Human-derived lactobacillus johnsonii HA1108 and application thereof in preparation of biological agent and / or medicine for treating or relieving Parkinson's disease

Abstract

The invention discloses human-derived lactobacillus johnsonii HA1108 and application thereof in preparation of biological agents and / or medicines for treating or relieving Parkinson's disease, and belongs to the technical field of biological medicines. The invention provides a strain of lactobacillus johnsonii, which is classified and named as Lactobacillus johnsonii, and the preservation number of the lactobacillus johnsonii is CGMCC (China General Microbiological Culture Collection Center) No.35865. Based on'flora-intestine-brain axis', the lactobacillus johnsonii HA1108 can improve dyskinesia in Parkinson's disease, relieve deletion of dopaminergic neurons of nigra in the brain, increase protein expression of tyrosine hydroxylase at the nigra part in the brain, and improve the immunity of the Parkinson's disease. The microbial inoculum prepared from the lactobacillus johnsonii HA1108 does not affect the body weight after being eaten for a long time, and a new choice is provided for patients with Parkinson's disease. The screened lactobacillus johnsonii HA1108 has important significance in preparation of biological agents or medicines for treating or relieving Parkinson's disease, improving dyskinesia and improving tyrosine hydroxylase expression, has safety and application value, and is wide in market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a human Lactobacillus johnsonii HA1108 and application thereof in preparing a biological preparation and / or a medicine for treating or relieving Parkinson's disease. BACKGROUND

[0002] Parkinson's disease is a neurodegenerative disease that occurs in middle-aged and elderly people, and its etiology is complex and there is currently no effective treatment method. The core feature of Parkinson's disease is the massive loss of dopaminergic neurons in the substantia nigra of the midbrain. It is known in the art that most Parkinson's disease patients have gastrointestinal symptoms (including constipation, delayed gastric emptying, slowed intestinal peristalsis, etc.) in the early stage. In addition, the composition of the intestinal flora of Parkinson's disease patients is significantly different from that of healthy controls, and the intestinal flora colonizing Parkinson's disease patients can exacerbate the motor dysfunction of mice, indicating that intestinal flora disorder is closely related to the occurrence and development of Parkinson's disease. Based on the "flora-gut-brain axis", exploring the feasibility of using human intestinal probiotics to treat or relieve Parkinson's disease is expected to provide a new idea for the treatment of Parkinson's disease.

[0003] Currently, oral drugs are mainly used for the treatment of Parkinson's disease in the clinic, such as levodopa, dopamine receptor agonists, and monoamine oxidase B inhibitors. However, long-term drug use can cause varying degrees of side effects (nausea, vomiting, visual hallucinations, on-off effect, dyskinesia, etc.), and as the disease progresses, the continuous loss of dopaminergic neurons leads to a gradual decrease in the therapeutic effect of the drug. In contrast, the use of human intestinal probiotics and other microecological therapies for the intervention treatment of Parkinson's disease has higher safety and application value, and the intestinal flora therapy for Parkinson's disease patients is a new trend in the clinical treatment of Parkinson's disease. SUMMARY

[0004] Based on the above needs, the present application aims to provide a human Lactobacillus johnsonii HA1108 and application thereof in preparing a biological preparation and / or a medicine for treating or relieving Parkinson's disease. The Lactobacillus johnsonii HA1108 screened in the present application has the function of improving motor dysfunction and can increase the expression of tyrosine hydroxylase and reduce the loss of dopaminergic neurons in the brain. Moreover, the Lactobacillus johnsonii HA1108 can maintain body weight when consumed for a long time, has safety, provides a new idea for preparing a biological preparation and / or a medicine for preventing and treating Parkinson's disease, and has a broad application prospect.

[0005] In order to achieve the above-mentioned purpose, the present application can adopt the following technical solutions:

[0006] The application provides a human Lactobacillus johnsonii HA1108, which is classified as Lactobacillus johnsonii and is preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.35865.

[0007] Further, the Lactobacillus johnsonii HA1108 has a short rod shape with blunt ends.

[0008] The application also provides a bacterial agent containing the human Lactobacillus johnsonii HA1108.

[0009] The application also provides application of the Lactobacillus johnsonii HA1108 or the bacterial agent thereof in preparation of a biological agent and / or a medicine for treating or relieving Parkinson's disease.

[0010] The application also provides application of the Lactobacillus johnsonii HA1108 or the bacterial agent thereof in preparation of a biological agent and / or a medicine for treating and improving motor dysfunction.

[0011] The application also provides application of the Lactobacillus johnsonii HA1108 or the bacterial agent thereof in preparation of a biological agent and / or a medicine for increasing expression of tyrosine hydroxylase.

[0012] Further, the Lactobacillus johnsonii HA1108 can reduce loss of dopaminergic neurons in the substantia nigra and significantly increase protein expression of tyrosine hydroxylase in the substantia nigra.

[0013] Further, the biological agent or the medicine containing the Lactobacillus johnsonii HA1108 can maintain body weight when consumed.

[0014] Further, the biological agent or the medicine contains the Lactobacillus johnsonii HA1108 with a viable bacterial amount of 2*10 10 CFU or more.

[0015] Further, the biological agent or the medicine is consumed once a day and the consumption cycle is 6 weeks.

[0016] Further, the method for long-term preservation of the bacterial solution of the Lactobacillus johnsonii HA1108 is as follows: 4 DEG C, centrifugation at 10,000 rpm for 2 minutes to collect cells, washing with a PBS buffer solution, resuspension in a 15%-25% skimmed milk powder solution at a ratio of 5:1, freeze-drying and sealing preservation, or preservation in a preservation solution containing 15% glycerol and 85% serum, liquid nitrogen freezing and-80 DEG C preservation.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] 1. The Lactobacillus johnsonii HA1108 strain provided by the application has a rising effect on the host tyrosine hydroxylase of substantia nigra, and plays a therapeutic role on Parkinson's disease through the above-mentioned regulating effect, and the Lactobacillus johnsonii HA1108 can be used for preparing a medicine for treating or preventing Parkinson's disease.

[0019] 2. When the Lactobacillus johnsonii HA1108 provided by the application is eaten to alleviate and improve the motor dysfunction of the Parkinson's disease model, no significant change in body weight occurs within 6 weeks of eating, further verifying that the Lactobacillus johnsonii HA1108 does not cause other side effects when preventing and treating the motor dysfunction of Parkinson's disease.

[0020] 3. The application provides a new way for treating or alleviating Parkinson's disease model motor dysfunction, based on the "microbiota-gut-brain axis", the screened Lactobacillus johnsonii HA1108 can realize the reduction of the loss of dopaminergic neurons in the substantia nigra of the brain, and increase the protein expression of tyrosine hydroxylase in the substantia nigra of the brain, thereby providing a new direction for the research or application of treating Parkinson's disease. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a bacterial body morphology diagram of Lactobacillus johnsonii HA1108 under transmission electron microscope.

[0022] Figure 2 It is a design flowchart of the live bacteria of Lactobacillus johnsonii HA1108 gavaged in animal experiments.

[0023] Figure 3 It is the influence of the live bacteria of Lactobacillus johnsonii HA1108 gavaged on the body weight of mice.

[0024] Figure 4 It is the pole climbing experiment mouse head turning time of the improvement effect of the live bacteria of Lactobacillus johnsonii HA1108 gavaged on the motor dysfunction of Parkinson's disease mice.

[0025] Figure 5 It is the time from the top to the bottom of the pole climbing experiment mouse of the improvement effect of the live bacteria of Lactobacillus johnsonii HA1108 gavaged on the motor dysfunction of Parkinson's disease mice.

[0026] Figure 6 It is the rotating rod experiment of the improvement effect of the live bacteria of Lactobacillus johnsonii HA1108 gavaged on the motor dysfunction of Parkinson's disease mice.

[0027] Figure 7 It is the open field experiment of the improvement effect of the live bacteria of Lactobacillus johnsonii HA1108 gavaged on the motor dysfunction of Parkinson's disease mice.

[0028] Figure 8Effects of live Lactobacillus johnsonii HA1108 gavage on tyrosine hydroxylase positive neurons in the substantia nigra of mice.

[0029] Figure 9 Effects of live Lactobacillus johnsonii HA1108 gavage on tyrosine hydroxylase protein expression in the substantia nigra of mice, tyrosine hydroxylase protein expression banding diagram.

[0030] Figure 10 Effects of live Lactobacillus johnsonii HA1108 gavage on tyrosine hydroxylase protein expression in the substantia nigra of mice, statistical analysis results of tyrosine hydroxylase protein expression. DETAILED DESCRIPTION

[0031] The application will be described more fully below with reference to the drawings and specific examples. The described examples are only a part of the embodiments of the application, not all the embodiments. Based on the examples in the application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0032] Example 1, strain isolation, identification and preservation

[0033] 1. Isolation and identification

[0034] The strain was obtained by the inventors of the present application on April 18, 2022, from fresh feces provided by a feces donor after signing an informed consent form, in the Shandong University Qingdao Campus, No. 72 Binhai Road, Jimo District, Qingdao City, Shandong Province, and was labeled as strain HA1108.

[0035] Colony morphology: fast growth in MRS medium, 37℃ anaerobic conditions for 8 hours to reach the plateau. The transmission electron microscopy morphology of the strain HA1108 is shown in Figure 1 The bacterial body is generally short rod-shaped with blunt ends.

[0036] Molecular identification: full sequence analysis of 16S rRNA, the sequence is shown in the sequence table SEQ ID NO. 1. The strain HA1108 has 99.73% homology with the annotated 16S rRNA sequence of Lactobacillus johnsonii. The identification classification is named Lactobacillus johnsonii, and the corresponding Chinese name is Lactobacillus johnsonii.

[0037] The Lactobacillus johnsonii HA1108 is preserved by the China General Microbiological Culture Collection Center (CGMCC) at 1 Qingliang Road, No. 3, Haidian District, Beijing, China, on September 9, 2025, and the preservation number is CGMCC No. 35865.

[0038] 2. Cultivation and preservation method:

[0039] The Lactobacillus johnsonii HA1108 is activated once in MRS medium. Then, it is inoculated into MRS liquid medium with the same composition at an inoculation amount of 0.1%-10%, and cultured anaerobically at 37°C overnight.

[0040] Long-term preservation method of bacteria: The bacterial solution is centrifuged at 10,000 rpm for 2 minutes at 4°C to collect cells, and then washed with PBS buffer solution. After resuspension in a solution containing 15%-25% skimmed milk powder at a ratio of 5:1, freeze-drying, and sealing, or in a preservation solution containing 15% glycerol and 85% serum, freezing in liquid nitrogen, and storing at -80°C.

[0041] Example 2, Animal experiment of Lactobacillus johnsonii HA1108 live bacteria in treating Parkinson's disease

[0042] 1. Materials and methods:

[0043] (1) Cultivation method of live bacteria of strain HA1108:

[0044] The strain HA1108 is activated on MRS solid medium and cultured anaerobically at 37°C overnight. Then, a single colony is picked and inoculated into MRS liquid medium for seed culture at a temperature of 37°C for 8-12 hours. Subsequently, the seed liquid is inoculated into MRS liquid medium with the same composition at an inoculation amount of 1-5%, and cultured anaerobically at 37°C overnight.

[0045] (2) Animal experiment design:

[0046] C57BL / 6J mice, male, 3 months old, are used in the experiment. A Parkinson's disease model is prepared by intraperitoneal injection of rotenone. The injection frequency is once a day, and the gavage frequency is once a day. The model treatment group (rotenone+HA1108) (10) is administered with HA1108 live bacteria at a dose of 2x10 10 CFU per day at the same time as the injection of rotenone. The model group (rotenone) (10) is administered with the same dose of PBS buffer solution at the same time as the injection of rotenone. The control group (10) is injected / gavaged with the same dose of PBS buffer solution every day. The total experimental period is 6 weeks. The specific animal experiment design is as follows:Figure 2 Results are shown in Figure 1.

[0047] 2. Experimental results:

[0048] (1) Live L. johnsonii HA1108 gavage did not affect the body weight of mice

[0049] To evaluate the effect of live L. johnsonii HA1108 bacterial agent on the body weight of mice, the body weight was weighed every day during gavage, and the body weight was calculated every week. The results are shown in Figure 2. Figure 3 Compared with the control group, the body weight of mice in the rotenone group decreased, and live L. johnsonii HA1108 gavage had no significant effect on the body weight of mice.

[0050] Table 1 Body weight of mice in each group (unit: g)

[0051]

[0052] (2) Live L. johnsonii HA1108 gavage improved the motor dysfunction of Parkinson's disease mice

[0053] After 6 weeks of gavage, the motor function of mice was detected, and the pole climbing experiment, rotating rod experiment and open field experiment were performed, respectively.

[0054] The results of pole climbing experiment showed that live L. johnsonii HA1108 gavage could reduce the head turning time of Parkinson's disease mice on the pole ( Figure 4 ) and the time from the top to the bottom of the pole ( Figure 5 ).

[0055] Table 2 Head turning time of mice on the pole (unit: s)

[0056]

[0057] Table 3 Time of mice from the top to the bottom of the pole (unit: s)

[0058]

[0059] The results of rotating rod experiment showed that live L. johnsonii HA1108 gavage could increase the duration of Parkinson's disease mice on the rotating rod ( Figure 6 ).

[0060] Table 4 Duration of mice on the rotating rod (unit: s)

[0061]

[0062] The results of open field experiment showed that live L. johnsonii HA1108 gavage could increase the total distance of Parkinson's disease mice in the open field ( Figure 7 ).

[0063] Table 5 Total distance traveled by mice in open field (unit: cm)

[0064]

[0065] The above behavioral experiment results show that L. johnsonii HA1108 live bacteria gavage can improve the motor dysfunction of Parkinson's disease mice.

[0066] (3) L. johnsonii HA1108 live bacteria gavage reduces the loss of dopaminergic neurons in the substantia nigra of Parkinson's disease mice

[0067] Parkinson's disease patients have a severe lack of dopaminergic neurons in the substantia nigra compact part, and the expression of tyrosine hydroxylase decreases. By observing the changes of tyrosine hydroxylase positive neurons in the substantia nigra of mice through immunofluorescence staining, green fluorescence marks tyrosine hydroxylase, and blue fluorescence marks the nucleus. L. johnsonii HA1108 live bacteria gavage significantly increased the number of tyrosine hydroxylase positive neurons in the substantia nigra of Parkinson's disease mice in the brain ( Figure 8 ).

[0068] Western blot results show that L. johnsonii HA1108 live bacteria gavage can significantly increase the protein expression of tyrosine hydroxylase in the substantia nigra of Parkinson's disease mice in the brain ( Figure 9 and Figure 10 ).

[0069] Table 6 Protein level of tyrosine hydroxylase in the substantia nigra of mice in the brain

[0070]

[0071] Table 7 Protein level of β-actin in the substantia nigra of mice in the brain

[0072]

[0073] Table 8 Protein level of tyrosine hydroxylase / β-actin in the substantia nigra of mice in the brain

[0074]

[0075] In summary, in combination with the above experimental verification, the L. johnsonii HA1108 screened by the application can treat or prevent or relieve Parkinson's disease, can improve motor dysfunction including Parkinson's disease model, can achieve alleviation of loss of dopaminergic neurons in the substantia nigra of the brain, increase protein expression of tyrosine hydroxylase in the substantia nigra of the brain, and the bacterial agent prepared from the L. johnsonii HA1108 does not affect body weight when consumed for a long time, thus providing a new choice for Parkinson's disease patients in the early stage with gastrointestinal symptoms (including constipation, delayed gastric emptying, slowed intestinal peristalsis, etc.).

[0076] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified or some technical features can be replaced by equivalents for ordinary skilled persons in the art; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A strain of Lactobacillus johnsonii of human origin HA1108, characterized in that, The classification name is Lactobacillus johnsonii, which is preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No.35865.

2. Lactobacillus johnsonii HA1108 according to claim 1, characterized in that, The cell morphology of the Lactobacillus johnsonii HA1108 is short rod-shaped with blunt ends.

3. A bacterial agent containing the human-derived Lactobacillus johnsonii HA1108 of claim 1.

4. Use of the Lactobacillus johnsonii HA1108 of claim 1 or the bacterial agent of claim 3 in the preparation of a biological agent and / or a medicine for treating or alleviating Parkinson's disease.

5. Use of the Lactobacillus johnsonii HA1108 of claim 1 or the bacterial agent of claim 3 in the preparation of a biological agent and / or a medicine for treating and improving motor dysfunction.

6. Use of the Lactobacillus johnsonii HA1108 of claim 1 or the bacterial agent of claim 3 in the preparation of a biological agent and / or a medicine for increasing the expression of tyrosine hydroxylase.

7. Use according to claim 6, characterized in that, The Lactobacillus johnsonii HA1108 can reduce the loss of dopaminergic neurons in the substantia nigra and significantly increase the protein expression of tyrosine hydroxylase in the substantia nigra.

8. Use according to claim 4, characterized in that, The biological agent or the medicine containing the Lactobacillus johnsonii HA1108 can maintain the body weight when consumed.

9. Use according to claim 4, characterized in that, The biological preparation or the medicine contains the viable bacteria of Lactobacillus johnsonii HA1108 in an amount of 2 x 10 10 CFU or more.

10. Use according to claim 4, characterized in that, The consumption frequency of the biological agent or the medicine is once a day, and the consumption period is 6 weeks.

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