CN102600370A - 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 used 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 reach the nigrostriatum path dopamine mediator that causes thus and reduce relevant with black substance dense area dopaminergic neuron degeneration disappearance.The traditional Chinese medical science thinks that PD belongs to " tremor syndrome ", and pilosity is born in person in middle and old age's " menses " when exhausting, and " menses " exhausts that to characterize kidney essense deficient, and Rong Wei is two empty, on not nourishing the liver wood, Ru Jinmai not outward, water files to nourish wood, and then hyperactivity of yang due to yin deficiency, palpitaition, sweating, hectic fever, agitation take place frequently; The muscle arteries and veins loses deadlock in gentle existing morning, spasm of limbs, trembles, tetanic, pain, bradykinesia.Take a broad view of the experience of ancient Chinese medicine doctor, take up modern medicine study, observe through facing card, 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 that levodopa is still and the most effectively prevents the parkinson disease medicine.But behind the levodopa prolonged application 3-5; About 5% PD patient can produce gradually symptom fluctuation with (or) levodopa " long-term syndrome " such as the dyskinesia, i.e. PD motor complication, laboratory is found high concentration dopamine (dopamine in addition; DA) and levodopa can be because autoxidation produces free radical; Can cause the neurocyte degeneration necrosis, therefore, the associating other drug prevents the PD motor complication very urgent jointly.
In recent years research shows; PD takes place to be activated in close relations with the direct path and the signal transduction pathways such as downstream cAMP deopendent protein kinases (PKA), ERK thereof of expressing the D1 receptor; (dopamine and cAMP-regulated phosphoprotein of Mr 32000, the change of albumen Thr75 site Expression of phosphorylated DARPP-32) possibly participated in the morbidity of motor complication to the phosphorylated protein-32 that its downstream signal transducin dopamine and adenosine cyclophosphate are regulated.
Aspect the prevention of PD motor complication, mainly adopt COMT inhibitor, amantadine, DR agonist, L-dopa methyl ester or ethyl ester etc. at present.Like Chinese patent document CN 200880108979.7; Date of publication on August 18th, 2010; A kind of " being used to improve 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 the agonist effect, has for the motor complication of supervening when the repetitively administered levodopa and improves and the effect of delayed onset.But Western medicine prevention side effect is bigger, and it is safe and reliable to be not so good as Chinese herb on the prevention.China is accumulating rich experiences aspect the Chinese medicine prevention PD, and summing up its prevention principle has the QI invigorating activating blood circulation method, 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., activating blood and removing stasis Method and collateral dredging rule such as relieve dizziness, high fever, infantile convulsions, epilepsy, etc. is used to prevent the Chinese medicine composition of PD also to have multiple; But but seldom about the Chinese medicine composition of prevention PD motor complication; Like Chinese periodical " Chinese Chinese medicine journal, 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, do not formed and proportioning but fully disclose the particulate medicine of described invigorating kidney, promoting blood circulation.Therefore, must try to explore effectively to prevent parkinson disease motor complication, safe and reliable medicine, but also not appear in the newspapers about this type medicine at present.
Summary of the invention
The objective of the invention is provides a kind of purposes of Chinese medicine composition to deficiency of the prior art.
For realizing above-mentioned purpose, the technical scheme that the present invention takes is:
The purposes of a kind of Chinese medicine composition in preparation prevention parkinson disease medicine, described Chinese medicine composition is to be processed by following bulk drugs: 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 processed by following bulk drugs: 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 processed by following bulk drugs: 15 parts in 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 reduces phlegm, easing the affected liver relieves dizziness, high fever, infantile convulsions, epilepsy, etc. method ", starts with from cause of disease at all, suits the remedy to the case, and auxiliary prevention parkinson disease effect is obvious;
Advantages such as 2, this Chinese medicine composition prevention parkinson disease have and have no side effect, and price is low are easy to 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 prospects for application arranged.
Description of drawings
Accompanying drawing 1 is the influence of Chinese medicine composition to PD rat model AIM total points.
Accompanying drawing 2 is Chinese medicine composition influences to PD rat model agent peak number of revolutions.
Accompanying drawing 3 is SABC figure of PD rat model striatum district GRK6.
Accompanying drawing 4 is SABC block diagrams of PD rat model striatum district GRK6.
Accompanying drawing 5 is Western collection of illustrative plates that PD rat model striatum district GRK6 expresses.
Accompanying drawing 6 is quantitative figure that PD rat model striatum district GRK6 expresses.
Accompanying drawing 7 is SABC figure of PD rat model model striatum district β-arrestin1.
Accompanying drawing 8 is Western collection of illustrative plates that PD rat model model striatum district β-arrestin1 expresses.
Accompanying drawing 9 is SABC figure of PD rat model striatum district phosphorylation DARPP-32 (Thr75).
Accompanying drawing 10 is Western collection of illustrative plates that PD rat model striatum district phosphorylation DARPP-32 (Thr75) expresses.
Accompanying drawing 11 is SABC figure of PD rat model striatum district phosphorylation ERK1/2.
Accompanying drawing 12 is Western collection of illustrative plates that PD rat model striatum district 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 in 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 in 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 in 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 in 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 in 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 in 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 in 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 in 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 in 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 in 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.
Need to prove that it is the conventional manufacture method of Chinese medicine decoction that the described conventional method of embodiment 1-10 decocts, and is about to described crude drug decocte with water and becomes decoction.
The preparation of the Parkinsonian Chinese medicine composition tablets/capsules of embodiment 11 preventions
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decocted 1-3 hour, leach medicine juice; Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night; Get supernatant, be concentrated into thick extractum; Add pharmaceutic adjuvant (method according to routine is selected pharmaceutic adjuvant for use), vacuum drying is pulverized and is granulated, and is pressed into tablet or fills encapsulated.
The particulate preparation of the embodiment 12 Parkinsonian Chinese medicine compositions of prevention
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decocted 1-3 hour, leach medicine juice; Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night; Get supernatant, be concentrated into thick extractum; Add pharmaceutic adjuvant (method according to routine is selected pharmaceutic adjuvant for use), vacuum drying is pulverized and is granulated, drying, and granulate gets 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, decocted 1-3 hour, leach medicine juice; Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night; Get supernatant, be concentrated into thick extractum; Add suitable pharmaceutical aids (method according to routine is selected pharmaceutic adjuvant for use), process mixture, oral liquid or syrup.
The preparation of the Parkinsonian Chinese medicine composition microcapsule formulation of embodiment 14 preventions
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decocted 1-3 hour, leach medicine juice; Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night; Get supernatant, be concentrated into thick extractum; Select pharmaceutically acceptable capsule material for use, process microcapsule formulation.
The preparation of the Parkinsonian Chinese medicine composition composition injection of embodiment 15 preventions
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decocted 1-3 hour, leach medicine juice; Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night; Get supernatant, 80 ℃ of distillations are reclaimed, and are equipped with water for injection, process injection.
The preparation of the Parkinsonian Chinese medicine composition suppository of embodiment 16 preventions
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decocted 1-3 hour, leach medicine juice; Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night; Get supernatant, be concentrated into thick extractum; Add pharmaceutic adjuvant (method according to routine is selected pharmaceutic adjuvant for use), vacuum drying is pulverized and is granulated, and injection molding is processed suppository.
The preparation of the Parkinsonian Chinese medicine composition composition spray of embodiment 17 preventions
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decocted 1-3 hour, leach medicine juice; Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night; Get supernatant, be concentrated into thick extractum; Add medicinal liquefier, inject nonmetal aerosol apparatus, process spray.
The preparation of the Parkinsonian Chinese medicine composition ointment of embodiment 18 preventions
Get the arbitrary described Chinese medicine composition of embodiment 1-10, add 6-10 times of water gaging, decocted 1-3 hour, leach medicine juice; Add 6-10 times of water gaging again, decocted 0.5-2 hour, leach medicine juice; Merge the secondary decocting liquid, leave standstill, the leaching supernatant concentrates, and puts coldly, adds concentrated solution 2-3 and doubly measures ethanol, stirs deposition and spends the night; Get supernatant, be concentrated into thick extractum; Add suitable substrate and mix, process ointment.
Embodiment 19 these Chinese medicine compositions prevent Parkinsonian preclinical test
One, experimental technique
1.PD rat model preparation
Get 65 rats and get into experiment, wherein 5 is sham operated rats, and promptly 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 are shown rat brain stereotaxic atlas (DeWire SM in stereo brain orienting instrument with reference to the bag new people; Lefkowitz RJ; Shenoy SK, et al. Beta arrestins and cell signaling. Annu Rev Physiol. 2007,69:483-510.); Confirm right side medial forebrain bundle coordinate: 1. 3.7 mm behind the bregma; Sagittal suture right side 1.7 mm, 7.8 mm under the cranial periosteum, front tooth line 2.4 mm; 2. 4.4 mm behind the bregma, sagittal suture right side 1.2 mm, 7.8 mm under the cranial periosteum, front tooth line 2.4 mm.By the boring of above-mentioned definite injection site, extract 6-OHDA 6 μ l (containing 0.2% ascorbic normal saline configuration, concentration 4 μ g/ μ l) with the microsyringe of 10 μ l, the withdraw of the needle behind 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.
Divide into groups and treatment
The PD rat model of 25 successes is divided into 3 groups greatly at random.(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; ID is 50 mg/kg LDMEs and 25 mg/kg benserazides; The two all is dissolved in and contains in the 0.2% ascorbic sterilization normal saline, 2 times/d at (in the morning with afternoons 5 point), continues 29d at 9; (3) dose groups, the heavy dose of group of Chinese medicine composition in Chinese medicine composition small dose group, the Chinese medicine composition: give to add respectively on the basis like the LDME/benserazide in the group (2) 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; The dose groups using dosage is 9 ml/kg in the Chinese medicine composition; The heavy dose of group of Chinese medicine composition using dosage is 10.8 ml/kg, 1 filling every day stomach, continuous 4 weeks.
Behavioristics measures
In therapeutic process, after medication in the morning in the 2nd, 8,15,22,29 day, 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 portion, axle property AIM and rotation AIM) to be evaluated, and every part has or not with the order of severity according to it again and is divided into 5 grades (0-4): 0 does not have; 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.Every 20min assessment is once observed 1min at every turn after the medication.The AIM total points is added up by the meansigma methods of total mark in observing time, and each component AIM maximum scores is 4 minutes behind 1 rat a drug in theory, and total AIM scoring is 16 minutes.Agent peak number of revolutions: behind the injection levodopa, every 5min record number of revolutions, maximum numbers of revolutions is an agent peak number of revolutions.
SABC shows the variation of striatum β-arrestin1, GRK6, DARPP-32 (Thr75) and ERK1/2
Finish injection back 12h, the anesthesia of 3% pentobarbital sodium, left ventricle is poured into 4% paraformaldehyde and is fixed, and broken end is got behind the brain in the same fixed liquid fixedly 8h of back, and piece, gradient alcohol dehydration, xylene are transparent through repairing, use FFPE behind the 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-hydrogen chloride (EDTA-Tris-HCl) microwave heating reparation of pH7.7; 1% bovine serum albumin room temperature envelope 20min; Incubated overnight in 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) 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 flushing, dehydration, transparent, mounting afterwards.All use the abundant rinsing of 0.01mol/l TBS between each step, TBS replaces one to resist as negative control.Examine under a microscope the SABC section; Each is observed the district and gets 5 not overlapping visuals field at random; Observe down in high power lens (10 * 40); 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 cell area * correction OD value (measurement zone optical density-background indensity).
Method detects the expression of β-arrestin1, GRK6, DARPP-32 (Thr75) and ERK1/2
Last injection back 3h, the anesthesia of 3% pentobarbital sodium, 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 subsequent use.40 μ g albumen samples separate through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), change the film appearance with electricity and are transferred on polyvinylidene fluoride (PVDF) film.Confining liquid (5% defatted milk powder) room temperature sealing 2h; According to respective concentration dilute anti-GRK6 antibody (1: 500), anti-β-arrestin1 antibody (1: 500), anti-total DARPP-32 antibody (1: 1000) or anti-phosphorylation DARPP-32 (Thr75) 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 incubated overnight.Add two next day and resist, shaking table is hatched 1 h under the room temperature; Drip ECL colour developing mixed liquor, the exposure of Bio-Rad gel imaging appearance, 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.
Statistical analysis technique
Experimental data adopts SPSS 13.0 statistical softwares to analyze.The gained measurement data is with (
± S) expression of mean ± standard deviation; The relatively employing one factor analysis of variance of a plurality of sample averages relatively adopts the t check in twos.< 0.05 is that difference has statistical significance with P.
Two, experimental result
1. Chinese medicine composition is to the influence of PD rat model behavior
1.1 Chinese medicine composition is to the influence of each component AIM scoring of PD rat model
Dose groups in Chinese medicine composition small dose group, the Chinese medicine composition, the heavy dose of group of Chinese medicine composition and Western medicine processed group to each the component AIM scoring of PD rat model to influence the result as shown in table 1.
With regard to the AIM of actinal surface portion scoring, average does not have significant difference between the 2nd day each group.8th, average significant difference (F=4.98, F=22.70, F=30.96, F=23.27, P < 0.01) between 15,22,29 days each groups.Wherein, the Chinese medicine composition small dose group reduces than the Western medicine processed group in the 22nd day AIM of actinal surface portion scoring, and difference has statistical significance (t=2.50, P < 0.05), all the other several days equal not statistically significants of difference; The 8th, 15,22,29 days AIM of actinal surface portion of dose groups scoring obviously reduces than the Chinese medicine composition small dose group in the Chinese medicine composition, difference have statistical significance (t=2.86, t=3.80, t=4.38, t=6.54, all P 0.05); In the Chinese medicine composition the heavy dose of group of dose groups and Chinese medicine composition relatively, only in the 22nd day heavy dose of group of Chinese medicine composition than Chinese medicine composition in the dose groups actinal surface AIM of portion scoring reduce, difference has statistical significance (t=2.50, P < 0.05).
With regard to upper limb AIM scoring, the 2nd, 8 day each class mean no significant difference, average difference has statistical significance (F=15.12, F=32.86, F=35.80, P < 0.01) between the 15th, 22,29 day each group.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; Be lower than the Western medicine processed group to the 15th, 22,29 days Chinese medicine composition small dose group upper limb of medication AIM scoring; Difference have statistical significance (t=4.45, t=4.47, all P 0.05).15th, dose groups obviously reduces than Chinese medicine composition small dose group upper limb AIM scoring in 22,29 days Chinese medicine compositions, difference have statistical significance (t=3.40, t=4.04, t=3.95, all P 0.01).Compare the difference no difference of science of statistics between the dose groups in heavy dose of group of Chinese medicine composition and the Chinese medicine composition.
With regard to axial AIM scoring, average difference has statistical significance (F=3.30, F=25.06, F=36.41, P < 0.01) between the 15th, 22,29 day each group.22,29 days axial AIM values of Chinese medicine composition small dose group medication to the reduce than Western medicine processed group, difference have statistical significance (t=3.93, t=3.04, all P 0.05); Dose groups and Chinese medicine composition small dose group relatively only have statistical significance (t=3.05, P < 0.05) in the 29th day difference in the Chinese medicine composition; The heavy dose of group of Chinese medicine composition has obviously reduced axial AIM value, but with Chinese medicine composition in the dose groups comparison, only variant in the 29th day (t=2.83, P < 0.05) puts the difference no difference of science of statistics At All Other Times.
With regard to rotation AIM scoring, average significant difference between the 8th, 15,22,29 day each group (F=10.50, F=11.62, F=18.92, F=61.26, P 0.01).The difference not statistically significant is compared in Chinese medicine composition small dose group and Western medicine processed group rotation AIM scoring.Dose groups medication to the is 15,22,29 days in the Chinese medicine composition, and rotation AIM scoring obviously reduces than Western medicine processed group, difference have 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 the Chinese medicine composition dose groups relatively, the 8th, 15,29 day obvious difference (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 divides)
| Time | Group | Mus number (only) | Actinal surface portion | 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 Chinese medicine composition is to the influence of 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) are divided; Than Western medicine processed group (10.48 ± 0.40), (11.41 ± 0.17) divide and (11.58 ± 0.24) divide obviously and reduce (t=2.88, t=9.25, t=7.77, all p 0.05).And the 8th, 15,22,29 days AIM total points of dose groups medication are respectively (8.82 ± 0.36), (8.71 ± 0.47), (8.13 ± 0.37) and (7.89 ± 0.57) in the Chinese medicine composition; All be lower than Chinese medicine composition small dose group total points; Difference have statistical significance (t=3.32, t=3.19, t=7.56, t=7.85, all p 0.05); In the 8th, 15,22 day; 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) are divided; Low than dose groups in the Chinese medicine composition, difference have statistical significance (t=3.12, t=3.44, t=2.57, all p 0.05); Dose groups (7.89 ± 0.57) compares in (7.71 ± 1.13) branch and the Chinese medicine composition and the 29th day Chinese medicine composition heavy dose organized; (see Fig. 1, # representes Chinese medicine composition small dose group and Western medicine processed group relatively to the difference not statistically significant, all P < 0.05; * represent dose groups and the comparison of Chinese medicine composition small dose group, all P < 0.05 in the Chinese medicine composition; + represent that dose groups compares in heavy dose of group of Chinese medicine composition and the Chinese medicine composition, equal P 0.05).
Chinese medicine composition is to the influence of PD rat model agent peak number of revolutions
Carried out agent peak number of revolutions detected in the 8th, 15,22,29 day; 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 dose groups and the comparison of Chinese medicine composition small dose group in the Chinese medicine composition; Dose groups agent peak number of revolutions is respectively (85.4 ± 15.60), (82.0 ± 21.76), (87 ± 10.20) and (73 ± 15.18) in the Chinese medicine composition; All be lower than the Chinese medicine composition small dose group; Difference have statistical significance (t=4.13, t=4.57, t=7.51, t=10.96, all p 0.05); Dose groups relatively in heavy dose of group of Chinese medicine composition and the Chinese medicine composition; 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 dose groups in the Chinese medicine composition, difference have statistical significance (t=6.73, t=2.52, all p 0.05) (see Fig. 2; # representes Western medicine processed group and Chinese medicine composition small dose group relatively, all P < 0.05; * represent dose groups and the comparison of Chinese medicine composition small dose group, all P < 0.01 in the Chinese medicine composition; + represent that dose groups compares in heavy dose of group of Chinese medicine composition and the Chinese medicine composition, equal P 0.05).
Chinese medicine composition is to the influence of PD rat model striatum district signal protein
2.1 Chinese medicine composition is to the influence of PD rat model striatum district 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 injury in 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, promptly the rat GRK6 of Western medicine processed group expresses and further is reduced to (3.53 ± 0.71) * 10
3, comparing with the rat of PD matched group, difference has statistical significance (t=2.99, P<0.05); And add rat with Chinese medicine composition of the present invention prevention; Comprise dose groups and the heavy dose of group of Chinese medicine composition in Chinese medicine composition small dose group, the Chinese medicine composition; Striatal damage side GRK6 expresses with the PD control rats and compares; All do not have further and reduce, compare Chinese medicine composition small dose group (4.13 ± 1.20) * 10 with the rat of Western medicine processed group
3With Western medicine processed group zero difference, the expression of dose groups, the heavy dose of group damage of Chinese medicine composition side GRK6 is respectively (4.31 ± 0.95) * 10 in the Chinese medicine composition
3, (4.68 ± 1.24) * 10
3, compare GRK6 expression obviously more (t=3.79, t=4.84, all P with the Western medicine processed group<0.05) (see Fig. 3 and Fig. 4, among Fig. 3: 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 dose groups in the Chinese medicine composition, and 6 is the heavy dose of group of Chinese medicine composition; U is not for damaging side, and L is the damage side; * represent relatively P of PD matched group and sham group<0.01 # representes relatively P of Western medicine processed group and PD matched group<0.05+expression Chinese medicine composition is little, in, heavy dose of group and Western medicine processed group relatively, equal P<0.05).
Western result and SABC be basically identical as a result.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 confidential reference items.Compare with (100.48 ± 5.57) % of sham group, 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 rat with Chinese medicine composition prevention of the present invention, and promptly dose groups and Chinese medicine composition are heavy dose of in Chinese medicine composition small dose group, the Chinese medicine composition 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, dose groups striatum GRK6 expression (83.68 ± 4.50) % is obviously higher in the Chinese medicine composition, difference has statistical significance (t=7.26; P 0.01), and Chinese medicine composition small dose group (77.12 ± 3.15) % and Chinese medicine composition heavy dose of group (80.36 ± 4.74) % and its compare, and difference significantly (is not seen Fig. 5 and Fig. 6; Among Fig. 5: 1,2 are the sham group; 3,4 is the PD matched group, and 5,6 is the Western medicine processed group, and 7,8 is the Chinese medicine composition small dose group; 9,10 is dose groups in the 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; * expression is compared with the sham group, and p < 0.01; # representes to compare with the PD matched group, and p < 0.05; + expression Chinese medicine composition is little, in, heavy dose of group and Western medicine processed group relatively, p 0.01).
Chinese medicine composition is to the influence of PD rat model model striatum district 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 proteic positive cell index of the rat β-arrestin1 of PD matched group is (3.27 ± 0.75) * 10
4, be lower than (4.54 ± 0.63) * 10 that sham organizes rat
4, but the difference not statistically significant.Be the rat of Western medicine processed group after the levodopa long-term treatment, β-arrestin1 expresses further and reduces, and the positive cell index is (2.54 ± 0.49) * 10
4, comparing with the rat of PD matched group, difference has statistical significance (t=3.99, P<0.05).Add rat with Chinese medicine composition of the present invention; Be dose groups and the heavy dose of group of Chinese medicine composition in Chinese medicine composition small dose group, the Chinese medicine composition; It is (3.57 ± 0.56) * 10 that dose groups rat β in the further decline, particularly Chinese medicine composition-arrestin1 positive cell index does not appear in β-arrestin1 positive cell index
4, increase (t=6.80, P than the rat of PD matched group<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, comparing with the PD matched group, (see Fig. 7, among Fig. 7: 1 is the sham group to no significant difference, 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 dose groups in the Chinese medicine composition, and 6 is the heavy dose of group of Chinese medicine composition; U is not for damaging side, and L is the damage side).
Western result and SABC be basically identical as a result.Every rat Western result is with damage side/do not damage the ratio value representation of side, and β-actin is as confidential reference items.Rat β-arrestin1 the expressing quantity of PD matched group is (76.46 ± 5.12) %, 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 the Chinese medicine composition of the present invention prevention, Chinese medicine composition small dose group β-arrestin1 expression be (69.54 ± 3.36) %, than the rat reduction of PD matched group (t=3.42, P < 0.05), demonstrates the trend similar with the Western medicine processed group; And the heavy dose of group of dose groups and Chinese medicine composition is respectively (73.52 ± 3.82) % and (72.36 ± 6.66) % in the Chinese medicine composition, occurs further descending.Simultaneously, 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 dose groups and Chinese medicine composition striatum in the Chinese medicine composition, and difference has statistical significance (t=3.11, t=3.70, P < 0.05).Compare between the heavy dose of group of dose groups and Chinese medicine composition in the Chinese medicine composition; (see Fig. 8, among Fig. 8: 1,2 are the sham group to the difference not statistically significant, and 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 dose groups in the 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).
The influence that Chinese medicine composition is expressed PD rat model striatum district phosphorylation DARPP-32 (Thr75)
Each group damage side average significant difference of SABC demonstration (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) is expressed as (3.35 ± 0.30) * 10
6, obviously reducing than PD matched group, 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 dose groups phosphorylation of Chinese medicine composition DARPP-32 (Thr75) expresses and is respectively (12.35 ± 0.19) * 10
6, (11.90 ± 0.47) * 10
6(7.30 ± 0.27) * 10
6, significantly reducing does not all appear in the heavy dose of group of dose groups and Chinese medicine composition in the Chinese medicine composition, and only the Chinese medicine composition small dose group reduces.Compare between the dose groups in heavy dose of group of Chinese medicine composition and the Chinese medicine composition; (see Fig. 9, among Fig. 9: A is the sham group to the difference not statistically significant, and 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 a dose groups in the Chinese medicine composition, and F is the heavy dose of group of Chinese medicine composition).
Western result and SABC be basically identical as a result, and the gray value that sham group rat phosphorylation DARPP-32 (THr75) expresses 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).Add with low dose of Chinese medicine composition of the present invention and do not play preventive effect, Chinese medicine composition small dose group phosphorylation DARPP-32 (THr75) expression is (1.80 ± 0.15) * 10
6, reduce (t=19.77, P than the rat of PD matched group<0.01); Rat striatum phosphorylation DARPP-32 (THr75) expression that adds dosage Chinese medicine composition prevention of the present invention in using is (3.15 ± 0.15) * 10
6, compare difference not statistically significant (P > with the rat of PD matched group; 0.05).The heavy dose of group of Chinese medicine composition phosphorylation DARPP-32 (THr75) expression is (3.81 ± 0.17) * 10
6, have slightly than the rat of PD matched group and to increase (t=38.57, P<0.01), simultaneously, the heavy dose of group of Chinese medicine composition is than dose groups increasing expression (t=21.78, P in the Chinese medicine composition<0.01) (see Figure 10, among Figure 10: 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 dose groups in the Chinese medicine composition, and 6 is the heavy dose of group of Chinese medicine composition).
The influence that Chinese medicine composition is expressed PD rat model striatum district phosphorylation ERK1/2
Each group damage side average significant difference of SABC demonstration (F=117.49, P 0.01).The rat phosphorylation ERK1/2 expression of PD matched group is (4.37 ± 0.23) * 10
4, be lower than (5.23 ± 0.34) * 10 that sham organizes rat
4, difference has statistical significance (t=15.32, P<0.01).Be the rat of Western medicine processed group after the levodopa long-term treatment, phosphorylation ERK12 expresses and is increased to (8.09 ± 0.37) * 10
4, comparing with the PD matched group, 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 ERK12 is expressed as (6.69 ± 0.36) * 10
4, in rising trend, the heavy dose of group of dose groups and Chinese medicine composition expression is (4.49 ± 0.34) * 10 in the Chinese medicine composition
4(4.23 ± 0.25) * 10
4, all do not occur significantly rising.Compare between the dose groups in heavy dose of group of Chinese medicine composition and the Chinese medicine composition, difference is not obvious.(see Figure 11, among 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 a dose groups in the Chinese medicine composition, and F is the heavy dose of group of Chinese medicine composition).
Western result and SABC be basically identical as a result, 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); The gray value that adds the rat striatum phosphorylation ERK12 expression that prevents with large, medium and small dosage Chinese medicine composition of the present invention is 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, Dose groups phosphorylation ERK12 expression rising amplitude is minimum in the Chinese medicine composition, and Chinese medicine composition small dose group rising amplitude is maximum, and (see Figure 12, among 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 dose groups in the Chinese medicine composition, and 5 is the heavy dose of group of Chinese medicine composition).
Originally the Chinese medicine composition of the present invention of dosage can suppress the carrying out property rising of PD rat model actinal surface portion, upper limb AIM scoring in discovering, 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 marking with rotation AIM, the 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.The dosage Chinese medicine composition can obviously reduce the generation of PD rat model motor complication in the explanation, and heavy dose of Chinese medicine composition to axially with 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 not to have difference with middle dosage, and the dosage Chinese medicine composition is remarkable and stable to the AIM effect that reduces the levodopa generation in the explanation.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 that along with PD course of disease progress, compensation constantly appears in Basal ganglia DA, and the early stage Basal ganglia DA receptor of PD patient occurs ultra quick, and the receptor number increases (rise effect).The DA receptor belongs to GPCRs family, when DA receptor long term exposure in agonist, the agonist of same level can not make the effector of receptor activate, and after promptly agonist has activated GPCRs, also start a degenerative process, 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.This result of study shows that the GRK6 expression reduces than the sham group during PD, and prompting DA receptor is ultra quick closely related with reduction GRK6.In addition, Western medicine processed group GRK6 expression further reduces than the PD matched group.This possibly be that GRK6 reduces along with the life-time service of levodopa, can't suppress the activation of ultra 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 ultra quick effect of DA receptor, have stoped the transmission of abnormal signal, thereby play the effect that the prevention motor complication takes place, and dosage increases more significantly preventive effect of not appearance.Equally, β during PD-arrestin1 protein expression reduces than the sham group, possibly be because the PD dopaminergic neuron is lost and degeneration in a large number, and residual dopamine neuron performance compensation increases the DA receptor sensitivity.And the Western medicine processed group β-arrestin1 expression of life-time service levodopa treatment further reduces than the PD matched group; Transduction suppressed to weaken β-arrestin1 to abnormal signal when this possibly be PD; Thereby caused the abnormal signal activation, caused motor complication.Add 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, and the effect that prevention unusual fluctuation complication takes place is more remarkable, and more significantly preventive effect does not appear in heavy dose of Chinese medicine composition.The dose groups Chinese medicine composition can increase β-arrestin1 protein expression in the explanation, plays the effect that the prevention motor complication takes place.
Phosphorylated protein-32 (the dopamine and cAMP-regulated phosphoprotein of Mr 32000 that dopamine and adenosine cyclophosphate (cAMP) are regulated; 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; Simultaneously possibly pass through 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 influences the downstream signal conduction.Become the inhibitor of PKA after the Thr75 site phosphorylation of DARPP-32, the reduction of its phosphorylation degree has alleviated the inhibitory action to PKA.The difference that this shows phosphorylation site can be given DARPP-32 with opposite physiological effect.Early-stage Study finds that the phosphorylation of motor complication rat model striatum district ERK strengthens, and DARPP-32Thr75 site phosphorylation reduces, and the ERK and the DARPP-32 Mediated Signal Transduction of prompting PKA signal path strengthen.Extracellular signal-regulated kinase (Extracellular signal-regulated kinases; ERK) be the activated protein kinase of mitogen (Mitogen activated protein kinase; MAPK) member in the family is considered to the important modulation point from cell surface to nuclear signal transduction.ERK through the kinases MEK of MAPK on Thr202 and Tyr204 site phosphorylation modification (claim ERK1/2 again, P44/P42), after can participate in the adjusting of intracellular signal transduction widely.The medicine of alleviating motor complication is (like adenosine A
2AReceptor antagonist, cannabinoid CB 1 receptor agonist) can weaken the unusual 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; Influence the gene expression of striatum district, some important proteic phosphorylation levels that also adjustable participation striatum function is regulated.Therefore the present invention observes the influence of total DARPP-32 of rat striatum Qu (Thr75) and phosphorylation DARPP-32 (Thr75) protein expression with regard to Chinese medicine composition, finds that respectively organizing the total DARPP-32 of striatum Qu (Thr75) expression does 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 the reduction of phosphorylation DARPP-32 (Thr75) expression.The DA receptor sensitivity increased when reason possibly be the motor complication generation; The PKA path activates; Thereby reduced the Expression of phosphorylated in DARPP-32 albumen Thr75 site; The expression of phosphorylation DARPP-32 (Thr75) has been raised in the use of Chinese medicine composition, and the PKA path is suppressed to increase, and has reduced unusual motor behavior.Simultaneously, the present invention finds that through the variation of SABC and Western blot technology observation PD total ERK1/2 of rat striatum Qu and phosphorylation ERK1/2 protein expression respectively organizing total ERK protein expression does not have significant change.With regard to phosphorylation ERK1/2 albumen, the proteic expression of PD matched group phosphorylation ERK1/2 reduces than sham group rat, and phosphorylation ERK1/2 expresses significantly and raises after the levodopa long-term treatment, and there is the activation of ERK path in the prompting motor complication when taking place.After the Chinese medicine composition auxiliary treatment of the present invention, middle dose groups does not all occur significantly rising with heavy dose of group expression, and dosage Chinese medicine composition of the present invention has suppressed the abnormal activation of ERK path in showing, 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 AIM scoring of PD rat model; Reduced PD rat model agent peak number of revolutions; Through increasing GRK6, β-arrestin1 expressing quantity, the reduction and the phosphorylation ERK1/2 that reverse phosphorylation DARPP-32 (Thr75) expression express significantly rising, have improved the function of anomalous signals transduction molecule 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 the disease progression.
From traditional Chinese medical science angle, Chinese medicine composition compatibility of the present invention meets " monarch " principle, and benefiting QI and nourishing blood, the endogenous wind stopping that reduces phlegm, QI and blood fill then wind-phlegm to be ended, and irritability is dredged and then trembled flatly, follows the Therapeutic Principle of " QI invigorating reduces phlegm, easing the affected liver relieves dizziness, high fever, infantile convulsions, epilepsy, etc. method ".
The above only is a preferred implementation of the present invention; 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 replenish, these improvement and replenish and also should be regarded 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 processed by following bulk drugs: 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 processed by following bulk drugs: 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 processed by following bulk drugs: 15 parts in 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, oral liquid, mixture, syrup, microcapsule formulation, injection, suppository, spray or ointment.
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| CN103800703A (en) * | 2014-02-27 | 2014-05-21 | 成都中医药大学 | Pharmaceutical composition for treating Parkinson disease and preparation method of composition |
| CN104800604A (en) * | 2015-04-20 | 2015-07-29 | 韩萍 | Traditional Chinese medicine formula for treating Parkinsonism |
| CN109432306A (en) * | 2018-12-20 | 2019-03-08 | 上海交通大学医学院附属新华医院 | Pharmaceutical composition and its application |
| CN113633721A (en) * | 2021-07-28 | 2021-11-12 | 安徽中医药大学第一附属医院(安徽省中医院) | Traditional Chinese medicine composition for treating Parkinson's disease and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103800703A (en) * | 2014-02-27 | 2014-05-21 | 成都中医药大学 | Pharmaceutical composition for treating Parkinson disease and preparation method of composition |
| CN103800703B (en) * | 2014-02-27 | 2016-03-02 | 成都中医药大学 | A kind of pharmaceutical composition for the treatment of parkinsonism and preparation method thereof |
| CN104800604A (en) * | 2015-04-20 | 2015-07-29 | 韩萍 | Traditional Chinese medicine formula for treating Parkinsonism |
| CN109432306A (en) * | 2018-12-20 | 2019-03-08 | 上海交通大学医学院附属新华医院 | Pharmaceutical composition and its application |
| CN113633721A (en) * | 2021-07-28 | 2021-11-12 | 安徽中医药大学第一附属医院(安徽省中医院) | Traditional Chinese medicine composition for treating Parkinson's disease and preparation method thereof |
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