CN113304248B - Application of IL-2 in preparation of medicine for relieving side effect of glucocorticoid medicine - Google Patents

Abstract

The invention provides an application of IL-2 in the preparation of drugs for alleviating the side effects of glucocorticoid drugs, which belongs to the technical field of medicine. Low-dose IL-2 therapy can reduce the incidence of CD25 ^high CD127 ^low Treg, active Treg, resting Treg and non-Treg percentage decline and the incidence of CD4+Treg cell CD25 expression decline during SLE patients receiving glucocorticoid therapy, and It can promote the increase of the above-mentioned Treg cell subsets and CD25 molecules in a considerable proportion of patients; IL-2 can activate the pSTAT5 pathway of CD4+Treg cells; IL-2 can increase the expression of anti-virus-related genes in CD4+T cells, and make CD4 +T cell immune response negatively regulates genes related to upregulation, and can increase the expression of anti-apoptotic molecule Bcl‑2.

Description

Application of IL-2 in the preparation of drugs for alleviating the side effects of glucocorticoids

technical field

The invention belongs to the technical field of medicines, and in particular relates to an application of IL-2 in the preparation of medicines for alleviating the side effects of glucocorticoids.

Background technique

Glucocorticoid (GC) is an extremely important class of regulatory molecules in the body. It plays an important role in regulating the development, growth, metabolism and immune function of the body. It is the most important regulatory hormone for the body's stress response. The most widely used and effective anti-inflammatory and immunosuppressant. In urgent or critical situations, glucocorticoids are often the first choice. Common clinical glucocorticoid drugs include prednisone, methylprednisone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone, dexamethasone, etc., which have anti-inflammatory, anti-toxic, Anti-allergic, anti-shock, non-specific immune suppression and antipyretic effects, etc., can prevent and prevent the occurrence of immune inflammatory response and pathological immune response, suitable for the treatment of systemic lupus erythematosus, systemic sclerosis, dry syndrome Syndrome, dermatomyositis and other diseases. However, the treatment of glucocorticoids is often accompanied by some side effects, such as osteoporosis, induction or aggravation of infection, mental disorders and other side effects. Moreover, studies have found that the use of glucocorticoids can excessively inhibit immune cell function and promote Treg apoptosis and other side effects. These side effects will lead to an increase in the incidence of infection in patients, as well as anti-autoimmunity or inflammation due to the decrease in the proportion of Treg and the decrease in the expression of related molecules. The curative effect is weakened.

At present, there are few drugs that relieve the side effects of glucocorticoids, and generally they can only be avoided in advance. Avoid high-sugar foods; regularly review electrocardiograms; avoid using them as much as possible for pregnant women, patients with diabetes and other endocrine diseases, hypertension, cardiovascular diseases, tumors, etc. . Dermatology Bulletin, 2015, 032(003):248-252). Therefore, there is an urgent need to invent a related drug that can effectively reduce the side effects of glucocorticoid drugs.

Contents of the invention

In view of this, the purpose of the present invention is to provide the application of IL-2 in the preparation of drugs for alleviating the side effects of glucocorticoid drugs.

In order to realize the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

Application of IL-2 in the preparation of medicines for alleviating the side effects of glucocorticoids.

Preferably, the side effects of the glucocorticoids include inhibition of Treg cells and increased risk of infection.

Preferably, the drug is used for patients receiving glucocorticoid drug treatment.

Preferably, the medicament is used for treating systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, myositis, IgG4-related diseases, rheumatoid arthritis, and vasculitis.

Preferably, the IL-2 can reduce the incidence of decreased percentages of CD4+CD25 ^high CD127 ^low Treg, active Treg, resting Treg and non-Treg cells in patients receiving the glucocorticoid therapy.

Preferably, the IL-2 can reduce the incidence of decreased expression of CD25 in CD4+Treg cells in patients receiving the glucocorticoid treatment.

Preferably, the IL-2 can increase the pSTAT5 level of CD4+Treg cells.

Preferably, the IL-2 can increase the expression of anti-virus-related genes of CD4+T cells, and up-regulate the genes related to the negative regulation of CD4+T cell immune response.

The invention also provides a pharmaceutical composition, which comprises effective doses of IL-2 and glucocorticoid drugs.

Preferably, the effective dose of IL-2 is 250,000 IU to 3 million IU.

Compared with the prior art, the present invention has the following beneficial effects:

(1) In addition to glucocorticoid therapy, low-dose IL-2 therapy can reduce the proportion of CD4+CD25 ^high CD127 ^low Treg, active Treg, restingTreg, non-Treg and other cells in patients receiving glucocorticoid therapy decreased incidence and further increased the proportion of these Treg cell subsets.

(2) At the same time of glucocorticoid treatment, adding low-dose IL-2 therapy can reduce the incidence of decreased CD25 expression in CD4+ Treg cells in patients receiving glucocorticoid therapy, and further increase the CD25 expression of CD4+ Treg cells. Express.

(3) IL-2 significantly reversed the down-regulation of active Treg cells and the reduction of CD25 expression caused by glucocorticoids, and activated the pSTAT5 pathway of CD4+Treg cells.

(4) For PBMCs of patients, IL-2 can increase the expression of Treg anti-apoptotic molecule Bcl-2.

(5) Glucocorticoids can reduce the expression of anti-virus related genes in CD4+ T cells, and IL-2 can reverse this effect, and up-regulate the genes related to the negative regulation of CD4+ T cell immune response. It can be seen that IL-2 can alleviate the side effects of glucocorticoid drugs, and the combined use of the two has a clinical application prospect.

Description of drawings

Figure 1 shows the effect of Prednisolone and IL-2 on the ratio of CD4+Foxp3+Treg: A. Gating strategy: Circle lymphocytes, single cells, live cells and CD3+CD4+T cells from left to right; B. Prednisolone Treatment of PBMCs alone can greatly reduce the expression of Foxp3 in CD4+ T cells, and the addition of IL-2 can significantly reverse this phenomenon;

Figure 2 is the effect of Prednisolone and IL-2 on the ratio of CD4+Foxp3+Treg: C. Changes of Treg subgroups and CD25, CD127 expression after PBMCs treatment of healthy people; D. Treg subgroups and CD25, CD127 expression after PBMCs treatment of SLE patients Variety;

Figure 3 is the effect of Prednisolone and IL-2 on CD4+Foxp3+Treg function, proliferation and expression of anti-apoptotic molecules: Prednisolone or Prednisolone combined with IL-2 on the expression of CD4+Foxp3+ cells A.CD39, B.CTLA-4, C .The influence of Bcl-2, D.Ki-67, E.ICOS; the left side is the result of PBMCs processing of healthy people, and the right side is the result of PBMCs processing of SLE patients;

Figure 4 is the effect of Prednisolone and IL-2 on the phosphorylation level of STAT5 in CD4+Foxp3+ cells;

Figure 5 principal component analysis: ZHT, JJY, YXJ are three healthy people, P is Prednisolone alone treatment group, H is Prednisolone combined with IL-2 treatment group, C is the control group;

Figure 6 is differential gene clustering analysis: heat map (Heatmap); ZHT, JJY, YXJ are three healthy people, P is Prednisolone alone treatment group, H is Prednisolone combined with IL-2 treatment group, C is the control group;

Figure 7 is the BPGO analysis: A. Prednisolone alone treatment group compared with the BPGO analysis of the control group down-regulated genes; B. Prednisolone combined with IL-2 treatment group compared with the BPGO analysis of Prednisolone alone treatment group up-regulated genes; C. Prednisolone combined with IL -2 BPGO analysis of up-regulated genes in the treatment group compared to the control group;

Figure 8 shows the changes in the proportion of CD25 ^high CD127 ^low Treg cells in SLE patients before and after treatment with glucocorticoids or glucocorticoids combined with low-dose IL-2: A. The changes in the proportions of Tregs in the two groups of patients before and after treatment. Changes in the proportion of CD25 ^{high CD127 low Treg cells before and after dose IL-2 treatment, the right figure shows the changes in the proportion of CD25 high CD127 low Treg cells} before and after glucocorticoid treatment alone; B. Schematic diagram of the changes in the proportion of Treg in the two groups after treatment; the proportion of Treg increased after treatment More than 10% is defined as an up-regulation, a decrease of more than 10% is defined as a down-regulation, and the rest of the situation is defined as a constant ratio;

Figure 9 shows the changes in the proportion of Treg cell subsets and CD25 expression in SLE patients before and after treatment with glucocorticoids or glucocorticoids combined with low-dose IL-2: A. The first row: glucocorticoids combined with low-dose IL-2 treatment group, the first row The second row: glucocorticoid treatment group; B. Changes in the proportion of CD4+CD25++ and Treg subgroups before and after treatment: after treatment, the proportion increased by more than 10% as up-regulation, the proportion decreased by more than 10% as down-regulation, and the rest remained unchanged.

detailed description

The invention provides the application of IL-2 in the preparation of medicines for alleviating the side effects of glucocorticoids.

The present invention has no special limitation on the side effects of the glucocorticoid drugs, including the suppression of Treg cells and the increase of infection risk, preferably the suppression of Treg cells. The medicine of the present invention is preferably used for treating systemic lupus erythematosus, sclerosis, Sjögren's syndrome and dermatomyositis.

The IL-2 of the present invention can increase the ratio of CD25 ^high CD127 ^low Treg, active Treg, resting Treg, and non-Treg cells; increase the expression of CD25 in CD4+T cells; activate the pSTAT5 pathway of CD4+T cells; increase the CD4+T cell The expression of anti-virus-related genes, and the up-regulation of genes related to negative regulation of CD4+T cell immune response; increase the expression of anti-apoptotic molecule Bcl-2.

The invention also provides a pharmaceutical composition, which comprises effective doses of IL-2 and glucocorticoid drugs.

In the present invention, the glucocorticoid drug preferably includes one of Prednisolone, prednisone or methylprednisolone.

Further, the drug of the present invention includes an effective amount of IL-2 and glucocorticoid drugs and pharmaceutically acceptable adjuvants or carriers; the effective amount means that it can produce functions or activities on humans and/or animals. In a preferred embodiment of the present invention, the effective dose of IL-2 is preferably 250,000 IU to 3 million IU; the pharmaceutically acceptable adjuvant Or carrier refers to the adjuvant or carrier used for the administration of therapeutic agents, including various excipients and diluents, which themselves are not necessary active ingredients, and there is no excessive toxicity after administration, the present invention is for the adjuvant and carrier There is no special limitation, and conventional pharmaceutical excipients and carriers in this field can be used. Furthermore, the preparation of the medicament for alleviating the side effects of glucocorticoid drugs according to the present invention also includes auxiliary substances, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, and the like.

The present invention has no special limitation on the sources of raw materials mentioned, and conventional commercially available products can be used.

The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

In the embodiment of the present invention, SPSS 20.0 software was used for statistical analysis. For the comparison of two sets of data, the Shapiro-Wilk normality test was performed first. If the data conformed to a normal distribution, the independent sample T test was used; if the data did not conform to a normal distribution, the Mann-Whiney test was used. For comparisons of multiple pairs of samples, pairwise t-tests were used. The chi-square test was used to compare the number of Treg cells up-regulated, down-regulated or remained unchanged before and after treatment between the two groups of patients.

Example 1

research object

From June 2016 to April 2017, 28 SLE patients admitted to a tertiary hospital were divided into two groups, 11 of whom were given glucocorticoid therapy, and the other 17 patients were given glucocorticoid combined with low-dose IL- 2 treatments.

The glucocorticoids used in the treatment of patients were prednisone or methylprednisolone. Prednisone is given orally and methylprednisolone is given intravenously. The daily dosage of glucocorticoids varies depending on the condition, and oral prednisone ranges from less than 7.5mg/day to 50mg/day. In severe cases, intravenous methylprednisolone pulse therapy can be used, up to 200mg-1g/day, and gradually reduce the dose after 3 days of medication. The dosage of IL-2 is not more than 3 million IU/day, and the course of treatment is determined according to the condition and changes of Treg. By studying the changes in the ratio of Treg, Treg three subpopulations and CD25++ subpopulation in CD4+Treg after 2 weeks of treatment in the two groups of patients, the effect of IL-2 on the effect of glucocorticoids on Treg was explored.

From January 2018 to December 2018, several cases of SLE patients admitted to a tertiary hospital, or peripheral blood of healthy people were collected, and PBMCs were isolated for in vitro experiments on Prednisolone and IL-2.

The markers of each subpopulation of immune cells are shown in Table 1.

Table 1 Markers of immune cell subsets

Example 2

Predinisolone and IL-2 stimulate PBMCs experiment in vitro

1. Use 4ml EDTA anticoagulant blood collection tubes to take peripheral blood from healthy people or SLE patients before treatment in January 2018 to December 2018 in Example 1.

2. Add 6mL of Human Peripheral Blood Lymphocyte Separation Medium into a 15mL centrifuge tube.

3. Use a sterile Pasteur pipette to mix and absorb the blood, tilt the centrifuge tube at 45°, slowly add 4 mL of blood along the lower wall about 1 cm away from the human peripheral blood lymphocyte separation solution, and be careful not to damage the liquid surface.

4. Slowly return the centrifuge tube to vertical, and centrifuge at 1800r20min.

5. Discard the upper layer of plasma, and use a Pasteur pipette to transfer the buffy coat to another clean 15mL centrifuge tube, add normal saline to 14mL, centrifuge at 1600r for 15min, and discard the supernatant to obtain PBMCs.

6. Use 2mL 1640 complete medium to concentrate all PBMCs into a 15mL centrifuge tube and mix well.

7. Take 10 μL cells and add 90 μL 1640 complete medium, vortex to mix and use a hemocytometer to count.

8. Use 1640 complete medium to adjust the volume of the cell suspension to a cell concentration of 200,000/100 μL, and calculate the number of duplicate wells in each group: there are 4 sets of stimulation conditions in total, and the number of cells in each well is 200,000, so the number of duplicate wells in each group is The total number of cells/800,000, rounded down.

9. Take four 15mL centrifuge tubes and use 1640 complete medium and reagent or reagent solvent to prepare in vitro stimulating solution with the following concentration (multiple wells in each group + 1) × 100 μL volume.

Group P: Prednisolone=10 μM, IL-2=0 IU/mL;

Group H: Prednisolone=10μM, IL-2=100IU/mL;

Group L: Prednisolone=10μM, IL-2=10IU/mL;

Group C: Prednisolone=0 μM, IL-2=0 IU/mL.

10. Add 100 μL of cell suspension to each well of a 96-well flat bottom plate, and add 100 μL of each group of stimulators to each well. Mark the position of each group on the cover.

11.37°C in a CO ₂ incubator for 4 days.

12. Collect cell pellets, perform Treg cell subsets and Treg function, anti-apoptosis indicator staining, Th1 and Th17 cell staining, Treg cell pSTAT5 signaling pathway staining, Treg staining, and then detect. Since the expression of CD4+ T cells with CD25++ or above is positively correlated with the expression of Foxp3 and CD25, Treg cells based on the expression of Foxp3 can also be divided into three groups: CD45RA-Foxp3high active Treg, CD45RA-Foxp3int non-Treg and CD45RA+Foxp3intresting Treg .

In the results, the control group was L group: Prednisolone=10 μM, IL-2=10 IU/mL.

It can be seen from Figure 1 and Figure 2 that compared with the control group, Prednisolone down-regulates the expression of Foxp3 in CD4+T cells (that is, the level of CD4+Foxp3+Treg), and IL-2 can significantly reverse the decrease in Foxp3 expression induced by Prednisolone. Compared with the control group, Prednisolone significantly down-regulated the proportion of active Treg, and had a certain up-regulation effect on the proportion of non-Treg; while IL-2 could significantly reverse the down-regulation of the proportion of active Treg by Prednisolone, and significantly down-regulated the proportion of non-Treg. Further analysis of the expression of IL-2 receptor CD25 and IL-7 receptor CD127 on the surface of Treg revealed that Prednisolone can increase the expression of CD127 in Foxp3+ cells, while IL-2 can significantly up-regulate the expression of Foxp3 in CD4+ T cells and increase the expression of CD25 in Foxp3+ cells. Express and downregulate the expression of CD127.

It can be seen from Figure 3 that Prednisolone significantly down-regulates the expression of CD39 in Foxp3+ cells in healthy people, while IL-2 significantly reverses the effect of Prednisolone; IL-2 can also regulate the expression of CD39 in Foxp3+ cells in SLE patients to a certain extent. IL-2 can up-regulate the expression of anti-apoptotic molecule Bcl-2 in CD4+Foxp3+Treg cells. In addition, IL-2 could reverse the down-regulation effect of Prednisolone on ICOS of CD4+Foxp3+Treg cells.

It can be seen from Figure 4 that the CD4+Foxp3+Treg cells in the healthy control group had a certain degree of STAT5 phosphorylation, and the phosphorylation level of STAT5 decreased after Prednisolone treatment, which marked the decrease in the activation level of Treg; while the CD4+Foxp3+Treg cells in the SLE patient control group The phosphorylation level of STAT5 in Treg cells was low, and it also decreased after Prednisolone treatment. IL-2 can reverse the effect of Prednisolone, and significantly increase the phosphorylation level of STAT5 in CD4+Foxp3+ cells, whether it is PBMCs from healthy people or SLE patients.

Example 3

Select 3 cases of healthy human cells treated in Example 2 except L group, use magnetic beads to negatively select CD4+ T cells for ClariomD chip sequencing, and perform principal component analysis, differential gene clustering analysis and functional enrichment analysis.

Principal component analysis (PCA) is a bioinformatics analysis method to evaluate the difference between sample groups and the repeatability within a group. It performs dimensionality reduction operations on thousands of gene expression variables and extracts principal components. The form will sample as shown in the figure. Samples with close distances have higher similarity, while samples with farther distances have greater differences. It can be seen from Figure 5 that the gene expression of cells treated with glucocorticoid combined with IL-2 was significantly different from that of the non-drug treatment group, while the gene expression of cells treated with glucocorticoid alone was significantly different from that of the other two groups of cells.

Clustering analysis is also a bioinformatics analysis method to evaluate the difference in gene expression between samples. Using unsupervised clustering analysis (Hierarchical Clustering), samples with similar gene expression patterns can be clustered together to find differential genes between samples; and these differences Genes are often related, may have similar functions, and may also participate in a certain same pathway of signal transduction. It can be seen from Figure 6 that IL-2 can reverse the effect of Prednisolone on CD4+ T cells, which is more similar to the non-drug treatment group, except for one case of Prednisolone alone treatment group. It may be that different people's cells have different responses to Prednisolone. Compared with the non-drug treatment group, Prednisolone alone treatment group had 2400 genes up-regulated and 2079 genes down-regulated; while IL-2 combined with Prednisolone group had 2614 genes up-regulated and 3230 genes down-regulated compared with prednisolone alone treatment group. The group had 1798 genes upregulated and 1612 genes downregulated. There were certain gene expression differences among the three groups.

In order to explore the functions of these differential genes, the present invention carried out Gene Ontology (Gene Ontology, GO) functional enrichment analysis, and compared them in terms of biological processes. It can be seen from Figure 7 that in CD4+ T cells, prednisolone treatment alone can reduce T cell signaling pathway and NF-κB inflammatory pathway genes, but also significantly down-regulate antiviral response genes; while IL-2 has the greatest effect on Prednisolone reversal Notable were the antiviral response genes of CD4+ T cells. Compared with the non-drug treatment group, when IL-2 and Prednisolone were co-treated, although the immune response-related genes of CD4+ T cells were up-regulated, the genes related to the negative regulation of immune response were also significantly up-regulated. Because CD4+ T cells are mainly effector T cells, most of the differential genes suggested by transcriptome sequencing are effector T cell genes, but even if the proportion of Treg cells is relatively small, the negative regulation of immune response can also be significantly observed. raised.

Example 4

Treg staining before and after treatment of patients between June 2016 and April 2017 in Example 1

1. Isolation of PBMCs.

2. Use PBS to transfer the cells to flow tubes, 100 μL per tube.

3. Add the flow cytometry antibody according to the following scheme, and incubate at room temperature for 30 minutes in the dark.

CD3 Alexa Fluor 7002μL

CD4 FITC 2 μL

CD8-PerCP 2 μL

CXCR5 APC 2 μL

PD-1 Biotin 2μL

CCR7 Brilliant Violet 421 2μL

CCR6 Brilliant Violet 650 2μL

CXCR3 PE-CF594 2μL

CD127 Brilliant Violet 605 1μL

CD25 PE 3 μL

CD45RA Brilliant Violet 510 2 μL.

4. Add 2mL PBS, 1800r 6min, and discard the supernatant.

5. Add 1 μL of StreptavidinPE-Cy7 and incubate at room temperature for 30 minutes in the dark.

6. Add 2mL PBS, 1800r 6min, and discard the supernatant.

7. Add 200 μL PBS to resuspend the cells, and test on the machine.

It can be seen from Figure 8 that the proportion of CD25 ^high CD127 ^low Treg in SLE patients treated with glucocorticoid combined with low-dose IL-2 was significantly up-regulated, while the proportion of CD25 ^high CD127 ^low Treg in a considerable number of patients in the glucocorticoid treatment group was significantly decreased, indicating that in the glucocorticoid At the same time of hormone therapy, adding low-dose IL-2 therapy can reduce the incidence of decreased CD4+CD25 ^high CD127 ^low Treg ratio in patients receiving glucocorticoid therapy, and can further increase the ratio of these Treg cells. (Treg proportion increased by more than 10% was defined as significantly up-regulated, decreased by more than 10% as significantly down-regulated, and increased or decreased within 10% was defined as no significant change in proportion).

It can be seen from Figure 9 that glucocorticoid treatment led to a significant decrease in the proportion of Tregs with high expression of CD25 (CD25++) and the proportions of active Treg, resting Treg, and non-Treg in a considerable number of patients; while IL-2 and The combined use of glucocorticoids can significantly increase the proportion of patients with high Treg expression of CD25 (ie CD25++) and the proportion of three types of Treg increased after treatment, and the proportion increase has a statistical difference.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (1)

1. The application of IL-2 in the preparation of drugs for alleviating the side effects of glucocorticoids; The side effect of the glucocorticoid is the inhibition of Treg cells and the increase of infection risk; The medicine is used for the treatment of systemic lupus erythematosus; The IL-2 can reduce the incidence of CD4+CD25 ^high CD127 ^low Treg, active Treg, resting Treg, and non-Treg cell ratio decline in patients receiving the glucocorticoid therapy; The IL-2 can reduce the incidence of decreased CD25 expression in CD4+ Treg cells in patients receiving the glucocorticoid therapy; The IL-2 can enhance the pSTAT5 level of CD4+ Treg cells; The IL-2 can increase the expression of anti-virus related genes of CD4+ T cells, and up-regulate the genes related to the negative regulation of CD4+ T cell immune response.

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