CN101049295A - Application of serine in preparing medication for treating cerebral ischemia - Google Patents
Application of serine in preparing medication for treating cerebral ischemia Download PDFInfo
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Abstract
An application of L-serine in preparing the slow-release or release controllable medicines in the form of solid, liquid, or spray for treating cerebral ischemia is disclosed.
Description
Technical field:
The present invention relates to the novel medical use of L-serine.
Background technology:
Cerebrovascular is the disordered brain function that is caused by various angiogenic cerebral lesions, and apoplexy (claiming apoplexy again) is that acute brain circulatory disturbance causes limitation or the damaged clinical events of diffusivity brain function rapidly.Apoplexy comprises ischemic and hemorrhagic apoplexy, and the former accounts for 70%.Its principal character is the focal neurologic impairment in lesion vessels domination district, as consciousness obstacle, hemiplegia, hemianopsia, hemidysesthesia and aphasia etc.According to statistics, China's stroke onset rate is 120～1,80/,100,000 people, and annual New Development apoplexy is greater than 1,500,000 people, and apoplexy mortality rate second place of the world (mortality rate 80～1,20/,100,000 people) is thus, annual because of apoplexy death toll about 1,200,000.So high stroke onset rate, mortality rate and quite surprising medical care expenses thereof caused white elephant for society and family.At cerebral infarction, except that thromboembolism treatment had sure curative effect, nerve protection medicine failed to show definite clinical effectiveness always at present.Therefore, Ischemic Stroke is prevented and treated the research utmost point need innovative research thinking and the research method of aspect.
Studies show that, after the cerebral ischemia, take place in the cascade reaction process of development at the ischemic hypoxia brain injury, excitatory amino acids such as neuron and a large amount of release glutamate of glial cell, and reuptake reduces, ECS glutamic acid is piled up, excitatory amino acid/inhibitory aminoacid ratio increases greatly, activate excitatory amino acid receptor, cause that excititoxic is considered to extremely crucial mechanism, and overload as cellular calcium with other mechanism, free radical and nitric oxide generation increase etc. are associated, and cause acute at last or delayed neuronal death, injured brain tissue.The success or not for the treatment of after the cerebral ischemia, depend on can in time set up again perfusion and effectively neuroprotective unit avoid the hypoxic-ischemic damage, the use in conjunction of neuroprotective and thromboembolism treatment is considered to a comparatively reasonably solution.Neuroprotective can improve the toleration of neurocyte to hypoxic-ischemic, prolongs the thromboembolism treatment time window, races against time for effectively setting up perfusion again, can also stop or alleviate the reperfusion injury that thromboembolism treatment brings.Even, use neuroprotective for the forfeiture thromboembolism treatment patient on opportunity, also can protect the neuron in the ischemic penumbra, suppress delayed neuronal death.The excititoxic of antagonism or inhibition glutamic acid becomes an important point of penetration of this respect treatment with the protection cerebral tissue.Yet at present excitatory amino acid receptor blocker or release inhibitor are unsatisfactory to the therapeutic effect of ischemia brain injury, the one, treatment time window narrow, the 2nd, the side effect of the spiritual aspect of potential nerve is bigger.We start with from brand-new angle; inhibitory aminoacid-Glycine Receptors in the exciting brain; reduce neuronic irritability; suppress glutamic acid to neuronic excitatory toxicity; thereby play the effect of protection brain, ischemia resisting anoxic brain injury, cause the some diseases of ischemic hypoxia brain injury with treatment cerebral infarction etc.Research report exciting γ-An Jidingsuan (GABA) receptor is arranged, on the whole animal model, can suppress excititoxic, and the ischemia brain is had good protective effect.
There are Gabanergic and glycine (Gly) two class inhibitory nerve mediator systems in the central nervous system.It has been generally acknowledged that GABA mediator system brings into play main effect in brain, and the Gly (Xu Tianle that can the mediator system mainly in spinal cord, plays a role.The research of maincenter Glycine Receptors molecular characterization.The physiological science progress, 2000,31 (4): 373-380).Yet not only there are some Gly serotonergic neurons in studies show that in recent years in the brain, and the distribution of Gly receptor is very extensive especially.Existing research data shows that (as midbrain ventral tegmental area, hypothalamus, Hippocampus, striatum, volt nuclear, corpus amygdaloideum, cerebral cortex) has the distribution of functional Gly receptor to the cerebral cortex many places from brain stem.Therefore, Gly energy inhibitory nerve mediator system, Gly receptor and the effect of part in brain more and more are subjected to people's attention.Studies show that sophisticated neuron in brain uses Gly can cause the hyperpolarization of neuron film.So promptly can reduce neuronic irritability, and stop Mg in the nmda receptor channel
2+Shift out, suppress the opening of its passage, suppress glutamic acid to neuronic excitation.Use during cerebral ischemia, be expected to suppress excitatory amino acids such as glutamic acid, play the cranial nerve protective effect neuronic excitatory toxicity.
According to this imagination, glycine (Glycine), taurine (Taurine), Beta-alanine have been compared in our research, and ((five kinds of Glycine Receptors agonist such as L-α-alanine) are to the protective effect of intraluminal middle cerebral artery occlusion in rats focal brain ischemia-reperfusion injury for β-alanine), L-serine (L-serine), L-α-Bing Ansuan.In five kinds of Glycine Receptors agonist, the ischemia resisting brain injury best results of L-serine; Its protective effect to the experimental cerebral ischemia reperfusion injury has dose dependent; The effect of L-serine may with the Glycine Receptors that activates in the brain, suppress neuronic excitatory toxicity behind the ischemic injuries, stop apoptosis in neuronal and downright bad relevant.
In the treatment of acute ischemic cerebral apoplexy, adopt medicine or intervening measure to disturb each link of ischemia waterfall reaction, prolongation neuronal survival to be called neuroprotective.Acute ischemia causes the cellular energy dysbolismus, and cause a series of ischemia waterfalls to react, comprise the neuron membrane depolarization, excitatory amino acid discharges to be increased, calcium ion enters in the cell by NMDA/AMPA receptor, metabotropic glutamate receptor and voltage dependent channel etc. in a large number, activator protein enzyme, lipase, various kinases, nuclease and nitric oxide (NO) synthase, free radical generates to be increased, and destroys the normal configuration and the function of cell.Simultaneously, inflammatory reaction, leukocytic adhesion and immersion, the cytokine effect etc. followed of perfusion will further aggravate brain tissue damage behind the ischemia again.Said process not only causes neuronic acute degeneration necrosis, also starts apoptosis process, causes delayed neuronal death.Just because of its pathophysiological mechanism is very complicated, it is very big therefore to treat difficulty.Existing more than the 50 kind of nerve protection medicine of hitherto reported carried out clinical trial in the Ischemic Stroke patient, but do not have a kind of medicine to obtain gratifying curative effect.The reason of neuroprotective clinical trial failure is numerous, thinks that at present main cause has: untoward reaction is heavy; Research type, sample size and observation index are incorrect; The therapeutic dose and the course of treatment are improper; Pharmacokinetics and pharmacodynamics are not good; Treatment time, window was narrow etc.
Compare with a large amount of exogenous medicines, in recent years some endogenous compound as: albumin, citicoline, erythropoietin etc. have been obtained certain effect in trial test, cause researcher extensive concern and profound thinking.These endogenous material of one side, body under normal circumstances just can produce, and exogenous use generally is unlikely to produce serious adverse effects; On the other hand, under the situation of acute ischemia damage, these materials can reactively discharge to be increased, and this may be to be forced to a kind of self-protective mechanism of carrying out under the body ischemia stress state.
Though the L-serine belongs to non essential amino acid, have many important physical functions and effect, therefore, in medicine, food, cosmetics, all have comparatively widely and use.But up to now not at the report of aspect cerebral ischemia, using.
Summary of the invention:
The object of the present invention is to provide a kind of novel medical use of L-serine, is the application of L-serine in the medicine of preparation treatment cerebral ischemia
The preparation type of L-serine comprises special injection or infusion preparation, comprises slow release or controlled-release pharmaceutical formulation, comprises enteric cavity administration, spray delivery, Transdermal absorption or oral pharmaceutical preparation.
We have carried out the experimentation of aftermentioned relevant L-serine anti-cerebral ischemia/reperfusion injury and neural tissue injury, confirmed that the L-serine has the clear and definite anti-cerebral ischemia damnification and the effect of neural tissue injury, for it provides more complete experimental basis as a kind of novel anti central nervous system injury medicine and clinical practice thereof.
For L-serine treating cerebral ischemia,, obtain to draw a conclusion by the focal cerebral ischemia in rats model:
I. in five kinds of Glycine Receptors agonist, except that taurine, the L-serine can obviously improve the neurobehavioral obstacle that focal brain ischemia-reperfusion injury in rats causes, reduces the cerebral infarction volume.
II.MRI dynamic observes the influence of L-serine to ischemia-reperfusion rat cerebral infarction volume, shows the sustainable performance cerebral ischemia of L-serine protective effect.
The III.L-serine reduces the cerebral tissue edema that ischemia causes and the destruction of blood brain barrier (BBB): the L-serine can obviously reduce the brain water content of ischemia hemisphere, and the L-serine can obviously reduce the destruction of the BBB of structure and hemisphere under cortex, the cortex simultaneously.
The effect of IV.L-serine strengthens with dosage, 168mg.kg
-1.12h
-1Shi Xiaoguo is near saturated.
The degeneration necrosis that the V.L-serine can reduce Hippocampus CA1 district pyramidal cell behind the cerebral ischemia re-pouring with lose; SABC, Western-blot prompting L-serine can suppress the expression of apoptosis-related genes (Caspase-3) behind the cerebral ischemia.
Advantage of the present invention: prove L-serine ischemia resisting brain injury best results by experiment; Its protective effect to the experimental cerebral ischemia reperfusion injury has dose dependent; The effect of L-serine may with the Glycine Receptors that activates in the brain, suppress neuronic excitatory toxicity behind the ischemic injuries, stop apoptosis in neuronal and downright bad relevant.So far the L-serine is applicable to and makes pharmaceutical preparation, can be made into slow release or controlled-release pharmaceutical formulation; Can be made into coelenteron administration, spray delivery, Transdermal absorption or oral pharmaceutical preparation; Can be made into ejection preparation or infusion preparation; Also can form compound medicinal formulation with other medicines.Above pharmaceutical preparation has clear and definite neuroprotective, can be used for making the pharmaceutical preparation of various nerve degenerations of treatment or injury disease, for example is used for the treatment of apoplexy brain injury, senile dementia and multiple sclerosis etc.
According to this imagination, glycine (Glycine), taurine (Taurine), Beta-alanine have been compared in our research, and ((five kinds of Glycine Receptors agonist such as L-α-alanine) are to the protective effect of intraluminal middle cerebral artery occlusion in rats focal brain ischemia-reperfusion injury for β-alanine), L-serine (L-serine), L-α-Bing Ansuan.In five kinds of Glycine Receptors agonist, the ischemia resisting brain injury best results of L-serine; Its protective effect to the experimental cerebral ischemia reperfusion injury has dose dependent; The effect of L-serine may with the Glycine Receptors that activates in the brain, suppress neuronic excitatory toxicity behind the ischemic injuries, stop apoptosis in neuronal and downright bad relevant.
Studies have shown that five kinds of inhibitory aminoacids of exciting Glycine Receptors have ischemia resisting brain injury effect in various degree, wherein with effect the best of L-serine.After the Focal Cerebral Ischemia Reperfusion, use the L-serine treatment, can obviously dwindle the cerebral infarction volume of rat, improve the neuroethology symptom, suppress the formation of cerebral edema, neuronic degeneration behind the prevention ischemic injuries, necrosis and Neuron Apoptosis.This neuroprotective of L-serine can be suppressed by Glycine Receptors blocker strychnine, illustrates that the effect of L-serine realizes by Glycine Receptors in the exciting brain really.
The possible mechanism of L-serine anti-cerebral ischemia reperfusion injury is: improve inhibitory aminoacid concentration in the brain; excitatory amino acid/inhibitory aminoacid is out of proportion when reversing cerebral ischemia; exciting Glycine Receptors; reduce neuronic irritability; suppress glutamic acid to neuronic excitatory toxicity, reduce the expression of ischemia half dark space Caspase-3, reduce neuronic acute necrosis and the death of tardy property; thereby alleviate the damage that ischemia-reperfusion brings, the protection cerebral tissue.
Description of drawings:
The invention will be further described below in conjunction with drawings and Examples.
Fig. 1 tests in the experiment just of the present invention ischemia 2h to pour into 24h again and respectively organize rat brain sheet TTC dyeing illustration.
Annotate: the infarcted region slough is pale asphyxia, and normal cerebral tissue takes on a red color, and the brain district basically identical of infarcted region and middle cerebral artery domination mainly is positioned at cortex and striatum district.L-serine group, each aspect brain sheet of taurine group are compared than other processed group, and it is less that infarcted region seems.A: sham operated rats; B: taurine group; C:L-serine group; D: glycine group; E:L-α-Bing Ansuan group; F: Beta-alanine group; G: cerebral ischemia matched group.
Fig. 2 is that ischemia cerebral infarction typical mr I changes diagrammatic sketch
Annotate: back 2 hours of MCAO (middle cerebral artery thromboembolism), DWI (Diffusionweightedimaging, the diffusion-weighted picture) upward just can reflect the ishemic part high density signal, L-serine group this moment (L-ser) does not have significant difference with the cerebral infarction volume of matched group (Vehicle); But behind MCAO 5 hours, can observe L-serine group cerebral infarction volume and compare obvious minimizing than matched group.Behind MCAO 2 hours, T
2WI (T
2Weighted imaging, T
2The weighting picture) do not have tangible signal change on; 5 hours, the high density signal of visible ishemic part, this moment, L-serine group cerebral infarction volume obviously dwindled than matched group; Behind MCAO 24,48 hours, matched group cerebral infarction volume continued to increase, and the sustainable performance cerebral ischemia of L-serine group protective effect, the cerebral infarction volume maintenance is constant.
Fig. 3 is that ischemia 2h pours into 24h again and respectively organizes rat Nissl's staining figure
Annotate: A: matched group bilateral Hippocampus, picture right side are ischemic region; B:L-serine group bilateral district Hippocampus, the picture right side is an ischemic region; C: sham operated rats (sham-operated) bilateral district Hippocampus; D: matched group ischemia side Hippocampus CA1 district; E:L-serine group ischemia side Hippocampus CA1 district; F: sham operated rats ischemia side Hippocampus CA1 district; G: matched group ischemia side cortex; H:L-serine group group ischemia side cortex; I: sham operated rats ischemia side cortex.Except that sham operated rats, Nissl's staining shows in various degree painted thin out of matched group, L-serine group rat ischemia side cerebral hemisphere, striatum, Hippocampus and cerebral cortex portion neuron dwindle distortion, as seen karyolysis, karyorrhexis, karyopycnosis, ischemia surrounding zone injured neurons presents the cellular swelling, the feature (Fig. 3) of neuronal degenerations such as Nissl body disappearance.Matched group ischemia side hippocampal tissue has obvious damage, mainly shows CA1, a large amount of pyramidal cell disappearances in CA2 district, and residual pyramidal cell is arranged loose irregular, the pyknosis of most cell karyon, and it is clear that kernel is owed, and Nissl body reduces or disappear (Fig. 3 A, D); Ischemia side cortex portion is painted obviously thin out, and neuron dwindles distortion, visible karyolysis, karyorrhexis, karyopycnosis, the feature (Fig. 3 G) of neuronal degenerations such as Nissl body disappearance.L-serine group ischemia side hippocampal pyramidal cell is arranged relatively more neat fine and close, and karyon is full, and kernel is more clear, and the CA1 district pyramidal cell disappearance or the karyon pyknosis (Fig. 3 B, E) that are dispersed in are on a small quantity only arranged; Ischemia side cortex portion neuron morphology is complete substantially, and karyon is fuller, and kernel is clear, and Nissl body enriches (Fig. 3 H).The sham operated rats hippocampal tissue does not have obvious damage, and pyramidal cell nuclear in CA1 district is justified greatly, and kernel is obvious, the projection of visible understain, and the pyramidal cell layer 2-3, the marshalling densification, complete form, Nissl body enriches (Fig. 3 C, F); Cortex portion neuron morphology Non Apparent Abnormality is arranged closelyr, and karyon is full, kernel clear (Fig. 3 I).
Fig. 4 is rat Caspase-3 SABC figure
Annotate: A: parietal cortex ischemia half blanking bar district, boxed area is for selecting counting Caspase-3 positive cell zone; B:L-serine group has a spot of Caspase-3 positive cell between normal neurons; C: matched group, visible a large amount of Caspase-3 positive cell has a spot of normal neurons; D: sham operated rats, see normal neurons, accidental Caspase-3 positive cell more.The black arrow indication is a normal neurons, and the red arrow indication is the Caspase-3 positive cell.
Fig. 5 is a rat Caspase-3 Western-bolt analysis chart
Annotate: β-actin has the expression than homogeneous as confidential reference items in each group.It is deep mixed that each tests processed group Caspase-3 band, and the expression of Caspase-3 is obvious in the matched group; Then express obviously downward modulation in the L-serine group; In the sham operated rats (sham), Caspase-3 does not see Table substantially and reaches.
The specific embodiment:
The application of a kind of L-serine in the medicine of preparation treatment cerebral ischemia.
Specifically just test and test:
One, experiment material and method
1 material
1.1 laboratory animal: the male Sprague-Dawley of healthy adult (SD) rat, the cleaning level, body weight 240-260g, Nantong University's Experimental Animal Center provides.
1.2 main agents and solution: taurine (Taurine) powder is available from Sigma-Aldrich; Glycine: Chemical Reagent Co., Ltd., Sinopharm Group; L-serine: China Medicine (Group) Shanghai Chemical Reagent Co.; Beta-alanine: Sigma company (U.S.); L-α-Bing Ansuan: China Medicine (Group) Shanghai Chemical Reagent Co.; Red tetrazolium (2,3,5-triphenyltetrazolium, TTC) powder is available from Shanghai Ling Jin Fine Chemical Co., Ltd; Anti-β-actin monoclonal antibody is available from Sigma company; The anti-mouse monoclonal antibody of Cleaved Caspase-3 (Asp175) rabbit is available from CellSignaling company; Two anti-are the SP of Bioisystech Co., Ltd of China fir Golden Bridge immunohistochemical staining test kit in Beijing.
2 methods
2.1 model preparation: with reference to middle cerebral artery line bolt method (middle cerebral artery occlusion, MCAO) the preparation Focal Ischemia-Reperfusion in Rats model of Longa EZ method and do improvement slightly.Nylon wire insertion depth average out to 18.5 ± 0.5mm (counting) from the ECA crotch.Ligation is nylon wire fixedly, skin suture, and the bolt line stays 1cm the end of a thread outward.Cervical region need not anaesthetized and cut to perfusion more once more behind the 2h, lifts the bolt line and show that the nylon wire head end has reached ICA bifurcated incision when resistance is arranged, and blood flow is logical again.Sham operated rats bolt line only inserts 10cm, the same model group of all the other steps.The homonymy Hornor that occurs blood vessel embolism after animal the revives contralateral limbs dyskinesia of seeking peace is the model success.
2.2 neuroethology scoring: with reference to Zea Longa system scoring in 6 fens.Scoring mark Huaihe River: 0 minute: impassivity damage symptom; 1 minute: can not full extension offside fore paw; 2 minutes: turn-take laterally; 3 minutes: topple over to offside; 4 minutes: can not spontaneously walk loss of consciousness; 5 minutes: death.
2.3 the cerebral infarction kitchen range is measured: rat is got brain rapidly, removes rhinencephalon, cerebellum and low brain stem, freezing 10min, from the antinion to the occipital pole, do the continuous volume shape section of 2mm thickness, section is put in the 0.5%TTC solution, hatches 30 minutes in 37 ℃ of lucifuges, shakes section at interval in 5 minutes.Again section is placed 4% paraformaldehyde buffer fixing.Take pictures behind the 24h and import computer, use Image J﹠amp; Scion Image image processing software calculates infarct size, and (pink zone is a normal cerebral tissue, white area is an infarcted region), infarct size=non-ischemia side hemisphere area-ischemia side hemisphere is the infarcted region area not, each brain sheet infarct size sum multiply by thickness (2mm) and is total Infarction volume, and the long-pending and full brain volume of cerebral infarction stove is than being the cerebral infarction percent by volume.
2.4 brain water content is measured: after ischemia 2h pours into 24h again, the rapid broken end of rat is got brain, survey the cerebral tissue moisture content with dried wet method, on ice cube, get brain rapidly, the cerebral tissue that takes out is placed in the culture dish that the moistening qualitative filter paper of normal saline is arranged in, in case water evaporates, the while is removed the pia mater encephali and the sludged blood on brain cortex surface fast.With analyzing the weight that Libra accurately takes by weighing two hemisphere, place 100 ℃ of baking boxs after 24 hours then, weigh once more, be relative water content with ((weight in wet base-dry weight)/weight in wet base) * 100%.
2.5 the penetrating rate of blood brain barrier: ischemia 2h pours into 22h again, inject 2% Azo-Blue (evans blue through rat tail vein, EB) physiological salt liquid (3ml/Kg), behind the circulation 2h, after the excessive anesthesia of rat, pour into normal saline fast till the effusive liquid of right atrium is colourless through heart, broken end is got brain rapidly.From the antinion to the occipital pole, do the continuous transverse section section of 2mm thickness, and be divided into cerebral tissue under damage side cortex, the cortex and strong side cortex, cortex under 4 parts, insert in the 50% trichloroacetic acid solution after on analytical balance, weighing.Homogenate and centrifugal (10000r/min, 20min), get supernatant in 1: 2 ratio with ethanol dilution after, adopt Tianjin, island UV-2450 type uv-spectrophotometric instrument in λ=620nm place colorimetric, draw the content of EB in the cerebral tissue according to standard curve, with amount (μ g) expression of contained EB in every gram weight in wet base cerebral tissue.
2.6 MRI checks: adopt the capable MRI of U.S. GE Healthcare 3.0T nuclear magnetic resonance machine and shoulder joint crossed coil to check.Behind the rat anesthesia, the ventricumbent position, head places coil central authorities, autonomous respiration.Except that sham operated rats, each group is randomly drawed 2h, 5h (DWI, T behind 4 rat METHOD FOR CONTINUOUS DETERMINATION ischemia-reperfusions
2WI); 24h, 48h (T
2The dynamic change of ischemic infarction kitchen range WI).T
2Descriptions such as the Infarction volume assay method adopting by reference Heiland on WI and the DWI are used Image-Pro Plus 5.1 image analysis software, Infarction volume=infarct size * (bed thickness+spacing).
2.7 date processing and statistical analysis: all data are represented (x ± SE), adopt Stata 8.0 statistical software deal with data with mean ± standard error.Relatively adopt the t check between two groups, group difference adopts the variance analysis of design in groups, relatively uses Scheff é method in twos.
Two, result
The therapeutic evaluation (drug screening) of 1 five kinds of anti-focal brain ischemia-reperfusion injury of Glycine Receptors agonist
1.1 neuroethology scoring
Sham operated rats (Sham) animal impassivity functional impairment, other each group all has neurologic impairment score value in various degree.After ischemia 2h poured into 24h again, Taurine (taurine) group (Tau), L-serine group (L-ser) had significant difference (p＜0.01) with the scoring of matched group (Veh) neuroethology; Glycine group (Gly) also has statistical significance (p＜0.05) with matched group difference; And L-α-alanine group (L-α-Ala), β-alanine group (β-Ala) and there was no significant difference (p＞0.05) between matched group.Illustrate that Taurine, L-serine and Glycine can obviously improve the function of nervous system of rat, like effect the best with L-serine.And L-α-alanine, β-alanine can not improve rat function of nervous system (referring to table 1).
Table 1: five kinds of Glycine Receptors agonist are learned the influence of scoring to function of nervous system.
| Group | Number of animals (only) | The neuroethology scoring |
| Cerebral ischemia matched group Taurine group L-ser group Gly group L-α-A1a group β-Ala organizes sham operated rats | 7 7 7 7 7 7 7 | 3.142857±0.2608 1.571429±0.2020 ** 1.285714±0.1844 ** 1.857143±0.2608 * 2.714286±0.2857 3.000000±0.2182 0±0 ** |
Annotate: compare with matched group,
*P＜0.05;
*P＜0.01.
1.2 the mensuration of cerebral infarction volume
After ischemia 2h pours into 24h again, row TTC dyeing, slough is pale asphyxia in the infarcted region, and slough does not take on a red color.The brain district basically identical of infarcted region and MCA domination is found in observation, compares with matched group, and each medicine group cerebral infarction volume and infarction percentage volume all dwindle to some extent, and be the most remarkable with the L-serine effect, is Taurine (p＜0.05) secondly; The difference of other treatment group and matched group does not have significance meaning (p＞0.05, table 2, Fig. 1).
Five kinds of Glycine Receptors agonist of table 2 are to the influence of cerebral infarction volume
| Group | Number of animals (only) | Infarction volume (mm 3) | Cerebral infarction percentage volume (%) |
| Cerebral ischemia matched group Taurine group L-ser group Gly group L-α-Ala group β-Ala organizes sham operated rats | 7 7 7 7 7 7 7 | 242.16±13.16 187.49±12.68 * 166.36±11.39 * 200.62±10.68 218.54±10.10 223.78±13.86 0±0 ** | 34.11±3.33 24.41±3.20 * 23.43±2.88 * 28.26±2.70 30.78±2.55 31.52±3.49 0±0 ** |
Annotate: compare with matched group,
*P＜0.05;
*P＜0.01.
The further investigation of the 2 pairs of L-serine drug effects and pharmacological action
2.1 influence to Focal Cerebral Ischemia Reperfusion rat cerebral tissue water content
Table 3 shows that compare with matched group, L-serine treatment group brain water content obviously reduces, and difference has statistical significance (p＜0.05), shows that L-serine can suppress the formation of cerebral ischemia reperfusion injury associated with hydrocephalus.
Table 3 L-serine is to the influence of rat brain water content
| Group | Number of animals (only) | Brain moisture content (%) |
| Cerebral ischemia matched group L-ser group | 6 6 | 0.810±0.012 0.792±0.014 * |
| Sham operated rats | 6 | 0.787±0.008 ** |
Annotate: compare with matched group,
*P＜0.05;
*P＜0.01.
2.2 L-serine is to the influence of blood-brain barrier disruption
The results are shown in Table 4.From the result, use L-serine behind the ischemia and can reduce the caused blood-brain barrier disruption of ischemia, especially to reduce cortical areas's blood-brain barrier disruption (the L-serine group EB of cortical areas content is compared obvious minimizing than the ischemic control group, and difference has statistical significance); Though L-serine group cortex inferior segment EB content also reduces, and compares with matched group, still not statistically significant.
The influence of the blood-brain barrier disruption that table 4 L-serine causes ischemia
| Group | Blood brain barrier is to the penetrating rate (x ± SE, n=5, μ g/g) of EB | |
| Hinder side cortical areas | Hinder side cortex inferior segment | |
| Cerebral ischemia matched group L-ser organizes sham operated rats | 62.31±2.62 38.17±2.94 ** 5.36±0.23 ** | 77.74±3.90 62.39±5.34 △ 7.41±0.25 ** |
Annotate: compare with matched group,
*P＜0.01;
△P＞0.05.
2.3 MRI (nuclear magnetic resonance, NMR) dynamic observes the influence of L-serine to ischemia-reperfusion rat cerebral infarction volume
Behind MCAO 2 hours, DWI is last just can to reflect the ishemic part high density signal, and the cerebral infarction volume that records L-serine group and matched group this moment does not have significant difference; But behind MCAO 5 hours, can observe L-serine group cerebral infarction volume and compare obvious minimizing (p＜0.05) than matched group.Behind MCAO 2 hours, T
2Do not have tangible signal change on the WI; 5 hours, the high density signal of visible ishemic part, this moment, L-serine group cerebral infarction volume reduced before to some extent, compared than matched group, and difference has statistical significance (p＜0.05); Behind MCAO 24,48 hours, matched group cerebral infarction volume continued to increase, and L-serine organizes sustainable performance cerebral ischemia protective effect, and comparing difference with matched group all has significance statistical significance (p＜0.01) (Fig. 2, table 5).
Table 5 L-serine is to the influence of ischemia hindbrain Infarction volume
| Group | DWI | TWI | |||
| 2 hours | 5 hours | 5 hours | 24 hours | 48 hours | |
| Matched group L-ser group | 176±13.2 167±6.85 | 208± 11.25 160± 6.65 * | 223± 13.35 182± 9.85 * | 241±11.00 171± 8.50 ** | 231±9.80 170± 6.00 ** |
Annotate: compare with matched group,
*P＜0.05;
*P＜0.01.
2.4 the effective dosage ranges of the anti-focal brain ischemia-reperfusion injury of L-serine
2.4.1 neuroethology scoring
After ischemia 2h pours into 24h again, L-serine 56mg.kg
-1.12h
-1The scoring of dosage group neuroethology decreases than matched group, but does not have significance meaning (p＞0.05); 168mg.kg
-1.12h
-1Group, 504mg.kg
-1.12h
-1Group is compared with matched group, and the neuroethology scoring significantly reduces, and difference all has statistical significance (p＜0.01), but no significant difference between these two dosage groups.Illustrate 168,504mg.kg
-1.12h
-1The L-serine of dosage can obviously improve the function of nervous system of rat, and this effect has dose-dependence, promptly is lower than 56mg and will produce obvious effect, is higher than 504mg and then acts on and be tending towards saturated (referring to table 6).
Table 6: various dose L-ser learns the scoring influence to Focal Cerebral Ischemia Reperfusion rat function of nervous system.
| Group | Number of animals (only) | The neuroethology scoring |
| Cerebral ischemia matched group 56mg group 168mg group | 7 7 7 | 3.14±0.26 2.29±0.18 1.29±0.18 ** |
| The 504mg group | 7 | 1.29±0.29 ** |
Annotate: compare with matched group,
*P＜0.05;
*P＜0.01.
2.4.2 cerebral infarction volume
After ischemia 2h pours into 24h again, 56mg.kg
-1.12h
-1The cerebral infarction volume that dosage group TTC method records decreases than matched group, but difference not statistically significant (p＞0.05), this is consistent with the neuroethology appraisal result; 168mg.kg
-1.12h
-1Group, 504mg.kg
-1.12h
-1Group is compared with matched group, and the cerebral infarction volume significantly reduces, and difference has statistical significance (p＜0.05), but no significant difference between these two dosage groups.L-serine 168,504mg.kg are described
-1.12h
-1Two dosage can obviously reduce the cerebral infarction volume of Focal Cerebral Ischemia Reperfusion rat, and effect is similar; But dosage is at 56mg.kg
-1.12h
-1The time, also do not demonstrate the ability of remarkable minimizing cerebral infarction volume.The effect that Infarction volume forms behind the prompting L-serine minimizing rat cerebral ischemia exists dose-dependence, to 504mg.kg
-1.12h
-1Effect has reached degree of saturation (referring to table 7) during dosage.
Table 7: various dose L-ser is to the influence of Focal Cerebral Ischemia Reperfusion rat cerebral infarction percent by volume.
| Group | Number of animals (only) | Cerebral infarction percentage volume (%) |
| Cerebral ischemia matched group 56mg group 168mg group 504mg group | 7 7 7 7 | 34.01±2.63 28.29±3.21 24.69±2.23 * 23.98±2.58 * |
Annotate: compare with matched group,
*P＜0.05.
2.5 tectology is observed (Nissl's staining)
2.5.1 the neuron number purpose is influenced
Matched group Hippocampus CA1 district pyramidal cell is sparse, and the lamellar disappearance is arranged, the other free district of dying of the cell that has, and visible karyopycnosis, kytoplasm is dense to be dyed, the cell that cell space diminishes; The cell level is unclear, and neuron density obviously reduces.L-serine group hippocampal tissue feature is similar to sham operated rats, neuron density is compared remarkable increase than matched group, difference has statistical significance (p＜0.01), show that the neuron loss that the L-serine group can partly stop focal brain ischemia-reperfusion injury in rats to cause (the results are shown in Table 8, Fig. 3).
Table 8: Hippocampus CA1 district pyramidal layer cell density
| Group | The neuron number |
| Sham operated rats cerebral ischemia matched group L-ser group | 181±7 44±5 ** 154±4 |
Annotate: with other two groups comparisons,
*P＜0.01
2.5.2 influence to neuron morphology
Rat right side MCAO2h, again pour into 24h after, the row Nissl's staining.Except that sham operated rats; Nissl's staining shows in various degree painted thin out of matched group, taurine treatment group rat ischemia side cerebral hemisphere; striatum, Hippocampus and cerebral cortex portion neuron dwindle distortion; as seen karyolysis, karyorrhexis, karyopycnosis; ischemia surrounding zone injured neurons presents the cellular swelling, the feature of neuronal degenerations such as Nissl body disappearance.Matched group ischemia side hippocampal tissue has obvious damage, mainly shows CA1, a large amount of pyramidal cell disappearances in CA2 district, and residual pyramidal cell is arranged loose irregular, the pyknosis of most cell karyon, and it is clear that kernel is owed, and Nissl body reduces or disappears; Ischemia side cortex portion is painted obviously thin out, and neuron dwindles distortion, visible karyolysis, karyorrhexis, karyopycnosis, the feature of neuronal degenerations such as Nissl body disappearance.L-serine group ischemia side hippocampal pyramidal cell is arranged relatively more neat fine and close, and karyon is full, and kernel is more clear, and the CA1 district pyramidal cell disappearance or the karyon pyknosis that are dispersed in are on a small quantity only arranged; Ischemia side cortex portion neuron morphology is complete substantially, and karyon is fuller, and kernel is clear, and Nissl body is abundant.The sham operated rats hippocampal tissue does not have obvious damage, and pyramidal cell nuclear in CA1 district is justified greatly, and kernel is obvious, the projection of visible understain, and the pyramidal cell layer 2-3, the marshalling densification, complete form, Nissl body is abundant; Cortex portion neuron morphology Non Apparent Abnormality is arranged closelyr, and karyon is full, kernel clear (as shown in Figure 3).
2.6 the expression of apoptosis-related genes Caspase-3 (activated form)
2.6.1 SABC detects
Every routine animal is got successive 6 sections, and numeration parietal cortex ischemia half blanking bar district Caspase-3 positive cell number under 10 * 40 times of light microscopics is got its meansigma methods and be every positive cell number.Matched group Caspase-3 positive cell number is more, and a spot of normal neurons is only arranged; L-serine treatment group positive cell number reduces obviously than matched group that (Fig. 4), more a spot of positive cell is between normal neurons for p＜0.01, table 9.This experiment prompting L-serine can make apoptosis-related genes Caspase-3 express obviously downward modulation, illustrates that L-serine can partly suppress cerebral ischemia half blanking bar district Neuron Apoptosis, reduces delayed neuronal death, thus performance cerebral ischemia protective effect.
Table 9 high power lens hypodermal layer ischemia half blanking bar district Caspase-3 positive cell number
| Group | The Caspase-3 positive cell number |
| Sham operated rats cerebral ischemia matched group L-ser group | 3.8±0.3 22.2±0.8 ** 9.8±0.7 |
Annotate: with other two groups comparisons,
*P＜0.01
2.6.2 Western-blot analyzes
β-actin has the expression than homogeneous as confidential reference items in each group.It is deep mixed that each tests processed group Caspase-3 band, and the expression of Caspase-3 is obvious in the matched group; Then express obviously downward modulation in the L-serine group; In the sham operated rats, Caspase-3 (cysteine aspartase) does not see Table substantially and reaches.Through gel images analytical system scanning, (ratio of β-actin), data are represented with x ± SE respectively to organize Caspase-3 expression and confidential reference items expression with four stars image processing system mensuration.L-serine group is compared with matched group, difference have the significance statistical significance (p＜0.01, table 10, Fig. 5).Further specify L-serine can suppress focal brain ischemia-reperfusion injury in rats the expression of inductive apoptosis-related protein Caspase-3.
Table 10:Caspase-3 expresses and quantizes figure
| Group | Gray level ratio |
| Sham operated rats cerebral ischemia matched group L-ser group | 0.3±0.035 0.91±0.015 0.72±0.02 ** |
Annotate: compare with matched group,
*P＜0.01
Claims (1)
1, the application of a kind of L-serine in the medicine of preparation treatment cerebral ischemia.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106361738A (en) * | 2016-08-25 | 2017-02-01 | 南通大学 | Medicinal composition for therapy and prevention of white matter injury (WMI), and medicinal application thereof |
| CN108740354A (en) * | 2018-06-05 | 2018-11-06 | 江西省农业科学院畜牧兽医研究所 | A kind of anti-Crowding Stress feed addictive of cray and preparation method |
| CN105963287B (en) * | 2016-07-04 | 2019-05-24 | 南通大学 | Compound and its medical usage |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105963287B (en) * | 2016-07-04 | 2019-05-24 | 南通大学 | Compound and its medical usage |
| CN106361738A (en) * | 2016-08-25 | 2017-02-01 | 南通大学 | Medicinal composition for therapy and prevention of white matter injury (WMI), and medicinal application thereof |
| CN108740354A (en) * | 2018-06-05 | 2018-11-06 | 江西省农业科学院畜牧兽医研究所 | A kind of anti-Crowding Stress feed addictive of cray and preparation method |
| CN108740354B (en) * | 2018-06-05 | 2021-07-06 | 江西省农业科学院畜牧兽医研究所 | Crayfish crowding stress resistant feed additive and preparation method thereof |
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