CN111529532A

CN111529532A - Application of trametinib in preparation of medicine for treating lung inflammatory diseases and medicine for promoting Tfh cell differentiation

Info

Publication number: CN111529532A
Application number: CN202010380712.6A
Authority: CN
Inventors: 吴杰; 夏家红
Original assignee: Union Hospital Tongji Medical College Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology; Union Hospital Tongji Medical College Huazhong University of Science and Technology
Priority date: 2020-05-05
Filing date: 2020-05-05
Publication date: 2020-08-14

Abstract

The invention relates to an application of trametinib in preparing a medicament for treating lung inflammatory diseases and promoting Tfh cell differentiation, wherein the chemical formula is as follows: c₂₆H₂₃FIN₅O₄The application of trametinib in promoting Tfh cell differentiation and the application of trametinib in preparing a medicament for treating lung inflammatory diseases are disclosed. The beneficial effects are that: can better inhibit 'cytokine storm' caused by activation of T cells and mononuclear macrophages, promote Tfh differentiation, mediate B cell maturation and antibody production; as a broad-spectrum immunoregulation medicine, the medicine has the effect of promoting the generation of antibodies for other viral pneumonia, virus infection or seedling inoculation.

Description

Application of trametinib in preparation of medicine for treating lung inflammatory diseases and medicine for promoting Tfh cell differentiation

Technical Field

The invention belongs to the technical field of medicine, and particularly relates to application of trametinib in preparation of a medicine for treating lung inflammatory diseases and promotion of Tfh cell differentiation.

Background

In order to obtain high-quality blocking antibodies, different subtypes of T cells perform different functions, the Tfh subtype is most important in the process, but simultaneously, excessive T cells of other proinflammatory subtypes can secrete a large amount of inflammatory factors to induce excessive inflammatory response of an organism, and complications occur: fever, organ damage, and even multiple organ failure, which is also the most serious complication of new coronary pneumonia (inflammatory factor storm). The technical problems at present are as follows:

1. viral pneumonia suppresses the immune system (hormonal drugs), does not give high quality protective antibodies, can be used in emergency, but is detrimental to the overall prognosis of the disease;

2. for the treatment of new coronary lung, specific treatment proposes that IL-6 antibody is used for reducing T cell reactivity and reducing the critical rate, but IL-6 has direct influence on Tfh function and development and directly influences the generation of protective antibody, and no related report currently indicates that IL-6 has protective effect on the long-term recovery of new coronary lung;

3. at present, only drugs for enhancing or inhibiting the overall functions of T cells and antibodies for single cytokines are clinically used, the proportion of Tfh cannot be changed for overall intervention of the T cells, viral immunity cannot be enhanced and unnecessary inflammatory reactions cannot be inhibited at the same time, the single cytokines play multiple roles, the use of the corresponding antibodies for one purpose can lead to unnecessary complications, and no more suitable monoclonal antibody is used for viral immunity.

Trametinib is mainly applied to the aspect of tumor treatment at present, and is not used for treating inflammation.

Disclosure of Invention

Aiming at the problems, the invention provides the application of trametinib in preparing the medicaments for treating pneumonia, and mainly solves the blank of the application of trametinib medicaments in treating pneumonia, lung injury and virus infection in the prior art.

In order to solve the problems, the invention adopts the following technical scheme:

trametinib, the chemical formula is: c₂₆H₂₃FIN₅O₄。

Application of trametinib in medicine for promoting Tfh cell differentiation.

Application of trametinib in preparation of medicines for treating lung inflammatory diseases.

In one form, the inflammatory disease of the lung comprises viral pneumonia.

One way, the pulmonary inflammatory disease includes lung injury.

One approach, the lung injury comprises viral lung injury.

In one mode, the medicament for treating the lung inflammatory diseases is in the form of tablets, granules, capsules, oral liquid or injection.

One mode is the application of trametinib serving as a Tfh cell differentiation promoting agent in a medicine for treating pneumonia.

Application of trametinib in preparation of medicaments for treating viral inflammation.

The invention has the beneficial effects that:

1. can better inhibit 'cytokine storm' caused by activation of T cells and mononuclear macrophages, promote Tfh differentiation, mediate B cell maturation and antibody production;

2. as a broad-spectrum immunoregulation medicament, has better treatment effect on other viral pneumonia and virus infection lung injury.

Drawings

FIG. 1 is a graph of the results of trametinib decreased IRF4 expression;

FIG. 2 is a graph showing the results of inhibition of total T cell (T lymphocyte) proliferation by trametinib;

FIG. 3 is a graph showing the results of trametinib inhibiting the differentiation of the proinflammatory T cell subsets Th1, Th17 and increasing the differentiation of the proinflammatory cell subset Treg;

FIG. 4 is a graph of the results of trametinib promoting Tfh differentiation, increasing the Tfh cell proportion;

figure 5 is a graph of the results of trametinib increasing the level of protective antibodies;

FIG. 6 is a graph of the survival rate of Tremellatinib in protecting mice against influenza virus pneumonia;

FIG. 7 is a graph showing that trametinib can reduce the occurrence of pulmonary inflammation in an LPS (lipopolysaccharide) -induced acute lung injury model;

FIG. 8 is a graph showing the result of LPS-induced acute lung injury model simulating macrophage injury to lung.

Detailed Description

The following is further described in conjunction with the detailed description:

application of trametinib in promoting Tfh cell differentiation.

The application of trametinib in preparing the medicine for treating the lung inflammatory diseases is that an adult takes 2mg each time 1 time per day.

The pulmonary inflammatory disease includes viral pneumonia.

The pulmonary inflammatory disease includes viral lung injury.

The medicament for treating the lung inflammatory diseases is in the dosage form of tablets, granules, capsules, oral liquid or injection.

Example 1: trametinib (Trametinib) reduced IRF4 expression.

The method comprises the following steps:

taking one spleen of a mouse in a sterile environment, adding a proper amount of PBS buffer solution in a sterile platform for grinding, filtering an unground part through a 40-micron screen, centrifuging for 3 minutes at 2000 r/min, discarding a supernatant, cracking red blood cells by using 1ml of ACK lysate for 3 minutes, adding the PBS buffer solution for dilution, and centrifuging again to obtain the lymphocytes. After cell counting lymphocytes were seeded in flat bottom 96 well plates with 1640 complete medium at a density of about 0.5 x 106 cells/well, CD28 was added to a final concentration of 1ug/ml, and Trametinib was added at various concentrations. (96-well plates were coated with 4ug/ml CD3 in advance). After 2-3 days of activation, flow-type staining is carried out, live T cells positive for CD4 are circled, and the IRF4 expression amount is detected (left in figure 1) and counted (right in figure 1).

And (4) analyzing results:

as shown in FIG. 1, the IRF4 expression level is reduced with the increase of Trametinib concentration, the interference of IRF4 expression level is significantly different when the concentration is more than 40nM, and Trametinib reduces the IRF4 expression.

Example 2: trametinib inhibits the proliferation of total T cells.

The method comprises the following steps:

t cell culture medium was mixed at 1: adding CTV cell cycle marker staining (invitrogen C34557) in a ratio of 1000, staining for 20 minutes in a dark place, adding complete culture medium, incubating for 5 minutes, centrifuging cells, removing supernatant, re-suspending cells with the complete culture medium, adding 100nM Trametinib or DMSO (equivalent solvent) in two groups respectively, and performing flow detection on cell proliferation after three days.

And (4) analyzing results:

as shown in fig. 2, T cells stimulated by Trametinib proliferated significantly slower, with the passage number of T cell proliferation being much lower than that of the control group.

Example 3: trametinib inhibits the differentiation of proinflammatory T cell subsets Th1 and Th17, and increases the differentiation of proinflammatory cell subset Treg.

T cell sorting:

to obtain purified CD4+ T cells and T cell depleted (CD3 negative) splenocytes, sorting of specific cell subsets was performed using a levitated and whirlpool magnetic bead sorting system. All manipulations were performed aseptically to prevent cell contamination and sorted according to the instructions of the America whirly and relevant kit.

1. Preparing a cell suspension:

after the operating room is disinfected by ultraviolet irradiation, the mouse is killed by using a cervical vertebra dislocation method and is soaked in 75% alcohol for 5 minutes to reduce pollution; rapidly taking out spleen tissues of the mouse after the abdominal median incision is performed, and putting the spleen tissues into a culture dish which is prepared in advance and is filled with a culture medium; the culture dish was transferred to a clean bench, the spleen was placed in a 200 mesh sieve and gently ground using a 1ml syringe piston, and the filtered single cell suspension was centrifuged, split red and counted for future use.

2. Labeling magnetic beads:

sucking the required amount of cell suspension, and washing the cells for 3 times by using ice PBS; adding 90 ul PBS and 10 ul corresponding labeled magnetic beads into each 107 total cells, mixing uniformly, and incubating for 10 minutes at 4 ℃; after 10 minutes, the cell suspension was removed and washed 3 times with 1ml PBS per 107 cells for use.

3. Sorting cells by a sorter:

placing a specific sorter in a matched magnet, wetting the sorter by PBS, and transferring the cell suspension marked by the magnetic beads into the sorter; collecting the effluent cells by using a centrifugal tube below the sorter, wherein the collected cells are cells which are not labeled by magnetic beads; and taking the sorter out of the magnet, adding a proper amount of PBS, and collecting cells flowing out of the lower part of the sorter, wherein the collected cells are cells marked by the magnetic beads.

Induction of Th cells:

sorted T cells were stimulated for 3 days as follows to obtain different subsets of Th cells. During induction, 100nM Trametinib or DMSO, an equivalent amount, was added to each group and induction was monitored after three days.

And (4) analyzing results:

as shown in fig. 3, the Trametinib intervention has an inhibitory effect on the differentiation of the pro-inflammatory cell subsets Th1 and Th17 and has a promoting effect on the differentiation of Treg suppressor T cells. Trametinib can reduce the proportion of T cells secreting proinflammatory factors and reduce inflammatory factor storm and nonspecific cell injury in the virus immune process.

Example 4: trametinib promotes Tfh differentiation, increasing the proportion of Tfh cells.

The method comprises the following steps:

construction of experimental animal and viral pneumonia models: BALB/c mice were randomly divided into normal group, model PBS group, and model Trametinib group, each group consisting of 36 mice under ether light anesthesia. Each mouse in both model groups was infected nasally with 25 μ L of 50LD50 virus droplets, and the normal group was nasally infected with an equal amount of sterile Phosphate Buffered Saline (PBS). 1h after infection, the normal group and the model group were subjected to sterile PBS intragastric administration, the Trametinib group was subjected to intragastric administration of 3 mg/(kg. d), each group was administered 1 time/d, and the administration was continued until the detection time point. Taking the spleen of a mouse, and detecting the Tfh subgroup proportion.

And (4) result sorting:

as shown in figure 4, the Tfh cell ratio in the mouse viral pneumonia model is obviously higher than that of the model-free model, the model is successfully formed, and the mouse is infected with the viral pneumonia and makes a related immune response. The Tfh cell subset in the Trametinib group is much higher than that in the PBS group, which proves that the Trametinib can induce the Tfh differentiation and increase the Tfh ratio.

Example 5: trametinib increased protective antibody levels.

The method comprises the following steps:

plasma total IgG levels were measured from the mice on

days

0, 9, and 15 of the model of example 4.

And (4) analyzing results:

as shown in fig. 5, the content of IgG antibody in mice in the Trametinib group is significantly higher than that in the control group at different time points, indicating that the immune system has stronger resistance to virus, faster antibody production speed and higher content.

Example 6: trametinib protects survival rates of influenza virus pneumonia in mice.

The method comprises the following steps:

and (5) carrying out the modeling method, and counting the death rate of the mice.

And (4) analyzing results:

as shown in figure 6, the death rate of the mice treated by Trametinib is obviously reduced, and the mice have good therapeutic effect on viral pneumonia.

Example 7:

the experimental method comprises the following steps: c57 mice aged 8 weeks, were treated by gavage with and without corn oil and 3mg/kg/d trametinib, and 2 hours later, were intratracheally treated with LPS (5mg/kg body weight) for 3 consecutive days. The mice were then sacrificed and the lungs were sectioned for paraffin observation for inflammatory response.

And (4) analyzing results:

trametinib can reduce the occurrence of pulmonary inflammation in an LPS (lipopolysaccharide) -induced acute lung injury model. Figure 7 is a section of a lung specimen from a model of acute lung injury induced by LPS and a pathological score based on the severity of inflammation, with significantly less lung inflammation (protective in inflammatory lung disease) using the trametinib group.

Example 8:

the experimental method comprises the following steps:

and (3) taking mouse abdominal cells by a flushing method, planting the mouse abdominal cells in a 6-well plate by using a DMEM complete culture medium, and flushing to remove nonadherent cells after 1 hour to obtain the primary mouse macrophage. Culture was performed in DMEM complete medium. Cells were given trametinib (100nM), or control DMSO for two hours, stimulated with LPS (100 ng/ml).

After 12 hours, cell mRNA is collected for fluorescent quantitative PCR, cytokine secretion is detected, and supernatant is collected for detecting the level of the cytokine protein. IL-1 β, MCP1, TNF- α were increased at mRNA levels and protein levels by LPS stimulation, which is protected by trametinib, as shown in FIG. 8.

And (4) analyzing results:

the acute lung injury model induced by LPS is a model that mimics the damage of macrophages to the lung. Macrophages were extracted, treated with LPS and treated with trametinib, and found to reduce inflammatory factors secreted by macrophages. Trametinib can reduce the degree of natural immunity to protect lung injury, viral immunity belongs to humoral immunity, and natural immunity belongs to complications in the humoral immunity.

It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.

Claims

1. Application of trametinib in medicine for promoting Tfh cell differentiation.

2. Application of trametinib in preparation of medicines for treating lung inflammatory diseases.

3. Use of trametinib according to claim 2, in the manufacture of a medicament for the treatment of pneumonia, wherein the pulmonary inflammatory disease comprises viral pneumonia.

4. Use of trametinib according to claim 2, in the manufacture of a medicament for the treatment of pneumonia, wherein the pulmonary inflammatory disease comprises viral lung injury.

5. The use of trametinib according to any one of claims 2-4 in the preparation of a medicament for treating pneumonia, wherein the medicament for treating pulmonary inflammatory diseases is in the form of tablets, granules, capsules, oral liquid or injection.

6. Use of trametinib according to claim 2, in the preparation of a medicament for treating pneumonia, wherein trametinib is used as a Tfh cell differentiation promoting agent in a medicament for treating pneumonia.

7. Application of trametinib in preparation of medicaments for treating viral inflammation.