Use of bacillus calmette-guerin vaccine in preparation of drug for preventing and treating cerebral infarction

Abstract

Use of a Bacillus Calmette-Guerin vaccine in the preparation of a drug for preventing and treating cerebral infarction. The Bacillus Calmette-Guerin vaccine exerts preventive and therapeutic effects on cerebral infarction by means of inducing immune tolerance memory in microglia. It can reduce the apoptosis rate of mice with cerebral infarction, decrease the cerebral infarction volume of mice, and significantly promote neurological function recovery.

Description

Application of BCG vaccine in the preparation of drugs for the prevention and treatment of cerebral infarction Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the application of BCG vaccine in the preparation of drugs for the prevention and treatment of cerebral infarction by inducing microglial immune tolerance memory. Background Technology

[0002] Cerebral infarction (ischemic stroke) is a major cause of central nervous system injury, and its pathological mechanism involves an acute ischemic-hypoxic injury and a chronic neuroinflammatory cascade. Current clinical treatments (such as thrombolysis and neuroprotective agents) are limited by a narrow time window (rt-PA must be administered within 4.5 hours) and immunosuppressive side effects (such as the risk of infection induced by glucocorticoids), yet two-thirds of patients still suffer from varying degrees of sequelae. Studies have shown that microglia, as innate immune cells in the brain, exhibit a double-edged sword effect in the inflammatory regulation after cerebral infarction: early M1 polarization exacerbates neuroinflammatory inflammation, while M2 polarization promotes repair. Therefore, precise regulation of microglia phenotypic transformation has become a new therapeutic target.

[0003] Bacillus Calmette-Guérin (BCG), a classic tuberculosis vaccine, has recently been found to possess immune memory induction capabilities. This means that through epigenetic reprogramming (such as histone modification and upregulation of metabolic enzyme expression), it enhances or strengthens the tolerance of innate immune cells (such as monocytes and macrophages) to subsequent stimuli. For example, BCG vaccination significantly improves the response of peripheral blood monocytes to β-glucan and reduces cytokine storms in sepsis models. While BCG applications have focused on the prevention of infectious diseases or the treatment of bladder cancer, its potential in regulating central nervous system immune memory has long been overlooked, particularly due to a lack of targeted gene regulation research. This hinders more effective intervention in signaling pathways to determine the mechanisms controlling anti-inflammatory polarization and apoptosis in microglia.

[0004] In recent years, there has been considerable research on drugs for treating cerebral infarction. For example, prior art document CN117257803A discloses the application of lurasidone in the preparation of drugs for treating or preventing ischemia / reperfusion injury and cell-protective drugs. Lurasidone can reduce the area and volume of myocardial infarction and cerebral infarction, reduce serum creatine kinase activity and improve neurological function, and reduce cardiomyocyte and neuronal cell death, thus exhibiting cardiomyocyte and neuronal cell protective effects. Prior art document CN103467578A discloses a short peptide, copper-loaded nanobiomaterials, and their application in the preparation of drugs for treating cerebral infarction.

[0005] Currently, there are no records of the use of BCG in the preparation of drugs for the prevention and treatment of cerebral infarction, nor are there any studies on the mechanism of action of BCG as a drug for the prevention and treatment of cerebral infarction. Summary of the Invention

[0006] The technical problem to be solved by this invention is:

[0007] Currently, there are no drugs available in clinical practice that can prevent or treat cerebral infarction by inducing immune tolerance memory in microglia.

[0008] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0009] This invention provides the application of BCG in the preparation of drugs for the prevention and treatment of cerebral infarction. The mechanism of the application is to use BCG to induce microglial immune tolerance memory to prevent and treat cerebral infarction.

[0010] Furthermore, BCG is used to reduce the volume of cerebral infarction and improve neurological function.

[0011] Furthermore, the application of BCG in reducing cleaved-caspase 3 protein levels.

[0012] Furthermore, the application of BCG in promoting the expression of TNFAIP3 gene mRNA in microglia.

[0013] Furthermore, the drug is BCG vaccine; or the drug is a drug prepared by using BCG vaccine as the main component and pharmaceutically acceptable excipients.

[0014] In vivo studies in mice have demonstrated that administration of BCG (1 mg / ml) at 5 μl / g can induce microglial immune tolerance memory, thereby preventing and treating cerebral infarction, reducing cerebral infarction damage, and enhancing neuroprotective effects.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] This invention provides the application of BCG in the preparation of drugs for the prevention and treatment of cerebral infarction. BCG reduces cerebral infarction damage by inducing immune tolerance memory in microglia. Experiments show that pre-injection of BCG can reduce the apoptosis rate of cells in mice with cerebral infarction, decrease the infarct volume, and significantly improve neurological function recovery. BCG induces microglia to favor anti-inflammatory M2 polarization, reduces microglia activation in ischemic areas, inhibits the release of pro-inflammatory cytokines, and increases the expression of anti-inflammatory factors, thereby enhancing neuroprotective effects.

[0017] This study found that TNFAIP3 expression is upregulated in BCG-induced immune-tolerant microglia, reducing pro-inflammatory responses and cell damage by regulating the NF-κB signaling pathway. This targeted regulation of a specific gene provides a more effective molecular basis for neuroprotection. BCG treatment significantly increased protein expression levels, promoted NF-κB activation in microglia, and significantly inhibited NF-κB phosphorylation in microglia. Attached Figure Description

[0018] Figure 1 shows a comparison of the effects of BCG on neurological function recovery and infarct volume in mice with cerebral ischemia-reperfusion injury in the embodiments of the present invention. Among them, (A) is the TTC staining result, (B) is the quantitative analysis result of infarct volume, (C) is the mNSS score result, (D) is the Bederson score result, and (E) is the neurological function assessment result of the staggered step experiment on mice 1, 3, and 7 days after MCAO surgery (n = 6. *P < 0.05, **P < 0.01, ****P < 0.0001, ns for no significance).

[0019] Figure 2 is a comparative diagram of the effects of BCG on microglial activation and polarization in the embodiments of the present invention; where (A) is the immunofluorescence intensity result of Arg1+ / Iba-1+ detected by immunostaining in each group, and (B) is the immunofluorescence intensity result of Iba-1 detected by immunostaining 24h after MCAO / R in each group (n=6, scale bar bar=50μm, ****P<0.0001);

[0020] Figure 3 shows a comparison of the effects of BCG pretreatment on cells in the brain of mice after MCAO / R in the embodiments of the present invention; where (A) is the TUNEL and DAPI immunostaining image of each group 24h after MCAO / R, (B) is the quantitative analysis image of TUNEL immunopositive cells, (C) is the TUNEL, Iba-1 and DAPI immunostaining image of each group 24h after MCAO / R, (D) is the quantitative analysis image of TUNEL immunopositive cells Iba-1+, and (E, F) are the expression and quantitative analysis images of cleaved-caspase3 protein 24h after MCAO / R in each group (n=6, scale bar bar=50μm, green represents TUNEL, red represents Iba-1, blue represents DAPI, ****P<0.0001, ns for no significance);

[0021] Figure 4 is a diagram illustrating the drug administration and cellular oxygen and glucose deprivation / reoxygenation (OGD / R) model construction process in an embodiment of the present invention.

[0022] Figure 5 shows the BCG-constructed microglial immune tolerance memory model in this embodiment of the invention and its effect on OGD / R. (A, B) represent the relative expression levels of Il-10 and TGF-β mRNA in BV2 cells after BCG treatment by RT-qPCR; (C, D) represent the relative expression levels of Il-10 and TGF-β mRNA in BV2 cells after OGD / R treatment by RT-qPCR; (E) represents the effect of BCG pretreatment on cell viability of BV2 cells after OGD / R; and (F) represents the expression of the apoptosis protein cleaved-caspase 3 in BV2 cells after OGD / R due to BCG pretreatment (n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns for no significance).

[0023] Figure 6 shows the verification results of the regulatory mechanism of BCG pretreatment on apoptosis-related genes after cerebral ischemia-reperfusion injury in the embodiments of the present invention; where (A) is the detection diagram of TNFAIP3 mRNA after BCG treatment, (B) is the detection diagram of TNFAIP3 mRNA after plasmid overexpression of TNFAIP3, (C) is the cell apoptosis detection diagram by TUNEL method, (D) is the cell apoptosis detection diagram by flow cytometry, and (E) is the cleaved-caspase3 protein assay diagram (n=4, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns for no significance);

[0024] Figure 7 shows the verification results of the pathways related to the increased expression regulation of TNFAIP3 gene by BCG pretreatment in the embodiments of the present invention; wherein, (A) is the detection diagram of TNFAIP3 mRNA after lentiviral knockdown of TNFAIP3 gene, (B) is the expression diagram of cleaved-caspase3 protein after BCG pretreatment and OGD / R after knockdown of TNFAIP3, and (C) is the change of NF-κB phosphorylation level after BCG pretreatment and OGD / R after knockdown of TNFAIP3 (n=4, *P<0.05, **P<0.01, ****P<0.0001, ns for no significance). Detailed Implementation

[0025] To enable those skilled in the art to better understand the present invention, exemplary embodiments or examples of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described embodiments or examples are merely some, not all, of the embodiments or examples of the present invention. All other embodiments or examples obtained by those skilled in the art based on the embodiments or examples of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0027] Example 1

[0028] Eighteen male C57BL / 6J mice were randomly divided into three groups: a control group, a PBS (buffered saline) + MCAO / R group, and a BCG + MCAO / R group. Mice in the BCG + MCAO / R group were intraperitoneally injected with 5 μl / g of BCG (1 mg / ml), while mice in the PBS + MCAO / R group were intraperitoneally injected with 5 μl / g of PBS. Fourteen days later, MCAO / R models were established in the PBS + MCAO / R and BCG + MCAO / R groups through cerebral ischemia-reperfusion surgery. Specifically, MCAO / R was induced in the mouse models using a suture occlusion method, and the reperfusion process was monitored using laser speckle Doppler flowmeter to control the dynamic changes in cerebral blood flow before and after the surgery. On the first day after establishing the MCAO / R model, TTC staining of brain tissue and infarct volume were performed on mice in each group.

[0029] Neurological function was assessed using the mNSS score, Bederson score, and staggered gait test on days 1, 3, and 7 after cerebral ischemia-reperfusion in mice.

[0030] The presence of significant infarct foci after cerebral ischemia-reperfusion indicates the successful establishment of the MCAO / R model. Pre-intervention with BCG reduced infarct volume (****P<0.0001, Figure 1B). Furthermore, except for no statistically significant difference in Bederson score on day 3, the mNSS, Bederson score, and misstep percentage of mice treated with BCG after MCAO / R were significantly lower than those in the PBS+MCAO / R group at all three time points (Figures 1C, D, E). This demonstrates that BCG can significantly improve neurological function in MCAO / R mice. It also indicates that BCG pretreatment can improve infarct volume and functional scores after MCAO / R in mice.

[0031] In this embodiment, the BCG vaccine used is 50mg of attenuated bovine tuberculosis BCG freeze-dried powder (Ruichu Biotechnology).

[0032] Example 2

[0033] In Example 1, immunostaining was performed on each group on day 14 of the experiment and 24 hours after the establishment of the MCAO / R model.

[0034] As can be seen, compared with the PBS+MCAO / R group, the BCG pretreatment group showed an increase in the anti-inflammatory phenotype (Argl / Iba-1+) of microglia (Figure 2A), indicating that BCG pretreatment can promote the transformation of microglia to the anti-inflammatory phenotype. Simultaneously, 24 hours after cerebral ischemia-reperfusion, the number of activated microglia in the ischemic hemisphere cortex of the BCG+MCAO / R group was significantly reduced compared with the PBS+MCAO / R group (****P<0.0001, Figure 2B), indicating that BCG pretreatment can significantly alleviate the excessive activation of microglia after cerebral ischemia-reperfusion injury.

[0035] Example 3

[0036] In Example 1, 24 hours after the MCAO / R model was established, TUNEL immunostaining and expression and quantification analysis of cleaved-caspase 3 protein were performed on each group.

[0037] The number of apoptotic cells in the ischemic hemisphere cortex was significantly increased in the PBS+MCAO / R group, indicating that BCG pretreatment significantly reduced apoptosis (****P<0.0001, Figures 3A, B). Twenty-four hours after cerebral ischemia-reperfusion, the number of apoptotic microglia in the ischemic hemisphere cortex was significantly increased in the PBS+MCAO / R group, and BCG pretreatment significantly reduced microglia apoptosis (****P<0.0001, Figures 3C, D). After pretreatment with BCG, the cleaved-caspase 3 protein level was significantly lower in the BCG+MCAO / R group than in the MCAO / R group (****P<0.0001, Figures 3E, F).

[0038] Example 4

[0039] The BV2 cell line was divided into four groups: control (PBS), BCG, PBS+OGD, and BCG+OGD. The control group was administered PBS at a concentration of 1 μl / ml, and the BCG group was administered BCG at a concentration of 50 ng / ml, once on day 1 and once on day 6, respectively. The OGD / R model was constructed in the PBS+OGD and BCG+OGD groups on day 8 after the completion of administration (Figure 4).

[0040] It was observed that repeated stimulation with 50 ng / ml BCG induced high levels of anti-inflammatory genes IL-10 and TGF-β in microglia. These high levels of expression remained statistically significant even after OGD / R treatment, indicating that specific doses and timing of BCG could induce anti-inflammatory gene expression in microglia (*P < 0.05, Figures 5A and 5B). Furthermore, the anti-inflammatory gene phenotype was maintained after OGD / R, suggesting the successful establishment of a microglia immune tolerance memory model (*P < 0.05, **P < 0.01, Figures 5C and 5D). BCG administration also increased microglia cell survival after OGD / R (**P < 0.01, ***P < 0.001, ****P < 0.0001, Figure 5E) and reduced the expression of the apoptotic protein cleaved-caspase 3 (**P < 0.01, ***P < 0.001, Figure 5F).

[0041] Example 5

[0042] RNA sequencing analysis was performed on BV2 microglia after OGD / R treatment to screen for differentially expressed genes.

[0043] It was observed that repeated stimulation with 50 ng / ml BCG promoted the expression of TNFAIP3 gene mRNA in microglia (**P<0.01, Fig. 6A). Overexpression of TNFAIP3 gene via plasmid significantly increased the relative survival rate of microglia in the OGD / R group (****P<0.0001, Fig. 6B). Furthermore, TUNEL staining and flow cytometry analysis showed a significant decrease in the apoptosis rate of microglia after OGD / R (*P<0.05, ****P<0.0001, Fig. 6C, D), and a significant decrease in the level of the apoptotic protein cleaved-caspase 3 (**P<0.01, ****P<0.0001, Fig. 6E).

[0044] Example 6

[0045] TNFAIP3 gene knockdown was performed on BV2 microglia after OGD / R treatment to examine the expression of apoptosis-related proteins in BV2 microglia.

[0046] Transfecting BV2 microglia with TNFAIP3 knockdown lentivirus significantly reduced TNFAIP3 gene mRNA expression (*P<0.05, **P<0.01, Figure 7A). After BCG pretreatment, the OGD / R model was performed. The expression level of the apoptosis protein cleaved-caspase 3 was significantly increased in the knockdown TNFAIP3 genome (Sh-TNFAIP3 group) (*P<0.05, Figure 7B). OGD / R promoted the activation of NF-κB in microglia. BCG pretreatment significantly inhibited the phosphorylation of NF-κB in microglia after OGD / R. TNFAIP3 knockout significantly reversed the inhibitory effect of BCG on NF-κB phosphorylation in microglia after OGD / R (****P<0.0001, Figure 7C).

[0047] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. The use of Bacille Calmette-Guerin in the preparation of a drug for preventing and treating cerebral infarction, characterized in that, The mechanism of this application is to use BCG to induce microglial immune tolerance memory to prevent and treat cerebral infarction.

2. The use of BCG vaccine according to claim 1 for the preparation of a medicament for the prevention and treatment of cerebral infarction, characterized in that, Application of BCG vaccine in reducing cerebral infarction volume and improving neurological function.

3. The use of BCG vaccine according to claim 1 for the preparation of a medicament for the prevention and treatment of cerebral infarction, characterized in that, Application of BCG in reducing cleaved-caspase 3 protein levels.

4. The use of BCG vaccine according to claim 1 for the preparation of a medicament for the prevention and treatment of cerebral infarction, characterized in that, Application of BCG in promoting the expression of TNFAIP3 gene mRNA in microglia.

5. The application of BCG vaccine according to claim 1 in the preparation of drugs for the prevention and treatment of cerebral infarction, characterized in that, The drug is BCG vaccine; or the drug is a drug prepared by using BCG vaccine as the main component and pharmaceutically acceptable excipients.

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