Methods for determining the appropriate dosage in MTX-induced myelosuppression rat models

Abstract

This application relates to a method for determining the appropriate dosage of methotrexate (MTX) in a rat model of myelosuppression. By establishing two novel rat models of chronic myelosuppression induced by methotrexate (MTX), and through comparative experiments with several animal models, it was found that in the rat model of chronic myelosuppression, the medium dose of methotrexate was the optimal concentration of MTX; in the CIA rat model of myelosuppression, the CMM group was the optimal concentration of MTX; and in the Wistar rat model of myelosuppression, the MM group was the optimal concentration of MTX. Therefore, this invention can investigate and clarify which concentration of MTX has a significantly greater therapeutic effect on rheumatoid arthritis (RA) than the adverse effects of myelosuppression, and explore the specific mechanism by which MTX induces myelosuppression, which has practical clinical significance for the treatment of RA with MTX.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, and in particular to a method for determining the appropriate dosage of MTX-induced myelosuppression in rats. Background Technology

[0002] Methotrexate (MTX) is the first-line DMARD for the treatment of rheumatoid arthritis (RA) and plays a certain therapeutic role in RA.

[0003] However, although MTX can treat RA, adverse reactions can occur in clinical practice. Among these, bone marrow damage is one of the top three common adverse reactions when using MTX to treat RA. The clinical dosage for RA patients is 15mg-30mg / week, but the specific dosage for each patient remains unclear. Currently, no research has reached a definitive conclusion on which concentration of MTX has a significantly greater therapeutic effect on RA than the adverse reaction of bone marrow damage.

[0004] Therefore, it is urgent to determine which concentration of MTX has a therapeutic effect on RA that far outweighs the adverse effects of bone marrow damage and to clarify the specific mechanism by which MTX causes bone marrow damage. This has practical clinical significance for the treatment of RA with MTX. Summary of the Invention

[0005] To address the aforementioned issues, this application proposes a method for determining the appropriate dosage in an MTX-induced myelosuppression rat model.

[0006] A method for determining the appropriate dosage of MTX in a rat model of myelosuppression, used for studying the optimal dosage of MTX for the treatment of rheumatoid arthritis (RA), includes the following steps:

[0007] S1. The first dose of MTX used in clinical RA patients was converted into the second dose corresponding to the rat coefficient, and the second dose was divided into three different MTX gavage doses of low, medium and high according to the dosage from low to high.

[0008] S2. Prepare several female Wistar rats and CIA rats, and feed them by gavage with sterile water and three different doses of MTX (low, medium and high) to establish animal models.

[0009] The animal models include the Wistar rat chronic bone marrow injury model and the CIA rat bone marrow injury model, wherein:

[0010] The Wistar rat chronic bone marrow injury model includes the following four groups: control (CT) group, low-dose MTX (ML) group, medium-dose MTX (MM) group, and high-dose MTX (MH) group, which were fed by gavage with sterile water and three different doses of MTX: control (CT) group, low-dose MTX (ML) group, medium-dose MTX (MM) group, and high-dose MTX (MH) group.

[0011] The CIA rat bone marrow injury model included four groups obtained by gavage feeding with sterile water and three different MTX doses (low, medium, and high): control (CIA) group, low-dose CIA-MTX (CML) group, medium-dose CIA-MTX (CMM) group, and high-dose CIA-MTX (CMH) group.

[0012] S3. After feeding the animal to the predetermined feeding plan, stop feeding and sacrifice the model. Extract animal model samples and, based on the predetermined comparative analysis method, explore the optimal applicable dose of MTX for the treatment of RA.

[0013] Technical effects of the present invention:

[0014] This application establishes two novel methotrexate (MTX)-induced chronic bone marrow injury models in rats. Through comparative experiments with several animal models, it was found that in the rat model of chronic bone marrow injury, a medium dose of methotrexate represents the optimal concentration of MTX; in the CIA rat model of bone marrow injury, the CMM group represents the optimal concentration of MTX; and in the Wistar rat model of bone marrow injury, the MM group represents the optimal concentration of MTX. Therefore, this invention can investigate and clarify which concentration of MTX has a significantly greater therapeutic effect on RA than the adverse effects of bone marrow injury, and explore the specific mechanism by which MTX induces bone marrow injury. This has practical clinical significance for the treatment of RA with MTX.

[0015] Other features and aspects of this disclosure will become clear from the following detailed description of exemplary embodiments with reference to the accompanying drawings. Attached Figure Description

[0016] The accompanying drawings, which are included in and form part of this specification, illustrate exemplary embodiments, features, and aspects of this disclosure together with the specification and serve to explain the principles of this disclosure.

[0017] Figure 1 These are the experimental protocols for the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model;

[0018] Figure 2 This study compares the blood biochemical levels of different groups in the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model. Blood was collected from the heart on day 42 to obtain whole blood from rats, which was then centrifuged to obtain serum for blood biochemical analysis.

[0019] Figure 3 This is a comparison of visceral index levels in different groups of the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model.

[0020] Figure 4 compares the serum-specific antibody levels of different groups in a CIA rat bone marrow injury model.

[0021] Figure 5 This is a comparison of 2D planar images and CT data analysis of the distal femur in different groups of the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model.

[0022] Figure 6 This study compares the blood routine levels of different groups in the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model. Blood was collected from the heart of the rats on day 42 for blood routine testing.

[0023] Figure 7 This is a comparison of femoral bone marrow smears from different groups of the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model.

[0024] Figure 8 This study compares the erythroid colony formation capacity of femoral bone marrow in different groups of CIA rat bone marrow injury models and Wistar rat bone marrow injury models.

[0025] Figure 9 This study compares the femoral bone marrow granulocyte colony formation capacity of different groups in the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model.

[0026] Figure 10 This study compares the levels of bone marrow cell apoptosis in different groups of the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model. Detailed Implementation

[0027] Various exemplary embodiments, features, and aspects of this disclosure will now be described in detail with reference to the accompanying drawings. The same reference numerals in the drawings denote elements that have the same or similar functions. Although various aspects of the embodiments are shown in the drawings, they are not necessarily drawn to scale unless specifically indicated otherwise.

[0028] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0029] Furthermore, to better illustrate this disclosure, numerous specific details are set forth in the following detailed description. Those skilled in the art will understand that this disclosure can be practiced without certain specific details. In some instances, means, components, and circuits well known to those skilled in the art have not been described in detail in order to highlight the main points of this disclosure.

[0030] The experimental reagents and equipment involved in this embodiment can all be provided by the laboratory or purchased from the market; this embodiment does not impose any limitations.

[0031] The experimental methods / steps involved in this embodiment can be implemented with reference to clinical or laboratory procedures or methods, such as blood routine data collection. This embodiment does not limit them.

[0032] The rat model of arthritis (CIA), namely the rat model of bone marrow injury in CIA rats, can be prepared directly in the laboratory.

[0033] like Figure 1 As shown, this invention first requires the construction of animal models with different MTX levels, and then explores the optimal applicable dose of MTX for treating RA based on the different experimental data from these animal models. The main animal models constructed consist of the following two types:

[0034] 1. Establish animal models of chronic bone marrow injury induced by MTX based on the CIA model: CIA group, low-dose CIA-MTX (CML) group, medium-dose CIA-MTX (CMM) group and high-dose CIA-MTX (CMH) group;

[0035] 2. Establish an animal model of chronic bone marrow injury induced by MTX in normal Wistar rats: control (CT) group, low-dose MTX (ML) group, medium-dose MTX (MM) group, and high-dose MTX (MH) group.

[0036] Specifically,

[0037] First, dosage conversion is required. Based on the MTX treatment doses of 15mg / 70kg, 30mg / 70kg, and 60mg / 70kg for RA patients, these were converted into three corresponding rat coefficients: low (1.35mg / kg), medium (2.7mg / kg), and high (5.4mg / kg). To further investigate the therapeutic effect of MTX on RA and its adverse effects on bone marrow damage, this invention established animal models of chronic bone marrow damage based on a CIA model and a normal Wistar rat model. This clarified which concentration of MTX had a therapeutic effect far greater than its adverse effects on bone marrow damage and elucidated the specific mechanism by which MTX induced bone marrow damage.

[0038] Secondly, the design is as follows. Figure 1The model culture plan is shown below. Forty 6-week-old female Wistar rats were randomly divided into 8 groups:

[0039] Wistar rat chronic bone marrow injury model: Four groups were established: control (CT), low-dose MTX (ML), medium-dose MTX (MM), and high-dose MTX (MH).

[0040] and

[0041] CIA rat bone marrow injury model: four groups, namely, control (CIA), low-dose CIA-MTX (CML), medium-dose CIA-MTX (CMM), and high-dose CIA-MTX (CMH).

[0042] After one week of acclimatization, four groups of rats were established to form a rat model of bone marrow injury with CIA on days 7 and 14. Rats underwent CIA modeling on days 7 and 14. Starting from day 21, all rats in all eight groups were administered MTX via gavage twice weekly. The CT and CIA groups were administered sterile water via gavage, while the other groups were administered different concentrations of MTX via gavage. Rats were sacrificed on day 42, and samples were collected for subsequent experiments.

[0043] The extraction and preservation of animal model samples can be carried out by laboratory personnel according to the laboratory's extraction and processing requirements.

[0044] Next, comparative analysis will be conducted to explore the optimal MTX concentration for treating RA and to clarify the specific mechanism by which MTX causes bone marrow damage.

[0045] To explore a chronic model of bone marrow injury, this invention selected CIA rats and normal Wistar rats to establish a bone marrow injury model, and explored the model from three aspects: general condition assessment, CIA model assessment, and bone marrow injury-related detection.

[0046] Example 1: Comparative Analysis of Blood Biochemistry Levels

[0047] like Figure 2The figures show a comparison of blood biochemical levels in different groups of the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model. Whole blood was collected from the heart on day 42, centrifuged, and serum was obtained for blood biochemical analysis. Blood biochemical levels in different groups of the CIA rat bone marrow injury model included: ALT (A), AST (B), BUN (C), CREA (D), and TBIL (E). Blood biochemical and complete blood count levels in different groups of the normal Wistar rat bone marrow injury model included: ALT (F), AST (G), BUN (H), CREA (I), and TBIL (J). Data are presented as mean ± standard error and analyzed using one-way ANOVA. "ns" indicates P > 0.05. Five samples were used in each group.

[0048] Example 2: Comparative Analysis of Visceral Indices

[0049] like Figure 3 The figures show a comparison of visceral index levels in different groups of the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model. The visceral index levels in different groups of the CIA rat bone marrow injury model are: Liver index (A), Spleen index (B), Kidney index (C), and Thymus index (D). The blood biochemistry and complete blood count levels in different groups of the normal Wistar rat bone marrow injury model are: Liver index (E), Spleen index (F), Kidney index (G), and Thymus index (H). Data are presented as mean ± standard error and analyzed using one-way ANOVA. "ns" represents P > 0.05, "**" represents P < 0.01, and "***" represents P < 0.001. Five samples were used in each group.

[0050] Example 3: Comparative Analysis of 2D Plan Views and CT Data of the Distal Femur

[0051] Figure 4 shows a comparison of serum-specific antibody levels in different groups based on a rat bone marrow injury model with CIA: anti-CCP antibody (A) and anti-CoI II antibody (B). Data are presented as mean ± standard error and analyzed using one-way ANOVA. "ns" represents P > 0.05, "*" represents P < 0.05, "**" represents P < 0.01, and "****" represents P < 0.0001. Five samples were used in each group.

[0052] Example 4: Comparative Analysis of Blood Routine Tests

[0053] like Figure 5The image shows a comparison of 2D planar images and CT data analysis of the distal femur in different groups of the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model. On day 42, the right femur of each group of rats was collected, fixed with 4% paraformaldehyde for 24 hours, and then immersed in 75% alcohol for CT scanning and analysis. Micro-CT scans (SkyScan 1176, Bruker-MicroCT, Kontich, Belgium) with a resolution of 10 μM were used to analyze the distal femur of each group; three-dimensional reconstruction was performed under the same conditions, and morphometric analysis was performed using the evaluation software of the μCT system. Data from different groups in the CIA rat bone marrow injury model: relative trabecular volume (BV / TV) (I), number of trabeculae (Tb.N) (J), trabecular thickness (Tb.Th) (K), and bone mineral density (BMD) (L). Data from different groups in the normal Wistar rat bone marrow injury model: relative trabecular volume (BV / TV) (M), number of trabeculae (Tb.N) (N), trabecular thickness (Tb.Th) (O), and bone mineral density (BMD) (P). The data provided are mean ± standard error and are analyzed using one-way ANOVA. “ns” represents P>0.05, “**” represents P<0.01, and “***” represents P<0.001. There are five samples in each group.

[0054] Example 5: Comparative Analysis of Femoral Bone Marrow Smears

[0055] like Figure 6The figures show a comparison of complete blood count (CBC) levels in different groups of the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model. Whole blood was collected from the heart on day 42 for CBC analysis. CBC levels in different groups of the CIA rat bone marrow injury model included: White Blood Cell Count (A), Red Blood Cell Count (B), HGB (C), Platelet Count (PLT) (G), Neutrophil Count (NEUT) (H), Lymphocyte Count (I), and Monocyte Count (J). Blood biochemistry and CBC levels in different groups of the normal Wistar rat bone marrow injury model included: White Blood Cell Count (D), Red Blood Cell Count (E), HGB (F), Platelet Count (PLT) (K), Neutrophil Count (NEUT) (L), Lymphocyte Count (M), and Monocyte Count (N). The data provided are mean ± standard error and are analyzed using one-way ANOVA. “ns” represents P>0.05, “*” represents P<0.05, “**” represents P<0.01, “***” represents P<0.001, and “****” represents P<0.0001. There are five samples in each group.

[0056] Example 6: Comparative Analysis of Femoral Bone Marrow Erythroid Colony Formation Capacity

[0057] like Figure 7 The image shows a comparison of femoral bone marrow smears from different groups of CIA rat bone marrow injury models and Wistar rat bone marrow injury models. Bone marrow smears were taken from the right femur of each group of rats. The bone marrow was removed by cutting open both ends of the femur, diluted twice with physiological saline, and then dropped onto a glass slide. The slide was quickly spread, stained with Swiss Giessam, and the staining was observed using a X400 microscope.

[0058] Example 7: Comparative analysis of femoral bone marrow granulocyte colony-forming ability based on animal model samples extracted according to claim 1

[0059] like Figure 8 The image shows a comparison of erythroid colony-forming ability in femoral bone marrow from different groups in CIA rat bone marrow injury models and Wistar rat bone marrow injury models. Single-cell suspensions of bone marrow cells from both tibias were prepared at a concentration of 5 × 10⁻⁶. 6 Cells / sample (3 replicates / sample). Incubate the suspension in 24-well plates using a suitable culture system. After 7-10 days, photograph typical colonies under a light microscope according to morphological criteria.

[0060] Example 8: Comparative analysis of bone marrow cell apoptosis based on animal model samples extracted according to claim 1

[0061] like Figure 9 The image shows a comparison of femoral bone marrow granulocyte colony-forming ability in different groups of CIA rat bone marrow injury models and Wistar rat bone marrow injury models. Single-cell suspensions of bone marrow cells from both tibias were prepared at a concentration of 5 × 10⁻⁶. 6 Cells / sample (3 replicates / sample). Incubate the suspension in 24-well plates using a suitable culture system. After 7-10 days, photograph typical colonies under a light microscope according to morphological criteria.

[0062] Example 9: Comparative analysis of serum-specific antibodies based on animal model samples extracted according to claim 1

[0063] like Figure 10 The figure shows a comparison of bone marrow cell apoptosis levels in different groups of the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model. Bone marrow cell samples were collected after blood collection from the heart on day 42, and apoptosis rates were detected by flow cytometry. Data are presented as mean ± standard error and analyzed using one-way ANOVA. "***" represents P < 0.001, and "****" represents P < 0.0001. Five samples were used in each group.

[0064] Figure 3 Analysis showed that compared with the CIA group, there were no statistically significant differences in liver, spleen, and kidney indices among the other groups, but the thymus index showed a significant decrease in CML / CMM / CMH. Compared with the control group, there were no statistically significant differences in liver, spleen, and kidney indices among the other groups, but the thymus index showed a significant decrease in the CMH group. Figure 4 analysis showed that compared with the CT group, after CIA modeling, the anti-CCP and anti-COII antibodies were significantly increased in the CIA, CML, CMM, and CMH groups, indicating that CIA modeling was successful. Figure 5 MicroCT scans of the distal femur in rats revealed a significant reduction in bone mass after CIA modeling, with no significant difference compared to the CIA group after treatment with low, medium, and high concentrations of methotrexate (MTX). Compared to the Control group, no significant difference in bone mass was observed after MTX modeling with only low, medium, and high concentrations. Figure 6 After establishing CIA and Wistar rat models, compared with the CT group, the number of erythrocytes, leukocytes, hemoglobin, neutrophils, monocytes, and lymphocytes in the ML, MM, and MH groups was significantly reduced. Compared with the CIA group, the number of the above cell types was also significantly reduced in the CML, CMM, and CMH groups. Figure 7These are femoral bone marrow smears, stained with Swiss Giemsa stain and photographed under a light microscope at 400x magnification. Blue-purple indicates nucleated cells; pale yellow biconcave round cells are erythrocytes. The results show that normal Wistar and CIA rats, after being induced by MTX, exhibited a decrease in hematopoietic nucleated cells and disordered hematopoietic structure. Figure 8 The study was a femoral bone marrow erythroid colony formation experiment. The control group had a large number of erythroid colonies, while the number of erythroid colonies in other groups decreased, especially in the MM, MH, CIA-MM, and CIA-MH groups. Figure 9 The femoral bone marrow granulocyte colony formation experiment showed that the results were consistent with the trend of the erythroid colony formation experiment. Figure 10 This refers to bone marrow cell apoptosis. The gating point for the apoptosis assay was determined using Blank tubes (A), FITC-stained tubes (B), and PI-stained tubes (C). Combined with... Figure 10 As shown in CIA group (D), the proportion of early-withering Q1-LR was 4.27%, and the proportion of late-withering Q1-UR was 0.69%. Figure 10 As shown in the CML group (E), the proportion of early-withering Q1-LR was 12.03%, and the proportion of late-withering Q1-UR was 1.05%. Figure 10 As shown in the CMM group (F), the proportion of early-withering Q1-LR was 19.25%, and the proportion of late-withering Q1-UR was 1.50%. Figure 10 As shown in the CMH group (G), the proportion of early-withering Q1-LR was 20.73%, and the proportion of late-withering Q1-UR was 4.78%. Figure 10 As shown in the CMH group (G), the proportion of early-withering Q1-LR was 20.73%, and the proportion of late-withering Q1-UR was 4.78%. Figure 10 As shown in CT group (H), the proportion of early-maturing Q1-LR was 1.68%, and the proportion of late-maturing Q1-UR was 0.38%. Figure 10 As shown in group ML(I), the proportion of early-withering Q1-LR was 17.04%, and the proportion of late-withering Q1-UR was 1.64%. Figure 10 As shown in group MM(J), the proportion of early-withering Q1-LR was 24.49%, and the proportion of late-withering Q1-UR was 2.81%. Figure 10 As shown in the MH group (K), the proportion of early apoptosis Q1-LR was 26.45%, and the proportion of late apoptosis Q1-UR was 7.89%. Compared with the Control group, the myeloid cell apoptosis rate was significantly increased in the ML / MM / MH group, with a statistically significant difference between the MM and MH groups; compared with the CIA group, the myeloid cell apoptosis rate was significantly increased in the CML / CMM / CMH group, with a statistically significant difference between the CMM and MH groups.

[0065] Conclusion: Based on general assessments (blood biochemistry levels, visceral indices) and classic indicators of bone marrow injury (complete blood count, bone marrow smears, erythroid colony formation assay, granulocyte colony formation assay, and bone marrow cell apoptosis assay), significant changes were observed in these indicators after CIA rat and Wistar rat modeling. Specifically, the ML / MM / MH and CML / CMM / CMH groups showed significantly reduced numbers of erythrocytes, leukocytes, hemoglobin, neutrophils, monocytes, and lymphocytes. Bone marrow smears indicated a significantly decreased number of nucleated cells, disordered hematopoietic structure, reduced erythroid and granulocyte colony formation capacity, and a significantly increased bone marrow cell apoptosis rate. Notably, this invention found that the MM and CMM groups better reflected the specific phenotype of bone marrow injury in these indicators. In representative bone marrow smears, the MM and CMM groups showed a significantly lower number of nucleated cells than the ML and CML groups, but not significantly different from the MH and CMH groups. Furthermore, there were no significant differences between these two groups in peripheral blood detection, bone marrow smears, colony formation, and bone marrow cell apoptosis.

[0066] Therefore, this invention concludes that methotrexate is the optimal concentration of methotrexate (MTX) in establishing a rat model of chronic bone marrow injury. In the CIA rat bone marrow injury model, the CMM group was the optimal concentration of MTX, while in the Wistar rat bone marrow injury model, the MM group was the optimal concentration of MTX.

[0067] The various embodiments of this disclosure have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for determining the appropriate dosage of MTX in a rat model of myelosuppression, used for studying the optimal dosage of MTX for treating rheumatoid arthritis (RA), characterized in that... Includes the following steps: S1. The first dose of MTX used in clinical RA patients was converted into the second dose corresponding to the rat coefficient, and the second dose was divided into three different MTX gavage doses of low, medium and high according to the dosage from low to high. S2. Prepare several female Wistar rats and CIA rats, and feed them by gavage with sterile water and three different doses of MTX (low, medium and high) to establish animal models. The animal models include the Wistar rat chronic bone marrow injury model and the CIA rat bone marrow injury model, wherein: The Wistar rat chronic bone marrow injury model includes the following four groups: control (CT) group, low-dose MTX (ML) group, medium-dose MTX (MM) group, and high-dose MTX (MH) group, which were fed by gavage with sterile water and three different doses of MTX: control (CT) group, low-dose MTX (ML) group, medium-dose MTX (MM) group, and high-dose MTX (MH) group. The CIA rat bone marrow injury model included four groups obtained by gavage feeding with sterile water and three different doses of MTX (low, medium, and high): control (CIA) group, low-dose CIA-MTX (CML) group, medium-dose CIA-MTX (CMM) group, and high-dose CIA-MTX (CMH) group. S3. After gavage feeding to the predetermined feeding plan, feeding was stopped and the model was euthanized. Animal model samples were extracted and, based on a predetermined comparative analysis method, the optimal applicable dose of MTX for the treatment of RA was explored. The feeding plan is as follows: Female Wistar rats were randomly divided into the following groups: Wistar rat chronic bone marrow injury model: control (CT) group, low-dose MTX (ML) group, medium-dose MTX (MM) group, high-dose MTX (MH) group, and CIA rat bone marrow injury model: control (CIA) group, low-dose CIA-MTX (CML) group, medium-dose CIA-MTX (CMM) group and high-dose CIA-MTX (CMH) group; After one week of adaptive feeding, four groups of rats were established to form a CIA rat bone marrow injury model on day 7 and day 14. The rats underwent CIA modeling on day 7 and day 14. Starting from day 21, all rats in the eight groups were administered MTX by gavage twice a week. The CT group and the CIA group were administered sterile water by gavage, while the other groups were administered different concentrations of MTX by gavage.

2. The establishment method according to claim 1, characterized by, The process of extracting animal model samples and exploring the optimal dosage of MTX for treating RA based on a pre-defined comparative analysis method includes: exploring the optimal dosage of MTX for treating RA based on blood biochemical comparisons of the extracted animal model samples.

3. The establishment method according to claim 1, characterized by, The process of extracting animal model samples and exploring the optimal dosage of MTX for treating RA based on a pre-defined comparative analysis method includes: exploring the optimal dosage of MTX for treating RA based on a comparison of visceral indices of the extracted animal model samples.

4. The establishment method according to claim 1, characterized by, The process of extracting animal model samples and exploring the optimal MTX dose for treating RA based on a pre-defined comparative analysis method includes: exploring the optimal MTX dose for treating RA based on a comparison of 2D femoral head images and CT data of the extracted animal model samples.

5. The establishment method according to claim 1, characterized by, The process of extracting animal model samples and exploring the optimal dosage of MTX for treating RA based on a pre-defined comparative analysis method includes: exploring the optimal dosage of MTX for treating RA based on a comparison of blood routine data of the extracted animal model samples.

6. The establishment method according to claim 1, wherein, The process of extracting animal model samples and exploring the optimal dosage of MTX for treating RA based on a pre-defined comparative analysis method includes: exploring the optimal dosage of MTX for treating RA based on the comparison of femoral bone marrow smears from the extracted animal model samples.

7. The establishment method according to claim 1, characterized by, The method of extracting animal model samples and exploring the optimal applicable dose of MTX for treating RA based on a preset comparative analysis method includes: exploring the optimal applicable dose of MTX for treating RA based on the comparison of femoral bone marrow erythroid colony formation ability of the extracted animal model samples.

8. The establishment method according to claim 1, characterized by, The method of extracting animal model samples and exploring the optimal applicable dose of MTX for treating RA based on a preset comparative analysis method includes: exploring the optimal applicable dose of MTX for treating RA based on the comparison of femoral bone marrow granulocyte colony formation ability of the extracted animal model samples.

9. The establishment method according to claim 1, characterized by, The method of extracting animal model samples and exploring the optimal applicable dose of MTX for treating RA based on a pre-defined comparative analysis method includes: exploring the optimal applicable dose of MTX for treating RA based on the comparison of bone marrow cell apoptosis in the extracted animal model samples.

10. The method for establishing according to claim 1, characterized in that, The process of extracting animal model samples and exploring the optimal dosage of MTX for treating RA based on a pre-defined comparative analysis method includes: exploring the optimal dosage of MTX for treating RA based on the comparison of serum-specific antibodies in the extracted animal model samples.

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