Application of chicken mesenchymal stem cells in preparation of medicine for treating avian tuberculosis

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of chicken mesenchymal stem cells in preparation of drugs for treating avian tuberculosis. In order to provide a new intervention means for avian tuberculosis, the application uses Mycobacterium avium-chicken tuberculosis natural host model to evaluate chicken mesenchymal stem cells (cMSCs). The specific method is as follows: 5x10 6 CFU of M. avium is used to intravenously infect 42-day-old SPF male white leihang chickens, and 3x10 6 cMSCs are respectively injected by intravenous injection at 3 weeks and 4 weeks after infection. The results show that, compared with the M. avium infection group, the cMSCs can significantly increase the survival rate of the chickens, reduce the bacterial load of the tissues and pharyngeal swabs, and reduce the tissue inflammatory damage.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of chicken mesenchymal stem cells in the preparation of drugs for treating avian tuberculosis. Background Technology

[0002] Tuberculosis (TB) is a chronic and serious zoonotic infectious disease caused by Mycobacterium tuberculosis (Mtb). Bovine tuberculosis (bTB) is caused by Mycobacterium bovine (M. bovis), causing severe economic losses to the dairy and beef cattle industries. Avian tuberculosis (aTB) is caused by Mycobacterium avium (M. avium). Mycobacterium avium is closely related to Mtb. Both natural infection pathology studies and previous experiments by our research group have found that chickens, as the natural host of M. avium, exhibit typical tuberculosis-like lesions in multiple organs after infection, making them a suitable natural host model for mycobacterial infection.

[0003] Up to 10% of human tuberculosis cases are caused by infection with *M. bovis* or *M. avium*. Therefore, animal-borne TB poses a significant threat to public health and has become a key obstacle to ending TB globally. The emergence and spread of drug-resistant, multidrug-resistant, and extensively drug-resistant TB have increased the difficulty of TB prevention and control. The complexity of drug resistance mechanisms means that strategies for developing novel antimicrobial drugs are ineffective against drug-resistant TB.

[0004] In summary, elucidating the pathological mechanisms of tuberculosis (TB) and developing new intervention technologies for TB have become urgent issues that need to be addressed. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides the application of chicken mesenchymal stem cells in the preparation of drugs for treating avian tuberculosis. Using a Mycobacterium avium-chicken tuberculosis model, the therapeutic effect of cMSCs on avian tuberculosis was evaluated, and its anti-tuberculosis mechanism was preliminarily explored.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] This invention provides the application of chicken mesenchymal stem cells in the preparation of drugs for treating avian tuberculosis.

[0008] Furthermore, the avian tuberculosis is caused by infection with M. avium.

[0009] Furthermore, the treatment includes improving the survival rate of chickens infected with avian tuberculosis.

[0010] Furthermore, the treatment includes reducing the bacterial load in the tissues and / or the bacterial load in the pharyngeal and anal swabs of chickens infected with avian tuberculosis.

[0011] Furthermore, the treatment includes reducing tissue inflammatory damage caused by avian tuberculosis infection.

[0012] The present invention also provides a drug for treating avian tuberculosis, comprising chicken mesenchymal stem cells.

[0013] Furthermore, the drug is administered via intravenous injection.

[0014] Furthermore, the dosage of the drug is 3 × 10⁻⁶. 6 Item / time.

[0015] Furthermore, the drug is administered once at 3 weeks and once at 4 weeks post-infection.

[0016] This invention also provides a method for evaluating the anti-avian tuberculosis effect of chicken mesenchymal stem cells, using a Mycobacterium avium-chicken tuberculosis natural host model, comprising the following steps:

[0017] 5×10 6 CFU of Mycobacterium avium was administered intravenously to 42-day-old SPF White Leghorn chickens, followed by intravenous injections of 3×10⁻⁶ at 3 and 4 weeks post-infection. 6 Chicken mesenchymal stem cells were analyzed to determine their survival rate, bacterial load, and inflammatory damage indicators.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] This invention is the first to validate the therapeutic effect of cMSCs in a chicken natural host model of M. avium. Results showed that cMSCs significantly improved the survival rate of infected chickens, reduced bacterial load in tissues and pharyngeal swabs, and alleviated tissue inflammatory damage.

[0020] This invention expands the application of MSCs in the field of anti-infection, and provides new strategies and experimental evidence for host-targeted treatment of avian tuberculosis and human tuberculosis, which has important scientific research and translational value. Attached Figure Description

[0021] Figure 1 The effect of cMSCs treatment on the survival rate of chickens infected with M. avium;

[0022] Figure 2 The effect of cMSCs treatment on bacterial load in pharyngeal and anal swabs from M. avium infected chickens;

[0023] Figure 3 The effect of cMSCs treatment on histopathological damage in chickens infected with M. avium;

[0024] Figure 4 is the effect of cMSCs therapy on tissue bacterial load in chickens infected with M. avium (acid-fast staining);

[0025] Figure 5 is the effect of cMSCs therapy on tissue bacterial load in chickens infected with M. avium (qRT-PCR);

[0026] Figure 6 is the effect of cMSCs therapy on the content of inflammatory cytokines in tissues of M. avium-infected chickens.

[0027] In all the figures, Ctrl represents the uninfected group, MSC represents the stem cell therapy group, M. avium represents the infection group, and M. avium+MSC represents the stem cell therapy group after M. avium infection. All results are expressed as mean ± standard deviation, and tested by t-test. Detailed Description of the Embodiments

[0028] The invention discloses an application of chicken mesenchymal stem cells in preparation of a drug for treating avian tuberculosis. A person skilled in the art can refer to the content of the invention and appropriately improve process parameters to achieve the application. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously make modifications or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention, so as to implement and apply the technology of the present invention.

[0029] The Mycobacterium avium (M. avium) strain (CVCC68202) used in the present invention was purchased from China Veterinary Drug Inspection Institute; SPF White Leghorn chickens were purchased from Beijing Boehringer Ingelheim Vital Biotechnology Co., Ltd., with license number SCXK (Jing) 2024-0002; chicken mesenchymal stem cells (cMSCs) were preserved in our laboratory. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0030] In order to further elaborate the technical solution of the present invention, the present invention is further illustrated by the following examples.

[0031] Example 1

[0032] I. Experimental animals

[0033] Forty-five 42-day-old male White Leghorn chickens were taken and randomly divided into 3 groups with 15 chickens in each group:

[0034] M. avium group: intravenously injected with 5×10 6 CFU of M. avium;

[0035] M. avium + MSC group: 5×10 6 CFU-containing M. avium was administered intravenously at 3 and 4 weeks post-infection. 6 One cMSC;

[0036] Ctrl group: Intravenous injection of the same volume of PBS at infection and at 3 and 4 weeks post-infection;

[0037] MSC group: Infection was achieved by intravenous injection of an equal volume of PBS, followed by intravenous injection of 3×10⁻⁶ PBS at 3 and 4 weeks post-infection. 6 One cMSC.

[0038] II. Infection and Treatment Plan

[0039] Chickens in the M. avium group were injected intravenously with 5×10 6 Chickens in the CFU (Cultural Fuminous Mycoplasma avium) group and the M. avium + MSC group were intravenously injected with 5×10 6 CFU's M. avium was administered intravenously at 3 weeks and 4 weeks post-infection. 6 Chickens in the cMSC group were infected with PBS via intravenous injection. Three and four weeks post-infection, they were injected intravenously with 3×10⁶ PBS. 6 One cMSC. Continuously observe and record mortality rates, and regularly collect pharyngeal and anal swabs to detect bacterial load.

[0040] III. Experimental Results

[0041] 1. The effect of MSC treatment on the mortality rate of chickens infected with *M. avium*. Results showed that no deaths occurred in the uninfected control group (Ctrl, MSC) during the observation period, with a survival rate consistently at 100%; after *M. avium* infection, the survival rate of chickens continuously decreased, reaching 25%; after MSC intervention, the survival rate increased to 80%. Figure 1 ).

[0042] The results showed that M. avium infection could lead to the death of experimental chickens, and the survival rate increased significantly after MSC treatment.

[0043] 2. Effect of MSC treatment on bacterial load in chicken pharyngeal and anal swabs caused by *M. avium* infection. Results showed that viral load in the *M. avium* infection group initially increased and then decreased with the progression of infection, peaking at 21 days. Although it subsequently declined, it remained at a high level overall. MSC intervention reduced viral load, showing a significantly lower level at week 4 compared to the *M. avium* infection group. Figure 2 ).

[0044] Example 2

[0045] I. Laboratory Animals

[0046] Forty-five 42-day-old male Leghorn chickens were randomly divided into three groups of 15 each:

[0047] M. avium group: 5×10 intravenous injections 6 M. avium of CFU;

[0048] M. avium + MSC group: 5×10 6 CFU-containing M. avium was administered intravenously at 3 and 4 weeks post-infection. 6 One cMSC;

[0049] Ctrl group: Intravenous injection of the same volume of PBS at infection and at 3 and 4 weeks post-infection;

[0050] MSC group: Infection was achieved by intravenous injection of an equal volume of PBS, followed by intravenous injection of 3×10⁻⁶ PBS at 3 and 4 weeks post-infection. 6 One cMSC.

[0051] II. Infection and Treatment Plan

[0052] Chickens in the M. avium group were injected intravenously with 5×10 6 Chickens in the CFU (Cultural Fuminous Mycoplasma avium) group and the M. avium + MSC group were intravenously injected with 5×10 6 CFU M. avium was administered intravenously at 3 weeks and 4 weeks post-infection. 6 Chickens in the cMSC group were infected with PBS via intravenous injection. Three and four weeks post-infection, they were injected intravenously with 3×10⁶ PBS. 6 One cMSC was collected. Mortality was continuously observed and recorded, and pharyngeal and anal swabs were collected regularly to detect bacterial load. Lung, liver, and spleen tissues were collected for HE staining to detect tissue damage and the size and number of granulomas, acid-fast staining was used to detect bacterial distribution and content, and qRT-PCR was used to detect bacterial load; lung mRNA was collected, and qRT-PCR was used to detect the content of inflammatory factor mRNA.

[0053] III. Experimental Results

[0054] 1. Effects of MSC treatment on chicken tissue damage. Five weeks post-infection, lungs, livers, and spleens were necropsy-examined for HE staining to assess tissue damage and the morphology and number of granulomas. Results showed that *M. avium* infection resulted in granulomatous necrotic foci of varying sizes in the lungs, liver, and spleen, with significant thickening of the alveolar walls and near-complete loss of liver and spleen structure. After MSC intervention, the number of granulomas was significantly reduced. Figure 3 ).

[0055] The results showed that the number of granulomas in the lungs, liver, and spleen of chickens was significantly reduced after MSC treatment.

[0056] 2. Effect of MSC treatment on bacterial load in chicken tissues. Lung, liver, and spleen tissues from chickens were collected and subjected to acid-fast staining. The results showed that significant lesions were observed in the lung, liver, and spleen tissues of the M. avium infected group, with numerous granulomas containing obvious acid-fast positive bacteria. After MSC intervention, the number of bacteria in the granulomas was significantly reduced. Figure 4 Subsequently, mRNA was extracted from various organs, and bacterial load was detected; the results showed that, compared with M. avium infection alone, bacterial load in the lungs and spleen decreased after MSC intervention, and bacterial load in the liver decreased significantly. Figure 5 ).

[0057] The results showed that MSC treatment significantly reduced the bacterial load in chickens.

[0058] 3. The effect of MSC treatment on inflammatory response in chicken tissues.

[0059] Lung tissue was collected from chickens, and the levels of inflammatory factors (IL-1β, IL-6, and TNF-α) were measured. Results showed that, compared to M. avium infection alone, MSC intervention resulted in a decreasing trend in IL-1β and TNF-α mRNA levels, and a significant decrease in IL-6, but all levels were significantly higher than in the uninfected group. Figure 6 ).

[0060] The results showed that MSCs could reduce inflammation levels in chickens.

[0061] The foregoing has shown and described the main features and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0062] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Application of chicken mesenchymal stem cells in the preparation of drugs for treating avian tuberculosis.

2. The application according to claim 1, characterized in that, The avian tuberculosis is caused by infection with Mycobacterium avium.

3. The application according to claim 1 or 2, characterized in that, The treatment includes improving the survival rate of chickens infected with avian tuberculosis.

4. The application according to claim 1 or 2, characterized in that, The treatment includes reducing the bacterial load in the tissues and / or in the pharyngeal and anal swabs of chickens infected with avian tuberculosis.

5. The application according to claim 1 or 2, characterized in that, The treatment includes reducing tissue inflammation and damage caused by avian tuberculosis infection.

6. A drug for treating avian tuberculosis, characterized in that, Including chicken mesenchymal stem cells.

7. The drug according to claim 6, characterized in that, The drug is administered via intravenous injection.

8. The drug according to claim 6, characterized in that, The dosage of the drug is 3 × 10⁻⁶. 6 Item / time.

9. The drug according to claim 6, characterized in that, The drug is administered once at 3 weeks and once at 4 weeks post-infection.

10. A method for evaluating the anti-avian tuberculosis effect of chicken mesenchymal stem cells, characterized in that, Using the Mycobacterium avium-chicken tuberculosis natural host model, the following steps are included: 5×10 6 CFU of Mycobacterium avium was administered intravenously to 42-day-old SPF White Leghorn chickens, followed by intravenous injections of 3×10⁻⁶ at 3 and 4 weeks post-infection. 6 Chicken mesenchymal stem cells were analyzed to determine their survival rate, bacterial load, and inflammatory damage indicators.

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