CN103933073B - Application of the long-nosed pit viper snake venom in preparing anti-angiogenic medicaments - Google Patents
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Abstract
The invention belongs to technical field of Chinese medicine, more particularly, to a kind of application of long-nosed pit viper snake venom in preparing anti-angiogenic medicaments.Its anti-angiogenic curative effect of medication of solution is not notable, the technical problems such as expensive.The present invention provides a kind of application of long-nosed pit viper snake venom in preparing anti-angiogenic medicaments, there is provided it is a kind of it is quality controllable, dosage is small, the anti-angiogenic medicaments esy to use, safe and reliable containing long-nosed pit viper snake venom, a kind of application of anti-angiogenic medicaments containing long-nosed pit viper snake venom as treatment relevant disease is provided.The advantage of the invention is that：Long-nosed pit viper snake venom price is cheap compared with the biological agent of anti-angiogenesis class, it is easy to get, desivac is easy to operate, it is easy to get purer long-nosed pit viper lyophilized venom, blood vessel formation against function is notable, can be applied to a variety of and relevant disease of blood vessel hyperplasia, have a wide range of application, it is significant in efficacy, it is highly practical, it is conducive to industrialized production and popularization.
Description
Technical field
The invention belongs to technical field of Chinese medicine, more particularly, to a kind of long-nosed pit viper snake venom in preparing anti-angiogenic medicaments
Application.
Background technology
Angiogenesis is the coefficient result of many factors.By taking the angiogenesis at inflammatory lesions position as an example, new green blood
3 steps of the formation of pipe point：Inflammatory mediator activates endothelial cell；Proteasome degradation blood vessel endothelium matrix；Endothelial cell chemotactic and
New vessels occur.VEGF is the letter of vascular endothelial growth factor (vascular endothelial growth factor)
Claim, early stage is also referred to as vascular permeability factor (vascular permeability factor), is vascular endothelial cell specificity
Heparin binding growth factor, can in vivo induction of vascular it is newborn.VEGF is main angiogenic factor, can stimulate blood
Endothelial cell survival, mobile, proliferation.In the maturation of blood vessel, angiogenin (angiopoietins, Ang) 1 is negative
Duty recruits the mesenchymal cell of surrounding and it is promoted to be divided into vascular smooth muscle cells, significantly enhances the blood vessel of new vessels
Stability.Therefore VEGF has mediated migrating and being proliferated for endothelial cell, and Ang, especially Ang-1 promote the stabilization of vascular wall
Property, corporate action makes new vessels stablize to be formed.The two plays main progradation in blood vessel hyperplasia.
Rheumatoid arthritis (rheumatoid arthritis, RA) is a kind of common chronic progressive autoimmunity
Property disease.Its pathological characteristic be joint inflammation repeatedly, synovial cell's " tumour sample " hyperplasia and thus caused by cartilage and bone
Destruction, eventually lead to joint deformity.It is embodied in synovial hyperplasia and inflammatory cell infiltration.The synovial membrane of rheumatoid arthritis changes
Change can be divided into inflammatory phase, pannus forms phase and fibrosis phase.Pannus formation is the important pathology of rheumatoid arthritis synovial membrane
Feature plays a significant role during rheumatoid arthritis cartilage and osteoclasia.The main pathological basis of extra-articular manifestation is
Vasculitis.Rheumatoid nodules is its feature sex expression, and nodule center is fiber element sample slough, around there is " palisade " arrangement
Histocyte, fibroblast and macrophage etc..VEGF and Ang-1 promotes the formation of pannus in RA, promotes synovial membrane
Inflammation generates and destroys bone and cartilage, and the overpreading of blood vessel is promoted in tumour.
Existing anti-angiogenic medicaments are considerably less and expensive, such as bevacizumab, by intravascular with people
Skin growth factor (VEGF) combines and vegf blocker (VEGF) bioactivity, to reduce the life of blood vessel
At.For rheumatoid arthritis, biological agent is current key agents, can reduce osteoclasia, reduces hormone
Dosage and osteoporosis.The biological agent for treating rheumatoid arthritis includes mainly tumor necrosis factor (TNF)-α antagonisms
Agent, interleukins (IL)-l and IL-6 antagonists, anti-CD20 monoclonal antibody and T cell costimulatory signal inhibitor etc., but this
A little biological agents are of high cost, expensive.
In order to be improved to the prior art, people have carried out long-term exploration, it is proposed that various solutions.
For example, Chinese patent literature discloses a kind of application of the cobra-venom after physical modification in preparing medicament for treating arthritis
[application number：CN201110361926.X], the invention is animal experiment proves that the total poison of Chinese cobra after physical modification exists
(10-3000 μ g/kg) range interior energy inhibits inflammatory pain, inflammatory hyperplasia and adjuvant-induced arthritis to have apparent therapeutic effect,
Arthrocele, the symptoms such as pain and joint injury can be mitigated.
Said program although disclose the cobra-venom after physical modification to treatment inhibit inflammatory pain, inflammatory hyperplasia and
Adjuvant-induced arthritis therapeutic effect, but the application range treated is small, and preparation process is complicated, needs by alcohol detoxification, practicability
Difference is unfavorable for industrialized production and popularization.
Invention content
Regarding the issue above, the present invention provides prepared by a kind of long-nosed pit viper snake venom easy to implement, safe and reliable
Application in anti-angiogenic medicaments.
In order to achieve the above objectives, present invention employs following technical proposals：This long-nosed pit viper snake venom is preparing anti-angiogenic drugs
Application in object, which is characterized in that the Ang-1 inhibitor when long-nosed pit viper snake venom is as anti-angiogenesis.
In above-mentioned long-nosed pit viper snake venom in the application in preparing anti-angiogenic medicaments, this drug includes long-nosed pit viper snake venom and medicine
Acceptable carrier on.
In above-mentioned long-nosed pit viper snake venom in the application in preparing anti-angiogenic medicaments, the long-nosed pit viper snake venom is by rising
The dry freeze-dried powder formed of China.
In above-mentioned long-nosed pit viper snake venom in the application in preparing anti-angiogenic medicaments, the freeze-dried powder is made containing begging
Calmy poison amount is the injection of 0.1-1.0mg/kg weight.
3 steps of the formation of new vessels point：Inflammatory mediator activates endothelial cell；Proteasome degradation blood vessel endothelium matrix；
Endothelial cell chemotactic and new vessels occur.New vessels occur it is initial, VEGF can stimulate vascular endothelial cell survival,
Mobile, proliferation, is most effective angiogenic growth factor, plays the role of in angiogenesis very big, passes through inhibition
VEGF can effectively treat corresponding disease.Still very fragile after blood vessel just generates, a ripe process is needed, and
It is played a significant role in the maturation angiopoietin (angiopoietins, Ang) 1 of blood vessel, is mainly responsible for trick
The mesenchymal cell for surrounding of raising simultaneously promotes it to be divided into vascular smooth muscle cells, significantly enhances the stabilization of vascular of new vessels
Property.Ang-1/Tie-2 accesses can lure that smooth muscle cell, neutrophil leucocyte, eosinophils generate chemotactic effect into, be filled between promotion
Cell plastid is converted to smooth muscle cell, and can induce the secretion of thrombocytin, matrix metalloproteinase, fibrinolysin, right
The inhibition of Ang-1 has interrupted the access of Ang-1/Tie2 indirectly, so that capilary substantially reduces.From the angle for the treatment of disease
It sees, the maturation by destroying blood vessel can also achieve the purpose that anti-angiogenesis.Long-nosed pit viper snake venom can be used to prepare VEGF inhibitor,
Ang-1 inhibitor is can be used for, and both VEGF inhibitor and Ang-1 inhibitor are combined, and can either prevent the first generation of blood vessel,
Also the maturation of blood vessel, the two can be prevented to generate synergistic effect, the effect of anti-angiogenesis is stronger.In the difference of angiogenesis
Phase, there is some difference for the effect that long-nosed pit viper snake venom is played.Obviously, in angiogenesis period, long-nosed pit viper snake venom primarily serves VEGF
The effect of inhibitor；In angiogenesis and maturation period, long-nosed pit viper snake venom primarily serves the effect of Ang-1 inhibitor；Both
Having new vessels again has maturation medium vessels period, and long-nosed pit viper snake venom primarily serves the effect that VEGF and Ang-1 collaborations inhibit.
Above-mentioned containing in the anti-angiogenic medicaments of long-nosed pit viper snake venom, the long-nosed pit viper snake venom is by lyophilization shape
At freeze-dried powder.Vacuum Freezing & Drying Technology, is referred to as lyophilized, and also known as lyophilization is widely used in drug, biological products, change
Work and food industry, to heat-sensitive substance such as antibiotic, vaccine, blood product, enzyme hormone and other biological tissue, freeze drying technology
It is applicable in very much.Because long-nosed pit viper snake venom is biological agent, there are a large amount of protein, to ensure drug effect, preferably with freeze-drying legal system
It is standby, it is the preparation method of desivac below：The first step, filtering are removal impurity, get clarified solution, are freezed, wait and build-up ice
It is dried again after block.The snake venom ice cube of freezing is positioned in vacuum desiccator, is dried in vacuo by second step, and vacuum is dry
It is dry generally thoroughly to have been dried in 6 hours.Snake venom after drying it is powdered or blocky, agkistrodon acutus venom is white powder, eye
Mirror snake venom is faint yellow powdery.Snake venom water content must not exceed 5% after drying, and such snake venom is refined lyophilized venom.
The above-mentioned anti-angiogenic medicaments for containing long-nosed pit viper snake venom as treatment relevant disease application in, the phase
Related disorders include intraocular neovascularization formation, psoriasis, tumour, artery sclerosis, inflammatory disease and endometriosis, glycosuria
Sick complication.
Further, the preferred rheumatic arthritis of the inflammatory disease and rheumatoid arthritis.In rheumatism joint
In scorching and rheumatoid arthritis, long-nosed pit viper snake venom is other than it can inhibit VEGF and Ang-1, additionally it is possible to come from other links of RA
Block the process of disease.Mainly there is the following aspects：The characteristic pathological change of first, RA originates in synovitis, with disease
There is had significant proliferation in the progress of journey, synovial membrane, form the villus with invasion, dash forward to articular cavity or invade under cartilage and cartilage
Sclerotin, that is, form synovial membrane pannus, there is a large amount of neovascularization resulting and various inflammatory cell infiltrations inside it, discharge
A variety of hydrolases finally completely lose destruction of joint, tetanic, function, and during osteoclasia, TNF-α triggers inflammatory cell
The expression of the factor and adhesion molecule, activation clostridiopetidase A and induction osteoclast differentiation.Researcher has found during studying RA
Long-nosed pit viper snake venom has good inhibiting effect to tumor necrosis factor-alpha.Second, in RA researchs, MMPs can be adjusted extracellularly
The degradation of matrix components plays determinant normal person, progressivity bone especially in arthritis disease to the degradation of cartilage matrix
There is the expression of the characteristic of MMP (8 kinds) and TIMP (4 kinds) in the synovium of joint liquid of arthritis (OA) and RA patient, and MMP contains
Amount increase is closely related with the severity of progressivity OA and RA, and long-nosed pit viper snake venom has good inhibiting effect to MMP-9.The
Three, as the TIMP-1 of MMP-9 specific inhibitors, the joint of low expression and the mediation of early stage synovitis in human serum
Surrounding osteoporosis is proportionate, it is believed that is likely to become the biomarker of the periarticular osteoporosis of early stage RA.Promote
The release of TIMP-1 then can effectively inhibit MMP-9 to achieve the purpose that treat rheumatic arthritis, and long-nosed pit viper snake venom energy
Effectively facilitate the release of TIMP-1.So there is unique curative effect for rheumatic arthritis and rheumatoid arthritis.
Compared with prior art, this long-nosed pit viper snake venom is in the advantages of application in preparing anti-angiogenic medicaments：It begs
Calmy poison price is cheap compared with the biological agent of anti-angiogenesis class, is easy to get, and desivac is easy to operate, is easy to get purer
Long-nosed pit viper lyophilized venom, blood vessel formation against function is notable, can be applied to it is a variety of with the relevant disease of blood vessel hyperplasia, using model
It encloses extensively, it is significant in efficacy, it is highly practical, it is conducive to industrialized production and popularization.
Description of the drawings
Fig. 1 is each group rat ankle joint Synovial histopathology change figure that zoopery 1 provides in the embodiment of the present invention 1
(HE×400).In figure, A：Normal group；B：Model group；C：Meloxicam group；D：Snake venom low dose group；E：Snake venom middle dose group；
F：Snake venom high dose group.
Fig. 2 is each group rat ankle joint synovial tissue vWF immunohistochemistry that zoopery 1 provides in the embodiment of the present invention 1
Colored graph (× 400).In figure, A：Normal group；B：Model group；C：Meloxicam group；D：Snake venom low dose group；E：Snake venom middle dosage
Group；F：Snake venom high dose group.
Fig. 3 is each group rat ankle joint synovial tissue VEGF immunohistochemistry that zoopery 1 provides in the embodiment of the present invention 1
Colored graph (× 400).In figure, A：Normal group；B：Model group；C：Meloxicam group；D：Snake venom low dose group；E：Snake venom middle dosage
Group；F：Snake venom high dose group.
Fig. 4 is that zoopery 2 provides each group rat ankle joint Synovial histopathology change (HE in the embodiment of the present invention 2
×400).In figure, A：Normal group；B：Model group；C：Meloxicam group；D：Snake venom low dose group；E：Snake venom middle dose group；F：Snake
Malicious high dose group.
Specific implementation mode
Embodiment 1
Application of the long-nosed pit viper snake venom in preparing anti-angiogenic medicaments.More specifically, long-nosed pit viper snake venom is as anti-angiogenic life
At when VEGF inhibitor；Or long-nosed pit viper snake venom as anti-angiogenesis when Ang-1 inhibitor；Or long-nosed pit viper snake venom is as anti-
VEGF and Ang-1 when angiogenesis cooperate with inhibitor.
A kind of anti-angiogenic medicaments containing long-nosed pit viper snake venom.This drug includes long-nosed pit viper snake venom and pharmaceutically acceptable load
Body.In the present embodiment, long-nosed pit viper snake venom is the freeze-dried powder formed by lyophilization.Freeze-dried powder is made is containing long-nosed pit viper snake venom amount
The injection of 0.1-1.0mg/kg weight.
A kind of application of anti-angiogenic medicaments containing long-nosed pit viper snake venom as treatment relevant disease.Relevant disease includes eye
Interior new vessels formation, psoriasis, tumour, artery sclerosis, inflammatory disease and endometriosis, diabetic complication.More
Further, the preferred rheumatic arthritis of the inflammatory disease and rheumatoid arthritis.Since intraocular neovascularization is formed, silver
Bits disease, tumour, artery sclerosis, inflammatory disease (such as rheumatic arthritis and rheumatoid arthritis) and endometriosis,
The diseases such as diabetic complication all have the similar features of angiogenesis, thus long-nosed pit viper snake venom treat above-mentioned disease mechanism it is basic
It is identical, it does not repeat herein.
One, the zoopery 1 of the embodiment of the present invention 1 is following (by taking rat rheumatoid arthritis as an example)：
1 zoopery
48 female SPF Lewis rats of 1.1 experimental animals and reagent (8~9 week old, 150 ± 10g of weight), are purchased from
Beijing Vital River Experimental Animals Technology Co., Ltd., raising in Zhejiang University of Traditional Chinese Medicine's Experimental Animal Center, free diet into
Water.II Collagen Type VI of ox (C II), complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) are purchased from Chondrex companies of the U.S.；
Rabbit-anti rat vWF is purchased from millipore companies；Rabbit anti-VEGF antibody, Ang-1ELISA kits are purchased from Santa cruz companies；
Long-nosed pit viper lyophilized venom is purchased from Huangshan class Science Institute；Shanghai Boehringer Ingelheim is not purchased from than (meloxicam tablet)
Pharmaceutcal corporation, Ltd.Vacuum Freezing & Drying Technology, referred to as be lyophilized, also known as lyophilization, be widely used in drug, biological products,
Skill is lyophilized to heat-sensitive substance such as antibiotic, vaccine, blood product, enzyme hormone and other biological tissue in chemical industry and food industry
Art is applicable in very much.Because long-nosed pit viper snake venom is biological agent, there are a large amount of protein, to ensure drug effect, preferably with desivac
It prepares, is the preparation method of desivac below：The first step, filtering are removal impurity, get clarified solution, are freezed, wait and form
It is dried again after ice cube.The snake venom ice cube of freezing is positioned in vacuum desiccator, is dried in vacuo, vacuum by second step
Drying has generally thoroughly been dried in 6 hours.Snake venom after drying it is powdered or blocky, agkistrodon acutus venom is white powder,
Cobra venom is faint yellow powdery.Snake venom water content must not exceed 5% after drying, and such snake venom is refined snake poison lyophilized
Powder.
II 10mg of C are made into concentration 2mg/mL by 1.2 modelings and grouping with 0.05mol/L glacial acetic acid solution 5mL mixings,
Set 4 DEG C of refrigerator overnights.The CFA of 4mg/mL is taken, is mixed in equal volume with the glacial acetic acid containing C II in ice bath, is made after fully emulsified
At the emulsion of II final concentration of l mg/mL of C as the 1st inoculation agent.Ibid method takes IFA and the ice vinegar containing C II
Sour isometric mixing, is made the emulsion of II final concentration of l mg/mL of C as the 2nd inoculation agent.With micro- after rat anesthesia
Syringe is measured in intradermal 3~4 points of root of the tail portion, the 1st immune emulsion 0.2mL/ is injected only, is injected the 2nd time in same area after 7d
0.15mL/ booster immunization of immune emulsion.Normal group is with method with injection location normal saline.Time 21d is modeled, except normal
Group is outer, sub-model group, Meloxicam group, snake venom low, middle and high dose groups after modeling success, every group 8.
1.3 medication each groups start to be administered on the 1st day after modeling success, and snake venom low, middle and high dose groups are respectively at abdomen
Chamber injection 0.1,0.33,1mg/kg weight, Meloxicam group press 0.072mg/kg weight gavages, normal group and model group abdominal cavity note
It penetrates and waits capacity physiological saline, 1 time/d, continuous 21d.
1.4 synovial membrane pathology detect and scoring left foot ankle-joint is fixed, decalcification, paraffin embedding, slice it is laggard
Row HE dyeing.With reference to standards such as Takyanagi, joint pathology injury severity score is carried out using double-blind study：According to synovial cell
3 hyperplasia, synovial membrane lower layer inflammatory cell infiltration and angiogenesis indexs, each by it is light, in, weight degree be chosen as 0~3 point respectively, 3
Item index summation is the total histological scores of rat arthritis tissue.
1.5 microvessel densities observe vWF immunohistochemistry positives region, evaluate new capillary vessel situation.
Stained slice first scans whole visual field under 100 times of low-powered microscopes, and endothelial cell is selected to dye 3 most intensive regions
(hotspot, i.e. capilary form most abundant region) then goes to counting 6 visuals field (each visual field under 200 power microscopes
0.16mm2) number of microvessels, take its average value, as the blood vessel number (MVD) per high power field.Any one endochylema is contaminated in palm fibre
Single endothelial cell or endothelial cell cluster, have an apparent boundary with surrounding its hetero-organization, no matter whether there is or not lumen of vessels, no matter in tube chamber
Be defined as a capilary whether there is or not red blood cell, tube chamber be more than 8 red blood cell diameters or have apparent muscle layer person without counting including.
1.6 immunohistochemistry views vegf proteins are expressed and scoring uses EnVisionTM two step methods immuning tissue
Method detects the expression of vegf protein.VEGF positive cells are that cytoplasm is in brown color or sepia.Vegf protein expression uses
Sxemiquantitative scoring method judges：It scores by positive coloring degree：0 point of non-coloring；1 point light yellow, slightly above background colour；2 points pale brown
Color, hence it is evident that be higher than background colour；3 points of sepias.It scores by positive cell proportion：0 second property；1 point 10% or less；2 points
11%~50%；3 point 51%~75%；4 point 75% or more.The two product is positive findings.
After 1.7 ELISA detect serum Ang-1 content last doses 1h, rat sterile abdominal aorta after intraperitoneal anesthesia
Blood sampling stands 1h, and 3000r/min centrifuges 15min, takes supernatant, is placed in -80 DEG C of refrigerators and preserves.ELISA detects concrete operations step
It is rapid with reference to kit specification, measure absorbance value under 450nm microplate reader wavelength.
1.8 statistical methods are analyzed using SPSS20.0 statistical packages, and each group of data is indicated with ± s, using One
Way ANOVA (Student-Newman-Keuls) carry out comparison among groups, and P < 0.05 are that difference is statistically significant.
2 results
2.1 long-nosed pit viper snake venom are on the pathological influence of lymphocyte of adjurant arthritis rat
Normal rat synovium of joint is made of 1~3 layer of flat synovial cell, no cell infiltration and blood vessel hyperplasia, joint
Structural integrity, cartilage layers are thicker, and surface is smooth.18~21d Articular swellings are most after immune for CIA rat models in this research
Height, synovial cell's number of plies increase to 10~15 layers, cell infiltration, synovial membrane pannus hyperplasia, articular cartilage surface damage；It is closed after 21d
Section swelling is gradually subsided stiff, and the visible necrosis of articular cartilage local fibrosis falls off, arthrostenosis, is soaked with extensive inflammatory cell
Profit, a large amount of fibroplasias of synovial membrane lower layer and new vessels are formed, and are in tumprigenicity invasive growth.Such as Fig. 1 and table 1, with model group
It compares, the histological scores of the rat ankle joint of Meloxicam group and the middle and high dosage group of snake venom significantly reduce (P<0.05).See figure
1, table 1.
Influence of the 2.2 long-nosed pit viper snake venom to lymphocyte of adjurant arthritis rat microvessel density
CIA model group rats synovial tissue capilary is abundant, and capilary number is intensive, and MVD is expressed in high；As shown in Fig. 2,
Compared with model group, the MVD of Meloxicam group and snake venom high dose group is substantially reduced (P<0.05), and snake venom is low, middle dose group
MVD reduction there is no statistical significance.See Fig. 2, table 1.
The influence that 2.3 long-nosed pit viper snake venom express vegf protein in lymphocyte of adjurant arthritis rat
The expression of normal rats vegf protein is rare, and vegf protein expression is apparent in CIA models, is expressed in cytoplasm
In.Compared with model group, the vegf protein positive expression amount of the rat ankle joint of Meloxicam group and the middle and high dosage group of snake venom is aobvious
Writing reduces (P<0.05).See Fig. 3, table 1.
Influence of the 2.4 long-nosed pit viper snake venom to RA rat blood serum Ang-1 contents
Compared with normal group, remaining each group serum Ang-1 contents significantly increase (P<0.01)；Compared with model group, Mei Luo
The Ang-1 contents of the rat ankle joint of former times health group and snake venom high dose group significantly reduce (P<0.05).It is shown in Table 1.
1 long-nosed pit viper snake venom of table is to the total histological scores of CIA rat ankle joint synovial membranes and VEGF score positives, Microvessel Count
It influences
Compared with normal group, * P<0.05, * * P<0.01；Compared with model group, #P<0.05, ##P<0.01.
3 steps of the formation of new vessels point：Inflammatory mediator activates endothelial cell；Proteasome degradation blood vessel endothelium matrix；
Endothelial cell chemotactic and new vessels occur.New vessels occur it is initial, VEGF can stimulate vascular endothelial cell survival,
Mobile, proliferation, is most effective angiogenic growth factor, plays the role of in angiogenesis very big, passes through inhibition
VEGF can effectively treat corresponding disease.Still very fragile after blood vessel just generates, a ripe process is needed, and
It is played a significant role in the maturation angiopoietin (angiopoietins, Ang) 1 of blood vessel, is mainly responsible for trick
The mesenchymal cell for surrounding of raising simultaneously promotes it to be divided into vascular smooth muscle cells, significantly enhances the stabilization of vascular of new vessels
Property.Ang-1/Tie-2 accesses can lure that smooth muscle cell, neutrophil leucocyte, eosinophils generate chemotactic effect into, be filled between promotion
Cell plastid is converted to smooth muscle cell, and can induce the secretion of thrombocytin, matrix metalloproteinase, fibrinolysin, right
The inhibition of Ang-1 has interrupted the access of Ang-1/Tie2 indirectly, so that capilary substantially reduces.From the angle for the treatment of disease
It sees, the maturation by destroying blood vessel can also achieve the purpose that anti-angiogenesis.Long-nosed pit viper snake venom can be used to prepare VEGF inhibitor,
Ang-1 inhibitor is can be used for, and both VEGF inhibitor and Ang-1 inhibitor are combined, and can either prevent the first generation of blood vessel,
Also the maturation of blood vessel, the two can be prevented to generate synergistic effect, the effect of anti-angiogenesis is stronger.In the difference of angiogenesis
Phase, there is some difference for the effect that long-nosed pit viper snake venom is played.Obviously, in angiogenesis period, long-nosed pit viper snake venom primarily serves VEGF
The effect of inhibitor；In angiogenesis and maturation period, long-nosed pit viper snake venom primarily serves the effect of Ang-1 inhibitor；Both
Having new vessels again has maturation medium vessels period, and long-nosed pit viper snake venom primarily serves the effect that VEGF and Ang-1 collaborations inhibit.
Embodiment 2
Two, the zoopery 1 of the embodiment of the present invention 2 is following (by taking rat rheumatoid arthritis as an example)：
Zoopery 1
1.1 SPF grades of animal Lewis rats 48, female, 8~9 week old, weight (150 10) g are logical purchased from Beijing dimension
Li Hua experimental animals Technology Co., Ltd. raises in Zhejiang University of Traditional Chinese Medicine's Experimental Animal Center.20 DEG C of room temperature, relative humidity
40%~60%, daily illumination 12h, free diet water inlet.
1.2 drug long-nosed pit viper lyophilized venoms are purchased from Huangshan class Science Institute；Not than (meloxicam tablet
7.5mg, batch number 284177) it is Shanghai Boehringer Ingelheim pharmaceutcal corporation, Ltd product.
1.3 reagents and II Collagen Type VI of instrument ox (C II), complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA)
It is U.S.'s Chondrex Products；TNF-α, MMP-9, TIMP-1ELISA kit are Santa cruz Products；
YLS-7A toes capacity measurers, Shandong Province instrumental science institute equipment station；Mike Audi BA210 biomicroscopes, instrument in Beijing
Guang Ke developments in science and technology Co., Ltd；550 type microplate reader, Bio-Rad companies of the U.S..
1.4 modelings and grouping take II 10mg of C, and with 0.05mol/L glacial acetic acid solution 5mL mixings, a concentration of 2mg/mL is set
4 DEG C of refrigerator overnights.By the CFA of 4mg/mL, mixed in equal volume with the glacial acetic acid containing C II under ice bath, syringe aspirates repeatedly
To milky thick liquid, the emulsion of II final concentration of l mg/mL of C is made as the 1st inoculation agent.Ibid method will
IFA is mixed in equal volume with the glacial acetic acid containing C II, and the emulsion of II final concentration of l mg/mL of C is made as the 2nd immune note
Penetrate agent.In addition to normal group, remaining each group is after adaptable fed 1w, with micro syringe in the intradermal 3-4 points in root of the tail portion, injection
1st immune emulsion 0.2mL/ only, 0.15mL/ booster immunization of the 2nd immune emulsion is injected after 7d in same area.When modeling
Between 21d, point normal group after model success, model group, Meloxicam group, snake venom low, middle and high dose groups, every group 8.
1.5 medication each groups start to be administered on the 1st day after modeling is successfully grouped, snake venom low, middle and high dose groups difference
In intraperitoneal injection 0.1,0.33,1mg/kg weight, Meloxicam group presses 0.072mg/kg weight gavages, normal group and model group abdomen
The capacity PBS such as chamber injection, 1 time/d, continuous 21d.
1.6 arthritis index (Arthritic Index, AI) scoring uses 0~4 grade of The joint evaluation method, measures joint
Scorching index.Arthritis score of the sum of the accumulative extremities joint scoring as each rat, every highest 16 are divided.
1.7 volumetric methods measure double hind leg paw swellings and are measured with toes volume measuring apparatus, and per 7d, measurement is primary, calculate double
Hind leg swelling average.
After the detection of 1.8 Synovial histopathologies carries out HE dyeing to left foot ankle-joint, the standards such as reference Takyanagi,
Joint pathology injury severity score is carried out using double-blind study：According to 2 indexs of inflammatory cell infiltration and synovial cell proliferation degree,
Each by it is light, in, weight degree be chosen as 0~3 point respectively, 2 index summations are the rat arthritis total tissue histological scores.
After 1.9 Serum TNF-α, MMP-9, TIMP-1 content detection last dose 1h, intraperitoneal anesthesia, aseptic condition lower abdomen
Aorta is taken a blood sample, and 1h is stood, and 3000r/min centrifuges 15min, takes supernatant, is placed in -80 DEG C of refrigerators and is preserved.It is examined using ELISA method
Serum cytokines content is surveyed, concrete operation step measures extinction with microplate reader with reference to kit specification under 450nm wavelength
Angle value.
1.10 statistical methods are analyzed using SPSS20.0 statistical packages, and each group of data is indicated with ± s, arthritis
Index and Synovial histopathology scoring are examined with Mann-Whitney U, remaining uses One way ANOVA (Student-
Newman-Keuls comparison among groups) are carried out, P < 0.05 are that difference is statistically significant.
2 results
Influence of the 2.1 long-nosed pit viper snake venom to RA rat arthritis index (AI)
It is administered the 7th, 14 and 21 day, compared with normal group, model group and the AI scorings of administration group rat dramatically increase (P<
0.01)；Compared with model group, the AI of the middle and high dosage group of snake venom and Meloxicam group scores than modeling group and snake venom low dose group
It substantially reduces, statistically significant (P<0.01).It is shown in Table 2.
The influence that 2 snake venom of table scores to CIA rats AI
Compared with normal group, * P<0.01；Compared with model group,△P<0.05,△△P<0.01；
Influence of the 2.2 long-nosed pit viper snake venom to swelling degree of the paw
It is administered the 7th, 14 and 21 day, compared with normal group, model group and the double hind leg pedal swellings of administration group rat significantly increase
Add (P<0.01)；Compared with model group, double hind leg pedal swelling ratio modeling groups of the middle and high dosage group of snake venom and Meloxicam group and
Snake venom low dose group substantially reduces, statistically significant (P<0.01).It is shown in Table 3.
Influence of 3 snake venom of table to the double hind leg swelling degree of the paw of CIA rats
Compared with normal group, * P<0.01；Compared with model group,△P<0.01；
Influence of the 2.3 long-nosed pit viper snake venom to RA rat articular Synovial histopathologies
Under light microscopic, the joint structure of rats in normal control group is normal, and synovial tissue is without congestion and edema, and synovial cell is without hyperplasia
Phenomenon.The visible apparent synovial cell proliferation of model group, disorganized, synovial tissue's congestion and edema, blood capillary proliferation, and can
See cell infiltration.Compared with model group, the rat ankle joint pathology damage of Meloxicam group and the middle and high dosage group of snake venom is scored
Significantly reduce (P<0.05) 4, Fig. 4, be the results are shown in Table.
4 ankle-joint Synovial histopathology of table scores
Compared with model group, * P<0.05；**P<0.01
Influence of the 2.4 long-nosed pit viper snake venom to RA rat blood serums TNF-α, MMP-9, TIMP-1 content
Compared with normal group, remaining each group Serum TNF-α, MMP-9, TIMP-1 content all significantly increase (P<0.05)；With
Model group compares, TNF-α in the snake venom administration group and Meloxicam group serum of various dose, MMP-9 expression all reduce,
And the expression of TIMP-1 increases.Wherein, the expression of TNF-α is more notable than model group in each dosage group rat blood serum of snake venom
It reduces, MMP-9 expressions are significantly reduced than model group in snake venom high dose group serum, and TIMP-1 expressions then significantly rise
Height (P<0.01).It is shown in Table 5.
TNF-α, MMP-9, TIMP-1 content in 5 each group rat blood serum of table
Compared with normal group, * P<0.05, * * P<0.01；Compared with model group,△△P<0.01；
This experiment shows that snake venom can significantly reduce TNF-α in serum, MMP-9 expressions, significantly improves in serum
Expression in TIMP-1, snake venom can immunotherapy targeted autoantibody rheumatoid arthritis, for such arthritis have it is unique
Curative effect.
Specific embodiment described herein is only an example for the spirit of the invention.Technology belonging to the present invention is led
The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method
In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.
Although term is used more herein, it does not preclude the possibility of using other terms.Use these terms
It is only for the convenience of describing and explaining the nature of the invention；Be construed as any one of the additional limitations all and be with
What spirit of that invention was disagreed.
Claims (4)
1. a kind of application of long-nosed pit viper snake venom in preparing anti-angiogenic medicaments, which is characterized in that the long-nosed pit viper snake venom conduct
Ang-1 inhibitor when anti-angiogenesis.
2. application of the long-nosed pit viper snake venom according to claim 1 in preparing anti-angiogenic medicaments, which is characterized in that this medicine
Object includes long-nosed pit viper snake venom and pharmaceutically acceptable carrier.
3. application of the long-nosed pit viper snake venom according to claim 2 in preparing anti-angiogenic medicaments, which is characterized in that described
Long-nosed pit viper snake venom be the freeze-dried powder formed by lyophilization.
4. application of the long-nosed pit viper snake venom according to claim 3 in preparing anti-angiogenic medicaments, which is characterized in that described
Freeze-dried powder be made containing long-nosed pit viper snake venom amount be 0.1-1.0mg/kg weight injection.
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| 尖吻蝮蛇毒活性肽 K组分的研究;雷丹青等;《时珍国医国药》;20071231;第18卷(第7期);第1608-1610页 * |
| 广西尖吻蝮蛇毒K组分抗血管生成活性研究;李映新等;《医药导报》;20110228;第30卷(第2期);第157-159页，尤其是第157页左栏第1段 * |
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