Application of 5-methyltetrahydrofolic acid in regulating effect CD8 + T cells

Abstract

The invention discloses application of 5-methyltetrahydrofolic acid in regulating effect CD8 + T cells, belongs to the technical field of biological medicines, and provides and verifies the application of the 5-methyltetrahydrofolic acid in regulating effect CD8 + T cells for the first time. PBS-treated wild type NOD mouse CD8 + T primary cells, 5-methyltetrahydrofolic acid-treated wild type NOD mouse CD8 + T primary cells, common feed-fed wild type NOD mice and 5-methyltetrahydrofolic acid-containing feed-fed wild type NOD mice are taken as research objects, and the effect of the 5-methyltetrahydrofolic acid effect CD8 + T cells is detected and explored through flow cytometry; according to the application of 5-methyltetrahydrofolic acid to the treatment of the CD8 + T cells, the 5-methyltetrahydrofolic acid is found to have an inhibition effect on activation and proliferation of the effect CD8 + T cells, a new thought is provided for a disease treatment scheme mainly based on the targeting effect CD8 + T cells, and the 5-methyltetrahydrofolic acid is expected to become a novel treatment scheme aiming at autoimmune diseases caused by the effect CD8 + T cells and has very wide market prospects and great significance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of 5-methyltetrahydrofolate in regulating effector CD8+T cells. Background Art

[0002] CD8+T cells are an important type of immune cell, whose main functions and effects include killing infectious pathogens, participating in anti-tumor immune responses, participating in the occurrence and development of autoimmune diseases, and participating in transplant rejection reactions.

[0003] 1. Participate in the occurrence and development of autoimmune diseases: In some cases, CD8+ T cells mistake their own normal tissues as foreign bodies and attack them, leading to the occurrence and development of autoimmune diseases.

[0004] 2. Killing infected pathogens: CD8+T cells can recognize and kill infected cells, such as virus-infected cells, cancer cells, etc.

[0005] 3. Participate in anti-tumor immune response: CD8+T cells are one of the main effector cells in the body's anti-tumor immune response. They can directly kill tumor cells or regulate the functions of other immune cells by secreting cytokines, thereby exerting an anti-tumor effect.

[0006] 4. Participate in transplant rejection reaction: After organ transplantation, due to tissue differences between the donor and the recipient, CD8+T cells will be activated and attack the graft, leading to the occurrence of transplant rejection reaction.

[0007] In short, CD8+T cells play an important role in the body's immune defense and the occurrence and development of diseases.

[0008] Type 1 Diabetes (T1D) is a metabolic disease characterized by absolute insulin deficiency, caused by the body's immune system disorder attacking its own pancreatic β cells, resulting in massive β cell death. Nearly 40 years of continuous systematic research has revealed that once the immune response against one's own β cells is initiated, it is difficult to prevent the occurrence and development of T1D, resulting in the lack of effective treatment measures to date. Therefore, once diagnosed, lifelong dependence on exogenous insulin is required, which makes it difficult to maintain blood sugar homeostasis and induces a variety of metabolic complications. T1D is more common in adolescents, and with the tremendous changes in people's lifestyles and environments, the incidence of T1D in my country has been rising year by year, and it has now become the country with the largest number of T1D patients.

[0009] Autoreactive effector T cells infiltrate the pancreatic islets in T1D. In the non-obese diabetic (NOD) mouse model, the dysfunction of CD8+T cells infiltrating the pancreatic islets is a key factor leading to the massive destruction of β cells. Antigen-specific CD8+T cell subsets, namely type 1 diabetes-causing CD8+T cells, induce apoptosis of pancreatic β cells in a contact-dependent or contact-independent manner, including the secretion of IFN-γ, granzyme B, perforin, TNF-α and other cytokines. Studies have shown that the number and proportion of effector CD8+T cells in patients with autoimmune diseases such as type 1 diabetes increase. Therefore, inhibiting the activation and proliferation of effector CD8+T cells is of great significance for reducing their pathogenicity and treating effector CD8+T cell-related diseases.

[0010] 5-Methyltetrahydrofolate (5-MTHF) is the biologically active form of folic acid, the main form of folic acid in human plasma, and the only form that can cross the blood-brain barrier. Folic acid is mainly supplemented through diet and intestinal microbial synthesis, and it acts as a carrier of one-carbon units in metabolic reactions. Studies have shown that folic acid can only have a protective effect on diseases such as neural tube defects, megaloblastic anemia, and cancer when it is converted into 5-MTHF in the presence of methylenetetrahydrofolate reductase. Possible explanations include the regulation of signaling pathways such as lowering homocysteine ​​concentration and oxidative stress. However, about 26% of Chinese people lack this methylenetetrahydrofolate reductase, which reduces the benefits of folic acid supplementation, and the bioavailability of 5-MTHF is about 7 times that of folic acid. Therefore, direct supplementation of 5-MTHF is the key to exerting its beneficial effects.

[0011] Currently, there is no literature report on the application of 5-methyltetrahydrofolate in regulating effector CD8+T cells. Summary of the invention

[0012] The purpose of the present invention is to provide an application of 5-methyltetrahydrofolate in regulating effector CD8+T cells, to provide a new idea for disease treatment programs mainly targeting effector CD8+T cells, and is expected to become a new treatment program for autoimmune diseases caused by effector CD8+T cells, which has a very broad market prospect and extremely significant significance.

[0013] In order to achieve the above purpose, this application adopts the following technical solutions:

[0014] In a first aspect, the present invention provides a use of 5-methyltetrahydrofolate in regulating effector CD8+T cells, wherein the 5-methyltetrahydrofolate inhibits the activation and proliferation of effector CD8+T cells.

[0015] In a second aspect, the present invention provides the use of 5-methyltetrahydrofolate in the preparation of a drug for treating diseases associated with excessive activation of CD8+T cells.

[0016] In the above technical solution, the diseases associated with excessive activation of CD8+T cells include type 1 diabetes, rheumatoid arthritis and systemic sclerosis.

[0017] In the above technical solution, the drug is a pharmaceutical composition composed of a therapeutically effective amount of 5-methyltetrahydrofolate as the only active ingredient and pharmaceutical excipients.

[0018] In the above technical solution, the drug is an oral administration preparation, an injection administration preparation, a mucosal administration preparation or a transdermal administration preparation.

[0019] In the above technical solution, the pharmaceutical excipient is selected from one or more of a disintegrant, a filler, a binder, a flavoring agent, a lubricant, a preservative, an antioxidant, and a colorant.

[0020] In a third aspect, the present invention provides a drug for preventing and treating diseases related to excessive activation of CD8+T cells, wherein the drug is a pharmaceutical composition consisting of a therapeutically effective amount of 5-methyltetrahydrofolate as the sole active ingredient and pharmaceutical excipients.

[0021] In the above technical solution, the diseases associated with excessive activation of CD8+T cells include type 1 diabetes, rheumatoid arthritis and systemic sclerosis.

[0022] In the above technical solution, the drug inhibits the activation and proliferation of effector CD8+T cells.

[0023] The beneficial effect of the present invention is that the present invention proposes and confirms the application of 5-methyltetrahydrofolate in regulating effector CD8+T cells for the first time. Using wild-type NOD mouse CD8+T primary cells treated with PBS and wild-type NOD mouse CD8+T primary cells treated with 5-methyltetrahydrofolate, wild-type NOD mice fed with ordinary feed, and wild-type NOD mice fed with feed containing 5-methyltetrahydrofolate as research objects, the effect of 5-methyltetrahydrofolate on effector CD8+T cells was explored by flow cytometry detection, and it was found that 5-methyltetrahydrofolate had an inhibitory effect on the activation and proliferation of effector CD8+T cells, providing new ideas for disease treatment schemes mainly targeting effector CD8+T cells, and is expected to become a new treatment scheme for autoimmune diseases caused by effector CD8+T cells, which has a very broad market prospect and extremely important significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the monitoring results of the incidence of mice in each group in the embodiment of the present invention;

[0025] Figure 2 H&E staining of pancreatic tissue pathological sections of mice in each group in the embodiment of the present invention;

[0026] Figure 3 This is a graph showing the CD8+T cell factor levels in each group of the in vitro model in the embodiments of the present invention;

[0027] Figure 4 This is a graph showing the proliferation level of CD8+T cells in each group of the in vitro model in the embodiment of the present invention;

[0028] Figure 5 This is a graph showing the CD8+T cell factor levels in each group of the in vivo model in the examples of the present invention. DETAILED DESCRIPTION

[0029] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0030] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0031] 1. Experimental process

[0032] 1. The effect of 5-MTHF on delaying the onset of type 1 diabetes

[0033] Mouse model establishment: 5-MTHF reagent was purchased from Macklin, with the catalog number L879618. Thirty 6-week-old wild-type NOD mice were divided into two groups, a control group and an experimental group, with 15 mice in each group. The control group was given ordinary mouse maintenance feed and the experimental group was fed with 100 mg / kg 5-methyltetrahydrofolate mouse feed, and both groups of mice were raised in an SPF-grade experimental animal room. Blood glucose was measured from the end of the mouse limbs weekly using a blood glucose meter, and the blood glucose concentration was ≥13.8 mmol / L as the onset. The weekly incidence of the two groups of mice was calculated and compared.

[0034] 2. Effect of 5-MTHF on Alleviating Insulitis in Type 1 Diabetes

[0035] H&E staining of mouse pancreatic tissue pathology sections: 3 mice in each of the control group and experimental group fed for 12-15 weeks were euthanized, and the pancreas of the mice was surgically removed and placed in a paraffin embedding frame. After fixation with 10% neutral formaldehyde for 24 hours, it was dehydrated using a tissue dehydration instrument. The dehydrated tissue was embedded in paraffin, and then cut into 4-micron slices using a slicer and adhered to an adhesive slide. 3-4 slices were made for the pancreas of each mouse. The slices were placed in a fully automatic H&E staining machine for staining. The stained slices were placed under an Olympus microscope for observation and photographed for record.

[0036] 3. Effect of 5-MTHF on the inhibition of effector CD8+T cell activation in vitro

[0037] MojoSort TM Mouse CD8+T Cell Isolation Kit was purchased from BioLegend with catalog number 480008.

[0038] Cytokine detection experiment: After euthanizing 6-week-old wild-type NOD mice, the spleen and pancreas were removed respectively. Use a 70-micron filter to grind the mouse spleen and pancreas to obtain a cell suspension, and add RPMI-1640 culture medium during the process. Centrifuge the obtained cell suspension at 300×g for 5 minutes. Discard the supernatant, add 1ml of red blood cell lysis buffer to the cell pellet and resuspend it, and let it stand at room temperature for 5 minutes. Add 5ml of RPMI-1640 culture medium to each tube to terminate the lysis, and centrifuge the mixture at 300×g for 5 minutes. Discard the supernatant, and centrifuge it for 10 minutes. 7 Add 100ul MojoSort TM Buffer was resuspended, the cell suspension was transferred to a sterile flow cytometry tube, and then 10ul antibody cocktail was added, and the cells were incubated on ice for 15 minutes. After the incubation, Streptavidin Nanobeads with the same volume as antibody cocktail was added to the cell suspension and mixed, and the cells were incubated on ice for 15 minutes. After the incubation, 2.5ml MojoSort TM Buffer to terminate the reaction. Place the sterile flow tube in a magnet for 5 minutes. Pour out and collect the liquid in the tube, centrifuge at 300×g for 5 minutes to obtain CD8+T cell precipitate. Use an appropriate volume of T cell culture medium (RPMI-1640 culture medium + 10% serum + 1% double antibody + 1% GlutaMAX TM +1% β-mercaptoethanol) to resuspend CD8+ T cells.

[0039] A control group and an experimental group were set up, with 5 replicate wells in each group. 10 ng / ml mouse IL-2 was added to both the experimental group and the control group, and 400 nM 5-MTHF and the same volume of sterile PBS were added respectively. The cells were plated in a 96-well cell culture plate that had been coated with 5 ug / ml anti-CD3 and 5 ug / ml anti-CD28 at 4 degrees overnight in advance and cultured for 48 hours.

[0040] 4-6 hours before the end of 48 hours, add 500× cell stimulation cocktail (a mixture of phorbol, 12-myristic acid, 13-acetate (PMA), ionomycin, Brefeldin A and monensin). After 48 hours of culture, collect cells into flow tubes and set up blank control and single staining groups. Add 1ml PBS to the cells, centrifuge at 300×g for 5 minutes, and discard the supernatant. At the same time, prepare the surface flow staining antibody solution, according to 100ul PBS and 0.3ul antibody per tube. The antibodies used are purchased from Biolegend, including FITC anti-mouse CD8a Antibody (100706). Add 100ul antibody solution to each tube, add 100ul1640 without antibody to the blank control, and add 100ul antibody solution containing 0.3ul single antibody to the single staining group, and stain on ice for 20 minutes. After staining, add 1ml PBS to stop staining, centrifuge at 300×g for 5 minutes, and discard the supernatant. Add 100ul of diluted ELISA to each tube and fix on ice for 30 minutes. After fixation, add 1ml of diluted ELISA to each tube, centrifuge at 300×g for 5 minutes, and discard the supernatant. At the same time, prepare the intracellular staining antibody solution. Prepare the antibody solution according to 100ul of diluted ELISA per tube and 0.3ul of antibody. The antibodies used were purchased from Biolegend, including PE anti-mouse Perforin Antibody (154306); APC anti-human / mouse Granzyme B Recombinant Antibody (372204); PE / Cyanine7 anti-mouse IFN-γAntibody (505826); Brilliant Violet 510 TM Anti-mouseTNF-αAntibody(506339). Add 100ul antibody solution to each tube, add 100ul1640 without antibody to blank control, add 100ul antibody solution containing 0.3ul single antibody to single staining group, and incubate at 4 degrees overnight.

[0041] After incubation, 1 ml of PBS was added to each tube to terminate staining, and the tube was centrifuged at 300 × g for 5 minutes. The supernatant was discarded, and 200 μl of 1% paraformaldehyde was added to each tube for fixation, followed by detection using an Attune NxT flow cytometer (Thermo Fisher). The fluorescence of FITC was detected in the BL1 channel; the fluorescence of PE was detected in the BL3 channel; the fluorescence of APC was detected in the RL1 channel; the fluorescence of PE-CY7 was detected in the BL4 channel; and the fluorescence of BV510 was detected in the VL2 channel.

[0042] 4. Effect of 5-MTHF on the proliferation inhibition of effector CD8+T cells

[0043] MojoSort TM Mouse CD8+T Cell Isolation Kit was purchased from BioLegend with catalog number 480008.

[0044] After euthanizing 6-week-old wild-type NOD mice, the spleen and pancreatic draining lymph nodes were removed. Use a 70-micron filter to grind the mouse spleen and pancreatic draining lymph nodes to obtain a cell suspension, and add RPMI-1640 culture medium during the process. Centrifuge the obtained cell suspension at 300×g for 5 minutes. Discard the supernatant, add 1ml of red blood cell lysis buffer to the cell pellet and resuspend it, and let it stand at room temperature for 5 minutes. Add 5ml of RPMI-1640 culture medium to each tube to terminate the lysis, and centrifuge the mixture at 300×g for 5 minutes. Discard the supernatant and centrifuge it for 10 minutes. 7 Add 100ul MojoSort TM Buffer was resuspended, the cell suspension was transferred to a sterile flow cytometry tube, and then 10ul antibody cocktail was added, and the cells were incubated on ice for 15 minutes. After the incubation, Streptavidin Nanobeads with the same volume as antibody cocktail was added to the cell suspension and mixed, and the cells were incubated on ice for 15 minutes. After the incubation, 2.5ml MojoSort TM Buffer to terminate the reaction. Place the sterile flow tube in a magnet for 5 minutes. Pour out and collect the liquid in the tube, centrifuge at 300×g for 5 minutes to obtain CD8+T cell precipitate. Use an appropriate volume of T cell culture medium (RPMI-1640 culture medium + 10% serum + 1% double antibody + 1% GlutaMAX TM +1% β-mercaptoethanol) to resuspend CD8+ T cells.

[0045] A control group and an experimental group were set up, with 5 replicates in each group. 10ng / ml mouseIL-2 was added to both the experimental group and the control group, and 400nM 5-MTHF and the same volume of sterile PBS were added respectively. The cells were plated in a 96-well cell culture plate coated with 5ug / ml anti-CD3 and 5ug / ml anti-CD28 at 4 degrees overnight and cultured for 48 hours. Cell proliferation level detection experiment: After 48 days of culture, some cells were collected into flow tubes, and blank controls and single staining groups were set up. 1ml PBS was added to the cells, centrifuged at 300×g for 5 minutes, and the supernatant was discarded. At the same time, the surface flow staining antibody solution was prepared, and the antibody solution was prepared according to 100ul PBS and 0.3ul antibody per tube. The antibodies used were purchased from Biolegend, including FITC anti-mouse CD8a Antibody (100706). Add 100ul antibody solution to each tube, add 100ul 1640 without antibody to the blank control, add 100ul antibody solution containing 0.3ul single antibody to the single staining group, and stain on ice for 20 minutes. After staining, add 1ml PBS to stop staining, centrifuge at 300×g for 5 minutes, and discard the supernatant. Add 100ul diluted to each tube and fix on ice for 30min. After fixation, add 1ml diluted to stop fixation to each tube, centrifuge at 300×g for 5 minutes, and discard the supernatant. At the same time, prepare the intracellular staining antibody solution, according to 100ul diluted, 0.3ul antibody to each tube. The antibodies used are purchased from Biolegend, including PE anti-mouse Ki-67 Antibody (652404). Add 100ul antibody solution to each tube, add 100ul 1640 without antibody to the blank control, and add 100ul antibody solution containing 0.3ul single antibody to the single staining group, and incubate overnight at 4 degrees.

[0046] CTV-labeled proliferating cell flow cytometry: After CD8+T cells are taken out, a blank control is first collected and then labeled. The cell concentration is adjusted to 106cell / ul, and CTV is prepared with 1640 or PBS at a concentration of 0.25uM, added in a volume of 0.2ul / ml, and stored in the dark. Add 1ml of diluted dye to each tube of cells, wrap with tin foil, and incubate at 37 degrees for 3-5min for staining. After staining, add 4ml of 1640 to each tube of cells to terminate the staining, centrifuge at 400×g for 4 minutes, discard the supernatant, resuspend with an appropriate volume of T cell culture medium, and then plate. After culturing in the dark for 48h, collect the cells and detect them in the VL2 channel of the flow cytometer.

[0047] 5. The inhibitory effect of 5-MTHF on effector CD8+ T cell activation in vivo

[0048] Mouse model establishment: 5-MTHF reagent was purchased from Macklin, with the catalog number L879618. Thirty 6-week-old wild-type NOD mice were divided into two groups, a control group and an experimental group, with 15 mice in each group. The control group was fed with ordinary mouse maintenance feed, and the experimental group was fed with mouse feed containing 52 mg / kg 5-methyltetrahydrofolate. Both groups of mice were kept in an SPF-grade experimental animal room.

[0049] After euthanizing 6-week-old wild-type NOD mice, the spleen and pancreatic draining lymph nodes were removed. The spleen and pancreatic draining lymph nodes of mice were ground using a 70-micron filter to obtain a cell suspension, and RPMI-1640 medium was added during the process. The obtained cell suspension was centrifuged at 300×g for 5 minutes. The supernatant was discarded, and 1ml of red blood cell lysis buffer was added to the cell pellet to resuspend it and placed at room temperature for 5 minutes. 5ml of RPMI-1640 medium was added to each tube to terminate the lysis, and the mixture was centrifuged at 300×g for 5 minutes. The supernatant was discarded, the cells were plated into a 96-well plate, and 500× cell stimulation cocktail (a mixture of phorbol, 12-myristic acid, 13-acetate (PMA), ionomycin, Brefeldin A and monensin) was added, and incubated at 37° in an incubator for 4-6 hours. After incubation, the cells were collected into a flow tube, and a blank control and a single staining group were set up. Add 1ml PBS to the cells, centrifuge at 300×g for 5 minutes, and discard the supernatant. At the same time, prepare the surface flow staining antibody solution, prepare the antibody solution according to 100ul PBS and 0.3ul antibody per tube. The antibodies used are purchased from Biolegend, including FITC anti-mouse CD8a Antibody (100706). Add 100ul antibody solution to each tube, add 100ul1640 without antibody to the blank control, add 100ul antibody solution containing 0.3ul single antibody to the single staining group, and stain on ice for 20 minutes. After staining, add 1ml PBS to stop staining, centrifuge at 300×g for 5 minutes, and discard the supernatant. Add 100ul diluted to each tube and fix on ice for 30min. After fixation, add 1ml diluted to stop fixation to each tube, centrifuge at 300×g for 5 minutes, and discard the supernatant. At the same time, the intracellular staining antibody solution was prepared. After dilution, 0.3ul antibody was added to each tube. The antibodies used were purchased from Biolegend, including PE anti-mouse Perforin Antibody (154306); APC anti-human / mouse Granzyme B Recombinant Antibody (372204); PE / Cyanine7 anti-mouse IFN-γ Antibody (505826); BrilliantViolet 510TM Anti-mouse TNF-αAntibody (506339). Add 100ul antibody solution to each tube, add 100ul 1640 without antibody to the blank control, add 100ul antibody solution containing 0.3ul single antibody to the single staining group, and incubate at 4 degrees overnight.

[0050] After incubation, 1 ml of PBS was added to each tube to terminate staining, and the tube was centrifuged at 300 × g for 5 minutes. The supernatant was discarded, and 200 μl of 1% paraformaldehyde was added to each tube for fixation, followed by detection using an Attune NxT flow cytometer (Thermo Fisher). The fluorescence of FITC was detected in the BL1 channel; the fluorescence of PE was detected in the BL3 channel; the fluorescence of APC was detected in the RL1 channel; the fluorescence of PE-CY7 was detected in the BL4 channel; and the fluorescence of BV510 was detected in the VL2 channel.

[0051] 2. Experimental Results

[0052] 1. Compared with the control group, 5-methyltetrahydrofolate significantly delayed the onset of type 1 diabetes in NOD mice

[0053] The incidence of type 1 diabetes (blood sugar>13.8mmol / L) in mice fed with ordinary maintenance diet increased with the early onset of type 1 diabetes, while the development of type 1 diabetes in mice fed with 5-methyltetrahydrofolate diet was relatively delayed. At the same time, the incidence of type 1 diabetes in mice fed with 5-methyltetrahydrofolate diet was significantly lower than that in mice fed with ordinary maintenance diet over time. The experimental results show that supplementation with 5-methyltetrahydrofolate can significantly delay the onset of type 1 diabetes in NOD mice and reduce the incidence ( Figure 1 ).

[0054] 2. Compared with the control group, 5-methyltetrahydrofolate significantly alleviated insulitis in NOD mice

[0055] H&E staining of pancreatic islet histopathological sections showed that in the late diabetic stage (12-15 weeks), the islet infiltrative inflammation (infiltration in the islets > 50%) in the mice fed with 5-methyltetrahydrofolate was less than that in the control group ( Figure 2 A), as assessed by the insulitis score, the control group showed a higher degree of insulitis cell infiltration ( Figure 2 B).

[0056] 3. Compared with the control group, 5-methyltetrahydrofolate significantly inhibited the activation of in vitro effector CD8+T cells

[0057] Compared with the control group, in the CD8+T cell effector activation model established in vitro, the spleen (Spleen) ( Figure 3 A) and pancreatic draining lymph nodes (PLN) ( Figure 3 B) The levels of inflammatory factors such as Granzyme B, Perforin, IFN-γ, and TNF-a in CD8+T cells derived from the cells were significantly reduced. The CD8+T cells in the control group showed a clear immune activation phenotype. The concentration of 5-methyltetrahydrofolate used here was 400nM

[0058] 4. Compared with the control group, 5-methyltetrahydrofolate significantly inhibited the proliferation of in vitro effector CD8+T cells

[0059] Compared with the control group, in the CD8+T cell effector activation model established in vitro, the proportion of CD8+T cells from spleen and PLN of wild-type NOD mice labeled with CTV was significantly lower than that of the control group after 5-methyltetrahydrofolate treatment ( Figure 4 A), 5-methyltetrahydrofolate also significantly reduced the proportion of Ki-67 positive CD8+T cells ( Figure 4 B). This indicates that 5-methyltetrahydrofolate inhibits the proliferation of type 1 diabetic CD8+ T cells.

[0060] 5. Compared with the control group, 5-methyltetrahydrofolate significantly inhibited the activation of effector CD8+T cells in PLN. Compared with the control group, in the NOD mouse animal model treated with 5-methyltetrahydrofolate, the spleen (Spleen) ( Figure 5 A) and pancreatic draining lymph nodes (PLN) ( Figure 5 B) The levels of inflammatory factors such as Granzyme B, Perforin, IFN-γ, and TNF-a in effector CD8+T cells were significantly reduced. CD8+T cells in the control group showed a clear immune activation phenotype.

[0061] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of 5-methyltetrahydrofolate in regulating effector CD8+T cells, wherein 5-methyltetrahydrofolate inhibits the activation and proliferation of effector CD8+T cells. Application of 2,5-methyltetrahydrofolate in the preparation of drugs for treating diseases associated with excessive activation of CD8+T cells.

3. The application according to claim 2, characterized in that: The 5-methyltetrahydrofolate inhibits effector CD8+ T cell activation and proliferation.

4. The application according to claim 2, characterized in that: Diseases associated with excessive activation of CD8+ T cells include type 1 diabetes, rheumatoid arthritis and systemic sclerosis.

5. The application according to claim 2, characterized in that: The medicine is a pharmaceutical composition comprising a therapeutically effective amount of 5-methyltetrahydrofolate as the only active ingredient and pharmaceutical excipients.

6. The application according to claim 2, characterized in that: The medicine is an oral preparation, an injectable preparation, a mucosal preparation or a transdermal preparation.

7. The use according to claim 5, characterized in that: The pharmaceutical excipients are selected from one or more of disintegrants, fillers, binders, flavoring agents, lubricants, preservatives, antioxidants, and colorants.

8. A drug for preventing and treating diseases related to excessive activation of CD8+T cells, characterized in that: The medicine is a pharmaceutical composition comprising a therapeutically effective amount of 5-methyltetrahydrofolate as the only active ingredient and pharmaceutical excipients.

9. The drug according to claim 8, characterized in that: Diseases associated with excessive activation of CD8+ T cells include type 1 diabetes, rheumatoid arthritis and systemic sclerosis.

10. The drug according to claim 8, characterized in that: The drug inhibits effector CD8+ T cell activation and proliferation.

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