CN102600370B - Application of Chinese medicinal composition in preparing medicament for preventing and treating Parkinsonism - Google Patents
Application of Chinese medicinal composition in preparing medicament for preventing and treating Parkinsonism Download PDFInfo
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Abstract
The invention relates to application of a Chinese medicinal composition in preparing a medicament for preventing and treating Parkinsonism. The Chinese medicinal composition is prepared from the following bulk pharmaceutical chemicals in parts by weight: 10 to 20 parts of gastrodia elata, 10 to 20 parts of astragalus mongholicus, 15 to 25 parts of prepared rehmannia root, 15 to 25 parts of white peony root, 15 to 25 parts of Chinese angelica, 10 to 20 parts of uncaria, 7 to 13 parts of stiff silkworm, and 7 to 13 parts of rhizoma arisaematis. The invention has the following advantages: the compatibility of Chinese medicinal composition is in accordance with the principle of 'monarch, minister, assistant and guide' of Chinese medicaments, treatment is started from the pathogen fundamentally, and the Chinese medicament is applied according to symptoms and has an exact curative effect in assisting and preventing the Parkinsonism; the Chinese medicinal composition for preventing the Parkinsonism has the advantages of no toxic or side effect, low price and the like, and is easily accepted by patients; and the Chinese medicinal composition has few drugs, rich raw materials, simple preparation process, environmental friendliness, and excellent application prospect in preventing the Parkinsonism.
Description
Technical field
The present invention relates to a kind of purposes of Chinese medicine composition, specifically, is that a kind of Chinese medicine composition is applied in preparation prevention parkinson disease medicine.
Background technology
Parkinson disease (Parkinson ' s disease, PD) be the common nervous system degeneration illness of middle-aged and elderly people, mainly with black substance dense area degeneration of dopaminergic neurons disappearance, reach the nigrostriatum path dopamine mediator that causes thus and reduce relevant.The traditional Chinese medical science thinks that PD belongs to " tremor syndrome ", and the multiple person in middle and old age of being born in " menses " exhaust Time, and " menses " exhausts and characterize the kidney essense scarcity, and Rong Wei is two empty, on not nourishing the liver wood, Ru Jinmai not outward, the kidney failing to nourish the liver hyperactivity of YANG due to deficiency of YIN, cardiopalmus, sweating, hectic fever, agitation takes place frequently; The muscle arteries and veins loses deadlock in gentle existing morning, spasm of the limbs, trembles, tetanic, pain, bradykinesia.Take a broad view of the experience of ancient Chinese medicine doctor, take up modern medicine study, by facing card, observe, we think, control with QI invigorating reduce phlegm, easing the affected liver relieves dizziness, high fever, infantile convulsions, epilepsy, etc. method.Vital energy benefiting and the kidney invigorating is turbid with expectorant, and the kind high and level tone of easing the affected liver trembles surely.
Through clinical practice for many years, it is generally acknowledged, levodopa is still and the most effectively prevents the parkinson disease medicine.But after levodopa prolonged application 3-5, about 5% PD patient can produce gradually symptom fluctuation and (or) levodopa " long-term syndrome " such as the dyskinesia, it is the PD motor complication, laboratory is found high concentration dopamine (dopamine in addition, DA) and levodopa can produce free radical due to autoxidation, can cause the neurocyte degeneration necrosis, therefore, the associating other drug prevents the PD motor complication very urgent jointly.
Research in recent years shows, PD occurs to be activated in close relations with direct path and the signal transduction pathways such as downstream cAMP deopendent protein kinase (PKA), ERK thereof of expressing the D1 receptor, the phosphorylated protein that its downstream signal transducin dopamine and adenosine cyclophosphate are regulated-32(dopamine and cAMP-regulated phosphoprotein of Mr 32000, the change of albumen Thr75 site Expression of phosphorylated DARPP-32) may participate in the morbidity of motor complication.
Aspect the prevention of PD motor complication, mainly adopt COMT inhibitor, amantadine, DR agonist, L-dopa methyl ester or ethyl ester etc. at present.As Chinese patent literature CN 200880108979.7, date of publication on August 18th, 2010, a kind of " be used to improving the medicament of Parkinsonian motor complication or mental symptom " disclosed, described medicament has the effect of 5-hydroxy tryptamine 1A acceptor portion agonist, simultaneously dopamine D 3 receptor is had to the agonist effect, for the motor complication of supervening when the repetitively administered levodopa, have and improve and the effect of delayed onset.But Western medicine prevention side effect is larger, is not so good as Chinese herb on the prevention safe and reliable.China is accumulating rich experiences aspect Chinese medicine prevention PD, sum up its Precautionary Principle the QI invigorating activating blood circulation method is arranged, the nourishing YIN for attracting YANG method, the suppressing the hyperactive liver to relieve the wind syndrome method, rub the muscle method that relieves dizziness, high fever, infantile convulsions, epilepsy, etc., the rules such as activating blood and removing stasis Method and collateral dredging relieve dizziness, high fever, infantile convulsions, epilepsy, etc., for preventing the Chinese medicine composition of PD also to have multiple, but about the Chinese medicine composition of prevention PD motor complication but seldom, as Chinese periodical " Chinese Chinese medicine magazine ", the 26th the 6th phase of volume of June in 2011, " clinical research of invigorating kidney, promoting blood circulation granule prevention parkinson disease motor complication " published, disclose the invigorating kidney, promoting blood circulation granule and can improve PD patient moving complication symptom, but ingredients and the proportioning of described invigorating kidney, promoting blood circulation granule are not fully disclosed.Therefore, must try to explore effectively to prevent parkinson disease motor complication, safe and reliable medicine, but about this class medicine, yet there are no report at present.
Summary of the invention
The objective of the invention is, for deficiency of the prior art, provides a kind of purposes of Chinese medicine composition.
For achieving the above object, the technical scheme taked of the present invention is:
The purposes of a kind of Chinese medicine composition in preparation prevention parkinson disease medicine, described Chinese medicine composition is to be made by the crude drug of following weight portion: Rhizoma Gastrodiae 10-20 part, Radix Astragali 10-20 part, Radix Rehmanniae Preparata 15-25 part, Radix Paeoniae Alba 15-25 part, Radix Angelicae Sinensis 15-25 part, Ramulus Uncariae Cum Uncis 10-20 part, Bombyx Batryticatus 7-13 part, Rhizoma Arisaematis 7-13 part.
Described Chinese medicine composition is to be made by the crude drug of following weight portion: Rhizoma Gastrodiae 12-18 part, Radix Astragali 12-18 part, Radix Rehmanniae Preparata 19-21 part, Radix Paeoniae Alba 18-22 part, Radix Angelicae Sinensis 19-21 part, Ramulus Uncariae Cum Uncis 12-18 part, Bombyx Batryticatus 8-12 part, Rhizoma Arisaematis 8-12 part.
Described Chinese medicine composition is to be made by the crude drug of following weight portion: 15 parts, Rhizoma Gastrodiae, 15 parts of the Radixs Astragali, 20 parts of Radix Rehmanniae Preparata, 20 parts of Radix Paeoniae Alba, 20 parts of Radix Angelicae Sinensis, 15 parts of Ramulus Uncariae Cum Uncis, 10 parts of Bombyx Batryticatus, 10 parts of Rhizoma Arisaematiss.
The medicament of described Chinese medicine composition is capsule, granule, tablet, oral liquid, mixture, syrup, microcapsule formulation, injection, suppository, spray or ointment.
The invention has the advantages that:
1, its compatibility of this Chinese medicine composition meets Chinese medicine " monarch " principle, follows the Therapeutic Method of " QI invigorating is reduced phlegm, easing the affected liver relieves dizziness, high fever, infantile convulsions, epilepsy, etc. method ", from cause of disease, starts with at all, suits the remedy to the case, auxiliary prevention parkinson disease successful;
The advantages such as 2, this Chinese medicine composition prevention parkinson disease have and have no side effect, and price is low, be easy to be accepted by the patient;
3, this Chinese medicine composition flavour of a drug number is few, abundant raw materials, and preparation technology is simple, and is environmentally friendly, in the prevention parkinson disease, good application prospect arranged.
The accompanying drawing explanation
Accompanying drawing 1 is the impact of Chinese medicine composition on PD rat model AIM total points.
Accompanying drawing 2 is Chinese medicine composition impacts on PD rat model agent peak number of revolutions.
Accompanying drawing 3 is SABC figure of PD rat model Striatum GRK6.
Accompanying drawing 4 is SABC block diagrams of PD rat model Striatum GRK6.
Accompanying drawing 5 is Western collection of illustrative plates that PD rat model Striatum GRK6 expresses.
Accompanying drawing 6 is quantitative figure that PD rat model Striatum GRK6 expresses.
Accompanying drawing 7 is SABC figure of PD rat model model Striatum β-arrestin1.
Accompanying drawing 8 is Western collection of illustrative plates that PD rat model model Striatum β-arrestin1 expresses.
Accompanying drawing 10 is PD rat model Striatum phosphorylation DARPP-32(Thr75) the Western collection of illustrative plates of expressing.
Accompanying drawing 11 is SABC figure of PD rat model Striatum phosphorylation ERK1/2.
Accompanying drawing 12 is Western collection of illustrative plates that PD rat model Striatum phosphorylation ERK1/2 expresses.
The specific embodiment
Below in conjunction with accompanying drawing, the specific embodiment provided by the invention is elaborated.
The preparation (one) of embodiment 1 prevention parkinson disease Chinese medicine composition
10 parts, Rhizoma Gastrodiae, 20 parts of the Radixs Astragali, 15 parts of Radix Rehmanniae Preparata, 25 parts of Radix Paeoniae Alba, 15 parts of Radix Angelicae Sinensis, 20 parts of Ramulus Uncariae Cum Uncis, 7 parts of Bombyx Batryticatus, 13 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (two) of embodiment 2 prevention parkinson disease Chinese medicine compositions
20 parts, Rhizoma Gastrodiae, 10 parts of the Radixs Astragali, 25 parts of Radix Rehmanniae Preparata, 15 parts of Radix Paeoniae Alba, 25 parts of Radix Angelicae Sinensis, 10 parts of Ramulus Uncariae Cum Uncis, 13 parts of Bombyx Batryticatus, 7 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (three) of embodiment 3 prevention parkinson disease Chinese medicine compositions
10 parts, Rhizoma Gastrodiae, 10 parts of the Radixs Astragali, 25 parts of Radix Rehmanniae Preparata, 25 parts of Radix Paeoniae Alba, 15 parts of Radix Angelicae Sinensis, 10 parts of Ramulus Uncariae Cum Uncis, 13 parts of Bombyx Batryticatus, 13 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (four) of embodiment 4 prevention parkinson disease Chinese medicine compositions
20 parts, Rhizoma Gastrodiae, 10 parts of the Radixs Astragali, 25 parts of Radix Rehmanniae Preparata, 25 parts of Radix Paeoniae Alba, 15 parts of Radix Angelicae Sinensis, 10 parts of Ramulus Uncariae Cum Uncis, 7 parts of Bombyx Batryticatus, 13 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (five) of embodiment 5 prevention parkinson disease Chinese medicine compositions
20 parts, Rhizoma Gastrodiae, 20 parts of the Radixs Astragali, 25 parts of Radix Rehmanniae Preparata, 25 parts of Radix Paeoniae Alba, 15 parts of Radix Angelicae Sinensis, 10 parts of Ramulus Uncariae Cum Uncis, 7 parts of Bombyx Batryticatus, 7 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (six) of embodiment 6 prevention parkinson disease Chinese medicine compositions
12 parts, Rhizoma Gastrodiae, 18 parts of the Radixs Astragali, 19 parts of Radix Rehmanniae Preparata, 22 parts of Radix Paeoniae Alba, 19 parts of Radix Angelicae Sinensis, 18 parts of Ramulus Uncariae Cum Uncis, 8 parts of Bombyx Batryticatus, 12 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (seven) of embodiment 7 prevention parkinson disease Chinese medicine compositions
18 parts, Rhizoma Gastrodiae, 12 parts of the Radixs Astragali, 21 parts of Radix Rehmanniae Preparata, 18 parts of Radix Paeoniae Alba, 21 parts of Radix Angelicae Sinensis, 12 parts of Ramulus Uncariae Cum Uncis, 12 parts of Bombyx Batryticatus, 8 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (eight) of embodiment 8 prevention parkinson disease Chinese medicine compositions
12 parts, Rhizoma Gastrodiae, 12 parts of the Radixs Astragali, 21 parts of Radix Rehmanniae Preparata, 22 parts of Radix Paeoniae Alba, 19 parts of Radix Angelicae Sinensis, 12 parts of Ramulus Uncariae Cum Uncis, 12 parts of Bombyx Batryticatus, 12 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (nine) of embodiment 9 prevention parkinson disease Chinese medicine compositions
18 parts, Rhizoma Gastrodiae, 12 parts of the Radixs Astragali, 19 parts of Radix Rehmanniae Preparata, 22 parts of Radix Paeoniae Alba, 19 parts of Radix Angelicae Sinensis, 12 parts of Ramulus Uncariae Cum Uncis, 8 parts of Bombyx Batryticatus, 8 parts of Rhizoma Arisaematiss, conventional method decocts.
The preparation (ten) of embodiment 10 prevention parkinson disease Chinese medicine compositions
10 parts, Rhizoma Gastrodiae, 18 parts of the Radixs Astragali, 25 parts of Radix Rehmanniae Preparata, 18 parts of Radix Paeoniae Alba, 21 parts of Radix Angelicae Sinensis, 10 parts of Ramulus Uncariae Cum Uncis, 13 parts of Bombyx Batryticatus, 8 parts of Rhizoma Arisaematiss, conventional method decocts.
It should be noted that, it is the manufacture method of Chinese medicine decoction routine that the described conventional method of embodiment 1-10 decocts, and is about to described crude drug and decocts with water into decoction.
The preparation of the Parkinsonian Chinese medicine composition tablet/capsule of embodiment 11 prevention
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decoct 1-3 hour, leach medicine juice; Add again 6-10 times of water gaging, decoct 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, standing, the leaching supernatant, concentrated, let cool, add concentrated solution 2-3 and doubly measure ethanol, stir precipitation and spend the night; Get supernatant, be concentrated into thick extractum; Add pharmaceutic adjuvant (method according to routine is selected pharmaceutic adjuvant), vacuum drying, pulverize and granulate, and is pressed into tablet or fills encapsulated.
The preparation of the Parkinsonian Chinese medicine composition composition granule of embodiment 12 prevention
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decoct 1-3 hour, leach medicine juice; Add again 6-10 times of water gaging, decoct 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, standing, the leaching supernatant, concentrated, let cool, add concentrated solution 2-3 and doubly measure ethanol, stir precipitation and spend the night; Get supernatant, be concentrated into thick extractum; Add pharmaceutic adjuvant (method according to routine is selected pharmaceutic adjuvant), vacuum drying, pulverize and granulate, drying, and granulate, obtain the 20g granule, packing 10g/ bag.
The preparation of embodiment 13 prevention Parkinsonian Chinese medicine composition mixture/oral liquid/syrups
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decoct 1-3 hour, leach medicine juice; Add again 6-10 times of water gaging, decoct 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, standing, the leaching supernatant, concentrated, let cool, add concentrated solution 2-3 and doubly measure ethanol, stir precipitation and spend the night; Get supernatant, be concentrated into thick extractum; Add suitable pharmaceutical aids (method according to routine is selected pharmaceutic adjuvant), make mixture, oral liquid or syrup.
The preparation of the Parkinsonian Chinese medicine composition microcapsule formulation of embodiment 14 prevention
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decoct 1-3 hour, leach medicine juice; Add again 6-10 times of water gaging, decoct 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, standing, the leaching supernatant, concentrated, let cool, add concentrated solution 2-3 and doubly measure ethanol, stir precipitation and spend the night; Get supernatant, be concentrated into thick extractum; Select pharmaceutically acceptable capsule material, make microcapsule formulation.
The preparation of the Parkinsonian Chinese medicine composition composition injection of embodiment 15 prevention
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decoct 1-3 hour, leach medicine juice; Add again 6-10 times of water gaging, decoct 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, standing, the leaching supernatant, concentrated, let cool, add concentrated solution 2-3 and doubly measure ethanol, stir precipitation and spend the night; Get supernatant, 80 ℃ of Distillation recoveries, be equipped with water for injection, makes injection.
The preparation of the Parkinsonian Chinese medicine composition suppository of embodiment 16 prevention
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decoct 1-3 hour, leach medicine juice; Add again 6-10 times of water gaging, decoct 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, standing, the leaching supernatant, concentrated, let cool, add concentrated solution 2-3 and doubly measure ethanol, stir precipitation and spend the night; Get supernatant, be concentrated into thick extractum; Add pharmaceutic adjuvant (method according to routine is selected pharmaceutic adjuvant), vacuum drying, pulverize and granulate, and injection molding, make suppository.
The preparation of the Parkinsonian Traditional Chinese medicine composition-type spray of embodiment 17 prevention
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decoct 1-3 hour, leach medicine juice; Add again 6-10 times of water gaging, decoct 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, standing, the leaching supernatant, concentrated, let cool, add concentrated solution 2-3 and doubly measure ethanol, stir precipitation and spend the night; Get supernatant, be concentrated into thick extractum; Add medicinal liquefier, inject nonmetal aerosol apparatus, make spray.
The preparation of the Parkinsonian Chinese medicine composition ointment of embodiment 18 prevention
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decoct 1-3 hour, leach medicine juice; Add again 6-10 times of water gaging, decoct 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, standing, the leaching supernatant, concentrated, let cool, add concentrated solution 2-3 and doubly measure ethanol, stir precipitation and spend the night; Get supernatant, be concentrated into thick extractum; Add suitable substrate to mix, make ointment.
The Parkinsonian preclinical test of embodiment 19 this Chinese medicine composition prevention
One, experimental technique
1.PD rat model preparation
Get 65 rats and enter experiment, wherein 5 is sham operated rats, and namely sham organizes, in medial forebrain bundle (right medical forebrain, MFB) injecting normal saline.Lumbar injection 3% pentobarbital sodium anesthetized rat, strict tack cranium position fixedly rat in stereo brain orienting instrument, with reference to the bag new people, show rat brain stereotaxic atlas (DeWire SM, Lefkowitz RJ, Shenoy SK, et al. Beta arrestins and cell signaling. Annu Rev Physiol. 2007,69:483-510.), determine right side medial forebrain bundle coordinate: 1. 3.7 mm after bregma, sagittal suture right side 1.7 mm, 7.8 mm under cranial periosteum, front tooth line 2.4 mm; 2. 4.4 mm after bregma, sagittal suture right side 1.2 mm, 7.8 mm under cranial periosteum, front tooth line 2.4 mm.By the boring of above-mentioned definite injection site, with the microsyringe of 10 μ l, extract 6-OHDA 6 μ l(and contain 0.2% ascorbic normal saline configuration, concentration 4 μ g/ μ l), the withdraw of the needle after every some injection 3 μ l, let the acupuncture needle remain at a certain point 10 min, stitching wound surface.After 3 weeks, rats by intraperitoneal injection apomorphine (0.5 mg/kg), average speed > 7 times/min is successful PD model.
2. grouping and treatment
The PD rat model of 25 successes is divided into to 3 at random to be organized greatly.(1) PD matched group: PD rat model lumbar injection 0.2% vitamin C liquid 29 days; (2) Western medicine processed group: lumbar injection LDME/benserazide, injected dose is 50 mg/kg LDME and 25 mg/kg benserazides, the two all is dissolved in and contains in 0.2% ascorbic sterilization normal saline, at 9 in 2 times/d(morning and afternoons 5 point), continue 29d; (3) dosage group, the heavy dose of group of Chinese medicine composition in Chinese medicine composition small dose group, Chinese medicine composition: give on the basis as the LDME/benserazide in group (2) to add respectively the Chinese medicine composition with embodiment 10 preparations, the ratio that is concentrated into 100ml according to the 50g crude drug decocts, Chinese medicine composition small dose group using dosage is 7.2 ml/kg, in Chinese medicine composition, dosage group using dosage is 9 ml/kg, the heavy dose of group of Chinese medicine composition using dosage is 10.8 ml/kg, every day 1 gavage, continuous 4 weeks.
3. behavioristics measures
In therapeutic process, after medication in the morning in the 2nd, 8,15,22,29 days, carry out rat behavior and learn observation and scoring.AIM evaluation: AIM is divided into 4 components (upper limb AIM, the AIM of actinal surface section, axle AIM and rotation AIM) to be evaluated, and every part has or not with the order of severity and is divided into 5 grades (0-4) according to it again: 0 nothing; 1 persistent period is less than 30s; 2 persistent period are greater than 30s, less than 60s; 3 persistent period, environmental stimuli can make it to stop greater than 60s; 4 persistent period, environmental stimuli can not make it to stop greater than 60s.After medication, every 20min assessment once, is observed 1min at every turn.The AIM total points is added up by the meansigma methods of total mark in observing time, and after 1 rat a drug, each component AIM maximum scores is 4 minutes in theory, and total AIM scoring is 16 minutes.Agent peak number of revolutions: after the injection levodopa, every 5min records number of revolutions, and maximum numbers of revolutions is agent peak number of revolutions.
4. SABC shows striatum β-arrestin1, GRK6, DARPP-32(Thr75) and the variation of ERK1/2
Finish 12h after injection, 3% pentobarbital sodium anesthesia, left ventricle perfusion 4% paraformaldehyde is fixed, broken end fixing 8h after getting after brain in identical fixative, piece, gradient alcohol dehydration, dimethylbenzene are transparent through repairing, use paraffin embedding after waxdip.The row paraffin section, slice thickness is 5 μ m.Paraffin section de-waxing is to water; 3% hydrogen peroxide room temperature lucifuge is hatched 5min, to eliminate the endogenous catalase activity; 1nmol/l ethylenediaminetetraacetic acid-Tris of pH7.7-hydrogen chloride (EDTA-Tris-HCl) microwave heating reparation; 1% bovine serum albumin room temperature envelope 20min; The anti-Mus GRK6 of rabbit antibody, the anti-Mus β of rabbit-arrestin1 polyclonal antibody, the anti-Mus DARPP-32 of rabbit antibody, anti-phosphorylation DARPP-32(Thr75) overnight incubation in antibody, the total ERKl/2 of rabbit Chinese People's Anti-Japanese Military and Political College Mus and 4 ℃ of wet boxes of the Mus phosphorylation ERKl/2 of rabbit Chinese People's Anti-Japanese Military and Political College monoclonal antibody; The biotin labeling two that drips dilution is anti-, hatches 20min for 37 ℃; Drip the horseradish peroxidase-labeled strepto-avidin of dilution, hatch 20min for 37 ℃; Benzidine (3,3 ˊ-diaminobenzidine tetrahydrochloride, DAB) chromogenic reagent, tap water rinses, afterwards dehydration, transparent, mounting.Between each step, all use the abundant rinsing of 0.01mol/l TBS, TBS replaces primary antibodie as negative control.Examine under a microscope the SABC section, each observation area is got 5 not overlapped views at random, in high power lens (10 * 40) is lower, observe, adopt OLYMPUS-IX50 to become phase system to take, Image-Pro Plus 5.1 imgae processing softwares carry out semi-quantitative analysis, calculate positive cell index (IOD)=Positive area * correction optical density value (measurement zone optical density-background indensity).
5.Western the blotting method detects β-arrestin1, GRK6, DARPP-32(Thr75) and the expression of ERK1/2
3h after last injection, 3% pentobarbital sodium anesthesia, broken end is got brain rapidly, peels off the bilateral striatum on ice, and ultrasonic degradation extracts total protein.After measuring protein concentration, put-80 ℃ frozen standby.40 μ g albumen samples separate through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with electric transferring film instrument, are transferred on polyvinylidene fluoride (PVDF) film.Confining liquid (5% defatted milk powder) room temperature sealing 2h, according to respective concentration dilution anti-GRK6 antibody (1: 500), anti-β-arrestin1 antibody (1: 500), anti-total DARPP-32 antibody (1: 1000) or anti-phosphorylation DARPP-32(Thr75) and antibody (1: 1000), anti-total ERKl/2 antibody (1: 1000) or anti-phosphorylation ERKl/2 antibody (1: 500) or anti-β-actin (l: 1000), 4 ℃ of jog overnight incubation.Add next day two to resist, under room temperature, shaking table is hatched 1 h; Drip ECL colour developing mixed liquor, the exposure of Bio-Rad gel imaging instrument, video picture.Western blot image adopts the SmartView2000 image analysis software to calculate the product of each sample destination protein band OD value and area value, thereby it is quantitative to carry out protein band density.
6. statistical analysis technique
Experimental data adopts SPSS 13.0 statistical softwares to analyze.The gained measurement data with mean ± standard deviation (
± S) expression, the relatively employing one factor analysis of variance of a plurality of sample averages, relatively adopt the t check in twos.Take P<0.05 as difference, has statistical significance.
Two, experimental result
1. the impact of Chinese medicine composition on the behavior of PD rat model
1.1 the impact of Chinese medicine composition on each component of PD rat model AIM scoring
Dosage group in Chinese medicine composition small dose group, Chinese medicine composition, the heavy dose of group of Chinese medicine composition and Western medicine processed group on each component of PD rat model AIM scoring to affect result as shown in table 1.
With regard to the AIM of actinal surface section scoring, between the 2nd day each group, average is without significant difference.8th, 15,22,29 days each the group between average significant difference (F=4.98, F=22.70, F=30.96, F=23.27, P<0.01).Wherein, the Chinese medicine composition small dose group reduces than the Western medicine processed group the AIM of actinal surface section scoring in the 22nd day, and difference has statistical significance (t=2.50, P<0.05), all the other several days equal not statistically significants of difference; In Chinese medicine composition, the AIM of the actinal surface section scoring in the 8th, 15,22,29 days of dosage group obviously reduces than the Chinese medicine composition small dose group, and difference has statistical significance (t=2.86, t=3.80, t=4.38, t=6.54, all P<0.05); In Chinese medicine composition, relatively, only the dosage group actinal surface AIM of section scoring reduces the heavy dose of group of dosage group and Chinese medicine composition in the 22nd day Chinese medicine composition heavy dose organized than Chinese medicine composition, and difference has statistical significance (t=2.50, P<0.05).
With regard to upper limb AIM scoring, the 2nd, 8 days each class mean no significant differences, between the 15th, 22,29 days each groups, average difference has statistical significance (F=15.12, F=32.86, F=35.80, P<0.01).Medication the 2nd day and Chinese medicine composition small dose group upper limb AIM scoring in the 8th day are compared there was no significant difference with the Western medicine processed group, the 15th, 22,29 days Chinese medicine composition small dose group upper limb AIM mark lower than the Western medicine processed group to medication, difference has statistical significance (t=4.45, t=4.47, all P<0.05).15th, in 22,29 days Chinese medicine compositions, the dosage group obviously reduces than Chinese medicine composition small dose group upper limb AIM scoring, and difference has statistical significance (t=3.40, t=4.04, t=3.95, all P<0.01).In the heavy dose of group of Chinese medicine composition and Chinese medicine composition, between the dosage group, compare the difference no difference of science of statistics.
With regard to axial AIM scoring, between the 15th, 22,29 days each groups, average difference has statistical significance (F=3.30, F=25.06, F=36.41, P<0.01).22,29 days axial AIM values of Chinese medicine composition small dose group medication to the reduce than Western medicine processed group, and difference has statistical significance (t=3.93, t=3.04, all P<0.05); In Chinese medicine composition, dosage group and Chinese medicine composition small dose group relatively, only have statistical significance (t=3.05, P<0.05) in the 29th day difference; The heavy dose of group of Chinese medicine composition has obviously reduced axial AIM value, but with Chinese medicine composition in the comparison of dosage group, only variant in the 29th day (t=2.83, P<0.05), put the difference no difference of science of statistics At All Other Times.
With regard to rotation AIM scoring, average significant difference (F=10.50, F=11.62, F=18.92, F=61.26, P<0.01) between the 8th, 15,22,29 days each groups.Chinese medicine composition small dose group and Western medicine processed group rotation AIM scoring are compared, the difference not statistically significant.In Chinese medicine composition, the medication to the of dosage group is 15,22,29 days, and rotation AIM scoring obviously reduces than Western medicine processed group, and difference has statistical significance (t=3.43, t=5.11, t=7.63, all P<0.01); In the heavy dose of group of Chinese medicine composition and Chinese medicine composition, the dosage group compares, the 8th, 15,29 days obvious differences (t=3.81, t=2.52, t=3.58, all P<0.05).
The comparison of table 1 Chinese drug-treated group and Western medicine group AIM scoring (
± S, minute)
| Time | Group | Mus number (only) | Actinal surface section | Upper | Axially | Rotation | |
| 2d | Chinese medicine composition- |
5 | 1.40±0.24 | 1.61±0.26 | 2.37±0.15 | 2.60±0.23 | |
| ? | Chinese medicine composition- |
5 | 1.39±0.19 | 1.50±0.28 | 2.50±0.22 | 2.70±0.34 | |
| ? | Chinese medicine composition- |
5 | 1.48±0.17 | 1.54±0.19 | 2.40±0.16 | 2.52±0.23 | |
| ? | The Western medicine processed |
5 | 1.44±0.18 | 1.51±0.22 | 2.49±0.17 | 2.67±0.28 | |
| 8d | Chinese medicine composition- |
5 | 1.50±0.22 | 1.64±0.22 | 2.42±0.24 | 2.60±0.06 | |
| ? | Chinese medicine composition- |
5 | 1.52±0.18 | 1.66±0.16 | 2.58±0.29 | 2.93±0.19 | |
| ? | Chinese medicine composition- |
5 | 1.84±0.21 | 1.81±0.19 | 2.70±0.41 | 3.14±0.20 | |
| ? | The Western medicine processed |
5 | 1.82±0.17 | 1.93±0.20 | 2.71±0.41 | 3.20±0.25 | |
| 15d | Chinese medicine composition- |
5 | 1.30±0.11 | 1.50±0.22 | 2.37±0.20 | 2.56±0.15 | |
| ? | Chinese medicine composition- |
5 | 1.47±0.14 | 1.71±0.22 | 2.69±0.24 | 2.80±0.19 | |
| ? | Chinese medicine composition- |
5 | 1.82±0.17 | 2.09±0.12 | 2.71±0.37 | 3.00±0.24 | |
| ? | The Western medicine processed |
5 | 2.11±0.22 | 2.17±0.15 | 2.90±0.28 | 3.34±0.28 | |
| 22d | Chinese medicine composition- |
5 | 1.29±0.13 | 1.49±0.23 | 2.24±0.18 | 2.53±0.15 | |
| ? | Chinese medicine composition- |
5 | 1.44±0.13 | 1.65±0.20 | 2.27±0.13 | 2.57±0.11 | |
| ? | Chinese medicine composition- |
5 | 1.96±0.20 | 2.13±0.14 | 2.62±0.28 | 3.11±0.21 | |
| ? | The Western medicine processed |
5 | 2.40±0.37 | 2.56±0.16 | 3.18±0.15 | 3.38±0.29 | |
| 29d | Chinese medicine composition- |
5 | 1.20±0.09 | 1.53±0.21 | 2.11±0.78 | 2.29±0.10 | |
| ? | Chinese medicine composition- |
5 | 1.31±0.21 | 1.60±0.18 | 2.33±0.16 | 2.56±0.14 | |
| ? | Chinese medicine composition- |
5 | 2.04±0.13 | 2.13±0.20 | 2.76±0.26 | 3.24±0.14 | |
| ? | The Western medicine processed |
5 | 2.36±0.36 | 2.60±0.13 | 3.16±0.13 | 3.47±0.23 |
1.2 the impact of Chinese medicine composition on PD rat model AIM total points
Medication was marked in the 8th, 15,22,29 days, find Chinese medicine composition small dose group medication to the 15,22,29 days, the AIM total points is respectively (9.73 ± 0.52), (9.80 ± 0.37) and (10.15 ± 0.32) minute, than Western medicine processed group (10.48 ± 0.40), (11.41 ± 0.17) minute, reach (11.58 ± 0.24) minute and obviously reduce (t=2.88, t=9.25, t=7.77, all p<0.05).And in Chinese medicine composition, the 8th, 15,22,29 days AIM total points of dosage group medication are respectively (8.82 ± 0.36), (8.71 ± 0.47), (8.13 ± 0.37) and (7.89 ± 0.57), all lower than Chinese medicine composition small dose group total points, difference has statistical significance (t=3.32, t=3.19, t=7.56, t=7.85, all p<0.05); In the 8th, 15,22 days, the heavy dose of group of Chinese medicine composition AIM total points is respectively (8.20 ± 0.27), (7.74 ± 0.34), (7.55 ± 0.38) minute, low than dosage group in Chinese medicine composition, difference has statistical significance (t=3.12, t=3.44, t=2.57, equal p<0.05), and in the heavy dose of group of the 29th day Chinese medicine composition (7.71 ± 1.13) minute and Chinese medicine composition dosage group (7.89 ± 0.57) is relatively, the difference not statistically significant (is shown in Fig. 1, # represents Chinese medicine composition small dose group and Western medicine processed group relatively, all P<0.05; * represent dosage group and the comparison of Chinese medicine composition small dose group, all P<0.05 in Chinese medicine composition; In the heavy dose of group of+expression Chinese medicine composition and Chinese medicine composition, the dosage group compares, all P<0.05).
1.3 the impact of Chinese medicine composition on PD rat model agent peak number of revolutions
In the 8th, 15,22,29 days, carrying out agent peak number of revolutions detects, find that the scoring of Chinese medicine composition small dose group is respectively that (123.6 ± 11.91), (132 ± 10.07), (151 ± 15.73) and (155.8 ± 7.40) are inferior, little with Western medicine processed group difference; And dosage group and the comparison of Chinese medicine composition small dose group in Chinese medicine composition, in Chinese medicine composition, dosage group agent peak number of revolutions is respectively (85.4 ± 15.60), (82.0 ± 21.76), (87 ± 10.20) and (73 ± 15.18), all lower than the Chinese medicine composition small dose group, difference has statistical significance (t=4.13, t=4.57, t=7.51, t=10.96, all p<0.05); In the heavy dose of group of Chinese medicine composition and Chinese medicine composition, the dosage group relatively, 22nd, 29 days Chinese medicine compositions heavy dose of group agent peak number of revolutions (51.2 ± 6.76) and (53.8 ± 7.79) is inferior, low than dosage group in Chinese medicine composition, difference has statistical significance (t=6.73, t=2.52, equal p<0.05) (see Fig. 2, # represents Western medicine processed group and Chinese medicine composition small dose group relatively, all P<0.05; * represent dosage group and the comparison of Chinese medicine composition small dose group, all P<0.01 in Chinese medicine composition; In the heavy dose of group of+expression Chinese medicine composition and Chinese medicine composition, the dosage group compares, all P<0.05).
2. the impact of Chinese medicine composition on PD rat model Striatum signal protein
2.1 the impact of Chinese medicine composition on PD rat model Striatum GRK6
Showed by immune group result GRK6 expresses on cell membrane.Each group is not damaged the side average relatively, the difference not statistically significant (F=0.17, P > 0.05), and each group damage side average significant difference (F=4.48, P<0.05).The Damage of Rats side GRK6 expression of PD matched group is (4.50 ± 0.85) * 10
3, than (6.10 ± 0.55) * 10 of the normal rat of sham group
3Obviously reduce (t=9.99, P<0.01); The life-time service levodopa treatment, namely the rat GRK6 of Western medicine processed group expresses and further is reduced to (3.53 ± 0.71) * 10
3, with the rat of PD matched group, comparing, difference has statistical significance (t=2.99, P<0.05); And add the rat with Chinese medicine composition of the present invention prevention, comprise dosage group and the heavy dose of group of Chinese medicine composition in Chinese medicine composition small dose group, Chinese medicine composition, striatal damage side GRK6 expresses with the PD control rats and compares, all without further reducing, with the rat of Western medicine processed group, compare Chinese medicine composition small dose group (4.13 ± 1.20) * 10
3With Western medicine processed group zero difference, in Chinese medicine composition, the expression of dosage group, the heavy dose of group damage of Chinese medicine composition side GRK6 is respectively (4.31 ± 0.95) * 10
3, (4.68 ± 1.24) * 10
3With the Western medicine processed group, compare, the GRK6 expression is more (t=3.79, t=4.84 obviously, equal P<0.05) (see Fig. 3 and Fig. 4, in Fig. 3: 1 is the sham group, and 2 is the PD matched group, 3 is the Western medicine processed group, 4 is the Chinese medicine composition small dose group, and 5 is dosage group in Chinese medicine composition, and 6 is the heavy dose of group of Chinese medicine composition; U is for not damaging side, and L is the damage side; * represent relatively P<0.01 of PD matched group and sham group, # represents relatively P<0.05 of Western medicine processed group and PD matched group ,+expression Chinese medicine composition is little, in, heavy dose of group and Western medicine processed group relatively, equal P<0.05).
Western result and ImmunohistochemistryResults Results are basically identical.With regard to every rat, testing result is with damage side/do not damage the ratio value representation of side, and β-actin is as internal reference.With (100.48 ± 5.57) % of sham group, compare, the rat GRK6 expression of PD matched group is reduced to (81.32 ± 5.94) %, significant difference (t=7.37, P<0.01); The levodopa long-term treatment is the rat of Western medicine processed group, and the GRK6 expression further is reduced to (73.66 ± 3.43) %, and difference has statistical significance (t=3.11, P<0.05); And add the rat with Chinese medicine composition prevention of the present invention, and namely in Chinese medicine composition small dose group, Chinese medicine composition, dosage group and Chinese medicine composition are heavy dose of organizes, and the rat of striatum GRK6 expression and PD matched group is compared, the difference not statistically significant.with the rat of life-time service levodopa be the Western medicine processed group relatively, in Chinese medicine composition, dosage group striatum GRK6 expression (83.68 ± 4.50) % is obviously higher, difference has statistical significance (t=7.26, P<0.01), Chinese medicine composition small dose group (77.12 ± 3.15) % and Chinese medicine composition heavy dose of group (80.36 ± 4.74) % and its comparison, difference significantly (is not shown in Fig. 5 and Fig. 6, in Fig. 5: 1, 2 are the sham group, 3, 4 is the PD matched group, 5, 6 is the Western medicine processed group, 7, 8 is the Chinese medicine composition small dose group, 9, 10 is dosage group in Chinese medicine composition, 11, 12 is the heavy dose of group of Chinese medicine composition, 1,3,5,7,9,11 for not damaging side, and 2,4,6,8,10,12 are the damage side, * expression is compared with the sham group, p<0.01, # represents to compare with the PD matched group, p<0.05, + expression Chinese medicine composition is little, in, heavy dose of group and Western medicine processed group relatively, p<0.01).
2.2 the impact of Chinese medicine composition on PD rat model model Striatum β-arrestin1
SABC shows that β-arrestin1 is expressed in cell membrane, each group damage side average significant difference (F=5.76, P<0.05).The positive cell index of the rat β of PD matched group-arrestin1 albumen is (3.27 ± 0.75) * 10
4, lower than (4.54 ± 0.63) * 10 of sham group rat
4, but the difference not statistically significant.After the levodopa long-term treatment, be the rat of Western medicine processed group, β-arrestin1 expresses further and reduces, and the positive cell index is (2.54 ± 0.49) * 10
4, with the rat of PD matched group, comparing, difference has statistical significance (t=3.99, P<0.05).Add the rat with Chinese medicine composition of the present invention, be dosage group and the heavy dose of group of Chinese medicine composition in Chinese medicine composition small dose group, Chinese medicine composition, β-arrestin1 positive cell index does not occur that in further decline, particularly Chinese medicine composition, dosage group rat β-arrestin1 positive cell index is (3.57 ± 0.56) * 10
4, than the rat of PD matched group, increase (t=6.80, P<0.01).The heavy dose of group of Chinese medicine composition, Chinese medicine composition small dose group β-arrestin1 positive cell index are respectively (3.24 ± 0.68) * 10
4(3.10 ± 0.59) * 10
4, with the PD matched group, compare, (see Fig. 7, in Fig. 7: 1 is the sham group, and 2 is the PD matched group, and 3 is the Western medicine processed group, and 4 is the Chinese medicine composition small dose group, and 5 is dosage group in Chinese medicine composition, and 6 is the heavy dose of group of Chinese medicine composition without significant difference; U is for not damaging side, and L is the damage side).
Western result and ImmunohistochemistryResults Results are basically identical.Every rat Western result is with damage side/do not damage the ratio value representation of side, and β-actin is as internal reference.The rat β of PD matched group-arrestin1 expressing quantity is (76.46 ± 5.12) %, and % obviously reduces (t=11.48, P<0.01) than sham group rat (101.56 ± 6.79).The levodopa long-term treatment, i.e. the rat of Western medicine processed group, β-arrestin1 expression further reduces, and is (65.40 ± 6.68) %, with the rat of PD matched group relatively, difference has statistical significance (t=8.31, P<0.01).Add with after Chinese medicine composition prevention of the present invention, Chinese medicine composition small dose group β-arrestin1 expression is (69.54 ± 3.36) %, than the rat of PD matched group, reduces (t=3.42, P<0.05), presents the trend similar to the Western medicine processed group; And in Chinese medicine composition, the heavy dose of group of dosage group and Chinese medicine composition is respectively (73.52 ± 3.82) % and (72.36 ± 6.66) %, occurs further descending.Simultaneously, in Chinese medicine composition, β-the arrestin1 expression is the rat of Western medicine processed group apparently higher than the life-time service levodopa to the heavy dose of group of dosage group and Chinese medicine composition striatum, and difference has statistical significance (t=3.11, t=3.70, P<0.05).In Chinese medicine composition, between the heavy dose of group of dosage group and Chinese medicine composition, compare, the difference not statistically significant (is shown in Fig. 8, in Fig. 8: 1,2 are the sham group, 3,4 is the PD matched group, 5,6 is the Western medicine processed group, 7,8 is the Chinese medicine composition small dose group, and 9,10 is dosage group in Chinese medicine composition, and 11,12 is the heavy dose of group of Chinese medicine composition; 1,3,5,7,9,11 for not damaging side, and 2,4,6,8,10,12 are the damage side).
2.3 Chinese medicine composition is on PD rat model Striatum phosphorylation DARPP-32(Thr75) impact expressed
SABC shows each group damage side average significant difference (F=472.10, P<0.01).Sham group rat phosphorylation DARPP-32(Thr75) expression is (7.06 ± 0.47) * 10
6, the PD matched group is increased to (11.44 ± 0.16) * 10
6, difference has statistical significance (t=14.07, P<0.01).Be the Western medicine processed group after the levodopa long-term treatment, phosphorylation DARPP-32(Thr75) be expressed as (3.35 ± 0.30) * 10
6, than PD matched group, obviously reducing, difference has statistical significance (t=39.99, P<0.01).Add the rat with Chinese medicine composition of the present invention, the large, medium and small dosage group of Chinese medicine composition phosphorylation DARPP-32(Thr75) express and to be respectively (12.35 ± 0.19) * 10
6, (11.90 ± 0.47) * 10
6(7.30 ± 0.27) * 10
6, in Chinese medicine composition, significantly reducing does not all appear in the heavy dose of group of dosage group and Chinese medicine composition, and only the Chinese medicine composition small dose group reduces.In the heavy dose of group of Chinese medicine composition and Chinese medicine composition, between the dosage group, compare, the difference not statistically significant (is shown in Fig. 9, in Fig. 9: A is the sham group, B is the PD matched group, C is the Western medicine processed group, D is the Chinese medicine composition small dose group, and E is dosage group in Chinese medicine composition, and F is the heavy dose of group of Chinese medicine composition).
Western result and ImmunohistochemistryResults Results are basically identical, sham group rat phosphorylation DARPP-32(THr75) gray value of expressing is (2.04 ± 0.24) * 10
6, the PD control rats is (3.00 ± 0.20) * 10
6, two groups of comparing differences have statistical significance (t=28.95, P<0.01).Adding with low dose of Chinese medicine composition of the present invention and not playing preventive effect, Chinese medicine composition small dose group phosphorylation DARPP-32(THr75) expression is (1.80 ± 0.15) * 10
6, than the rat of PD matched group, reduce (t=19.77, P<0.01); The rat striatum phosphorylation DARPP-32(THr75 that adds dosage Chinese medicine composition prevention of the present invention in using) expression is (3.15 ± 0.15) * 10
6, with the rat of PD matched group, compare, the difference not statistically significant (P>0.05).The heavy dose of group of Chinese medicine composition phosphorylation DARPP-32(THr75) expression is (3.81 ± 0.17) * 10
6Than the rat of PD matched group, slightly have and increase (t=38.57, P<0.01), simultaneously, the heavy dose of group of Chinese medicine composition is than dosage group increasing expression (t=21.78 in Chinese medicine composition, P<0.01) (see Figure 10, in Figure 10: 1 is the sham group, and 2 is the PD matched group, 3 is the Western medicine processed group, 4 is the Chinese medicine composition small dose group, and 5 is dosage group in Chinese medicine composition, and 6 is the heavy dose of group of Chinese medicine composition).
2.4 the impact that Chinese medicine composition is expressed PD rat model Striatum phosphorylation ERK1/2
SABC shows each group damage side average significant difference (F=117.49, P<0.01).The rat phosphorylation ERK1/2 expression of PD matched group is (4.37 ± 0.23) * 10
4, lower than (5.23 ± 0.34) * 10 of sham group rat
4, difference has statistical significance (t=15.32, P<0.01).After the levodopa long-term treatment, be the rat of Western medicine processed group, phosphorylation ERK1/2 expresses and is increased to (8.09 ± 0.37) * 10
4, with the PD matched group, comparing, difference has statistical significance (t=34.13, P<0.01).Add the rat with Chinese medicine composition of the present invention, Chinese medicine composition small dose group phosphorylation ERK1/2 is expressed as (6.69 ± 0.36) * 10
4, in rising trend, in Chinese medicine composition, the heavy dose of group of dosage group and Chinese medicine composition expression is (4.49 ± 0.34) * 10
4(4.23 ± 0.25) * 10
4, all do not occur significantly rising.In the heavy dose of group of Chinese medicine composition and Chinese medicine composition, between the dosage group, compare, difference is not obvious.(see Figure 11, in Figure 11: A is the sham group, and B is the PD matched group, and C is the Western medicine processed group, and D is the Chinese medicine composition small dose group, and E is dosage group in Chinese medicine composition, and F is the heavy dose of group of Chinese medicine composition).
Western result and ImmunohistochemistryResults Results are basically identical, and the gray value that the rat phosphorylation ERK1/2 of PD matched group expresses is (2.85 ± 0.17) * 10
6, than (3.51 ± 0.12) * 10 of sham group rat
6Reduce (t=20.77, P<0.01); Add with the gray value that the rat striatum phosphorylation ERK1/2 that large, medium and small dosage Chinese medicine composition of the present invention prevents expresses and be respectively (3.30 ± 0.19) * 10
6, (3.15 ± 0.16) * 10
6(4.40 ± 0.16) * 10
6, with the rat of PD matched group, rising in various degree being arranged more all, difference all has statistical significance (t=13.81, t=3.41, t=45.12, P<0.01).Wherein, dosage group phosphorylation ERK1/2 expression rising amplitude minimum in Chinese medicine composition, Chinese medicine composition small dose group rising amplitude is maximum (sees Figure 12, in Figure 12: 1 is the sham group, 2 is the PD matched group, 3 is the Chinese medicine composition small dose group, and 4 is dosage group in Chinese medicine composition, and 5 is the heavy dose of group of Chinese medicine composition).
In this research discovery, the Chinese medicine composition of the present invention of dosage can suppress the carrying out property rising of PD rat model actinal surface section, upper limb AIM scoring, and long-term observation finds that heavy dose of Chinese medicine composition and middle dosage Chinese medicine composition do not have difference.And with regard to axially with rotation AIM, marking, result shows that heavy dose of Chinese medicine composition has the effect that suppresses the rising of carrying out property of AIM scoring, and the Chinese medicine composition of middle dosage is more effective.In explanation, the dosage Chinese medicine composition can obviously reduce the generation of PD rat model motor complication, and heavy dose of Chinese medicine composition is to axially and the more remarkable treatment effect of rotation symptom and stable.The medication to 2 of small dose group Chinese medicine composition has suppressed the carrying out property rising of AIM overall score during week, brought into play preventive effect, middle dosage Chinese medicine composition can reduce the AIM that Dopar produces in early days, and the long-term observation discovery is heavy dose of does not have difference with middle dosage, and in explanation, the dosage Chinese medicine composition is remarkable and stable to the AIM effect that reduces the levodopa generation.In a word, by the have preventive effect of the visible Chinese medicine composition of the present invention of AIM appraisal result for the PD motor complication.
In research previously, most scholars think, along with the PD course advancement, compensation constantly appears in Basal ganglia DA, and the early stage Basal ganglia DA receptor of PD patient occurs super quick, and the receptor number increases (rise effect).The DA receptor belongs to GPCRs family, and in agonist, the agonist of same level can not make the effector of receptor activate, and after namely agonist has activated GPCRs, also start a degenerative process when DA receptor long term exposure, and the mistake that is called receptor is quick.This process makes the film signal weakening, the potential injury of having avoided undue irritation cell to cause.The demonstration of this result of study, during PD, the GRK6 expression reduces than the sham group, and prompting DA receptor is super quick closely related with reduction GRK6.In addition, Western medicine processed group GRK6 expression further reduces than the PD matched group.This may be that GRK6 reduces along with the life-time service of levodopa, can't suppress the activation of super quick receptor abnormality signal, finally causes the generation of motor complication.What is more important, middle dosage Chinese medicine composition can suppress the reduction that GRK6 expresses, and have suppressed the super quick effect of DA receptor, have stoped the transmission of abnormal signal, thereby play the effect that the prevention motor complication occurs, and more obvious preventive effect does not appear in the dosage increase.Equally, β during PD-arrestin1 protein expression reduces than the sham group, may be to lose and degeneration because the PD dopaminergic neuron is a large amount of, and residual dopamine neuron performance compensation, increase the DA receptor sensitivity.And the Western medicine processed group β of life-time service levodopa treatment-arrestin1 expression further reduces than the PD matched group, when this may be PD, transduction suppressed to weaken β-arrestin1 to abnormal signal, thereby caused the abnormal signal activation, caused motor complication.Add the rat with Chinese medicine composition of the present invention, low dose of Chinese medicine composition can prevent the further reduction of β-arrestin1 protein expression, and middle dosage Chinese medicine composition not only suppresses the further reduction of β-arrestin1 protein expression, this value is increased, the effect that prevention unusual fluctuation complication occurs is more remarkable, and more obvious preventive effect does not appear in heavy dose of Chinese medicine composition.In explanation, dosage group Chinese medicine composition can increase β-arrestin1 protein expression, plays the effect that the prevention motor complication occurs.
The phosphorylated protein that dopamine and adenosine cyclophosphate (cAMP) are regulated-32(dopamine and cAMP-regulated phosphoprotein of Mr 32000, DARPP-32) be expressed in the striatum projection neuron, in the signal conduction of dopamine, play an important role.PKA can promote the phosphorylation in the Thr34 site of DARPP-32; May pass through simultaneously activator protein phosphatase-2A(PP-2A), make Thr75 site dephosphorylation.After the Thr34 site phosphorylation of DARPP-32, change the highly efficient depressor of protein phosphatase 1 (PP-1) into, thereby the phosphorylation state of modulin further affects the downstream signal conduction.After the Thr75 site phosphorylation of DARPP-32, become the inhibitor of PKA, the reduction of its phosphorylation degree has alleviated the inhibitory action to PKA.The difference of phosphorylation site can be given DARPP-32 with opposite physiological effect as can be seen here.Early-stage Study is found, the phosphorylation enhancing of motor complication rat model Striatum ERK, and DARPP-32Thr75 site phosphorylation reduces, and the ERK of prompting PKA signal path and the signal transduction of DARPP-32 mediation strengthen.Extracellular signal-regulated kinase (Extracellular signal-regulated kinases, ERK) be protein kinase (the Mitogen activated protein kinase that mitogen activates, MAPK) member in family, be considered to the important modulation point from cell surface to nuclear signal transduction.ERK is through the kinases MEK of MAPK phosphorylation modification (claiming again ERK1/2, P44/P42) on Thr202 and Tyr204 site, after can participate in the adjusting of intracellular signal transduction widely.Alleviate the medicine of motor complication (as adenosine A
2AReceptor antagonist, cannabinoid CB 1 receptor agonist) can weaken the Abnormal Phosphorylation of ERK and DARPP-32, prompting ERK and DARPP-32 have participated in the generation of motor complication, the enhancing of its function can activate the transcription factor in downstream, affect Striatum gene expression, also can regulate and control to participate in the phosphorylation level of some important albumen of striatum function adjusting.Therefore the present invention with regard to Chinese medicine composition on the total DARPP-32(Thr75 of Striatum of Rats) and phosphorylation DARPP-32(Thr75) impact of protein expression observes, the total DARPP-32(Thr75 of Striatum is respectively organized in discovery) expression do not change.After the levodopa long-term treatment, phosphorylation DARPP-32(Thr75) expression ratio PD matched group obviously reduces, add with in dosage Chinese medicine composition of the present invention can reverse phosphorylation DARPP-32(Thr75) reduction of expression.When reason may be the motor complication generation, the DA receptor sensitivity increased, the PKA Pathway Activation, thereby reduced the Expression of phosphorylated in DARPP-32 albumen Thr75 site, phosphorylation DARPP-32(Thr75 has been raised in the use of Chinese medicine composition) expression, the PKA path is suppressed to increase, reduced abnormal motor behavior.Simultaneously, the present invention observes the variation of the total ERK1/2 of PD Striatum of Rats and phosphorylation ERK1/2 protein expression by SABC and Western blot technology, finds respectively to organize total ERK protein expression without significant change.With regard to phosphorylation ERK1/2 albumen, the expression of PD matched group phosphorylation ERK1/2 albumen reduces than sham group rat, and after the levodopa long-term treatment, phosphorylation ERK1/2 expresses significantly and raises, and there is the activation of ERK path in the prompting motor complication while occurring.After Chinese medicine composition auxiliary treatment of the present invention, significantly rising does not all appear in middle dosage group and heavy dose of group expression, shows that middle dosage Chinese medicine composition of the present invention has suppressed the abnormal activation of ERK path, has played the effect of prevention motor complication.
In a word, middle dosage Chinese medicine composition of the present invention has reversed the carrying out property rising of each component of PD rat model AIM scoring, reduced PD rat model agent peak number of revolutions, by increasing GRK6, β-arrestin1 expressing quantity, reverse phosphorylation DARPP-32(Thr75) reduction of expression and phosphorylation ERK1/2 express significantly and raise, improved the function of signal transducers abnormal in the motor complication born of the same parents, reduced the generation of PD motor complication, prevented PD and delaying to have great using value aspect disease progression.
From traditional Chinese medical science angle, Chinese medicine composition compatibility of the present invention meets " monarch " principle, benefiting QI and nourishing blood, the endogenous wind stopping that reduces phlegm, and QI and blood is only filled wind-phlegm, and irritability is dredged and is trembled flatly, follows the Therapeutic Principle of " QI invigorating is reduced phlegm, easing the affected liver relieves dizziness, high fever, infantile convulsions, epilepsy, etc. method ".
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and supplement, these improvement and supplement and also should be considered as protection scope of the present invention.
Claims (4)
1. a Chinese medicine composition prevents the purposes in the parkinson disease medicine in preparation, it is characterized in that, described Chinese medicine composition is to be made by the crude drug of following weight portion: Rhizoma Gastrodiae 10-20 part, Radix Astragali 10-20 part, Radix Rehmanniae Preparata 15-25 part, Radix Paeoniae Alba 15-25 part, Radix Angelicae Sinensis 15-25 part, Ramulus Uncariae Cum Uncis 10-20 part, Bombyx Batryticatus 7-13 part, Rhizoma Arisaematis 7-13 part.
2. purposes according to claim 1, it is characterized in that, described Chinese medicine composition is to be made by the crude drug of following weight portion: Rhizoma Gastrodiae 12-18 part, Radix Astragali 12-18 part, Radix Rehmanniae Preparata 19-21 part, Radix Paeoniae Alba 18-22 part, Radix Angelicae Sinensis 19-21 part, Ramulus Uncariae Cum Uncis 12-18 part, Bombyx Batryticatus 8-12 part, Rhizoma Arisaematis 8-12 part.
3. purposes according to claim 1 and 2, it is characterized in that, described Chinese medicine composition is to be made by the crude drug of following weight portion: 15 parts, Rhizoma Gastrodiae, 15 parts of the Radixs Astragali, 20 parts of Radix Rehmanniae Preparata, 20 parts of Radix Paeoniae Alba, 20 parts of Radix Angelicae Sinensis, 15 parts of Ramulus Uncariae Cum Uncis, 10 parts of Bombyx Batryticatus, 10 parts of Rhizoma Arisaematiss.
4. purposes according to claim 1 and 2, is characterized in that, the medicament of described Chinese medicine composition is capsule, granule, tablet, mixture, syrup, microcapsule formulation, injection, suppository, spray or ointment.
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