Method for determining appropriate administration dosage of MTX-induced myelosuppression rat model

Abstract

The invention relates to a method for determining an appropriate administration dosage of an MTX (Methotrexate)-induced myelosuppression rat model, through establishing two novel rat chronic bone marrow injury models induced by methotrexate (MTX) and through experimental comparison of a plurality of animal models, it is found that in establishing the rat chronic bone marrow injury models, the dosage in methotrexate is MTX with the optimal concentration; in a C IA rat bone marrow injury model, a CMM group is an optimal concentration MTX group; and in a Wi star rat bone marrow injury model, the MM group is the MTX group with the optimal concentration. Therefore, which concentration of MTX has the treatment effect far greater than the adverse reaction of bone marrow injury on RA can be studied and determined, and the specific mechanism of bone marrow injury caused by MTX can be explored and determined, which has practical clinical significance on RA treatment by MTX.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to a method for establishing a suitable dosage for an MTX-induced bone marrow suppression rat model. Background Art

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

[0003] However, although MTX can treat RA, adverse reactions still occur clinically. Among them, 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 patients is still unclear. There is no relevant research to determine which concentration of MTX has a greater therapeutic effect on RA than the adverse reaction of bone marrow damage.

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

[0005] In order to solve the above problems, the present application proposes a method for establishing an appropriate dosage for an MTX-induced myelosuppression rat model.

[0006] A method for establishing an appropriate dosage for a rat model of myelosuppression induced by MTX, which is used for studying the optimal dosage of MTX for treating RA, comprises the following steps:

[0007] S1. Convert the first dose of MTX for RA patients clinically into the second dose corresponding to the rat coefficient, and divide the second dose into three different MTX gavage doses of low, medium and high according to the dosage from low to high;

[0008] S2. Prepare several Wistar female rats and CIA rats, and feed them by gavage with sterile water and three different MTX doses of low, medium and high to construct an animal model;

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

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

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

[0012] S3. When the preset feeding plan is reached by oral feeding, feeding is stopped and the model is killed. Samples of the animal model are extracted and the optimal dose of MTX for the treatment of RA is explored based on the preset comparative analysis method.

[0013] Technical effects of the present invention:

[0014] This application establishes two new rat chronic bone marrow injury models induced by methotrexate (MTX). After experimental comparison of several animal models, it is found that in the establishment of the rat chronic bone marrow injury model, the methotrexate medium dose is the best concentration of MTX; in the CIA rat bone marrow injury model, the CMM group is the best concentration MTX group; and in the Wistar rat bone marrow injury model, the MM group is the best concentration MTX group. Therefore, the present invention can study and clarify which concentration of MTX has a therapeutic effect on RA that is far greater than the adverse reactions of bone marrow injury and explore the specific mechanism of MTX causing bone marrow injury, which has practical clinical significance for the treatment of RA with MTX.

[0015] Further features and aspects of the present disclosure will become apparent from the following detailed description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0017] Figure 1 This is the experimental protocol for the CIA rat bone marrow injury model and the Wistar rat bone marrow injury model;

[0018] Figure 2 It is a comparison of blood biochemical levels in different groups of CIA rat bone marrow injury model and Wistar rat bone marrow injury model. Blood was collected from the heart on the 42nd day to obtain rat whole blood, which was centrifuged and then the serum was obtained for blood biochemical testing;

[0019] Figure 3 It is a comparison of the visceral index levels of different groups in the bone marrow injury model of CIA rats and the bone marrow injury model of Wistar rats;

[0020] FIG4 is a comparison of serum specific antibody levels in different groups based on the CIA rat bone marrow injury model;

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

[0022] Figure 6 It is a comparison of the blood routine levels of different groups in the bone marrow injury model of CIA rats and the bone marrow injury model of Wistar rats. Blood was collected from the heart on the 42nd day to obtain whole blood for blood routine testing;

[0023] Figure 7 It is a comparison of femoral bone marrow smears of different groups in the bone marrow injury model of CIA rats and the bone marrow injury model of Wistar rats;

[0024] Figure 8 It is a comparison of the erythroid colony-forming ability of femoral bone marrow in different groups of CIA rat bone marrow injury model and Wistar rat bone marrow injury model;

[0025] Fig. 9 This is a comparison of the granulocyte colony-forming ability of femoral bone marrow in different groups of CIA rat bone marrow injury model and Wistar rat bone marrow injury model;

[0026] Fig.10 It is a comparison of the apoptosis levels of bone marrow cells in different groups of the bone marrow injury model of CIA rats and the bone marrow injury model of Wistar rats. DETAILED DESCRIPTION

[0027] Various exemplary embodiments, features and aspects of the present disclosure will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0028] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0029] In addition, in order to better illustrate the present disclosure, numerous specific details are given in the following specific embodiments. It should be understood by those skilled in the art that the present disclosure can also be implemented without certain specific details. In some examples, means, components and circuits well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present disclosure.

[0030] The experimental reagents, equipment, etc. involved in this embodiment can be provided by the laboratory or purchased on the market, and this embodiment does not limit them.

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

[0032] The CIA rat model, namely the CIA rat bone marrow injury model, can be directly prepared by the laboratory.

[0033] like Figure 1 As shown, the present invention first needs to construct different MTX animal models, and explore the optimal MTX dose required for treating RA based on different experimental data of the animal models. The animal model mainly consists of the following two animal models:

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

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

[0036] Specifically,

[0037] First, dose conversion is needed. Based on the 15mg / 70kg, 30mg / 70kg and 60mg / 70kg doses of MTX for treating RA patients, the three doses of low (1.35mg / kg), medium (2.7mg / kg) and high (5.4mg / kg) corresponding to the rat coefficient are converted. In order to further explore the therapeutic effect of MTX on RA and the adverse reaction to bone marrow injury, the present invention establishes an animal model of chronic bone marrow injury based on the CIA model and the normal Wistar rat model, so as to clarify which concentration of MTX, the therapeutic effect is far greater than the adverse reaction of bone marrow injury and clarify the specific mechanism of MTX causing bone marrow injury.

[0038] Secondly, the design is as shown in the attached Figure 1Forty 6-week-old female Wistar rats were randomly divided into 8 groups:

[0039] The Wistar rat chronic bone marrow injury model was divided into four groups: control (CT), MTX low-dose (ML), MTX medium-dose (MM), and MTX high-dose (MH).

[0040] and

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

[0042] After one week of adaptive feeding, four groups of CIA rat bone marrow injury models were established on the 7th and 14th days. Rats were modeled with CIA on the 7th and 14th days. From the 21st day, all rats in the eight groups were gavaged with MTX twice a week. The CT group and the CIA group were gavaged with sterile water, and the other groups were gavaged with different concentrations of MTX. The rats were killed on the 42nd day, and the samples were collected for subsequent experiments.

[0043] The extraction and preservation of animal model samples can be performed by the experimenter according to the laboratory extraction and processing requirements.

[0044] Next, a comparative analysis method was used to explore the optimal MTX concentration for the treatment of RA and to clarify the specific mechanism by which MTX causes bone marrow damage.

[0045] In order to explore the chronic model of bone marrow injury, the present invention selected CIA rats and normal Wistar rats for bone marrow injury modeling, and explored the model from three aspects: general condition evaluation, CIA model evaluation and bone marrow injury related detection.

[0046] Example 1: Comparative analysis of blood biochemical levels

[0047] like Figure 2The figure shows the comparison of blood biochemical levels in different groups of bone marrow injury model of CIA rats and Wistar rats. Blood was collected from the heart on the 42nd day to obtain whole blood of rats and centrifuged to obtain serum for blood biochemical detection. Blood biochemical levels of different groups in bone marrow injury model of CIA rats: ALT (A), AST (B), BUN (C), creatinine CREA (D) and total bilirubin TBIL (E). Blood biochemical and blood routine levels of different groups in bone marrow injury model of normal Wistar rats: ALT (F), AST (G), BUN (H), creatinine CREA (I) and total bilirubin TBIL (J). The data given are mean ± standard error and analyzed by one-way ANOVA. "ns" means P>0.05, five samples in each group.

[0048] Example 2: Comparative analysis of visceral index

[0049] like Figure 3 The figure shows the comparison of visceral index levels in different groups of bone marrow injury model of CIA rats and Wistar rats. Visceral index levels in different groups of bone marrow injury model of CIA rats: liver index Liverindex (A), spleen index Spleen index (B), kidney index Kidney index (C) and thymus index Thymusindex (D). Blood biochemistry and blood routine levels in different groups of bone marrow injury model of normal Wistar rats: liver index Liver index (E), spleen index Spleen index (F), kidney index Kidney index (G) and thymus index Thymus index (H). The data given are mean ± standard error and analyzed by one-way ANOVA, "ns" represents P>0.05, "**" represents P<0.01, "***" represents P<0.001, five samples in each group.

[0050] Example 3: Comparative analysis of distal femur 2D plan view and CT data

[0051] As shown in Figure 4, it is a comparison of serum specific antibody levels in different groups based on the CIA rat bone marrow injury model, anti-CCP antibody (A), anti-CoII antibody (B). The data given are mean ± standard error and one-way ANOVA, "ns" represents P>0.05, "*" represents P<0.05. "**" represents P<0.01, "****" represents P<0.0001, five samples in each group.

[0052] Example 4: Comparative analysis of blood routine tests

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

[0054] Example 5: Comparative analysis of femoral bone marrow smears

[0055] like Figure 6The following table shows the comparison of blood routine test levels in different groups of bone marrow injury model of CIA rats and Wistar rats. Blood was collected from the heart on the 42nd day to obtain whole blood for blood routine test. Blood routine test levels in different groups of bone marrow injury model of CIA rats: White Blood Cell Count (A), Red Blood Cell Count (B), HGB (C), Platelet Count (PLT) (G), Neutrophil Count (NEUT) (H), Lymph Count (I) and Monocyte Count (J). Blood biochemistry and blood routine test levels in different groups of bone marrow injury model of normal Wistar rats: White Blood Cell Count (D), Red Blood Cell Count (E), HGB (F), Platelet Count (PLT) (K), Neutrophil Count (NEUT) (L), Lymph Count (M) and Monocyte Count (N). The data presented are mean ± SD and analyzed by 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, with five samples in each group.

[0056] Example 6: Comparative analysis of femoral bone marrow erythroid colony-forming ability

[0057] like Figure 7 The figure shows the comparison of femoral bone marrow smears of different groups in the bone marrow injury model of CIA rats and Wistar rats. The right femur of each group of rats was selected for bone marrow smear, and the two ends of the femur were cut to pick out the bone marrow, which was diluted 2 times with normal saline and dripped onto the slide. The slide was pushed quickly, Swiss Guisam staining was performed, and the staining was observed using a microscope X400.

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

[0059] like Figure 8 The figure shows the comparison of the colony-forming ability of femoral bone marrow erythroid cells in different groups of CIA rat bone marrow injury model and Wistar rat bone marrow injury model. Single cell suspension was prepared from bone marrow cells of bilateral tibia at a concentration of 5×10 6 cells / sample (repeated 3 times / sample). The suspension was cultured in a 24-well plate according to an appropriate culture system. After 7-10 days, typical colonies were photographed under a light microscope according to morphological standards.

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

[0061] like Fig. 9 The figure shows the comparison of the granulocyte colony-forming ability of femoral bone marrow in different groups of CIA rat bone marrow injury model and Wistar rat bone marrow injury model. Single cell suspension was prepared from bone marrow cells of bilateral tibia at a concentration of 5×10 6 cells / sample (repeated 3 times / sample). The suspension was cultured in a 24-well plate according to an appropriate culture system. After 7-10 days, typical colonies were photographed under a light microscope according to morphological standards.

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

[0063] like Fig.10 The figure shows the comparison of the apoptosis level of bone marrow cells in different groups of the bone marrow injury model of CIA rats and the bone marrow injury model of Wistar rats. After blood was taken from the heart on the 42nd day, bone marrow cell samples were collected and the apoptosis rate was detected by flow cytometry. The data given are mean ± standard error and analyzed by one-way ANOVA, "***" represents P < 0.001, "****" represents P < 0.0001, and there are five samples in each group.

[0064] Figure 3 The analysis showed that compared with the CIA group, the other groups had no statistical differences in liver, spleen, and kidney indexes, and the thymus index of CML / CMM / CMH was significantly decreased. Compared with the Control, the other groups had no statistical differences in liver, spleen, and kidney indexes, and the thymus index of the CMH group was significantly decreased; Figure 4 analysis showed that compared with the CT group, after CIA modeling, the anti-CCP and anti-COII antibodies of the CIA group, CML group, CMM group, and CMH group were significantly increased, indicating that the CIA modeling was successful; Figure 5 MicroCT scanning of the distal femur of rats revealed that bone mass was significantly reduced after CIA modeling, and there was no significant difference compared with the CIA group after treatment with low, medium and high concentrations of MTX. Compared with the Control group, there was no significant difference in bone mass after simple MTX low, medium and high concentrations modeling; Figure 6 It was found that after CIA rat modeling and Wistar rat modeling, the number of red and white blood cells, hemoglobin, neutrophils, monocytes, lymphocytes, etc. in the ML group, MM group, and MH group were significantly reduced compared with the CT group. Compared with the CIA group, the number of the above cell types in the CML group, CMM group, and CMH group also decreased significantly. Figure 7This is a femoral bone marrow smear, which was stained with Swiss Giemsa and photographed under a light microscope at x400 times. The blue-purple color is the result of nucleated cell staining; the pale yellow biconcave round cells are red blood cells. The results showed that after normal Wistar and CIA rats were modeled with MTX, there was a decrease in hematopoietic nucleated cells and disordered hematopoietic structures; Figure 8 This is a femoral bone marrow erythroid colony formation experiment, in which the number of erythroid colonies in the blank group was large, while the number of erythroid colonies formed in other groups decreased, especially in the MM, MH, CIA-MM and CIA-MH groups, where the number of colonies decreased significantly. Fig. 9 It is a femoral bone marrow granulocyte colony formation experiment. The results analysis found that the trend was consistent with the erythroid colony formation experiment. Fig.10 The apoptosis of bone marrow cells was detected by using Blank tube (A), FITC single staining tube (B) and PI single staining tube (C). Fig.10 As shown in the CIA group (D), the proportion of early withering Q1-LR was 4.27%, and the proportion of late withering Q1-UR was 0.69%. Fig.10 As shown in the CML group (E), early withering Q1-LR accounted for 12.03%, late withering Q1-UR accounted for 1.05%, combined with Fig.10 As shown in the CMM group (F), early withering Q1-LR accounted for 19.25%, late withering Q1-UR accounted for 1.50%, combined with Fig.10 As shown in the CMH group (G), early withering Q1-LR accounted for 20.73%, late withering Q1-UR accounted for 4.78%, combined with Fig.10 As shown in the CMH group (G), early withering Q1-LR accounted for 20.73%, late withering Q1-UR accounted for 4.78%, combined with Fig.10 As shown in the CT group (H), the proportion of early withering Q1-LR was 1.68%, and the proportion of late withering Q1-UR was 0.38%. Fig.10 As shown in the ML group (I), early withering Q1-LR accounted for 17.04%, late withering Q1-UR accounted for 1.64%, combined with Fig.10 As shown in the MM group (J), early withering Q1-LR accounted for 24.49%, late withering Q1-UR accounted for 2.81%, combined with Fig.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 apoptosis rate of bone marrow cells in the ML / MM / MH group was significantly increased, and there was a statistical difference between the MM and MH groups; compared with the CIA group, the apoptosis rate of bone marrow cells in the CML / CMM / CMH group was significantly increased, and there was a statistical difference between the CMM and MH groups.

[0065] Conclusion: From the evaluation of general conditions (blood biochemical levels, visceral index) and classic indicators of bone marrow injury (blood routine, bone marrow smear, erythroid colony formation experiment, granulocyte colony formation experiment and bone marrow cell apoptosis experiment), it was found that after CIA rat modeling and Wistar rat modeling, the above indicators showed significant changes, such as the number of red and white blood cells, hemoglobin, neutrophils, monocytes, lymphocytes, etc. in the ML / MM / MH group and the CML / CMM / CMH group was significantly reduced, the bone marrow smear showed a significant decrease in the number of nucleated cells, hematopoietic structure disorder, erythroid colony and granulocyte colony formation ability decreased, and bone marrow cell apoptosis rate increased significantly. It is worth noting that the present invention found that the MM group and the CMM group were more in line with the phenotypic embodiment of bone marrow injury in the above indicators. In the representative smear of bone marrow smear, the bone marrow nucleated cells in the MM and CMM groups were significantly reduced compared with the ML and CML groups, but not much different from the MH and CMH groups, and the two groups had little difference in peripheral blood detection, bone marrow smear, colony, and bone marrow cell apoptosis.

[0066] Therefore, the present invention draws a conclusion that in establishing a rat chronic bone marrow injury model, the medium dose of methotrexate is the best concentration of MTX. In the CIA rat bone marrow injury model, the CMM group is the best concentration MTX group, and in the Wistar rat bone marrow injury model, the MM group is the best concentration MTX group.

[0067] The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for establishing an appropriate dosage for a rat model of myelosuppression induced by MTX, for use in the study of the optimal dosage of MTX for the treatment of RA, characterized in that: The steps include: S1. Convert the first dose of MTX for RA patients clinically into the second dose corresponding to the rat coefficient, and divide the second dose into three different MTX gavage doses of low, medium and high according to the dosage from low to high; S2. Prepare several Wistar female rats and CIA rats, and feed them by gavage with sterile water and three different MTX doses of low, medium and high to construct an animal model; The animal models include a Wistar rat chronic bone marrow injury model and a CIA rat bone marrow injury model, wherein: The Wistar rat chronic bone marrow injury model includes the following four groups obtained by gavage feeding with sterile water and three different MTX gavage doses: a control (CT) group, a MTX low dose (ML) group, a MTX medium dose (MM) group, and a MTX high dose (MH) group; The CIA rat bone marrow injury model includes four groups obtained by gavage feeding with sterile water and three different MTX doses: control (CIA) group, CIA-MTX low dose (CML) group, CIA-MTX medium dose (CMM) group and CIA-MTX high dose (CMH) group. S3. When the preset feeding plan is reached by oral feeding, feeding is stopped and the model is killed. Samples of the animal model are extracted and the optimal dose of MTX for the treatment of RA is explored based on the preset comparative analysis method.

2. Based on the blood biochemistry comparison of the animal model samples extracted according to claim 1, explore the optimal dose of MTX for the treatment of RA.

3. Based on the comparison of visceral indexes of the animal model samples extracted according to claim 1, explore the optimal dose of MTX for the treatment of RA.

4. Based on the comparison of the 2D plane image of the distal femur and the CT data of the animal model sample extracted in claim 1, the optimal applicable dose of MTX for the treatment of RA is explored.

5. Based on the comparison of blood routine of animal model samples extracted according to claim 1, explore the optimal dosage of MTX for treating RA.

6. Based on the comparison of femoral bone marrow smears of the animal model samples extracted according to claim 1, explore the optimal dosage of MTX for treating RA.

7. Based on the comparison of femoral bone marrow erythroid colony-forming ability of the animal model samples extracted according to claim 1, the optimal dosage of MTX for treating RA is explored.

8. Based on the comparison of femoral bone marrow granulocyte colony-forming ability of the animal model samples extracted according to claim 1, explore the optimal dosage of MTX for treating RA.

9. Based on the comparison of bone marrow cell apoptosis in the animal model samples extracted according to claim 1, explore the optimal dosage of MTX for treating RA.

10. Based on the comparison of serum specific antibodies of the animal model samples extracted according to claim 1, explore the optimal dosage of MTX for treating RA.

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