Medical use of a terpenoid compound

Abstract

The present invention belongs to the field of biopharmaceuticals and relates to the medical uses of terpenoid compounds, specifically their use in the preparation of immunomodulatory or antioxidant drugs or foods. The terpenoid compound is a compound represented by the following structure, or a pharmaceutically acceptable salt thereof, wherein R1, R2, and R3 are as described in the claims and description. The terpenoid compound or a composition containing the terpenoid compound described herein has antioxidant or immunomodulatory effects and can be used to prepare antioxidant or immunomodulatory drugs or foods.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the medical use of a terpenoid compound, and particularly relates to the use of a terpenoid compound in the preparation of immunomodulatory and antioxidant drugs. Background Art

[0002] The function of the immune system is closely linked to human health. The pathogenesis of the vast majority of human diseases, including inflammation, infection, tumors, aging, reproduction, allergic diseases, and autoimmune diseases, involves immune function. With the accelerated pace of modern life, irregular work and rest schedules, unbalanced diets, lack of exercise, and air pollution, the incidence of immune system dysfunction is on the rise, and the ability to resist pathogens is also declining. Therefore, improving immunity is crucial for preventing disease, responding to viral infections, and promoting overall health.

[0003] Macrophages are important innate immune cells. They are specialized, long-lived, phagocytic cells of the innate immune system and serve as the first responders to pathogens. They bridge the gap between innate and adaptive immune responses. Macrophages participate in both nonspecific defense (innate immunity) and specific defense (cellular immunity). They recognize, phagocytose, and degrade cellular debris and pathogens. They also play a role in presenting antigens to T cells and inducing the expression of co-stimulatory molecules by other antigen-presenting cells, thereby initiating adaptive immune responses. Furthermore, they play a crucial role in the early stages of pathogen invasion by releasing cytokines and chemokines, which in turn recruit other immune cells to sites of inflammation. In addition to initiating immune and inflammatory responses against pathogens, macrophages also play a role in maintaining tissue homeostasis, as well as repair and remodeling. Unfortunately, this function is associated with numerous diseases, including metabolic and autoimmune disorders, cancer, infection, obesity, and fibrosis. Therefore, macrophages have become therapeutic targets for a variety of diseases, and the development of drugs that modulate macrophage function holds significant clinical value.

[0004] Reactive oxygen species (ROS), such as hydrogen peroxide, singlet oxygen, hydroxyl radicals, and superoxide anions, are natural byproducts of normal oxygen metabolism in the body. ROS act as signaling molecules to regulate various cellular physiological functions, playing a vital role in cell signaling and homeostasis. ROS can directly affect the activity of pathogens, cancer cells, and various immune cells.

[0005] The production and clearance of ROS are tightly regulated to maintain homeostasis. Appropriate levels of ROS in the body serve as key signaling molecules that regulate various physiological functions, including inflammatory responses, and can also activate the immune system, thereby generating immune protection against pathogens or tumor cells. However, excessive and uncontrolled ROS can trigger harmful oxidative stress responses in the body, including lipid peroxidation, DNA oxidative damage, protein oxidation, and monosaccharide oxidation, and damage cellular structures, such as mitochondria, leading to the development of various diseases. Reactive oxygen species have been widely implicated in a variety of health-threatening conditions, including cancer, cardiovascular disease, diabetes, neurodegeneration (such as Parkinson's disease and Alzheimer's disease), autoimmune diseases, stroke, aging, and inflammatory disorders. Therefore, finding ways to eliminate intracellular ROS has become an important strategy for restoring ROS homeostasis, limiting inflammatory responses, addressing immune imbalance, and treating disease.

[0006] ROS play a crucial role in regulating macrophage function. However, excessive ROS can damage mitochondrial structure, induce lipid peroxidation, promote macrophage immunosuppression or inflammatory responses, and contribute to the development and progression of diseases such as tumors, aging, and inflammation. Therefore, targeting ROS levels in cells is an important therapeutic strategy for a variety of diseases. However, currently, there are limited reagents and drugs that regulate intracellular ROS production or clearance.

[0007] Certain small molecule compounds play an important regulatory role in disease treatment. They have the advantage of entering cells to regulate ROS levels, can better regulate the production or clearance of ROS in cells, and can be used to prepare immunomodulatory and antioxidant drugs. Summary of the Invention

[0008] In order to overcome the defects in the prior art, the present invention provides an application of a heteroterpenoid compound in the preparation of immunomodulatory and antioxidant drugs.

[0009] The present invention is achieved through the following technical solutions:

[0010] Application of heteroterpenoid compounds in the preparation of immunomodulatory drugs.

[0011] Application of heteroterpenoid compounds in the preparation of antioxidant drugs.

[0012] The structure of the heteroterpenoid compound is as follows:

[0013]

[0014] in,

[0015] R1, R2, and R3 are H, C1-C6 alkyl, C1-C6 alkoxy, hydroxy, halogen, amino, or cyano;

[0016] Further,

[0017] R1, R2, and R3 are H, C1-C4 alkyl, or C1-C4 alkoxy;

[0018] Furthermore,

[0019] The structure of the heteroterpenoid compound is:

[0020]

[0021] The heteroterpenoid compounds achieve antioxidant effects and enhance immune responses by reducing the production of reactive oxygen species (ROS) in cells.

[0022] The heteroterpenoid compounds achieve the effects of anti-oxidation and enhancing immunity by regulating the function of macrophages.

[0023] The heteroterpenoid compounds can regulate the functions of macrophages, including regulating the redox homeostasis of macrophages, regulating the ROS level of macrophages, reducing the ROS level in macrophage mitochondria, and regulating the production of macrophage cytokines.

[0024] The heteroterpenoid compounds of the present invention can be prepared into compositions with acceptable carriers or excipients.

[0025] The heteroterpenoid compound or the composition containing the heteroterpenoid compound provided by the present invention has an antioxidant or immunomodulatory effect and can be used to prepare antioxidant and immunomodulatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The enhancement effect of the terpenoid compound CND on cellular immune response;

[0027] A is a terpenoid compound CND that regulates macrophages to enhance CD8 + Schematic diagram of the T cell-mediated immune response assay;

[0028] B is the effect of terpenoid compound CND on the CD8 + Representative peak graphs of T cell proliferation;

[0029] C is the macrophages and CD8 + T cells co-cultured, CD8 + Statistical analysis of the proportion of T cells;

[0030] D is the macrophages and CD8 + After co-culture of CD8 T cells + Statistical analysis of changes in T cell numbers.

[0031] Figure 2 The effect of CND on CD8 macrophages + The level of interferon IFN expressed by T cells.

[0032] Figure 3 This is a statistical analysis of the effects of CND treatment on the expression levels of different inflammatory factors and chemokines in mouse macrophages;

[0033] A: Inflammatory factors B: Chemokines.

[0034] Figure 4 This is a diagram showing the regulation and statistical analysis of the terpenoid compound CND on ROS levels in macrophages. DETAILED DESCRIPTION

[0035] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments.

[0036] Intracellular reactive oxygen species (ROS) play a crucial regulatory role in cellular function and the development of disease. Macrophages are an important immune cell subset involved in a variety of inflammatory diseases. Maintaining redox homeostasis in macrophages is key to their normal physiological function. Under stressful conditions, macrophages can become dysfunctional, leading to the development of disease. Intracellular ROS can regulate macrophage polarization, and polarized macrophages differ in their ability to produce ROS. The present invention evaluated the antioxidant and immunomodulatory activities of heteroterpenoid compounds after H2O2 treatment of macrophages.

[0037] Tumor-associated macrophages (TAMs) in the tumor microenvironment suppress anti-tumor immune responses under the regulation of reactive oxygen species (ROS). This study treated macrophages with tumor cell culture supernatant (TCM) to induce ROS production in macrophages. The study then evaluated the inhibitory activity of the compounds on ROS levels, their ability to regulate macrophage redox homeostasis, and the ability of terpenoid compounds to protect macrophages from ROS damage, which can lead to disruption of cytoplasmic membrane and mitochondrial structures.

[0038] Macrophages regulate the type and intensity of immune responses and influence disease progression by expressing various cytokines. Furthermore, we evaluated changes in the expression of different cytokines in macrophages after treating them with terpenoids, assessing their ability to regulate inflammatory responses.

[0039] The preparation method of the terpenoid compound CND involved in each embodiment of the present invention can be referred to the literature: Construction of a meroterpenoid-like compound collection by precursor-assisted biosynthesis[J]. Organic & Biomolecular Chemistry, 2020, 18. DOI:10.1039 / D0OB01235A.

[0040] Example 1

[0041] Enhancement of cellular immune response by terpenoid compound CND

[0042] 1.1 Obtain wild-type adult C57BL / 6J mice, sacrifice them, and disinfect them by immersing them in 75% alcohol for 2-3 minutes. Make an abdominal incision, separate the muscles from the bones with scissors, and remove the tibia and femur. These bones are then immersed in phosphate-buffered saline (PBS). All subsequent steps should be performed under a sterile operating hood.

[0043] 1.2 After removing the tibia and femur with sterile forceps, cut off small pieces on both sides with scissors. Use a 20 mL syringe filled with PBS to repeatedly flush the bone marrow up and down. Collect the flushed bone marrow cells through a 70 μm cell sieve into a 50 mL sterile centrifuge tube and centrifuge at 2000 rpm for 4 min at 4°C.

[0044] 1.3 Add 1 mL of 1× red blood cell lysis buffer to the cell pellet to suspend the cells. After standing for 2 min, add 10 mL of PBS to wash and centrifuge at 2000 rpm at 4°C for 4 min.

[0045] 1.4 Resuspend the cells in RPMI 1640 medium (containing 5% mouse fibroblast L929 conditioned medium) and adjust the cell density of bone marrow-derived macrophages (BMDM) to 2×10 6 / well, plated into untreated 6-well cell culture plates and cultured at 37°C, 5% CO2. On the 4th day of cell culture, bone marrow-differentiated macrophages were observed under a microscope.

[0046] 1.5 BMDM cells were cultured at 1×10 4 Each well was plated in a 96-well plate and divided into three groups for the following treatments:

[0047] A: Fresh culture medium (FCM) was used for 12 h, followed by DMSO (0.1%) treatment for 24 h.

[0048] B: Tumor cell culture supernatant (TCM) was treated for 12 h, and then DMSO (0.1%) was added for 24 h.

[0049] C: Tumor cell culture supernatant (TCM) was treated for 12 h, followed by addition of 1 μM compound CND for 24 h.

[0050] The TCM preparation method is as follows: 3 × 10 6 Hepa1-6LM cells were mixed in 10 ml of culture medium and plated into a 10 cm culture dish. After culturing for 36 h, the cell culture supernatant was collected.

[0051] Compound CND was dissolved in DMSO (dimethyl sulfoxide).

[0052] 1.6 Discard the culture medium from the previous step, add 100 ng / ml ovalbumin (OVA) to stimulate BMDM cells for 2 h, and wash twice with PBS.

[0053] 1.7 OT1 mouse CD8 + T cells and BMDM were co-cultured at a ratio of 1:10 for 2-3 days, and the expression of CTV in CD8 + Evaluation of expression intensity in T cells, CD8 + T cell proliferation effect.

[0054] 1.8 CD8 T cells were isolated from OT1 mice by flow cytometry. + T cells were labeled with CTV, and then CD8 + T cells and BMDM were co-cultured at a ratio of 1:10 for 2-3 days.

[0055] 1.9 Flow cytometry detection of CTV in CD8 + The expression intensity in T cells was calculated and CD8 + The number of T cells was used to evaluate CD8 + T cell proliferation.

[0056] 1.10 Collect the supernatant of the co-cultured cells and detect the IFN level using ELISA.

[0057] See the results Figure 1-2 .

[0058] like Figure 1 As shown in Figure B, tumor supernatant treatment of macrophages inhibited the macrophages' CD8 + The activation ability of T cells (simulating poor immune status of the body), the addition of compound CND-treated macrophages significantly promoted OT1 CD8 +T cell proliferation (ability to divide).

[0059] Figure 1 C shows the statistical analysis of CD8 proliferation more than 4 times + The proportion of T cells;

[0060] Figure 1 D in shows CD8 + Total number of T cells;

[0061] Figure 2 It was shown that tumor cell culture supernatant can inactivate CD8 + The ability of T cells to produce IFNγ, and CND treatment reversed this effect;

[0062] The above results show that the terpenoid compound CND of the present invention can significantly promote the proliferation of CD8 + The proliferation capacity of T cells is shown by a significant increase in the number of divisions and the total number of T cells. + T cell suppression, thereby promoting CD8 + T cells produce effector molecules that boost the immune response to cells.

[0063] Example 2

[0064] Terpenoid compound CND regulates the expression of different cytokines in macrophages

[0065] The specific steps are as follows:

[0066] 2.1 The preparation method of macrophages was the same as steps 1.1-1.4 of Example 1.

[0067] 2.2 Place BMDM cells in 6-well plates and perform the following treatments:

[0068] A: control (0.1% DMSO)

[0069] B: Lipopolysaccharide (LPS) solution (final concentration 500 ng / ml) + 0.1% DMSO

[0070] C: Lipopolysaccharide (LPS) solution (500 ng / ml) + CND (1 µM)

[0071] 2.3 After 6 hours of cell treatment, discard the culture medium, wash the cells with PBS, add Trizol, pipette and harvest the cells, and extract mRNA.

[0072] 2.3.1 Discard the cell culture supernatant, add PBS buffer to the cell culture dish, wash the cells three times, add 1 mL of Trizol solution, pipette the cells until they are completely removed, and transfer the mixture into a 1.5 mL nuclease-free centrifuge tube.

[0073] 2.3.2 Incubate at room temperature for 5 min, add 200 μL of chloroform, vortex for 15 s, and incubate on ice for 3 min.

[0074] 2.3.3 Centrifuge at 12,000 rpm for 15 min at 4°C in a high-speed centrifuge.

[0075] 2.3.4 Pipette the supernatant into a new 1.5 mL nuclease-free centrifuge tube, add isopropanol equal to the volume of the supernatant, mix thoroughly by inverting the tube 8-10 times, and place on ice for 30 min.

[0076] 2.3.5 Centrifuge at 12,000 rpm for 10 min at 4°C.

[0077] 2.3.6 Discard the supernatant, add 1 mL of 75% ethanol, gently blow up the cell pellet, and centrifuge at 12,000 rpm at 4°C for 3 min.

[0078] 2.3.7 Discard the supernatant, place on ice for 10 min, add 30 μL of DEPC water to dissolve and mix.

[0079] 2.3.8 Use a spectrophotometer to measure RNA concentration and OD value and store at -80℃.

[0080] 2.4 Convert mRNA to cDNA.

[0081] 2.4.1 Using reverse transcription reagents, mix the reactants according to the system in Table 1 in a 0.2 mL nuclease-free centrifuge tube:

[0082]

[0083] 2.4.2 Place the 0.2 mL centrifuge tube in a 42°C water bath for 2 min. Add 8 μL of 5× HiscriptII qRT Supermix II and perform reverse transcription according to the procedure in Table 2:

[0084]

[0085] 2.4.3 After the reaction is completed, store the cDNA sample at -20℃.

[0086] 2.5 The expression of different factors was detected using Roche LightCycler 480II real-time fluorescence quantitative PCR instrument.

[0087] 2.5.1 Use autoclaved ddH2O to dilute the cDNA template 5-fold, mix well and set aside.

[0088] 2.5.2 Using a qPCR plate, mix the reaction components according to the system in Table 3:

[0089]

[0090] 2.5.3 Centrifuge at 1500 rpm at 4°C for 3 min.

[0091] 2.5.4 Place the qPCR plate in the instrument and amplify according to the reaction program in Table 4:

[0092]

[0093] qPCR result calculation method: Using Actb gene as internal reference, correct the Ct value of target gene of each sample and calculate value, with The relative expression levels of genes were calculated.

[0094] The qPCR primer sequences used are shown in Table 5:

[0095]

[0096] See the results Figure 3 .like Figure 3 As shown in A and 3B, CND significantly increased the expression levels of different inflammatory-related factors (IL6, IL12b, NOS2, COX2) and chemokines (CCL2, CXCL9) in macrophages, indicating that CND has the ability to enhance the inflammatory response of macrophages to pathogens and recruit other immune cells to the lesion site to control pathogens. CND is a potential immunomodulatory active molecule.

[0097]

[0098] Example 3 Antioxidant activity of heteroterpenoid compound CND

[0099] The experimental steps are as follows:

[0100] 1.1 Preparation and treatment of mouse macrophages

[0101] 1.1.1 Obtain wild-type adult C57BL / 6J mice, sacrifice them, and disinfect them by immersing them in 75% alcohol for 2-3 minutes. Make an abdominal incision, separate the muscles from the bones with scissors, and remove the tibia and femur. Then, immerse them in PBS buffer. All subsequent steps should be performed under a sterile operating table.

[0102] 1.1.2 After removing the tibia and femur with sterile forceps, cut off small pieces on both sides with scissors. Use a 20 mL syringe filled with PBS to repeatedly flush the bone marrow up and down. Collect the flushed bone marrow cells through a 70 μm cell sieve into a 50 mL sterile centrifuge tube and centrifuge at 2000 rpm for 4 min at 4°C.

[0103] 1.1.3 Add 1 mL of 1× red blood cell lysis buffer to the cell pellet to suspend the cells. After standing for 2 minutes, add 10 mL of PBS to wash and centrifuge at 2000 rpm at 4°C for 4 minutes.

[0104] 1.1.4 Resuspend the cells in RPMI 1640 medium (containing 5% L929 supernatant) and adjust the cell density to 2 × 106 / well. Plate the cells into untreated 6-well cell culture plates and culture at 37°C, 5% CO2. On day 4 of culture, observe the differentiated bone marrow-derived macrophages (BMDM) under a microscope.

[0105] 1.1.5 Place BMDM cells in 6-well plates and perform the following treatments:

[0106] Control (0.1% DMSO) and terpenoid compound CND (1 μM) were treated for 12 h.

[0107] 1.1.6 Add H2O2 (0.25 µM) to each group and treat for 1 h.

[0108] 1.1.7 Discard the supernatant, wash once with PBS, resuspend in 200 μl 2% FACS Buffer per well in a 96-well U-bottom plate, and centrifuge at 2000 rpm for 4 min.

[0109] 1.1.8 ROS measurement: discard the supernatant, add PBS to wash once, add ROS probe (1000x) and treat at 37°C for 30 min (1640 serum-free and without double antibody).

[0110] 1.1.9 Wash once with PBS. Add 150 μl of 7AAD (400x) to each well in 2% FACS buffer, mix well, filter into a flow cytometer, and analyze using a Canto II flow cytometer and FlowJo software.

[0111] The results are as follows Figure 4 As shown in Table 7, the heteroterpenoid compound CND can effectively reduce the level of reactive oxygen species (ROS) in macrophages after H2O2 treatment, indicating that CND has good antioxidant activity and is an effective antioxidant molecule.

[0112]

[0113] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Accordingly, the specification and drawings are to be regarded as illustrative rather than restrictive.

Claims

1. Use of the following heteroterpenoid compounds or pharmaceutically acceptable salts thereof in the preparation of immune-enhancing drugs: 。 2. The use according to claim 1, characterized in that The heteroterpenoid compound or a pharmaceutically acceptable salt thereof enhances immunity by regulating the function of macrophages.

3. The use according to claim 1, characterized in that The heteroterpenoid compound or a pharmaceutically acceptable salt thereof can enhance immunity by reducing the generation of active oxygen in cells.

4. The use according to claim 1, characterized in that The heteroterpenoid compound or a pharmaceutically acceptable salt thereof enhances the immune response mediated by T cells by enhancing the immune activation ability of macrophages.

5. The use according to claim 1, characterized in that The heteroterpenoid compound or a pharmaceutically acceptable salt thereof enhances immunity by increasing the cytokines and chemokines produced by macrophages.

6. The use according to claim 1, characterized in that The heteroterpenoid compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient are prepared into a composition.

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