CN103316027B - Application of catalpol and retinoic acid medicine composition in preparing medicine used for preventing or treating white matter damage - Google Patents
Application of catalpol and retinoic acid medicine composition in preparing medicine used for preventing or treating white matter damage Download PDFInfo
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Abstract
The invention belongs to the field of medicine, and relates to the field of medicine preparation treatment activity. The invention specifically relates to a catalpol and retinoic acid medicine composition CAT-RA and an application thereof in preparing medicines used for preventing and treating with white matter damage after chronic cerebral ischemia. As a result of experiments of CAT-RA in cells and in chronic cerebral ischemia disease animal models, CAT-RA assists in reducing brain tissue inflammatory responses after brain damage, reducing white matter pathological changes, inhibiting oligodendrocyte apoptosis and myelin damage in white matters, and promoting oligodendrocyte precursor cell differentiation and maturation. CAT-RA has the effects of protecting white matters and promoting white matter remyelination. Therefore, CAT-RA can be used as an active component for preparing medicines used for preventing and treating white matter damage after chronic cerebral ischemia, and can also be used for preparing medicines used for treating other cerebral vascular related diseases characterized in white matter damages. The invention provides a novel solution for prevention and treatment of white matter damage related diseases.
Description
Technical field
The invention belongs to field of medicaments, relate to the therapeutic activity field of pharmaceutical preparation, be specifically related to catalpol and retinoic acid pharmaceutical composition thing CAT-RA and prevent in preparation or treat the application in the medicine of chronic cerebral white matter lesions.
Background technology
Central nervous system, accumulation place of pericaryon and dendron, because being rich in blood vessel, color is gloomy is called grey matter for fresh specimen, nerve fiber gather place, myelin color and luster brilliant white is called white matter, is also called medullary substance in the white matter of the dark side of brain inner cortex.Alba is formed primarily of nerve fiber and glial cell, central nervous system nerve fiber myelin has oligodendrocyte to be formed, Adult Human Brain white matter accounts for 50% of full brain volume, tissue metabolism leads only slightly lower than grey matter, alba itself has ischemia vulnerability, mild cerebral ischemic can cause damage, and the vascular anatomy factor in alba district causes the main cause of this regioselectivity ischemia rapid wear simultaneously.All there is Infant Injury in White Matter in a lot of diseases seeing central nervous system clinically, from the apoplexy of neonatal periventricular leukomalacia, manhood, sudden cardiac arrest to the vascular dementia of old people, alba is all a target spot of hypoxic-ischemic damage, and also finds a large amount of Infant Injury in White Matter in normal old people.
Current research shows, Infant Injury in White Matter is the main result of chronic cerebral ischemia effect.Ischemic white matter lesions can destroy the signal transmission between neuron, between cortex and subcortical center, and pathological change mainly comprises microglial activation Reactive astrocytes proliferation and hypertrophy, oligodendrocyte reduce, white matter is loose and demyelination.Microglia is the immune effector cell in central nervous system, is activated in pathological conditions, and produce cytokine profiles, release cells toxin, causes Infant Injury in White Matter.The clinical common neurobehavioral Novel presentation of patient with ischaemic leukoaraiosis damage is the exception of focal neurosigns, consciousness, emotion and behavioral function.
The mechanism of Infant Injury in White Matter is very complicated, thinks that immunoinflammatory damage is the important mechanisms causing white matter lesions after ischemia at present.A large amount of inflammatory glial cells is contained in alba district, mainly refers to microglia and astrocyte.Inflammatory glial cell activates in a large number after ischemia, the activated leukocyte, immunoglobulin, complement etc. that enter brain essence with the blood brain barrier by damaging cause Immune inflammatory reaction jointly, by producing toxic protein enzyme and the TNF secretion such as serine protease, urokinase, collagenase, elastoser
-α, nitric oxide, the inflammatory mediator such as interleukin-1 beta draw Infant Injury in White Matter.Alba district inflammatory reaction can increase the weight of the ischemia injury of aixs cylinder and oligodendrocyte, and Bing Shigai district vascular endothelial cell is impaired, blood vessel wall collagen deposition, angiostenosis, causes cerebral blood flow Progressive symmetric erythrokeratodermia to decline, and worsens the blood supply in white matter district further.Under the pathologic conditions such as cerebral ischemia, response to oxidative stress also strengthens, cause reactive oxygen free radical too much, and free radical scavenging enzyme such as superoxide dismutase isoreactivity reduces, dynamic equilibrium destroys, start free radical chain reactions, cause tissue ischemia to damage and worsen further.In addition, after white matter ischemia, Immune inflammatory reaction discharges a large amount of inflammatory cytokines, protease and oxygen-derived free radicals etc., and these materials directly or indirectly promote oligodendrocyte death.Oligodendrocyte numbers reduces can cause Demyelination, and severe dysfunction appears in the aixs cylinder that myelin is held.
Adopt pharmaceutical intervention treatment in the laboratory research of chronic cerebral ischemia white matter lesions at present more.Microglial activation can be suppressed as immunosuppressant Tacrolimus and reduce white matter lesions; Phosphodiesterase inhibitor ibudilast is by suppressing TNF
-αgeneration, alleviate the Demyelination of 2VO (the permanent ligation of bilateral common carotid arteries) postoperative white matter; III type phosphodiesterase inhibitor cilostazol causes transcription factor cyclic adenosine monophosphate response element in conjunction with activator protein phosphorylation by signal path, plays the protective effect of the postoperative white matter of 2VO; Cyclooxygenase2 inhibitor nimesulide can make rat immunity activity decrease, and the microglia quantity be activated reduces, and White matters lesions alleviates etc.But these medicines do not demonstrate in clinical practice show in laboratory research obvious effect, and this kind of drug side effect is larger, pneumonia can be caused as taken Tacrolimus, peritonitis and biliary tract inflammation infection rate are about 20%-38%, lymphadenosis incidence rate is about 0.7%-1.6%, when neurotoxicity is more common in quiet note, akinetic mutism can be shown as, expressive aphasia, be full of epilepsy outbreak, mental disorder, encephalopathy, persistent coma, tremble, headache, sleep disordered, nightmare, insensitive and photophobia etc., hyperglycemia and diabetes incidence rate about 17%, majority needs insulinize.Thus these medicines not extensive use.In addition; some patients morbidity early symptom is very light and not obvious clinically; easy recurrence and tardy nerve damage once again, can invade whole marrowbrain and cause nerve function lesion to occur to paralyse and crisis life time serious, and most of patient is caused by genetic immunization infects by particular virus extremely.Early stage treatment is mainly with hormone and trophotherapy treatment, but curative effect is difficult to control admittedly, and because primary disease causes myelinoclasis to cause function of nervous system, panimmunity is extremely not low, and the even and state of an illness infects disease relapse makes function of nervous system's symptom increase the weight of further.If can not get correct treatment, can recur and soften with tardy many focus sclerosis or focus, and injured nerve secondary dementia or paralysis once again.Therefore, at present safe and effective treatment means is still lacked for such disease.
The apoptosis of the oligodendrocyte (OL) that the damage of alba and inflammatory reaction are induced and myelin damage closely related.There is oligodendrocyte precursors (OPC) static on a small quantity in central nervous system, these OPC can also grow for ripe OL by proliferation and differentiation under certain condition, participate in the reparation of Infant Injury in White Matter.Therefore, using effective medicine to react by inflammation-inhibiting the survival promoting OL on the one hand, on the other hand by promoting that the OPC differentiation of resting state carrys out the OL of supplementary apoptosis, the effect of the Infant Injury in White Matter of prevention or the induction for the treatment of chronic cerebral ischemia can be reached.
Catalpol (CAT) is a kind of iridoid monoglycosides compounds, molecular formula C
15h
22o
10.Catalpol is distributed widely in Metachlamydeae, Loganiaceae Herba Buddlejae Lindleyanae belongs to, Scrophulariaceae bubble Chinese parasol tree belongs to and in the plant such as Radix Rehmanniae genus, bavin Wei section Cinnamomum, Orobanchaceae Cistanche deserticola, Plantaginaceae Plantago, originate and enrich.Catalpol has anticancer, neuroprotective, antiinflammatory, diuresis, the multiple pharmacotoxicological effect such as blood sugar lowering and hepatitis virus resisting.Research shows, catalpol has obvious anti-inflammatory activity, and catalpol can pass through to suppress microvascular permeability thus the effect reaching hemostasis.Application number be 201210239357.6 patent of invention disclose catalpol preparation anti-ovarian-senescence drug in application.Application number be 201110184792.9 patent of invention disclose a kind of novelty teabag of catalpol, the i.e. application of catalpol in preparation treatment ischemic cerebral apoplexy sequelae medicine, this purposes of catalpol based on principle be that catalpol can shorten tactual stimulation incubation period, promote the recovery of ischemic cerebral apoplexy sequelae antennal nerve function.
Retinoic acid (RA) is the metabolic intermediate of vitamin A in body, the metabolism such as the growth of major effect bone and promotion epithelial hyperplasia, differentiation, cutin dissolving.The retinoic acid of effective dose can induce liver precursor to be divided into quasi-liver cell; Induced by Retinoic Acid Neonatal Rat Neural Stem Cells/precursor vitro differentiation neuron cell etc.Existing research has confirmed that retinoic acid has stabilized cell structure, promotes the effect of precursor cell differentiation.
Summary of the invention
In view of this, the invention provides the compositions CAT-RA based on above-mentioned catalpol and retinoic acid two kinds of components, and it is for the preparation of the application in prevention or treatment chronic cerebral white matter lesions medicine.
For achieving the above object, technical scheme of the present invention is:
Alba itself has ischemia vulnerability, and mild cerebral ischemic can cause damage, and the vascular anatomy factor in alba district causes the main cause of this regioselectivity ischemia rapid wear simultaneously.All there is Infant Injury in White Matter in a lot of diseases seeing central nervous system clinically, from the apoplexy of neonatal periventricular leukomalacia, manhood, sudden cardiac arrest to the vascular dementia of old people, alba is all a target spot of hypoxic-ischemic damage, and also finds a large amount of Infant Injury in White Matter in normal old people.Therefore, effectively the research of the medicine of prevention or treatment Infant Injury in White Matter has very important effect.
An object of the present invention there are provided a kind of compositions CAT-RA, and its two kinds of components contained have synergism when treating Infant Injury in White Matter.
Pharmaceutical composition, is made up of under effective dose catalpol and retinoic acid.
Further, described in described pharmaceutical composition ratio, the ratio of catalpol and described retinoic acid is 50 ~ 2000:0.5 ~ 2.
Further, described in described pharmaceutical composition ratio, the ratio of catalpol and described retinoic acid is 500:1.
Two of object of the present invention there are provided the one application of catalpol, and this is applied as Infant Injury in White Matter treatment and provides new thinking.
The application of catalpol in the medicine for the preparation of prevention or treatment chronic cerebral white matter lesions.Catalpol has anticancer, neuroprotective, antiinflammatory effect, the apoptosis of the oligodendrocyte (OL) that the damage of alba is induced with inflammatory reaction again and myelin damage closely related.In cell experiment, research shows that being used alone CAT can have significant facilitation to oligodendrocyte precursor cells survival after Anoxia, suppress damage or the apoptosis of oligodendrocyte precursor cells after Anoxia, therefore, catalpol can be used for the research of the medicine preventing or treat Infant Injury in White Matter.
Further, described catalpol and retinoic acid combine the application in the medicine for the preparation of prevention or treatment chronic cerebral white matter lesions.Retinoic acid has confirmed to have stabilized cell structure, promotes the effect of precursor cell differentiation.Oligodendrocyte precursors (OPC) static is on a small quantity there is in central nervous system, these OPC can also grow for ripe oligodendrocyte (OL) by proliferation and differentiation under certain condition, supplement the apoptosis of the oligodendrocyte (OL) of inflammatory reaction induction, participate in the reparation of Infant Injury in White Matter.In experiment, research shows that CAT-RA can promote the survival of Anoxia OPC, the apoptosis of OL or OPC after Anoxia can be suppressed, and conbined usage CAT and RA has synergism to cell survival effect, the effect of both conbined usage is better than being used alone CAT or being used alone the attainable effect of RA.Therefore, catalpol and retinoic acid compositions can be used for the research of the medicine preventing or treat Infant Injury in White Matter.
Further, described catalpol and retinoic acid are preparing the application in oligodendrocyte precursor cells differentiation and maturation promoter.
Further, described catalpol and retinoic acid are at the inhibitor preparing the inflammatory reaction that oligodendrocyte precursors Anoxia is induced, or the application in the inhibitor of the apoptosis of oligodendrocyte precursor cells Anoxia induction.
Further, described catalpol and the retinoic acid application in preparation alba in oligodendrocyte inhibitors of apoptosis or alba in oligodendrocyte myelin damage inhibitor.
The application of the compositions that described catalpol and retinoic acid form under effective dose in the medicine for the preparation of prevention or treatment chronic cerebral white matter lesions.
Three of object of the present invention is the preparation providing a kind of aforementioned pharmaceutical compositions to prepare, and dosage form can have multiple, and tablet or capsule or granule or ejection preparation, it is easy to use, and absorption efficiency is high.
Preparation prepared by the compositions that described catalpol and retinoic acid form under effective dose.
Beneficial effect of the present invention is: CAT-RA compositions of the present invention is not known to prior art, two kinds of components being combined under effective dose has synergism when preventing or treat Infant Injury in White Matter, application safety in the medicine of its Infant Injury in White Matter after preparation prevention or treatment chronic cerebral ischemia, effectively, have no side effect, for the prevention and therapy of Infant Injury in White Matter after chronic cerebral ischemia provides a kind of new solution, the cerebrovascular relevant disease taking also simultaneously Infant Injury in White Matter as feature for other prevents or treatment provides new thinking, in addition, also for prevention or treatment relate to the oligodendrocyte of Anoxia induction or the inflammatory reaction of oligodendrocyte precursor cells, apoptosis, the disease of myelin damage etc. provides new thinking.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described.
Fig. 1 is used alone CAT or is used alone the impact of RA on Anoxia oligodendrocyte precursor cells survival rate;
Fig. 2 CAT-RA conbined usage is on the impact of the survival rate of oligodendrocyte precursor cells after Anoxia;
Fig. 3 CAT-RA is to the inhibitory action of the inflammatory reaction of Anoxia induction oligodendrocyte precursor cells;
Fig. 4 CAT-RA can suppress the apoptosis of oligodendrocyte precursor cells after Anoxia;
Fig. 5 CAT-RA can promote the differentiation and maturation of oligodendrocyte precursor cells after Anoxia;
Fig. 6 CAT-RA on the impact of the alba pathological change that chronic cerebral ischemia is induced and CAT-RA to the inhibitory action of the alba inflammatory reaction that chronic cerebral ischemia is induced;
The inhibitory action of the oligodendrocyte apoptosis that Fig. 7 CAT-RA induces chronic cerebral ischemia and myelin damage;
Fig. 8 CAT-RA promotes the differentiation and maturation of oligodendrocyte precursor cells in alba after chronic cerebral ischemia.
Detailed description of the invention
In order to make the object, technical solutions and advantages of the present invention clearly, below in conjunction with accompanying drawing, the present invention is described in detail.
Immunoinflammatory damage is the important mechanisms causing white matter lesions after ischemia.The apoptosis of the oligodendrocyte (OL) that the damage of alba and inflammatory reaction are induced and myelin damage closely related.There is oligodendrocyte precursors (OPC) static on a small quantity in central nervous system, these OPC can also grow for ripe OL by proliferation and differentiation under certain condition, participate in the reparation of Infant Injury in White Matter.
As described above, the catalpol that the present invention relates to and retinoic acid pharmaceutical composition, wherein catalpol (CAT) is a kind of iridoid monoglycosides compounds, molecular formula C
15h
22o
10, there is anticancer, neuroprotective, antiinflammatory, diuresis, the multiple pharmacotoxicological effect such as blood sugar lowering and hepatitis virus resisting.Retinoic acid (RA) is the metabolic intermediate of vitamin A in body, and the metabolism such as the growth of major effect bone and promotion epithelial hyperplasia, differentiation, cutin dissolving, retinoic acid has stabilized cell structure, promotes the effect of precursor cell differentiation.
Shown by In vitro cell experiment: CAT-RA two kinds of combination of components effects are greater than and are used alone CAT or are used alone RA, CAT-RA has inflammation-inhibiting reaction, promote OL survival and promote the effect of OPC differentiation.
In embodiment, laboratory animal is: the healthy adult male Wistar rat (Field Surgery Inst. of The Third Military Medical Univ.'s Experimental Animal Center) of be born cleaning grade SD rat (Military Medical Univ No.3, P.L.A's Experimental Animal Center) in 72 hours and body weight 250 ~ 300g.
Catalpol and retinoic acid are from Nat'l Pharmaceutical & Biological Products Control Institute.
If without specified otherwise, be experimentally normal experiment method; The reagent used and material all obtain by commercial sources.
The In vitro cell experiment of CAT-RA
Experiment one is used alone CAT or is used alone the impact of RA on cell survival rate
Isolation and purification culture OPC SD rat brain tissue in birth 72 hours, by OPC with 1.5 × 10
4the density of/ml is inoculated in 96 orifice plates, and every hole chemically defined culture medium 200 μ l, is placed in 37 DEG C, 5%CO
2cultivate in incubator, choose differentiation 3 days, based on late period, the cell of OPC is for subsequent use.
Preparation containing the DMEM/F12 culture medium of CAT of 0.25mM, 0.5mM and 1mM, containing the DMEM/F12 culture medium of the RA of 0.5 μM, 1 μM and 2 μMs.
The CAT treatment group DMEM/F12 culture medium of the above-mentioned CAT containing 0.25mM, 0.5mM and 1mM hatches 1 hour, then be changed to DMEM/F12 culture medium in 37 DEG C of process after 20 minutes with the low sugar culture-medium containing 10mM sodium dithionite, each concentration arranges 8 multiple holes again.Establish anoxia matched group simultaneously, be changed to DMEM/F12 culture medium in 37 DEG C of process after 20 minutes with the low sugar culture-medium containing 10mM sodium dithionite.
The above-mentioned DMEM/F12 culture medium containing the RA of 0.5 μM, 1 μM and 2 μMs of RA treatment group hatches 1 hour, then be changed to DMEM/F12 culture medium in 37 DEG C of process after 20 minutes with the low sugar culture-medium containing 10mM sodium dithionite, each concentration arranges 8 multiple holes again.Establish anoxia matched group simultaneously, be changed to DMEM/F12 culture medium in 37 DEG C of process after 20 minutes with the low sugar culture-medium containing 10mM sodium dithionite.
After cultivation after a while, adopting tetrazolium bromide (MTT) test kit to detect cell survival, respectively to adding 1mg/ml MTT20 μ l in every hole, cultivating 4 hours for 37 DEG C.After centrifugal, every hole adds DMSO150 μ l, and microplate reader measures the absorbance at 570nm wavelength place, calculates cell survival rate, and experimental data is with x ± s, and between group, significance test adopts variance analysis and t inspection, and result as shown in Figure 1.
Result shows, being used alone CAT has obvious facilitation to cell survival, and when CAT concentration is 0.5mM, survivaling cell ratio is close to 80%, and cell survival significantly increases (p<0.01).Be used alone the facilitation of RA to cell survival not obvious, with anoxia group (matched group) no difference of science of statistics between 3 concentration, but after anoxia cell survival 1 μM of concentration RA compared with 0.5 μM and 2 μMs of effects slightly good.
Test two CAT-RA conbined usage to the impact of OPC cell survival rate
The research of CAT-RA conbined usage on the impact of cell survival is by establishing the mono-medicine group of RA (concentration 0.5,1,2,4 μM), the mono-medicine group of CAT (concentration 0.001,0.01,0.05,0.1,0.25,0.5,1,2,5,10mM), then combine the CAT of variable concentrations through the RA of 4 concentration respectively, establish the positive and negative control to study simultaneously.Here choose wherein one group illustrate.
The isolation and purification culture of OPC is with experiment one, and choose differentiation 3 days, based on late period, the cell of OPC is divided into normal group, anoxia group and treatment group.Preparation is containing the DMEM/F12 culture medium of variable concentrations CAT-RA, and CA concentration is 1 μM, and the concentration of catalpol is respectively: 0.001,0.01,0.05,0.1,0.25,0.5,1,2,5,10mM.
Normal group, with DMEM/F12 culture medium culturing, does not carry out Anoxia and CAT-RA process.
The low sugar culture-medium of anoxia group containing 10mM sodium dithionite is changed to DMEM/F12 culture medium in 37 DEG C of process after 20 minutes.
The treatment group DMEM/F12 culture medium containing variable concentrations CAT-RA of above-mentioned preparation hatches 1 hour, and be then changed to DMEM/F12 culture medium in 37 DEG C of process after 20 minutes with the low sugar culture-medium containing 10mM sodium dithionite, each concentration arranges 8 multiple holes again.
After cultivating a period of time, carry out the detection of cell survival rate, method is with experiment one, and experimental data is with x ± s, and between group, significance test adopts variance analysis and t inspection, and experimental result as shown in Figure 2.Cell survival rate (52.5 ± 2.8%) the compared with normal group (100.0 ± 1.0%) of anoxia group obviously reduces (p<0.01).The impact of CAT-RA on cell survival rate of variable concentrations has amphicheirality, when the catalpol concentration in CAT-RA is lower than 5mM, cell survival can be promoted, but only have, containing the cell survival rate of the CAT-RA of 0.05-2mM catalpol and anoxia group, there is notable difference, and when the catalpol concentration in CAT-RA is 0.5mM, cell survival rate the highest (85.6 ± 5.2%) (p<0.01).When the catalpol concentration in CAT-RA is 10mM, cell survival rate (46.6 ± 2.4%) is lower than anoxia group, and the catalpol of prompting high concentration can produce toxic action to cell.
The above results shows, for the OPC of In vitro culture, CAT optium concentration in compositions CAT-RA is 0.5mM, RA optium concentration is 1 μM, now cell survival rate the highest (85.6 ± 5.2%) (p<0.01), the effect of CAT and RA conbined usage is better than being used alone CAT or being used alone the attainable effect of RA, then show that CAT and RA conbined usage has synergism.
According to the result of this experiment, the cell experiment below carried out all selects CAT-RA combination (CAT optium concentration is 0.5mM, RA optium concentration is 1 μM) to study.
Test the inhibitory action of three CAT-RA to the inflammatory reaction that Anoxia is induced
TNF-a is a kind of many active cytokines of the secretion such as a kind of macrophage by activation by lipopolysaccharide and lymphocyte.TNF-a can participate in the immunopathogenesis damage of body, in the pathogenesis of immune disease, have important clinical value.The TNF-a of normal level can immunity moderation response, infection, promotion tissue repair, cause apoptosis of tumor cells etc., but to produce and release then can destroy the immunologic balance of body in a large number, produce multiple pathology damage together with other inflammatory factor.TNF-a especially comes into one's own as a kind of important inflammatory mediator, other cytokines can be stimulated as the release of IL-4, IL-6 etc., expand its biological effect, thus Cellular inflammatory reaction after mediation wound, cause multiple organ tissue injury.Therefore, by can reflect the inflammatory reaction degree of different experiments group to the detection of TNF-alpha content.
Choose the OPC broken up 3 days in experiment one and be divided into normal group, anoxia group and treatment group.
The process of normal group and anoxia group is with the operation of testing two.Treatment group is 1 μM with being the most concentration of 0.5mM, RA containing CAT-RA(CAT concentration) DMEM/F12 culture medium hatch 1 hour, be then again changed to DMEM/F12 culture medium in 37 DEG C of process after 20 minutes with the low sugar culture-medium containing 10mM sodium dithionite.
Cultivate to collect respectively after a period of time normal group, anoxia group, treatment group cell conditioned medium liquid for measuring the content of TNF-α in supernatant.The rabbit TNF-α ELISA kit of R & D company is adopted to measure.Experimental data is with x ± s, and between group, significance test adopts variance analysis and t inspection, and result as shown in Figure 3.
In anoxia group, the expression of TNF-α obviously raises (237.5 ± 18.2%), compares with normal group (100%), has significant difference (p<0.01).The expression of the TNF-α for the treatment of group obviously reduces (124.3 ± 10.7%), compares with anoxia group, has significant difference (p<0.05), shows the inflammatory reaction that CAT-RA can suppress Anoxia to be induced effectively.
Test the inhibitory action of four CAT-RA to the apoptosis of OPC after Anoxia
Apoptosis process is with the change of mitochondrial membrane potential, and can measure with JC-1 dyeing, when mitochondrial membrane potential is higher, JC-1 produces red fluorescence; When mitochondrial membrane potential is lower, JC-1 produces green fluorescence, utilizes the relative scale of red green fluorescence to carry out the early apoptosis of measure of cell.
The OPC of 24 orifice plates (Anoxia and CAT-RA process operation are with experiment two) after Anoxia and CAT-RA process will be incubated at, JC-1 mitochondrial membrane potential detection kit is adopted to detect OPC mitochondrial membrane potential, 1 time is washed with PBS, add 0.5ml culture medium and the 0.5ml JC-1 working solution that dyes respectively and hatch 20 minutes in 37 DEG C, absorb supernatant, wash 2 times with JC-1 dye solution, under laser confocal microscope, after being changed to 1ml culture medium, observe the transformation of red green fluorescence.Experimental data is with x ± s, between group, significance test adopts variance analysis and t inspection, result as shown in Figure 4, the red fluorescence of anoxia group is less, green fluorescence is more, and green/red Fluorescence Ratio (72.6 ± 8.3%) compared with normal group (18.3 ± 2.2%) obviously raises (p<0.01).The red fluorescence for the treatment of group increases, green fluorescence reduces, and green/red Fluorescence Ratio (35.8 ± 4.7%) comparatively anoxia group obviously declines (p<0.05), shows that CAT-RA can suppress the decline of mitochondrial membrane potential and the apoptosis of OPC cell.
Test the differentiation that five CAT-RA promote OPC after Anoxias
OPC cell refractivity after Anoxia weakens, and projection bounces back, and changes in sections sample, or projection fracture, and cell differentiation is lower.
Be inoculated in the OPC of 24 orifice plates (Anoxia and CAT-RA process operation are with experiment two) after Anoxia and CAT-RA process, continue cultivation 7 days, by the cellular morphology of phase contrast microscope observe differentiation 3 days and 6 days.As shown in Figure 5, after treatment group CAT-RA process, projection retraction capable of inhibiting cell and fracture, promote projection branch and cell differentiation to result, shows that CAT-RA has the effect of the differentiation and maturation promoting OPC.
The In vitro cell experiment of CAT-RA shows, CAT-RA has promotion cell survival, can react, suppresses OL apoptosis and myelin damage by inflammation-inhibiting, and can promote OPC differentiation and maturation.
According to In vitro cell experiment result of study, and then carried out zooperal research further, result shows in chronic cerebral ischemia disease animal model, use the alba inflammatory reaction that the CAT-RA of effective dose can suppress chronic cerebral ischemia to be induced, suppress OL apoptosis and the myelin damage of chronic cerebral ischemia induction, promote chronic cerebral ischemia alba OPC differentiation and maturation.Be described in further detail below in conjunction with embodiment.
The pharmaceutical composition of embodiment 1CAT-RA
The pharmaceutical composition of CAT-RA, gets catalpol 100mg, retinoic acid 0.3mg, and the mol ratio of catalpol and retinoic acid is 280:1, with physiological saline solution to 100ml.
The pharmaceutical composition of embodiment 2CAT-RA
The pharmaceutical composition of CAT-RA, gets catalpol 500mg, retinoic acid 0.45mg, and the mol ratio of catalpol and retinoic acid is 1380:1.5, with physiological saline solution to 100ml.
The impact of CAT-RA on cell survival rate of variable concentrations has amphicheirality, when the catalpol concentration in CAT-RA is lower than 5mM, can promote cell survival; Along with the catalpol concentration in CAT-RA higher than 5mM time, along with the increase of concentration, cell survival rate reduce, show that the catalpol of high concentration can produce toxic action to cell.Before carrying out animal model experiment research, experimentally result combines and consults pertinent literature report, and the amount of application of below catalpol 10mg/kg is its safe dose.
Embodiment 3CAT-RA is on the impact of rat brain white matter pathological change
Thirty male rats is divided into normal group, ischemia group and treatment group, catalpol and retinoic acid are with physiological saline solution.Ischemia group is the chronic cerebral ischemia model set up by permanent Banded Rats bilateral common carotid arteries, postoperative lumbar injection 5mg/kg normal saline.Treatment group is the CAT-RA in the postoperative lumbar injection embodiment 1 of permanent Banded Rats bilateral common carotid arteries, application dosage with catalpol dosage for 1mg/kg, every day 1 time, continuous 10 days.Within postoperative 30 days, put to death all animals.Carrying out paraffin section (thick 5 μm) and H & E dyes to comprising callosal cerebral tissue block, measuring callosal cavity area under microscope, experimental data is with x ± s, and between group, significance test adopts variance analysis and t inspection.
Result as shown in Figure 6, under the corpus callosum of ischemia group rat, cortex, the ventricles of the brain are other etc. there is significantly loose and cavity sample change in white matter region, its corpus callosum cavity area accounts for corpus callosum gross area percentage ratio (20.1 ± 4.3%) and compares with normal group (2.4 ± 0.8%), has notable difference (p<0.01).The corpus callosum cavity area percentage (15.6 ± 2.0%) of 1mg/kg treatment group compares with ischemia group, no significant difference, but alleviates to some extent through the treatment group alba pathological change degree of CAT-RA treatment.
Embodiment 4CAT-RA is on the impact of rat brain white matter pathological change
Thirty male rats is divided into normal group, ischemia group and treatment group, catalpol and retinoic acid are with physiological saline solution.Ischemia group is the chronic cerebral ischemia model set up by permanent Banded Rats bilateral common carotid arteries, postoperative lumbar injection 5mg/kg normal saline.Treatment group is the CAT-RA in the postoperative lumbar injection embodiment 1 of permanent Banded Rats bilateral common carotid arteries, application dosage with catalpol dosage for 5mg/kg, every day 1 time, continuous 10 days.Within postoperative 30 days, put to death all animals.Carrying out paraffin section (thick 5 μm) and H & E dyes to comprising callosal cerebral tissue block, measuring callosal cavity area under microscope, experimental data is with x ± s, and between group, significance test adopts variance analysis and t inspection.
Result as shown in Figure 6, under the corpus callosum of ischemia group rat, cortex, the ventricles of the brain are other etc. there is significantly loose and cavity sample change in white matter region, its corpus callosum cavity area accounts for corpus callosum gross area percentage ratio (20.1 ± 4.3%) and compares with normal group (2.4 ± 0.8%), has notable difference (p<0.01).The above-mentioned pathological change degree of 5mg/kg treatment group alba is obvious, the corpus callosum cavity area percentage of 5mg/kg treatment group be 9.6 ± 1.5% comparatively ischemia group obviously reduce (p<0.05).
Embodiment 5CAT-RA is on the impact of rat brain white matter pathological change
Thirty male rats is divided into normal group, ischemia group and treatment group, catalpol and retinoic acid are with physiological saline solution.Ischemia group is the chronic cerebral ischemia model set up by permanent Banded Rats bilateral common carotid arteries, postoperative lumbar injection 5mg/kg normal saline.Treatment group is the CAT-RA in the postoperative lumbar injection embodiment 2 of permanent Banded Rats bilateral common carotid arteries, application dosage with catalpol dosage for 10mg/kg, injection every day 1 time, continuous 10 days.Within postoperative 30 days, put to death all animals.Carrying out paraffin section (thick 5 μm) and H & E dyes to comprising callosal cerebral tissue block, measuring callosal cavity area under microscope, experimental data is with x ± s, and between group, significance test adopts variance analysis and t inspection.
Result as shown in Figure 6, under the corpus callosum of ischemia group rat, cortex, the ventricles of the brain are other etc. there is significantly loose and cavity sample change in white matter region, its corpus callosum cavity area accounts for corpus callosum gross area percentage ratio (20.1 ± 4.3%) and compares with normal group (2.4 ± 0.8%), has notable difference (p<0.01).The above-mentioned pathological change degree of 10mg/kg treatment group alba is obvious, and the corpus callosum cavity area percentage of 10mg/kg treatment group is 8.3 ± 1.0%, and comparatively ischemia group obviously reduces (p<0.05).
Embodiment 6CAT-RA suppresses the alba inflammatory reaction of chronic cerebral ischemia induction
The experimental technique of the present embodiment, with embodiment 4, puts to death all animals in postoperative 30 days.Be separated callus tissue, extract supernatant after homogenate, use ELISA kit, detect the expression of TNF-α, experimental data is with x ± s, and between group, significance test adopts variance analysis and t inspection.
As shown in Figure 6, in ischemia group, the expression of TNF-α obviously raises (300.5 ± 39.8%) result, compares, have significant difference (p<0.01) with normal group (100%).The expression of the TNF-α for the treatment of group obviously reduces (157.8 ± 15.1%), compares with ischemia group, has significant difference (p<0.05).CAT-RA treats the alba inflammatory reaction that chronic cerebral ischemia can be suppressed to induce.
Embodiment 7CAT-RA suppresses OL apoptosis and the myelin damage of chronic cerebral ischemia induction
The present embodiment experimental technique is with embodiment 4, within postoperative 30 days, put to death all animals, carrying out frozen section (thick 20 μm) to comprising callosal cerebral tissue block, jointly hatching APC antibody and caspase-3 antibody, after the anti-labelling of fluorescence two, analyze the two positive cell number of APC and caspase-3; MBP antibody is hatched in section, and immunoenzyme mark observes the average optical density value of MBP dyeing.In addition, be separated callus tissue, extract albumen, detect the expression of caspase-3 and MBP albumen at callus tissue respectively by Western blot technology after homogenate, experimental data is with x ± s, and between group, significance test adopts variance analysis and t inspection.
As shown in Figure 7, in Showed by immune group result normal group alba, APC(is red for result) and caspase-3(green) less (3.2 ± 1.1/0.25mm of OL apoptosis cell of two positive (Huang)
2), after ischemia, the two positive cell number of APC and caspase-3 obviously raises (64.2 ± 8.0/0.25mm
2), compare with normal group and have significant difference (p<0.01).The two positive cell number of APC and caspase-3 for the treatment of group obviously reduces (32.0 ± 5.0/0.25mm
2), compare with ischemia group and have significant difference (p<0.05).The MBP of ischemia group average optical density value (0.15 ± 0.02) the compared with normal group (0.31 ± 0.04) that dyes obviously reduces (p<0.01), the MBP for the treatment of group dye average optical density value (0.24 ± 0.03) comparatively ischemia group obviously raise (p<0.05).Western blot result also show chronic cerebral ischemia and can significantly improve the expression of caspase-3 albumen and reduce the expression (p<0.01) of MBP albumen, and CAT-RA then can reduce the expression of caspase-3 and improve the expression (p<0.05) of MBP albumen.CAT-RA treats the OL apoptosis and myelin damage that chronic cerebral ischemia can be suppressed to induce.
Embodiment 8CAT-RA promotes chronic cerebral ischemia alba OPC differentiation and maturation
The present embodiment experimental technique is with embodiment 4.Three groups of rats in postoperative 24 hours intracerebroventricular injection 50mg/kg BrdU(with physiological saline solution), every day 1 time, continuous 7 days.Within postoperative 30 days, put to death all animals.Carrying out frozen section (thick 20 μm) to comprising callosal cerebral tissue block, cutting into slices after calf serum is closed, jointly hatching BrdU antibody and CNPase antibody, after the anti-labelling of fluorescence two, analyze the two positive cell number of BrdU and CNPase.
Result as shown in Figure 8, experimental data is with x ± s, and between group, significance test adopts variance analysis and t inspection, and in normal group alba, the proliferative cell of the BrdU positive (green) is less, the OL of the CNPase positive (red) is more, and the percentage ratio that two positive cell number accounts for the total cell number of BrdU is 5.0 ± 1.4%.The percentage ratio that the two positive cell number of ischemia group BrdU and CNPase accounts for the total cell number of BrdU is 5.7 ± 1.6%, compares no significant difference with normal group.The percentage ratio that in CAT-RA treatment group alba, the two positive cell number of BrdU and CNPase accounts for the total cell number of BrdU is 17.2 ± 3.9%, compares have significant difference (p<0.05) with ischemia group.CAT-RA treatment can promote the differentiation and maturation of OPC in chronic cerebral ischemia alba.
The using dosage of CAT-RA compositions defined above can between 1mg catalpol/kg body weight to 10mg catalpol/kg body weight, and preferred 5mg catalpol/kg body weight is to 10mg catalpol/kg body weight.
What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (9)
1. pharmaceutical composition, is characterized in that: be made up of under effective dose catalpol and retinoic acid.
2. pharmaceutical composition according to claim 1, is characterized in that: the ratio of catalpol described in ratio and described retinoic acid is 50 ~ 2000:0.5 ~ 2.
3. pharmaceutical composition according to claim 2, is characterized in that: the ratio of catalpol described in ratio and described retinoic acid is 500:1.
4. catalpol and retinoic acid combine the application in the medicine for the preparation of prevention or treatment chronic cerebral white matter lesions.
5. application according to claim 4, is characterized in that: described catalpol and retinoic acid are preparing the application in oligodendrocyte precursor cells differentiation and maturation promoter.
6. application according to claim 4, it is characterized in that: described catalpol and retinoic acid are at the inhibitor preparing the inflammatory reaction that oligodendrocyte precursors Anoxia is induced, or the application in the inhibitor of the apoptosis of oligodendrocyte precursor cells Anoxia induction.
7. application according to claim 4, is characterized in that: the application in preparation alba in oligodendrocyte inhibitors of apoptosis or alba in oligodendrocyte myelin damage inhibitor of described catalpol and retinoic acid.
8. the application of compositions according to claim 1 in the medicine for the preparation of prevention or treatment chronic cerebral white matter lesions.
9. the preparation prepared of pharmaceutical composition according to claim 1.
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| CN101250205A (en) * | 2008-03-12 | 2008-08-27 | 谢鹏 | Catalpol and use of its derivatives in preparation of medicine controlling cerebrovascular disease |
| US20090131498A1 (en) * | 2005-01-18 | 2009-05-21 | Danishefsky Samuel J | Enantioselective Synthesis of Merrilactone and Its Analogs |
| CN101843630A (en) * | 2010-06-11 | 2010-09-29 | 首都医科大学宣武医院 | Composite containing iridoid compound and application thereof |
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| US20090131498A1 (en) * | 2005-01-18 | 2009-05-21 | Danishefsky Samuel J | Enantioselective Synthesis of Merrilactone and Its Analogs |
| CN101250205A (en) * | 2008-03-12 | 2008-08-27 | 谢鹏 | Catalpol and use of its derivatives in preparation of medicine controlling cerebrovascular disease |
| CN101843630A (en) * | 2010-06-11 | 2010-09-29 | 首都医科大学宣武医院 | Composite containing iridoid compound and application thereof |
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