CN101108180A - Application of gingkgo lactones B in preparing medicament for preventing and curing retinosis illness - Google Patents
Application of gingkgo lactones B in preparing medicament for preventing and curing retinosis illness Download PDFInfo
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Abstract
The invention relates to the application of gingko lactone B in preventing retinosis disease. The gingko lactone B has protective effect for the damage to the retina photoreceptor cell and damage to visual performance of bandicoot resulted from retinosis and the effect relies on the dosage. Therefore, the invention provides a new and effective treatment means of retinosis disease.
Description
Technical field
The present invention relates to the application of ginkalide B in the preparation opthalmological, relate to the application of ginkalide B in the medicine of preparation control retinal degenerative disease specifically.
Background technology
Along with the control of infectious disease, the retinal degeneration disease has become one of main diseases causing blindness.According to estimates, at present about in the world 2 * 10
7The people suffers from various retinal degeneratioies.What this class degenerative disease was mainly impaired is photoreceptor cell, and photoreceptor cell is considered to the unrenewable neurocyte in impaired back, so still there is not ideal Therapeutic Method at present.
For the retinal degeneration treatment of diseases, mainly contain gene therapy at present, artificial retina, retina transplantation, pharmaceutical intervention etc.Gene therapy has brought dawn for the human final retinal degeneration disease of curing, but the road of the final clinical practice of gene therapy distance is also very very long, also has many obstacles.One, gene loci is many.Find that at present certified human photoreceptor degeneration gene is above 80.Two, gene imports the screening problem of good carrier.Three, how to improve the genes of interest transfection efficiency and reach how to prolong expression time.Four, the safety of clinical practice.Artificial retina still exists a lot of problems to be difficult to solve at present.Concrete mechanism as visual system information coding? the electronic eye long-term existence is in body, can take place to change in quality and lose efficacy? whether retina or visual cortex neurocyte can stand its long-time stimulus, does this stimulation cause neurocyte form and changing function? do electronic eye itself and implantation technique thereof damage the remaining peripheral visual acuity of patient? retina transplantation will be promoted and be used for the treatment of, and still has: many problems such as rejection, postoperative complication, long-term efficacy, reconstruction need further research and inquire into.So first three methods clinical less application at present.Drug therapy: somatomedin and neurotrophic factor, its deficiency are most oral can not absorptions of somatomedin, also can't be by blood a glance barrier, and it is renderd a service the time of keeping in animal body and has only one month usually.Regular application intraocular injection administration meeting causes complication such as infection, detachment of retina.Nutrient substance clinical practices such as vitamin are more, and curative effect is not remarkable.More about the report of Chinese traditional treatment RP in recent years, and receive effect preferably, but mostly be case report and retrospective study, less about the report of its mechanism of action aspect, clinical expansion theoretical foundation deficiency, this is the weak point of traditional Chinese medical science prescription.
Semen Ginkgo (Ginkgo biloba L.) is a Ginkgoaceae perennial woody plant, and only a section one belongs to, really, leaf is all pharmaceutically acceptable.Gingko leaf preparation is one of focus of current plant amedica developmental research, and it derives from the leaf of Semen Ginkgo, and effective ingredient is mainly bilobalide (Ginkgolides).Bilobalide is a terpenoid, main component is ginkalide A, B, C, M, J and bilobalide, owing to have height specificity antagonism platelet activating factor (Platelet-activatingfactor, PAF) effect and cause the concern of international the world of medicine, the now clinical treatment of conditions such as cerebral thrombosis, cerebral ischemia, nervous system disease that are used for.Clear and definite along with to effective ingredient in the Folium Ginkgo, exploitation Semen Ginkgo class single active ingredient new drug becomes the target of international research over past ten years, the bilobalide kind new medicine that is carrying out clinical experiment mainly contains: bilobalide mixture (BN52063) and the pure product of ginkalide B (BN52021), the latter renders a service is much higher than the former.
The neurocyte experimental study of effect is shown by the research table: the cerebral tissue middle reaches were from fatty acid (FFA) content and to the active inhibition of mouse brain ischemia-reperfusion phase cerebral tissue glutathione peroxidase after bilobalide can significantly reduce mice ECS and cerebral ischemia and brain injury; forebrain and midbrain FFA content in the time of simultaneously can also reducing pallasiomy cerebral ischemia re-pouring 90min, thus show that bilobalide is to protecting neuron from acute.Monsoon waits clearly and adopts the cortical neurogenic cell of gestational age 15~17d rat to set up anoxia reoxygenation neuronal damage model, by observing the dynamic expression of neurocyte NOS, from cultured cell in vitro level research Folium Ginkgo extract to protecting neuron from acute.Found that Folium Ginkgo extract can be by suppressing the NOS activity of neurocyte in the anoxia reoxygenation process, the effect of neural cell injury due to its protection anoxia is brought into play in the generation that reduces NO.
Wu Xiaomei etc. studies show that the anoxybiotic protective effect of mouse cortex neurocyte; bilobalide has significant protective effect to the cortical neuron of anoxia-induced apoptosis; mechanism of action may be with its raising neurocyte oxidation resistance, reduce the generation of free radical, and the 26S Proteasome Structure and Function of protection cell is relevant.
Bilobalide has neuro-protective and anti-apoptotic two aspect effects, and Folium Ginkgo extract have convenient sources, cheap, can be absorbed, see through easily plurality of advantages such as blood brain barrier by body.Clinically be used for treatment of conditions such as cerebral thrombosis, cerebral ischemia, nervous system disease, but still do not have the application of retina degenerative disease aspect.
Summary of the invention
The purpose of this invention is to provide the application of ginkalide B in the medicine of preparation control retinal degenerative disease.The mechanism of action is as follows:
Ginkalide B energy dose dependent is protected retinal neuronal cell to the antiglutamic acid excitatory toxicity.
This effect may be that platelet activating factor receptor is blocked the excitatory neuron poison signal transduction process of PAF mediation, lifting bcl-2 expression, reduction bax expresses and rising retinal neuronal cell mitochondrial membrane potential is realized by competing to ginkalide B to the antiglutamic acid excitatory toxicity.
Ginkalide B has protective effect to damage of rat retina photoreceptor cell and the visual function infringement due to the experimental rat retinal degeneration, and this effect is dose dependent.
Ginkalide B can suppress the apoptosis of the photoreceptor cell of N-methyl-inductive retina injury rat of N-Nitrosourea.
Ginkalide B can suppress the apoptosis of the photoreceptor cell of the inductive retina injury rat of MNU.Its mechanism of action may with the expression that raises Bcl-2, slightly reduce Bax and express, it is relevant to improve Bcl-2/Bax ratio.
Ginkalide B has protective effect to damage of rat retina photoreceptor cell and the visual function infringement due to the retinal degeneration, and this effect is dose dependent.The application of ginkalide B in the medicine of preparation control retinal degenerative disease, for retinal degenerative disease provide a kind of new, effectively treat approach.
Description of drawings
Fig. 1 is the influence result schematic diagram of each drug treating group to the retinal neuronal cell survival rate;
Fig. 2 is that flow cytometer detects different pharmaceutical effect group retinal neuronal cell apoptosis result schematic diagram;
Fig. 3 is the result of variations sketch map of Bcl-2/Bax ratio in each drug treating group retinal neuronal cell;
Fig. 4 is rat retina neurocyte Rh123 marker image (under the Laser Scanning Confocal Microscope 400 *) result schematic diagram, wherein Fig. 4 A: retinal neuronal cell Rh123 marker image under the ginkalide B effect; Fig. 4 B: retinal neuronal cell Rh123 marker image under the glutamic acid effect; Fig. 4 C: left side figure is retinal neuronal cell Rh123 marker image behind the time adding glutamic acid for 60S; Right figure is a retinal neuronal cell Rh123 marker image behind the continuation adding ginkalide B;
Fig. 5 is that ginkalide B is to retinal neuronal cell mitochondrion Rh123 fluorescence intensity influence curve figure;
Fig. 6 is that glutamic acid is to retinal neuronal cell mitochondrion Rh123 fluorescence intensity influence curve figure;
Fig. 7 be ginkalide B to the glutamic acid effect after the influence curve figure of retinal neuronal cell mitochondrion Rh123 fluorescence intensity;
Fig. 8 is the MNU model control group and the result schematic diagram of the ERG of GB (40mg/kg) treatment different periods of group;
Fig. 9 is rat retina form (HE * 200) sketch map of model group and ginkalide B (40mg/kg) treatment group different time, wherein, Fig. 9 a, b, c are respectively the rat retina form of model group 3d, 5d, 7d, and Fig. 9 d, e, f are respectively the rat retina form of ginkalide B (40mg/kg) treatment group 3d, 5d, 7d;
Figure 10 is an outer retina thickness comparative result sketch map of respectively organizing different time sections central retina after the MNU administration;
Figure 11 is that the TUNEL method detects retina cell apoptosis (TUNEL * 200) result schematic diagram;
Figure 12 is model control group and the GB treatment group result schematic diagram in different time sections rat retina Bcl-2 changes of expression level;
Figure 13 is the expression comparative result sketch map of GB treatment group and model group different time Bcl-2/ β-actin;
Figure 14 is the expression comparative result sketch map of GB treatment group and model group different time Bax/ β-actin;
Figure 15 is that GB treatment group and model group different time Bcl-2/Bax ratio change result schematic diagram.
The specific embodiment
Embodiment 1: ginkalide B is to the influence of the rat retina neuronal apoptosis of In vitro culture
Material and method:
Material: SD (Sprague-Dawley) neonatal rat gave birth to back 8 days, and male and female are regardless of.(Ginkgolide B, GB), Guangzhou medicine inspecting institute provides ginkalide B.
Method: the 1. in vitro study of the anti-retinal neuronal cell apoptotic effect of ginkalide B
Adopt external former 15d rat retina neurocyte of being commissioned to train foster, be divided into six groups: the A group is the normal control group; The B group is the glutamic acid model group; The C group is ginkalide B (50 μ mol/L)+glutamic acid group; The D group is ginkalide B (100 μ mol/L)+glutamic acid group; The E group is ginkalide B (50 μ mol/L) pretreatment+C group; The F group is ginkalide B (100 μ mol/L) pretreatment+D group.When cultivating 14d, E, F group adds ginkalide B 50,100 μ mol/L respectively in advance and cultivates 24h, organizing the ginkalide B and the final concentration that add respective concentration with B~D then when 15d is 0.8mmol/L glutamic acid, sets up the retinal neuronal cell apoptosis model of glutamic acid damage.Measure the neurocyte survival rate with tetrazolium bromide (MTT) method, flow cytometer is observed neuronal apoptosis and related gene B cl-2 and Bax protein expression and is used laser confocal microscope (Confocal) and carry out mitochondrial membrane potential (MMP) mensuration.
2. ginkalide B is to the influence of retinal neuronal cell mitochondrial function
Get and prepare retinal neuronal cell and (contain 1 * 10
6Individual/the ml living cells) each 0.15ml of suspension, be inoculated in respectively in the Petri culture dish (the ware bottom scribbles 0.1% poly-D-lysine) of diameter 35mm, leave standstill 15min, make cell closely stick at the culture dish bottom, add the DMEM/F12 that contains 10% calf serum, 37 ℃, cultivate in the 5%CO2 incubator.Every 3d half amount is changed liquid 1 time, wait to grow to 70% when merging when 10d (usually after cultivation), cell to be measured is washed 3 times with the DMEM/F12 culture fluid of serum-free, the DMEM/F12 culture fluid 800 μ l that add serum-free, 0.1% rhodamine 123 (Rh123) fluorescent dye, 2 μ l labelling 1h, supernatant inclines, clean 1 time with same medium, the DMEM/F12 culture fluid 800 μ l that add serum-free, detect with laser scanning co-focusing microscope (Confocal), carry out fluorescent strength determining, the random labelling retinal neuronal cell, after prescan treats that curve steadily, carry out the drug effect test.
4~6 in each random labelling cell, repeated experiments is 4 times altogether.Retinal neuronal cell carries out each retinal neuronal cell fluorescent strength determining under the drug effect after prescan treats that fluorescence intensity curves is stable.Dosing as follows is limited to 180s during observation.
The A group: when 60s, adding final concentration is the ginkalide B 10 μ l of 100 μ mol/L.
The B group: when 60s, adding final concentration is the glutamic acid 10 μ l of 2mmol/L.
The C group: when 60s, adding final concentration is the glutamic acid 10 μ l of 2mmol/L, treats that adding final concentration after figure steadily is the ginkalide B 10 μ l of 100 μ mol/L.
Carry out statistical analysis according to every group of fluorescence intensity level that is scanned cell.Be directly proportional with fluorescence intensity based on Rh123 concentration height, can judge the Rh123 situation of change according to fluorescence intensity.With 10S is one at interval, and dynamically recording is each processed group fluorescence intensity level, the relatively situation of change of fluorescence intensity curves before and after the dosing during from 0S to 180S.Because sample and drug effect effect time-histories difference, the observation of experimental effect variation tendency is only done in this research.
Statistical analysis:
All statistics adopt the one factor analysis of variance method (ANOVA) in SPSS12.0 software kits that data are analyzed, the result with average ± standard deviation (
) expression.Have the significance meaning with P<0.05 expression difference, P<0.01 expression difference has the highly significant meaning.
The result
1.MTT method detects ginkalide B the retinal neuronal cell survival rate is influenced
B~D group compares unknown significance difference with the A group as seen from Figure 1.F~L group and E (glutamic acid 8mmol/L) group more all have between significant difference: F, G, three groups of H and I, J, three groups of groups of K significant difference (P<0.01) are relatively arranged, as seen GB is between 10~100 μ mol/L, increase with concentration, cell survivaling number increases, and has concentration dependent.Compare P<0.0 5 between F and I group, G and J compare P<0.01 between H and K group, and visible GB processed group is better than the GB pretreated group, the cell survival rate height.Compare P<0.05 between L and K group, as seen unite the pretreated group effect and be better than the individual processing group.
2. the in vitro study of the anti-retinal neuronal cell apoptotic effect of ginkalide B
The percentage comparisons of each drug treating group retinal neuronal cell apoptosis sees Table 1-1 and Fig. 1-10.
By showing 1-1 and Fig. 2 as seen: C~F group more all has significant difference (P<0.01) with the B group, compares P<0.01 between C and D group, and prompting GB can resist the retinal neuronal cell apoptosis of glutamate induction, and this effect has concentration dependent.Between C and E, D and F group significant difference (P<0.01) is arranged relatively, prompting is handled cell with GB in advance before glutamic acid acts on cell, can obviously reduce apoptosis rate.
Group | Sample number (n) | Apoptosis cell (%) |
A (normal control group) B (glutamic acid processed group) C (GB50 μ mol/L processed group) D (GB100 μ mol/L processed group) E (GB50 μ mol/L pretreatment+C group) F (GB100 μ mol/L pretreatment+D group) | 6 6 6 6 6 6 | 2.3±0.3 49.6±2.0 27.1±0.6 ** 12.9±0.2 ** 11.4±0.5 ** 8.2±0.3 ** |
Annotate:
*Compare P<0.01 with the B group
Bcl-2 and Bax protein expression and the odds ratio of the two see Table 1-2 and Fig. 3 in each drug effect group retinal neuronal cell:
The variation of Bd-2 and expression of Bax positive protein and ratio thereof in each drug treating group retinal neuronal cell of table 1-2 (
%, n=6)
Group | Bcl-2 protein expression (%) | Bax protein expression (%) | Bcl-2/Bax |
A (normal control group) B (glutamic acid processed group) C (GB50 μ mol/L processed group) D (GB100 μ mol/L processed group) E (GB50 μ mol/L pretreatment+C group) F (GB100 μ mol/L pretreatment+D group) | 2.7±0.2 2.2±0.2 3.6±0.3 ** 5.7±0.3 ** 8.1±0.2 **?11.1±0.2 ** | 2.9±0.4 10.4±0.4 7.6±0.4 ** 5.3±0.3 ** 4.4±0.4 ** 3.3±0.3 ** | 0.95 0.21 0.48 ** 1.08 ** 1.81 ** 3.36 ** |
Annotate:
*Compare P<0.01 with the B group
Table 1-2 and Fig. 3 show: the expression of Bcl-2 and Bax positive protein and the two odds ratio more all have significant difference (P<0.01) in GB different time and various dose effect group and the glutamic acid processed group, retinal neuronal cell.The Bcl-2 protein expression strengthens with the GB activity, carries out the GB pretreated group, strengthens significantly.The Bax protein expression then increases with the GB activity and lowers, and carries out the GB pretreated group, descends significantly.The variation tendency of the two ratio also increases with the GB activity, carries out the GB pretreated group, rises significantly.
3. ginkalide B is to the influence of retinal neuronal cell mitochondrial function
Each processed group rat retina neurocyte Rh123 marker image is seen Figure 14.
Fig. 5 shows: A organize when 60s, and adding final concentration is the ginkalide B 10 μ l of 100 μ mol/L, and fluorescence intensity curves is moved on gradually, and fluorescence intensity rises to 1669.9 ± 5.3 during 96S, and begins decline gradually, and 120S returns to normal level, up to EOT.
Fig. 6 shows: B organizes when 60s, and adding final concentration is the glutamic acid 10 μ l of 2mmol/L, and fluorescence intensity curves moves down gradually, and fluorescence intensity drops to 7.4 ± 2.8 during 120S, and keeps this fluorescence intensity up to EOT.
Fig. 7 shows: C organizes when 60s, and adding final concentration is the glutamic acid 10 μ l of 2mmol/L, and fluorescence intensity curves moves down gradually, and fluorescence intensity drops to 81.6 ± 3.5 during 80S, and keeps this fluorescence intensity.It is the ginkalide B 10 μ l of 100 μ mol/L that 120S adds final concentration, and fluorescence intensity curves is moved on gradually, and fluorescence intensity rises to 784.6 ± 5.2 during 130S, and keeps this fluorescence intensity up to EOT.
This result of study shows, uses ginkalide B that cultured cell is not had obvious influence separately.Only after adding the glutamic acid processing, could significant protective effect be arranged to neurocyte.This shows that indirectly PAF may participate in the signal transduction process of glutamate induction retinal neuronal cell damage as one of second message,second messenger.Ginkalide B has the effect of height specificity antagonism PAF, and it may block the excitatory neuron poison signal transduction process of PAF mediation by the competition paf receptor, thus the neuroprotective of providing.
Experiment shows: rat retina neurocyte generation hyperpolarization behind the adding glutamic acid, and MMP (mitochondrial membrane potential) descends; Add GB, the rapid depolarization of cell, MMP rises, and returns to normal level subsequently.After adding glutamic acid, add GB again, MMP is gone up, prompting GB can be to antiglutamic acid excitatory toxicity protection retinal neuronal cell, and this effect may realize thereby influence mitochondrial function by rising retinal neuronal cell MMP.
In sum; GB can protect retinal neuronal cell to the antiglutamic acid excitatory toxicity by dose dependent, and this effect may be that paf receptor is blocked the excitatory neuron poison signal transduction process of PAF mediation, lifting bcl-2 expression, reduction bax expresses and rising retinal neuronal cell MMP realizes by competing.
Embodiment 2: ginkalide B is to the protective effect of the inductive rat retina degeneration of MNU
Laboratory animal and reagent
84 of SD rats gave birth to back 46 days, and male and female are regardless of.All animals is raised in the standard mouse cage, keeps 12h daytime, the time cycle at 12h night.Ginkalide B (Ginkgolide B, GB) Guangzhou medicine inspecting institute provides, face with preceding with physiological saline solution, be diluted to required mass concentration.
Method
Get and give birth to 86 of back 46 days SD rats, randomly draw 6 and be normal control group (A group); Remaining 80 is experimental group, being divided into 4 again at random organizes greatly, every group 20, wherein B organizes: model control group (lumbar injection MNU 40mg/kg), C group: the heavy dose of group of ginkalide B (lumbar injection GB 60mg/kg), D: dosage group in the ginkalide B (lumbar injection GB40mg/kg), E: ginkalide B small dose group (lumbar injection GB 20mg/kg).Above-mentioned rat is from giving birth to the administration of back 46 days beginning stomaches, and ginkalide B treatment group gives the GB of corresponding dosage, and model control group is given equivalent normal saline, every day 1 time, continuous 7 days.The 50th day model control group and all disposable lumbar injection MNU of ginkalide B treatment group 40mg/kg set up rat retina degeneration model.Each is organized each 4 and checks respectively at 24h, 48h, 3d, 5d, the capable right eye flash of light of 7d ERG, and 3d, 5d, 7d put to death animal behind lumbar injection MNU, get eyeball and do pathological section.
Statistical analysis
All statistics adopt the one factor analysis of variance method (ANOVA) in SPSS12.0 software kits that data are analyzed, the result with average ± standard deviation (
) expression.Have the significance meaning with P<0.05 expression difference, P<0.01 expression difference has the highly significant meaning.
The result
1. electroretinogram detects
Each variation of organizing ERGa, b wave-amplitude sees Table 3-1, and 3-2 is shown in Figure 8:
Rats in normal control group ERG waveform is clear, and a wave-amplitude is 105.4 ± 16.8 μ V, and a ripple incubation period is 22.8 ± 1.3ms; The b wave-amplitude is 292.6 ± 19.6 μ V, and b ripple incubation period is 38.8 ± 3.7ms.After model control group MNU40mg/kg handled 24h, a ripple disappeared, and 72hb ERG is and extinguishes type.
GB (20mg/kg) treatment group is after MNU40mg/kg handles 2d, and a ripple disappears, and the b wave-amplitude drops to normal 32%; During 3d, wherein 2 rat ERG are and extinguish type, and 2 b wave-amplitudes drop to normal 8% in addition; 5d ERG is and extinguishes type.
GB (40mg/kg) treatment group is after MNU40mg/kg handles 3d, and a ripple disappears, and the b wave-amplitude drops to normal 22%; During 5d, wherein 1 rat ERG is and extinguishes type, and 3 b wave-amplitudes drop to normal 14% in addition; 7d ERG is and extinguishes type.
GB (60mg/kg) treatment group is after MNU40mg/kg handles 3d, and a ripple disappears, and the b wave-amplitude drops to normal 20%; During 5d, the b wave-amplitude drops to normal 14%; The b wave-amplitude drops to normal 8% during 7d.
Table 3-2 respectively organize different time sections ERGb wave-amplitude after the MNU modeling (
μ m, n=8)
Group | The b wave- | ||||
|
|
3d | |||
5d | |||||
7d | |||||
Normal control group model group (MNU40mg/kg) GB (20mg/kg) treatment group GB (40mg/kg) treatment group GB (60mg/kg) treatment group | 309.6±49.0 121.8±27.4 146.6±35.2 262.4±16.4 265.4±37.8 ? | ? 56.3±9.4 99.8±19.9 157.6±25.0 145.1±25.8 ? | ? 0 25.0±27.5 67.2±11.5 60.9±14.1 ? | ? 0 0 42.2±28.3 42.2±16.3 ? | ? 0 0 0 25.0±27. 5 |
2. the variation of retina form
We are the variation that the object of observation is observed each processed group retina form with central retina (adjacent with optic nerve), show as Fig. 9: the rat retina of model control group is 3d photoreceptor cell considerable damage after processing, the obvious attenuation of outer nuclear layer (3-2a), the 5d photoreceptor cell obviously reduces, and outer nuclear layer is left 2~4 layers (3-2b); The photoreceptor cell layer disappears behind the 7d, but still has a small amount of outer nuclear layer (3-2c).
3d after GB (40mg/kg) the treatment group MNU40mg/kg administration, the light damage of visible light receptor cell, the slight attenuation of outer nuclear layer (3-2d); 5d does not see significant change (3-2e), photoreceptor cell considerable damage behind the 7d, the obvious attenuation of outer nuclear layer, about 4~5 layers (3-2f).
Model control group and GB treatment are organized each time period outer retina thickness and are relatively seen Table 3-3, visible GB is little, in, the more visible significant difference (P<0.01) of heavy dose of treatment group and model group.And the GB treatment is organized more all significant difference (P<0.01) between three groups, and visible dosage is dose dependent between 20~60mg/kg.
Studies show that: the outer retina thickness of normal rats central retina is 98.4 ± 1.8 μ m.It is 17.8 ± 2.1 μ m that MNU handles 7 days B group outer retina thickness, and GB is little, in, heavy dose of group is respectively 25.2 ± 2.7 μ m, 38.3 ± 2.3 μ m, 45.8 ± 2.3 μ m, with the more visible significant difference (P<0.01) of model group outer retina thickness.And relatively have significant difference (P<0.01) between three groups, prompting GB has the certain protection effect to the central retina damage that MNU causes; Dosage is dose dependent between 20~60mg/kg.
In sum, ginkalide B has protective effect to damage of rat retina photoreceptor cell and the visual function infringement due to the inductive experimental rat retinal degeneration of MNU, and this effect is dose dependent.
Table 3-3 respectively organize different time central retina after the administration outer retina thickness (
μ m, n=6)
Group | Outer retina thickness (μ m) | ||
| 5d | 7d | |
Normal control group model group (MNU40mg/kg) GB (20mg/kg) treatment group GB (40mg/kg) treatment group GB (60mg/kg) treatment group | 98.4±1.8 57.8±2.9 64.7±1.7 ** 70.7±2.1 ** 79.8±1.9 ** | 42.0±2.7 50.0±2.0 ** 59.4±2.0 ** 65.9±2.6 ** | 17.8±2.1 25.2±2.7 ** 38.3±2.3 ** 45.8±2.3** |
Annotate:
*Compare P<0.01 with model group (MNU 40mg/kg)
Embodiment 3: ginkalide B is to the protective effect Study on Mechanism material of the inductive rat retina degeneration of MNU:
26 of SD rats were born back 46 days, and male and female are regardless of, and were provided by Zhongshan University's Experimental Animal Center, and word 2004A089 checks and affirm in credit number Guangdong.Main medicine and reagent: (Ginkgolide B, GB), Guangzhou medicine inspecting institute provides ginkalide B.
Method:
26 of the SD rats that is born back 46 days are divided into A at random: the normal control group; B: model control group; C:GB treatment group, 6 of A groups, all the other two groups each 10.The GB treatment is organized in giving birth to back 46 days GB40mg/kg gastric infusions, model control group irritates stomach for the equivalent normal saline, once a day, gavages 7 continuously, and pressed the body weight disposable celiac respectively with 2 groups in back the 50th day in life and inject MNU40mg/kg, set up rat retina degeneration model.Each is organized each 2 and puts to death animal respectively at 24h, 48h, 3d, 5d, 7d behind the lumbar injection MNU, gets 1 rat eye and does pathological section, and TUNEL detects apoptosis and goes immunohistochemical staining simultaneously and observe the expression of retina Bcl-2 and Bax; Isolate retina extraction RNA for 1 in addition, RT-PCR detects the expression of retina Bcl-2 and Bax.
Statistical analysis:
All statistics adopt the one factor analysis of variance method (ANOVA) in SPSS12.0 software kits that data are analyzed, the result with average ± standard deviation (
) expression.Have the significance meaning with P<0.05 expression difference, P<0.01 expression difference has the highly significant meaning.
The result:
1.TUNEL detect the apoptosis (Figure 11) of rat retina
Do not see apoptotic nucleus in the normal rats retina.TUNEL positive cell nuclear appears in model control group MNU effect 1d, outer nuclear layer, increases in a large number during 2d, reaches the peak, reduces gradually subsequently, still sees a small amount of TUNEL positive cell nuclear during 5d.GB40mg/kg treatment group and model group comparison outer nuclear layer apoptotic index have significant difference (P<0.01) (seeing Table 4-1).GB40mg/kg treatment group is not seen TUNEL positive cell nuclear during 5d after the MNU modeling.
The table 4-1 respectively organize rat retina outer nuclear layer apoptotic index (
%)
Group | The eye number | Outer nuclear layer apoptotic index (%) | ||||
| |
3d | 5d | 7d | ||
Normal control group model matched group GB treatment group | 6 6 6 | 0 22.4±5.3 14.0±5.6 ** | 38.8±3.5 25.5±5.0 ** | 14.0±5.6 7.9±2.8 ** | 6.8±4.3 0 ** | 0 0 |
*Compare P<0.01 with model group
2.RT-PCR detect the expression of retina Bcl-2 and Bax
The expression of treatment group and model control group Bcl-2, Bax and internal reference β-actin mRNA is seen Fig. 4-2, wherein, and A: normal control group; B:GB40mg/kg treatment back 12h; C:GB40mg/kg treatment back 1d; D:GB40mg/kg treatment back 2d; E:GB40mg/kg treatment back 3d; F:GB40mg/kg treatment back 5d; G: model control group 12h; H: model control group 1d; I: model control group 2d; J: model control group 3d; K: model control group 5d.According to the integral optical density value (OPTDI) of band, calculate respectively organize Bcl-2, Bax mRNA with respect to β-actin mRNA expression over time situation see Table 4-2,4-3, Fig. 4-3,44:
Table 4-2 respectively organize different time retina Bcl-2mRNA with respect to the expression of β-actin mRNA (Bcl-2/ β-actin,
N=3)
Group | Different time after the MNU administration |
| |
2d | 3d | 5d | ||||
Normal control group GB treatment group model matched group | ? 0.44±0.08 ? ? ? | ? ? 0.98±0.07 0.39±0.09 ? | ? ? 1.19±0.26 0.14±0.08 ? | ? ? 0.81±0.18 0.61±0.11 ? | ? ? 0.48±0.11 0.71±0.12 ? | ? ? 0.67±0.18 0.81±0.19 ? |
As shown in figure 13, the normal control group can detect the expression of Bcl-2mRNA.Model control group Bcl-2mRNA expression in the 12h retina of MNU effect back descends, and it is minimum that 1d reduces to, and it is normal 1/4 that expression is low to moderate approximately, bottom out after this.When GB treatment group was handled 12h at MNU, the expression of Bcl-2mRNA was higher than normal 2 times, reaches the highest during 24h, after this gradually descended, and 3d reduces the most remarkable, bottom out again during 5d.
As shown in figure 14, normal group can detect the expression of Bax mRNA.Bcl-2mRNA expression rising in the 12h retina behind the model group MNU lumbar injection, it is the highest that 2d rises to, and after this expression begins to descend approximately to normal 9 times, during 5d still is normal 5 times.When GB treatment group was handled 12h at MNU, the expression of Bax mRNA raise, and was higher than normally, and 2d rises to the highest, and after this expression gradually descends approximately to normal 6 times, during 5d still is normal 4 times.
Bcl-2/Bax ratio (Figure 15): model control group 12h reduces to 0.36, and 1d reduces to 0.15, and 2d rises to 0.29, and 3d and 5d are respectively 0.42 and 0.64.GB treatment group is when MNU handles 12h, 1d, 2d, 3d, 5d, and Bcl-2/Bax ratio is respectively 0.98,0.92,0.53,0.45,0.68, is higher than model group.
3. immunohistochemical staining (seeing Table 4-4)
Each layer of normal control group retina there is no Bcl-2 and Bax positive expression.Behind the model control group 1d, the visible Bax positive expression of outer nuclear layer is expressed the byest force during 2d, after this express gradually and weaken, and still has a small amount of positive expression during 5d.Each time point of MNU processed group there is no retina Bcl-2 positive expression.
GB treatment group Bcl-2 expresses the strongest at 1d, the 2d positive expression descends, and positive expression disappears behind the 3d.Bax expresses the strongest at 2d, the 3d positive expression descends, and still has positive expression behind the 5d.
The expression of table 4-4 model group and treatment group different time retina Bcl-2 and Bax (
N=6)
Group | Different time after the MNU administration |
| 2d | 3d | 5d | |||
Model control group Bcl-2 Bax GB treatment group Bcl-2 Bax | ? 0 20.1±3.2 ? 30.5±3.4 15.5±2.4 | ? 0 38.6±3.4 ? 16.1±2.3 23.8±2.9 | ? 0 25.8±2.9 ? 0 10.2±1.9 | ? 0 13.2±2.2 ? 0 6.2±1.5 |
The result shows: do not see apoptotic nucleus in the rats in normal control group retina.During model control group 1d, TUNEL positive cell nuclear appears in outer nuclear layer, increases in a large number during 2d, reaches the peak, reduces gradually subsequently, still sees a small amount of TUNEL positive cell nuclear during 5d.GB40mg/kg treatment group and model group comparison outer nuclear layer apoptotic index have significant difference (P<0.01) GB40mg/kg treatment group not see TUNEL positive cell nuclear during 5d after the MNU modeling.Prompting GB can suppress the apoptosis of the photoreceptor cell of the inductive retina injury rat of MNU.
Two kinds of methods and resultses of RT-PCR and immunohistochemistry show that the trend of the expression of Bcl-2 and Bax has concordance in two kinds of methods.
RT-PCR result shows: model control group handles at MNU that the Bcl-2mRNA expression descends in the 12h retina, and it is minimum that 1d reduces to, and it is normal 1/4 that expression is low to moderate approximately, bottom out after this.When GB treatment group was handled 12h at MNU, the expression of Bcl-2mRNA was higher than normal 2 times, reaches the highest in the time of 1 day, after this gradually descended, and reduction in the 3rd day is the most remarkable, bottom out again in the time of the 5th day.
Bcl-2mRNA expression rising in the 12h retina behind the model control group lumbar injection MNU, it is the highest that 2d rises to, and after this expression begins to descend approximately to normal 9 times, still is normal 5 times in the time of the 5th day.When GB treatment group was handled 12h at MNU, the expression of Bax mRNA raise, and was higher than normally, and 2d rises to the highest, and after this expression gradually descends approximately to normal 6 times, in the time of the 5th day still is normal 4 times.
Bcl-2/Bax ratio: 12h reduces to 0.36 behind the MNU lumbar injection, and 1d reduces to 0.15, and 2d rises to 0.29, and 3d and 5d are respectively 0.42 and 0.64.GB treatment group is when MNU handles 12h, 1d, 2d, 3d, 5d, and Bcl-2/Bax ratio is respectively 0.98,0.92,0.53,0.45,0.68, is higher than model group.
The visible GB treatment of immunohistochemical analysis group Bcl-2 positive expression 1d after the MNU administration expresses the strongest, and the 2d positive expression descends, and positive expression disappears behind the 3d.Model group is not seen retina Bcl-2 positive expression.Bax 2d after the MNU administration expresses the strongest, and the 3d positive expression descends, and still has positive expression behind the 5d.With the apparent in view attenuating of model group.The prompting ginkalide B suppress retinal photoreceptor cell generation mechanism of apoptosis may with the expression that raises Bcl-2, slightly reduce Bax and express, it is relevant to improve Bcl-2/Bax ratio.
1) GB energy dose dependent is protected retinal neuronal cell to the antiglutamic acid excitatory toxicity.And before GB and glutamic acid combined effect, give GB pretreatment to retinal neuronal cell, action effect is better separately.
2) this effect may be that paf receptor is blocked the excitatory neuron poison signal transduction process of PAF mediation, lifting bcl-2 expression, reduction bax expresses and rising retinal neuronal cell MMP realizes by competing to GB to the antiglutamic acid excitatory toxicity.
3) MNU optionally induces retinal photoreceptor cell generation apoptosis, causes the change of electroretinogram, and effect is dosage and time dependence.Mechanism of action may be expressed relevant with rise Bax and Caspase-3.
4) simple, the good reproducibility of the inductive rat retina degeneration of MNU model manipulation is the research medicine to live body photoreceptor cell apoptosis inhibitory action model preferably; The optimal dose of analogue formation is 40mg/kg.
5) GB has protective effect to damage of rat retina photoreceptor cell and visual function infringement due to the inductive experimental rat retinal degeneration of MNU, and this effect is dose dependent.
6) GB can suppress the apoptosis of the photoreceptor cell of the inductive retina injury rat of MNU.Its mechanism of action may with the expression that raises Bcl-2, slightly reduce Bax and express, it is relevant to improve Bcl-2/Bax ratio.
Claims (1)
1. the application of ginkalide B in the medicine of preparation control retinal degenerative disease.
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CN102716117A (en) * | 2012-07-03 | 2012-10-10 | 广州中医药大学 | Application of naringenin in preparing medicines for resisting apoptosis of retinal photoreceptor cell |
CN110876746A (en) * | 2018-09-05 | 2020-03-13 | 江苏康缘药业股份有限公司 | Ginkgo diterpene lactone eye preparation and preparation method and application thereof |
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CN102716117A (en) * | 2012-07-03 | 2012-10-10 | 广州中医药大学 | Application of naringenin in preparing medicines for resisting apoptosis of retinal photoreceptor cell |
CN110876746A (en) * | 2018-09-05 | 2020-03-13 | 江苏康缘药业股份有限公司 | Ginkgo diterpene lactone eye preparation and preparation method and application thereof |
CN110876746B (en) * | 2018-09-05 | 2022-04-22 | 江苏康缘药业股份有限公司 | Ginkgo diterpene lactone eye preparation and preparation method and application thereof |
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