Macrophage activation inhibitor and application thereof

Abstract

The invention provides a macrophage activation inhibitor and application thereof, and belongs to the technical field of medicines. As a substance naturally generated in a human body, HMB is extremely high in safety, free of side effects and free of damage to body health. Experiments prove that HMB can inhibit activation of macrophages, can reduce infiltration of the macrophages and inflammatory response by reducing generation of pro-inflammatory cytokines such as IL-6, TNF-alpha, IL-1beta and iNOS in the macrophages, further can regulate functions of an immune system, can be used for preventing or treating macrophage activation related diseases, and has a good application prospect. A novel treatment medicine with high safety is provided for clinical treatment of subsequent inflammation and macrophage activation related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a macrophage activation inhibitor and application thereof. Background Art

[0002] Macrophages (Mφ) are a type of innate immune cells that are widely present in the body. They have multiple biological functions such as phagocytosis and clearance of pathogens, processing and presenting antigens, releasing inflammatory cytokines, and mediating immune responses. They are an important subject for studying cellular phagocytosis, cellular immunity, and molecular immunology.

[0003] Macrophages are distributed in various tissues of the body. They can fight against different types of pathogenic microorganisms by executing specific immune responses in the early stages of development, so they are an indispensable part of the host defense system. Macrophages have been found to play an important regulatory role in inflammatory responses. As the main phagocytes in the inflammatory stage, they are responsible for clearing necrotic fragments of tissues and cells at the site of injury and pathogens, etc. These substances have an important regulatory effect on the wound healing process. However, after the inflammatory response ends, if macrophages continue to be activated, they will also have adverse effects on the body. For example, Ehrentraut et al. found that changing the activation state of macrophages can regulate the pathological occurrence of acute lung injury; Barreto et al. found that photoprotein polysaccharides can regulate macrophage polarization and promote the occurrence of osteoarthritis. Therefore, how to regulate the activation of macrophages is of great significance for the subsequent in-depth study of the clinical treatment of diseases related to inflammation and macrophage activation.

[0004] β-Hydroxy-β-methylbutyrate (HMB) is a metabolite with certain biological activity formed after the decomposition of the branched-chain amino acid leucine. It is a substance produced naturally in the human body and is safe and has no side effects. HMB can promote protein synthesis and inhibit protein breakdown in the body, thereby helping to increase muscle mass and strength. It can be used as a dietary supplement and as an ingredient in certain medical foods designed to promote wound healing. However, there is currently no existing technology that reveals the effect of HMB on macrophage activation. Summary of the invention

[0005] The purpose of the present invention is to provide a macrophage activation inhibitor and its application, using HMB to inhibit macrophage activation, and providing a new therapeutic drug for the subsequent clinical treatment of inflammation and macrophage activation-related diseases.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a macrophage activation inhibitor, characterized in that the macrophage activation inhibitor contains HMB.

[0007] Preferably, the macrophage activation inhibitor further comprises one or more pharmaceutically acceptable excipients.

[0008] Preferably, the macrophage activation inhibitor can prevent, improve or treat diseases caused by macrophage activation.

[0009] The present invention also provides an application of a macrophage activation inhibitor in the preparation of a medicine for treating diseases related to macrophage activation.

[0010] Preferably, the macrophage activation-related diseases include: acute lung injury, infectious diseases, and inflammatory bowel disease.

[0011] The present invention also provides an application of exogenously adding a macrophage activation inhibitor HMB to alleviate macrophage activation, comprising the following steps: (1) Cell culture: Isolate and culture macrophages, use LPS to induce macrophage activation, and then add exogenous HMB to continue culturing; (2) Cell proliferation activity detection: After exogenous HMB was added to the macrophages in step (1) and continued to be cultured, CCK8 was used to detect cell proliferation activity; (3) Detection of CD80 and CD86 expression levels: Perform flow cytometry detection on the expression levels of CD80 and CD86 of macrophages after the exogenous addition of HMB and continued cultivation in step (1); (4) qRT-PCR detection of inflammatory factor levels: The total RNA of macrophages after the exogenous addition of HMB in step (1) was extracted and the expression levels of pro-inflammatory cytokines IL-6, TNF-α, IL-1β and iNOS were detected.

[0012] Preferably, when LPS is used to induce macrophage activation in step (1), the dosage of LPS is 100 ng / ml and the induction time is 24 h; when exogenously adding HMB and continuing the culture in step (1), the concentration of HMB is 25 μM and the culture time is 2 h.

[0013] Preferably, the flow cytometry method in step (3) is: After the culture, the macrophages were washed twice in FACS buffer and resuspended, and incubated in FACS buffer supplemented with Fc-γ receptor CD16 / CD32 antibody for 30 minutes. Then, at 4°C in the dark, the macrophages were incubated with PE-labeled anti-CD86 antibody and APC-labeled anti-CD80 antibody, with fluorescently labeled isotype IgG as isotype control, at a dilution of 1:100, incubated in FACS buffer for 30 minutes, and the cells were analyzed using an Agilent NovoCyte flow cytometer.

[0014] Preferably, the specific steps of detecting the level of inflammatory factors by qRT-PCR in step (4) are: The total RNA of macrophages was extracted by TRIzol method, the total RNA concentration was measured by nucleic acid protein analyzer, and the mRNA level of target gene was detected by real-time fluorescence quantitative PCR system according to TaKaRa SYBR Premix Ex Taq procedure.

[0015] The beneficial effects of the present invention compared with the prior art are: As a substance naturally produced in the human body, HMB is extremely safe, has no side effects, and does not harm health. The present invention has experimentally confirmed that HMB can inhibit the activation of macrophages, and reduce the infiltration of macrophages by reducing the production of proinflammatory cytokines such as IL-6, TNF-α, IL-1β and iNOS in macrophages, reduce the inflammatory response, and thus regulate the function of the immune system. It can be used to prevent or treat diseases related to macrophage activation, and provides a new and safe therapeutic drug for the subsequent clinical treatment of inflammation and macrophage activation-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0017] Figure 1 The effect of HMB on the viability of RAW264.7 cells in Example 1 of the present invention; Figure 2 The effect of HMB on BMDM cell viability in Example 1 of the present invention; Figure 3 The effect of HMB on CD80 and CD86 of RAW264.7 cells in Example 1 of the present invention; Figure 4The effect of HMB on BMDM cells CD80 and CD86 in Example 1 of the present invention Figure 5 The effect of HMB on the expression of inflammatory factors in RAW264.7 cells in Example 1 of the present invention; Figure 6 This is the effect of HMB on the expression of inflammatory factors in BMDM cells in Example 1 of the present invention. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] The present invention provides an application of exogenously adding a macrophage activation inhibitor HMB to alleviate macrophage activation, which specifically comprises the following steps: (1) Cell culture: Macrophages were isolated and cultured, and 100 ng / mL LPS was used to induce macrophage activation for 24 h. 25 μM HMB was exogenously added and cultured for another 2 h. (2) Cell proliferation activity detection: After exogenous HMB was added to the macrophages in step (1) and continued to be cultured, the cell proliferation activity was detected by CCK8 method; (3) Detection of CD80 and CD86 expression levels: After the exogenous addition of HMB in step (1) was completed, the macrophages were washed twice in FACS buffer, resuspended, and incubated in FACS buffer supplemented with Fc-γ receptor CD16 / CD32 antibody for 30 minutes. Then, at 4°C in the dark, the macrophages were incubated with PE-labeled anti-CD86 antibody and APC-labeled anti-CD80 antibody, with fluorescently labeled isotype IgG as isotype control, at a dilution of 1:100, incubated in FACS buffer for 30 minutes, and the cells were analyzed using an Agilent NovoCyte flow cytometer.

[0024] (4) qRT-PCR detection of inflammatory factor levels: TRIzol extraction steps (1) After exogenous HMB was added and cultured, the total RNA of macrophages was measured by nucleic acid protein analyzer, and the mRNA level of the target gene was detected using real-time fluorescence quantitative PCR system according to TaKaRa SYBR Premix Ex Taq steps. Example 1

[0025] Example 1 of the present invention detects the effect of HBM in inhibiting macrophage activation, which specifically includes the following steps: (1) Culture of mouse RAW264.7 cells The cells were cultured in DMEM medium containing 10% FBS at 37°C with 5% CO 2 After the cells proliferate to 70%-80% fusion, discard the original culture medium, add pre-cooled phosphate buffered saline (PBS), gently blow the culture dish with a 1 mL pipette and transfer the cell suspension to a 10 mL centrifuge tube, centrifuge it at 1000 r / min for 5 min, discard the supernatant, resuspend the cells with 5 mL of DMEM medium containing 10% FBS, and inoculate them in the culture dish.

[0026] (2) Mouse BMDM cell extraction and culture A. Collection of L929 cell supernatant L929 cells were routinely revived and cultured in DMEM medium containing 10% FBS for 24 h. The culture medium was replaced once and cultured for another 24 h before digestion and subculturing. During the subculturing process, the cell supernatant (L929 cell conditioned medium) was collected by centrifugation (1000 r / min for 5 min), filtered through a 0.22 µm filter membrane, and stored in a −80°C refrigerator.

[0027] B. Mouse BMDM cell extraction and culture C. BMSCs were isolated from the femur and tibia of 6-8 week-old wild-type C57BL / 6J mice and lysed with red blood cell lysis buffer (155 mM NH 4 Cl, 12 mM NaHCO 3 , and 0.1 mM EDTA), suspended in 2 ml pre-cooled PBS, and then transferred to 25% L929 conditioned medium. After 24 h, the suspended cells were transferred to culture dishes at 5.0-7.5 × 10 5 cells, differentiated for 7 days, and added 2 ml of fresh culture medium on the 2nd, 4th, and 6th days respectively.

[0028] (3) Cell proliferation activity detection RAW264.7 cells and BMDM cells were collected by trypsin digestion after 7 days of primary culture and plated at the same density (1×10 4 Cells were seeded in 96-well plates with 200 μL of cell suspension in each well.

[0029] RAW264.7 cells were pretreated with different concentrations of HMB (10 / 25 / 40 μM) for 2 hours, and then treated with LPS 100 ng / ml for 6 hours and 24 hours, and then the cell viability was measured by CCK8 method. Primary BMDM cells were pretreated with different concentrations of HMB (10 / 20 / 40 / 80 / 150 / 300 μM) for 2 hours, and then treated with LPS 100 ng / ml for 24 hours, and then the cell viability was measured by CCK8 method.

[0030] Using the CCK8 method, 20 µL of CCK8 solution was added to each well and incubated in a cell culture incubator for 2 h. The OD value at 450 nm was measured using a full-wavelength multifunctional detector to detect cell activity and proliferation. The results are as follows: Figure 1 , 2 shown.

[0031] Depend on Figure 1 , 2It can be seen that different concentrations of HMB (10 / 25 / 40 μM) had no significant effect on the viability of RAW264.7 cells; while high concentrations of HMB (40 / 80 / 150 / 300 μM) downregulated the viability of primary BMDM cells. Based on the results of CCK8 in the two cells, we chose HMB 25 μM as the dosage for subsequent experiments.

[0032] (4) Flow cytometry detection of CD80 and CD86 expression The RAW264.7 cells and BMDM cells cultured in steps (1) and (2) were digested with trypsin, and the primary cultured cells after 7 days were collected and plated at the same density (1×10 4 / mL) were inoculated in 96-well plates, with 200 μL of cell suspension in each well. The cells were divided into 4 groups, namely blank group (CON), HMB group, LPS group, and LPS+HMB group. 25 μM HMB was added to the HMB group and cultured for 2 hours; 100 ng / mL of LPS was added to the LPS group to induce macrophage activation for 24 hours; 100 ng / mL of LPS was first added to the LPS+HMB group to induce macrophage activation for 24 hours, and 25 μM HMB was added exogenously and continued to be cultured for 2 hours; the blank group was cultured with an equal amount of culture medium.

[0033] After the culture was completed, the cells were taken separately, washed twice in FACS buffer (PBS containing 1% BSA and 1% FBS, pH 7.2), resuspended, and incubated in FACS buffer supplemented with Fc-γ receptor CD16 / CD32 antibody for 30 minutes. Subsequently, the cells were incubated in FACS buffer for 30 minutes at 4°C in the dark with PE-labeled anti-CD86 antibody and APC-labeled anti-CD80 antibody, with fluorescently labeled isotype IgG as isotype control (dilution of 1:100). The antibodies used were purchased from Invitrogen eBioscience, and cell analysis was performed using an Agilent NovoCyte flow cytometer. Finally, the results were analyzed using Flow Jo V10 software, as shown in the following figure. Figure 3 , 4 shown.

[0034] Depend on Figure 3 , 4 It can be seen that in both cell types, the expression of CD80 and CD86 in the HMB-treated group was significantly lower than that in the LPS group. Furthermore, the use of HMB after LPS-induced activation of RAW264.7 cells and BMDM cells could significantly reduce the levels of RAW264.7 cells and BMDM cells and inhibit the activation of RAW264.7 cells and BMDM cells.

[0035] (4) qRT-PCR detection of inflammatory factor levels The total RNA of RAW264.7 cells and BMDM cells was extracted by TRIzol method, and the total RNA concentration was measured by nucleic acid protein analyzer. The total RNA amount of each sample was 1ug. The target gene mRNA level was detected by real-time fluorescence quantitative PCR system according to the TaKaRa SYBR PremixEx Taq procedure. Each sample was repeated at least three times. The relative expression of the target gene mRNA was measured by the CT value, and the CT value of GAPDH was used as the internal reference. -△△ct The calculation results are as follows Figure 5 , 6 shown.

[0036] Depend on Figure 5 , 6 It can be seen that HMB treatment reduced the expression of IL-6, TNF-α, IL-1β and iNOS in LPS-induced RAW264.7 cells and BMDM cells. The results suggest that HMB can reduce the infiltration of macrophages and inhibit the activation of macrophages by inhibiting the production of proinflammatory cytokines.

[0037] Statistical analysis GraphPad Prism9.5 statistical software was used for statistical analysis. Measurement data were expressed as mean±SEM. One-way analysis of variance was used for comparison between multiple groups, and t-test was used for pairwise comparison. Differences were considered statistically significant when p<0.05.

[0038] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A macrophage activation inhibitor, characterized in that The macrophage activation inhibitor includes HMB.

2. The macrophage activation inhibitor according to claim 1, characterized in that The macrophage activation inhibitor further comprises one or more pharmaceutically acceptable excipients.

3. The macrophage activation inhibitor according to any one of claims 1 or 2, characterized in that The macrophage activation inhibitor can prevent, improve or treat diseases caused by macrophage activation.

4. Use of the macrophage activation inhibitor according to any one of claims 1 to 3 in the preparation of a medicament for treating diseases related to macrophage activation.

5. Use of the macrophage activation inhibitor according to claim 4 in the preparation of a drug for treating macrophage activation-related diseases, characterized in that: The macrophage activation-related diseases include: acute lung injury, infectious diseases, and inflammatory bowel disease.

6. A use of exogenously adding a macrophage activation inhibitor HMB to alleviate macrophage activation, characterized in that: The steps include: (1) Cell culture: Isolate and culture macrophages, use LPS to induce macrophage activation, and then add exogenous HMB to continue culturing; (2) Cell proliferation activity detection: After exogenous HMB was added to the macrophages in step (1) and continued to be cultured, the cell proliferation activity was detected by CCK8 method; (3) Detection of CD80 and CD86 expression levels: Perform flow cytometry detection on the expression levels of CD80 and CD86 of macrophages after the exogenous addition of HMB and continued cultivation in step (1); (4) qRT-PCR detection of inflammatory factor levels: The total RNA of macrophages after the exogenous addition of HMB in step (1) was extracted and the expression levels of pro-inflammatory cytokines IL-6, TNF-α, IL-1β and iNOS were detected.

7. The use of exogenously added macrophage activation inhibitor HMB to alleviate macrophage activation according to claim 6, characterized in that: When LPS is used to induce macrophage activation in step (1), the dosage of LPS is 100 ng / ml, and the induction time is 24 h. When exogenously adding HMB and continuing the culture in step (1), the concentration of HMB is 25 μM, and the culture time is 2 h.

8. The use of exogenously adding macrophage activation inhibitor HMB to alleviate macrophage activation according to claim 6, characterized in that: Step 3) The method of flow cytometry is: After the culture, the macrophages were washed twice in FACS buffer and resuspended, and incubated in FACS buffer supplemented with Fc-γ receptor CD16 / CD32 antibody for 30 minutes. Then, at 4°C in the dark, the macrophages were incubated with PE-labeled anti-CD86 antibody and APC-labeled anti-CD80 antibody, with fluorescently labeled isotype IgG as isotype control, at a dilution of 1:100, incubated in FACS buffer for 30 minutes, and the cells were analyzed using an Agilent NovoCyte flow cytometer.

9. The use of exogenously adding the macrophage activation inhibitor HMB to alleviate macrophage activation according to claim 6, characterized in that: The specific steps of the qRT-PCR detection of inflammatory factor levels in step (4) are as follows: The total RNA of macrophages was extracted by TRIzol method, the total RNA concentration was measured by nucleic acid protein analyzer, and the mRNA level of target gene was detected by real-time fluorescence quantitative PCR system according to TaKaRa SYBR Premix Ex Taq procedure.

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