CN101683348B - Application of cholestane-3 beta, 5 alpha, 6 beta-triol in preparation of neuronal protection medicine - Google Patents
Application of cholestane-3 beta, 5 alpha, 6 beta-triol in preparation of neuronal protection medicine Download PDFInfo
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Abstract
The invention reveals that endogenous cholesterol metabolic product cholestane-3 beta, 5 alpha, 6 beta-triol (YC-5) has protection function for neuronal damage caused by cerebral ischemia, ischemia of spinal cord, epileptic seizure and convulsion and relates to an application of YC-5 in preparation of neuronal protection medicine. The YC-5 can be prepared from cholesterol through two steps. The primitive cell culture in vitro and spinal cord ischemia animal model proves that YC-5 has protection function for neuronal damage caused by cerebral ischemia, ischemia of spinal cord, epileptic seizure and convulsion. YC-5 in effective dose has no toxic and side reactions. YC-5 is an effective neural protectant which aims to multiple molecular mechanism and cures cerebral ischemia damage, spinal cord ischemia damage, epileptic seizure and convulsion.
Description
Technical field
The present invention relates to endogenous steroidal compounds cholestane-3 β, 5a, 6 beta-triols be at cerebral ischemia, ischemia of spinal cord, epilepsy with faint from fear in the protective effect of neuronal damage.
Background technology
The release of most quick irritability transmission and glutamate receptor and relevant neurotransmitter glutamate is relevant among the central nervous system (CNS).The early development of glutamate receptor participant CNS plays a decisive role in synaptic plasticity such as learning and memory.Though glutamic acid is to keep the active important medium of normal neurocyte; But a large amount of excitatory neurotransmitters are (like glutamic acid; Glu) discharge, the overstimulation glutamate receptor is known from experience initiation nerve cell death (being called as excitatory toxicity effect (excitotoxicity)).The nerve injury that this excitatory toxicity causes is usually with over-drastic flow of calcium ions, and the generation of oxygen-derived free radicals (ROS) and nitrogen free radical (RNS) can cause the damage of cell inner membrance afterwards, and activation causes the signal of apoptosis and causes tardy cell death.
Intracellular free calcium [the Ca that comprises stream in the calcium ion cell
2+] variation of i, representing certain cell function to start, strengthen or suppress, significant in the generation of some disease, development, cell [Ca
2+] variation of i possibly be " inspiring a little " that a series of pathology damage and pathophysiological mechanism change.The content of intracellular calcium and [Ca
2+] variation of i can provide related positive evidence between the conduction of calcium ion signal and disease.And the nerve excitability toxic damages that glutamic acid causes has stimulated ionotropic receptor, and main be the depolarization that activating nmda receptor causes cell membrane, cause a large amount of flow of calcium ions.When In vitro culture neuron and glial cell, if there is not calcium ion in the culture medium, can block the cell death that digenic acid (KA) causes, this shows that flow of calcium ions is very necessary to the process of damaging due to the excitatory toxicity.Simultaneously verified, the outer calcium ion concentration of born of the same parents reduces can make the toxic damages of the cortical neuron that KA receptor and ampa receptor cause obviously alleviate.Therefore, flow of calcium ions is to cause indispensable factor in neuron and the glial cell death process, also is the effective ways of inquiring into Neuroprotective Mechanisms.
The excessive release of glutamic acid be can cause behind brain and the ischemia of spinal cord, neuronic damage even death caused through a plurality of chain order reactions.Along with the aging of society, this type CNS disease incidence is in rising trend, and people's life and health in serious threat.Ischemia brain, spinal cord and neurocyte are protected the research focus that becomes current nervus centralis ischemic injuries treatment.The dominant mechanism of neuroprotective treatment acute ischemic damage has: Ca
2+Channel blocker, removing free radical, use excitatory amino acid receptor antagonists, neurotrophic factor and gaba receptor agonist etc.It is the dihydropyridines medicine and the magnesium sulfate of representative that calcium channel blocker mainly contains with the nimodipine.Acute ischemia causes a large amount of free radicals to produce in a large number, makes that membrane structure is destroyed, the neuron infringement, but also induction of vascular spasm and intravascular coagulation enlarge infarction size.So the free radical treatment is very important.Vitamin E, vitamin C, superoxide dismutase (SOD) are free radical scavenger commonly used.
It is closely related with convulsions with epilepsy that glutamic acid is also thought always; The excessive release of glutamic acid can cause the startup and the propagation of epilepsy and convulsions on the one hand; Can cause the excessive release of glutamic acid on the other hand after epilepsy and the convulsions again; Then produce the exitotoxicity infringement, cause neuronal necrosis or apoptosis.And glutamic acid causes epilepsy and faints from fear possibly be owing to synthesize increase, the outer a large amount of results that discharge of later stage born of the same parents in its early stage born of the same parents.The glutamate, Glu of discovering extracellular high concentration can trigger the temporal lobe epilepsy outbreak, produce and faint from fear, and has identical pathologic basis with brain and ischemia of spinal cord.
Seek the dream that effective neuroprotective is whole world neuroscientists always.But because the pathomechanism of central nervous tissue and cell injury relates to complicated time and spatial cascade reaction and reciprocal action; So the treatment of intervening to wherein single link or individual molecule mechanism is at present only attempted producing little effect, reason possibly be to have ignored the cell injury process that causes to have multistage, multicenter mechanism and polydirectional characteristics.Therefore, the pharmacologist prediction is arranged, the neuroprotective that possesses a plurality of links, a plurality of target spot mechanism of action is the medicine that is hopeful to obtain desirable clinical effectiveness most.
In numerous chemical compounds, neuroactive steroid has extensive effect to neuroprotective and receives publicity day by day.These neuroactive steroid levels are relevant with the incidence and development of CNS disease, and neuronal damage, death and multiple central nervous system disease are had remarkable regulating action.The eighties in last century, scientists begins these natural existence or synthetic, nervous tissue have active steroid hormone called after neuroactive steroid (neuroactive steroids, NAS).Apply to these steroid hormones clinical as alternative medicine clinically at present.Estrogen is the strongest NAS of known neuroprotective, and the menopausal women ovary stops to produce estrogen, can cause A β deposition, causes Alzheimer (AD).And give the A β level that estrogen can obviously reduce brain, and clinically estrogen is used for the treatment of AD, receive good effect.There are some researches show that allopregnenolone can be protected the irreversible neurotoxic injury of In vitro culture hippocampal cell that is caused by hypoxia and glutamic acid.Dehydroepiandrosterone can reduce the neurotoxic effect of glutamic acid analog and corticosteroid.Up till now for this reason, research report cholestane-3 β also, 5a, 6 beta-triols (YC-5) have activity or as the research of neuroactive steroid in nervous tissue.
YC-5 extensively is present in the mammalian body, has detected the existence of YC-5 among the human normal plasma.Confirmed that YC-5 is one of basal metabolism product of cholesterol.Cholesterol forms cholesterol-5 through autoxidation or enzyme catalysis under the environment of aerobic, the 6-epoxide, and the latter is at cholesterol-5, and aquation under the effect of 6-Cycloxygenase forms YC-5.In addition, people's tissue or body fluid also detect multiple " oxidation sterol ", such as the 7-hydroxy cholesterol, and 24-hydroxy cholesterol and 27-hydroxy cholesterol etc.In hypercholesterolemia crowd's blood plasma and LDL in " oxidation sterol " content far above normal population.It is one of toxicity maximum " oxidation sterol " that YC-5 is considered in the body.The source of YC-5 mainly contains two in the body: the one, food source property, promptly directly eaten the food that contains YC-5 via the cholesterol oxidation; The 2nd, cholesterol-5,6-epoxide generate YC-5 through the catalysis of cholesterol-5,6 Cycloxygenase in vivo.Cholesterol-5, the 6-epoxide is mainly produced through non-oxydasis by cholesterol.Participate in the oxygen-derived free radicals that is mainly of non-oxydasis.These many causes of free radical lipid peroxidations, another part then comes from the activated product in macrophage and the neutrophil cell.Non-oxydasis process is accounting for very important position in the forming process of " oxidation steroidal ".Because give the animal antioxidant, the level of the intravital oxidation sterol of animal promptly descends.YC-5 distributes the abundantest in the atherosis speckle of coronary heart disease human coronary artery.
As stated; Between the decades in past; The research of YC-5 launches around the oxidation of being rich in cholesterol food, hypercholesterolemia and atherosclerosis always, and the dependency between cholesterol oxide and the atherosclerosis was once becoming the hot research field of the atherosclerosis origin cause of formation.Big quantity research confirms that secular hypercholesterolemia has increased atherosclerotic sickness rate.In view of cholesterol itself does not have atherogenic effect, we infer that the cholesterol metabolism product comprises that its oxidation product (i.e. " oxidation sterol ") possibly be to cause atherosclerotic reason.The oxidation sterol is transported under the inner membrance by LDL and deposition, and the formation in the gangrene district of atherosclerotic plaque has been played important function.These zones are accompanied by the deposition and the necrocytosis of lipid, will cause the irreversible damage of endotheliocyte for a long time, platelet adhesion, gathering and thrombosis.The oxidation sterol that comprises YC-5 is hanging down the apoptosis that uM concentration gets final product inducing endothelial cell, and this is main cell death way, also is atherosis main pathological process.In high uM concentration then but trigger cell is downright bad.The oxidation sterol only deposits in the atherosclerotic plaque district on a small quantity, comprising YC-5.Can find the crystallization that one deck is formed by YC-5 or its cholesterol of 25-hydroxyl, cholesterol in atherosis district.Atherosclerotic formation is also raised relevant with the endothelial cell inflammation cytokine.Post-depositional oxidation sterol is induced and is raised the inflammatory cytokine of oxidation LDL to vascular system, and induce these adhesion factor high expresseds as an inflammatory stimulus, promotes apoptosis, necrosis and cytokine to produce.But blended " oxidation steroidal " to the toxicity of endotheliocyte than pure, single " oxidation steroidal " low.
What is interesting is, prolong the Calculus Bovis (cholelithiasis of cattle or sheep) of having used several thousand at the nations of China and India, contain abundant cholesterol and metabolite thereof, proving has the good curing effect to CNS disease such as acute shock, epilepsy, convulsions.Clinical research in recent years shows, the normal cholesterin disease people of blood plasma hypercholesterolemia patient suffer stroke mortality rate low and prognosis is better.Because cholesterol itself, can think that there are certain dependency in the endogenous metabolism product of cholesterol and the treatment and the prevention of apoplexy to former neuron unprotect effect of being commissioned to train foster, also promptly, the endogenous metabolism product of cholesterol possibly have neuroprotective.Through inventor's research that experimentizes, confirm that first in mammalian body, distributing all has a neuroprotective than the cholesterol endogenous metabolism product YC-5 body of horn of plenty is inside and outside.
Summary of the invention
YC-5 is the endogenous metabolism product of cholesterol.Cholesterol can autoxidation in the presence of free radical that lipid peroxidation forms or through NADPH/Fe
2+The catalysis of/ADP enzyme system forms cholesterol-5, the 6-epoxide, and the latter is at cholesterol-5, and aquation under the effect of 6-epoxide hydrase generates YC-5.Research shows that YC-5 can be transported to the tunica intima deposit by LDL, and the damage of hyperamization endothelial tube, and induced platelet adhesion, gathering and thrombosis are atherosclerotic risk factors.Up till now for this reason, the research report that does not also have YC-5 that nervous tissue is had activity or have neuroprotective.
Inventor's research shows that as the endogenous metabolism product of cholesterol, YC-5 maybe be through the negativity regulation and control performance physiological neuroprotective to nmda receptor and non-NMDA receptor; 2) concentration of YC-5 performance neuroprotective does not have obvious toxic and side effects to animal subject, and prompting YC-5 is the neuroprotective drug of CNS diseases such as a kind of potential effective treatment cerebral ischemia, ischemia of spinal cord damage, epilepsy and convulsions.
YC-5 can obviously suppress the toxic action of cerebellar granule neuron, cortical neuron and the damage of dynamoneure irritability of glutamate induction, improves neuron count, neuronal survival, reduces the lactic acid dehydrogenase release value, and is dose dependent.Confocal measures Ca in the cortical neuron cell
2+Concentration change [Ca
2+] i and full cell patch tongs technology mensuration cell membrane Ca
2+Electric current (I
NMDA) variation show that this effect possibly be to activate through suppressing nmda receptor, and then suppresses the Ca that it causes
2+Concentration raises and realizes.
In order to confirm that YC-5 has or not neuroprotective in vivo, we adopt the spinal cord injury model of blocking-up ventral aorta, and YC-5 is to the neuronic protective effect of rabbit ischemia of spinal cord model in research.Implement ischemia of spinal cord and injected YC-5 in preceding 30 minutes in advance, its function of nervous system marks apparently higher than untreated matched group, and does not have animal appearance paralysis.And the matched group all animals presents paralysis.Histopathology shows that the normal quantity of cells of YC-5 group is obviously more than matched group, and further specifying YC-5 has the significant protection effect to neuronal damage.
YC-5 can obviously suppress the toxic action of inductive cerebellar granule neuron of digenic acid and the damage of hippocampal neuron irritability, improves neuron metabolic activity and survival rate, reduces the release value of lactic acid dehydrogenase, and is the dosage reliability.Confocal measures [Ca
2+] i and full cell patch tongs technology mensuration I
NMDAShow that this effect possibly be to activate through the non-NMDA receptor that the inhibition digenic acid causes, and then suppresses the Ca that it causes
2+(data slightly) that concentration raises and realizes.Therefore, YC-5 is for cerebral ischemia, ischemia of spinal cord damage, and epilepsy and convulsions have neuroprotective.
In sum, cholestane-3 β, 5a, 6 beta-triols can be applicable to the preparation of neuro-protective medicaments, can be applicable to the preparation of anti-cerebral ischemia damnification, anti-ischemia of spinal cord damage, epilepsy and anticonvulsant drug.
Description of drawings
Fig. 1 YC-5 is to the protective effect of the cerebellar granule neuron irritability damage of glutamate induction
A. phase contrast microscope photo; B.FDA fluorescent staining photo; C.Hoechst33258 fluorescent staining photo;
D. neuron survival rate; The E.LDH release value
Fig. 2 YC-5 is to the protective effect of the cortical neuron irritability damage of glutamate induction
A. phase contrast microscope photo; B.FDA fluorescent staining photo; C.Hoechst33258 fluorescent staining photo;
D. neuron survival rate; The E.LDH release value
Fig. 3 YC-5 suppresses the cortical neuron cell [Ca of glutamate induction
2+] the i increase
A. neuronal cell [Ca
2+] the i change curve; B. neuronal cell [Ca
2+] the i changing value
Fig. 4 YC-5 suppresses the I of the cortical neuron cell membrane of glutamate induction
NMDA
A. neuronal cell I
NMDAChange curve; B. neuronal cell I
NMDAChanging value
Fig. 5 YC-5 is to the protective effect of the dynamoneure irritability damage of glutamate induction
A. phase contrast microscope photo; B. neuron survival rate
Fig. 6 YC-5 closes the neuroprotective that causes rabbit ischemia of spinal cord model to the ventral aorta folder
A. function of nervous system's scoring; B.FDA fluorescent staining photo; C. normal neurons quantity
Fig. 7 YC-5 is to the protective effect of the irritability damage of the inductive cerebellar granule neuron of digenic acid
A. phase contrast microscope photo; B.FDA fluorescent staining photo; C.Hoechst33258 fluorescent staining photo;
D. neuron survival rate; The E.LDH release value
Fig. 8 YC-5 is to the protective effect of the irritability damage of the inductive hippocampal neuron of digenic acid
A. phase contrast microscope photo; B.FDA fluorescent staining photo; C.Hoechst33258 fluorescent staining photo;
D. neuron survival rate; The E.LDH release value
The specific embodiment
One, by the synthetic YC-5 of cholesterol
YC-5's
13C-NMR (DMSO)
11.578(18-CH3)，18.107(21-CH3)，20.535(26-CH3)，21.988(27-CH3)，23.581(24-CH3)，66.374(3-CH)，74.782(6-CH)，75.005(5-C)
Two, neuronic cultivation
1. the neuronic cultivation of rat cerebellum granule
Take out and give birth to 7～8d; The removal meninges of the about 15～20g rat of body weight and the XIAONAO of blood vessel; After the 0.25g/L trypsinization, use the liquid that dispels of 0.05g/L DNase I cell is dispelled to be single cell suspension, centrifugal; Get deposition, usefulness contains the BME culture medium dilution of 10% (v/v) FBS and 25mM KCl and is inoculated in the culture dish that poly-D-lysine encapsulates in advance.Inoculate after 24 hours and to add 10 μ M Ara-C to suppress the growth and the propagation of non-neuronal cell, the purity that makes cerebellar granule neuron is more than 95%.Add glucose during cultivation and replenish cellular metabolism institute energy requirement.Experimentized in the 8th day.
2. the neuronic cultivation of rat layer
Take out the removal meninges of the rat that gives birth to 1d and the cortex of blood vessel; After the 0.25g/L trypsinization; Use the liquid that dispels of 0.05g/L DNase I cell is dispelled to be single cell suspension; Centrifugal, get deposition, usefulness contains the DMEM-F12 culture medium dilution of 5% (v/v) FBS and 2%B27 and is inoculated in the culture dish that poly-D-lysine encapsulates in advance.Inoculate after 24 hours and to add 10 μ M Ara-C, measure for 2-3 time half weekly later on and change liquid to suppress the growth and the propagation of non-neuronal cell.Experimentized in the 8th day.
3. the cultivation of dynamoneure
Pregnant 15 days SD rats separate spinal cord, remove spinal meninges and blood film, get tire Mus myeloid tissue with 0.125% trypsinization after; Centrifugal, get and contain the middle level of enriching motor neuron, centrifugal remove cell debris after; The adherent 1h of differential, choose adherent slow-footed, the dynamoneure of suspension.Add cytosine arabinoside behind the inoculation 24h, full dose was changed liquid in the 3rd day, continued to cultivate with the L-15 serum-free medium, and half amount was changed liquid in every 2-3 days.Experimentized in 3-5 days.
4. the cultivation of hippocampal neuron
Take out the removal meninges of the rat that gives birth to 1d and the hippocampal tissue of blood vessel, cultivate, experimentized in the 8th day with cortical neuron.
Three, YC-5 is to the protective effect and the mechanism of action of cerebral ischemia
1.YC-5 protective effect to the damage of the cerebellar granule neuron irritability of glutamate induction
Cultivate 8 days cerebellar granule neuron, establish normal control group, glutamic acid group, MK-801+ glutamic acid, YC-5+ glutamic acid group respectively.The normal control group is left intact, and the glutamic acid group adds 200 μ M glutamic acid, and other group adds YC-5 and MK801 in advance, hatch after 30 minutes for 37 ℃ to add glutamic acid, behind the 24h with phase contrast microscope neurocyte form; With FDA dyeing, inverted fluorescence microscope is observed, and carries out cell counting, and calculates neuronal survival:
Survival rate=respectively organize viable count/matched group viable count * 100%.
Hoechst33258 dyeing, inverted fluorescence microscope is observed the neuronal kernel form.And mensuration is respectively organized the activity of lactic acid and lactalase.
As shown in Figure 1; The most of cerebellar granule neuron of YC-5+ glutamic acid group and MK-801+ glutamic acid group can keep the complete of cell space and projection; FDA dyeing showed cell survival rate increases, and nucleus and chromatin are normal morphology, and the LDH release value reduces; Relatively present notable difference (P < 0.05) with the glutamic acid group, and all results show all with YC-5 and have the concentration dependence.And normal neuron survival rate is not had influence at above-mentioned dosage range YC-5.
2.YC-5 protective effect to the damage of the cortical neuron irritability of glutamate induction
Cultivate 10 days cortical neuron, establish normal control group, glutamic acid group, MK-801+ glutamic acid, YC-5+ glutamic acid group respectively.The normal control group is left intact, and the glutamic acid group adds 200 μ M glutamic acid, and other group adds solvent, YC-5 and MK801 in advance; Hatch after 30 minutes for 37 ℃ and add glutamic acid; With phase contrast microscope neurocyte form, with FDA dyeing, inverted fluorescence microscope is observed behind the 24h; Carry out cell counting, and calculate neuronal survival:
Survival rate=respectively organize viable count/matched group viable count * 100%.
Hoechst33258 dyeing, inverted fluorescence microscope is observed the neuronal kernel form.And mensuration is respectively organized the activity of lactic acid and lactalase.
As shown in Figure 2; The most of cortical neuron of YC-5+ glutamic acid group and MK-801+ glutamic acid group can keep the complete of cell space and projection; FDA dyeing showed cell survival rate increases, and nucleus and chromatin are normal morphology, and the LDH release value reduces; Relatively present notable difference (P < 0.05) with the glutamic acid group, and all results show all with YC-5 and have the concentration dependence.And normal neuron survival rate is not had influence at above-mentioned dosage range YC-5.
3.YC-5 concentration dependent ground suppresses the rat layer neuronal cell [Ca that glutamic acid causes
2+] the i increase
On behalf of certain cell function, the variation of intracellular free calcium start, strengthen or suppress, and [Ca
2+] variation of i and the variation of Mediated Signal Transduction thereof be significant in the generation of some disease, development, cell [Ca
2+] variation of i possibly be " inspiring a little " that a series of pathology damage and pathophysiological mechanism change.Therefore understand [Ca
2+] variation of i, have many-sided meaning, and related positive evidence between calcium ion signal transduction and disease can be provided.
Fluo-3 is Ca of new generation
2+Fluorescence indicator, the calcium ion probe that excites by visible light (488nm), under the situation that does not combine calcium ion, usually can fluorescence excitation.Fluo-3 and Ca
2+In conjunction with after, fluorescence intensity increases by 60～80 times.Measured fluorescence intensity changes can reflect [Ca
2+] variation of i.In neuronal cell, add 200 μ M glutamic acid earlier, measure cell fluorescence intensity and be designated as F0, measure cell fluorescence intensity after the administration and be designated as Ca in the F born of the same parents
2+Concentration change degree (△ [Ca
2+] i) represent in order to the percent value of fluorescence intensity level before fluorescence intensity changing value before and after the administration and the administration, that is:
△[Ca
2+]i＝(F-F0)/F0×100％。
As shown in Figure 3, give solvent or YC-5 separately and do not change calcium fluorescence intensity in the neuronal cell; After giving 5～15 μ M YC-5 pretreatment 30min, give 200 μ M glutamic acid and YC-5 more simultaneously, then can suppress the increase of this intracellular Ca2+ fluorescence intensity that glutamic acid causes significantly.The increase of the cortical neuron cellular calcium concentration that the special noncompetitive antaganist MK-801 (50 μ M) of nmda receptor causes glutamic acid then mainly shows as the intracellular Ca2+ fluorescence intensity that has increased is reduced rapidly; Though and YC-5 mainly suppresses the concentration of the cellular calcium that glutamic acid causes and increases that prompting YC-5 is the same with MK-801 to bring into play effect through the activation that suppresses nmda receptor, Confocal measures [Ca
2+] result of i shows that the YC-5 negativity is regulated the activated mechanism of nmda receptor maybe be incomplete same with MK-801.
4.YC-5 the cortical neuron cell membrane that concentration dependent ground inhibition NMDA causes is interior to Ca
2+Electric current (I
NMDA)
In order to prove that further YC-5 suppresses the nerve excitability toxic action of glutamate induction and the relation of nmda receptor, we utilize full cell patch tongs technology, the cortical neuron I that record YC-5 causes NMDA
NMDAVariation.As shown in Figure 4, cortical neuron gives NMDA and YC-5, I after incubating in advance with YC-5 more simultaneously
NMDACurrent peak reduces to some extent.The YC-5 of 5,10,15 μ M can make cell that the reaction of NMDA is reduced to 0.82 ± 0.12,0.67 ± 0.11,0.57 ± 0.05 of NMDA group respectively.Wherein 10 compare with the NMDA group with 15 μ M YC-5 group and to have remarkable significant difference (p 0.01).Thereby proof YC-5 is to the activation performance negativity regulating action of nmda receptor.
To sum up, YC-5 has protective effect to cerebral ischemia, and is through the activation performance negativity regulating action to nmda receptor.
Four, YC-5 is to the protective effect and the mechanism of action of ischemia of spinal cord damage
1.YC-5 protective effect to the damage of the dynamoneure irritability of glutamate induction
Cultivate 5 days dynamoneure, establish normal control group, glutamic acid group, MK-801+ glutamic acid, YC-5+ glutamic acid group respectively.The normal control group is left intact; The glutamic acid group adds 200 μ M glutamic acid; Other group adds MK-801 and YC-5 in advance, hatch after 30 minutes for 37 ℃ to add glutamic acid again, behind the 24h with phase contrast microscope neurocyte form; Carry out cell counting with FDA dyeing inverted fluorescence microscope, and calculate neuronal survival:
Survival rate=respectively organize viable count/matched group viable count * 100%.
As shown in Figure 5, inverted phase contrast microscope is observed down, and the dynamoneure number of matched group survival is many, and cell space is plentiful, and triangular in shape, polygon, third dimension are strong, and visible branch of halation and projection are arranged; Glutamic acid group survival dynamoneure number is few, but has projection to form, and cell is badly damaged; And YC-5+ glutamic acid and MK-801+ glutamic acid group dynamoneure number showed increased (P 0.05), and a lot of projections are arranged, but still have the minority dead cell.With the matched group ratio, the dynamoneure survival rate of its excess-three group all has in various degree and to descend, and compares YC-5+ glutamic acid group survival rate be significantly improved (P < 0.05) with the glutamic acid group.And all results all show with YC-5 and have the concentration dependence.And normal neuron survival rate is not had influence at above-mentioned dosage range YC-5.
2.YC-5 the ventral aorta folder is closed the neuroprotective that causes rabbit ischemia of spinal cord model
24 male new zealand white rabbits are divided into 4 groups (n=6): matched group does not carry out other processing before ischemia of spinal cord; The YC-5 group is at ischemia of spinal cord auricular vein injection in preceding 30 minutes 8mg/Kg YC-5; The DMSO group is at preceding 30 minutes capacity DMSO (1mg/Kg) such as the same manner injection of ischemia of spinal cord; The Sham group promptly only exposes ventral aorta, and does not block it.The ischemia of spinal cord model carries out with reference to the method for Johnson etc.At once, 10min reaches each physiologic parameters between perfusion back 20min mensuration group again behind the ischemia before the ischemia.Respectively organize the function scoring of rabbit with the Talov point system.
As shown in Figure 6, before the ischemia at once, behind the ischemia 10min and again perfusion back 20min measure physiologic parameters no significant difference between each group (P>0.05).The scoring of YC-5 group function is apparently higher than matched group, pour into 48 hours again after, 6 rabbit hind legs of matched group are paralysed fully, and the YC-5 group does not have a paralysis fully, all can beat or have tangible hind leg motion.And function of nervous system's scoring of DMSO group is compared no significant difference with matched group.Sham group and YC-1 group can be observed more polymorphic normal anterior angle motor neurocyte, and these neurocyte structural integrities have, and kernel is clear, visible Nissl body; And matched group and the normal neurocyte of DMSO group form are rare, and spinal cord injury is heavier, and tangible vacuolar degeneration is arranged.Sham group and the normal neurocyte number of YC-5 ventricornu are compared no significant difference with matched group.YC-5 can significantly suppress the variation (P < 0.05) of myeloid tissue's lactic acid and lactic acid dehydrogenase activity behind the ischemia.
To sum up, damage has protective effect to YC-5 to ischemia of spinal cord, and is through the activation performance negativity regulating action to nmda receptor.
Five, YC-5 is to the protective effect and the mechanism of action of epilepsy
1.YC-5 to the inductive protective effect that relates to the damage of granule neuronal excitability of digenic acid
The 8th day rat cerebellum granule neuron of In vitro culture.Establish normal control group, digenic acid group, YC-5+ digenic acid group, CNQA+ digenic acid group respectively.The normal control group is left intact; The digenic acid group adds 200 μ M digenic acids; Other group adds YC-5 and CNQA in advance, hatch after 30 minutes for 37 ℃ to add digenic acid again, behind the 24h with phase contrast microscope neurocyte form; Carry out cell counting with FDA dyeing inverted fluorescence microscope, and calculate neuronal survival:
Survival rate=respectively organize viable count/matched group viable count * 100%.
Hoechst33258 dyeing, inverted fluorescence microscope is observed the neuronal kernel form.And mensuration is respectively organized the activity of lactic acid and lactalase.
As shown in Figure 7; YC-5+ digenic acid group (gives YC-5 earlier; Give 200 μ M digenic acid and YC-5 afterwards simultaneously); After cultivating 24h, phase contrast microscope is observed down and found: cerebellar granule neuron can keep complete, the nucleus of cell space and projection and chromatin to be normal morphology to some extent to some extent; FDA dyeing showed cell survival rate increases; The LDH release value reduces; And these phenomenons all with the concentration of giving YC-5 show dose dependence to a certain extent.And the YC-5 of above-mentioned dosage range does not have influence to the survival rate of normal cerebellar granule neuron.This shows that the YC-5 of 5～15 μ M can obviously suppress the toxic action of the inductive cerebellar granule neuron of digenic acid, improves neuronal survival.The YC-5 (64.05 ± 6.31%) of the dosage 15 μ M of maximum protection effect wherein.Because the special blocker CNQX of non-NMDA receptor has significant protective effect (CNQA+ digenic acid group to the inductive cerebellar granule of digenic acid through the toxic damages of unit; Neuron survival rate is 97.78 ± 3.11%); The inductive cerebellar granule of prompting digenic acid mainly is through the activation non-NMDA receptor through the toxic damages of unit, and the irritability damage of the inductive cerebellar neuron of YC-5 inhibition digenic acid maybe be relevant with the activation that suppresses non-NMDA receptor.
2.YC-5 protective effect to the damage of the irritability of the inductive hippocampal neuron of digenic acid
10 days hippocampus of rats of In vitro culture is established normal control group, digenic acid group, YC-5+ digenic acid group, CNQA+ digenic acid group respectively.The normal control group is left intact; The digenic acid group adds 200 μ M digenic acids; Other group adds solvent, YC-5 and CNQA in advance, hatch after 30 minutes for 37 ℃ to add digenic acid again, behind the 24h with phase contrast microscope neurocyte form; Carry out cell counting with FDA dyeing inverted fluorescence microscope, and calculate neuronal survival:
Survival rate=respectively organize viable count/matched group viable count * 100%.
Hoechst33258 dyeing, inverted fluorescence microscope is observed the neuronal kernel form.And mensuration is respectively organized the activity of lactic acid and lactalase.
As shown in Figure 8; YC-5+ digenic acid group (gives YC-5 earlier; Give 200 μ M digenic acid and YC-5 afterwards simultaneously); After cultivating 24h, phase contrast microscope is observed down and found: hippocampal neuron can keep complete, the nucleus of cell space and projection and chromatin to be normal morphology to some extent to some extent; FDA dyeing showed cell survival rate increases; The LDH release value reduces; And these phenomenons all with the concentration of giving YC-5 show dose dependence to a certain extent.And the YC-5 of above-mentioned dosage range does not have influence to the survival rate of normal hippocampal neuron.This shows that the YC-5 of 5～15 μ M can obviously suppress the toxic action of the inductive hippocampal neuron of digenic acid, improves neuronal survival.The YC-5 (62.12 ± 5.1%) of the dosage 15 μ M of maximum protection effect wherein.Because the special blocker CNQX of non-NMDA receptor has significant protective effect (CNQA+ digenic acid group to the inductive Hippocampus of digenic acid through the toxic damages of unit; Neuron survival rate is 97.89 ± 7.11%); The inductive Hippocampus of prompting digenic acid mainly is through the activation non-NMDA receptor through the toxic damages of unit, and the irritability damage of the inductive hippocampal neuron of YC-5 inhibition digenic acid maybe be relevant with the activation that suppresses non-NMDA receptor.
To sum up, YC-5 has protective effect to epilepsy and convulsions.
Claims (4)
1. cholestane-3 β, 5a, the application of 6 beta-triols in the preparation anti-cerebral ischemia damnification medicament.
2. cholestane-3 β, 5a, the application of 6 beta-triols in the anti-ischemia of spinal cord damage medicine of preparation.
3. cholestane-3 β, 5a, the application of 6 beta-triols in the preparation antiepileptic.
4. cholestane-3 β, 5a, the application of 6 beta-triols in the preparation anticonvulsant drug.
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| CN105012313B (en) * | 2014-04-25 | 2018-03-16 | 广州市赛普特医药科技股份有限公司 | The application of the β of 5 α androstanes 3,5,6 β triols and the like in preventing or treating altitude sickness caused by hypobaric hypoxia |
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| CN109985046B (en) * | 2017-12-29 | 2021-07-27 | 广州市赛普特医药科技股份有限公司 | 5 alpha-androst-3 beta, 5,6 beta-triol for the treatment of inflammation mediated optic neuropathy |
| CN108659089B (en) * | 2018-07-31 | 2020-08-25 | 温州医科大学 | Sterol compound with antioxidant effect and application thereof in preparation of medicines |
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