Application of compound K67 in the preparation of anti-tuberculosis drugs

Abstract

The present invention provides an application of a compound K67 in the preparation of an anti-tuberculosis drug, belonging to the technical field of chemical drugs. Experiments show that K67 can inhibit the proliferation of Mycobacterium tuberculosis, promote macrophage autophagy to enhance the bactericidal effect, and can also reduce the oxidative stress level of macrophages, inhibit the expression of inflammatory factor genes and proteins in macrophages, and reduce the inflammatory damage caused by Mycobacterium tuberculosis infection. Therefore, K67 has potential research and application value in the field of anti-tuberculosis.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical drugs, and particularly relates to application of compound K67 in preparing anti-tuberculosis drugs. Background Art

[0002] In recent years, anti-tuberculosis drugs have been widely used and abused. The combined use of some first-line anti-tuberculosis drugs, such as isoniazid (INH), ethambutol (EMB), rifampicin (RIF), pyrazinamide (PZA), etc., can exert a good anti-tuberculosis effect, but the abuse of drugs makes pathogens prone to drug resistance. In terms of second-line anti-tuberculosis drugs, only two new drugs, bedaquiline and delamanid, have been launched in recent decades. [1] The emergence of drug-resistant strains and the many side effects of drug use make the task of developing new anti-tuberculosis drugs urgent. [2] .

[0003] Macrophages are sentinel cells that detect invading microorganisms. They have strong phagocytic ability and are the main target cells for infection by Mycobacterium tuberculosis (M.tb). When M.tb infects macrophages, macrophages can phagocytize M.tb, produce immune responses, and produce a large amount of reactive oxygen species (ROS), and synthesize and secrete a variety of proinflammatory cytokines, such as TNF-α, IL-6, IL-1β, etc. At this time, macrophages kill intracellular M.tb through autophagy, strong self-oxidation, and other pathways. However, higher ROS levels in macrophages can cause oxidative stress damage, and the secretion of proinflammatory cytokines can also activate a variety of immune cells, promoting the occurrence and development of inflammatory responses.

[0004] The compound 2-acetyl-1,4-di[(4-methoxyphenylsulfonyl)amino]naphthalene (K67) is a small molecule compound obtained by Japanese scholars through high-throughput screening and identification. [3] Limited experimental data show that it can not only inhibit the proliferation of liver cancer cells and the tolerance of anticancer agents [4] , and can also inhibit the proliferation of esophageal squamous cell carcinoma cells and enhance radiosensitivity [5] , but it has not yet been applied in the field of anti-tuberculosis.

[0005] References

[0006] [1] Wu Jiang, Cui Jiawei, Xue Yunxin, Wang Dai: Research progress on anti-tuberculosis drug targets. Chinese Journal of Antibiotics, 2022, 47(11): 1119-1127.

[0007] [2]. Liang Chen, Tang Shenjie, Lin Minggui: Research progress on comprehensive treatment of tuberculosis. Journal of Tuberculosis and Lung Diseases: 1-23.

[0008] [3].Yasuda D,Obata R,Takahashi K,Ohe T,Mashino T:[Hit-to-Lead inAcademia:Discovery of a Protein-Protein Interaction Inhibitor of Keap1-Nrf2].Yakugaku Zasshi 2018,138(8):1059-1065.

[0009] [4]. Saito T, IchimuraY, Taguchi K, Suzuki T, Mizushima T, Takagi K, HiroseY, Nagahashi M, Iso T, Fukutomi T et al: p62 / Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming. Nat Commun 2016,7:12030.

[0010] [5]. Wang Zhe: Research on small molecule compound K67 mediating radiosensitization of esophageal squamous cell carcinoma by regulating p62-Keap1-Nrf2 pathway. PhD. Shandong University; 2019. Summary of the invention

[0011] The purpose of the present invention is to provide an application of a compound K67 in the preparation of an anti-tuberculosis drug, wherein K67 can not only inhibit the proliferation of Mycobacterium tuberculosis, but also increase the level of macrophage autophagy, reduce the level of oxidative stress of macrophages, and inhibit the secretion of pro-inflammatory factors by macrophages, thereby reducing the degree of inflammatory damage to the body.

[0012] The present invention provides an application of a compound K67 in the preparation of an anti-tuberculosis drug.

[0013] Preferably, the anti-tuberculosis effect includes inhibiting the proliferation of Mycobacterium tuberculosis in the body.

[0014] The present invention provides an application of a compound K67 in preparing a medicine for repairing body damage caused by mycobacterium infection.

[0015] Preferably, the compound K67 regulates at least one of the following reactions: increasing the autophagy level of macrophages, reducing the oxidative stress level of macrophages, and inhibiting the inflammatory response in macrophages;

[0016] The macrophages are macrophages infected with Mycobacterium tuberculosis.

[0017] Preferably, increasing the autophagy level of macrophages includes promoting the expression of autophagy-related proteins and / or increasing the ratio of LC3Ⅱ / LC3Ⅰ.

[0018] Preferably, reducing the oxidative stress level of macrophages includes reducing the level of reactive oxygen species in macrophages and / or reducing the expression level of NADPH oxidase 2 in macrophages.

[0019] Preferably, the inhibiting of inflammatory response in macrophages comprises inhibiting the expression of genes and / or proteins of pro-inflammatory factors in macrophages.

[0020] Preferably, the pro-inflammatory factors include at least one of the following: tumor necrosis factor-α, interleukin-6 and interleukin-1β.

[0021] The present invention provides an application of a compound K67 in preparing an antibacterial agent for inhibiting mycobacteria in vitro.

[0022] Preferably, the mycobacterium comprises Mycobacterium tuberculosis.

[0023] Beneficial effects:

[0024] The present invention provides an application of a compound K67 in the preparation of an anti-tuberculosis drug. The embodiments of the present invention show that K67 has the effect of inhibiting the proliferation of M.tb in vitro; K67 can effectively kill M.tb in macrophages at the cellular level, reduce the load of M.tb in macrophages, promote the expression of autophagy-related proteins p-p62 and p62, and increase the ratio of LC3Ⅱ / LC3Ⅰ, increase the autophagy level of macrophages, and reduce the level of reactive oxygen and the expression level of NADPH oxidase 2 in macrophages, inhibit the expression of genes and proteins of pro-inflammatory factors tumor necrosis factor-α, interleukin-6 and interleukin-1β in macrophages, and reduce the inflammatory damage caused by M.tb infection. At the same time, the present invention verifies that K67 has no cytotoxicity to macrophages in the concentration range of 0 to 20 μM and has good safety in use. In addition, the present invention verifies that K67 does not affect the phagocytic function of THP-1-derived macrophages to M.tb. Therefore, K67 has potential application value for preparing new anti-tuberculosis drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] Figure 1 The CCK8 method was used to detect the survival rate of macrophages after K67 treatment;

[0027] Figure 2 This is an extracellular K67 antibacterial experiment; Figure a shows the growth of M.tb colonies on the plate before and after the action of K67, and Figure b shows the colony count (CFU×10 4 ) and statistical analysis;

[0028] Figure 3 The effect of K67 treatment on the phagocytic function of macrophages; the MFI value is the mean fluorescence intensity, which reflects the phagocytic function of THP-1-derived macrophages on M.tb;

[0029] Figure 4 Figure 1 shows the killing activity of K67 pretreated macrophages on intracellular M.tb; Figure a shows the effect of K67 pretreated macrophages on the growth of M.tb colonies on the plate, and Figure b shows the colony counts (CFU×10 4 ) and statistical analysis;

[0030] Figure 5 Figure 1 shows the killing activity of K67 against M.tb in infected macrophages; Figure a shows the effect of K67 treatment on the growth of M.tb colonies on the plate, and Figure b shows the colony count (CFU×10 4 ) and statistical analysis;

[0031] Figure 6 Figure 1 is the effect of K67 pretreatment on autophagy in macrophages; Figure a is the Western blot analysis of the effect of K67 pretreatment on the expression level of autophagy-related proteins in macrophages, and Figure b is the statistical analysis of the grayscale value of the immunoblot in Figure a, where LC3Ⅱ represents the ratio of LC3Ⅱ / LC3Ⅰ;

[0032] Figure 7 Figure 1 is the effect of K67 pretreatment on ROS level in macrophages; Figure a is the flow cytometry analysis of the effect of K67 pretreatment on ROS level in macrophages, and Figure b is the statistical analysis of the mean fluorescence intensity (MFI) of ROS in Figure a;

[0033] Figure 8The effect of K67 pretreatment on the expression level of NADPH oxidase 2 (NOX2) mRNA in macrophages; Figure a is the RT-qPCR analysis of NOX2 mRNA expression level, showing the amplification curve (Ct value is between 15-35, representing reliable values) and melting curve (single peak indicates unique product, good amplification specificity), Figure b is the statistical analysis of NOX2 mRNA expression level;

[0034] Fig. 9 Figure 1 shows the effect of K67 pretreatment on the expression levels of proinflammatory cytokines TNF-α, IL-6, and IL-1β in macrophages; Figure a shows the statistical analysis of the mRNA expression levels of each proinflammatory cytokine detected by RT-qPCR, and Figure b shows the statistical analysis of the protein expression levels of each proinflammatory cytokine in the cell culture supernatant detected by ELISA;

[0035] Note: Figures 1 to 9 n=3, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001. DETAILED DESCRIPTION

[0036] The present invention provides an application of a compound K67 in the preparation of an anti-tuberculosis drug.

[0037] In the present invention, the anti-tuberculosis preferably includes inhibiting the proliferation of Mycobacterium tuberculosis in the body.

[0038] In the present invention, the Chinese name of the compound K67 is 2-acetyl-1,4-di[(4-methoxyphenylsulfonyl)amino]naphthalene, and the molecular formula is C 29 H 30 N2O7S2, molecular weight is 582.69, and the structural formula is shown in Formula I.

[0039]

[0040] In the embodiment of the present invention, the K67 is purchased from Sigma-Aldrich Company with the product number SML1922, and is a powdery substance that can be dissolved in DMSO.

[0041] In the embodiment of the present invention, the effect of K67 on the activity of THP-1-derived macrophages was detected, and different doses of K67 were added to THP-1-derived macrophages to make their final concentrations of 10 μM, 20 μM, 40 μM and 80 μM, respectively. The results show that within the concentration range of 0 to 20 μM, K67 has no cytotoxicity to macrophages and has good safety in use. The present invention further verifies the effect of K67 on the function of THP-1-derived macrophages to phagocytose Mycobacterium tuberculosis. After K67 is added to THP-1-derived macrophages for culture, THP-1-derived macrophages are infected with RFP-H37Ra bacterial suspension, and the content of RFP-H37Ra in macrophages is detected by flow cytometry. The results show that K67 does not affect the phagocytic function of THP-1-derived macrophages to M.tb.

[0042] The present invention also conducted an experiment on the killing effect of K67 on M.tb in macrophages. THP-1-derived macrophages were first treated with 20 μM K67 and then infected with RFP-H37Ra bacterial suspension to detect the effect of K67 on M.tb in macrophages. The results showed that the number of M.tb colonies on the agar plate of the group first treated with K67 was significantly reduced, indicating that K67 can first act on macrophages that are not infected with M.tb, regulate the immune response of macrophages to M.tb, thereby effectively killing M.tb that invaded macrophages at a later stage and reducing the load of M.tb in macrophages. At the same time, the present invention first infected THP-1-derived macrophages with RFP-H37Ra bacterial suspension and then treated with 20 μM K67 to detect the effect of K67 on M.tb in macrophages. The results showed that the number of colonies on the agar plate of the group infected first and then treated with K67 was significantly reduced, indicating that K67 can also act on macrophages that have been infected with M.tb, change the immune response of infected macrophages to M.tb, thereby effectively killing M.tb that has invaded macrophages and reducing the load of M.tb in macrophages.

[0043] In the present invention, the compound K67 regulates at least one of the following reactions: increasing the autophagy level of macrophages, reducing the oxidative stress level of macrophages and inhibiting the inflammatory response in macrophages; the macrophages are macrophages infected with Mycobacterium tuberculosis. The increasing the autophagy level of macrophages preferably includes promoting the expression of autophagy-related proteins and / or increasing the ratio of LC3Ⅱ / LC3Ⅰ; the promoting the expression of autophagy-related proteins preferably includes promoting the expression of autophagy-related proteins p-p62 and p62. Autophagy is an important means for macrophages to perform defense, and macrophages can kill invading pathogenic microorganisms through autophagy. During the infection of Mycobacterium tuberculosis, autophagy can not only play an important role in the antibacterial mechanism of intracellular pathogen clearance, but also prevent excessive inflammation and avoid damage to the host.

[0044] In the embodiment of the present invention, the effect of K67 on autophagy after THP-1-derived macrophages were infected with M.tb was detected. The present invention used K67 to pretreat THP-1-derived macrophages, and then added H37Ra bacterial suspension to infect THP-1-derived macrophages. The results of detecting the expression level of autophagy-related proteins showed that compared with the control group, there was no significant difference in the expression of autophagy-related proteins in the K67 group alone (P>0.05), while the expression of autophagy-related proteins in the K67+M.tb group was increased compared with the M.tb infection group alone, and the expression of autophagy-related proteins p-p62 and p62 and / or the ratio of LC3Ⅱ / LC3Ⅰ were significantly increased. This shows that K67 can enhance the level of autophagy in macrophages after infection with M.tb and improve the immune response of cells against M.tb.

[0045] In the present invention, the reduction of the oxidative stress level of macrophages preferably includes reducing the level of reactive oxygen in macrophages and / or reducing the expression level of NADPH oxidase 2 in macrophages. In the embodiment of the present invention, the effect of K67 on the ROS level of THP-1-derived macrophages was detected, and the ROS level of THP-1-derived macrophages pretreated with K67 was detected by flow cytometry. The results showed that K67 can significantly inhibit the production of macrophage ROS regardless of whether it is infected with M.tb. This shows that K67 can reduce the oxidative stress level caused by macrophages themselves and M.tb infection to cells, and has a protective effect on cells. The present invention further determined the effect of K67 on the expression level of NADPH oxidase 2 (NOX2) mRNA after THP-1-derived macrophages were infected with M.tb. The results showed that compared with the control group, M.tb infection caused NOX2 mRNA expression to be significantly upregulated (P<0.05), and K67 significantly downregulated the high expression of NOX2 mRNA caused by M.tb infection (P<0.05). Therefore, K67 can reduce the expression level of NOX2 in macrophages infected with M.tb and inhibit the oxidative stress response caused by M.tb infection.

[0046] In the present invention, the inhibition of inflammatory response in macrophages preferably includes inhibiting the expression of genes and / or proteins of proinflammatory factors in macrophages. The proinflammatory factors preferably include at least one of the following: tumor necrosis factor-α, interleukin-6 and interleukin-1β; the genes preferably include mRNA. In the embodiment of the present invention, the effect of K67 on the expression levels of TNF-α, IL-6 and IL-1β mRNA and protein after THP-1-derived macrophages were infected with M.tb was determined. The results showed that M.tb infection caused a significant increase in the expression levels of TNF-α, IL-6 and IL-1β mRNA and protein (P<0.001), while K67 significantly downregulated the high expression of TNF-α, IL-6 and IL-1β mRNA and protein caused by M.tb infection. This shows that K67 can inhibit the high inflammatory state caused by M.tb infection and protect cells from oxidative stress damage. Therefore, K67 has potential application value for preparing new anti-tuberculosis drugs.

[0047] Based on the fact that the compound K67 has the effect of inhibiting the proliferation of Mycobacterium tuberculosis and also has the performance of reducing body damage, the present invention provides an application of the compound K67 in the preparation of a drug for repairing body damage caused by mycobacterium infection.

[0048] The present invention provides an application of a compound K67 in preparing an antibacterial agent for inhibiting mycobacteria in vitro.

[0049] In the present invention, the mycobacterium preferably includes Mycobacterium tuberculosis.

[0050] In the present invention, the dosage form of the antibacterial agent preferably includes powder and aqueous solution, more preferably aqueous solution. When the antibacterial agent is an injection, the effective dose of K67 in the antibacterial agent is preferably 10 to 20 μM, more preferably 20 μM. The present invention has no special restrictions on the preparation method of the antibacterial agent, and the preparation method of the antibacterial agent known in the art can be used.

[0051] In the embodiment of the present invention, 20 μM K67 was added to the culture system of the attenuated strain RFP-H37Ra of M.tb. The results showed that the number of colonies in the K67 group was significantly reduced compared with the control group. Therefore, K67 has a direct antibacterial effect.

[0052] In order to further illustrate the present invention, the application of the compound K67 provided by the present invention in the preparation of anti-tuberculosis drugs is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Sources of materials and reagents

[0054] Human M.tb standard attenuated strain H37Ra (CAT: 9302025, purchased from China Medical Bacteria Collection Center), M.tb attenuated strain H37Ra expressing red fluorescent protein (RFP-H37Ra) (containing kanamycin resistance gene) see the prior art Jie Zhou, Fang Fang, Jinying Qi, et al. Activation of Nrf2 modulates protective immunity against Mycobacterium tuberculosis infection in THP1-derived macrophages. Free Radical Biology and Medicine, 193 (2022) 177-189. Donated by Professor Li Baiqing (Bengbu Medical University, Anhui Key Laboratory of Basic and Clinical Immunology of Chronic Diseases);

[0055] Human acute monocytic leukemia cells (THP-1, TIB-202) were purchased from ATCC (Manassas, USA);

[0056] K67: 2-acetyl-1,4-di[(4-methoxyphenylsulfonyl)amino]naphthalene, product number: SML1922, powdery substance, soluble in DMSO, purchased from Sigma-Aldrich;

[0057] PMA: phorbol ester, product number: P1585, powdery substance, soluble in DMSO, purchased from Sigma-Aldrich.

[0058] Example 1

[0059] K67 Safety Evaluation

[0060] Methods for macrophage culture and differentiation

[0061] THP-1 cells were revived and added to RPMI 1640 medium containing 10% fetal bovine serum (FBS) and a mixture of penicillin / streptomycin / gentamicin antibiotics (final concentration of penicillin was 100 U / mL, final concentration of streptomycin was 0.1 mg / mL, and final concentration of gentamicin was 50 μg / mL), and cultured in a constant temperature incubator at 37°C and 5% CO2. THP-1 cells in good cell condition were selected and cultured until the density reached 5×10 6 / mL, the cells were divided into cell culture plates according to the groups, and 100ng / mL phorbol 12-myristate 13-acetate (PMA) was added to each well to stimulate THP-1 cells for 24h. THP-1 cells differentiated into spindle-shaped macrophages attached to the wall (THP-1-derived macrophages). After washing with PBS to remove the residual culture medium, PMA and undifferentiated THP-1 cells, the culture medium was replaced with RPMI 1640 medium containing 10% FBS and the density was adjusted to 1×10 6 / mL for future use.

[0062] Detection of the effect of K67 on the activity of THP-1-derived macrophages

[0063] CCK8 method to detect cell activity: add THP-1 derived macrophage test wells (containing 10 μL of THP-1 derived macrophages prepared in Example 1, 10 4 Different doses of K67 were added to the cells (10 cells / well) to make the final concentrations 10 μM, 20 μM, 40 μM and 80 μM, respectively (the solvent was DMSO). An equal volume of DMSO was added to the control group. After incubation at 37°C for 24 h, 10 μL of CCK-8 solution was added (avoiding bubbles). After being placed in a 37°C incubator for 3 h, the absorbance at 450 nm was measured with a microplate reader, and the cell survival rate was calculated according to Formula I.

[0064] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100% Formula I

[0065] In formula I, As is the absorbance of the experimental well (containing cells, culture medium, CCK-8 solution and drug solution), Ac is the absorbance of the control well (containing cells, culture medium, CCK-8 solution, but not containing drugs), and Ab is the absorbance of the blank well (containing culture medium, CCK-8 solution, but not containing cells or drugs).

[0066] Finally, GraphPad 9.1.0 was used for statistical analysis.

[0067] The results of CCK8 assay showed that there was no statistical difference in cell activity between the K67 group and the control group at 10 μM and 20 μM concentrations (P>0.05, Figure 1 ), indicating that within a certain drug concentration range (0-20 μM), K67 is non-toxic to macrophages and has good safety for use.

[0068] Example 2

[0069] Extracellular K67 antibacterial assay

[0070] Pick out an inoculation loop of the attenuated strain of M.tb RFP-H37Ra and inoculate it into Sutong medium (2g / L citric acid, 0.05g / L ammonium ferric citrate, 1.025g / L magnesium sulfate, 0.5g / L potassium dihydrogen phosphate, 4g / L asparagine, 0.06% (v / v) glycerol, 50μg / mL kanamycin), and place it in a 37°C constant temperature bacterial incubator for 3 to 4 weeks. When RFP-H37Ra in Sutong medium grows to the logarithmic growth phase, pick 3 to 4 inoculation loops and grind them thoroughly in 300μL phosphate Tween buffer (PBST solution), centrifuge at 1500rpm for 5min, draw the supernatant into a 1.5mL EP tube, repeat the operation 3 times, and you can get a single bacterial suspension. Use a spectrophotometer to measure the live bacterial concentration of the bacterial suspension at a wavelength of 600nm, and adjust its concentration to 1×10 9 CFU / mL is reserved.

[0071] K67 (solvent: DMSO) with a final concentration of 20 μM was added to 1 mL of bacterial suspension and placed in a 37°C bacterial incubator for 12 hours. The bacterial suspension was diluted 100 times with PBST and 100 μL was spread on an agar plate containing kanamycin (50 μg / mL). After being placed upright for 30 minutes, it was inverted and cultured, which was counted as the K67 group. The difference between the control group and the K67 group was that the K67 solution was replaced with an equal volume of DMSO solution. After 3 weeks, the growth of tuberculosis colonies on the agar plate was observed and photographed, and the colony count and statistical analysis were performed.

[0072] The results showed that compared with the control group, the number of colonies in the K67 group was significantly reduced, and the difference was significant (P<0.05, Figure 2 ), indicating that K67 has a direct antibacterial effect.

[0073] Example 3

[0074] Effect of K67 on phagocytic function of macrophages

[0075] Methods for infection of THP-1 derived macrophages by M.tb

[0076] Preparation of H37Ra bacterial suspension and RFP-H37Ra bacterial suspension: Pick an inoculation loop of the attenuated strain H37Ra of M.tb and inoculate it into Sutong medium (2g / L citric acid, 0.05g / L ammonium ferric citrate, 1.025g / L magnesium sulfate, 0.5g / L potassium dihydrogen phosphate, 4g / L asparagine, 0.06% (v / v) glycerol), and place it in a 37°C constant temperature bacterial incubator for 3 to 4 weeks. For M.tb (RFP-H37Ra) expressing red fluorescent protein, 50μg / mL of kanamycin is additionally added to the culture medium. When M.tb grows to the logarithmic growth phase, pick 3 to 4 inoculation loops into 300μL phosphate Tween buffer (PBST solution) and grind it thoroughly, centrifuge at 1500rpm for 5min, aspirate the supernatant into a 1.5mL EP tube, repeat the operation 3 times, and a single bacterial suspension can be obtained. The concentration of live bacteria in the bacterial suspension was measured by a spectrophotometer at a wavelength of 600 nm. The concentration of the bacterial suspension was adjusted to 1×10 9 CFU / mL of H37Ra bacterial suspension and 1×10 9 CFU / mLRFP-H37Ra bacterial suspension, set aside.

[0077] With MOI = 10 (5 × 10 6 CFU / 5×10 5 cells) and infected THP-1 derived macrophages to establish a bacterial infection model.

[0078] The treatment method of the K67 group was as follows: 0.5 ml of THP-1 derived macrophages prepared in Example 1 was added with K67 at a final concentration of 20 μM, and then 5 μL of the above RFP-H37Ra bacterial suspension was added at an MOI of 10 (5×10 6 CFU / 5×10 5 THP-1 derived macrophages were infected with 100 μL of 5% EDTA (100 μL each) and cultured for 3 h, 6 h, 12 h and 24 h, respectively. Then, 300 μL of trypsin digestion solution containing 0.25% EDTA was added and incubated in a 37 ° C incubator for 5 min. After the incubation, 1 mL of culture medium without triple antibodies was added to each well to terminate the digestion. The digested cells were transferred to the corresponding flow tube, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 300 μL PBS. Repeat the above washing operation twice, add 300 μL PBS to each tube to resuspend the cells, use a flow cytometer to detect the FL3 positive area, and record and statistically analyze the mean fluorescence intensity (MFI). The difference between the control group and the K67 group is that the K67 solution was replaced with an equal volume of DMSO solution.

[0079] The results showed that there was no statistically significant difference in MFI between the K67 group and the control group at 3h, 6h, 12h and 24h (P>0.05, Figure 3). This indicates that K67 did not affect the phagocytic function of THP-1-derived macrophages against M.tb.

[0080] Example 4

[0081] Effect of K67 pretreatment on the killing activity of intracellular M.tb in macrophages

[0082] The K67 group was prepared by adding K67 at a final concentration of 20 μM to 0.5 ml of THP-1 derived macrophages prepared in Example 1 and culturing for 1 h, and then adding 5 μL of the RFP-H37Ra bacterial suspension prepared in Example 3 at an MOI of 10 (5×10 6 CFU / 5×10 5 THP-1 derived macrophages were infected with 100 μL of K67 (100 μL / mL) and cultured for 12 h. After culturing, the cells were washed twice with PBS to remove extracellular K67 and unphagocytosed RFP-H37Ra. The cells were lysed with 0.05% SDS solution. The lysate was diluted 10 times with ultrapure water and 100 μL was applied to an agar plate containing kanamycin (50 μg / mL). After 30 min of upright culture, the agar plate was inverted for culture. After 3 weeks, the growth of tuberculosis colonies on the agar plate was observed and photographed, and the colony count and statistical analysis were performed. The difference between the control group and the K67 group was that the K67 solution was replaced with an equal volume of DMSO solution.

[0083] The results showed that compared with the control group, the number of colonies in the K67 group was significantly reduced, and the difference was significant ( Figure 4 ), indicating that K67 can cross the cell membrane into macrophages, act on macrophages that are not infected with M.tb, regulate the immune response of macrophages to M.tb, thereby effectively killing M.tb that invade macrophages in the later stage and reducing the load of M.tb in macrophages.

[0084] Example 5

[0085] Killing activity of K67 against M.tb in infected macrophages

[0086] 5 μL of the RFP-H37Ra bacterial suspension prepared in Example 3 was added at an MOI of 10 (5×10 6 CFU / 5×10 50.5 ml of THP-1 derived macrophages prepared in Example 1 were infected with 100 μL of THP-1 macrophages (100 μL / mL) and cultured for 12 h. After culturing, the cells were washed twice with PBS to remove the unphagocytosed RFP-H37Ra outside the cells. 1640 medium (10% FBS) containing 20 μM K67 was added and cultured for 12 h. The cells were washed twice with PBS to remove the K67 outside the cells. The cells were lysed with 0.05% SDS solution. After the lysate was diluted 10 times with ultrapure water, 100 μL was aspirated and applied to an agar plate containing kanamycin (50 μg / mL). After 30 min of upright placement, the agar plate was inverted for culture. After 3 weeks, the growth of tuberculosis colonies on the agar plate was observed and photographed, and the colony count and statistical analysis were performed. The difference between the control group and the K67 group is that the K67 solution was replaced with an equal volume of DMSO solution.

[0087] The results showed that compared with the control group, the number of colonies in the K67 group was significantly reduced, and the difference was significant ( Figure 5 ), indicating that K67 can cross the cell membrane into macrophages, act on macrophages that have been infected with M.tb, change the immune response of infected macrophages to M.tb, thereby effectively killing M.tb that have invaded macrophages and reducing the load of M.tb in macrophages.

[0088] Example 6

[0089] Effects of K67 pretreatment on autophagy in macrophages

[0090] The experimental groups were set as: control group, K67 group, M.tb group and K67+M.tb group.

[0091] The K67+M.tb group was prepared by adding K67 with a final concentration of 20 μM to 0.5 ml of THP-1 derived macrophages prepared in Example 1 and culturing for 1 h, then adding 5 μL of the H37Ra bacterial suspension prepared in Example 3 at an MOI of 10 (5×10 6 CFU / 5×10 5 THP-1 derived macrophages were infected with 100 cells and cultured for 12 h, then washed twice with PBS to remove extracellular K67 and unphagocytosed H37Ra. Total cell protein was extracted, and Western blot was used to analyze the effect of K67 on the expression level of autophagy-related proteins.

[0092] Total cell protein was extracted, and the protein concentration was measured by BCA method. After boiling the protein, it was placed in a -80℃ refrigerator. According to the molecular weight of the protein, an SDS-PAGE gel of appropriate concentration was prepared, sampled, electrophoresed, wet-transferred, blocked, and placed in a 4℃ shaker to incubate the primary antibody (β-actin, p62, p-p62, LC3) overnight. After washing the membrane three times with TBST, the secondary antibody corresponding to the species of the primary antibody (1:5000) was incubated. After incubation at room temperature for 2 hours, the ECL supersensitive luminescent liquid was used for color development and the development image was collected. The grayscale value of the developed band was relatively quantitatively analyzed by Image J software;

[0093] The sources of primary antibodies are as follows: β-actinAntibody was purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd., catalog number 20536-1-AP;

[0094] SQSTM1 / p62 (D1Q5S) Rabbit mAb was purchased from Cell Signaling Techonology, catalog number #39749;

[0095] Phospho-P62 (Ser349) Antibody was purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd., catalog number 29503-1-AP;

[0096] LC3A / BAntibody was purchased from Cell Signaling Technology, catalog number #12741;

[0097] The sources of secondary antibodies are as follows: horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H+L) was purchased from Shanghai Biotime Biotechnology Co., Ltd. with the catalog number A0208.

[0098] Compared with the K67+M.tb group, THP-1-derived macrophages in the K67 group were not infected with H37Ra.

[0099] Compared with the K67+M.tb group, the K67 solution in the M.tb group was replaced with an equal volume of DMSO solution.

[0100] Compared with the K67+M.tb group, the K67 solution in the control group was replaced with an equal volume of DMSO solution, and THP-1-derived macrophages were not infected with H37Ra.

[0101] The results showed that compared with the control group, the expression of autophagy-related proteins in the K67 group alone was not significantly different (P>0.05); while the expression of autophagy-related proteins in the K67+M.tb group was significantly higher than that in the M.tb infection group alone ( Figure 6 ). This indicates that K67 can enhance the autophagy level of macrophages infected with M.tb, improve the immune response of cells against M.tb, and facilitate the clearance of intracellular M.tb.

[0102] Example 7

[0103] Effect of K67 pretreatment on ROS levels in macrophages

[0104] The experimental groups were set as: control group, K67 group, M.tb group and K67+M.tb group.

[0105] The K67+M.tb group was prepared by adding K67 with a final concentration of 20 μM to 0.5 ml of THP-1 derived macrophages prepared in Example 1 and culturing for 1 h, then adding 5 μL of the H37Ra bacterial suspension prepared in Example 3 at an MOI of 10 (5×10 6 CFU / 5×10 5 THP-1 derived macrophages were infected with 100 μL of 100 μL of EDTA-containing trypsin digestion solution, and the cells were digested into the corresponding flow tubes after PBS washing twice, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The fluorescent probe DCFH-DA (1:70000) diluted with basal 1640 was added to reach a final concentration of 0.14 μM, 1 mL per tube, and placed in a 37 ° C water bath for 20 min. The flow tube was shaken every 3-5 min to allow the cells and the probe to fully contact; after the incubation time was up, centrifuged at 1500 rpm for 5 min, and washed by centrifugation with ice PBS, the supernatant was discarded for the last time, and resuspended with 300 μL of ice PBS and placed on an ice box for flow detection; the flow detection used the FL1 channel, and the mean fluorescence intensity (MFI) was recorded. The experimental results were analyzed using FlowJo software. The fluorescent probe DCFH-DA was derived from the reactive oxygen species kit of Shanghai Biyuntian Biotechnology Co., Ltd., item number: S0033S.

[0106] Compared with the K67+M.tb group, THP-1-derived macrophages in the K67 group were not infected with H37Ra.

[0107] Compared with the K67+M.tb group, the K67 solution in the M.tb group was replaced with an equal volume of DMSO solution.

[0108] Compared with the K67+M.tb group, the K67 solution in the control group was replaced with an equal volume of DMSO solution, and THP-1-derived macrophages were not infected with H37Ra.

[0109] The results of flow cytometry detection of ROS levels showed that K67 could inhibit the production of ROS in macrophages regardless of whether they were infected with M.tb, and the difference was significant ( Figure 7 ). This indicates that K67 can reduce the oxidative stress level caused by macrophages themselves and M.tb infection to cells, and play a protective role on cells.

[0110] Example 8

[0111] Effect of K67 pretreatment on the expression level of NADPH oxidase 2 (NOX2) mRNA in macrophages

[0112] 1. THP-1 derived macrophage gene expression detection method

[0113] Cells were lysed with Trizol and total RNA was extracted using Thermo Scientific TM NanoDrop TM One micro-volume UV-Vis spectrophotometer was used to measure the total RNA concentration and purity. 2 μg RNA was quantified based on the RNA concentration, and the total RNA loading volume was calculated. The reverse transcription reaction system was as follows:

[0114] Table 1 Reverse transcription reaction system

[0115]

[0116] After adding the sample, gently mix and perform reverse transcription PCR: incubate at 50℃ for 5min; inactivate at 85℃ for 5s. RT / RI and gDNARemover, the product was diluted 5-fold and stored at -80°C for qPCR.

[0117] The primers used in this experiment were designed and synthesized by Shanghai Sangon. The specificity of the primer sequences has been verified by BLAST. The concentration of the primers after dissolution was 100 μM. They were diluted 10 times to 10 μM with enzyme-free water when used. Roche real-time fluorescence quantitative PCR instrument Light Cycler480 was used to amplify the single-stranded DNA template by qPCR. Each sample was performed in triplicate, and finally the expression of the target gene was determined by normalization using the internal reference gene as the standard.

[0118] Table 2 Primer sequences

[0119]

[0120] Table 3 qPCR system

[0121]

[0122] Table 4 qPCR instrument program settings

[0123]

[0124]

[0125] The annealing temperature in the three-step method was set according to the Tm of the primers; in addition, the melting and cooling programs used the instrument's native settings without modification.

[0126] Analytical methods:

[0127] Using LightCycler480 analysis software, the CT values ​​of the reference genes and target genes in each group were derived, and the mean CT values ​​of each group were calculated. The mean CT value of the reference gene was used to normalize the mean CT value of the target gene, that is, ΔCT = CT (mean value of the target gene) - CT (mean value of the reference gene). The ΔCT value of the target gene in the control group in the three repeated experiments was calculated. The ΔCT value of the control group was used to normalize the ΔCT value of each group, that is, ΔΔCT = ΔCT (control and treatment group) - ΔCT (control group), and finally 2 -ΔΔCT value.

[0128] The experimental groups were set as: control group, K67 group, M.tb group and K67+M.tb group.

[0129] The K67+M.tb group was prepared by adding K67 with a final concentration of 20 μM to 0.5 ml of THP-1 derived macrophages prepared in Example 1 and culturing for 1 h, then adding 5 μL of H37Ra bacterial suspension prepared in Example 3 at an MOI of 10 (5×10 6 CFU / 5×10 5 THP-1 macrophages were infected with 500 cells and cultured for 12 h. The cells were washed twice with PBS to remove extracellular K67 and unphagocytosed H37Ra. Total cellular RNA was extracted and the expression level of NOX2 mRNA was detected by RT-qPCR.

[0130] Compared with the K67+M.tb group, THP-1-derived macrophages in the K67 group were not infected with H37Ra.

[0131] Compared with the K67+M.tb group, the K67 solution in the M.tb group was replaced with an equal volume of DMSO solution.

[0132] Compared with the K67+M.tb group, the K67 solution in the control group was replaced with an equal volume of DMSO solution, and THP-1-derived macrophages were not infected with H37Ra.

[0133] The results showed that compared with the control group, there was no significant difference in the expression of NOX2 mRNA in the K67-treated group (P>0.05); M.tb infection led to an upregulation of NOX2 mRNA expression, with a significant difference (P<0.05); in the presence of K67, K67 downregulated the high expression of NOX2 mRNA caused by M.tb infection, with a significant difference (P<0.05, Figure 8 ). The above results indicate that K67 can reduce the expression level of NOX2 in macrophages and inhibit the oxidative stress response caused by M.tb infection.

[0134] Example 9

[0135] Effect of K67 pretreatment of macrophages on the expression levels of proinflammatory cytokines TNF-α, IL-6, and IL-1β. The experimental groups were set as control group, K67 group, M.tb group, and K67+M.tb group.

[0136] The K67+M.tb group was prepared by adding K67 with a final concentration of 20 μM to 0.5 ml of THP-1 derived macrophages prepared in Example 1 and culturing for 1 h, then adding 5 μL of H37Ra bacterial suspension prepared in Example 3 at an MOI of 10 (5×10 6 CFU / 5×10 5 THP-1 derived macrophages were infected with 500 cells and cultured for 12 h, then washed twice with PBS to remove extracellular K67 and unphagocytosed H37Ra, total cell RNA was extracted using the method in Example 8, and the cell culture supernatant was aspirated. The expression levels of pro-inflammatory cytokine mRNA and protein were detected by RT-qPCR and ELISA, respectively.

[0137] Human tumor necrosis factor α (TNF-α) ELISA KIT was purchased from R&D Systems, catalog number DY210-05;

[0138] Human interleukin-6 (IL-6) ELISAKIT was purchased from R&D Systems, catalog number DY206;

[0139] Human interleukin 1β (IL-1β) ELISA KIT was purchased from Jiangsu Jingmei Biotechnology Co., Ltd. with the catalog number JM-03336H1.

[0140] The operation method of ELISA method is to go through the steps of coating, blocking, washing; adding samples and standards, detection antibodies, colorimetric substrates, stop solution, etc., and finally detecting the absorbance value at 450nm by an enzyme reader, drawing a standard curve and calculating the sample concentration.

[0141] Compared with the K67+M.tb group, THP-1-derived macrophages in the K67 group were not infected with H37Ra.

[0142] Compared with the K67+M.tb group, the K67 solution in the M.tb group was replaced with an equal volume of DMSO solution.

[0143] Compared with the K67+M.tb group, the K67 solution in the control group was replaced with an equal volume of DMSO solution, and THP-1-derived macrophages were not infected with H37Ra.

[0144] The results showed that compared with the control group, there was no significant difference in the mRNA and protein expression levels of TNF-α, IL-6 and IL-1β in the K67 treatment group alone (P>0.05), while M.tb infection led to an increase in the mRNA and protein expression levels of TNF-α, IL-6 and IL-1β, with a significant difference (P<0.001). In the presence of K67, K67 downregulated the high expression of mRNA and protein of various proinflammatory cytokines caused by M.tb infection, with a significant difference ( Fig. 9 ). This suggests that K67 can inhibit the high inflammatory state caused by M.tb infection and protect cells from damage by oxidative stress.

[0145] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of compound K67 in the preparation of anti-tuberculosis drugs, wherein the Chinese name of K67 is 2-acetyl-1,4-di[(4-methoxyphenylsulfonyl)amino]naphthalene, and the structural formula is shown in Formula I: Formula I.

2. The use according to claim 1, characterized in that: The anti-tuberculosis includes inhibiting the proliferation of Mycobacterium tuberculosis in the body.

3. The use according to claim 1 or 2, characterized in that: The compound K67 regulates at least one of the following reactions: increasing the autophagy level of macrophages, reducing the oxidative stress level of macrophages, and inhibiting the inflammatory response in macrophages; The macrophages are macrophages infected with Mycobacterium tuberculosis.

4. The use according to claim 3, characterized in that: The method of increasing the autophagy level of macrophages includes promoting the expression of autophagy-related proteins and / or increasing the ratio of LC3Ⅱ / LC3Ⅰ.

5. The application according to claim 3, characterized in that: The reducing the oxidative stress level of macrophages includes reducing the level of reactive oxygen species in macrophages and / or reducing the expression level of NADPH oxidase 2 in macrophages.

6. The use according to claim 3, characterized in that: The inhibiting of inflammatory response in macrophages includes inhibiting the expression of genes and / or proteins of pro-inflammatory factors in macrophages.

7. The use according to claim 6, characterized in that: The pro-inflammatory factors include at least one of the following: tumor necrosis factor-α, interleukin-6 and interleukin-1β.

8. Use of compound K67 in the preparation of an antibacterial agent for inhibiting mycobacteria in vitro, wherein the Chinese name of the compound K67 is 2-acetyl-1,4-di[(4-methoxyphenylsulfonyl)amino]naphthalene, and the structural formula is shown in Formula I: Formula I; The mycobacterium is Mycobacterium tuberculosis.

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