CN109966274B - Application of guaiol in preparation of medicine for inhibiting tumor-related M2-type macrophages - Google Patents

Abstract

The invention relates to the technical field of new indications of medicaments, in particular to application of guaiol in preparation of a medicament for inhibiting tumor-related M2 type macrophages, and also discloses a medicament for inhibiting tumor-related M2 type macrophages. Experiments show that guaiol can modify the phenotype of M2 macrophage, promote the conversion of M2 macrophage to macrophage without induced activation, has obvious inhibiting effect on the expression of macrophage surface marker, the expression of cytokine secreted by macrophage, the expression of M2 macrophage mRNA, the expression of M2 macrophage protein and the like, and achieves the purpose of inhibiting the growth, invasion and metastasis of tumor by inhibiting M2 macrophage, thereby realizing the treatment of tumor. The guaiol is a low-toxicity and high-efficiency anti-tumor substance, provides a new idea for treating tumors, and has great medicinal prospect.

Description

Application of guaiol in preparation of medicine for inhibiting tumor-related M2-type macrophages

Technical Field

The invention relates to the technical field of new indications of medicines, in particular to application of guaiol in preparation of medicines for inhibiting tumor-related M2 type macrophages.

Background

Tumors are a group of diseases that seriously threaten human health. It and cardiovascular disease have become the first two causes of death worldwide. In tumor tissue, there are relatively many non-tumor cells, including stromal cells (fibroblasts and endothelial cells) and leukocytes. Macrophages are the major component of leukocytes infiltrating tumors. The relationship of macrophages in Tumor tissues, i.e., Tumor-associated macrophages (TAMs), to tumors and the various functions performed in tumors are being intensively studied. The different activation states of tumor-associated macrophages determine whether they exert an antitumor or tumorigenic effect in tumors. On the one hand, can play immune surveillance and anti-tumor roles by phagocytizing and killing tumor cells and presenting tumor-associated antigens; on the other hand, tumor growth and angiogenesis can be promoted by releasing many chemokines and cytokines, and the matrix can be degraded to promote tumor invasion and metastasis.

Macrophages can be classified into at least two types: type M1, classically activated macrophages (classically activated macrophages) and type M2, Alternatively activated macrophages (Alternatively activated macrophages). M1-type macrophages are induced by bacteria or their products lipopolysaccharide or interferon- γ and appear as: the ability to highly present antigen; high secretion of interleukin 12 and interleukin 23 and their subsequent induction of a type I immune response are high: high ability to produce toxic intermediates (nitric oxide, active oxygen intermediates). Therefore, macrophages of type M1 are thought to have the ability to kill bacteria and tumor cells and to secrete a variety of proinflammatory cytokines. M2 type macrophages can be induced by interleukin 4, interleukin 13, glucocorticoid, transforming growth factor-beta, prostaglandin E2 and the like, and show low capability of presenting antigen; production of cytokines that inhibit cell proliferation and activity, such as interleukin 10; better debris removal capability; ability to enter angiogenesis and wound healing.

More and more studies in recent years have shown that TAMs do not exert an antitumor effect, but are involved in the processes of tumor development, growth, invasion and metastasis, and are particularly closely related to tumor angiogenesis and lymphangiogenesis. According to the role played by macrophages in tumors and the activation type of macrophages, the two types of macrophages are presumed to exist in the tumors, and the macrophages in the tumors are mainly of M1 type and play immune monitoring and anti-tumor roles in the early stage of tumor development; in the late stage of the tumor, macrophages in the tumor mainly take M2 type, and play a role in promoting the growth, invasion and metastasis of the tumor; macrophages in tumors undergo a type change in the early and late stages of the tumor, affecting tumor growth, invasion and metastasis.

Guaiol is a fat-soluble natural compound extracted from guaiacum, radix ophiopogonis, cinnamon, mangnolia officinalis, rhizoma atractylodis, notopterygium root, rhizoma acori graminei, artemisia capillaris, fructus amomi, eucalyptus globulus fruit, artemisia rupestris, blumea balsamifera, rhododendron, asafetida, propolis and other plants, is sesquiterpene alcohol, has a molecular formula of C15H26O and a molecular weight of 222.37, and is mainly used in the perfume industry at present.

Chinese patent document 201410032832.1 discloses a pharmaceutical use of guaiol, which is one of the active ingredients or the only active ingredient used for preparing a pharmaceutical preparation for treating tumor, and the research result shows that: the guaiol can obviously inhibit the activity and proliferation of tumor cells in vitro, but does not inhibit the survival of normal immune cells; the guaiol can obviously inhibit the growth of tumors in vivo; therefore, guaiol is an active substance expected to be developed into a low-toxicity and high-efficiency antitumor pharmaceutical preparation, and has a medicinal prospect. Chinese patent document 201110133892.9 discloses the use of guaiol in the preparation of botanical insecticides, and the results of insecticidal experiments show that: guaiol exhibits antifeedant, contact, fumigant and juvenile hormone analog activities on test insects, and the action mode of the guaiol is closely related to the application dosage; the contact toxicity LD50 to 4-year armyworm and 3-year diamondback moth is respectively 0.072 mg/head, 24h and 8.920 mg/head, 24 h; the fumigation virulence LC50 for the houseflies and the 4-year-old armyworm adults is respectively 3.52 mu L/L, 12h and 16.95 mu L/L, 12h, and the result shows that the compound has good insecticidal activity and can be used as an insecticidal active component to further prepare plant insecticides such as spray or fumigant.

However, the application of guaiol in inhibiting tumor-associated M2 macrophages has not been reported yet.

Disclosure of Invention

The first purpose of the present invention is to provide a new use of guaiol against the deficiencies of the prior art.

The second purpose of the invention is to provide a medicine for inhibiting tumor-associated M2 type macrophages.

In order to achieve the first purpose, the invention adopts the technical scheme that:

application of guaiol in preparing medicine for inhibiting tumor-related M2 type macrophage is provided.

As a preferred technical scheme of the invention, the guaiol is used as the only active ingredient of the medicament for inhibiting the tumor-related M2 type macrophages or is used as the active ingredient together with other medicaments for inhibiting the tumor-related M2 type macrophages.

More preferably, the use of said guaiol in the manufacture of a medicament for inhibiting the proliferative activity of macrophage M2.

More preferably, the use of said guaiol in the preparation of a medicament for promoting the conversion of macrophages of type M2 to macrophages that have not been induced to activate.

More preferably, the use of said guaiol in the manufacture of a medicament for inhibiting the expression of M2-type macrophages.

In order to achieve the second object, the invention adopts the technical scheme that:

a medicament for inhibiting tumor-associated M2 type macrophages, wherein the active ingredient of the medicament is guaiol.

As a preferable technical scheme of the invention, the medicament for inhibiting the tumor-related M2 type macrophages further comprises a pharmaceutically acceptable carrier.

As a preferred embodiment of the present invention, the pharmaceutically acceptable carrier includes an emulsifier, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption enhancer, a flavoring agent, a coloring agent, or a solubilizing agent.

As a preferred technical scheme of the invention, the medicament is in the form of powder, granules, tablets, capsules, pills, solution, suspension or injection.

By "pharmaceutically acceptable" is meant a substance that is not substantially biologically or otherwise undesirable, i.e., the substance can be administered to an individual without causing any substantially undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The "carrier" also referred to as "excipient" includes any of the usual excipients in pharmacy and should be selected based on compatibility and the desired release profile properties of the dosage form. Exemplary carrier materials include, for example, emulsifiers, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. "pharmaceutically acceptable carriers" may include, for example, gum arabic, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, dextrin-maltose complexing agent, glycerin, magnesium silicate, sodium caseinate, soybean lecithin, sodium chloride, tricalcium phosphate, dipotassium hydrogen phosphate, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, and the like.

As a preferred embodiment of the present invention, the pharmaceutical or pharmaceutical composition of the present invention is in various forms that are conventional in the art, preferably in solid, semi-solid or liquid form, and may be in the form of an aqueous solution, a non-aqueous solution or a suspension, and more preferably in the form of powder, granule, tablet, capsule, pill, solution, suspension or injection. The route of administration of the drug or pharmaceutical composition is preferably injection or oral administration. The injection administration preferably comprises intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection and the like.

Experiments show that guaiol can modify the phenotype of M2 macrophage, promote the conversion of M2 macrophage to macrophage without induced activation, has obvious inhibiting effect on sexual expression of macrophage surface marker, expression of cytokine secreted by macrophage, expression of M2 macrophage mRNA, expression of M2 macrophage protein and the like, and achieves the purpose of inhibiting tumor growth, invasion and metastasis by inhibiting M2 macrophage, thereby realizing the treatment of tumor. The guaiol is a low-toxicity and high-efficiency anti-tumor substance, provides a new idea for treating tumors, and has great medicinal prospect.

Drawings

FIG. 1 is a graph of the effect of various concentrations of guaiacol on the proliferative activity of M2 macrophages. Wherein A is CCK-8 for detecting the cell survival rate; b is the IC50 value for cell survival and guaiol effect on the 24 hour node.

Figure 2 is an in vitro study of the inhibitory effect of guaiol on the M2 macrophage phenotype. Wherein A is a morphological change; b, detecting the expression of the surface antigen CD206 of M2 by using a flow cytometry; and C is a statistical result.

FIG. 3 is a flow chart of a macrophage clearance model.

Figure 4 is an in vivo study of the inhibition of M2 macrophages by guaiol. Wherein A is the weight change of the mouse; b is the mouse peripheral blood IL-10 expression level; c is the mouse subcutaneous transplantation tumor macrophage surface antigen expression; d is mouse spleen macrophage surface antigen expression; e is the expression of mouse subcutaneous transplantation tumor M2 macrophage mRNA; f is the expression of macrophage protein of mouse subcutaneous transplantation tumor M2.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention; furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims. The reagent instruments which are not described in detail in the invention can be obtained commercially, and the operation of the method which is not described in detail is carried out according to the routine operation in the field or the instruction of manufacturers.

Experimental Material

1.1 medicaments

Guaiol (-) -Guaiol: chemical name: (3R,6S,10S) -6,10, α, α -tetramethylbicyclo [5.3.0] dec-1 (7) -ene-3-methanol of the formula: C15H26O, molecular weight: 222.37. sigma company, usa, cat #: 29242 and 250 MG. Prepared into 20 mu M mother liquor by adopting methanol and stored for standby at 4 ℃.

1.2 animal experiments

Healthy male C57BL/6 mice, clean grade, 18-22g body weight, 4-6 weeks old, Shanghai Slek laboratory animals Co., Ltd; liposome Clodronate/Liposome PBS: amsterdam Vrije University (https:// clodronelisposomes. com, the Netherlands) in the Netherlands, Cat #: 40337ES05/10, 40338ES 05/10.

1.3 cell culture

Lewis lung cancer cell line (LLC for short) and RAW264.7 (mouse leukemia mononuclear macrophage cell line): cell banks of the Shanghai Life sciences institute of Chinese academy of sciences; DMEM medium (1% cyan/streptomycin): hyclone, USA; fetal Bovine Serum (FBS): a complete culture medium containing 10% FBS is prepared before use by Gibco company in America, and is stored in a refrigerator at 4 ℃ for later use; M-CSF: U.S. Peprotech corporation, cat #: 315-02-50; IL-4: U.S. R & D, Cat number: BC 1817031.

1.4 flow cytometry (FACS)

FITC-F4/80, PE-CD206 antibody: peprotech, Inc., USA; tumor tissue dissociation kit: germany Miltenyi company, cat #: 130, 096, 730; spleen dissociation kit: germany Miltenyi company, cat #: 130, 095, 926; GentleMeACS 25C Tubes: miltenyi, Germany, Cat No. 130-; title MACS tissue processor: miltenyi, Germany; 100um filter screen: corning, USA.

1.5 real-time fluorescent quantitative PCR (RT-PCR)

PrimeScriptTM RT Master Mix (Perfect Real Time): TaKaRa, Japan, Cat No. RR 036A; SYBRPrime Ex TaqTM: TaKaRa, Japan, Cat. No. RR 420A.

1.6 Western immunoblotting (Western Blot)

GAPDH primary antibody: U.S. CST corporation, cat #: 2118S; CD206 primary antibody: immunoway, Cat No.: YT 0739; IL-10 primary antibody: abram corporation, cat # in usa: ab 189393; anti-rat IgG secondary antibody: U.S. CST corporation, cat #: # 7077; anti-rabbit IgG secondary antibody: U.S. CST corporation, cat #: # 7074.

Example 1 in vitro study of the inhibitory Effect of guaiol on the M2 macrophage phenotype

1 in vitro M2 macrophage model establishment

This was done using mouse Bone marrow-derived macrophages (BMDM). A healthy C57BL/6 male mouse is taken, the mouse is 6-8 weeks old and 18-22g in body mass, the cervical vertebra of the mouse is killed, the abdominal skin of the mouse is disinfected by 75% alcohol, then the mouse is in a supine position, four limbs of the mouse are unfolded, and the mouse is fixed on a dissection board. Opening the abdominal cavity of a mouse under the aseptic condition, taking a femur, removing muscles, shearing off two ends by an ophthalmic scissors, sucking DMEM culture solution containing 10% FBS by using an injector, blowing out bone marrow cells, repeatedly blowing and beating by using a pipette gun, filtering by using a filter screen with the aperture of 100um, removing impurities by screening, centrifuging for 10min by 300g, obtaining precipitates which are the bone marrow mononuclear cells of the mouse, inoculating the collected bone marrow mononuclear cells of the mouse into a 6cm culture dish, adding M-CSF to enable the final concentration to be 10ng/ml, and culturing in a cell culture box with the temperature of 37 ℃ and the concentration of 5% CO 2. Washing with PBS every day, discarding non-adherent cells, replacing culture solution, and continuously culturing for 7 days to obtain BMDM primary cells.

Collecting BMDM primary cells with good growth condition, and making cell concentration be 1 × 10⁶And adding IL-4 with the concentration of 20ng/mL to induce 24 hours to establish M2 type macrophage which induces activation.

2 results of the experiment

2.1 screening of the effective concentration of guaiol on macrophages

In vitro screening the effective action concentration of the guaiol on the macrophage to determine reasonable action concentration and time, wherein the selected concentration and time range can regulate the phenotype of the macrophage without causing the killing effect of the macrophage.

We adopt CCK-8 experiment to observe the inhibition effect of guaiol with different concentrations on the proliferation of the mononuclear macrophage strain RAW264.7 so as to determine the reasonable action concentration and time of further experiment of guaiol. The results show that as the concentration of guaiacol increases, the CCK-8 test results show that the concentration of the guaiacol is increased, and the result of the CCK-8 test is shown in figure 1A, and the IC50 value of 24 hours is 232.1 mu M, 95% CI (160.8, 396.2), and figure 1B. We selected the guaiacol concentration range from 20 μ M to 80 μ M with 24 hours of action as the next experimental study concentration range.

2.2 in vitro study of the inhibitory Effect of guaiol on the M2 macrophage phenotype

The activated M2 macrophage model was first induced in vitro with IL-4 and 24h later, guaiacol was administered at concentrations of 0. mu.M, 20. mu.M, 40. mu.M, 60. mu.M, 80. mu.M, respectively. We observed changes in macrophage morphology before and after induction and after guaiacol treatment on a high power microscope. Changes in macrophage morphology were photographed, see fig. 2A. The macrophage which is not induced and activated by IL-4 is round, a small amount of the macrophage is polygonal, the macrophage which is induced and activated by IL-4 is differentiated into M2 type, the cell is fusiform, a large amount of pseudopoda extends out, and the shape of the macrophage M2 after the treatment of the guaiol is similar to that of the macrophage which is not induced and activated. Thus, the guaiol has a regulating effect on the M2 macrophage phenotype.

Cell membrane antigen CD206⁺Watch (A)The expression of the protein is changed into a main marker of M2 macrophage, so that the flow cytometry is adopted to detect the double positive expression of M2 type macrophage surface marker F4/80 and CD 206. Flow cytometry results show that the guaiol in the 40 mu M and 60 mu M concentration groups has the most significant inhibition effect on M2 macrophage compared with the 0 mu M concentration group, and the flow cytometry results are respectively 64.6 +/-2.5% and 42.3 +/-2.8% in a figure 2B, and the differences have statistical significance (P)<0.001), fig. 2C. Thus, the guaiol has an inhibiting effect on the M2 macrophage phenotype.

Example 2 inhibition of Lewis Lung cancer mice subcutaneous graft tumor and spleen M2 macrophage by guaiol

1 course of the experiment

1.1 establishment and group administration of macrophage scavenging mouse model

Clean grade healthy male C57BL/6 mice were randomly assigned to 4 groups, each: liposome PBS-normal saline group (M +/Control), Liposome PBS-Guaiol group (M +/Guaiol), Liposome clone-normal saline group (M-/Control), Liposome clone-Guaiol group (M-/Guaiol), using LLC cells to inoculate C57BL/6 mice right shoulder blade part subcutaneous, the inoculated cell density is 1 × 10⁶Each cell is 0.2 mL/cell, and a subcutaneous transplantation tumor model is established. Macrophage scavenger (Liposome clone/Liposome PBS) was injected into the abdominal cavity of 4 groups of mice 2 days before LLC cell inoculation, 0.2 ml/mouse, 0.1 ml/mouse of Liposome clone/Liposome PBS was injected into the abdominal cavity every 4 days from 0 th day, 3-5mm tumor mass was observed under the right shoulder blade at about 1 week time, and 0.2ml of guaiol (8mg/kg/2 days) was injected into the abdominal cavity of 4 groups of mice. The macrophage clearance model flow is shown in figure 3.

1.2 specimen Retention

And (3) weighing the weights of the mice of all groups when the experiment end point is reached on the 28 th day, taking the peripheral venous blood of the mice by adopting an eyeball blood taking method, standing for 2 hours at 4 ℃, centrifuging for 30min at 3000rpm, taking the supernatant, and storing in a refrigerator at minus 80 ℃. Performing cervical dislocation, removing fresh spleen and tumor tissue, preparing single cell suspension in one part, storing at-80 deg.C in refrigerator for RNA and protein extraction, and fixing in 4% paraformaldehyde in one part.

1.3 flow cytometry (FACS)

Collecting cells, adjusting the number of cells to about 1 × 10⁶And (4) respectively. Dissociating the tumor tissue and the spleen, preparing a single cell suspension, and sequentially adding a mixed enzyme solution into a gene MACS C tube according to an instruction; shearing fresh mouse tumor tissue/spleen tissue into 2-4mm size, and adding into a C tube; installing the C tube into a cannula of a gentle MACS tissue processor, and running a gentle MACS program; after the program is finished, transferring the cell suspension from the tube C into a 15ml centrifuge tube, and centrifuging for 5min at 300 g; filtering with a 100um filter screen, and collecting single cell suspension; regulating cell number to about 1 × 10⁶And (4) respectively. Sequentially adding fluorescent antibody into each group, and incubating at 4 deg.C in dark for 30 min; washing the antibody with PBS, and performing on-machine detection with a flow meter; the results were analyzed using Flowjo software.

1.4 real-time fluorescent quantitative PCR (RT-PCR)

Cell collection/subcutaneous tumor tissue homogenization. Carrying out total RNA extraction on TRIZON; cDNA synthesis was performed according to kit instructions, setting reaction conditions: 15min at 37 ℃, 5s at 87 ℃ and an infinite value at 4 ℃; Real-Time PCR was performed according to the kit instructions, and the target gene primer sequences: arg-1: f (5 '-3') -GGTTCTGGGAGGCCTATCTT, R (5 '-3') -CACCTCCTCTGCTGTCTTCC; IL-10: f (5 '-3') -CTGCTATGCTGCCTGCTCTTACTG, R (5 '-3') -ATGTGGCTCTGGCCGACTGG, set up the amplification program: signals were detected at 95 ℃ for 30s, 95 ℃ for 5s, 60 ℃ for 30s, 95 ℃ for 30s, 37 cycles, and 72 ℃. Data processing uses 2^-ΔΔCtThe relative expression level of the gene was calculated.

1.5 Western immunoblotting (Western Blot)

Cell collection/subcutaneous tumor tissue homogenization. RIPA (containing 1% PMSF) is subjected to lysis, and the protein concentration is determined by a BCA protein quantitative method; equal amounts of protein (50. mu.g/lane) were loaded; electrophoresis was performed using 10% SDS-PAGE: the upper layer glue is 80V, and the lower layer glue is 120V; rotating the film at constant pressure of 100V on ice; incubating the primary antibody at 4 ℃ overnight, and incubating the secondary antibody at room temperature for 1 hour; ECL chemiluminescence exposure.

1.6 statistics of data

Performed using SPSS 20.0.

2 results of the experiment

2.1 Effect on mouse body weight

At the end of the experiment, the average body weight of each group of mice was: 23.3 +/-0.27 g, 23 +/-0.78 g, 23.27 +/-0.47 g and 22.9 +/-0.55 g. The results showed no significant difference in body weight (P >0.05) among the groups, fig. 4A. The guaiol has no obvious toxic and side effects on experimental mice.

2.2 Effect on expression levels associated with M2-type macrophages

IL-10 is a cytokine secreted by M2 macrophages and is closely related to M2 macrophages. Therefore, the expression level of mouse peripheral blood IL-10 is detected by adopting an ELISA technology, and the result shows that the guaiol has a remarkable inhibiting effect on the Lewis lung cancer mouse peripheral blood IL-10, and compared with a control group, the difference has statistical significance (P is less than 0.001), and fig. 4B. It shows that guaiol has obvious inhibition effect on IL-10.

The expression level of M2 type macrophages in mouse tumor and spleen is detected by flow cytometry, and the result shows that guaiol has obvious inhibition effect on M2 type macrophages in Lewis lung cancer mouse tumor and spleen, and the expression level of M2 type macrophage surface antigen CD206 in each tumor is as follows: 23.78 +/-5.78%, 14.9 +/-3.91%, 12.31 +/-3.34% and 12.875 +/-4.97%. The expression levels of the spleen M2 type macrophage surface antigen CD206 are respectively as follows: 23.09 +/-1.05%, 5.43 +/-2.34%, 5.26 +/-1.09% and 2.34 +/-0.93%. Compared with the Liposome PBS-normal saline group, the expression level of CD206 in each experimental group is remarkably reduced, (P <0.001), and the difference has statistical significance, and the images are shown in FIGS. 4C-D. It shows that guaiol has obvious inhibition effect on M2 macrophage.

RT-PCR and Western Blot techniques are adopted to detect the expression of mRNA and protein, and the results show that guaiol has significant inhibition effect on the expression of mRNA and protein of M2 type macrophages and related cytokines IL-10 in the tumor body and spleen of Lewis lung cancer mice, and fig. 4E-F shows that the expression of mRNA and protein is obvious. It shows that guaiol has obvious inhibition effect on M2 macrophage.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims (2)

1. The application of guaiol in preparing an experimental reagent for regulating the phenotype of M2 type macrophages in vitro.

2. Use according to claim 1, characterized in that the guaiol inhibits the M2-type macrophage phenotype.

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