Application of LFA-1 neutralizing antibody in medicine for relieving neuroinflammation of Parkinson's disease
Treatment of Teff cells through LFA-1 neutralizing antibodies solved the problems of limited efficacy and drug resistance in Parkinson's disease treatment, significantly alleviating the decline in cell viability, apoptosis and necrosis of Parkinson's cell model, and providing more effective nerves Inflammation treatment plan.
Patent Information
- Application Number
- CN202411413930.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-11
- Publication Date
- 2025-01-10
AI Technical Summary
The existing treatments for neuroinflammatory drugs for Parkinson's disease are limited in efficacy, strong resistance, and the toxic and side effects of temozolomide are relatively large, so patients are prone to drug resistance.
Teff cells were pretreated with LFA-1 neutralizing antibodies to reduce cell viability, apoptosis and necrosis in Parkinson's cell model, and reduce apoptotic protein expression, thereby reducing neuroinflammatory response.
Through LFA-1 neutralizing antibody treatment, the apoptosis and necrosis of DA neurons caused by Teff cells were significantly alleviated, and the neuroinflammatory response was alleviated, providing a theoretical basis for clinical practice.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of LFA-1 neutralizing antibodies in drugs for alleviating neuroinflammation in Parkinson's disease. Background Art
[0002] Parkinson's disease (PD) is a common chronic neurodegenerative disease. Recent studies have shown that the loss of dopaminergic neurons in the brain of PD model mice is accompanied by neuroinflammatory reactions. In addition to the activation of microglia, the innate immune cells residing in the brain, which can cause neuroinflammatory reactions, CD4+T cells invading the periphery are also involved in the neuroinflammation of PD. Effector T cells (Teff) are a subpopulation of CD4+T cells and are very important immune effector cells in the human body. Teff cells entering the central nervous system also have the function of aggregating chemokines, thereby aggravating microglia-induced neuroinflammatory reactions and leading to neuronal damage. Postmortem examinations of PD patients found that the loss of DA neurons was accompanied by obvious glial activation, Teff cells invading the central nervous system, and elevated inflammatory factors in cerebrospinal fluid and brain tissue. Studies have reported that the interaction between intercellular adhesion molecule-1 (ICAM-1) on vascular endothelial cells and lymphocyte function-associated antigen 1 (LFA-1) on immune cells mediates the adhesion and migration of leukocytes and the invasion of activated leukocytes into central inflammatory sites. LFA-1 is a target molecule for intercellular communication and is involved in a series of inflammatory and immune responses. Recent studies have shown that the LFA-1 binding protein of Teff cells is associated with the inflammatory response and apoptosis in PD brain tissue in which these cells participate. In model animal experiments, Teff cells have LFA-1 receptor activity. After drug intervention, this activity and the expression of Teff cells in the inflammatory and apoptotic states of the brain are significantly reduced, indicating that LFA-1-related Teff cells can regulate inflammatory responses and apoptosis in PD brains. Summary of the invention
[0003] Technical issues solved:
[0004] The present application aims at the deficiencies in the prior art and solves the technical problems faced by current glioma drugs in treatment, such as limited efficacy and drug resistance, strong toxic side effects of temozolomide, and patients are prone to develop drug resistance thereto. The application of LFA-1 neutralizing antibodies in drugs for alleviating neuroinflammation in Parkinson's disease is provided. After pretreatment with LFA-1 neutralizing antibodies, the cell viability of the PD cell model caused by Teff cells is decreased, the number of apoptotic and necrotic cells is increased, the LDH release is increased, and the expression of apoptotic proteins is upregulated, which indicates that LFA-1 neutralizing antibodies can reduce the apoptosis and necrosis of DA neurons caused by Teff cells, thereby reducing neuroinflammatory reactions. The present application provides more theoretical basis for the clinical practice of LFA-1 neutralizing antibodies.
[0005] Technical solution:
[0006] To achieve the above objectives, this application is implemented through the following technical solutions:
[0007] Application of LFA-1 neutralizing antibodies in drugs to reduce neuroinflammation in Parkinson's disease.
[0008] Furthermore, the application of LFA-1 neutralizing antibodies in alleviating cell damage in PD cell models.
[0009] Furthermore, LFA-1 neutralizing antibodies were used to reduce cell apoptosis and necrosis in PD cell models.
[0010] Furthermore, LFA-1 neutralizing antibodies were used to reduce the expression of apoptotic proteins in PD cell models.
[0011] Beneficial effects:
[0012] The present application provides the use of LFA-1 neutralizing antibodies in drugs for alleviating neuroinflammation in Parkinson's disease, which has the following beneficial effects compared with the prior art:
[0013] The present invention mainly studies the effect of Teff cells treated with LFA-1 neutralizing antibodies on cell viability, apoptotic and necrotic cell numbers, LDH release, and apoptotic protein expression in PD cell models. The study of this application shows that after pretreatment with LFA-1 neutralizing antibodies, the cell viability of the PD cell model caused by Teff cells decreased, the number of apoptotic and necrotic cells increased, the LDH release increased, and the up-regulation of apoptotic protein expression were all alleviated, indicating that LFA-1 neutralizing antibodies can reduce DA neuron apoptosis and necrosis caused by Teff cells, thereby reducing neuroinflammatory reactions. The present invention provides more theoretical basis for the clinical practice of LFA-1 neutralizing antibodies. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1The Teff cells pretreated with LFA-1 neutralizing antibody in this application are + Effect diagram of induced MN9D cell viability;
[0015] Figure 2 The Teff cells pretreated with LFA-1 neutralizing antibody in this application are + The effect diagram of induced apoptosis and necrosis of MN9D cells; the upper figure in A is a flow cytometry scatter diagram of apoptosis and necrosis of MN9D cells detected by flow cytometry, the lower left figure in A is a statistical diagram of the percentage of necrotic cells, the lower right figure in A is a statistical diagram of the percentage of apoptotic cells, the left figure in B is a green fluorescence diagram of TUNEL staining to detect apoptosis-positive cells, the right figure in B is a statistical diagram of the percentage of TUNEL-positive cells in the total cells, and C is a statistical diagram of LDH release experiment;
[0016] Figure 3 The Teff cells pretreated with LFA-1 neutralizing antibody in this application are + The effect diagram of induced apoptotic protein expression in MN9D cells, where A is a representative immunoblot of two apoptotic proteins, cleaved-caspase-3 and cleaved-caspase-9, and B is the corresponding statistical diagram. DETAILED DESCRIPTION
[0017] In this section, the specific embodiments of this patent application will be described in detail in conjunction with the accompanying drawings, aiming to provide a clear and comprehensive description of the technical solution. It should be clear that the embodiment described here only represents one of the possible applications of this patent and does not represent all possible implementation methods. Based on the content of this document, a person of ordinary skill in the art can derive all other potential embodiments covered by this application without performing any creative work. Therefore, these embodiments should all be deemed to be within the scope of protection of this patent.
[0018] The MN9D cell line is a hybrid cell line formed by the fusion of DA neurons of the primary mouse embryonic ventral midbrain and the N18TG2 neuroblastoma cell line. It has the characteristics of midbrain DA neurons, can synthesize, release and absorb DA, and can express tyrosine hydroxylase. Therefore, it is widely used in PD-related research. + ) stimulated MN9D cells to simulate the in vitro PD model. The LFA-1 molecule expressed on Teff cells is considered to be a key factor in mediating the invasion of immune inflammatory cells into the central nervous system and participating in the inflammatory response of the central nervous system during the course of PD.
[0019] This application adopts MPP +The MN9D cell line was stimulated to prepare a PD cell model, and the Teff cells separated and purified in vitro were added to the PD cell model to observe the effect of Teff cells on the aggravation of the inflammatory response of the PD cell model; then the Teff cells were pretreated with LFA-1 neutralizing antibody and then added to the PD cell model to observe the reduction of the inflammatory response. The results showed that after pretreatment with LFA-1 neutralizing antibody, the decrease in MN9D cell viability, increase in the number of apoptotic and necrotic cells, increase in LDH release, and upregulation of apoptotic enzyme expression caused by Teff cells were significantly reduced (P<0.05).
[0020] Example 1
[0021] The in vitro PD cell model of this example was established using MPP + Induce MN9D cell line.
[0022] In this example, LFA-1 neutralizing antibody was used to pretreat Teff cells to observe its effects on cell viability, the number of apoptotic and necrotic cells, LDH release, and expression of apoptotic proteins in the PD cell model, thereby observing the role of LFA-1 neutralizing antibody in alleviating the PD neuroinflammatory response caused by Teff cells.
[0023] The specific process is as follows:
[0024] 1. Establishment of in vitro PD cell model and co-culture with Teff cells pre-treated with LFA-1 neutralizing antibody
[0025] 1) Isolation of Teff cells: 7-8 week old male B6 mice were killed by cervical dislocation, and the spleen was removed by aseptic laparotomy and placed in RPMI-1640 (Sigma) basal medium, ground into a single cell suspension, and mouse Teff cells were sorted using magnetic beads (MACS). The T cells with adjusted density were inoculated into culture plates, and CD3 monoclonal antibody (1 μg / ml) and CD28 monoclonal antibody (2 μg / ml) (both purchased from BD) were added to activate Teff cells, and cultured in a 5% CO2, 37°C incubator for 4 days.
[0026] 2) Establishment of PD cell model and co-culture with Teff cells: MN9D cell line was purchased from Guangzhou Genio Biotechnology Co., Ltd. When the MN9D cells in the culture bottle grew to cover 80% of the bottom of the bottle, 2 ml of 0.25% trypsin solution was added for digestion at room temperature, and the number of cells was calculated as 1×10 5 / L cell density, inoculated in 6-well plates (pre-built sterile round glass slides for cell climbing growth). When MN9D cells grew to about 60%, the sorted activated Teff cell suspension was centrifuged to remove the supernatant, and the cells were resuspended in RPMI-1640 complete medium and counted. 1×106 Teff cells, and then add MPP 1 hour later + (final concentration is 50 μM), after 48 h, the Teff cells in the culture system were washed away, and the cultured MN9D cells were collected for subsequent detection.
[0027] 3) Teff cells were treated with LFA-1 neutralizing antibody and then co-cultured with PD cells: LFA-1 neutralizing antibody (Biolegend) was added to the cell culture system on the third day of Teff cell activation. The culture medium was replaced after 24 hours, and Teff cells were then co-cultured with MN9D cells.
[0028] 2. Observe the effect of LFA-1 antibody treatment on MN9D cell viability, cell apoptosis and necrosis ratio, and expression of apoptotic proteins after Teff cells were treated
[0029] 1) Cell viability detection: MN9D cell suspension was inoculated in a 96-well plate, with 6-8 parallel wells in each group. After 7 days of culture, the culture medium in the culture wells was aspirated, 5 mg / ml MTT was diluted with 0.01M PBS buffer at a ratio of 1:10, 100 ul of MTT solution was added to each well, and incubated at 37°C in the dark for 4 hours, the supernatant in the well was carefully aspirated, 100 ul of DMSO was added to each well, and incubated at 37°C for 30 minutes. After the crystals were fully dissolved, they were placed in an ELISA colorimeter (wavelength 570 nm), the absorbance value (OD value) of each well was measured, and the results were recorded.
[0030] 2) Annexin V / PI staining method to detect apoptosis and necrosis of MN9D cells (the operation steps were carried out according to the instructions of the kit): ① MN9D cells were digested with 0.25% trypsin and collected (the trypsin digestion time should not be too long, otherwise it is easy to cause false positives); ② Wash the cells twice with PBS, centrifuge (2000rpm 5min), and discard the supernatant; ③ Add 500μl of Binding Buffer to suspend the cells; ④ Add 5μl Annexin V-FITC and 5μl PI and mix well; ⑤ Incubate at room temperature in the dark for 15min; ⑥ Wash the cells twice with PBS, centrifuge (2000rpm 5min), and discard the supernatant; ⑦ Add 500μl of PBS to resuspend the cells and analyze them on a flow cytometer, excitation wavelength Ex=488nm; emission wavelength Em=530nm.
[0031] 3) TUNEL staining to detect apoptosis of MN9D cells: MN9D cells in each treatment group were washed with PBS for 3 times × 5 min, fixed with 4% formaldehyde solution at room temperature for about 30 min, washed with PBS for 3 times × 5 min, blocked with blocking solution for 30 min, then added with 50 μl TUNEL staining working solution, incubated at 37°C in the dark for 1 h, then washed with PBS for 3 × 5 min, and observed and counted TUNEL-positive cells under a fluorescence microscope (excitation wavelength of 488 nm, observation wavelength of 515-565 nm). Three fields of view were taken for each slide, and the percentage of TUNEL-labeled positive cells in each group to all cells was calculated.
[0032] 4) LDH release assay to detect cell death: MN9D cell culture fluid of each treatment group was collected, diluted with PBS at a ratio of 1:10, 10 μl of standard or sample was added to the microplate, substrate buffer and oxidized coenzyme I solution were mixed at a volume ratio of 5:1, 60 μl of the mixture was added to each well, mixed thoroughly, incubated at 37°C for 15 min, 50 μl of 2,4-dinitrophenylhydrazine solution was added, mixed thoroughly with the standard or sample, incubated at 37°C for 15 min, 150 μl of stop reagent was added, mixed thoroughly, left at room temperature for 3 minutes, the absorbance of the sample well was read at a wavelength of 440 nm, and the enzyme activity unit was calculated according to the standard curve, which was the measured enzyme activity unit, cell LDH release (%) = enzyme activity unit measured in cell culture medium / (enzyme activity unit measured in cell lysate + enzyme activity unit measured in cell culture medium) × 100%. Cell death is expressed by the percentage of LDH release, the larger the latter value, the worse the cell activity and the more serious the damage.
[0033] 5) Western blot method to detect the expression of apoptotic proteins: MN9D cells were washed twice with PBS and then lysed with 60 μl / well of 1×SDS cell protein lysis buffer. The cell lysate was collected into an Eppendorf tube, placed in an ice bath for ultrasonic cell crushing, centrifuged for 15 min (12000 rpm, 4°C), and the supernatant was aspirated into a new centrifuge tube. Protein standards and protein samples were diluted 10 times with physiological saline and mixed with Coomassie brilliant blue dye. The protein-free Coomassie brilliant blue dye was used as a blank control, and the OD was measured with a spectrophotometer. 595Value. Protein content (g / L) = absorbance of measuring tube / absorbance of standard tube × concentration of standard tube (g / L). Mix the protein sample with 4× loading buffer, boil in a 100℃ water bath for 10min, and store at -70℃ after aliquoting. Next is SDS-polyacrylamide gel electrophoresis: 12% SDS-PAGE vertical electrophoresis is used for separation, the sample amount is 45μg, the voltage is 70-110v, and the electrophoresis ends when bromophenol blue reaches the bottom. Then, the membrane was transferred using the Bio-rad wet transfer system at 250 mA for 60-120 min. The transferred PVDF membrane was washed with 1×TBS washing buffer for 5 min × 3 times, blocked with a blocking solution containing 5% skim milk powder at room temperature for 2 h, and then washed with TBS / T washing buffer for 5 min × 3 times, immersed in rabbit anti-caspase3 (1:1000), mouse anti-caspase9 (1:1000) or mouse anti-β-actin (1:2000) at 4°C overnight. Washed with TBS / T washing buffer for 5 min × 3 times, incubated with fluorescently labeled goat anti-rabbit IgG (1:5000), fluorescently labeled goat anti-mouse IgG (1:5000) or fluorescently labeled donkey anti-goat IgG (1:5000) at room temperature for 1 h. After washing with TBS / T for 5 min × 3 times, the Odyssey dual infrared laser scanning system was used to scan and analyze the average gray value of the bands. The relative expression levels of activated pro-apoptotic proteins caspase3 and caspase9 were expressed by the ratios of cleaved-caspase3 / pro-caspase3 and cleaved-caspase9 / pro-caspase9, respectively.
[0034] 3. Statistical analysis
[0035] All results are expressed as mean ± standard deviation (M ± SD). SPSS 21.0 statistical software was used for data analysis and processing, and one-way analysis of variance (ANOVA) was used to test the significance of differences between groups, and Student-Newman-Keul's test was used to test the significance of differences between groups. P < 0.05 was considered to be significantly different.
[0036] 1 is the effect of Teff cells pretreated with LFA-1 neutralizing antibody on MPP + Effect of induced MN9D cell viability. (++p<0.01 compared with Intact group. ##p<0.01 compared with Teff group; **p<0.01 compared with MPP group + Group comparison; &&p<0.01 vs. MPP + +Teff group comparison).
[0037] Figure 2 The Teff cells pretreated with LFA-1 neutralizing antibody responded to MPP +The effect of induced apoptosis and necrosis of MN9D cells. Among them: A is a flow cytometry detection of apoptosis and necrosis of MN9D cells, the upper figure in A is a flow cytometric scatter plot of apoptosis and necrosis of MN9D cells, the upper right quadrant is Annexin V / PI double positive cells, representing late apoptotic cells, the lower right quadrant is Annexin V single positive cells, representing early apoptotic cells, the upper left quadrant is PI single positive cells, representing necrotic cells, and the lower left quadrant is double negative cells, representing normal cells. The lower left figure in A is a statistical chart of the percentage of necrotic cells, and the lower right figure in A is a statistical chart of the percentage of apoptotic cells, that is, the sum of the proportions of cells in the upper right and lower right quadrants. B is a TUNEL staining detection of cell apoptosis, the left figure in B is a green fluorescence image of TUNEL staining detection of cell apoptosis positive cells, representing apoptotic cells, and the right figure in B is a statistical chart of the percentage of TUNEL positive cells in total cells. Figure C is a statistical chart of LDH release experiment. (++p<0.01 compared with Intact group; ##p<0.01 compared with Teff group; **p<0.01 compared with MPP + Group comparison; &&p<0.01 vs. MPP + +Teff group comparison).
[0038] Figure 3 The Teff cells pretreated with LFA-1 neutralizing antibody responded to MPP + The effect diagram of induced apoptotic protein expression in MN9D cells. A is a representative immunoblot of two apoptotic proteins, cleaved-caspase-3 and cleaved-caspase-9, and B is the corresponding statistical diagram. (++p<0.01 compared with the Intact group; #p<0.05 compared with the Teff group; ##p<0.01 compared with the Teff group; **p<0.01 compared with the MPP group + Group comparison; &&p<0.01 vs. MPP + +Teff group comparison).
[0039] The above description has fully disclosed the specific embodiments of the present invention. It should be pointed out that any changes made by those skilled in the art to the specific embodiments of the present invention do not deviate from the scope of the claims of the present invention. Accordingly, the scope of the claims of the present invention is not limited to the above specific embodiments.
Claims
1. Application of LFA-1 neutralizing antibodies in drugs to reduce neuroinflammation in Parkinson's disease.
2. The use according to claim 1, characterized in that: Application of LFA-1 neutralizing antibody in alleviating cell damage in PD cell models.
3. The use according to claim 1, characterized in that: Application of LFA-1 neutralizing antibody in alleviating cell apoptosis and necrosis in PD cell models.
4. The use according to claim 1, characterized in that: Application of LFA-1 neutralizing antibody in reducing the expression of apoptotic proteins in PD cell models.