Application of bacillus calmette guerin vaccine in preparation of medicine for preventing and treating cerebral infarction

Abstract

The invention discloses application of BCG (Bacillus Calmette Guerin) in preparation of a medicine for preventing and treating cerebral infarction, relates to the technical field of medicines, and aims to solve the problem that a medicine for preventing and treating cerebral infarction by inducing immune tolerance memory of microglial cells is lacked clinically at present. Experimental research proves that the bacillus calmette-guerin vaccine has prevention and treatment effects on cerebral infarction by inducing immune tolerance memory of microglial cells. By injecting the BCG, the apoptosis rate of cerebral infarction mice can be reduced, the cerebral infarction volume of the mice is reduced, and the neurological function recovery is remarkably improved. The BCG induces the microglial cells to deviate to anti-inflammatory M2 type polarization, reduces the activation of the microglial cells in an ischemic area, inhibits the release of proinflammatory cytokines, and increases the expression of anti-inflammatory factors, thereby enhancing the neuroprotective effect. The research finds that the expression of the TNFAIP3 in the immune tolerance microglial cells induced by the BCG is up-regulated, and the proinflammatory response and the cell damage are reduced by regulating and controlling an NF-kappa B signal channel.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of BCG in the preparation of a drug for inducing microglial immune tolerance memory to prevent and treat cerebral infarction. Background Art

[0002] Cerebral infarction (ischemic stroke) is the main cause of central nervous system damage, and its pathological mechanism involves acute ischemic hypoxia injury and chronic neuroinflammatory cascade. Current clinical treatment methods (such as thrombolysis and neuroprotectants) are limited by a narrow time window (rt-PA must be used within 4.5 hours) and immunosuppression-related side effects (such as the risk of infection induced by glucocorticoids), but two-thirds of patients still have sequelae of varying degrees. Studies have shown that microglia, as innate immune cells in the brain, have a double-edged sword effect in the regulation of inflammation after cerebral infarction: early M1 polarization exacerbates neuroinflammation, while M2 polarization promotes repair. Therefore, precise regulation of microglial phenotypic conversion has become a new therapeutic target.

[0003] Bacillus Calmette-Guérin (BCG) is a classic tuberculosis vaccine. In recent years, studies have found that it has the function of inducing immune memory, that is, through epigenetic reprogramming (such as histone modification and upregulation of metabolic enzyme expression), innate immune cells (such as monocytes and macrophages) produce enhanced or tolerant responses to subsequent stimuli. For example, BCG vaccination can significantly enhance the response of peripheral blood monocytes to β-glucan and reduce the inflammatory factor storm in the sepsis model. The application of BCG is concentrated in the prevention of infectious diseases or the treatment of bladder cancer. Its potential in the regulation of central nervous system immune memory has been neglected for a long time. In particular, there is a lack of targeted gene regulation research, which makes it difficult to achieve more effective signaling pathway intervention to determine the mechanisms that control the anti-inflammatory polarization and apoptosis of microglia.

[0004] In recent years, there have been many studies on drugs for treating cerebral infarction. For example, the prior art with document number CN117257803A discloses the use of lurasidone in the preparation of drugs for treating or preventing ischemia / reperfusion injury and cell protection drugs. Lurasidone can reduce myocardial infarction area and cerebral infarction volume, reduce serum creatine kinase activity and improve neurological function, reduce myocardial cell and nerve cell death, and has myocardial cell and nerve cell protection effects. The prior art with document number CN103467578A discloses a short peptide, copper-loaded nanobiomaterials and their use in the preparation of drugs for treating cerebral infarction.

[0005] At present, there is no record of the use of BCG in the preparation of drugs for the prevention and treatment of cerebral infarction, nor is there any relevant research on the mechanism of BCG as a drug for the prevention and treatment of cerebral infarction. Summary of the invention

[0006] The technical problems to be solved by the present invention are:

[0007] Currently, there is a lack of drugs in clinical practice that can prevent and treat cerebral infarction by inducing microglial immune tolerance memory.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] The present invention provides the use of BCG in the preparation of a drug for preventing and treating cerebral infarction, wherein the mechanism of the application is to prevent and treat cerebral infarction by inducing microglial immune tolerance memory with BCG.

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

[0011] Furthermore, BCG is used to reduce the level of cleaved-caspase3 protein.

[0012] Furthermore, BCG is used to promote the expression of TNFAIP3 gene mRNA in microglia.

[0013] Furthermore, the drug is BCG; or the drug is a drug prepared with BCG as the main ingredient and pharmaceutically acceptable excipients.

[0014] The present invention has been proved through in vivo studies on mice that administration of 5 μl / g of BCG (1 mg / ml) to mice can induce microglial immune tolerance memory to prevent and treat cerebral infarction, reduce cerebral infarction damage, and enhance the protective effect of nerves.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides the use of BCG in the preparation of drugs for the prevention and treatment of cerebral infarction. BCG reduces cerebral infarction damage by inducing microglia to produce immune tolerance memory. Experiments show that by pre-injection of BCG, the apoptosis rate of cells in mice with cerebral infarction can be reduced, the volume of cerebral infarction in mice can be reduced, and the recovery of neurological function can be significantly improved. BCG induces microglia to polarize toward the anti-inflammatory M2 type, reduces microglia activation in ischemic areas, inhibits the release of pro-inflammatory cytokines, and increases the expression of anti-inflammatory factors, thereby enhancing the neuroprotective effect.

[0017] The present study found that TNFAIP3 was upregulated in BCG-induced immune-tolerant microglia, reducing proinflammatory responses and cell damage by regulating the NF-κB signaling pathway. This targeted regulation of specific genes provides a more effective molecular basis for neuroprotection. The protein expression level was significantly increased after BCG treatment, promoting the activation of NF-κB in microglia and significantly inhibiting the phosphorylation of NF-κB in microglia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figures are comparative diagrams of the effects of BCG in the embodiments of the present invention on the recovery of neurological function and infarct volume of cerebral ischemia-reperfusion injury in mice, wherein (A) is the result of TTC staining, (B) is the result of quantitative analysis of infarct volume, (C) is the result of mNSS score, (D) is the result of Bederson score, and (E) is the result of neurological function evaluation of mice 1, 3, and 7 days after MCAO surgery by staggered experiment (n=6. *P<0.05, **P<0.01, ****P<0.0001, ns for no significance);

[0019] Figure 2 The figures are comparative diagrams of the effects of BCG on microglial activation and polarization in the examples of the present invention; wherein (A) is a graph showing the immunofluorescence intensity of Arg1+ / Iba-1+ detected by immunostaining in each group, and (B) is a graph showing the immunofluorescence intensity of Iba-1 detected by immunostaining 24 hours after MCAO / R surgery in each group (n=6, scale bar=50 μm, ****P<0.0001);

[0020] Figure 3 : The comparative diagram of the effect of BCG pretreatment on the cells in the brain of mice after MCAO / R in the embodiment of the present invention; wherein, (A) is the TUNEL and DAPI immunostaining diagram of each group 24 hours after MCAO / R surgery, (B) is the quantitative analysis diagram of TUNEL immunopositive cells, (C) is the TUNEL, Iba-1, and DAPI immunostaining diagram in the same visual field 24 hours after MCAO / R surgery in each group, (D) is the quantitative analysis diagram of TUNEL immunopositive cells Iba-1+, (E, F) are the expression and quantitative analysis diagrams of cleaved-caspase3 protein in each group 24 hours after MCAO / R surgery (n=6, scale bar=50μm, green represents TUNEL, red represents Iba-1, blue represents DAPI, ****P<0.0001, ns for no significance);

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

[0022] Figure 5The figure is a comparison of the BCG-treated microglial immune tolerance memory model and its effect after OGD / R in the embodiment of the present invention; wherein, (A, B) are the relative expression of I1-10 and TGF-β mRNA in BV2 cells after BCG treatment by RT-qPCR analysis, (C, D) are the relative expression of I1-10 and TGF-β mRNA in BV2 cells after OGD / R by RT-qPCR analysis, (E) is the effect of BCG pretreatment on the cell activity of BV2 cells after OGD / R, (F) is the expression of cleaved-caspase3 protein, apoptotic protein, in BV2 cells after OGD / R by BCG pretreatment (n=4, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns for no significance);

[0023] Figure 6 The results of the verification of the regulatory mechanism of BCG pretreatment on reducing apoptosis-related genes after cerebral ischemia-reperfusion injury in the embodiments of the present invention are shown in Figure 1; wherein, (A) is a graph of TNFAIP3 mRNA detection after BCG treatment, (B) is a graph of TNFAIP3 mRNA detection after overexpression of TNFAIP3 by plasmid, (C) is a graph of cell apoptosis detection by TUNEL method, (D) is a graph of cell apoptosis detection by flow cytometry, and (E) is a graph of cleaved-caspase3 protein determination (n=4, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns for no significance):

[0024] Figure 7 The figures are validation result diagrams of pathways related to increased TNFAIP3 gene expression regulation by BCG pretreatment in the embodiments of the present invention; wherein, (A) is a graph of TNFAIP3 mRNA detection after lentiviral knockdown of TNFAIP3 gene, (B) is a graph of cleaved-caspase3 protein expression after OGD / R detected by BCG pretreatment after TNFAIP3 knockdown, and (C) is a graph of changes in NF-κB phosphorylation levels after OGD / R detected by BCG pretreatment after TNFAIP3 knockdown (n=4, *P<0.05, **P<0.01, ****P<0.0001, ns for no significance). DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the scheme of the present invention, exemplary implementations or embodiments of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described implementations or embodiments are only implementations or embodiments of a part of the present invention, not all of them. Based on the implementations or embodiments of the present invention, all other implementations or embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present invention.

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are 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, namely, control group, PBS (buffer) + MCAO / R group and BCG + MCAO / R group. The mice in the BCG + MCAO / R group were intraperitoneally injected with BCG (1 mg / m1) 5 μl / g, and the mice in the PBS + MCAO / R group were intraperitoneally injected with PBS 5 μl / g. After 14 days, the MCAO / R model was established by cerebral ischemia reperfusion operation in the PBS + MCAO / R group and the BCG + MCAO / R group. Specifically, MCAO / R was induced in the mouse model using the thread embolization method, and the reperfusion process was monitored by laser speckle Doppler blood flowmeter to control the dynamic changes of cerebral blood flow before and after the operation. On the first day after the establishment of the (MCAO / R) model, TTC staining of brain tissue and determination of infarction volume of mice were performed on mice in each group.

[0029] The neurological function of mice was evaluated using mNSS score, Bederson score and staggered step test on the 1st, 3rd and 7th days after brain ischemia-reperfusion.

[0030] It was found that there were obvious infarcts after cerebral ischemia and reperfusion, indicating that the MCAO / R model was successfully established. The infarct volume was reduced after BCG pre-intervention (****P<0.0001, Figure 1B), and except for the Bederson score on the third day, there was no statistical difference. The mNSS, Bederson score, and percentage of staggered steps after MCAO / R in mice treated with BCG at three time points were significantly reduced compared with the PBS+MCAO / R group (Figure 1C, D, E). This shows that BCG can significantly improve the neurological function of MCAO / R mice. This shows that BCG pretreatment can improve the infarct volume and functional score of mice after MCAO / R.

[0031] The BCG vaccine in this embodiment adopts attenuated bovine tuberculosis BCG lyophilized powder 50 mg (Rui Chu Biological).

[0032] Example 2

[0033] On the 14th day of the experiment and 24 h after the MCAO / R model was established in Example 1, immunostaining was performed on each group.

[0034] It can be seen that the anti-inflammatory phenotype (Argl / Iba-1+) of microglia in the BCG pretreatment group increased compared with the PBS+MCAO / R group (Figure 2A), indicating that BCG pretreatment can promote the transformation of microglia to an anti-inflammatory phenotype. At the same time, it was found that 24 hours after cerebral ischemia-reperfusion, the number of activated microglia in the ischemic hemisphere cortex in 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 reduce the excessive activation of microglia after cerebral ischemia-reperfusion injury.

[0035] Example 3

[0036] 24 hours after the MCAO / R model was established in Example 1, TUNEL immunostaining and expression and quantitative analysis of cleaved-caspase3 protein were performed on each group.

[0037] The number of apoptotic cells in the ischemic hemisphere cortex of the PBS+MCAO / R group increased significantly, indicating that BCG pretreatment intervention can significantly reduce apoptosis (****P<0.0001, Figure 3A, B). 24 hours after cerebral ischemia-reperfusion, the number of apoptotic microglial cells in the ischemic hemisphere cortex of the PBS+MCAO / R group increased significantly, and BCG pretreatment intervention can significantly reduce microglial apoptosis (****P<0.0001, Figure 3C, D). After BCG intervention in advance, the level of cleaved-caspase3 protein in the BCG+MCAO / R group was significantly lower than that in the MCAO / R group (****P<0.0001, Figure 3E, F).

[0038] Example 4

[0039] The BV2 cell line was divided into control (PBS) group, BCG group, PBS+OGD group and BCG+OGD group. The control group was given PBS at a concentration of 1 μl / ml, and the BCG group was given BCG at a concentration of 50 ng / ml, once on the first day and the sixth day, respectively; the OGD / R model was established in the PBS+OGD group and the BCG+OGD group on the 8th day after the administration. Figure 4 ).

[0040] It can be seen that repeated administration of 50ng / ml BCG stimulation can induce microglia to express high levels of anti-inflammatory genes IL-10 and TGF-β. After OGD / R treatment, microglia can still maintain high levels of expression and are statistically significant. This indicates that BCG at a specific time and dose can induce microglia to express anti-inflammatory genes (*P<0.05, Figure 5A, B), and the anti-inflammatory gene phenotype can also be maintained after OGD / R, indicating that the microglial immune tolerance memory model was successfully constructed (*P<0.05, **P<0.01, Figure 5C, D), and BCG administration can increase the cell survival rate of microglia after OGD / R (**P<0.01, ***P<0.001, ****P<0.0001, Figure 5E), and reduce the expression of apoptotic protein cleaved-caspase3 protein (**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 differentially expressed genes.

[0043] It can be seen that repeated administration of 50ng / ml BCG stimulation promoted the expression of TNFAIP3 gene mRNA in microglia (**P<0.01, Figure 6A). Overexpression of TNFAIP3 gene by plasmid significantly increased the relative survival rate of microglia in the OGD / R group (****P<0.0001, Figure 6B). TUNEL staining and flow cytometry detection showed that the apoptosis rate of microglia after OGD / R was significantly decreased (*P<0.05, ****P<0.0001, Figure 6C, D), and the protein level of apoptotic protein cleaved-caspase3 was significantly reduced (**P<0.01, ****P<0.0001, Figure 6E).

[0044] Example 6

[0045] TNFAIP3 gene was knocked down in BV2 microglia after OGD / R treatment, and the expression of apoptosis-related proteins in BV2 microglia was examined.

[0046] By knocking down TNFAIP3 and lentiviral transfection of BV2 microglia, the mRNA expression of TNFAIP3 gene was significantly reduced (*P<0.05, **P<0.01, Figure 7A). After BCG pretreatment, the OGD / R model was treated, and the expression level of apoptotic protein cleaved-caspase3 was detected to be significantly increased in the TNFAIP3 genome knockdown group (Sh-TNFAIP3 group) (*P<0.05, Figure 7B). OGD / R promoted the activation of NF-κB in microglia. BCG pretreatment intervention could significantly inhibit the phosphorylation of NF-κB in microglia after OGD / R. TNFAIP3 knockout could significantly reverse the inhibitory effect of BCG on NF-κB phosphorylation in microglia under OGD / R (****P<0.0001, Figure 7C).

[0047] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. The use of BCG in the preparation of drugs for preventing and treating cerebral infarction, characterized in that: The mechanism of the 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 in the preparation of a drug for preventing and treating cerebral infarction, characterized in that: The application of BCG in reducing cerebral infarction volume and improving neurological function.

3. The use of BCG vaccine according to claim 1 in the preparation of a drug for preventing and treating cerebral infarction, characterized in that: Application of BCG in reducing cleaved-caspase3 protein levels.

4. The use of BCG vaccine according to claim 1 in preparing a drug for preventing and treating cerebral infarction, characterized in that: Application of BCG in promoting the expression of TNFAIP3 gene mRNA in microglia.

5. The use of BCG vaccine according to claim 1 in preparing a drug for preventing and treating cerebral infarction, characterized in that: The drug is BCG; or the drug is a drug prepared with BCG as a main component and pharmaceutically acceptable excipients.

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