CN108452301A - Antiangiogenic/anti-thymocyte globulin is preparing the application in treating neurological autoimmune disease medicament - Google Patents

Abstract

The invention discloses the application of anti-lymphoid/thymocyte globulin in the preparation of medicines for treating nervous autoimmune diseases, and belongs to the field of biomedicine. Through in vitro experiments and rat EAE model research, the present invention finds that the anti-lymphoid/thymocyte globulin can treat neurological autoimmune diseases by inhibiting T cell activation, and can be used for preparing medicines for treating neurological autoimmune diseases. Anti-lymphoid/thymocyte globulin has the following advantages as a drug for the treatment of neurological autoimmune diseases: (1) strong specificity: this type of drug mainly targets T cells; (2) significant effect: this type of drug can significantly improve EAE (3) Minor toxic and side effects: the drugs did not cause obvious toxic and side effects to EAE model rats.

Description

Anti-lymphoid/thymocyte globulin in the preparation of drugs for the treatment of neurological autoimmune diseases application in things

technical field

The invention belongs to the field of biomedicine, and in particular relates to the application of anti-lymphocyte/thymocyte globulin in the preparation of medicines for treating neurological autoimmune diseases.

Background technique

Nervous autoimmune disease is a disease in which the body's immune system has an abnormal immune response to the autologous nervous system, causing inflammatory damage to the nervous system, resulting in demyelination, neuron and axon damage as the main pathological changes. Lesions can involve the central nervous system, peripheral nervous system, and neuromuscular junction. Such diseases in the central nervous system are characterized by multiple sclerosis, neuromyelitis optica, autoimmune encephalitis [Leypoldt F, Armangue T, Dalmau J. Autoimmune encephalopathy [J]. Annals of the New York Academy of Sciences, 2015, 1338 (1 ):94-114.; Graus F, Titulaer M J, Balu R, et al.A clinical approach to diagnosis of autoimmune encephalitis[J].Lancet Neurology,2016,15(4):391.] as the main representative; in peripheral nerve System and neuromuscular junction are mainly represented by Guillain-Barre Syndrome (GBS) and myasthenia gravis.

Multiple sclerosis (MS) is an autoimmune disease of the nervous system characterized by inflammatory demyelination of the central nervous system. The etiology and pathogenesis are still unclear. It plays an important role in the autoimmune response of multiple sclerosis [Noseworthy J H, Lucchinetti C, Rodriguez M, et al. Multiple sclerosis. [J]. New England Journal of Medicine, 2000, 343 (13): 938-952. ]. Multiple sclerosis is more common in young and middle-aged people, and there are more women patients than men. Lesions mostly involve the white matter of the brain, optic nerve, spinal cord, brainstem, and cerebellum. The clinical manifestations vary depending on the location of the lesion. It often manifests as limb weakness, paresthesia, vision loss, and ataxia. In the advanced stage, it can lead to severe paralysis and sensory disturbance. , and even blindness.

At present, the treatment of multiple sclerosis is mainly based on drug therapy, including acute exacerbation treatment and remission disease regulating treatment. In the acute attack stage of multiple sclerosis, the main purpose of drug treatment is to relieve symptoms, shorten the course of the disease, and reduce disability. Usually, glucocorticoid shock is used as the first-line therapy, and patients who are not sensitive to steroid drugs are treated with plasma exchange and hematopoietic stem cell transplantation as the second-line therapy. Hormone drugs have serious side effects, and it is not clear whether the application of hormones in the acute phase can alleviate the long-term dysfunction of multiple sclerosis; the hematopoietic stem cell transplantation scheme also has the disadvantages of high short-term recurrence rate and high perioperative mortality. Disease-regulating treatment in remission is mainly aimed at reducing relapse, delaying disability accumulation, and improving quality of life. Clinically, immunosuppressive drugs such as interferon-β and glatiramer acetate are mainly used. This type of drug has disadvantages such as high price and reduced curative effect after long-term use [Feinstein A, Freeman J, Lo A C. Treatment of progressive multiple sclerosis: what works, what does not, and what is needed [J]. Lancet Neurology, 2015, 14(2):194.]. Due to the complexity of multiple sclerosis and other autoimmune diseases of the nervous system, the effect of drug treatment is often unsatisfactory, and it has high mortality and disability. Therefore, the development and research of new immunomodulatory drugs is very necessary.

Experimental autoimmune encephalomyelitis (EAE) animal model is a neurological autoimmune disease mediated by T cells produced by immunizing susceptible animals with nerve cell antigens. Neuroautoimmune diseases such as inflammation are very similar in clinical manifestations, immunity and pathology. EAE is internationally recognized as an ideal animal model for neurological autoimmune diseases such as multiple sclerosis, and it is of great value in elucidating the pathogenesis of the disease and evaluating the therapeutic effect of drugs.

Anti-lymphoid/thymocyte globulin is a common immunosuppressive polyclonal antibody drug. It was first applied to a case of kidney transplantation in 1966. It is now widely used in the treatment of transplanted organ rejection and severe aplastic anemia. Its mechanism of action It is mainly complement-mediated cytolysis of T cells and induction of T cell apoptosis [Mohty M. Mechanisms of action of antithymocyte globulin: T-cell depletion and beyond. [J]. .]. At the end of the last century, there were reports on the relevant clinical trials of anti-lymphoid/thymocyte globulin combined with cyclosporin A (CsA) in the pretreatment of hematopoietic stem cell transplantation for multiple sclerosis [Burt R K, Balabanov R, Han X, et al. al.Association of nonmyeloablative hematopoietic stemcell transplantation with neurological disability in patients with relapsing-remitting multiple sclerosis[J].Jama,2015,313(3):275.;Atkins H L,Bowman M,Allan D,et al.Immunoablation and autologous haemopoietic stem-celltransplantation for aggressive multiple sclerosis: a multicentre single-groupphase 2trial[J].Lancet,2016,388(10044):576.], its pharmacological effect is to reduce the immune rejection of the host after hematopoietic stem cell transplantation, but cut off At present, there is no relevant report on the direct application of anti-lymphoid/thymocyte globulin in the treatment of neuroautoimmune diseases such as multiple sclerosis.

Contents of the invention

The present invention aims at the problems existing in medicine and therapy for the treatment of nervous autoimmune diseases, and conducts experimental research on the possibility of direct application of antilymphoid/thymocyte globulin drugs in the treatment of neuroimmune diseases such as multiple sclerosis. Through cell experiments and animal model experiments, the present invention proves that this type of drug has important clinical application value for the treatment of neuroimmune diseases such as multiple sclerosis, and its pharmacological mechanism is different from that of anti-lymphoid/thymocyte globulin drugs in hematopoietic stem cells. The role played by transplantation therapy is to inhibit the inflammatory injury caused by autoimmunity by directly inhibiting the activation of self-reactive T cells. As a clinically approved drug, anti-lymphoid/thymocyte globulin drugs have clear active ingredients, significant curative effect and good safety, and also greatly save the cost of new drug development.

The purpose of the present invention is to provide the application of the anti-lymphoid/thymocyte globulin in the preparation of medicine for treating nervous autoimmune diseases.

The experimental research and results of using anti-lymphoid/thymocyte globulin in the treatment of neurological autoimmune diseases in the present invention are as follows:

1. Peripheral Blood Mononuclear Cell (PBMC) cells were isolated and cultured in vitro, and the immunosuppressive effect of anti-human T cell porcine immunoglobulin (ALG) was detected by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA).

The results of immunohistochemistry showed that ALG could specifically bind to the surface of T cells in PBMC. The results of ELISA experiments showed that ALG treatment could significantly reduce the secretion of inflammatory cytokines IL-2, IFN-γ and IL-17 after T cell activation.

2. Construction of rat EAE model: 42 SPF-level female SD rats weighing 170 g (purchased from Beijing Weitong Lihua Company) were selected and raised in the Experimental Animal Center of Wuhan University. The rats were randomly divided into 3 groups, 14 in each group, one of which was used as the normal control group, and the other two were used to construct the EAE model. Using the method of autogenous brain and spinal cord homogenate to make neuroantigen, immunization was carried out twice to construct the EAE model. After the second immunization, daily observation was started, the clinical behavioral performance was scored, and the clinical behavioral evaluation was performed on rats in each group. Symptoms began to appear on the 10th day after immunization, and the administration began after the incidence rate reached 30% on the 14th day. The rats in the model group were randomly divided into two groups according to the score, and ALG (ALG treatment group) and Pig IgG (model control group) was administered by tail vein injection for 7 days. On the 21st day after immunization, the rats in the three groups were killed under CO ₂ anesthesia, and the relevant indicators were detected.

The experimental results showed that the symptoms of EAE began to appear on the 10th day after the modeling, the incidence rate was >70%, and the modeling was successful. The rats in the normal control group had no abnormal neurological symptoms, the behavioral scores of the rats in the model control group were the highest, and the behavioral scores of the rats in the ALG treatment group were lower than those in the model control group, and the behavioral scores of the two groups were statistically statistically There were significant differences (P<0.05).

3. After 7 days of administration, the rats were sacrificed and the materials were collected. The spinal cords of the rats in each group were fixed and sectioned, and the rats in each group were evaluated pathologically by LuxolFast Blue and hematoxylin-eosin (HE) staining.

The experimental results showed that there was no demyelination and inflammatory infiltration in the spinal cord of the rats in the normal control group, and the demyelinating lesion area of the spinal cord was larger in the model control group of EAE rats, and there were more inflammatory cells infiltrated in the lesion area, while ALG treatment The area of spinal cord demyelinating lesion in rats in the treatment group was reduced, and the number of infiltrating inflammatory cells around the lesion was also significantly reduced compared with the model control group. Quantitative statistics showed that the demyelination and inflammatory infiltration of the model control group and the ALG treatment group The situation scores were statistically different (P<0.01).

4. The cerebrospinal fluid and peripheral blood of rats in each group were collected, centrifuged to remove cells, and the contents of cytokines IL-2, IFN-γ and IL-17 in cerebrospinal fluid and serum were detected by ELISA.

The experimental results showed that the contents of inflammatory cytokines interleukin-2 (IL-2), interferon-γ (IFN-γ) and interleukin-17 (IL-17) in the cerebrospinal fluid and serum of the rats in the EAE model control group were higher than those in the control group. High, while the content of inflammatory cytokines in cerebrospinal fluid and serum of rats treated with ALG was significantly reduced (P<0.01).

5. Collect the heart, liver, lung, spleen, kidney and other major organs of rats in each group, and fix and slice them. The slices were stained with HE, and the safety of ALG in EAE model rats was initially evaluated at the histopathological level.

The experimental results showed that there were no obvious pathological changes in the main internal organs of rats in the normal control group, the model control group and the ALG treatment group.

6. Statistical analysis, Graph Pad PRISM 5.0 software was used for statistical analysis, and the data were expressed as mean ± standard deviation (Mean ± Sem), and the significance level for comparing the differences between groups was P<0.05. Non-parametric statistical methods were used for non-normally distributed data, and the Kruskal-Wallis test was used for behavioral scores and histopathological scores; for other normally distributed data, one-way ANOVA and independent sample t test.

The above experimental studies and results show that anti-lymphoid/thymocyte globulin can treat neurological autoimmune diseases by inhibiting T cell activation, and its therapeutic mechanism is different from that of anti-lymphoid/thymocyte globulin in reducing host immune rejection after hematopoietic stem cell transplantation .

Based on above-mentioned experimental research and result, the present invention provides following application:

Application of anti-lymphoid/thymocyte globulin in the preparation of medicine for treating neurological autoimmune diseases.

The anti-lymphocyte/thymocyte globulin includes anti-human T cell pig immunoglobulin, rabbit anti-human thymocyte immunoglobulin, horse anti-human thymocyte globulin, rabbit anti-human lymphocyte immunoglobulin and the like.

The neurological autoimmune diseases include multiple sclerosis (Multiple Sclerosis, MS), optic neuromyelitis (Neuro Myelitis Optica, NMO), autoimmune encephalitis (Autoimmune Encephalitis, AE), diffuse sclerosis (Diffuse Sclerosis), acute Transverse myelitis (Acute Transverse myelitis, ATM), concentric sclerosis (Concentric Sclerosis) and myasthenia gravis (Myasthenia Gravis, MG) and other diseases.

The application of the anti-lymphoid/thymocyte globulin in the preparation of medicines for treating neurological autoimmune diseases does not include the preparation of medicines for hematopoietic stem cell transplantation (Hematopoietic Stemcell Transplantation, HSCT).

A medicine for treating nervous autoimmune diseases, comprising anti-lymphoid/thymocyte globulin and a pharmaceutically acceptable carrier or excipient of anti-lymphoid/thymocyte globulin.

The present invention has the following advantages: (1) strong specificity: anti-lymphoid/thymocyte globulin can be effectively combined on the surface of T cells, and specifically inhibit T cell activation in vitro and in vivo, so it can be used for neural autoimmunity such as multiple sclerosis Direct treatment of disease. Its administration method and mechanism of action are different from the previous use of this type of drug in hematopoietic stem cell transplantation therapy in the acute relapse stage of multiple sclerosis. Before hematopoietic stem cell transplantation, ALG combined with cyclosporine is used to pre-medicate the patient, only as a Clinical medicine against transplant rejection. (2) Remarkable effect: animal experiment results show that anti-lymphocyte/thymocyte globulin can significantly improve the condition of EAE model rats, effectively reduce behavioral scores and alleviate pathological changes. Since the EAE model is a classic animal model of autoimmune diseases of the nervous system, anti-lymphocyte/thymocyte globulin also has potential application value in the treatment of neuromyelitis optica and other neurological autoimmune diseases. (3) Minimal toxic and side effects: ALG did not cause obvious toxic and side effects to EAE model rats in animal experiments, indicating that the drug has low toxicity and can be safely used in the treatment of nervous system diseases.

Description of drawings

Fig. 1 is a graph showing the results of ALG specifically binding to T cells in PBMCs in vitro. A is the result of immunofluorescence of rat PBMC; B is the result of immunofluorescence of human PBMC.

Fig. 2 is a graph showing the results of ALG inhibiting the secretion of inflammatory cytokines after T cell activation in PBMCs. A is the quantitative statistical chart of rat PBMC cytokine results; B is the quantitative statistical chart of human PBMC cytokine results. (n=5; ns: nonsignificant vs non-stimulated group; NS: non significant vs single-stimulated group; ^# : P<0.05vs stimulated+IgG group; ^## : P<0.01vs stimulated+IgG group; ^### : P ＜0.001vs stimulation+IgG group; ***: P＜0.001vs unstimulated group)

Figure 3 is a graph showing the results of ALG improving spinal cord demyelination and inflammatory cell infiltration in the lesion area of EAE rats. A is the LFB (Luxol Fast Blue) and HE staining results of the spinal cord of EAE model rats; B is the quantification statistics. (n=6; ^## : P<0.01vs model control group; ***: P<0.001vs blank control group)

Fig. 4 is a graph showing the results of ALG reducing the content of T cell-related inflammatory cytokines in cerebrospinal fluid and peripheral blood of EAE rats. A is the statistical graph of the cytokines IFN-γ and IL-17 in the cerebrospinal fluid of rats in each group; B is the statistical graph of the cytokines IL-2, IFN-γ and IL-17 in the serum of rats in each group. (n=5; ^## : P<0.01vs model control group; **: P<0.01vs blank control group; ^### : P<0.001vs model control group; ***: P<0.001vs blank control group )

Fig. 5 is a graph showing HE staining results of heart, liver, lung, spleen, and kidney tissue sections of rats in each group.

Detailed ways

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of protection claimed by the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Experimental Research of Embodiment 1 ALG on Rat/Human PBMC Cell Pharmacodynamics

(1) For the separation of rat PBMC cells, GE PercollPLUS lymphocyte separation medium was used, and the steps were summarized as follows according to the instruction manual: A. SD rats were anesthetized with 10% chloral hydrate, and after complete anesthesia, the abdomen was disinfected, and the abdominal cavity was cut open to take the main body. Intravenously, extract 5mL of fresh anticoagulated peripheral blood from rats; B. transfer the anticoagulated blood into the ultra-clean bench, and dilute the blood by 1 times with 1×PBS; Solution (10×PBS) and 0.01M Phosphate Buffer Saline (1×PBS) were mixed according to the volume ratio of 6.9:0.1:3 to prepare the working solution of the cell separation solution, and 5mL of the mixed cell separation solution was added to a new 15mL free D. Slowly add diluted blood on the surface of the 5mL mixed cell separation solution, the action should be gentle, try not to disturb the liquid surface; E. Centrifuge at 400g for 30 minutes, and lower the acceleration and deceleration gears of the centrifuge ; F. Stratification can be seen after the centrifugation is completed, and the lymphocytes gather at the interface between the plasma and the separation liquid, showing a white mist. Aspirate the layer of cells, add PBS to wash, and centrifuge at 200g for 10 minutes; G. Repeat washing twice and pour Remove the supernatant, resuspend the cells with 1640 medium containing 10% FBS, and proceed to the next experiment after counting.

(2) Immunofluorescence to verify the interaction between ALG and rat T cells, the steps are as follows: A. Resuspend the isolated rat PBMCs with PBS, adjust the cell density to 1× ¹⁰⁵ cells per ml, and add poly-lysine The center of the coated confocal culture dish; B. After 4 hours, suck away the supernatant of the unattached cell suspension, add PBS to wash, and the action should be gentle; C. After washing 3 times, add different groups of cell samples respectively 15 μg/mL ALG or IgG, incubate at room temperature for 1 hour; D. remove the supernatant, add PBS to wash 3 times; E. add PE-labeled anti-rat CD3 fluorescent antibody and FITC-labeled anti-pig IgG antibody, avoid Co-incubate with light for 1 hour; F. remove the supernatant containing the fluorescent antibody, add PBS to wash 3 times, and avoid light; F. add DAPI for staining for 1 minute, remove the supernatant after the end, add PBS to wash 3 times , pay attention to avoid light; G. Add 1mL glycerin, and image detection with laser confocal microscope. Experimental results show that ALG can effectively bind to the surface of rat CD3 ⁺ T cells ( FIG. 1A ).

The cell immunofluorescence experiment method of human PBMC is the same as that of rat PBMC. The experimental results show that the red/green two-color fluorescence of cells in the ALG treatment group has co-localization, and the cells in the control IgG group have no green IgG fluorescence signal, which proves that ALG can be combined with CD3 ⁺ T cells specifically bound in human PBMCs (Fig. IB). The above results show that ALG can specifically bind to T cells in vitro.

(3) Activation of rat PBMC: A. resuspend the isolated PBMC cells with 1640 complete medium containing 10% FBS, adjust the cell concentration to 2× ¹⁰⁶ per milliliter, inoculate in 96-well plate, each well 100 μL; B. The cells were divided into blank group and stimulation group, the blank group was given PBS, and the cells in the stimulation group were stimulated with T cell stimulators PMA (tetradecanoylphorbol acetic acid) and Ionomycin (ionomycin) (the former was finally stimulated). The concentration was 50ng/mL, and the final concentration of the latter was 1μg/mL); C. In the stimulation group, different concentrations of ALG (1, 2, 7, 15μg/mL) and IgG were administered respectively, and ALG and IgG were administered before PMA and Ionomycin stimulation Add in 60min, each group set up three replicate wells, and add a group of IgG control in the stimulation group; D. Place the treated cells in a 37°C, 5% CO ₂ incubator for 24h, and prepare for later experiments.

(4) Verify that ALG inhibits the secretion of T cell-related inflammatory cytokines by ELISA experiments: centrifuge and collect the supernatant of the cell culture medium after (3) treatment, record the numbers, and follow the instructions (Enzyme-linked immunosorbent assay produced by Beijing Daktronics Co., Ltd.) Assay kit) operation steps ELISA experiment, the process is as follows: A. Before use, fully mix all reagents to avoid foam; B. According to the number of experimental wells (blank and standard), determine the number of strips required. Both the sample (including the standard) and the blank should be duplicated; C. Sample addition: add 100 μL/well of the diluted Cytokine standard (standard) to the standard well, add 100 μL/well of the sample to the sample well, and set a blank well. Use Dilution buffer R (1×) to replace samples and standards; D. Add detection antibody: 50 μL/well to add diluted Biotinylated antibody (biotin-labeled antibody). After mixing, cover the plate with sealing film and incubate at 37°C for 90 min; E. Wash the plate: remove the liquid in the well, add 1×washing buffer at 300 μL/well; discard the liquid in the well after staying for 1 min. Repeat 3 times, and dry it on the filter paper for the last time; F. Add enzyme: add diluted Streptavidin-HRP (streptavidin-horseradish peroxidase) at 100 μL/well. Cover the plate with sealing film and incubate at 37°C for 30 minutes; G. Wash the plate: repeat step E; H. Color development: add TMB at 100 μL/well, and incubate at 37°C in the dark for 5-30 minutes, according to the depth of the color in the well (dark blue) to determine the termination reaction. Usually the color development takes 10-20 minutes to achieve good results; I. Termination reaction: 100μL/well is quickly added Stop solution to terminate the reaction, within 10 minutes after termination, read the value with a detection wavelength of 450nm. The results showed that the contents of IL-2, IFN-γ and L-17 in the culture supernatant of unstimulated cells were lower than the detection limit. The levels of IL-2, IFN-γ and L-17 in the culture medium of the group stimulated with PMA and Ionomycin alone and pretreated with IgG were the highest, and there was no statistical difference between the two groups. The ALG pretreatment group could reduce the contents of cytokines IL-2, IFN-γ and L-17 in the supernatant of rat PBMC culture medium in a significant dose-dependent manner (Fig. 2A).

The experimental method of human PBMC activation was the same as that of rat PBMC. The experimental results showed that ALG could reduce the contents of cytokines IL-2, IFN-γ and IL-17 in the supernatant of human PBMC culture medium (Fig. 2B).

The above experiments proved that ALG can inhibit the secretion of related inflammatory cytokines IL-2, IFN-γ and IL-17 after T cell activation in PBMC.

Embodiment 2 ALG is to the experimental research of clinical behavior of EAE model rat

(1) Establishment of EAE model in rats: A. Prepared antigen: 10% chloral hydrate was taken to anesthetize SD rats by intraperitoneal injection, and the injection dose was 4 mL/kg. After complete anesthesia, cut open the chest cavity, take 0.9% normal saline for cardiac perfusion, perfuse 50-150mL until the blood is washed, decapitate the head, remove the cerebellar white matter and spinal cord with ophthalmic forceps, weigh and add Amount of sterilized PBS, transferred to a sterile centrifuge tube. Place the centrifuge tube in ice water and use an electric homogenizer to homogenize for 1 hour. The final concentration of the homogenate in the tube is 1 g/mL. Mycobacterium tuberculosis was inactivated in a water bath at 65°C for 30 min, and then added to incomplete Freund's adjuvant and mixed evenly at a concentration of 5 mg/mL to prepare complete Freund's adjuvant (CFA). The homogenate of the brain and spinal cord was slowly added to Freund's complete adjuvant at a volume ratio of 1:1, and was homogenized with an electric homogenizer without stopping to make a water-in-oil emulsion. Dilute pertussis toxin stock solution to 10 μg/mL with PBS for later use; B. Model construction: SD rats were randomly divided into 3 groups, rats in the model control group and ALG treatment group were given spinal cord homogenate and complete Freund's adjuvant The normal control group was immunized with the same amount of PBS plus complete Freund's adjuvant. During immunization, inject the needle from the hind foot pad, inject water-in-oil emulsion subcutaneously, the dose is 150μg/200g, and inject 0.1mL pertussis toxin into the dermis of the dorsum of the foot at the same time, press with a sterile cotton swab to prevent leakage, and take another one after seven days Enough for the second immunization with the same dose. After the second immunization, two observers used the double-blind method to score the behavioral changes of the rats every day. The behavioral scoring standards reported in the literature were used to score, and it was divided into 5 grades: 0 points: no clinical symptoms; 1 point: loss of tail tension; 2 points: weakness of both hind limbs, which can be recovered after passive turning; 3 points: paralysis of both hind limbs, which cannot be recovered after passive turning; 4 points: quadriplegia or weakened muscle strength with urinary and fecal incontinence; 5 points: Dying state or death; C. Grouping and administration, the onset of the disease began on the 10th day after the first immunization, and when the incidence rate rose to 30% (the 14th day after the first immunization), the rats in the model group were randomly divided It was two groups, one group was given 20 mg/kg body weight control product pig IgG (model control group), and the other group was given 20 mg/kg body weight ALG (ALG treatment group). During the administration period, continue to observe and record the clinical behavioral changes of the rats in each group every day. The experiment ends on the 21st day after the first immunization, and relevant indicators are collected and tested.

(2) Clinical behavioral evaluation of EAE model rats: The animals began to develop symptoms on the 10th day after the first immunization, and initially showed loss of tail tension and weakness of hind limbs, and then progressed to obvious limb paralysis and paralysis.

Note: *P<0.05

The results showed that: the normal control group had no abnormal nervous system symptoms, and the behavioral score was 0; the rats in the model control group had obvious neurological symptoms such as hindlimb weakness, and the behavioral score was the highest; the behavioral score of the rats in the ALG treatment group was lower than that of the model control group , and the statistical difference between the model control group and the ALG treatment group was statistically significant on the 20th day and the 21st day after immunization.

The experimental results proved that ALG treatment can effectively improve the clinical behavioral performance of EAE model rats.

Embodiment 3 ALG is to the experimental research of EAE model rat pathology

(1) Histopathological evaluation for EAE model rats: intraperitoneal injection of anesthesia (10% chloral hydrate solution) at a dose of 5 mL/kg body weight, after complete anesthesia, the chest cavity was cut open, and 100 mL of normal saline was taken for apical perfusion. After the blood cells were washed clean, the heart was perfused with formalin solution for fixation. After perfusion, the spinal cords of the rats in each group were removed and fixed in formalin solution overnight at 4°C. The fixed spinal cord sample is trimmed, and the trimmed tissue is placed in a pre-marked dehydration box, and the dehydration box containing the tissue is placed in a dehydrator, and dehydrated with gradient alcohol. The dehydrated tissues were treated with xylene and embedded in paraffin. The removed wax block was trimmed and placed on a paraffin microtome to cut continuous slices with a thickness of 4 μm. Float the slices in a water bath at 40°C, flatten the tissue, pick up the flattened tissue slices with a glass slide, bake the slices at 60°C, and store them for later use. Histological evaluation of spinal cord demyelination and inflammatory infiltration of rats in each group was performed by Luxol Fast Blue and HE staining on the processed sections. Through the observation of the spinal cord slices of rats in each group, it was found that the area of the spinal cord demyelinating lesion was smaller in the ALG treatment group, and the infiltration of inflammatory cells in the lesion area was lighter than that of the model control group (Figure 3). The experimental results proved that ALG can improve the demyelinating lesions of the spinal cord of EAE model rats and reduce the infiltration of inflammatory cells around the lesion.

(2) Diagnostic evaluation for EAE model rats: After the rats in each group were killed by CO ₂ , the cerebrospinal fluid and peripheral blood of the rats in each group were collected, centrifuged and the supernatant was stored at -20°C. The content of cytokines in cerebrospinal fluid and serum was detected by ELISA method, and the therapeutic effect of ALG on EAE model rats was evaluated. The results showed that the contents of IFN-γ and IL-17 in the cerebrospinal fluid of rats in the normal control group were very low, and the contents of IFN-γ and IL-17 in the cerebrospinal fluid of rats in the model control group were the highest, while the contents of IFN-γ and IL-17 in the cerebrospinal fluid of rats in the ALG treatment group and IL-17 levels were reduced (Fig. 4A). The serum of rats in the normal control group contained only a very small amount of IL-2, IFN-γ and IL-17; the contents of IL-2, IFN-γ and IL-17 in the serum of rats in the ALG treatment group were greater than those in the model control group. Rats are low (Fig. 4B). In the model control group, the levels of cytokines IFN-γ and IL-17 in the cerebrospinal fluid of rats were slightly higher than those in the serum, which further verified the existence of inflammation in the central nervous system.

The experimental results proved that ALG can reduce the secretion of T cell-related inflammatory cytokines in the cerebrospinal fluid and serum of EAE model rats.

The toxicity evaluation of embodiment 4ALG to EAE model rat

(1) Toxicity evaluation of ALG on EAE model rats: In order to further evaluate the safety of ALG in rats, the animals were sacrificed after drug treatment, and the hearts and livers of rats in the normal control group, model control group and ALG treatment group were collected. , spleen, lung, kidney.

(2) Fix and embed the collected tissues, and perform HE staining after sectioning.

(3) Pathological analysis was performed on the tissue sections of rats in each group, and the results showed that there was no significant difference in heart, liver, spleen, lung and kidney tissues of rats in each group ( FIG. 5 ).

The experimental results proved that intravenous administration of ALG had no obvious toxicity to EAE rats.

It should be understood that the embodiments of the present invention are not limited by the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods , are all included within the protection scope of the present invention.

Claims (6)

1. Application of anti-lymphoid/thymocyte globulin in the preparation of drugs for treating neurological autoimmune diseases. 2. application according to claim 1, is characterized in that: described anti-lymphoid/thymocyte globulin comprises anti-human T cell pig immunoglobulin, rabbit anti-human thymocyte immunoglobulin, horse anti-human thymocyte globulin protein and rabbit anti-human lymphocyte immunoglobulin. 3. The application according to claim 1, characterized in that: said nervous autoimmune diseases include multiple sclerosis, optic neuromyelitis, autoimmune encephalitis, diffuse sclerosis, acute transverse myelitis, concentric myelitis sclerosis and myasthenia gravis. 4. The application according to any one of claims 1-3, characterized in that: the application does not include the preparation of medicines for hematopoietic stem cell transplantation therapy. 5. A medicine for treating nervous autoimmune diseases, characterized in that it contains anti-lymphoid/thymocyte globulin. 6. The medicine according to claim 5, characterized in that it comprises anti-lymphoid/thymocyte globulin pharmaceutically acceptable carrier or excipient.