CN115721667A - Application of panax notoginseng saponins and acegastrodine in amyotrophic lateral sclerosis - Google Patents

Abstract

The invention discloses a new medical application of panax notoginseng saponins and acegastrodine, and relates to a new application of panax notoginseng saponins and acegastrodine in preparing a medicament for preventing and treating Amyotrophic Lateral Sclerosis (ALS) in a combined way. The experimental result shows that the combination of the panax notoginseng saponins and the acegastrodine plays roles in preventing and treating ASL, and has good clinical application value.

Description

Application of panax notoginseng saponins and acegastrodine in amyotrophic lateral sclerosis

Technical Field

The invention relates to a combined drug of panax notoginseng saponins and acegastrodine, in particular to an application of the combined drug of the panax notoginseng saponins and the acegastrodine in preparing a drug for preventing and treating amyotrophic lateral sclerosis.

Background

Amyotrophic Lateral Sclerosis (ALS) is a rare fatal neurological disease characterized by degenerative degeneration of motor neurons in the spinal cord and brain, manifested clinically as muscle weakness, atrophy, speech, swallowing, respiratory dysfunction, etc. (ZAREI S, CARR K, REILEY L, et al. A complex review of amyotrophic vascular disorders [ J ], surg Neurol, 2015,6 (1): 171), and is rare in the first disease catalogue united by 5 departments of the national institutes of health, 2018. ALS occurs at a rate of about 1.5/10 million worldwide, and most patients die due to respiratory failure caused by respiratory muscle weakness within 3-5 years from the first appearance of symptoms (MILLER RG, APPEL SH, introduction to supplement: the current status of treatment for ALS [ J ]; amyotrop Lateral scaler FRO-NTOTEMPLANT Degenerer, 2017, 18 (supply 1): S1-S4.). Approximately 10% of ALS cases are familial, usually with a dominant genetic profile, with the remaining approximately 90% of ALS cases being sporadic with no family history. The pathogenic mechanism of ALS is complex, and researches suggest that the pathogenic mechanism of ALS mainly comprises oxidative stress, gene mutation, excitotoxicity, virus infection, imbalance of metals and trace elements, mitochondrial abnormality, immune inflammatory response and the like (Guo Neng Qiang, lisen, zhang Haihong and the like, research progress of the pathogenic mechanism of amyotrophic lateral sclerosis [ J ]), chinese rehabilitation theory and practice, 2017, 23 (6): 685-689), and the functions of inflammation and oxidative stress in SOD 1-mediated motor neuron degeneration become a research hotspot recently. The pathogenesis of the disease is mutually linked and influenced, and finally, the multi-system lesion mainly comprising the motor nervous system is caused, and no cure or effective treatment method exists at present. Although riluzole (the chemical name of riluzole: 2-amino-6-trifluoromethoxybenzothiazole) has been the only drug approved by the FDA for the treatment of ALS for many years, it has been a limited drug that has been used for the treatment of ALS, and it is expensive, difficult to tolerate in most patient families, and it also causes damage to the liver and kidney functions, so that there is an urgent need to develop a safer, economical and effective drug.

Panax Notoginsenosides (PNS) have the effects of dilating blood vessels, improving microcirculation disturbance, reducing oxygen consumption of cardiac muscle, inhibiting platelet aggregation, prolonging blood coagulation time, reducing blood lipid, scavenging free radicals, resisting inflammation, resisting oxidation, etc., and can be widely used for treating and researching cardiovascular and cerebrovascular diseases, blood diseases, immune diseases, tumors, etc. The acetyl gastrodine is 4-hydroxymethyl benzene-2, 3,4, 6-tetra-O-acetyl-beta-D-glucopyranoside, is a similar derivative of gastrodine, has a sedative index 1.77 times stronger than that of gastrodine, and has better treatment effect on diseases such as neurasthenia, vascular headache and the like than gastrodine (X iong JM, moYQ, liang B, C hen Z H, deng SX. Infiluence of acetic-gastrodin on wounds of mice and rates [ J ]; zhongguo Yao Li Xue Bao,1987,8 (1): 57-59).

However, the effect of the two in ALS by single use or combined application is not reported, and the invention discloses the medical application of the combined application of the panax notoginseng saponins and the acegastrodine. Experiments show that the protective effect of the combined application of the panax notoginseng saponins and the acegastrodine on brain injury is stronger than that of the combined application of the panax notoginseng saponins and the acegastrodine; simultaneously, the combination of the two can obviously reduce the generation of MDA and LDH and improve the activity of SOD, thereby antagonizing the damage of oxygen free radicals generated during ischemia and hypoxia to brain cells; the combination of the two can also obviously reduce the contents of TNF-alpha and IL-6 in the brain homogenate of rats after cerebral ischemia, thereby resisting inflammatory injury caused by IL-6 and TNF-alpha. As the effects of inflammation and oxidative stress in SOD 1-mediated motor neuron degeneration are hot spots of recent research, the experiments of designing medicated serum cells show that the medicated serum obtained by combined application of panax notoginseng saponins and acegastrodine can obviously promote transfection of hSODl ^G93A NSC34 cells after plasmid survived and the activity was significantly increased (P)<0.001 ); and has MDA lowering effect (P)<0.01 ); and has protective effect on free radical injury and inflammatory injury. According to possible pathogenesis of ASL, the combination of the panax notoginseng saponins and the acegastrodine can intervene on the pathogenesis of ASL, and plays a role in preventing and treating the ASL.

The serum pharmacology method is proposed for the first time by Hiroko Iwama scholarly in the late 20 th century and 80 s, and great progress has been made through years of efforts, so the serum pharmacology method is considered to be a new thinking and a new breakthrough of traditional Chinese medicine research (Xiaoyarre, kangtai, wujing east, taohong four-drug decoction medicine-containing serum influence research on isolated cells [ J light, kangtai, wu Jingdong ] research general []Journal of internal medicine of practical TCM 2015,29 (2): 172 to 175; zhang J, zhang L, qiu J, et al, isobaric tags for relative and absolute quantification (i TRAQ) -based genomic analysis of Cryptococcus humus response to aluminum stress J Biosci biology 2015;120 359-363; medicated serum pairs of CoCl for addition and subtraction of Zhu Jing prescription, such as Gaonan, ganzeng and Broussonetia ₂ Induce ARPE-19 cells to express VEGF and HIF-1 alpha, 2016, 26 (6): 351-354, J.Med.J.Med.Med.J.Med.Med.Med.Med.Med.Med.Med.Med.Med.Med.Oz.

Serum pharmacology is not an independent subject, but an experimental technical method for applying drug-containing serum to an experimental reaction system to research the action effect, the pharmacological mechanism and the like of the drug (especially complex-component drugs such as traditional Chinese medicines, compound medicines and the like) is becoming mature at present. (Gao SY, gong YF, sun QJ, et al. Screening anticancer or biological active from Sauromatum giganteum. Engl.) using the method of pharmacological method and differentiation by UPLC-TOF-MS. Molecules.2015;20 (3): 4290; hanli, gong, yankee, etc.; study of the mechanism of protection of liver cells and Kupffer cells from inflammatory injury by serum containing drugs to liver cells and Kupffer cells under stimulation based on NF- κ B pathway [ J ], chinese patent medicine, 2015,37 (5): 1114-1119). The serum containing medicine is obtained after a certain amount of medicine is taken by animals, blood is collected for a period of time and the serum is centrifuged (systematic methodology of the basic research of the pharmacodynamic materials of the Chinese medicine of the Mingxinje, chinese medicine and evidence, metabonomics [ J ], china Chinese medicine journal, 2015, 40 (1): 13-17; chen Shaoyang, wanshichuan, wanzhglin, hepG2.2.15, the research on the related problems of the research on the hepatitis B by using the Chinese medicine compound [ J ], chinese and Western medicine combination journal, 2016, 36 (10): 1265-1267). Compared with other methods, the method can better reflect the drug effect and action environment of the drug, and is helpful for researching the drug effect substances of the traditional Chinese medicine and the in-vivo process thereof (Hongyongdun, li Ching, chen Shuang, etc. the compound for warming yang, activating blood and detoxifying has the effect on the function of human umbilical vein endothelial cells [ J ] the new drug and clinical pharmacology of traditional Chinese medicine, 2016, 27 (4): 519-5). Due to its unique advantages, it is widely used by many scholars and experts, and has been extensively explored and studied.

Disclosure of Invention

The invention provides application of panax notoginseng saponins and acegastrodine in preparation of a medicine for treating and/or preventing amyotrophic lateral sclerosis.

Further, the use is for reducing the level of MDA in a cell model of amyotrophic lateral sclerosis.

Further, the application is to improve the activity of nerve cells in amyotrophic lateral sclerosis.

Further, the use is to inhibit nerve excitotoxicity in amyotrophic lateral sclerosis, inhibit apoptosis of nerve cells, and/or promote proliferation of nerve cells.

Furthermore, the application is to promote cell survival in amyotrophic lateral sclerosis, resist oxidation and inflammation and play a role in protecting brain injury caused by free radicals.

Further, the use is that the mass ratio of the panax notoginseng saponins to the acegastrodine is preferably 1.

On the other hand, the invention provides a pharmaceutical composition of panax notoginseng saponins and acegastrodine for preparing a medicine for treating and/or preventing amyotrophic lateral sclerosis, wherein the pharmaceutical composition comprises panax notoginseng saponins, acegastrodine and pharmaceutically acceptable auxiliary materials. The pharmaceutical compositions may be administered to a subject in a single dose, either simultaneously or sequentially, or in combination, with an "effective amount" of the drug. By "effective amount" is meant a dosage of a drug that prevents, alleviates, retards, inhibits or cures a condition in a subject. The size of the administered dose is determined by the mode of drug administration, the pharmacokinetics of the agent, the severity of the disease, the individual signs of the subject (sex, weight, height, age), etc.

The administration route of the pharmaceutical composition can be intestinal or parenteral, such as oral administration, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs, respiratory tract, skin, vagina, rectum and the like; the preparation can be tablet, capsule, soft capsule, granule, pill, dripping pill, injection, lyophilized powder for injection, oral liquid, patch, unguent, cataplasma or sustained release preparation, controlled release preparation injection, tablet, oral liquid, capsule, soft capsule, dripping pill, sustained release preparation, and controlled release preparation.

In order to prepare the pharmaceutical composition of the present invention into a capsule, the pharmaceutical composition of the present invention may be mixed with a diluent and a glidant, and the mixture may be directly placed into a hard capsule or a soft capsule. The pharmaceutical composition can also be prepared into granules or pellets with a diluent, an adhesive and a disintegrating agent, and then placed into hard capsules or soft capsules. In order to prepare the pharmaceutical composition of the invention into injection, water, ethanol, isopropanol, propylene glycol, polyethylene glycol or a mixture thereof can be used as a solvent, and a proper amount of solubilizer, cosolvent, pH regulator and osmotic pressure regulator which are commonly used in the field can be added. The solubilizer or cosolvent can be ethanol, isopropanol, propylene glycol, polyethylene glycol, poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the pH regulator can be citrate, phosphate, carbonate, acetate, hydrochloric acid, hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, citrate, acetate, etc. For example, mannitol and glucose can be added as proppant for preparing lyophilized powder for injection. In addition, colorants, preservatives, flavors, flavorings, or other additives may also be added to the pharmaceutical preparation, if desired.

In another aspect, the present invention relates to a kit comprising a pharmaceutical composition of panax notoginseng saponins and acegastrodine, and instructions for the use of panax notoginseng saponins and acegastrodine in combination for the treatment and/or prevention of amyotrophic lateral sclerosis.

The invention has the following beneficial effects:

1. the invention provides application of the combination of panax notoginseng saponins and acegastrodine in preparing ASL medicaments;

2. experiments prove that the combination of the panax notoginseng saponins and the acegastrodine can play a role in protecting brain injury caused by free radicals by promoting cell survival, resisting oxidation and inflammation;

3. the combination of the panax notoginseng saponins and the acegastrodine can play a role in preventing and treating ASL, and has a synergistic effect.

Drawings

FIG. 1 is a schematic diagram showing the effect of the combined use of total saponins of panax notoginseng and acegastrodine containing serum on the activity of NSC34 cells;

FIG. 2 shows NSC34 cells and hSODl of the present invention ^G93A A schematic representation of intracellular SOD1 protein expression;

FIG. 3 shows the combination of total saponins of panax notoginseng and acegastrodine with medicated serum versus hSODl ^G93A And hSODl ^WT Effect of NSC34 cell viability a schematic;

FIG. 4 shows the combination of total saponins of Panax Notoginseng and acegastrodine with drug-containing serum versus hSODl ^G93A And hSODl ^WT Effect of MDA in NSC34 cells.

Detailed Description

The embodiments of the present invention will be described in further detail with reference to the drawings attached hereto.

Example 1 focal cerebral ischemia reperfusion injury test by rat wire-embolus method

1. Test sample

1.1 test article

Xuesaitong soft capsule, 1 granule of 100mg, batch number: 131009, kunmao group, inc.;

acetyl gastrodin tablets, 1 tablet 50mg, batch number: 12DP, kunzi group, inc.

1.2 0.9% sodium chloride injection (saline), batch No.: 2a13100708, source: yaowang pharmaceutical ltd, qingzhou.

2. Laboratory animal

SPF grade SD rat, male, 200-250 g, from Kunming medical university, production permit SCXK (Dian) K2015-0002. Animals are raised in polypropylene boxes (the specification of a cage is 485mm multiplied by 350mm multiplied by 200 mm), each cage does not exceed 5 per sex, an SPF animal house and an experimental animal use license are SYXK (Yunnan) K2014-0001) are well ventilated, the room temperature is controlled to be 20-25 ℃, the relative humidity is controlled to be 40-70%, and the light and shade are alternated after 12 hours of illumination.

3. Other reagents

Red tetrazolium (TTC), national chemical group agents corporation,batch number: 20200315. weighing 1.20g of red tetrazole and 0.286g of Na ₂ HPO ₄ 12H2O and 0.027g K ₂ HPO ₄ Adding double distilled water, and fixing the volume to 100mL and storing in dark.

Chloral hydrate, national drug group chemical reagent limited, lot # 20201026, was prepared at 12% concentration with normal saline at the time of use.

Nylon thread with diameter of 0.26mm, west Deng technology.

4. Contents of the experiment

4.1 animal groups

Healthy male SD rats, randomly and uniformly divided by body weight into 7 groups, which were: pseudo-operation group, model group, group with 50mg/kg of panax notoginseng saponins alone, group with 100mg/kg of panax notoginseng saponins alone, group with 50mg/kg of acegastrodine alone, group with 100mg/kg of acegastrodine alone, and group with 50mg/kg +50mg/kg of panax notoginseng saponins and acegastrodine.

4.2 route and time of administration

The administration is started 5 days before the operation, and the last gastric lavage is performed on the day of the operation when the reperfusion is performed, and the administration volumes are all 0.2mL/100g of body weight.

4..3 Experimental methods

Rats were anesthetized with 12% chloral hydrate by intraperitoneal injection (360 mg/kg) and fixed on the operating table supinely; maintaining the room temperature at about 25 deg.C, cutting the skin of the right neck, separating and ligating the right common carotid artery, external carotid artery and branch arteries; isolating the right Internal Carotid Artery (ICA) until its extracranial branch, the pterygopalatine artery, is visible at the bleb, and ligating the branch at the root; placing an artery clamp at the proximal end of the ICA and the distal end, cutting an incision at the bifurcation of the common carotid artery, inserting a nylon wire with the diameter of 0.26mm by 17-20 mm, leading a embolism wire to enter the ICA and pass through the starting end of the Middle Cerebral Artery (MCA) to the proximal end of the anterior cerebral artery, and blocking all blood flow sources of the MCA; fastening the prepared thread, pulling out the nylon thread after 1.5 hours to allow the blood flow to be communicated, ligating the prepared thread and suturing the skin, simultaneously giving each test sample, and breeding the operation rats in cages; the sham group isolated the vessels only and did not perform intubation.

4.4 evaluation index

Collecting blood from abdominal aorta 24h after operation, centrifuging to separate serum, and freezing at-20 deg.C to detect LDH, MDA, SOD, TNF-alpha and IL-6; cutting off the head and taking the brain, cutting the average coronal section of the brain into 5 slices, placing the slices in TTC solution, incubating at 37 ℃ for 10-15 min for staining, enabling the infarcted area not to be stained, staining the normal brain tissue into red, counting the areas of the whole brain slices and the infarcted area by ImageJ software after photographing, and calculating the infarcted percentage.

4.5 statistical methods

The mean value and standard deviation of each experimental group are calculated, data analysis is carried out by single-factor analysis of variance, and student Newman-Keuls analysis is adopted for comparison among groups.

4.6 statistical results

The statistical results are shown in Table 1.

5 results of the experiment

Rats have obvious behavioral defects and brain tissue necrosis after operation, and indexes such as LDH, MDA and the like are increased compared with a false operation group, which indicates that the model building is successful.

The infarction percentage of the administration group brain tablet is reduced compared with that of a model group, wherein the high dose of the panax notoginseng saponins and the combination group are obviously reduced, and the combination group is better, so that the combination of the panax notoginseng saponins and the acegastrodine has a certain protective effect on brain injury; the administration group can reduce the generation of MDA and LDH and improve the SOD activity, but the combination effect is more obvious, which suggests that the combination of the panax notoginseng saponins and the acegastrodine can enhance the scavenging ability of oxygen free radicals by weakening lipid peroxidation, thereby antagonizing the damage of the oxygen free radicals generated during ischemia and anoxia on brain cells; simultaneously, the administration group can reduce the contents of TNF-alpha and IL-6, the combined effect is more obvious, and the suggestion that the combined use of the panax notoginseng saponins and the acegastrodine participates in the immune response and the inflammatory reaction of the organism so as to resist the inflammatory injury caused by the IL-6 and the TNF-alpha.

Example 2 Effect on cell model of amyotrophic lateral sclerosis

1. Test article

Xuesaitong soft capsule, 1 granule of 100mg, batch number: 131009, kun medicine group, inc.;

acetyl gastrodin tablets, 1 tablet 50mg, batch number: 12DP, kunjin group ltd.

2. Experimental cells and plasmids

NSC34 murine neurons, purchased from Jannie Europe Biotechnology, inc., guangzhou; the cells were cultured in a 10-vol% FBS-containing DMEM medium at 37 ℃ in a 5-vol CO2 incubator; pF141 pAcFPl SOD1 ^WT And pF145 pAcGFPL SOD1 ^G93A Plasmids were constructed and sequenced by prohibitin biotechnology (beijing) limited.

3. Laboratory animal

SPF grade SD rat, male and female is unlimited, 200-220g, comes from Kunming medical university, production permit SCXK (Dian) K2015-0002; animals are raised in polypropylene boxes (with the specification of a cage: 485mm multiplied by 350mm multiplied by 200 mm), each cage does not exceed 5 animals per sex, an SPF-grade animal house and an experimental animal use license SYXK (Yunnan) K2014-0001 are well ventilated, the room temperature is controlled to be 20-25 ℃, the relative humidity is controlled to be 40-70%, and the light and shade are alternated after 12 hours of illumination.

4. Reagent and instrument

DMEM medium, polylysine, DMSO (Gibco, USA); lipofectamine 2000 (Invtrogen, USA); fetal bovine serum (FBS, hycolne, USA); penicillin-streptomycin (Sigma); ammonium persulfate, TEMED, thiazole blue (MTT) (Sigma, USA); acrylamide, N' -methylenebisacrylamide (Amersco, USA); SDS (Biomol, USA); PVDF film

(Millipore, USA); BCA egg self-quantification kit (bi yun day); lipofectamine TM 2000 (Invitrogen corporation); 5 × Loading Buffer (Fermentas, USA); MDA kit (Nanjing institute of bioengineering); the other reagents are imported or domestic analytically pure reagents.

The main apparatus is as follows: a ThermoForma model 3121 carbon dioxide incubator (Thermo, USA); biofuge benchtop refrigerated centrifuge (Thermo, USA)); an XSE type electronic analytical balance (mettler, switzerland); milli-Q Integral 5 ultra pure water system (Millipore, USA); ultra-low temperature refrigerators (Thermo, USA); synergy 2 microplate reader (BioTek, USA); V-GES electrophoresis apparatus (Wealtec Crop); STS-3 type decolorizing shaker (Shanghai Qitt Analyzer Co., ltd.); chemiluminescence imaging analysis System (UVP) model ChemiDoc-It Imager.

4. Test method

4.1 cell culture: frozen NSC34 cells were removed from liquid nitrogen, cultured in DMEM medium supplemented with 10% FBS serum, replaced every 2 days, and cultured in a 37 ℃ incubator containing 5% CO2.

4.2 cell transfection: one day prior to transfection NSC34 cells were trypsinized with trypsin containing 0.25% EDTA and plated in 6-well plates, the cultured cells were grown to 90% confluency, and hSODl was transfected using Lipofectamine 2000 as per the instructions ^WT And hSODl ^G93A Mu.g of each plasmid was transfected for 6 hours, the medium was changed to the normal medium, and the cells were further cultured and selected with G418 400 mg. Multidot.L-1.

4.3 preparation of serum containing medicine: SPF healthy adult SD rats, 12, were randomly divided into 2 groups, i.e., a blank group and a dosed group, each of which had 6 rats. Fasting is not forbidden for 12h before administration, feeding equal amount of physiological saline into the blank group, and feeding Panax notoginsenosides 50mg/kg and acegastrodine 50mg/kg into the administration group at an interval of 1h; the administration is performed 2 times a day at 6h intervals for 5 days. Collecting blood from abdominal aorta 1h after last administration, standing at room temperature for 4h, centrifuging at 3000r/min for 15min, separating serum, inactivating in 56 deg.C water bath for 30min, filtering with 0.22 μm microporous membrane for sterilization, mixing, subpackaging in 2mL EP tube, and storing at-20 deg.C for use.

4.4 treatment of drugs in pre: adding 100 mu L of 5X 104 mL-1 cell suspension into a coated 96-well plate, or adding 2mL of 2X 105. ML-1 cell suspension into a coated 6-well plate, culturing for 48h, and then performing drug intervention treatment; preparing culture solution containing 5%, 10% and 20% drug serum with DMEM medium in advance, and preparing culture solution containing 10% blank serum by the same method; adding 100 mu L of 96-well plate or 2mL of DMEM complete culture medium containing the medicines (experimental group) or not containing the medicines (control group) into 6-well plate, placing the DMEM complete culture medium in a cell incubator for culture, and observing the cell morphology and the cell viability after 48 hours.

4.5 MTT detection: after 48h of drug intervention, 10 mu L of MTT (50 mg. ML-1) is added into each well of a 96-well plate, after 4h, triple liquid is added for overnight incubation at 37 ℃, an OD value under the condition of 570nm is measured by an enzyme-labeling instrument, and the activity of cells is analyzed.

4.6 Western blot: sucking the cell to be treated out of the supernatant culture medium, washing twice with PBS, sucking, adding human cell lysate, ultrasonic crushing on ice, centrifuging at 40 deg.C at 12000 r.min-1 for 30min, and collecting the supernatant. Quantifying by using a BCA protein quantification kit according to an operation instruction, adjusting each group to have consistent protein concentration, adding 5 XLoadingBuffer with the volume of 1/4 of that of human, uniformly mixing, boiling for 5min to denature the protein, and storing at-70 ℃. Preparing 10% separation gel by adopting an egg self SDS-PAGE protein blotting method, loading the separation gel in an equal amount, carrying out electrophoresis, transferring a membrane, sealing, adding an antibody, developing by adopting ECL (electron cyclotron resonance imaging), and observing the expression of SOD1 protein in cells.

4.7 And (3) detecting an MDA kit: taking out a 6-well plate for cell culture, washing twice by PBS at 4 ℃, adding 1mL PBS, scraping the cells by a cell scraper, transferring the cells into a 1.5mL Ep tube, centrifuging for 5min at 1000 r.min < -1 >, carefully sucking out the supernatant, adding 200 mu L cell lysate, homogenizing in an ice-water bath, centrifuging for 10min at 2500 r.min < -1 >, and taking the supernatant to detect according to the specification of an MDA kit.

4.8 statistical processing methods the experimental data were analysed using Graphpad prism7.0 software and the data were recorded "

"indicates that a one-way analysis of variance (ANOVA) and Newman-keuls post-hoc were used for inter-group significance testing.

5 results of the test

5.1 Effect on NSC34 cells

In order to investigate whether the combined use of the total saponins of panax notoginseng and the acegastrodine containing the drug serum has the effects of promoting survival and cytotoxicity on normally cultured NSC34 cells, the NSC34 cells are cultured for 48 hours by 1 drug-containing serum through experiments, and the cell morphology and the activity change of the NSC34 cells are detected by adopting microscope observation and MTT (methyl thiazolyl tetrazolium); there was no significant difference in cell morphology and viability between the administered and control groups compared to normal cultured NSC34 cells (P > 0.05), see fig. 1; the result shows that in the NSC34 cells cultured normally, the combination of the panax notoginseng saponins and the acegastrodine has no obvious survival promotion or cytotoxicity effect.

5.2 establishment of ALS cell model

This experiment was carried out by transferring hSODl into NSC34 cells ^G93A And hSODl ^WT Plasmid to establish ALS experimental and control cell models. The results show that hSODl can be used in transfection ^G93A And hSODl ^WT After plasmid, normally cultured NSC34 and hSODl were examined ^G93A Expression level of SOD1 protein in NSC34 cells to further verify hSODl ^G93A The expression level of SOD1 protein in NSC34 cells is shown in FIG. 2; the results show that hSODl ^G93A SOD1 level in NSC34 cells is obviously increased, and ALS model is successfully established.

5.3 pairs of hSODl ^G93A NSC34 cell has protective effect

To further explore the effect of the combined use of total saponins of panax notoginseng and acegastrodine and the serum containing drug on ALS cell model, the hSODl ^G93A And hSODl ^WT The NSC34 cells are subjected to intervention treatment by adopting panax notoginseng saponins and acegastrodine with different concentrations and drug-containing serum, and the cell morphology and MTT activity change are observed. After 5%, 10% and 20% of the combined drug-containing serum is administered in the transfected NSC34 cells and cultured for 48 hours, the cell morphology is not obviously changed, but the cell number of the combination drug-containing serum stem prognosis of 10%, 20% of the panax notoginseng saponins and the acegastrodin is increased compared with the cell number of the non-administered group (P)<0.05 See fig. 3).

5.4 Quzhazhigan reduces MDA levels

Since the action mechanism of the combination of the panax notoginseng saponins and the acegastrodine on ALS is not clear, the experiment determines that NSC34 cells and hSODl are normally cultured ^G93A Changes in the levels of lipid peroxide Malondialdehyde (MDA) in each group of NSC34 cells; the results of the experiments found that hSODl was comparable to that of normally cultured NSC34 cells ^G93A The MDA content in NSC34 cell is obviously increased (P)<0.001 And after 20 percent of panax notoginseng saponins and acegastrodine are administered to the patient together with the drug-containing serum, the MDA content is obviously reduced (P)<0.01 And exhibit some dose dependence, see figure 4.

In conclusion, the combination of the panax notoginseng saponins and the acegastrodin can be used for hSODl ^G93A NSC34 cells are protectedThe effect is that the hSODl is obviously increased by combining high-dose drug-containing serum ^G93A NSC34 cell activity, has the function of obviously promoting cell survival and reducing MDA, and the experiment of an acute ischemia reperfusion model in a rat body shows that the combination of the panax notoginseng saponins and the acegastrodine has the protective effect on free radical injury and inflammatory injury. Therefore, the combination of the panax notoginseng saponins and the acegastrodine plays roles in preventing and treating ASL, and has good clinical application value.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (8)

1. The combined application of the panax notoginseng saponins and the acegastrodine in preparing the medicine for treating and/or preventing amyotrophic lateral sclerosis.

2. Use according to claim 1, characterized in that the level of MDA in a cell model of amyotrophic lateral sclerosis is reduced.

3. Use according to claim 1, characterized in that the nerve cell viability is increased in amyotrophic lateral sclerosis.

4. Use according to claim 1, characterized in that it inhibits neuroexcitotoxicity in amyotrophic lateral sclerosis, inhibits apoptosis of nerve cells and/or promotes proliferation of nerve cells.

5. The use according to claim 1, characterized by promoting cell survival in amyotrophic lateral sclerosis, antioxidant, anti-inflammatory, protective effect against free-radical induced brain damage.

6. The use according to claim 1, characterized in that the mass ratio of the panax notoginseng saponins to the acegastrodine is preferably 1.

7. A pharmaceutical composition of panax notoginseng saponins and acegastrodine for treating and/or preventing amyotrophic lateral sclerosis comprises panax notoginseng saponins, acegastrodine and pharmaceutical auxiliary materials.

8. A kit, which comprises a pharmaceutical composition of panax notoginseng saponins and acegastrodine, and a medical instruction book of combined use of panax notoginseng saponins and acegastrodine for treating and/or preventing amyotrophic lateral sclerosis.

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