CN107126445A - Caprophyl is implanted in the application in terms for the treatment of Parkinson's - Google Patents

Abstract

The invention discloses the application of fecal bacteria transplantation in treating Parkinson's disease, and belongs to the technical field of medical neuropharmacology. The present invention relates to the potential application of fecal flora in the clinical treatment of Parkinson's disease. The present invention establishes an animal model of Parkinson's disease, comprehensively uses various research methods such as behavior, immunology, and biochemical indicators, and experiments prove that this type of drug can improve the symptoms of dyskinesia in Parkinson's disease model mice; reduce dopamine in the substantia nigra compacta neuronal degeneration. Fecal flora raw materials are easy to source and have good effects, so they have certain development value and prospects in clinical application.

Description

Application of fecal microbiota transplantation in the treatment of Parkinson's disease

technical field

The invention relates to the application of fecal bacteria transplantation in treating Parkinson's disease, and belongs to the technical field of medical neuropharmacology.

Background technique

Parkinson's disease (PD) is the most common degenerative disease of the motor nervous system, the main clinical manifestations are: resting tremor, slowness of movement, unsteady standing and postural reflex disorder. The main lesions in the brain of patients with Parkinson's disease are the substantia nigra and striatum. The death and reduction of dopaminergic neurons in the substantia nigra compacta is the direct cause of the decrease in dopamine projected to the striatum. As the second largest neurodegenerative disease after Alzheimer's disease, the incidence and prevalence of Parkinson's disease increase sharply with age, and the prevalence rate reaches 1% among the elderly over 60 years old. People over the age of 85 account for 5%-6%. At present, the number of Parkinson's disease patients in my country exceeds 2 million. The pathogenesis of PD is related to aging, environmental and genetic factors, but the pathogenesis of PD is still unclear. Studies have shown that the interaction of pathological mechanisms such as oxidative stress injury, autophagy, immune inflammation, endoplasmic reticulum stress, and apoptosis may affect the occurrence and development of PD. Recent studies have shown that the imbalance of intestinal flora may be related to the occurrence and clinical phenotype of PD. At present, my country has gradually entered an aging society. With the increase of the elderly population, the number of PD patients will also increase rapidly. Due to the long course of the disease, high disability rate and lack of effective cure, the economic burden on patients, families and society is extremely heavy. Therefore, it is urgent to find effective treatments and drugs for PD.

The current clinical treatments for Parkinson's disease mainly include drug intervention, neuron nucleus destruction, deep brain stimulation, or nerve tissue and stem cell transplantation, as well as cell therapy and gene therapy, which are still in the experimental stage. Among them, drug treatment is mostly symptomatic treatment, which can temporarily relieve symptoms, but cannot inhibit or delay the progress of the disease, while surgical treatment has certain indications, and the cost is relatively high. Different from drug therapy, fecal microbiota transplantation (Fecal Microbiota Transplantation, FMT), as a special organ transplantation and therapy that can rebuild intestinal flora, has attracted clinical attention. Currently, it is mainly used in recurrent Clostridium difficile infection, inflammatory bowel disease, Irritable bowel syndrome and other intestinal diseases, as well as metabolic diseases and autism, have the advantages of high cure rate and few adverse reactions. There are very few reports on the treatment of PD, and its great potential remains to be discovered.

FMT is to transplant the functional flora in the feces of healthy people into the gastrointestinal tract of patients to rebuild the intestinal flora with normal functions, so as to treat intestinal and extraintestinal diseases. During the Eastern Jin Dynasty, "Behind the Elbow" edited by Ge Hong contained records about FMT. The "Compendium of Materia Medica" compiled by Li Shizhen during the Ming Dynasty also included human feces (Huanglong Decoction) to treat severe diarrhea and food poisoning. At present, FMT has begun to be used in clinical Clostridium difficile infection, inflammatory bowel disease, irritable bowel syndrome and other intestinal diseases. FMT may also be used to treat intestinal immune deficiency and allergy, obesity, non-alcoholic fatty liver, Disorders such as autism in children.

Contents of the invention

In order to overcome the deficiencies of the existing clinical treatment methods and medicines for Parkinson's disease, the primary purpose of the present invention is to provide a treatment method and medicine with a potential treatment effect on Parkinson's disease.

The present invention establishes a C57BL/6J mouse model of Parkinson's disease, observes the movement disorder symptoms of Parkinson's disease mice by fecal bacteria transplantation, and demonstrates through experiments that FMT can improve the slow movement symptoms of Parkinson's disease mice and improve the substantia nigra pars compacta neurons, thus proving that FMT has a neuroprotective effect on Parkinson's disease.

The first object of the present invention is to provide a drug or pharmaceutical composition for alleviating and/or assisting in the treatment and/or treatment of Parkinson's disease. The drug or pharmaceutical composition uses fecal flora as the active ingredient or the main active ingredient.

In one embodiment of the present invention, the drug or pharmaceutical composition further includes a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier that can be used in the pharmaceutical field.

In one embodiment of the present invention, the medicament or pharmaceutical composition contains more than 99% by mass of fecal flora and less than 1% by mass of pharmaceutically acceptable carriers or excipients.

In one embodiment of the present invention, the fecal flora of the present invention can be used in combination with other active ingredients, as long as they do not produce other adverse effects, such as allergic reactions.

In one embodiment of the present invention, the therapeutic substance of the present invention can be formulated into several dosage forms, which contain some excipients commonly used in the field of pharmacy; for example, oral preparations (such as tablets, capsules, solutions or suspensions) liquid).

In one embodiment of the present invention, the carrier used in the pharmaceutical composition of the present invention is a common type available in the field of pharmacy, including: binders for oral preparations, lubricants, disintegrants, solubilizers, diluents Agents, stabilizers, suspending agents, no coloring agents, flavoring agents, etc. Pharmaceutical preparations can be administered orally or parenterally (eg, enteral or parenteral nutrition), and if certain drugs are unstable under gastric conditions, they can be formulated as enteric-coated tablets.

The second object of the present invention is to provide a drug for improving exercise capacity, which uses fecal flora as the active ingredient or the main active ingredient.

The third object of the present invention is to provide a drug for increasing the expression of tyrosine hydroxylase in the striatum, and the drug uses fecal flora as the active ingredient or the main active ingredient.

The fourth object of the present invention is to provide a health food, drink, enteral nutrition preparation or dietary supplement for alleviating and/or assisting treatment and/or treatment of Parkinson's disease, the health food, drink, enteral nutrition preparation Or a dietary supplement with fecal flora as the main active ingredient.

The fifth object of the present invention is to provide a new application of fecal bacteria transplantation. The application is to prepare medicines, health food, drinks, enteral nutrition preparations, dietary supplements, veterinary drugs or feed additives for alleviating and/or assisting and/or treating Parkinson's disease.

In the present invention, the fecal flora refers to the fecal flora of normal animals or healthy people without Parkinson's disease.

In the present invention, the fecal flora refers to the flora in the feces of healthy people.

In the present invention, the fecal flora is obtained by taking the feces of normal animals, diluting, shaking, and filtering the bacterial liquid.

Beneficial effects of the present invention:

The present invention establishes a mouse model of Parkinson's disease, observes the movement disorder symptoms of Parkinson's disease mice by fecal bacteria transplantation, and demonstrates through experiments that fecal bacteria transplantation can improve the slow movement symptoms of Parkinson's disease mice and improve the nerve function of the substantia nigra compacta. Yuan, thus proving that fecal microbiota transplantation has a neuroprotective effect on Parkinson's disease. The invention uses fecal bacteria transplantation to significantly improve the dopamine content in the striatum, and the exercise ability of mice, tyrosine hydroxylase-positive neuron cells in the substantia nigra compacta, etc. have been significantly improved or improved.

Description of drawings

Figure 1: The effect of fecal bacteria transplantation on the body weight change of MPTP-induced Parkinson's model mice;

Figure 2: Experimental results of the effect of fecal bacteria transplantation on the motor behavior of MPTP-induced Parkinson's model mice; among them, Figure 2A is the result of climbing pole experiment; Figure 2B is the result of suspension experiment;

Figure 3: Effect of fecal bacteria transplantation on fecal short-chain fatty acid content of MPTP-induced Parkinson's model mice; among them, Figure 3A is the content of acetic acid, Figure 3B is the content of propionic acid, Figure 3C is the content of n-butyric acid, and Figure 3D is the content of n-pentanoic acid acid content;

Figure 4: Effect of fecal bacteria transplantation on intestinal inflammation in MPTP-induced Parkinson's model mice; wherein, Figure 4A is the content of TNFα mRNA, Figure 4B is the content of TRAF2 mRNA, Figure 4C is the content of TLR4 mRNA, and Figure 4D is the content of NFκB mRNA;

Figure 5: Neuroprotective effect of fecal bacteria transplantation on MPTP-induced PD mouse model;

Figure 6: Differences in fecal flora composition between blank group mice (C) and PD mouse model (M).

specific implementation plan

The method of the present invention will be further described below through specific implementation, but the embodiments of the present invention are not limited thereto.

Example 1: Establishment and grouping of animal models

Male C57BL/6J mice weighing 20-25g, 8 weeks old, were randomly divided into control group, MPTP model group (glycerol-PBS mixture was given after MPTP), FMT bacteria group (MPTP was given first, and normal mice were given feces bacteria liquid), 20 rats in each group. Placed at 23±2°C, the lighting time is 12h/day, and the animals are given drugs after one week of adaptation to the environment. The MPTP model group was intraperitoneally injected with MPTP (30 mg/kg) once a day for 5 consecutive days, and then intragastrically administered a mixture of glycerol and PBS once a day for 7 consecutive days. The administration group was intraperitoneally injected with MPTP (30 mg/kg) once a day for 5 consecutive days, and then intragastrically administered the fecal bacteria liquid of normal mice once a day for 7 consecutive days. The control group was given intraperitoneal injection of normal saline for 12 consecutive days. During the experiment, the weight changes of the mice in each group were recorded and the feces of the mice in each group were collected. Then the behavior of the mice was evaluated by climbing pole experiments, and then the content of fecal short-chain fatty acids was detected by GC-MS, and the expression of intestinal inflammatory factors was detected by qPCR. The neuroprotective effect of fecal microbiota transplantation on Parkinson's disease mice was evaluated by the above method.

1. Weight monitoring

The mice in each group were weighed and recorded before the drug administration operation, and the weight was continuously weighed until the end of the drug administration.

The results of body weight monitoring are shown in Figure 1. After the first day of administration of MPTP, the mice in the model group and the mice in the bacteria administration group all experienced weight loss, and the body weight increased slowly after adaptation. After the treatment with bacteria, the body weight of the mice in the bacteria-administered group was higher than that in the model group. Body weight monitoring confirmed that FMT can improve the body weight loss of Parkinson's mice.

2. Behavioral evaluation

The mice in each group were trained three days before the administration of the behavioral test, once a day, and the behavioral test was performed on the first day after the end of the administration.

Pole test (pole test): self-made pole climbing frame: choose a metal tube with a length of 55 cm and a diameter of 1 cm, which is tightly wrapped with gauze to prevent slipping, and the upper end is covered with a spherical protrusion with a diameter of 2 cm (as an attachment point for mice during the experiment). It is fixed to a metal base, placed in a mouse cage, and the base is covered with a dressing. During the test, the mouse was placed head-up on the spherical protrusion, and the time from placing the rod to climbing to the bottom of the rod and landing on the hind limbs was recorded, and the average value was measured 3 times, with an interval of more than 10 minutes between each time. If the mouse pauses midway or climbs backwards, measure again. The experimental animals were trained for 3 days in advance.

The exercise ability of the mice was detected by the rod climbing test, as shown in Figure 2A. The test results showed that the average time for the mice in the blank group, the model group, and the bacteria-treated group to climb the pole were 3.635s, 5.153s, and 4.325s, respectively. Compared with the mice in the blank group, the time required for the mice in the model group to climb the pole increased, and the speed of movement slowed down. The results confirmed that the mice injected with MPTP had reduced mobility. The time required for the mice in the bacteria-administered group was reduced and the speed was faster than that in the model group, and there was a significant difference compared with the model group (P<0.01). The results confirmed that FMT can improve the exercise capacity of Parkinson's disease mice.

Suspension test (traction test): A metal wire with a diameter of 1 mm is stretched horizontally between two metal uprights and 30 cm above the ground. During the experiment, let the mice hang on a horizontal metal wire with their two front paws and stay for 10 seconds, and take the average value for 3 measurements with an interval of more than 10 minutes each time. Scoring criteria for mouse hanging ability: within the test time, if the mouse can grasp the metal wire with two hind paws, it will be counted as 3 points; if the mouse can grasp the metal wire with only one hind paw, it will be counted as 2 points; 1 point for the metal wire, and 0 points for the mouse to fall. At the same time, before each experiment, observe whether each mouse has abnormal behaviors such as tremor, erect hair, raised front legs, vertical tail, slow movement, and reduction. The experimental animals were trained for 3 days in advance.

The exercise ability of the mice was detected by the suspension test, as shown in Figure 2B. The test results showed that the average scores of the suspension test of the mice in the blank group, the model group, and the bacteria-administered group were 2.788s, 2.091s, and 2.667s, respectively. Compared with the mice in the blank group, the mice in the model group had lower suspension scores and decreased motor flexibility. The results confirmed that the mice injected with MPTP had reduced mobility. Compared with the mice in the model group, the mice in the bacteria group had higher suspension scores and higher flexibility, and there was a significant difference compared with the model group (P<0.01). The results of the bacteria administration confirmed that FMT can improve the exercise capacity of Parkinson's disease mice.

3. GC-MS detection of short-chain fatty acid content in feces

(1) Sample processing

The fecal tissues of 8 mice in each group were collected in 1.5mL EP tubes, and the obtained samples were immediately transferred to -80°C for storage. Add 10 μL ddH ₂ O (10%, w/v) per 1 mg of feces during measurement, prepare a homogenate with a suspension apparatus, centrifuge at 13,000 r/min at 4°C for 5 min, filter with a 0.2 μm membrane filter, and store at 4°C for later use. The content of short-chain fatty acids in mouse feces was detected by GC-MS.

(2) GC-MS detection

(a) Chromatographic conditions: Japan Shimadzu Rtx-WAX (0.25mm×30.0m, 0.25μm); split injection; carrier gas, ultra-pure helium; column oven temperature: 100°C; inlet temperature: 240°C; The heating program is shown in the table below; split injection, total flow rate: 12.7mL/min, split ratio: 10.0:1; total program time: 9.67 min.

(b) Mass spectrometry conditions: ion source (EI source) temperature: 220°C; interface temperature: 250°C; solvent delay: 2.5min; selected ion scan: 43m/z, 74m/z; specific conditions are shown in Table 1.

Table 1

rate final temperature hold time 7.50 140.0 0.00 60.00 230.0 3.00

(c) Preparation of standard curve

Make a stock solution with a concentration of 1 mg/mL of the short-chain fatty acid product with ultrapure water, and then dilute it with ultrapure water to obtain 5, 10, 20, 40, 60, 80, and 100 μg/mL reference solution, take 10 μL and inject The chromatograph is analyzed, obtains the reference substance solution chromatogram. Take the concentration of each standard solution as the abscissa (X), and the peak area of each component as the ordinate (Y) to perform linear regression to obtain the standard curve equation.

(3) Experimental results

As shown in Figure 3, compared with the normal mice in the blank group, the content of short-chain fatty acids (acetic acid, propionic acid, n-butyric acid, n-valeric acid) in the feces of the Parkinson's disease mice in the model group increased significantly, and there was a significant difference (P <0.05, of which acetic acid P<0.01); the content of short-chain fatty acids (acetic acid, propionic acid, n-butyric acid, n-valeric acid) in the feces of mice in the bacteria-administered group was significantly lower than that of the model group (acetic acid P<0.001, propionic acid P <0.01, n-butyric acid P<0.05, n-valeric acid P<0.01), the results proved that the intestinal flora of mice in the model group was disordered, resulting in abnormal intestinal short-chain fatty acid metabolism, FMT can improve the imbalance of intestinal flora And regulate short-chain fatty acid metabolism.

4. qRT-PCR detection of changes in the level of colonic inflammation

(1) Sample processing

Take 8 mice in each group, use isoflurane inhalation anesthesia, cut the right atrial appendage, and perfuse normal saline with a needle from the left ventricle. After finding the anatomical position of the cecum, take 2 cm of colon tissue in 1.5 mL of EP from 1 to 2 cm below it. All operations were carried out on an ice bath, and the obtained samples were immediately transferred to -80°C for storage.

(2) Extraction of total RNA

(a) Weigh 10 mg of colon tissue into a 1.5 mL EP tube with a precision balance, add 500 μL of Buffer LY first, then add 10 μL of β-mercaptoethanol, and break the tissue with an ultrasonic breaker to completely lyse the tissue.

(b) Transfer the lysed tissue fluid into a DNA column (placed in a 2 mL collection tube), centrifuge at 13000 rpm for 2 min, discard the DNA column, and keep the liquid in the collection tube.

(c) Add 500 μL of ethanol to the collection tube and mix well.

(d) Transfer the liquid in the collection tube to the RNA column (placed in a 2 mL collection tube), centrifuge at 13000 rpm for 1 min, discard the collection tube, and put the RNA column into a new collection tube.

(e) Add 500 μL of Buffer RB to the RNA column, centrifuge at 13,000 rpm for 30 s, discard the liquid in the collection tube, and put the RNA column back into the collection tube.

(f) Add 500 μL of RNAWash Buffer to the RNA column, centrifuge at 13000 rpm for 1 min, discard the liquid in the collection tube, and put the RNA column back into the collection tube.

(g) Add 500 μL of RNAWash Buffer to the RNA column, centrifuge at 13000 rpm for 30 s, discard the collection tube, and put the RNA column into a new collection tube.

(h) Centrifuge at 13000 rpm for 2 min, open the cap of the centrifuge tube, and place it at room temperature for 10 min to ensure that the ethanol is completely evaporated.

(i) Transfer the RNA column to an RNase-free centrifuge tube (1.5 mL), add 30 μL of DEPC water, centrifuge at 13,000 rpm for 1 min, collect the RNA solution, and store it at -80°C for later use.

(3) Reverse RNA to cDNA

Reaction system (10 μL): 5×PrimeScript Buffer 2 μL, Total RNA 8 μL;

Reaction conditions: 15 minutes at 37°C, 5 seconds at 85°C, hold at 4°C.

(4) Fluorescent quantitative PCR reaction

Reaction system (10 μL): 5 μL of 2×SYBR Premix Ex TaqⅡ, 0.5 μL of upstream primer, 0.5 μL of downstream primer, 1 μL of cDNA template, 3 μL of sterile water;

Reaction conditions: 95°C for 30 seconds; 95°C for 5 seconds, 55°C for 30 seconds, 72°C for 30 seconds, 40 cycles, record the ct value stage; add the dissolution curve.

(5) Experimental results

The result is shown in Figure 4. Compared with the normal mice in the blank group, the inflammatory factors in the colon of Parkinson's disease mice without administration (TNFα, TRAF2, TLR4, NFκB, *p<0.05, **p<0.01, ***p<0.001) The expression was significantly increased, and the inflammatory factors (TNFα, TRAF2, TLR4, NFκB, *p<0.05, **p<0.01, ***p<0.001) in the colon of mice in the bacteria-administered group were significantly lower than those in the model group. The result proves that the colon of the mice in the model group is inflamed, and the fecal bacteria transplantation in the present invention helps to reduce the inflammation in the colon of the Parkinson's disease mice.

5. Neuroprotective effect of FMT on MPTP-induced PD mouse model

TH (a marker of dopaminergic neurons, green fluorescence) and Iba-1 immunofluorescent staining (a marker of activated microglia, red fluorescence) were performed on the substantia nigra of mice. The results of TH staining (Figure 5) showed that the number of TH-positive cells in the model group was less than that in the blank group, while the number of TH-positive cells in the bacterial treatment group was more than that in the model group, indicating that FMT can significantly improve the degeneration of dopaminergic neurons in the brain of PD mice induced by MPTP The phenomenon of death. The results of Iba-1 staining showed that the Iba-1 positive cells in the model group were significantly more than those in the blank group, while the Iba-1 positive cells in the bacterial treatment group were less than those in the model group, indicating that FMT can inhibit the microglia in the brain of PD mice induced by MPTP The phenomenon of cell activation has inhibitory effect on neuroinflammation (N=3~4).

6. Differences in the composition of fecal flora

As shown in Figure 6, square dots represent blank group mice, and circle dots represent PD mice. PC1 is the first principal coordinate, and PC3 is the ordinate. The distribution of the 10 samples included in the present invention in the PC1-PC3 coordinate system shows that the two groups of samples of blank mice and PD mice have an obvious tendency to separate, and the two groups of samples cluster significantly, indicating that the composition of the intestinal flora between the two groups has a significant difference. There are significant differences and similarities among the samples in the same group (N=5).

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. A medicine or pharmaceutical composition for relieving and/or adjuvant treatment and/or treating Parkinson's disease, characterized in that, the medicine or pharmaceutical composition uses fecal flora as an active ingredient or a main active ingredient. 2. The medicine or pharmaceutical composition according to claim 1, characterized in that, the medicine or pharmaceutical composition further comprises a pharmaceutically acceptable excipient. 3. The medicine or pharmaceutical composition according to claim 2, characterized in that, the pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier that can be used in the field of pharmacy. 4. The medicament or pharmaceutical composition according to claim 1, characterized in that, the medicament or pharmaceutical composition contains a mass percentage greater than 99% of fecal flora and a pharmaceutically acceptable carrier with a mass percentage of less than 1% or excipients. 5. The medicine or pharmaceutical composition according to claim 1, characterized in that, the medicine or pharmaceutical composition is medicine in various dosage forms. 6. The medicine or pharmaceutical composition according to claim 1, characterized in that, the fecal flora refers to the fecal flora of a normal animal without Parkinson's disease or a healthy person. 7. A drug for improving exercise capacity, characterized in that the drug uses fecal flora as an active ingredient or a main active ingredient. 8. A drug or pharmaceutical composition for increasing the expression of tyrosine hydroxylase in the striatum, characterized in that the drug or pharmaceutical composition uses fecal flora as the active ingredient or the main active ingredient. 9. A health food, drink, enteral nutrition preparation or dietary supplement for alleviating and/or adjuvant treatment and/or treatment of Parkinson's disease, characterized in that, the health food, drink, enteral nutrition preparation or dietary supplement The agent uses fecal flora as the main active ingredient. 10. The application of fecal bacteria transplantation, characterized in that the application is the preparation of medicaments, enteral nutrition preparations, dietary supplements, veterinary drugs or feed additives for alleviating and/or adjuvant treatment and/or treatment of Parkinson's disease.