CN112933117B - Medicine for preventing and/or treating Parkinson's disease and application thereof - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to a medicament for preventing and/or treating Parkinson's disease and application thereof. The invention provides a medicament for preventing and/or treating Parkinson's disease, which comprises inactivated lactobacillus murinus and pharmaceutically acceptable auxiliary materials. The inactivated lactobacillus murinus CICC23140 provided by the invention can protect dopaminergic neurons, improve the motor function and prevent and treat the Parkinson disease, and has a good effect.

Description

A kind of medicine and application thereof for preventing and/or treating Parkinson's disease

technical field

The invention relates to the technical field of microorganisms, in particular to a drug for preventing and/or treating Parkinson's disease and its application.

Background technique

Parkinson's disease (PD) is a neurodegenerative disease, and its typical clinicopathological features are degeneration and loss of dopaminergic neurons in the substantia nigra compacta of the midbrain and α-synuclein (α- Synuclein) abnormal accumulation of Lewy bodies in neurons, which usually leads to involuntary motor symptoms such as slowness of movement, muscle rigidity, resting tremor and abnormal posture and gait, accompanied by some non-motor symptoms such as stomach Bowel dysfunction, hyposmia, depressive symptoms, rapid eye sleep behavior disorder, etc. At present, the mechanism leading to the pathogenesis of PD is still unclear and there is a lack of safe and effective treatment methods, but it is of great clinical significance to find effective prevention and treatment options to relieve the pain of PD patients.

Gastrointestinal dysfunction is one of the non-motor symptoms with a high incidence rate, which can occur many years earlier than motor symptoms. At the same time, pathological studies have found that misfolding and aggregation of α-synuclein in the enteric nervous system can occur early Because of the pathogenesis of PD, this shows that PD may start in the gut. The microorganisms colonizing the human intestine are in a dynamic balance under normal conditions, and changes in the structure of the intestinal flora are closely related to various diseases. Probiotics have been extensively studied for their beneficial effects in regulating intestinal flora. In 1994, the definition of probiotics at an international conference held in Germany pointed out that probiotics are bacterial products containing live or dead bacteria, including their composition and products, which are administered orally or through other mucosal routes, with the purpose of improving mucosal surface microorganisms or bacteria. Enzyme balance, either stimulating specific or nonspecific immune mechanisms.

As a kind of probiotics, inactivated probiotics have similar physiological functions to live probiotics, and at the same time have higher safety and stability than live probiotics, so they have attracted extensive attention and research. After probiotic inactivation, dead cells can release bacterial components with key immunomodulatory and antipathogenic properties, such as peptidoglycan, polysaccharides, and teichoic acid in probiotic cell wall components, and related metabolites. These components can still function after the bacteria are inactivated, and there is a large amount of published evidence that preparations containing dead cells and their metabolites can also produce relevant biological responses to restore normal intestinal homeostasis, in many cases Similar to living cells, although there are potential differences.

Lactobacillus murinus is a type of lactic acid bacteria isolated from the intestinal tract of rats and mice. It has been reported to reduce the onset of sepsis in young mice, reduce intestinal permeability and systemic inflammatory markers in aged mice colonized with microorganisms, and improve offspring in offspring induced by antibiotic-based models of maternal dysbiosis. Neurobehavioral and microglial dysfunction. However, studies on the effects of probiotic strains on Parkinson's disease have not been reported.

Contents of the invention

In view of this, the object of the present invention is to provide a drug for preventing and/or treating Parkinson's disease and its application. The inactivated murine lactobacillus provided by the invention can protect dopaminergic neurons, improve motor function, prevent and treat Parkinson's disease, and has good effect.

The invention provides a medicine for preventing and/or treating Parkinson's disease, which comprises inactivated Lactobacillus murine and pharmaceutically acceptable auxiliary materials.

Preferably, the auxiliary material includes one or more of starch, maltodextrin, trehalose and lactose.

Preferably, the Lactobacillus murinus is Lactobacillus murinus, which is preserved in the China Industrial Microorganism Culture Collection Management Center with a preservation number of CICC23140.

Preferably, the dosage form of the drug includes suspension, suppository, tablet, capsule, soft capsule, prefilled injection, effervescent tablet and gel.

Preferably, the preparation method of described inactivated Lactobacillus murine comprises the following steps:

The Lactobacillus murine is sterilized to obtain an inactivated Lactobacillus murine.

Preferably, the concentration of inactivated Lactobacillus murine in the drug is 10 ⁶ -10 ¹² cfu/ml.

The invention also provides the application of the inactivated Lactobacillus murine in the preparation of medicaments for preventing and/or treating Parkinson's disease.

The invention also provides the application of the inactivated Lactobacillus murine in the preparation of the medicine for improving movement disorders.

The invention also provides the application of the inactivated type of Lactobacillus murine in the preparation of the medicine for preventing dopaminergic neuron damage.

The present invention also provides the application of the inactivated Lactobacillus murine in the preparation of the drug for rescuing the loss of the dopaminergic neuron marker tyrosine hydroxylase protein in the substantia nigra of the brain.

The invention provides a medicine for preventing and/or treating Parkinson's disease and its application. The inactivated Lactobacillus murine provided by the invention can protect dopaminergic neurons, improve motor function, prevent and/or treat Parkinson's disease, has good effect, no toxic and side effects, high safety and good stability; the source of raw materials is wide and easy to Acquisition and inactivation of probiotics is relatively mature, the preparation of the drug is simple and easy, and the cost is highly controllable. The manufacturer can use the existing equipment and technology for production and preparation. The test results show that the inactivated Lactobacillus murine of the present invention has a good improvement effect on the movement disorder induced by 6-OHDA; it can rescue the brain dopaminergic neuron marker tyrosine hydroxylase induced by 6-OHDA (tyrosine hydroxylase, TH) protein loss; and has preventive and protective effects on dopaminergic neuron damage caused by 6-OHDA.

Description of drawings

Fig. 1 is a histogram of rats' exercise time on a stick;

Fig. 2 is a histogram of the distance of rat spontaneous exploration activities;

Figure 3 is a histogram of the proportion of rats' impaired contralateral forelimb movement;

Fig. 4 is the Western blot protein expression band of rat midbrain substantia nigra tyrosine hydroxylase (TH);

Fig. 5 is a histogram of rat midbrain tyrosine hydroxylase (TH) protein expression;

Fig. 6 is the immunohistochemical picture of rat midbrain substantia nigra dopaminergic neurons;

Figure 7 is a histogram of the number of positive cells of dopaminergic neurons in the substantia nigra of the rat midbrain.

Detailed ways

The invention provides a medicine for preventing and/or treating Parkinson's disease, which comprises inactivated Lactobacillus murine and pharmaceutically acceptable auxiliary materials.

In the present invention, the Lactobacillus murinus is preferably Lactobacillus murinus, which is preserved in the China Industrial Microorganism Culture Collection Management Center with a preservation number of CICC23140. The Lactobacillus murine can improve the movement disorder induced by 6-OHDA after being inactivated, and rescue the loss of 6-OHDA-induced dopaminergic neuron marker tyrosine hydroxylase (TH) protein in the substantia nigra of the brain; The dopaminergic neuron injury caused by OHDA has a preventive and protective effect, and has a good preventive and therapeutic effect on Parkinson's disease.

In the present invention, the auxiliary material preferably includes one or more of starch, maltodextrin, trehalose and lactose, more preferably includes trehalose. In the present invention, the adjuvant can improve patient compliance and improve the stability of the inactivated Lactobacillus murine. The adjuvant of the present invention preferably also includes other emulsifiers, fillers, flavoring agents and suspending agents. In the present invention, there is no special limitation on the sources of the auxiliary materials, and conventional commercially available products well known to those skilled in the art can be used.

In the present invention, the dosage form of the drug includes suspension, suppository, tablet, capsule, soft capsule, prefilled injection, effervescent tablet and gel. The present invention has no special requirements on the preparation method of the above dosage form, and the preparation method well known in the art can be used.

In the present invention, the preparation method of the inactivated Lactobacillus murine preferably comprises the following steps:

The Lactobacillus murine is sterilized to obtain an inactivated Lactobacillus murine.

The present invention has no special limitation on the sterilization method, and conventional sterilization methods can be used, such as inactivation at 121°C for 20-30 minutes, more preferably at 121°C for 20 minutes.

In the present invention, the concentration of inactivated Lactobacillus murine in the drug is preferably 10 ⁶ -10 ¹² cfu/ml, more preferably 10 ⁹ cfu/ml. The inactivated Lactobacillus murine at said concentration can better prevent and treat Parkinson's disease.

The invention also provides the application of the inactivated Lactobacillus murine in the preparation of medicaments for preventing and/or treating Parkinson's disease. The inactivated murine lactobacillus has good preventive and therapeutic effects on Parkinson's disease.

The invention also provides the application of the inactivated Lactobacillus murine in the preparation of the medicine for improving movement disorders. In the present invention, the movement disorder is preferably a movement disorder induced by 6-OHDA. In the present invention, in the application, the dosage of the drug is preferably 10 ⁶ -10 ¹² cfu/ml, more preferably 10 ⁹ cfu/ml. In the embodiment of the present invention, taking 6-OHDA-induced rats as an example, the inactivated Lactobacillus murine can significantly increase the rod time and spontaneous exploration time of the mouse, and significantly increase the number of strides on the injured side forelimb, Improvement of motor impairment in mice.

The invention also provides the application of the inactivated type of Lactobacillus murine in the preparation of the medicine for preventing dopaminergic neuron damage. In the present invention, the dopaminergic neuron injury is preferably dopaminergic neuron injury induced by 6-OHDA. In the present invention, in the application, the dosage of the drug is preferably 10 ⁶ -10 ¹² cfu/ml, more preferably 10 ⁹ cfu/ml. In the embodiment of the present invention, taking 6-OHDA-induced rats as an example, the inactivated Lactobacillus murine can significantly improve the decrease in the number of positive cells of dopaminergic neurons and prevent damage to dopaminergic neurons in the substantia nigra of the midbrain.

The invention also provides the application of the inactivated Lactobacillus murine in the preparation of a drug for rescuing the loss of the dopaminergic neuron marker tyrosine hydroxylase protein in the substantia nigra of the midbrain. In the present invention, the loss of tyrosine hydroxylase protein, a marker of dopaminergic neurons in the substantia nigra of the midbrain, is preferably the loss of tyrosine hydroxylase protein induced by 6-OHDA. In the present invention, in the application, the dosage of the drug is preferably 10 ⁶ -10 ¹² cfu/ml, more preferably 10 ⁹ cfu/ml. In the embodiment of the present invention, taking 6-OHDA-induced rats as an example, the inactivated Lactobacillus murine can rescue the loss of the dopaminergic neuron marker tyrosine hydroxylase protein in rats caused by 6-OHDA.

The drug for preventing and/or treating Parkinson's disease and its application according to the present invention will be further described in detail below in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

Example 1

1 Experimental materials and instruments

1.1 Experimental animals

SD male rats, weighing 200-250g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. (permit: SCXK (Beijing) 2019-0008), and the animals were raised in the Key Laboratory of Basic Pharmacology, Ministry of Education, Zunyi Medical College SPF animals The experimental animals in each group had free access to food and water.

1.2 Main instruments and equipment

Fatigue rotarod instrument ZH-300 (Anhui Zhenghua Biological Instrument Equipment Co., Ltd.), rat brain stereotaxic instrument (Narishige, USA), OLYMPUS optical microscope (OLYMPUS, Japan), Bio-Rad chemiluminescence imaging system (Bio-Rad, USA) -Rad Company), BSS-110 Electronic Analytical Balance (Beijing Sartorius Electronic Balance Co., Ltd.), MilliQA Pure Water Processor (Millipore Company), etc.

1.3 Main experimental reagents

Lactobacillus murinus (Lactobacillus murinus, Lactobacillus murine, China Industrial Microbiology Culture Collection Management Center); MRS medium: 10.0g casein peptone, 10.0g beef extract, 4.0g yeast extract powder, 20.0g glucose, 5.0g sodium acetate , triammonium citrate 2.0g, Tween 801.0ml, K ₂ HPO ₄ 2.0g, MgSO ₄ ·7H ₂ O 0.2g, MnSO ₄ ·H2O 0.05g, distilled water 1000mL, pH 6.2 (Solarbio); 6-hydroxydopamine (6-OHDA), purity ≥ 98% (Sigma company); tyrosine hydroxylase (TH) rabbit polyclonal antibody (Proteintech Chinese company); broad-spectrum SP immunohistochemical detection kit (Beijing Boaosen Biology Technology Co., Ltd.); DAB chromogen (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.).

2. Experimental method

2.1 Experimental grouping

60 SD male rats, weighing 200-250g, were randomly divided into 6 groups, 10 rats in each group, respectively as sham operation group, sham operation + Lactobacillus murine (10 ⁹ cfu) group, model group, model + mouse milk Bacillus (10 ⁷ cfu) group (low dose), model + Lactobacillus murine (10 ⁹ cfu) group (high dose) and model + inactivated Lactobacillus murine (10 ⁹ cfu) group.

1) Sham operation group: after 5 weeks of treatment with drinking water, only stereotaxic injection of 0.2% ascorbic acid was given, and then drinking water was continued for 2 weeks;

2) Sham operation + Lactobacillus murine (10 ⁹ cfu) group: After pretreatment with Lactobacillus murine 10 ⁹ cfu/day for 5 weeks, only stereotaxic injection of 0.2% ascorbic acid was given, and then continued administration of Lactobacillus murine 10 ⁹ cfu/day 2 week;

3) Model group: After 5 weeks of treatment with drinking water, the Parkinson's model was established by stereotaxic, and then drinking water was continued for 2 weeks;

4) Model + Lactobacillus murine (10 ⁷ cfu) group: After pretreatment with Lactobacillus murine 10 ⁷ cfu/day for 5 weeks, the Parkinson's model was established by stereotaxic, and then continued administration of Lactobacillus murine 10 ⁷ cfu/day for 2 weeks;

5) Model + Lactobacillus murine (10 ⁹ cfu) group: After pretreatment with Lactobacillus murine 10 ⁹ cfu/day for 5 weeks, the Parkinson's model was established by stereotaxic, and then continued administration of Lactobacillus murine 10 ⁹ cfu/day for 2 weeks;

6) Model + inactivated Lactobacillus murine (10 ⁹ cfu) group: After pretreatment with inactivated Lactobacillus murine 10 ⁹ cfu/day for 5 weeks, the Parkinson's model was established by stereotaxic, and then continued administration of inactivated Lactobacillus murine 10 ⁹ cfu/day for 2 weeks;

2.2 Preparation of animal models

Anesthetize SD rats with 2% pentobarbital sodium (injection dose 0.3ml/100g) intraperitoneally, and then under sterile conditions, the bregma is the coordinate origin, 4.8mm behind the bregma, 1.8mm beside the midline, subdural 8.0 mm, inject 1.6 μL of 5 μg/μL 6-OHDA solution at a constant speed.

2.3 Culture and administration of Lactobacillus murine

Lactobacillus murine culture: first activate the strains in the freeze-dried tube, carry out subculture to restore vitality, inoculate Lactobacillus murine in MRS medium, culture at 37°C for 8 hours, and measure the bacterial liquid with a UV spectrophotometer under the condition of 600nm OD value, the OD value is about 0.8, the total number of bacteria is 2.8×10 ⁸ cfu/mL, take the suspension and centrifuge directly or under high pressure at 121°C for 20 minutes, then centrifuge at 4500 rpm for 30 minutes, discard the supernatant and resuspend with drinking water Rats were treated by gavage.

2.4 Behavioral testing

Rats were subjected to behavioral testing 14 days after modeling, and medication was continued during the behavioral period.

2.4.1 Rotating rod experiment

The initial rotation speed of the rotating rod experiment is set to 10rpm, and then the speed is increased by 5 rotations every 60s until the rat falls off the rotating rod shaft or after 300s, the system will automatically sense and record the entire experimental process, and compare the rats in each group. behavioral capacity.

2.4.2 Field experiment

The experiment was carried out in a quiet and dark environment, and the animals were placed in the center of the bottom of the box, and the camera and time were taken at the same time. After 10 minutes of observation, the camera was stopped. Spray alcohol to wipe the floor and inner wall, so as not to affect the next test result due to the last animal residue information.

2.4.3 Stride experiment

Hold up the second half of the rat's body so that the rat's body weight is only supported by one side of the forelimb, record the number of times the rat walks on this side of the forelimb within 10 seconds, and then detect the walking situation of the other forelimb in the same way to measure the damage. The proportion of contralateral forelimb walking times (the percentage of adjusted steps on the opposite side of the injury/adjusted steps on the injured side) was used as the evaluation standard.

2.5 Material collection and specimen preparation

After the behavioral experiment was completed, 5 rats were randomly selected from each group and anesthetized with 7% pentobarbital sodium, the abdominal cavity was opened, the abdominal aorta was clamped, the thorax was cut open, the heart was fully exposed, and the perfusion Insert the needle into the aorta through the apex of the heart, fix the hemostat and cut the right atrial appendage, perfuse 0.1MPBS to wash the blood in the brain, take the whole brain, separate the midbrain and store it at -80°C, or continue to permeabilize it with 4% paraformaldehyde. After perfusion, the whole brain was fixed in 4% paraformaldehyde and stored at 4°C when the limbs of the rats were stiff. After 48 hours, it was dehydrated, embedded in paraffin, sliced, and stored at room temperature.

2.6 Western blot experiment

1) Grind the midbrain on ice, add RIPA lysate (containing PMSF), and let stand for 30 minutes;

2) Use an ultra-low temperature centrifuge to centrifuge at a speed of 12,000 rpm for 10 minutes, and collect the supernatant;

3) Use the BCA protein quantification kit to quantify, prepare the loading system, denature and mix;

4) Add 10 μL per well to 10% precast gel, electrophoresis for 2 hours (voltage 70-110V);

5) After electroporation for 9 minutes, wash with TBST, block with 5% skimmed milk for 2 hours, and wash with TBST;

6) Add primary antibody overnight at 4°C, wash with TBST;

7) Incubate with secondary antibody for 40 min, wash with TBST;

8) Add exposure agent and expose.

2.7 Immunohistochemical experiments

From the preserved brain slices, one slice was taken every 6 slices, and 6 slices were taken from each group, and the damage of dopaminergic neurons was observed by immunohistochemical method. The operation steps are as follows:

1) Paraffin sections were dewaxed and hydrated in xylene and graded ethanol, rinsed with running water, dewaxed to water, rinsed with running water, washed with PBS for 5 min, and washed 3 times;

2) Treat with 3% H ₂ O ₂ at room temperature in the dark for 15 minutes, then wash with PBS for 5 minutes and repeat the washing 3 times;

3) Repair with high heat in citrate repair solution, cool naturally to room temperature, then wash with PBS for 5 minutes and repeat the wash 3 times;

4) Block with 100% goat serum at 37°C for 30 minutes;

5) Prepare TH antibody at a ratio of 1:500, overnight at 4°C;

6) Return to room temperature for 30 minutes, wash with PBS for 5 minutes, and repeat the washing 3 times;

7) Add the corresponding secondary antibody working solution, react at 37°C for 20 minutes, then wash with PBS for 5 minutes and repeat the washing 3 times;

8) Add horseradish enzyme-labeled streptavidin working solution dropwise and incubate at 37°C for 20 minutes, wash with PBS for 5 minutes, and repeat washing 3 times;

9) DAB color development for 1 min, then washed with PBS for 5 min and repeated washing 3 times;

10) Carry out gradient alcohol dehydration according to the alcohol concentration sequence of 80%-90%-100%, and dehydrate for 10s at each concentration;

11) Dry at room temperature first, then air-dry in a fume hood, and seal with neutral plastic;

12) Observe, take pictures and count under an optical microscope (4×).

2.8 Statistical analysis of data

The experimental data was counted by SPSS21.0 statistical software, and all the data were expressed as (mean ± standard deviation). One-way analysis of variance (ANOVA) was used to compare the group differences of the mean, and the LSD method was used for the homogeneity of variance test. Dunnett's T3 was used for comparison The group difference of mean, P<0.05 is statistically significant.

2. Experimental results

3.1 Improvement effect of inactivated Lactobacillus murine on 6-OHDA-induced dyskinesia in rats

The above-mentioned 2.4 behavioral experiment adopts the statistical analysis method of the above-mentioned 2.8 data to analyze the obtained data, so as to detect the improvement effect of Lactobacillus murine on the movement disorder induced by 6-OHDA in rats.

Figure 1, Figure 2 and Figure 3 are the results of three behavioral tests after 5 weeks of pretreatment with probiotics, followed by continuous administration of probiotics for two weeks. Fig. 1 is a histogram of rats' movement time on a rod, which is the result of a rat rotating rod experiment. The ordinate is the time of rod movement (s) of each group of rats, and the abscissa is the group of 6 groups of rats; Fig. 2 is The histogram of the spontaneous exploration activity distance of the rats is the result of the mine field experiment. The ordinate is the spontaneous exploration distance (mm) of each group of rats, and the abscissa is the group of 6 groups of rats; Figure 3 is the damaged contralateral side of the rats The histogram of the proportion of forelimb movement is the result of the striding experiment. The ordinate is the ratio of the number of adjusted steps of the damaged contralateral forelimb to the adjusted number of steps of the damaged forelimb of the rats in each group. group. The 6 groups of rats correspond to each group in 2.1 experimental grouping respectively, * indicates that this group is P<0.05 compared with the sham operation group, # indicates that this group is P<0.05 compared with the model group.

As can be seen from Figure 1, compared with the sham operation group, the model group had significantly shortened movement time on the rod and significantly impaired exercise ability, while the administration of Lactobacillus murine alone had no significant effect on the exercise ability of rats; the model was established with 6-OHDA After pretreatment with 10 ⁷ cfu/day and 10 ⁹ cfu/day of Lactobacillus murine for 5 weeks, the model + Lactobacillus murine low-dose group, model + Lactobacillus murine high-dose group were compared with the model group respectively, There was no significant change in the exercise time on the bar, and no significant improvement in the activity ability; compared with the model group, the inactivated Lactobacillus murine group had a significant increase in the exercise time on the bar, and the activity ability was significantly enhanced.

It can be seen from Figure 2 that compared with the sham operation, the spontaneous exploration distance of the rats in the model group was significantly shortened under dark conditions, while the single administration of Lactobacillus murine had no significant effect on the exploration ability of the rats; the model + low dose of Lactobacillus murine Compared with the model group, there was no significant change in the exploration distance, and no significant improvement in the exploration ability of the model group, the model+Lactobacillus murine high-dose group; the exploration ability of the inactivated Lactobacillus murine group was significantly enhanced compared with the model group.

Similarly, the same trend can be seen from Figure 3. Compared with the sham operation, the number of strides on the injured side of the rats in the model group was significantly reduced, while the number of strides on both sides of the rats given Lactobacillus murine alone was basically the same ; Model + Lactobacillus murine low-dose group, model + Lactobacillus murine high-dose group were compared with the model group, the number of forelimb strides on the injured side of the rats had no significant change; compared with the model group, the rats The number of strides of the forelimb on the injured side increased significantly.

The above behavioral results all suggested that the administration of inactivated Lactobacillus murine could significantly improve the movement disorder of rats with 6-OHDA injury in the substantia nigra.

3.2. Inactivated Lactobacillus murine protects 6-OHDA-induced loss of dopaminergic neuron marker tyrosine hydroxylase (TH) in the substantia nigra of rats

After calculating the gray value in the aforementioned 2.6 Western blot experiment, the statistical analysis method of the aforementioned 2.8 data was used to analyze the obtained data to detect the protective effect of Lactobacillus murine on the loss of 6-OHDA-induced loss of rat dopaminergic neuron marker protein .

Fig. 4 is the Westernblot protein expression band of rat midbrain substantia nigra tyrosine hydroxylase (TH), and Fig. 5 is the rat midbrain substantia nigra tyrosine hydroxylase (TH) obtained after statistics of the results in Fig. 4 TH) protein expression histogram, the ordinate is the ratio of TH protein in the substantia nigra of rats in each group to the internal reference GAPDH (based on the sham operation group), and the abscissa is the group of 6 groups of rats. The 6 groups of rats correspond to each group in 2.1 experimental grouping respectively, * indicates that this group is P<0.05 compared with the sham operation group, # indicates that this group is P<0.05 compared with the model group.

It can be seen from Figure 4 and Figure 5 that compared with the sham operation group, the expression of TH protein in the substantia nigra of the rat midbrain was significantly reduced after administration of 6-OHDA, while the expression of TH protein in the rat midbrain was slightly increased after the administration of Lactobacillus murine alone. Expression of TH protein in the substantia nigra; Model + Lactobacillus murine low-dose group, model + Lactobacillus murine high-dose group were compared with the model group respectively. After administration of Lactobacillus murine, the reduction of TH protein in the substantia nigra of the rat midbrain was not alleviated ; Compared with the model group, the inactivated Lactobacillus murine group can significantly rescue the loss of TH protein in the substantia nigra of the rat midbrain caused by 6-OHDA after administration of the inactivated Lactobacillus murine.

3.3 Inactivated Lactobacillus murine prevents 6-OHDA-induced degeneration of dopaminergic neurons in rats

Compare the photos of the above-mentioned 2.7 immunohistochemical experiments, and use the statistical analysis method of the above-mentioned 2.8 data to analyze the counting results, so as to detect the effect of inactivated Lactobacillus murine on 6-OHDA-induced rat dopamine Prevention of neuronal degeneration.

Figure 6 is an immunohistochemical diagram of dopaminergic neurons in the substantia nigra of the rat midbrain (the length of the scale in the lower right corner is 100 μm), and Fig. 7 is the positive cells of dopaminergic neurons in the substantia nigra of the rat midbrain obtained by statistics of the results in Figure 6 The number histogram, the ordinate is the number of positive cells of dopaminergic neurons in the substantia nigra of the rats in each group, and the abscissa is the group of 6 groups of rats. The 6 groups of rats correspond to each group in 2.1 experimental grouping respectively, * indicates that this group is P<0.05 compared with the sham operation group, # indicates that this group is P<0.05 compared with the model group.

As can be seen from Figure 6 and Figure 7, compared with the sham operation group, the number of dopaminergic neurons in the substantia nigra of the rat's midbrain was significantly reduced after the administration of 6-OHDA, showing that 6-OHDA has a significant effect on the effect of 6-OHDA on the substantia nigra of the rat. Significant damage of dopaminergic neurons in rats; after Lactobacillus murine was administered alone, dopaminergic neurons in the rat midbrain did not change significantly compared with the sham operation group; model + Lactobacillus murine low-dose group, model + Lactobacillus murine high-dose group Compared with the model group, the number of dopaminergic neurons in the substantia nigra of the rat midbrain was not significantly improved after being given Lactobacillus murine; Lactobacillus can significantly antagonize the degeneration of dopaminergic neurons caused by 6-OHDA, indicating that the administration of inactivated Lactobacillus murine has preventive and protective effects on dopaminergic neuron damage caused by 6-OHDA.

The above results show that the inactivated Lactobacillus murine CICC23140 of the present invention has a good effect on improving the movement disorder of rats induced by 6-OHDA; it can rescue the dopaminergic neuron markers in the substantia nigra of rats induced by 6-OHDA Loss of tyrosine hydroxylase (TH) protein; and has preventive and protective effects on dopaminergic neuron damage caused by 6-OHDA, and has good preventive and therapeutic effects on Parkinson's disease.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (4)

1. Application of inactivated Lactobacillus murinus in the preparation of a drug for preventing and/or treating Parkinson's disease induced by 6-OHDA; said Lactobacillus murine is Lactobacillus murinus ( Lactobacillus murinus ), preserved in China Industrial Microorganisms Deposit Management Center, the deposit number is CICC 23140. 2. Application of inactivated Lactobacillus murinus in the preparation of medicines for improving 6-OHDA-induced dyskinesia; the Lactobacillus murine is Lactobacillus murinus ( Lactobacillus murinus ), preserved in China Industrial Microorganism Culture Collection Management Center, preservation number for CICC 23140. 3. Application of inactivated Lactobacillus murinus in the preparation of medicines for preventing 6-OHDA-induced dopaminergic neuron damage; the Lactobacillus murine is Lactobacillus murinus , which is preserved in China Industrial Microorganism Culture Preservation Management Center, the deposit number is CICC 23140. 4. Application of the inactivated Lactobacillus murine in the preparation of a drug for rescuing the loss of the brain dopaminergic neuron marker tyrosine hydroxylase protein induced by 6-OHDA; the Lactobacillus murine is Lactobacillus murinus ), preserved in the China Industrial Microorganism Culture Collection Management Center, the preservation number is CICC 23140.

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