory cell, various cytokines and inflammatory mediator discharge in a large number, enlarge tissue injury, caused the interstitial lung hypertrophy, caused pulmonary fibrosis.Because the Fibrotic mechanism of lung is still not exclusively clear and definite, once it is often irreversible to develop into pulmonary fibrosis, serious danger side of body patient life.Therefore the injury of lung that how rationally effectively prevention and treatment chemotherapeutics and radiotherapy cause in time is very important clinically problem.Treatment for injury of lung and pulmonary fibrosis at present there is no definite effectively Therapeutic Method and medicine.
Summary of the invention
The object of the invention is to: a kind of Chinese medicine composition and preparation method of preventing and treating injury of lung and pulmonary fibrosis is provided, draws materials extensively, with low cost, effect is remarkable, has no side effect.
Technical solution of the present invention is: the Chinese medicine composition of this control injury of lung and pulmonary fibrosis is comprised of following weight percentages of components: Radix Platycodonis, Bulbus Fritillariae Thunbergii each 28.6%, Flos Lonicerae, Cortex Moutan each 21.4%.
The method that the Chinese medicine composition of this control injury of lung and pulmonary fibrosis is made oral liquid is: by above-mentioned mass percent weighing, get each component, co-grinding 100% is crossed 100 mesh sieves, the water that adds 1000ml in the mixed powder placing container, boil rear continuation 45 minutes, precipitation, filter, get filtrate, vacuum concentration is 100ml, the sub-bottle fill, 100 milliliters every bottle, the sterilizing sealing.
The method that the Chinese medicine composition of this control injury of lung and pulmonary fibrosis is made capsule is: get each component by above-mentioned mass percent weighing, co-grinding 100% is crossed 100 mesh sieves, 3/4 drug decocting 45 minutes, and precipitation, filter, and gets filtrate, and vacuum concentration is 100ml; 1/4 again pulverized 200 mesh sieves, with above-mentioned medicinal liquid, mix homogeneously, vacuum drying or oven drying at low temperature, sterilizing, pulverize, and incapsulates every capsules 0.25g.
Instructions of taking is: oral 20ml, every day 3 times; Or 4 of capsules, every day 3 times.
Chinese medicine composition protection lungs of the present invention are avoided chemotherapeutics and radiation injury, and pulmonary fibrosis resistant, have no side effect, and has no adverse reaction.
The accompanying drawing explanation
Fig. 1 is experiment 1 sham operated rats (HE * 400) photo.
Fig. 2 is experiment 1 model group (HE * 400) photo.
Fig. 3 is experiment 1 Dexamethasone group (HE * 400) photo.
Fig. 4 tests 1 Bulbus Fritillariae Uninbracteatae mixture group (HE * 400) photo.
Fig. 5 tests 1 for sham operated rats (Masson * 400) photo.
Fig. 6 tests 1 model group (Masson * 400) photo.
Fig. 7 tests 1 for Dexamethasone group (Masson * 400) photo.
Fig. 8 tests 1 Bulbus Fritillariae Uninbracteatae mixture group (Masson * 400) photo.
The specific embodiment
Embodiment 1: according to following methods oral liquid processed: get Radix Platycodonis, each 28.6g of Bulbus Fritillariae Thunbergii, Flos Lonicerae, each 21.4g of Cortex Moutan, co-grinding 100% is crossed 100 mesh sieves, adds the water of 1000ml in the mixed powder placing container, boiling rear slow fire continues 45 minutes, precipitation, filter, and gets filtrate, vacuum concentration is 100ml, the sub-bottle fill, 100 milliliters every bottle, the sterilizing sealing.
Embodiment 2: according to following methods glue capsule: get Radix Platycodonis, each 28.6g of Bulbus Fritillariae Thunbergii, and Flos Lonicerae, each 21.4g of Cortex Moutan, co-grinding 100% is crossed 100 mesh sieves, 3/4 heavy drug decocting 45 minutes, precipitation, filter, and gets filtrate, and vacuum concentration is 100ml; 1/4 heavy 200 mesh sieves of again pulverizing, mix homogeneously with above-mentioned medicinal liquid, vacuum drying or oven drying at low temperature, and sterilizing, pulverize, and incapsulates every capsules 0.25g.
Experiment 1: on the impact of alveolitis and pulmonary fibrosis
1. experiment material
1.1 laboratory animal is selected clean level SD rat, body weight 200 ± 20g, and male and female half and half, purchased from Shanghai Slac Experimental Animal Co., Ltd., the laboratory animal quality certification 0037253.
1.2 experimental drug
The oral liquid that the Bulbus Fritillariae Uninbracteatae mixture is embodiment 1, simmer down to is containing crude drug 0.315g/ml, and by Nanjing University of Traditional Chinese Medicine's tcm treatment method laboratory preparation, the prepared slices of Chinese crude drugs are provided by Nanjing Pharmaceutical limited company.
Dexamethasone tablet: the 0.75mg/ sheet, Tianjin Tian Yao pharmaceutcal corporation, Ltd produces, and is mixed with the solution of 0.0000405g/ml with distilled water.
Bleomycin A5: 8mg/ props up, and Tianjin Tai He pharmaceutical Co. Ltd produces.
1.3 experimental apparatus
Paraffin slicing machine: German Leica company produces.
Optical microscope: Japanese Olympus company produces.
Full-automatic image-taking system OlympusDP71: Japanese Olympus company produces.
experimental technique
2.1 animal grouping
After rat conventional adaptation raising 3d, be divided at random 4 groups: sham operated rats, model group, Dexamethasone group, Bulbus Fritillariae Uninbracteatae mixture group.
2.2 model preparation
Prepare model by literature method, Bleomycin A5 is mixed with to 5mg/ml concentration with normal saline, after 10% chloral hydrate 0.3ml/100g anesthetized rat, the skin of neck sterilization, cut off cervical region Mus hair, row neck median incision, separate and expose trachea, inject Bleomycin A5 5mg/kg through the centripetal end puncture of tracheal cartilages czermak space, immediately animal is upright and rotation, medicinal liquid is distributed fully, evenly in lung, then skin suture; Rats in sham-operated group injects the equivalent normal saline in trachea under same anesthesia; Each is organized rat and freely drinks water under the same conditions, ingests, and keeps natural lighting, observes and record the ordinary circumstances such as activity, feed, body weight of rat.
2.3 medication:
Modeling starts gastric infusion every day in postoperative the 2nd day, and sham operated rats and model group give 5 ‰ sodium carboxymethyl cellulose (CMC sodium) solution 1ml/100g body weight; Dexamethasone group gives isometric(al) dexamethasone distilled water solution 0.000405g/Kgd; Bulbus Fritillariae Uninbracteatae mixture group gives isometric(al) Bulbus Fritillariae Uninbracteatae mixture 3.15g/Kgd; Every day, gavage was 1 time, successive administration 28 days.
2.4 collection of specimens
Raise 28 days after modeling and administration, with 10% chloral hydrate 0.3ml/100g anesthetized rat, take body weight, carotid artery is got blood and is put to death rat, opens thoracic cavity and takes out lungs, extracts the tissues such as trachea, hilar lymph node, blot lungs surface blood with filter paper, take and record the lung weight; Left lung rinses 3 times with brine ice, and the filter paper suck dry moisture is fixing, embedded section, conventional dewaxing, row HE dyeing and Masson trichrome stain.
statistical method
All data all with mean ± standard deviation (
± s) mean, adopt the SPSS13.0 statistical software to analyze, adopt one factor analysis of variance between many groups.
experimental result
4.1 ordinary circumstance is observed
The modeling of model group rat tail and coolness of extremities occurred the same day, and the tail vein is purple darkly, bunched, towering hair phenomenon is comparatively obvious, and fur tarnishes, and after 3 days, symptom is progressively improved, the basic disappearance to the 7th day; Model group and Dexamethasone group rat be movable the minimizing all, and appetite reduces, and becomes thin gradually, and body weight to the is progressively recovered in 18～21 days, and the Dexamethasone group rat body weight alleviates more obvious; Sham operated rats and Bulbus Fritillariae Uninbracteatae mixture group have no obvious movable minimizing, appetite reduces and loses weight; Each dead 1 of model group and Dexamethasone group rat.
4.2 pathologic is observed
Each is organized the left lung tissue of rat and rinses 3 times with brine ice, the filter paper suck dry moisture, and 10% neutral formalin solution is fixed, paraffin embedding, with 4 μ m paraffin sections, conventional row HE dyeing and Masson trichrome stain, do PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM.
4.2.1 perusal result: sham operated rats lung tissue structure is normal, two lung pinkiness, and smooth surface, the quality softness, elasticity is good; The visible lung tissue volume-diminished of model group, there is the rough pale kitchen range of lamellar on surface, and indivedual grey black focuses are also arranged, and elasticity reduces, and quality is harder; Dexamethasone group has pale kitchen range in various degree, but obviously reduces than model group, and elasticity is stronger, and quality is more soft, but with sham operated rats, notable difference is arranged; Bulbus Fritillariae Uninbracteatae mixture group, close to sham operated rats, has no obvious change.
4.2.2HE colored light Microscopic observation result: sham operated rats lung morphology structure is without obvious change; The visible significantly interstitial lung pathological changes of model group lung tissue, alveolar septum broadening, interstitial edema, inflammatory cell infiltration increases, visible a large amount of cell infiltration in interstitial lung and alveolar space; Dexamethasone group and the above-mentioned pathological change of Bulbus Fritillariae Uninbracteatae mixture group alleviate to some extent, but with sham operated rats, notable difference are arranged, and see Fig. 1～Fig. 4.
4.2.3 Masson colored light Microscopic observation result: the rats in sham-operated group lung tissue contains a small amount of collagen fiber, is the key component of extracellular matrix (ECM); Typical interstitial pulmonary fibrosis, appear in model group rat collagen fiber showed increased; Dexamethasone group and Bulbus Fritillariae Uninbracteatae mixture group rat also occur that interstitial pulmonary fibrosis changes, and degree is evident as gently than model group, but with sham operated rats, notable difference is arranged, and sees Fig. 5～Fig. 8.
4.2.4 the scoring of the degree of alveolitis and pulmonary fibrosis relatively
Observe lung tissue of rats HE dyeing and Masson dyeing pathological section under light microscopic, with reference to Szapiel etc.
[2,3]method is determined alveolitis and pulmonary fibrosis degree, and its classification and standards of grading are as follows:
(1) alveolitis is divided 4 grades: without alveolitis, comment 1 minute; Slight alveolitis, show as monocyte infiltration and make alveolar septum broadening, only limits to local and nearly pleura section, and the area of getting involved is less than 20% of full lung, and alveolar structure is normal, comments 2 minutes; The moderate alveolitis, the area of getting involved is 20%～50%, closely pleura section is heavier, comments 3 minutes; The severe alveolitis, the area of getting involved is greater than 50%, and mononuclear cell and the hemorrhage consolidation that causes are arranged in accidental alveolar space, comments 4 minutes; The results are shown in Table 1.
(2) pulmonary fibrosis divides 4 grades: without fibrosis, comment 1 minute; Mild fibrosis, the area of getting involved is less than 20% of full lung, and fibrosis is involved pleura and subpleural interstitial lung, and the alveolar structure disorder is commented 2 minutes; The moderate fibrosis, the area of getting involved is 20%～50%, extend from pleura in the fibrosis zone, still belongs to locality, comments 3 minutes; The severe fibrosis, the area of getting involved is greater than 50%, and fusion is arranged, and the capsule air cavity differed in size is as seen commented 4 minutes; The results are shown in Table 2.
The impact that table 1 Bulbus Fritillariae Uninbracteatae mixture is marked on the induced lung injury in rats alveolitis (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
The impact that table 2 Bulbus Fritillariae Uninbracteatae mixture is marked on induced lung injury in rats interstitial lung inflammation (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
Alveolar inflammation and pulmonary fibrosis are marked and are relatively shown, model group alveolar inflammation and pulmonary fibrosis scoring, apparently higher than sham operated rats, have statistical significance (P<0.01); Dexamethasone group and Bulbus Fritillariae Uninbracteatae mixture group alveolar inflammation and pulmonary fibrosis scoring are starkly lower than model group, have statistical significance (P<0.01).
Experiment 2: on the impact of induced lung injury in rats lung index
1. experiment material
1.1 laboratory animal is selected clean level SD rat, body weight 200 ± 20g, and male and female half and half, purchased from Shanghai Slac Experimental Animal Co., Ltd., the laboratory animal quality certification 0037253.
1.2 experimental drug
The oral liquid that the Bulbus Fritillariae Uninbracteatae mixture is embodiment 1, containing crude drug 0.315g/ml, by Nanjing University of Traditional Chinese Medicine's tcm treatment method laboratory preparation, the prepared slices of Chinese crude drugs are provided by Nanjing Pharmaceutical limited company.
Dexamethasone tablet: the 0.75mg/ sheet, Tianjin Tian Yao pharmaceutcal corporation, Ltd produces, and is mixed with the solution of 0.0000405g/ml with distilled water.
Bleomycin A5: 8mg/ props up, and Tianjin Tai He pharmaceutical Co. Ltd produces.
experimental technique
2.1 animal grouping
After rat conventional adaptation raising 3d, be divided at random 4 groups: sham operated rats, model group, Dexamethasone group, Bulbus Fritillariae Uninbracteatae mixture group.
2.2 model preparation
Prepare model by literature method, Bleomycin A5 is mixed with to 5mg/ml concentration with normal saline, after 10% chloral hydrate 0.3ml/100g anesthetized rat, the skin of neck sterilization, cut off cervical region Mus hair, row neck median incision, separate and expose trachea, inject Bleomycin A5 5mg/kg through the centripetal end puncture of tracheal cartilages czermak space, immediately animal is upright and rotation, medicinal liquid is distributed fully, evenly in lung, then skin suture; Rats in sham-operated group injects the equivalent normal saline in trachea under same anesthesia; Each is organized rat and freely drinks water under the same conditions, ingests, and keeps natural lighting, observes and record the ordinary circumstances such as activity, feed, body weight of rat.
2.3 medication:
Modeling starts gastric infusion every day in postoperative the 2nd day, and sham operated rats and model group give 5 ‰ sodium carboxymethyl cellulose (CMC sodium) solution 1ml/100g body weight; Dexamethasone group gives isometric(al) dexamethasone distilled water solution 0.000405g/Kgd; Bulbus Fritillariae Uninbracteatae mixture group gives isometric(al) Bulbus Fritillariae Uninbracteatae mixture 3.15g/Kgd; Every day, gavage was 1 time, successive administration 28 days.
2.4 collection of specimens
Raise 28 days after modeling and administration, with 10% chloral hydrate 0.3ml/100g anesthetized rat, take body weight, carotid artery is got blood and is put to death rat, opens thoracic cavity and takes out lungs, extracts the tissues such as trachea, hilar lymph node, blot lungs surface blood with filter paper, take and record the lung weight.
2.5 statistical procedures
All data all with mean ± standard deviation (
± s) mean, adopt the SPSS16.0 statistical software to analyze, adopt one factor analysis of variance between many groups.
result
Calculate the lung index according to lung weight and weighing machine.
Heavy (the g)/body weight (g) * 100% of lung index=lung.
Experimental result shows, the Bulbus Fritillariae Uninbracteatae mixture can obviously reduce weight and the lung index that Bleomycin A5 causes lung after Rat Lung Injury, relatively has significant difference (P<0.01) with model group, in Table 3.
Table 3 Bulbus Fritillariae Uninbracteatae mixture on the impact of induced lung injury in rats lung index (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
Experiment 3: on the impact of induced lung injury in rats serum inflammatory mediator
1. experiment material
1.1 laboratory animal is selected clean level SD rat, body weight 200 ± 20g, and male and female half and half, purchased from Shanghai Slac Experimental Animal Co., Ltd., the laboratory animal quality certification 0037253.
1.2 experimental drug
The oral liquid that the Bulbus Fritillariae Uninbracteatae mixture is embodiment 1, containing crude drug 0.315g/ml, by Nanjing University of Traditional Chinese Medicine's tcm treatment method laboratory preparation, the prepared slices of Chinese crude drugs are provided by Nanjing Pharmaceutical limited company.
Dexamethasone tablet: the 0.75mg/ sheet, Tianjin Tian Yao pharmaceutcal corporation, Ltd produces, and is mixed with the solution of 0.0000405g/ml with distilled water.
Bleomycin A5: 8mg/ props up, and Tianjin Tai He pharmaceutical Co. Ltd produces.
1.3 experimental apparatus
γ-911 Full automatic is exempted from calculating instrument, and Chinese University of Science and Technology's industry head office is produced.
Enzyme is exempted from instrument: the full-automatic microplate reader of SATAT FAX2100, Awareness Technology.inc.USA.
1.4 experiment reagent:
IL-4, TNF-α test kit are provided by Beijing China English biotechnology research.
γ-IFN test kit by Beijing China English biotechnology research institute purchased from Adlitteram Diagnostic laboratories inc USA.
experimental technique
2.1 animal grouping
After rat conventional adaptation raising 3d, be divided at random 4 groups: sham operated rats, model group, Dexamethasone group, Bulbus Fritillariae Uninbracteatae mixture group.
2.2 model preparation
Prepare model by literature method, Bleomycin A5 is mixed with to 5mg/ml concentration with normal saline, after 10% chloral hydrate 0.3ml/100g anesthetized rat, the skin of neck sterilization, cut off cervical region Mus hair, row neck median incision, separate and expose trachea, inject Bleomycin A5 5mg/kg through the centripetal end puncture of tracheal cartilages czermak space, immediately animal is upright and rotation, medicinal liquid is distributed fully, evenly in lung, then skin suture; Rats in sham-operated group injects the equivalent normal saline in trachea under same anesthesia; Each is organized rat and freely drinks water under the same conditions, ingests, and keeps natural lighting, observes and record the ordinary circumstances such as activity, feed, body weight of rat.
2.3 medication:
Modeling starts gastric infusion every day in postoperative the 2nd day, and sham operated rats and model group give 5 ‰ sodium carboxymethyl cellulose (CMC sodium) solution 1ml/100g body weight; Dexamethasone group gives isometric(al) dexamethasone distilled water solution 0.000405g/Kgd; Bulbus Fritillariae Uninbracteatae mixture group gives isometric(al) Bulbus Fritillariae Uninbracteatae mixture 3.15g/Kgd; Every day, gavage was 1 time, successive administration 28 days.
2.4 collection of specimens
Raise 28 days after modeling and administration, with 10% chloral hydrate 0.3ml/100g anesthetized rat, take body weight, carotid artery is got blood and is put to death rat; Carotid artery is got after blood and is got the standby inspection of the freezing preservation of serum after 3000 rev/mins of centrifugal 10min.
statistical procedures
All data all with mean ± standard deviation (
± s) mean, adopt the SPSS16.0 statistical software to analyze, adopt one factor analysis of variance between many groups.
result
4.1 the impact of Bulbus Fritillariae Uninbracteatae mixture on induced lung injury in rats blood IL-4
Experimental result shows, rats in sham-operated group blood IL-4 level is starkly lower than model group, has statistical significance (P<0.01); Bulbus Fritillariae Uninbracteatae mixture group rat serum IL-4 level and model group be not statistically significant (P ﹥ 0.05) relatively, in Table 4.
Table 4 Bulbus Fritillariae Uninbracteatae mixture on the impact of induced lung injury in rats blood IL-4 (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
4.2 the impact of Bulbus Fritillariae Uninbracteatae mixture on induced lung injury in rats blood TNF-α
Experimental result shows, rats in sham-operated group blood TNF-alpha levels and model group comparison, not statistically significant (P ﹥ 0.05); Bulbus Fritillariae Uninbracteatae mixture group rat serum TNF-alpha levels and model group be not statistically significant (P ﹥ 0.05) relatively, in Table 5.
Table 5 Bulbus Fritillariae Uninbracteatae mixture on the impact of induced lung injury in rats blood TNF-α (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
4.3 the impact of Bulbus Fritillariae Uninbracteatae mixture on induced lung injury in rats blood IFN-γ
Experimental result shows, rats in sham-operated group blood IFN-γ level is starkly lower than model group, has statistical significance (P<0.05).Bulbus Fritillariae Uninbracteatae mixture group rat serum IFN-γ level is starkly lower than model group, has statistical significance (P<0.01), in Table 6.
Table 6 Bulbus Fritillariae Uninbracteatae mixture on the impact of induced lung injury in rats blood IFN-γ (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
The Bulbus Fritillariae Uninbracteatae mixture shows the experimental result of induced lung injury in rats blood inflammatory mediator, Bulbus Fritillariae Uninbracteatae mixture group and model group rat injury of lung in the time of 28 days blood TNF-α, IL-4 level have no notable difference, not statistically significant (P ﹥ 0.05).Rats in sham-operated group blood IFN-γ level is starkly lower than model group, has statistical significance (P<0.05); Bulbus Fritillariae Uninbracteatae mixture group rat serum IFN-γ level is starkly lower than model group, has statistical significance (P<0.01).
experiment 4:impact on induced lung injury in rats lung tissue intracellular signal transduction pathway
1. experiment material
1.1 laboratory animal is selected clean level SD rat, body weight 200 ± 20g, and male and female half and half, purchased from Shanghai Slac Experimental Animal Co., Ltd., the laboratory animal quality certification 0037253.
1.2 experimental drug
The capsule that the Bulbus Fritillariae Uninbracteatae mixture is embodiment 2, by Nanjing University of Traditional Chinese Medicine's tcm treatment method laboratory preparation, the prepared slices of Chinese crude drugs are provided by Nanjing Pharmaceutical limited company.
Dexamethasone tablet: the 0.75mg/ sheet, Tianjin Tian Yao pharmaceutcal corporation, Ltd produces, and is mixed with the solution of 0.0000405g/ml with distilled water.
Bleomycin A5: 8mg/ props up, and Tianjin Tai He pharmaceutical Co. Ltd produces.
1.3 experimental apparatus
Paraffin slicing machine: German Leica company produces.
Optical microscope: Japanese Olympus company produces.
Full-automatic image-taking system OlympusDP71: Japanese Olympus company produces.
1.4 experiment reagent:
The Mus ERK1/2 of the rabbit Chinese People's Anti-Japanese Military and Political College, NF-κ B, CTGF, TGF-β, Fas/Fasl polyclonal antibody, purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.
2 experimental techniques
2.1 animal grouping
After rat conventional adaptation raising 3d, be divided at random 4 groups: sham operated rats, model group, Dexamethasone group, Bulbus Fritillariae Uninbracteatae mixture group.
2.2 model preparation
Prepare model by literature method, Bleomycin A5 is mixed with to 5mg/ml concentration with normal saline, after 10% chloral hydrate 0.3ml/100g anesthetized rat, the skin of neck sterilization, cut off cervical region Mus hair, row neck median incision, separate and expose trachea, inject Bleomycin A5 5mg/kg through the centripetal end puncture of tracheal cartilages czermak space, immediately animal is upright and rotation, medicinal liquid is distributed fully, evenly in lung, then skin suture; Rats in sham-operated group injects the equivalent normal saline in trachea under same anesthesia; Each is organized rat and freely drinks water under the same conditions, ingests, and keeps natural lighting, observes and record the ordinary circumstances such as activity, feed, body weight of rat.
2.3 medication:
Modeling starts gastric infusion every day in postoperative the 2nd day, and sham operated rats and model group give 5 ‰ sodium carboxymethyl cellulose (CMC sodium) solution 1ml/100g body weight; Dexamethasone group gives isometric(al) dexamethasone distilled water solution 0.000405g/Kgd; Bulbus Fritillariae Uninbracteatae mixture group gives isometric(al) Bulbus Fritillariae Uninbracteatae mixture 3.15g/Kgd; Every day, gavage was 1 time, successive administration 28 days.
2.4 collection of specimens
Raise 28 days after modeling and administration, with 10% chloral hydrate 0.3ml/100g anesthetized rat, take body weight, carotid artery is got blood and is put to death rat, opens thoracic cavity and takes out lungs, extracts the tissues such as trachea, hilar lymph node, with filter paper, blots lungs surface blood; Left lung rinses 3 times with brine ice, and the filter paper suck dry moisture is fixing, embedded section, and conventional dewaxing, row HE dyeing and Masson dyeing, SABC SABC method detects the expression of lung tissue ERK1/2, NF-κ B, CTGF, TGF-β, Fas/Fasl.Adopt optical microscope and full-automatic image-taking system Olympus DP71 to choose at random 5 zones (visual field) under the high power lens visual field (* 400), measure and record average optical (IOD) value of each visual field positive staining, representative value as this sheet, calculate again the average optical of this group, carry out statistics relatively.
statistical method
All data all with mean ± standard deviation (
± s) mean, adopt the SPSS13.0 statistical software to analyze, adopt one factor analysis of variance between many groups.
result
4.1 the impact of Bulbus Fritillariae Uninbracteatae mixture on induced lung injury in rats lung tissue ERK1/2 level.
The level of model group lung tissue of rats ERK1/2, apparently higher than sham operated rats, has statistical significance (P<0.01); The level of Bulbus Fritillariae Uninbracteatae mixture group lung tissue ERK1/2 is starkly lower than model group, has statistical significance (P<0.01), in Table 7.
Table 7 Bulbus Fritillariae Uninbracteatae mixture on the impact of induced lung injury in rats lung tissue ERK1/2 (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
4.2 the impact of Bulbus Fritillariae Uninbracteatae mixture on induced lung injury in rats lung tissue NF-κ B level
Model group lung tissue of rats NF-κ B level, apparently higher than sham operated rats, has statistical significance (P<0.01); Bulbus Fritillariae Uninbracteatae mixture group lung tissue NF-κ B level is starkly lower than model group, has statistical significance (P<0.01), in Table 8.
Table 8 Bulbus Fritillariae Uninbracteatae mixture on the impact of induced lung injury in rats lung tissue NF-κ B level (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
4.3 the impact of Bulbus Fritillariae Uninbracteatae mixture on induced lung injury in rats lung tissue CTGF level
The level of model group lung tissue of rats CTGF, apparently higher than sham operated rats, has statistical significance (P<0.01); The level of Bulbus Fritillariae Uninbracteatae mixture group lung tissue CTGF is starkly lower than model group, has statistical significance (P<0.01), in Table 9.
Table 9 Bulbus Fritillariae Uninbracteatae mixture on the impact of induced lung injury in rats lung tissue CTGF level (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
4.4 the impact of Bulbus Fritillariae Uninbracteatae mixture on induced lung injury in rats lung tissue TGF-β level
The level of model group lung tissue of rats TGF-β, apparently higher than sham operated rats, has statistical significance (P<0.01); The level of Bulbus Fritillariae Uninbracteatae mixture group lung tissue TGF-β is starkly lower than model group, has statistical significance (P<0.01).In Table 10.
Table 10 Bulbus Fritillariae Uninbracteatae mixture on the impact of induced lung injury in rats lung tissue TGF-β level (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
4.5 the impact of Bulbus Fritillariae Uninbracteatae mixture on induced lung injury in rats lung tissue Fas/FasL level
The level of model group lung tissue of rats Fas, apparently higher than sham operated rats, has statistical significance (P<0.05); The level of Bulbus Fritillariae Uninbracteatae mixture group lung tissue Fas and model group compare, not statistically significant (P ﹥ 0.05); The level of model group lung tissue of rats FasL, apparently higher than sham operated rats, has statistical significance (P<0.01); The level of Bulbus Fritillariae Uninbracteatae mixture group lung tissue FasL, significantly lower than model group, has statistical significance (P<0.01), in Table 11.
Table 11 Bulbus Fritillariae Uninbracteatae mixture on the impact of induced lung injury in rats lung tissue Fas/FasL level (
± s)
Annotate: with model group, compare * P<0.05 * * P<0.01
The Bulbus Fritillariae Uninbracteatae mixture shows the impact experiment of induced lung injury in rats lung tissue intracellular signal transduction pathway, the level of model group lung tissue of rats ERK1/2, NF-κ B, CTGF, TGF-β, Fas/Fasl, apparently higher than sham operated rats, has statistical significance (P<0.01); The level of Bulbus Fritillariae Uninbracteatae mixture group lung tissue ERK1/2, NF-κ B, CTGF, TGF-β, Fasl is starkly lower than model group, has statistical significance (P<0.01).
experiment 5:impact on induced lung injury in rats lung tissue angiogenesis
1. experiment material
1.1 laboratory animal
Select clean level SD rat, body weight 200 ± 20g, male and female half and half, purchased from Shanghai Slac Experimental Animal Co., Ltd., the laboratory animal quality certification 0037253.
1.2 experimental drug
The capsule that the Bulbus Fritillariae Uninbracteatae mixture is embodiment 2, by Nanjing University of Traditional Chinese Medicine's tcm treatment method laboratory preparation, the prepared slices of Chinese crude drugs are provided by Nanjing Pharmaceutical limited company.
Dexamethasone tablet: the 0.75mg/ sheet, Tianjin Tian Yao pharmaceutcal corporation, Ltd produces, and is mixed with the solution of 0.0000405g/ml with distilled water.
Bleomycin A5: 8mg/ props up, and Tianjin Tai He pharmaceutical Co. Ltd produces.
1.3 experimental apparatus
Paraffin slicing machine: German Leica company produces.
Optical microscope: Japanese Olympus company produces.
Full-automatic image-taking system Olympus DP71: Japanese Olympus company produces.
1.4 experiment reagent
The Mus VEGF of the rabbit Chinese People's Anti-Japanese Military and Political College and CD34 polyclonal antibody, step neoplasm technology company limited purchased from Fujian.
experimental technique
2.1 animal grouping
After rat conventional adaptation raising 3d, be divided at random 4 groups: sham operated rats, model group, Dexamethasone group and Bulbus Fritillariae Uninbracteatae mixture group.
2.2 modeling and administration
Prepare the induced lung injury in rats model by literature method.Bleomycin A5 is mixed with to 5mg/ml concentration with normal saline, with after 10% chloral hydrate 0.3ml/100g anesthetized rat, the skin of neck sterilization, cut off cervical region Mus hair, row neck median incision, separate and expose trachea, inject Bleomycin A5 5mg/kg through the centripetal end puncture of tracheal cartilages czermak space, animal is upright and rotation immediately, make medicinal liquid in lung, distributes fully, even, skin suture then.The sham operated rats animal is injected the equivalent normal saline in trachea under same anesthesia; Postoperative the 2nd day starts gastric infusion every day, and sham operated rats and model group are given 5 ‰ sodium carboxymethyl cellulose (CMC sodium) solution 1ml/100g body weight; Dexamethasone group is given isometric(al) dexamethasone distilled water solution; The Bulbus Fritillariae Uninbracteatae mixture gives isometric(al) Bulbus Fritillariae Uninbracteatae mixture.
2.3 collection of specimens
Postoperative 28 days, to put to death after 10% chloral hydrate 0.3ml/100g anesthetized rat, cut open and get two lungs, wipe out trachea, main bronchus, left lung tissue is rinsed 3 times to the filter paper suck dry moisture with brine ice, 10% neutral formalin solution is fixed, specimens paraffin embedding slices, conventional dewaxing, detect the expression of lung tissue VEGF and CD34 by Use immunohistochemistrySP SP; Adopt optical microscope and full-automatic image-taking system Olympus DP71 to choose at random 5 zones (visual field) under the high power lens visual field (* 400), measure and record average optical (IOD) value and the CD34 number of each visual field positive staining, as the representative value of this sheet.
2.4 statistical procedures
All data all with mean ± standard deviation (
± s) mean, adopt the SPSS16.0 statistical software to analyze, adopt one factor analysis of variance between many groups.
result
3.1 the Bulbus Fritillariae Uninbracteatae mixture causes the impact of induced lung injury in rats vegf expression on Bleomycin A5
The expression of model group lung tissue of rats VEGF, apparently higher than sham operated rats, has statistical significance (P<0.01); The expression of Bulbus Fritillariae Uninbracteatae mixture group lung tissue VEGF is starkly lower than model group, has statistical significance (P<0.01), in Table 12.
Table 12 Bulbus Fritillariae Uninbracteatae mixture on Bleomycin A5 cause induced lung injury in rats VEGF impact (
± s)
Annotate: with matched group, compare * P<0.05 * * P<0.01
3.2 Bulbus Fritillariae Uninbracteatae mixture side causes the impact of induced lung injury in rats microvessel density on Bleomycin A5
Model group lung tissue of rats microvessel density is starkly lower than sham operated rats, has statistical significance (P<0.01); Bulbus Fritillariae Uninbracteatae mixture group lung tissue microvessel density, apparently higher than model group, has statistical significance (P<0.01), in Table 13.
Table 13 Bulbus Fritillariae Uninbracteatae mixture on Bleomycin A5 cause the Rat Lung Injury microvessel density impact (
± s)
Annotate: with matched group, compare * P<0.05 * * P<0.01
conclusion:
The Bulbus Fritillariae Uninbracteatae mixture can significantly alleviate the degree that Bleomycin A5 brings out induced lung injury in rats alveolar inflammation and interstitial lung inflammation, inflammatory factor TNF-α, IL-4 is had no significant effect in the time of 28 days at injury of lung, but can significantly reduce blood IFN-γ level.The Bulbus Fritillariae Uninbracteatae mixture can obviously reduce the level of induced lung injury in rats lung tissue ERK1/2, NF-κ B, CTGF, TGF-β, Fasl; The Bulbus Fritillariae Uninbracteatae mixture can reduce Bleomycin A5 to be brought out induced lung injury in rats lung tissue VEGF level and increases the lung tissue microvessel density, thereby alleviates the injury of lung degree, the control pulmonary fibrosis.Illustrate that this Traditional Chinese drug mixture has good control injury of lung and the effect of pulmonary fibrosis.
The above is only preferred implementation method of the present invention; it should be pointed out that the ordinary person for technical field, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (2)
1. the Chinese medicine composition of control injury of lung and pulmonary fibrosis, is characterized in that the oral liquid according to this Chinese medicine composition of following methods system: get Radix Platycodonis, each 28.6g of Bulbus Fritillariae Thunbergii, Flos Lonicerae, each 21.4g of Cortex Moutan, co-grinding 100% is crossed 100 mesh sieves, the water that adds 1000ml in the mixed powder placing container, boil rear slow fire and continue 45 minutes, precipitation, filter, get filtrate, vacuum concentration is 100ml, the sub-bottle fill, 100 milliliters every bottle, the sterilizing sealing.
2. prevent and treat the Chinese medicine composition of injury of lung and pulmonary fibrosis, it is characterized in that the capsule according to this Chinese medicine composition of following methods system: get Radix Platycodonis, each 28.6g of Bulbus Fritillariae Thunbergii, Flos Lonicerae, each 21.4g of Cortex Moutan, co-grinding 100% is crossed 100 mesh sieves, 3/4 heavy drug decocting 45 minutes, precipitation, filter, get filtrate, vacuum concentration is 100ml; 1/4 heavy 200 mesh sieves of again pulverizing, mix homogeneously with above-mentioned medicinal liquid, vacuum drying or oven drying at low temperature, and sterilizing, pulverize, and incapsulates every capsules 0.25g.
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