CN1903188B - Application of teprenone for preparing medicine for treating and/or preventing glaucoma - Google Patents
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- CN1903188B CN1903188B CN2006100891092A CN200610089109A CN1903188B CN 1903188 B CN1903188 B CN 1903188B CN 2006100891092 A CN2006100891092 A CN 2006100891092A CN 200610089109 A CN200610089109 A CN 200610089109A CN 1903188 B CN1903188 B CN 1903188B
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Abstract
An application of teprenone in preparing the medicine GGA for preventing and treating glaucomas is disclosed. Its advantages are high curative effect, high safety and low cost.
Description
Technical field
The present invention relates to novel application of compound, particularly relate to teprenone (Geranylgeranylacetone, GGA) preparation glaucoma prevention and/application in ("/" expression or) or the curative drug.
Background technology
Glaucoma be one group because absoluteness or the relative property intraocular pressure optic nerve injury disease due to raising.Effective control along with infectious diseases causing blindness, extensively carrying out of corneal transplantation, curing cataract operation, has glaucoma become modal irreversible diseases causing blindness (Foster PJ, Johnson GJ.Glaucoma in China:howbig is the problem in the global range? Br.J.Ophthalmol 2001; 85:1277-82.).
At present, the treatment means that raises at the glaucoma intraocular pressure mainly comprises medicine, laser, operation etc., and has obtained good curative effect in clinical practice.But, obtained good control even if find the intraocular pressure of glaucoma patient after deliberation, 40% carrying out property of the patient visual function of still having an appointment damages (Caprioli J.Neuroprotection of theoptic nerve in glaucoma.Acta Ophthalmol Scand.1997; 75:364-67.).
Studies show that, the glaucoma optic nerve lesion is meant by the ischemia due to pressure or the low blood perfusion pressure, anoxia etc. the optic nerve fiber axoplasmic flow is interrupted, cause the supply of target derived neurotrophic factor to interrupt retinal ganglial cells (retinal ganglion cells, the RGCs) death directly sustain damage.Owing to produce more excitotoxin,, activated some apoptosis-induced gene simultaneously, excited a series of tandem type reactions, finally caused dna break, caused that apoptosis takes place RGCs original complete around damaging by intracellular signal transduction.Therefore, treating glaucomatous key is to prevent the infringement of optic nerve and the apoptosis of RGCs.On the basis of control intraocular pressure, give optic nerve protection and be considered to the ideal therapeutic modality of glaucoma.
Present strategy mainly concentrates on blocking-up and starts apoptosis pathway and additional anti-apoptosis factor aspect.Because the startup of apoptosis is the process of a complexity, has numerous known paths and unknown path, so block any one path merely or several path can not stop glaucoma patient fully because the apoptosis of the retina cell that multiple factor causes.Though, aspect clinical practice, fail to obtain the convictive effect of tangible energy so this strategy has been obtained success at the experimental animal model and the experiment in vitro of various single factors.The supply treatment measure of anti-apoptosis factor comprises nerve growth factor supply therapy, and the using gene engineering method weakens apoptogene, and the strategy that strengthens anti-apoptotic genes expression has obtained certain success in zoopery.But how can continue for a long time, safety these factors are supplied with retinas and optic nerve is still an an open question.Yet, the problem that in the treatment research field of glaucomatous optic neuropathy, exists in addition extremely important and out in the cold at present.The optic nerve lesion that glaucoma caused not only comprises the retinal ganglion cell, also comprise other cell of retina, even more important problem is that the death of retinal ganglion cell finally can cause upper neuronal cell metabolic activity reduction, atrophy, even secondary lesion such as apoptosis, comprise LGN (Lateral geniculate nucleus, lateral geniculate body), occipital ctx neuron (Gupta, Neeru MD, Yucel YH.Glaucoma and the Brain. Journal of Glaucoma 2001; Supplement 1:S28-S29.).Evidence suggests, reduce nutritional support, induce RGCs born of the same parents' degeneration, damage (the Dandona L that can cause primary visual cortex and LGN, Hendrickson A, Quigley HA.Selective effects of experimental glaucoma on axonal transportby retinal ganglion cells to the dorsal lateral geniculate nucleus.InvestOphthalmol.Vis.Sci.1991; 32:1593-99; Anderson DR, Hendrickson A.Effect ofintraocular pressure on rapid axoplasmic transport in monkey optic nerve.Invest Ophthalmol 1974; 13:771-83.).As seen, after the optic nerve fiber loss, LGN and look the insecondary neuronal damage of cortex and can directly cause glaucomatous disease progression, and the RGCs that increases the weight of to survive is to the susceptibility of carrying out property injury.
The death of RGCs is to the neuronic influence of target, the target neuron of damage is to the influence of the ganglia retinae of survival, the damage that this mutual pathological effect can increase the weight of to have existed already, play an important role during sb.'s illness took a turn for the worse glaucomatous, this points out us to examine generation, the development mechanism that the glaucoma visual function is hindered again closely from whole aggregate level of looking the road, for clinical glaucomatous control provides new approaches.Therefore, seek and a kind ofly can act on whole pathways for vision, start the anti-damage of its endogenous neuron, the new therapeutic strategy of anti-apoptotic effect becomes the direction of glaucomatous optic neuropathy treatment research.
HSP claims stress protein again, is prevalent in prokaryotic cell and the eukaryotic cell, and under stress state, biological cell starts HSP gene, the most conservative albumen in the synthetic class biological evolution of encoding.According to its molecular weight, isoelectric point, IP difference, HSP is divided into: low-molecular-weight HSP family, HSP60 family, HSP70 family, HSP90 family and HSP110 family etc., each family has a plurality of member composition (GuptaM again, Vavasis C, Frishman WH.Heatshock proteins in cardiovascular disease a new therapeutic target.Cardio1.Rev.2004; 12:26-30.).
Lot of experiments shows that body is under stimulation states such as heat stress, anoxia, pathogen, cytokine and physics and chemistry harmful factor, and body cell will synthesize HSPs in a large number.HSPs expresses and increases, and cell is survived under stress state, and can promote the reparation of damaged cell self.Think that at present HSPs cytoprotective mechanism can be relevant with HSPs various biological function under the heat stress state, as help protein folding, assembling, to endoplasm Netease position and decompose misloading " molecular chaperones " functions such as albumen, improve the cell temperature capacity and participate in function of receptors etc.HSPs makes up the effect of playing stabilized cell by participating in cytoskeleton, can keep cell intermediate filament protein integrity under the stress state, prevents the expansion of cell protein, therefore can prevent apoptotic generation.In addition, HSPs can also suppress the generation of apoptosis by a plurality of aspects of apoptosis involvement approach, and for example: (1) is by suppressing the preceding Bcl-2 protein activation of apoptosis, the seepage and the release that causes antiapoptotic factors that stop mitochondrial outer membrane, thereby the generation of the endogenous apoptosis that the blocking-up mitochondrion relies on.
(2) blocking-up is by the exogenous apoptosis pathway of death receptor mediation.(3) by suppressing the active of caspase and, blocking the formation of apoptotic body to the apoptosis induced effect.(4) regulation and control participate in regulating some signal cascade courses of reaction of the key component in the apoptosis cascade reaction, comprise JNK, NF-
κ(Rokutan K.Role of heatshock proteins in gastric mucosal protect ion.J.Gastroenterol.Hepatol.2000 such as B and AKT; 15 Suppl:D12-D19.).That research is maximum in numerous HSP family members is (the Park KH of HSP70 family, Cozier F, Ong OC, Caprioli J.Induction of heat shock protein 72 protectsretinal ganglion cells in a rat glaucoma model.Invest Ophthalmol.Vis.Sci.2001; 42:1522-30.).
1988, can be observed the expression of HSP during report thermostimulations such as Barber in the retina, make sensor avoid the damage of light, and neuroprotective (the Barbe MF of HSPs proposed first, Tytell M, Gower DJ, Welch WJ.Hyperthermia protects against light damage in the rat retina.Science1988; 241:1817-20.).People have carried out a large amount of correlational studyes subsequently, found that: induce HSP70 to express increase or integrated the rat neuron that contains the HSP70 gene ischemia and damage are demonstrated higher toleration (KellyS, Yenari MA.Neuroprotection:heat shock proteins.Curr.Med.Res Opin.2002; 18 Suppl 2:s55-s60.).To cultivating that rat RGC carries out that heat (42 ℃) is handled and after the sublethal dose hypoxia handles, find that the two can induce all that HSP72's is synthetic, and the survival rate of GRC is far above the normal control group, prompting high temperature and sublethal dose hypoxia may be to produce by inductive endogenous HSP to have improved the toleration of RGC to anoxia and excitotoxin, thereby RGC is played a protective role, make it to avoid death (Caprioli J, Kitano S, Morgan JE.Hyperthermia and hypoxia increase tolerance of retinal ganglioncells to anoxia and excitotoxicity.Invest Ophthalmol.Vis.Sci.1996; 37:2376-81.).Calendar year 2001, Park etc. use rat glaucoma model, discovery can be induced the generation of endogenous HSP70 by heat stress and Zn agent, induce HSP72 can significantly improve survival rate (the Park KH of RGCs under the uncontrolled intraocular pressure in amphiblestroid expression in advance by heat stress and Zn agent, Cozier F, Ong OC, Caprioli J.Induction of heat shock protein 72 protects retinal ganglion cells in a ratglaucoma model.Invest Ophthalmol.Vis.Sci.2001; 42:1522-30.).This shows, may reach the purpose of control glaucoma optic neuropathy by inducing endogenous HSP.
But no matter be to adopt to improve body body temperature or cause the sublethal dose anaerobic condition all not to be suitable for the mankind with the means of bringing out this protective protein generation, also the someone proposes to adopt the warm stimulation of laser to induce retina to produce the strategy of above-mentioned protective protein (Mainster MA through pupil, Reichel E.Transpupillary thermotherapyfor age-related macular degeneration:long-pulse photocoagulation, apoptosis, and heat shock proteins.Ophthalmic Surg.Lasers 2000; 31:359-73.), still, this method can only be induced the generation of local this class protective protein of retina, and effect is not played in neuronic protection to whole pathways for vision.
Comprise relaying neuron and relay cell two big class neurons among the LGN.The neuronic aixs cylinder of relaying is projected to primary visual cortex, and the aixs cylinder of relay cell is confined among the LGN.A relaying LGN neuron can be accepted the input of a plurality of ganglionic cells, also accepts contiguous neuronic influence by relay cell.Little pure albumen is the neuronic specific marker albumen of labelling relaying, and alternative ground mark transfer vision information is to the relaying neuron of looking cortex.Little pure albumen is a kind of water miscible calbindin that has, acidity, and molecular weight is approximately 12kD.
(Geranylgeranylacetone is a kind of non-annularity isoprenoid complex GGA) to teprenone, now has been widely used in the treatment of digestive tract ulcer.
Summary of the invention
(Geranylgeranylacetone, new purposes GGA) promptly prevent and/or treat application in the glaucoma medicine in preparation to the purpose of this invention is to provide teprenone.
Application of the present invention is, and to be active component with GGA prevent and/or treat application in the glaucoma medicine in preparation.
When needing, in application of the present invention, can also add one or more acceptable accessories, described adjuvant comprises diluent, excipient, filler, binding agent, wetting agent, absorption enhancer, surfactant, lubricant, stabilizing agent of pharmaceutical field routine etc., also can add flavouring agent, sweeting agent and pigment etc. in case of necessity.
Application of the present invention except that making capsule, can also make tablet, powder, granule, oral liquid, injection, etc. multiple medicament forms.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field.
The oral consumption of the adult of this medicine is generally 50mg/ time, 3 times/day.
The invention provides the new purposes of a kind of GGA, promptly prevent and/or treat application in the glaucoma medicine in preparation.The zoopery result: 6 hours is that visible rat LGN position HSP 70 expresses rising behind (1) oral GGA, reaches the peak in the time of 24 hours, obviously reduces in the time of 48 hours.The expression of HSP70 increases along with the increase that gives GGA dosage, and is the most remarkable with 400mg/kg and two groups of 800mg/kg.It is 400mg/kg/d that GGA induces rat LGN position HSP 70 to express the best dosage that raises.(2) optic nerve cuts off and to add GGA treatment group and optic nerve and cut off the PV positive cell quantity that adds among the heat shock processed group bilateral dLGN and area and cut off than optic nerve cut-out group, optic nerve and add vehicle-treated groups and significantly increase.(3) optic nerve cuts off and to add GGA treatment group and optic nerve and cut off the PV positive cell quantity that adds among the heat shock processed group bilateral vLGN and area and cut off than optic nerve cut-out group, optic nerve and add vehicle-treated groups and significantly increase, and difference has significance meaning (p＜0.01).(4) optic nerve cuts off and to add GGA treatment group and optic nerve and cut off the CCX activity that adds among the heat shock processed group bilateral LGN and cut off than optic nerve cut-out group, optic nerve and add vehicle-treated groups and significantly increase, and difference has significance meaning (p＜0.01).(5) oral GGA or look heat shock and handle back 24 hours visible retina HSP 70 and express significantly and raise.(6) optic nerve cuts off and to add GGA treatment group and optic nerve and cut off and add heat shock processed group RGCs born of the same parents quantity and cut off than optic nerve cut-out group, optic nerve and add vehicle-treated groups and significantly increase, and difference has significance meaning (p＜0.01).(7) optic nerve cuts off and to add GGA treatment group and optic nerve and cut off and add heat shock processed group RGCs born of the same parents apoptosis quantity and cut off than optic nerve cut-out group, optic nerve and add vehicle-treated groups and significantly reduce, and difference has significance meaning (p＜0.01).Above-mentioned experimental result shows that oral GGA can effectively induce HSP70 expression in lateral geniculate body and the retina; and the rat optic nerve cut off back LGN neuron and RGCs born of the same parents have protective effect, therefore can GGA be that active component is prepared into and prevents and/or treats glaucomatous medicine.Compare with glaucoma treatment medicine commonly used clinically at present, medicine of the present invention has the following advantages: 1) curative effect is obvious, and total effective rate can reach more than 92.5%; 2) safe; 3) main route of administration is oral, and medication is convenient; 4) GGA is cheap, and the cost for preparing this medicine is lower, thereby can alleviate patient's financial burden.The present invention not only provides a new approach for glaucomatous control, also nervous system disease and carrying out property nervous system injury treatment of diseases and research is had reference function, having a extensive future of medicine and pharmacology field.
Below in conjunction with specific embodiment the present invention is described in further detail.
Description of drawings
Fig. 1 surveys the standard curve of protein quantification for the BCA method
Fig. 2 is Western blot and the semi-quantitative analysis result that oral vehicle, GGA and heat shock are handled rat LGN position, back HSP70 expression time effect
Fig. 3 is Western blot and the semi-quantitative analysis result that rat LGN position HSP70 expresses dosage effect behind the oral GGA
Fig. 4 is the immunohistochemistry testing result that oral vehicle, GGA or heat shock are handled rat LGN position, back HSP70 distribution situation
Fig. 5 is normal and optic nerve cuts off rat optical fundus, back
Fig. 6 is the immunohistochemical staining result of rat bilateral dLGN position PV positive cell distribution situation
Fig. 7 is the immunohistochemical staining result of rat bilateral vLGN position PV positive cell distribution situation
Fig. 8 is a rat lateral geniculate body position view
Fig. 9 A expresses the quantitative analysis results of quantity for PV positive cell among the operation homonymy dLGN
Fig. 9 B is the quantitative analysis results of PV positive cell area among the operation homonymy dLGN
Figure 10 A expresses the quantitative analysis results of quantity for PV positive cell among the operation offside dLGN
Figure 10 B is the quantitative analysis results of PV positive cell area among the operation offside dLGN
Figure 11 A expresses the quantitative analysis results of quantity for PV positive cell among the operation homonymy vLGN
Figure 11 B is the quantitative analysis results of PV positive cell area among the operation homonymy vLGN
Figure 12 A expresses the quantitative analysis results of quantity for PV positive cell among the operation offside vLGN
Figure 12 B is the quantitative analysis results of PV positive cell area among the operation offside vLGN
Figure 13 is the mitochondrial protein concentration standard curve
Figure 14 A is a rat operation homonymy LGN mitochondrion CCX expression
Figure 14 B is a rat operation offside LGN mitochondrion CCX expression
Figure 15 is the Western blot and the semi-quantitative analysis result of rat retina HSP70 expression behind the oral GGA
Figure 16 A cuts off the retrograde labelling result of FG of back RGCs for the rat optic nerve
Figure 16 B cuts off back RGCs counting and results of statistical analysis for the rat optic nerve
Figure 17 A cuts off retinal ganglial cells is detected in the back with dUTP breach latter end labelling method apoptosis situation for the rat optic nerve
Figure 17 B cuts off the results of statistical analysis of back retinal ganglion cells apoptosis number for the rat optic nerve
The specific embodiment
Method therefor is conventional method if no special instructions among the following embodiment.
Laboratory animal: healthy Sprague-Dawley (SD) rat, male, body weight 200-250g, available from Beijing dimension tonneau China experimental animal technology company limited, the animal quality certification is numbered SCXK (capital) 2002-0003.Illumination/12 hour dark condition was raised down in 12 hours.
GGA: available from Japanese Eisai Co., Ltd.
The influence that embodiment 1, the oral GGA of detection express rat lateral geniculate body position HSP70
One, Western blot detects oral GGA to rat lateral geniculate body (Lateral geniculate nucleus, LGN) influence of position HSP70 expression
1, Western blot detects oral vehicle, GGA and heat shock and handles the time graph that back rat LGN position HSP70 expresses
Experimental rat is divided into 2 groups at random: (the normal control group is n=6) with T group (processed group) for the N group; The T group is divided into again: VT group (excipient administration group), GT group (GGA administration group) and HT group (heat shock processed group).
Medication is: irritate stomach after GGA (400mg/kg) or excipient (containing 5% lactose and 0.008% vitamin E) are dissolved in the 5mL/kg normal saline, the normal control group is irritated stomach (5mL/kg) with normal saline.The heat shock processing method is: chloral hydrate (40mg/100g body weight) intraperitoneal anesthesia, after 5 minutes rat is placed the uncovered aluminum lunch box of 20 * 15 * 8 (cm), the lunch box that rat will be housed then is placed in 42 ℃ of water baths, timing 15min took out when measurement rat rectal temperature reached 40-42 ℃, treated under the room temperature that rat is clear-headed.
Respectively rat is carried out above-mentioned processing, in handling back 6h, 12h, 24h, 48h (each time point n=6) sacrificed by decapitation rapidly, get brain, separate bilateral LGN nuclear, frozen in the liquid nitrogen, detect oral vehicle, GGA and heat shock with Western blot and handle the time graph that back rat LGN position HSP70 expresses.The Western blot detection method may further comprise the steps:
(1) extraction of whole-cell protein
Above-mentioned frozen rat LGN tissue is transferred in the Eppendorf pipe, organize the ratio of 10 μ l to add full cell pyrolysis liquid (50mM TrisCl (pH 7.5) in every mg, 150mM NaCl, 5mM EDTA, 5mM EGTA, 1%SDS), with the homogenate of hand-held homogenizer, ultrasonication is dissolved histiocyte fully, being prepared into protein content is the electrophoresis sample of 5g/L, and-20 ℃ of preservations are standby.
(2) BCA method protein quantification
Adopt the BCA method to carry out protein quantification, the reactive group present principles is: under 37 ℃, alkaline environment condition, and peptide bond and Cu in the protein molecule
2+Form the tetramer of univalent copper ion, univalent copper ion forms a purple complex with dicinchonine acid (BCA) again, and this complex has the absorption maximum crest at wavelength 562nm place.Concrete grammar is: get 17 of eppendorf pipes, be numbered and dosing by table 1.After preparing, place 37 ℃ of water bath temperature to bathe 30 minutes, utilize the interior protein quantification program of BCA analysis of protein test kit (Pierce company) and ultraviolet spectrophotometer, the standard curve that is used to measure total protein content is seen Fig. 1.
The full total protein of cell standard curve liquid dosage of table 1
(3)SDS-PAGE
Get 10 μ l (50 μ g albumen) protein electrophoresis sample and molecular weight of albumen standard substance and carry out the 10%SDS-PAGE electrophoretic analysis jointly, deposition condition: 4 ℃, electric current 20-30mA.After electrophoresis finishes albumen is transferred to the NC film from the SDS-PAGE gel, changes the film condition: the aperture is the NC (Nitrocellulose filter, nitrocellulose filter, Bio-Rad company), 4 ℃ of 0.22 μ m, electric current 400mA, 3 hours time.
(4)Western-blot
The albumen that is transferred to the NC film is carried out Western-blot detect, concrete grammar may further comprise the steps:
1. the NC film is taken out, (pH7.5) middle rinsing is 5 minutes for 20mM Tris, 0.5M NaCl in the TBS buffer.
2. use 10% skim milk confining liquid (to take by weighing the 10g defatted milk powder, be dissolved among the 80mLTTBS (pH7.5), stir dissolving fully, the back adds TTBS and is settled to 100mL, add 10 μ l thiomersalates (Thimerosal, U.S. Sigma company) again, mixing) closing membrane 1 hour, reclaim confining liquid, to reuse.
3. after using distilled water flushing film 3 times, and reuse TTBS liquid (20mM Tris, 0.5M NaCl, 0.05%Tween-20, pH7.5) the rinsing film is three times, each 10 minutes.With HSP 70 monoclonal mouse antibody (Canadian Stressgen company) by 1: 1000 dilution proportion in 20mL TTBS, and add 0.01%Thimerosal.
4. the NC film after the rinsing is put into above-mentioned antibody diluent, hatch 3 hours under the room temperature after, reclaim antibody, preserve in 4 ℃ of environment, in order to repeated application.
5. use distilled water flushing film number all over after, wash film three times with TTBS liquid, each 10 minutes.With two anti-(lucky safe biotechnology company) of horseradish peroxidase-labeled by 1: 5000 dilution proportion in 20mL TTBS liquid, in hybridizing box, hatched film 1 hour.
6. abandon two anti-solution, use distilled water flushing film 5 times, wash film with TTBS liquid once more, totally 3 times, each 10 minutes.(annotate: above all processes are all carried out on shaking table)
7. also carry out following operation to specifications with Supersignal West Pico Chemiluminescent Substrate test kit: get each 1mL of luminous agent and reinforcing agent, mixed in 1: 1 ratio, pour in the hybridizing box that fills the NC film, with the moving hybridizing box of have gentle hands jog, make luminous agent and oxidant evenly mixed and fully contact with the film surface.
8. after 5 minutes, take out film, with the preservative film parcel, the emptying bubble is attached to the NC film on the magazine, enters the darkroom exposure.
9. get an X-mating plate, photosurface aimed at the NC film, be placed on film directly over, magazine closes, pick up counting, take out the X-mating plate after 1 minute, drop in the developer solution immediately, developed 5 minutes, and with water rinse X-mating plate for several times, then the X-mating plate was transferred in the fixative solution photographic fixing 10-20 minute.The X-mating plate that exposure finishes and can obtain clear HSP70 band, flowing water flushing 5 minutes, hang airing.
The Western blot testing result is seen the figure A (N: normal control group, VT: vehicle-treated group, GT:GGA processed group among Fig. 2, HT: the heat shock processed group), 6 hours is the expression rising of visible rat LGN position HSP70 behind the oral GGA, reaches the peak during 24h, obviously reduces during 48h; Visible rat LGN position HSP70 obviously expressed when heat shock was handled back 12h, 24 and during 48h expression descend, but still be significantly higher than normal level; The LGN position of matched group and excipient administration group does not see that the HSP70 expression changes.
The Quantity One image analysis software that above-mentioned Western blot testing result is used in the Gel-Doc gel imaging system is carried out semi-quantitative analysis, draw the density value of HSP70 band on the X-mating plate, expressing quantity with the normal control group is 1, with each experimental group HSP70 expressing quantity of ratio value representation of the density value of matched group HSP70 band; With β-actin is the density value that the Western blot result of confidential reference items measures HSP70 and corresponding β-actin band on the X-mating plate earlier, with β-actin is confidential reference items, calculate and respectively organize HSP70 expression (density value of the density value of HSP 70 bands/corresponding β-actin band), and then be 1 with the expressing quantity of normal control group, with each experimental group HSP70 expressing quantity of ratio value representation of the density value of matched group HSP70 band.Test data is carried out statistical procedures (SPSS11.5 software) with variance analysis, relatively uses q check between group, the result with
Expression, there is the significance meaning p＜0.05 for difference.The HSP70 expressing quantity is seen the figure B among Fig. 2, with N (the normal control group: 1) compare VT (vehicle-treated group), GT (GGA processed group) and HT (heat shock processed group) after processing when 6h, 12h, 24h, 48h the expressing quantity of rat LGN position HSP70 be respectively: vehicle group: (1.13 ± 0.10), (1.01 ± 0.24), (0.98 ± 0.14), (1.05 ± 0.23); GGA group: (4.19 ± 0.39) *, (7.24 ± 0.40) *, (17.28 ± 0.32) *, (2.53 ± 0.37) *; The heat treatment group: (0.99 ± 0.05), (15.01+0.52) *, (11.18+0.47) *, (9.12 ± 0.48) * (
*P＜0.01).Above-mentioned experimental result shows that GGA can induce HSP70 to express at the LGN position, makes that inducing endogenous protection mechanism to carry out the maincenter protection by safety, easy mode becomes possibility.HSP70 reached the peak in 24 hours after the GGA medication, obviously reduce in the time of 48 hours.
2, GGA induces the dose curve that rat LGN position HSP70 expresses
The dose curve that detection induces rat LGN position HSP70 to express with GGA, concrete grammar is: experimental rat is divided into 0 (contrast) at random, 100,200,400,800 (mg/kg of unit) GGA dosage group (each dosage group n=6), all rats in administration (medication see step 1) after 24 hours rapid sacrificed by decapitation, get brain, separate bilateral LGN nuclear, frozen in the liquid nitrogen, being used for Western blot detects, detection method and statistical analysis method are identical with step 1, wherein, it is anti-with the β-actin monoclonal mouse antibody amount of being ginseng when Western blot detects with one, being about to be used to detect film that GGA induces the dose curve that HSP70 expresses places and strips off liquid (62.5mM TrisCl (pH6.7), the 100mM2-mercaptoethanol, 2%SDS) 55 ℃ hatch 30-60min after, again carry out hybridization with β-actin monoclonal mouse antibody (1: 1000), all the other steps are identical.
The Western blot testing result is seen the figure A among Fig. 3, and the expression of HSP70 increases along with the increase that gives GGA dosage, and is the most remarkable with 400mg/kg and two groups of 800mg/kg.
Sxemiquantitative and statistic analysis result are seen the figure B among Fig. 3, compare with matched group, various dose GGA induces the proteic expression of rat LGN position HSP70 to be: (4.19 ± 0.39) *, (11.33 ± 0.52) *, (17.20 ± 0.45) *, (17.00 ± 0.14) * (* p＜0.01).100 or 200mg/kg and 400 or 800mg/kg between respectively comparing difference significance meaning (#p＜0.01) is arranged, the differential expression of HSP70 does not have the significance meaning between 400mg/kg and the 800mg/kg dosage group.
Above-mentioned experimental result shows that GGA can induce HSP70 to express at the LGN position, and 400mg/kg and 800mg/kg GGA dosage group induce the amount of HSP70 to be significantly higher than other group, but there was no significant difference between two groups.In addition, be 396.25mg/kg according to the standard dose of human therapy gastric ulcer by the consumption that bulk area converses rat, extremely near 400mg/kg, therefore with 400mg/kg/d as preferred GGA dosage.
Two, immunohistochemical method detects branch's situation that oral vehicle, GGA or heat shock are handled back rat LGN position HSP70
Immunohistochemical method detects branch's situation that oral vehicle, GGA or heat shock are handled back rat LGN position HSP70.Experimental rat is divided into 2 groups at random: (the normal control group is n=6) with T group (processed group) for the N group; The T group is divided into again: (GGA administration group is 400mg/kg) with HT group (heat shock processed group) for VT group (excipient administration group), GT group.Administration and heat shock processing method are identical with step 1.With above-mentioned processing deep anaesthesia rat in the time of back 24 hours, left ventricle ascending aorta intubate, pour into normal saline fast, blood-letting is cut off in the right auricle, treat that trickle is limpid after, pour into 4% paraformaldehyde fixative again and (take by weighing the 40g paraformaldehyde, place conical flask, add 500-800mL 0.1mol/L PBS (NaCl 0.8g, KCl 0.2g, KH
2PO
40.24g, Na
2HPO
41.44g, be dissolved in successively in the 900mL distilled water, after the dissolving, pH value is transferred to 7.5, after add water to 1000mL), be heated to about 60 ℃, continue to stir (or magnetic agitation) powder dissolved fully, usually needing to drip a little 1mol/L NaOH just can make solution limpid, the PBS that supplies 0.1mol/L at last is in 1000mL, fully mixing) 300mL, round brain, the paraffin section of preparation superior colliculus and LGN position carries out immunohistochemical staining, may further comprise the steps:
Roasting sheet is put into dimethylbenzene with section after 2 hours and is taken off each 15 minutes cured 2 times in (1) 60 ℃ of baking box.
(2) take off benzene with 100%, 95%, 85%, 75% gradient ethanol.At different levels is 2-5 minute.Distilled water flushing 2 times, each 5 minutes.
(3) use 3%H
2O
2The sealing endogenous peroxydase, 15 minutes, lucifuge.Distilled water flushing, 0.1M PBS (pH7.5) washed 5 minutes
(4) slide is placed the slide box that fills PBS, microwave reparation (95 ℃, 5 minutes) naturally cools to room temperature.10% normal goats serum room temperature sealing 15min, the serum deprivation that inclines is not washed.
(5) in the wet box of HSP 70 monoclonal mouse antibody (1: 100) 4 ℃ spend the night, replace an anti-section of hatching to make negative control with PBS.
(6) next day, PBS washes section, 3 * 5 minutes.Drip IgG (available from the safe biotechnology of the Ji company) working solution of biotinylated anti-mice, incubated at room 1 hour.PBS washes section 3 times, each 5 minutes.
(7) drip the strepto-avidin working solution (available from from lucky safe biotechnology company) of horseradish peroxidase-labeled, incubated at room 30 minutes.PBS washes section 3 times, each 5 minutes.
(8) with new DAB (DAB 6g, PBS 10mL, the 30%H for preparing
2O
210mL, fully mixing), light microscopic colour developing down.Fully wash with tap water, haematoxylin is redyed nucleus.
(9) 100%, 95%, 85%, 75% gradient alcohol dehydration, dimethylbenzene is transparent, and neutral gum (chemical plant, northern suburbs, Beijing City) mounting, microscopically are observed and are taken a picture.
The immunohistochemical staining result is (normal control group (A) as shown in Figure 4, vehicle-treated group (B), GGA processed group (C), heat shock processed group (D), scale=20 μ m), normal control group and vehicle-treated group are not seen obvious HSP70 positive cell, give GAA 400mg/kg or heat shock and handle after 24 hours, as seen be dispersed in the pale brown color HSP70 positive expression cell (shown in the arrow) of distribution at rat LGN tissue slice, prove that further GGA can induce HSP70 to express at the LGN position.
The oral GGA that experimental results show that implements in 1 can induce HSP70 to express at the LGN position, to have neuroprotective still indeterminate but GGA induces HSP70 whether to raise the LGN that optic nerve is cut off model, with little pure albumen (Parvalbumin, PV) labelling relaying neuron (Yucel YH optionally, Zhang Q, Gupta N, Kaufman PL, Weinreb RN.Loss of neurons in magnocellular and parvocellular layers of the lateralgeniculate nucleus in glaucoma.Arch.Ophthalmol 2000; 118:378-84.), by distribution situation and the quantity and the area of detection PV positive cell, and measure mitochondrial cytochrome oxidase (CCX) (the Wong-Riley MT.Cytochrome oxidase:an endogenousmetabolic marker for neuronal activity.Trends Neurosci.1989 that indicates neuron endogenous metabolism level; 12:94-101.) activity, detect GGA to optic nerve transection after the neuronic influence of LGN, specifically test as follows:
One, immunohistochemical method detects branch's situation of rat lateral geniculate body PV
At random rat is divided into: N (normal control group), T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), (optic nerve cuts off and adds GGA treatment group T+G, 400mg/kg/d), T+H (optic nerve cut off add heat shock processed group), each organizes n=12.Rhizoma Atractylodis Macrocephalae administration in preceding 24 hours once a day, continues 28 days after the operation; The heat shock processed group: preceding 24 hours of Rhizoma Atractylodis Macrocephalae once, after the operation week about once, continue 28 days, administration and heat shock processing method are identical with embodiment 1, following (the Clarke DB of optic nerve cutting-off method, Bray GM, Aguayo AJ.Prolonged administration of NT-4/5fails to rescue most axotomized retinalganglion cells in adult rats.Vision Res.1998; 38:1517-24.):
1. use chloral hydrate (40mg/100g body weight) intraperitoneal anesthesia rat, sterile drape;
2. cut off skin along margo orbitalis on the left eye, passivity is separated soft tissue to lachrymal gland, and the lachrymal gland major part is wiped out, and continues to be separated to superior rectus, and behind the disconnected superior rectus, passivity is separated backward, exposes optic nerve initial part and optic nerve;
3. stringer teasing optic nerve adventitia is cut off optic nerve fully in the position of the about 1mm of distance optic nerve initial part, and whole process will avoid damaging the Zinn's artery that is positioned at the optic nerve below;
4. eyeball resets, and sews up wound, flattens cornea with coverslip and observes optical fundus blood confession.Be coated with the antibiotic eye ointment, operation finishes.Check postoperative rat optical fundus, see Fig. 5 (A: normal optical fundus, B: optical fundus after optic nerve cuts off), will exist the rat of persistence retinal ischemia to reject.
In the time of back 28 days, respectively organize and get 6 rats respectively by deep anaesthesia in above-mentioned processing for rat, left ventricle ascending aorta intubate is poured into normal saline fast, and blood-letting is cut off in the right auricle, after treating that trickle is limpid, pour into 4% paraformaldehyde fixative 300mL again, round brain and put into 4 ℃ of 4% paraformaldehyde fixatives fixedly 12-24 hour, paraffin embedding, at the about 200um in every interval, LGN place, as thickness is the serial section of 7um, totally 6, is used for immunohistochemical analysis.The LGN of rat mainly is divided into dLGN (Dorsal lateral geniculate nucleus, lateral geniculate body dorsal part nuclear), vLGN (Ventral lateral geniculate nucleus, the lateral geniculate body dorsal part is examined) and IGL (Intergeniculate leaf, the intermediate layer) three parts, position view are seen Fig. 8 (Paxinos G, WatsonC.The rat brain in stereotaxic coordinates, Academic, San Diego, Ca, 1986).Now the method with immunohistochemical staining detects above-mentioned different disposal group rat lateral geniculate body PV at dLGN, vLGN and IGL branch situation, one anti-for the PV monoclonal mouse antibody (by 1: 200 dilution proportion, U.S. AffinityBioReagents company), two anti-are the IgG of biotinylated anti-mice (available from the safe biotechnology of Ji company) working solution, three anti-are the strepto-avidin working solution of horseradish peroxidase-labeled (available from from lucky safe biotechnology company), concrete steps are referring to embodiment 1, replace an anti-Incubating Solution with 0.1mol/L PBS, with the section of same procedure preparation as negative control.
The immunohistochemical staining result shows that the PV cell mainly is distributed among the dLGN and vLGN of LGN, the dyed dark-brown that is of PV positive cell, the cell space form differs, karyon is light to be dyed, the part cell process is obvious, the distribution situation of the PV positive cell at rat bilateral dLGN and vLGN position such as Fig. 6, (operation homonymy: A, C, E, G, I shown in Figure 7; Operation offside: B, D, F, H, J.A-B normal control group; C-D optic nerve cut-out group; The E-F optic nerve cuts off and adds vehicle-treated groups; The G-H optic nerve cuts off and adds GGA treatment group; The I-J optic nerve cuts off and adds the heat shock processed group.Scale=10 μ m), both compare, and the PV positive cell quantity among the vLGN is more.
Two, the PV positive cell number and the areal analysis thereof of dLGN, vLGN operation homonymy and offside
The immunohistochemical staining result of step 1 shows that the expression quantity variance of PV positive cell in dLGN and vLGN two positions is very big, so carry out date processing respectively: under high power lens, the PV positive cell that is positioned at dLGN, vLGN operation homonymy and offside is counted, calculated every mm
2Cell number, and use Leica Qwin image analysis software and carry out the measurement of LGN cell area, average neuron size tried to achieve.Simultaneously test data is used variance analysis and carries out statistical procedures (SPSS11.5 software), relatively use the q check between group, the result with
Expression, there is the significance meaning p＜0.05 for difference.
Wherein, the quantitative analysis results of PV positive cell expression quantity is seen (#p＜0.01) shown in Fig. 9 A among the operation homonymy dLGN, and the PV positive cell number among N (normal control group), T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups) T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the operation homonymy dLGN is followed successively by: (55.24 ± 3.32/mm
2), (44.33 ± 3.83/mm
2) *, (43.33 ± .27/mm
2) *, (51.33 ± 3.26/mm
2) * *, (50.83 ± 2.86/mm
2) * * (compares with matched group
*P＜0.01,
*P＜0.05), PV positive cell number among T+G (optic nerve cuts off and adds GGA treatment group) and T+H (optic nerve cuts off and adds the heat shock processed group) the homonymy dLGN significantly increases than T (optic nerve cut-out group) and T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (p＜0.01).The quantitative analysis results of PV positive cell area is seen (#p＜0.01) shown in Fig. 9 B among the operation homonymy dLGN, and the PV positive cell area among N, T, T+V, T+G and the T+H group operation homonymy dLGN is followed successively by: (180.437 ± 38.93/ μ m
2), (140.582 ± 17.924/ μ m
2) *, (141.439 ± 13.381/ μ m
2) *, (161.282 ± 21.257/ μ m
2) * *, (160.830 ± 25.269/ μ m
2) * * (compares with matched group
*P＜0.01,
*P＜0.05), PV positive cell area among T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the homonymy dLGN significantly increases than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (p＜0.01).
The quantitative analysis results of PV positive cell expression quantity is seen (#p＜0.01) shown in Figure 10 A among the operation offside dLGN, and the PV positive cell number among N, T, T+V, T+G and the T+H group operation offside dLGN is followed successively by: (55.24 ± 3.32/mm
2), (40.666 ± 2.50/mm
2) *, (41 ± 2.607/mm
2) *, (49.16 ± 4.91/mm
2) *, (48.5 ± 3.209/mm
2) * (compares with matched group
*P＜0.01), PV positive cell number among T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the offside dLGN significantly increases than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (p＜0.01).The quantitative analysis results of PV positive cell area is seen (#p＜0.01) shown in Figure 10 B among the operation offside dLGN, and the PV positive cell area among N, T, T+V, T+G and the T+H group operation offside dLGN is followed successively by: (180.437 ± 38.93/ μ m2), (134.54 ± 17.150/ μ m
2) *, (134.112 ± 11.203/ μ m
2) *, (160.837 ± 24.711/ μ m
2) *, (158.112 ± 21.198/ μ m
2) * (compares with matched group
*P＜0.01), PV positive cell area among T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the offside dLGN significantly increases than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (p＜0.01).
The quantitative analysis results of PV positive cell expression quantity is seen (#p＜0.01) shown in Figure 11 A among the operation homonymy vLGN, and the PV positive cell number among N, T, T+V, T+G and the T+H group operation homonymy vLGN is followed successively by: (325.16 ± 6.79/mm
2), (302 ± 9.89/mm
2) *, (303.83 ± 11.44/mm
2) *, (314.66 ± 8.59/mm
2) * *, (314.33 ± 5.16/mm
2) * * (compares with matched group
*P＜0.01;
*P＜0.05), PV positive cell number among T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the homonymy vLGN significantly increases than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (p＜0.01).The quantitative analysis results of PV positive cell area is seen (#p＜0.01) shown in Figure 11 B among the operation homonymy vLGN, and the PV positive cell area among N, T, T+V, T+G and the T+H group operation homonymy vLGN is followed successively by: (192.997 ± 46.851/ μ m
2), (151.160 ± 18.627/ μ m
2) *, (151.010 ± 16.682/ μ m
2) *, (173.375 ± 18.035/ μ m
2), (172.082 ± 24.805/ μ m
2) * * (compares with matched group
*P＜0.01;
*P＜0.05), PV positive cell area among T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the offside dLGN significantly increases than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (p＜0.01).
The quantitative analysis results of PV positive cell expression quantity is seen (#p＜0.01) shown in Figure 12 A among the operation offside vLGN, and the PV positive cell number among N, T, T+V, T+G and the T+H group operation offside vLGN is followed successively by: (325.16 ± 6.79/mm
2), (302.66 ± 9.309/mm
2) *, (303 ± 11.436/mm
2) *, (312.33 ± 4.546/mm
2) *, (312.33 ± 4.457/mm
2) * (compares with matched group
*P＜0.01).PV positive cell number among T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the offside vLGN significantly increases than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (p＜0.01).The quantitative analysis results of PV positive cell area is seen (#p＜0.01) shown in Figure 12 B among the operation offside vLGN, and the PV positive cell area among N, T, T+V, T+G and the T+H group operation offside vLGN is followed successively by: (192.997 ± 46.851/ μ m
2), (130.825 ± 9.083/ μ m
2) *, (135.153 ± 12.880/ μ m
2) *, (168.803 ± 24.027/ μ m
2), (167.796 ± 27.106/ μ m
2) * * (compares with matched group
*P＜0.01), PV positive cell area among T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the offside dLGN significantly increases than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (p＜0.01).
Compare with matched group; optic nerve cuts off back LGN relaying neuron number to be reduced; and the relaying neuron number of GGA administration group and heat shock processed group raises than surgery alone group and mouthful vehicle group; and optic nerve cuts off the not only neuronic quantity minimizing of relaying among the LGN of back; the area of cell also reduces; the neuronic area of the relaying of GGA administration group and heat shock processed group increases than surgery alone group and mouthful vehicle group, shows that GGA cuts off the LGN damage that causes to optic nerve and has protective effect.
Three, detect mitochondrion cytochrome oxidase activity in the rat lateral geniculate body
Cytochrome oxidase is present in mitochondrial inner membrane, is the terminal enzyme of respiratory chain, plays a decisive role in the ATP generative process.Neuron is well differentiated high energy consumption cell, is rich in mitochondrion.The aerobic that the high-energy phosphate compound of cerebral tissue forms main dependence glucose decomposes, and the consumption process of organism 90% molecular oxygen is all located to finish at mitochondrion cytochrome oxidase (CCX), so, CCX is guaranteeing the brain neuron normal energy metabolism, is keeping in the process of its normal function and play an important role, and CCX vigor and neuronic activity are proportionate, and are the signs of neuron endogenous metabolism.Oral GGA induces LGN position mitochondrial cytochrome oxidase active to raise, and whether the damage that optic nerve cuts off the LGN that causes is had protective effect to detect GGA.
With experimentize grouping and handle of the method identical with step 1, when handling back 28 days, rat is carried out deep anaesthesia, each group is got 6 rats respectively, the sacrificed by decapitation rat, extract test kit (Beijing Bao Sai Bioisystech Co., Ltd) and extract brain mitochondria with mitochondrion, and measure mitochondrion concentration with the BCA method according to the test kit description.
1, sets up mitochondrial protein concentration determination standard curve
Need to set up mitochondrial protein concentration determination standard curve before the concentration determination, method is: use the method identical with embodiment 1 to set up mitochondrial protein concentration determination standard curve: get 17 of Eppendorf pipes, be numbered and dosing by table 2.After preparing, place 37 ℃ of water bath temperature to bathe 30 minutes, use quant program in BCA assay kit and the ultraviolet spectrophotometer then, draw a mitochondrial protein concentration standard curve (seeing Figure 13), standby.
Table 2 mitochondrial protein concentration standard curve liquid dosage
2, cytochrome C oxidase determination of activity
Measure above-mentioned respectively organize rat brain mitochondria concentration after, with cytochrome C oxidase assay kit (U.S. sigma company) mensuration cytochrome C oxidase (CCX) activity wherein, concrete grammar is: earlier the 0.95mL analysis buffer is added cuvette, carry out the spectrophotometer zeroing.Then, add an amount of enzyme or mitochondrial suspension and 1 * enzyme dilution buffer liquid and make total liquid measure reach 1.05mL, and mixing.Add 50 μ l reduced form ferrocyanide C solution, mixing, reaction beginning.Begin after 5 seconds to measure.The 10 seconds spectrophotometers in every interval are measured once automatically, and totally six times, record data.Last following formula (the formula I that provides according to sigma company, Δ A represents: A/min (sample)-A/min (blank), dil represents: the enzyme or the dilution of sample factor) calculate enzymatic activity, be 100% with matched group CCX activity, represent each experimental group CCX live vol with percent.Test data is used variance analysis and is carried out statistical procedures (SPSS11.5 software), relatively uses the q check between group, the result with
Expression, there is the significance meaning p＜0.05 for difference.
Enzymatic activity
Formula I
The active testing result of rat operation homonymy geniculate body position CCX is seen Figure 14 A (#p＜0.01), compare with N (normal control group 100%), CCX activity level among T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the operation homonymy LGN is respectively: (57.07 ± 2.85) %*, (57.24 ± 3.84) %*, (88.36 ± 2.41) %*, (87.36 ± 3.72) %*, difference has significance meaning (* p＜0.01).The CCX activity that compares between group among T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the homonymy LGN significantly increases than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (p＜0.01).
The active testing result of rat operation CK shape body region CCX is seen Figure 14 B (#p＜0.01), compare with N (normal control group 100%), CCX activity among T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the operation offside LGN descends successively: (47.17 ± 4.00) %*, (47.46 ± 1.44) %*, (74.10 ± 4.50) %*, (72.77 ± 4.07) %*, difference has significance meaning (* p＜0.01).The CCX activity that compares between group among T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) the offside LGN significantly increases than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (#p＜0.01).
Above-mentioned experimental result shows that optic nerve cuts off the active obviously decline of back LGN cytochrome oxidase; neuronic active reduction of prompting LGN; the LGN cytochrome oxidase of GGA administration group and heat shock processed group is active obviously to raise, and further specifies the GGA LGN damage that cut-out causes to optic nerve and has protective effect.
It is that death of carrying out property of retinal ganglial cells (RGCs) and optic nerve fiber are lost that glaucoma causes the pathologic basis of visual function damage; the death of RGC often causes the infringement of visual function generation irreversibility; therefore, the key of glaucoma treatment is to stop optic nerve lesion, the protection visual function.Embodiment 2 experimental results show that GGA cuts off the LGN damage that causes to optic nerve and has protective effect; but whether GGA also can play a protective role to cut off inductive RGCs damage by optic nerve, now detects oral GGA cuts off back RGCs to the rat optic nerve influence with following experiment.
One, detects the influence that oral GGA expresses rat retina HSP70
Experimental rat is divided into 4 groups at random: (the GGA processed group, 400mg/kg) and H (heat shock processed group), each organizes n=6 for N (normal control group), V (vehicle-treated group), G.Rat is in handling back 24 hours rapid sacrificed by decapitation for excipient, GGA or heat shock, get eyeball, separate retina of both eyes, frozen in the liquid nitrogen, detect rat retina HSP70 expression with Western blotting, and the HSP70 expression carried out semi-quantitative analysis, concrete experimental technique is seen embodiment 1.
The Western blot of rat retina HSP70 expression the results are shown in the figure A among Figure 15, and oral GGA of rat or heat shock are handled back 24 hours visible retina HSP70 and expressed rising.The semi-quantitative analysis of rat retina HSP70 expression the results are shown in the figure B among Figure 15, compare (N: 1) with normal group, (the GGA processed group, the expression of HSP70 is respectively 400mg/kg) and in H (heat shock processed group) retina: (0.96 ± 0.12), (12.31 ± 0.68) *, (11.82 ± 0.75) * (* p＜0.01) for V (vehicle-treated group), G.
Above-mentioned experimental result shows that oral GGA can induce HSP70 in amphiblestroid expression safely, effectively, thereby RGCs is played a protective role.
Two, detect oral GGA cuts off the back retinal ganglial cells to the rat optic nerve influence
1, the retrograde labelling method counting of FG rat retinal ganglion cell
Rat is divided into 5 groups at random: N (normal control group), T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), T+G (optic nerve cuts off and adds GGA treatment group, GGA 400mg/kg), T+H (optic nerve cuts off and adds the heat shock processed group).Rhizoma Atractylodis Macrocephalae administration in preceding 24 hours, once a day later on.Heat shock is handled: preceding 24 hours of Rhizoma Atractylodis Macrocephalae once, later on week about once.Concrete administration and processing method are seen embodiment 1.The method for building up that optic nerve cuts off model is: chloral hydrate (40mg/100g body weight) intraperitoneal anesthesia, sterile drape, cut off skin and soft tissue along margo orbitalis on the left eye, expose optic nerve initial part and optic nerve, stringer teasing optic nerve adventitia, position at the about 1mm of distance optic nerve initial part is cut off optic nerve fully, when cutting off optic nerve, operation keeps somewhere the FG (Flurogold that a fritter is soaked with 50g/L in the socket of the eye side broken ends of fractured bone, fluorogold, U.S. Affinity company) gelfoam (Beijing chemical reagents corporation), otch resets, and sews up.The whole surgery process will avoid damaging the Zinn's artery that is positioned at the optic nerve below.
The method of the above-mentioned RGCs that respectively organizes rat being carried out the retrograde labelling of the retrograde labelling method of FG is: after the Animal Anesthesia, fixedly rat is on stereotaxic instrument (Japanese Narishige company), calvarium median line cutting perpendicularly scalp between ear, separate exposure skull sagittal suture and lambdoid suture, find the Bregma point, determine the bilateral superior colliculus) according to the rat brain stereotaxic atlas, every side superior colliculus inject 2 points (behind the bregma-5.9mm and-6.4mm, 1.4mm is opened on the side, dark 0.4mm), every some injection 1.5 μ l.Thoroughly hemostasis, pericranium, aponeurosis (aponeuroses) and skin are sewed up in layering.When handling back 14 days, each group is got 6 rats, and anesthesia is got eyeball after putting to death animal, fixes 1 hour after the paraformaldehyde solution lucifuge of immersion 40g/L.Wipe out cornea through 3 tailing edge limbus of corneae of PBS rinsing, separate retina, be tiled on the microscope slide, do the little otch in several places in retinal periphery and be convenient to calmly, use Vectashield (Vector company) mounting at last.
The above-mentioned FG that respectively organizes rat RGCs drives in the wrong direction, and (A-E figure is respectively the labelling result: N (normal control group), T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) shown in Figure 16 A, scale=30 μ m), as seen the retrograde labelling of FG can come out the rat retinal ganglion cell labelling well, and labelling is evenly clear.
Under fluorescence microscope, the above-mentioned RGCs that respectively organizes rat is counted, concrete grammar is: be the center with the optic disc on by the coordinate axes of looking nipple, respectively on the retina nose, under the nose, on the temporo, temporo is down apart from getting center 1mm (rear portion), 2mm (middle week), 3mm (periphery) locates, under the same microscopic fields of view high power field (400 *), RGCs number (Caprioli J in each visual field of manual counting, Ishii Y, Kwong JM.Retinal ganglion cellprotection with geranylgeranylacetone, a heat shock protein inducer, in a ratglaucoma model.Trans.Am.Ophthalmol Soc.2003; 101:39-50.).It is RGCs that circular cell space, diameter surpass 8Lm, and form is irregular, diameter is less is microglia, will not count (Cheng L, Sapieha P, Kittlerova P, Hauswirth WW, Di PA.TrkB gene transfer protectsretinal ganglion cells from axotomy-induced death in vivo.J.Neurosci.2002; 22:3977-86.), be 100% with matched group RGCs number, represent the survival volume of each experimental group RGCs with percent, simultaneously count results is carried out statistical analysis.
RGCs counting and results of statistical analysis (#p＜0.01) shown in Figure 16 B, compare with N (normal control group 100%), RGCs survival volume in T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and the adds the heat shock processed group) retina: (11.33 ± 0.99) %*, (11.39 ± 0.39) %*, (46.16 ± 4.10) %*, (47.91 ± 1.67) %*, difference has significance meaning (* p＜0.01).The RGCs survival volume that compares between group in T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and the adds the heat shock processed group) retina significantly increases than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), and difference has significance meaning (p＜0.01).
Studies show that RGCs cuts off back 4-5d in optic nerve usually and begins death, postoperative time lengthening conveniently, the RGCs number constantly reduces, RGCs number 7d after operation is about 50% of positive constant, about 90% RGCs death (Cheng L, Sapieha P, Kittlerova P during the 14d of operation back, Hauswirth WW, Di PA.TrkB gene transferprotects retinal ganglion cells from axotomy-induced death in vivo.J.Neurosci.2002; 22:3977-86.).Above-mentioned experimental result shows that the quantity of optic nerve cut-out back RGCs is 11.3% of normal control group approximately; consistent with existing result of study; and the simple optic nerve of quantity of GGA administration group and the heat shock processed group optic nerve RGCs after cutting off cuts off and neural cutting adds vehicle group and significantly increases, and proves that GGA has protective effect to RGCs.
2, dUTP breach latter end labelling method detects the apoptosis situation of retinal ganglial cells
Studies show that cutting off optic nerve can bring out a large amount of RGCs apoptosis, the experiment of embodiment 1 has also obtained further checking to this, but cause apoptosis former carry on as before unclear, studies show that may be relevant with following reason: the interruption of target derived neurotrophic factor, excessively release of excitatory amino acid, oxygen-derived free radicals and NO (nitric oxide, nitric oxide) etc. to neuronic toxic action, in addition, the state of neuron self also can affect the nerves the unit survival; Impaired RGCs may also be one of reason of RGCs apoptosis (Marcic TS to the reactivity reduction of target derived neurotrophic factor, Belyea DA, Katz B.Neuroprotection in glaucoma:a model for neuroprotectionin optic neuropathies.Curr.Opin.Ophthalmol 2003; 14:353-56.), now use TdT mediated dUTP nick end labeling breach end-labelling (Terminal deoxynucleotidyl transferase-mediated dUTPnick-end labeling) to detect optic nerve and cut off retinal ganglial cells ground, back apoptosis situation.
With experimentize grouping and handle of the method identical with step 1, each organizes rat deep anaesthesia when handling back 7 days, left ventricle ascending aorta intubate, pour into normal saline fast, blood-letting is cut off in the right auricle, after treating that trickle is limpid, pour into 4% paraformaldehyde 300mL again, getting eyeball puts into 4% paraformaldehyde and spends the night for 4 ℃, paraffin embedding, preparation are carried out TUNEL dyeing (TUNEL test kit through 4 μ m paraffin sections of optic nerve to it, Germany Roche company) and add up RGCs apoptosis number, concrete grammar may further comprise the steps:
(1) the conventional dewaxing of section, entry;
(2) dry moisture around the sample, add E.C. 3.4.21.64 (20 μ g/mL are dissolved among the TrisHCL, pH7.4-8.0) 50 μ l/ sheets, room temperature was placed in the wet box 10 minutes;
(3) PBS liquid cessation reaction is washed 3 times, each 5min;
(4) add TUNEL reactant mixture (mixture of reagent 1 and reagent 2, test kit carries) 50 μ l/ sheets, add behind the mixing on the sample strip, place wet box, room temperature labelling 60 minutes;
(5) PBS liquid cessation reaction is washed 3 times, each 5min;
(6) dry sample moisture on every side, add transforming agent-POD liquid 50 μ l/ sheets, room temperature reaction 30 minutes;
(7) PBS liquid cessation reaction is washed 3 times, each 5min;
(8) use freshly prepared 0.03%H
2O
2-0.5mg/mL DAB colour developing about 10 minutes, microscopically is observed the colour developing situation;
(9) haematoxylin is redyed;
(10) dehydration is transparent, conventional mounting, microscopy.
Apoptosis is criterion as a result: the apoptosis positive be endochylema and (or) after birth sepia dyeing, count apoptosis RGCs number in every section, each retina counting 3 section (n=6), calculate RGCs apoptotic cell sum, try to achieve the RGCs apoptosis number of average every section, and test data is used variance analysis carries out statistical procedures (SPSS11.5 software), relatively use the q check between group, the result with
Expression, there is statistical significance p＜0.05 for difference.
(A-E figure is respectively the TUNEL coloration result of RGCs: N (normal control group), T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and adds the heat shock processed group) shown in Figure 17 A, scale=10 μ m), the TUNEL positive cell be endochylema and (or) after birth sepia dyeing.Statistic analysis result is (#p＜0.01) shown in Figure 17 B, apoptosis cell is respectively (42.28 ± 5.15) *, (39.83 ± 5.63) *, (21.56 ± 4.12) *, (20.72 ± 3.27) * in T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and the adds the heat shock processed group) retina, and difference has significance meaning (* p＜0.01).Compare (1 ± 0.69) compares difference significance meaning (* P＜0.01) is arranged with N (normal control group).Between group relatively in T+G (optic nerve cuts off and adds GGA treatment group), T+H (optic nerve cuts off and the adds the heat shock processed group) retina apoptosis cell significantly reduce than T (optic nerve cut-out group), T+V (optic nerve cuts off and adds vehicle-treated groups), difference has significance meaning (p＜0.01).
Above-mentioned experimental result shows that the quantity of RGCs born of the same parents' apoptosis of GGA administration group and heat shock processed group reduces than surgery alone group and mouthful vehicle group; further proof GGA or heat shock are handled RGCs are had protective effect, and reason may be relevant with the inhibitory action of HSP70 pair cell apoptosis.
To be diagnosed as early, the patient of advanced glaucoma carries out Preliminary Clinical Observation after taking GGA, comprise curative effect and untoward reaction etc., successively early stage and patients with terminal is observed to glaucoma, and carried out preliminary analysis, the result shows, polypeptide compound of the present invention to early, advanced glaucoma curative effect preferably all arranged, wherein particularly outstanding, and do not see obvious adverse reaction to early stage curative effect.
Claims (1)
1. the application of teprenone in the medicine of lateral geniculate body neuron infringement due to preparation prevents and/or treats glaucoma.
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