Application of piperlongumine in enhancing inhibition of intracellular mycobacterium tuberculosis by isoniazide

Abstract

The invention relates to a novel application of piperlongumine in enhancing inhibition of isoniazide on intracellular mycobacterium tuberculosis, piperlongumine can specifically improve the level of ROS (reactive oxygen species) in macrophages infected by mycobacterium tuberculosis, and can enhance autophagy of the macrophages by improving the level of ROS, so that the aim of resisting mycobacterium tuberculosis is fulfilled; piperlongumine combined medication can significantly enhance the inhibition of isoniazid on the growth of mycobacterium tuberculosis in macrophages.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to use of piperlongum amide in enhancing the inhibition of intracellular mycobacterium tuberculosis by isoniazid. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by infection with Mycobacterium tuberculosis (Mtb) and primarily transmitted through respiratory droplets. Isoniazid (INH), currently one of the first-line drugs for TB treatment, is suitable for both the clinical treatment of active TB and the effective elimination of latent TB infection. Its minimum inhibitory concentration (MIC) against TB is 0.01-0.2 μg / ml (0.0729-1.458 μM). INH's anti-TB mechanism of action is to inhibit the biosynthesis of mycolic acids, achieving a bactericidal effect by disrupting the integrity of the bacterial cell wall. It is particularly effective against TB during its growth and reproduction phase, exhibiting high bactericidal activity and a favorable safety profile. However, due to long-term antimycobacterial treatment, the detection rate of isoniazid-resistant Mycobacterium tuberculosis continues to rise, posing a significant challenge to the clinical treatment of TB. Developing new anti-tuberculosis drugs or constructing a synergistic drug delivery system to reduce the clinical dosage of isoniazid has become an urgent need in the current field of tuberculosis prevention and control.

[0003] When Mycobacterium tuberculosis invades the host, it triggers activation of the NADPH oxidase complex in macrophages, leading to a significant increase in reactive oxygen species (ROS) levels. This oxidative stress not only causes molecular damage such as bacterial DNA strand breaks, protein denaturation, and lipid peroxidation, but also disrupts bacterial energy metabolism and activates autophagy-related signaling pathways (such as the mTOR-ULK1 pathway) to eliminate intracellular pathogens. However, Mycobacterium tuberculosis has evolved multiple immune evasion mechanisms. These include secreting virulence factors such as ESAT-6 to inhibit lysosome-phagosome fusion and block macrophage maturation and differentiation; utilizing cell wall components such as tuberculin-dimycolic acid (PDIM) to resist the host's innate immune response; and promoting granuloma formation by regulating the necroptosis pathway. Autophagy is a key immune mechanism for controlling intracellular infection in mammals, but M. tb can inhibit this process. Studies have shown that some compounds with anti-TB activity can also eliminate Mycobacterium tuberculosis by increasing endogenous ROS levels in infected cells and activating pathways such as autophagy.

[0004] Piperlongumine (PL) is an alkaloid isolated from the medicinal plant Piperlongum L. of the Piperaceae family. Its molecular structure features a characteristic α,β-unsaturated enone functional group and an amide bond. This unique electron conjugation system endows it with significant electrophilic reactivity, enabling it to undergo Michael addition reactions with sulfhydryl groups in biomacromolecules. Studies have shown that PL exhibits multiple effects, including anti-inflammatory, neuroprotective, and hypoglycemic properties. In recent years, it has become a hot topic in anticancer drug development due to its selective ability to induce tumor cell apoptosis. Regarding its antitumor mechanism of action, it has demonstrated potent antiproliferative effects in various tumor models, including breast, lung, and colon cancer, by depleting reduced glutathione (GSH) and inhibiting thioredoxin reductase (TrxR) activity, inducing ROS accumulation, inhibiting the NF-κB signaling pathway, and inducing cell apoptosis through irreversible oxidative stress. In the prior art, the detection rate of isoniazid-resistant Mycobacterium tuberculosis has been improved, and there has been no evidence that piperlongum amide combined with isoniazid can inhibit intracellular Mycobacterium tuberculosis. Based on this, the present application is proposed. Summary of the Invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and propose a new use of piperlongum amide, which relates to the use of piperlongum amide to enhance the inhibition of intracellular Mycobacterium tuberculosis by isoniazid.

[0006] The present invention provides the following technical solutions:

[0007] The present invention provides the use of piperlongum amide in enhancing the inhibition of intracellular mycobacterium tuberculosis by isoniazid.

[0008] Piperlongamin inhibits Mycobacterium tuberculosis by enhancing the autophagy level of Mycobacterium tuberculosis-infected macrophages after isoniazid treatment.

[0009] Furthermore, the concentration of piperlongum amide is 0.2 μM-1 μM; the concentration of isoniazid is 0.05-1.25 μg / mL.

[0010] Furthermore, the concentration of piperlongumamide was 0.5 μM.

[0011] Application of piperlongum amide combined with isoniazid in the preparation of medicines for treating tuberculosis.

[0012] The medicine contains therapeutically effective amounts of piperlongum amide, isoniazid and pharmaceutically acceptable carriers and / or excipients.

[0013] The present invention has the following beneficial effects:

[0014] Piper longum can specifically increase the level of ROS in macrophages infected with Mycobacterium tuberculosis, and can enhance macrophage autophagy by increasing the level of ROS, thereby achieving the purpose of anti-Mycobacterium tuberculosis; Piper longum has the effect of significantly enhancing the inhibitory effect of isoniazid on the growth of Mycobacterium tuberculosis in macrophages. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0016] Figure 1 This is a graph showing the effect of piperonyl longum (PL) on the viability of RAW264.7 cells;

[0017] Figure 2 This is a graph representing the effect of the combined use of piperlongum amide (PL) and isoniazid (INH) on the mean fluorescence intensity (MFI) of Mycobacterium tuberculosis in macrophages;

[0018] Figure 3 This is a graph representing the effect of 0.5 μM piperlongum amide (PL) combined with isoniazid (INH) on the proportion of GFP-positive (GFP+) cells;

[0019] Figure 4 This is a graph showing the effect of 0.5 μM piperlongum amide (PL) combined with isoniazid (INH) on GFP fluorescence intensity;

[0020] Figure 5 Figure 1 shows the effect of combined use of piperlongum amide (PL) and INH on the intracellular survival rate of Mycobacterium tuberculosis, including (A) CFU statistics after combined use of PL and INH; (B) bacterial survival statistics after combined use of PL and INH; (C) dilution plate results after combined use of PL and INH;

[0021] Figure 6 Figure 1 represents the effect of piperlongum amide (PL) on ROS levels in macrophages infected with Mycobacterium tuberculosis; (A) The effect of PL treatment at different times on ROS levels; (B) The effect of PL treatment for 6 days on ROS levels in infected and uninfected cells; (C) The result of flow cytometry detection of the percentage of ROS+ cells;

[0022] Figure 7Figure 3 represents the effect of combined use of piperlongum amide (PL) and isoniazid (INH) on LC3B protein expression; (A) original WB protein band image, (B) LC3BⅠ / LC3BⅡ protein expression ratio by quantitative analysis. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] The present invention provides the use of piperlongum amide in enhancing the inhibition of intracellular mycobacterium tuberculosis by isoniazid.

[0025] The molecular formula of piperlongum amide is C17H19NO5, with a relative molecular weight of 317.34. It exists in cis and trans isomers, with the trans structure being its active form. Its molecular structure contains characteristic α,β-unsaturated enone functional groups and amide bonds. Piperlongum amide has multiple effects such as anti-inflammatory, neuroprotective, and hypoglycemic, and is commonly used for anti-tumor effects.

[0026] The present invention finds that piperlongumide can specifically increase the level of ROS in macrophages infected with Mycobacterium tuberculosis, and can enhance macrophage autophagy by increasing the level of ROS, thereby achieving the purpose of anti-Mycobacterium tuberculosis; a new use of piperlongumide to enhance the inhibition of intracellular Mycobacterium tuberculosis by isoniazid is now proposed.

[0027] The present invention will be further described below by the following specific embodiments:

[0028] 1. Minimum inhibitory concentration of PL against different standard strains and clinical isolates

[0029] The microdilution method was used to determine the minimum inhibitory concentration (MIC) of PL against the test strains. PL was prepared as a stock solution and serially diluted twofold with 7H9 broth (supplemented with OADC enrichment solution). 100 μL was added to each well. The H37Ra bacterial solution in the logarithmic growth phase was diluted to 1×10 6After measuring the CFU / mL, add 100 μL to each well. After incubation for 5 days, add 10 μL of Lamar blue indicator (10%) to the positive control well and incubate at 37°C for 24 hours. If the positive control well changes from blue to pink, add 10 μL of Lamar blue indicator (10%) to the other test wells and incubate at 37°C for 24 hours to record the results. The minimum inhibitory concentration (MIC) is the lowest drug concentration that prevents the blue well from changing to pink.

[0030] PL was prepared into a stock solution and serially diluted twice with MH medium. 100 μL was added to each well. The bacterial solutions of Enterococcus faecalis ATCC29212, Salmonella typhimurium CVCC541, Streptococcus pneumoniae ATCC49619, Salmonella enteritidis CICC21527, Klebsiella pneumoniae ATCC13883, Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213, MRSAB4, MRSAB5, MRSAP2, MRSAP3, MRSAP4, and MRSAP5 in the logarithmic growth phase were diluted to 1×10 6 CFU / mL, 100 μL was added to each well, and the minimum inhibitory concentration was recorded after incubation for 1 day.

[0031] The results in Table 1 show that the MIC value of PL against H37Ra was 32 μg / mL, and the MIC values against the other tested strains were greater than or equal to 64 μg / mL. Therefore, Mycobacterium tuberculosis was selected for subsequent testing.

[0032] Table 1 MIC statistics of piperlongum amide (PL) against different strains

[0033]

[0034]

[0035] 2. Extracellular inhibitory effect of combined drug therapy on Mycobacterium tuberculosis

[0036] PL, INH, BDQ, DEA, PZA, and CFZ were prepared into stock solutions and serially diluted twice with 7H9 broth (with OADC enrichment solution added). 100 μL was added to each well. The H37Ra bacterial solution in the logarithmic growth phase was diluted to 1×10 6 After 100 μL of culture medium was added to each well, the MIC value was recorded after 7 days of incubation. The Fractional Inhibitory Concentration Index (FICI) of PL in combination with other drugs was then determined in a 96-well plate using the checkerboard method: FICI = (MIC A combined / MIC A alone) + (MIC B combined / MIC B alone).

[0037] The results showed that the MIC value of PL was 32 μg / mL, and the MIC values of INH, BDQ, DEA, PZA, and CFZ were 0.05, 0.06, 4, 40, and 0.5 μg / mL, respectively. The average FICI value was greater than 0.5, indicating that PL had no synergistic antibacterial effect when used in combination with the above drugs in extracellular space.

[0038] 3. Intracellular inhibitory effect of combined drug therapy on Mycobacterium tuberculosis

[0039] (1) Effect of PL on the survival rate of RAW264.7 cells

[0040] According to 2×10 4 RAW264.7 cells were seeded at a density of 100 μL / well in a 96-well cell culture plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. After removing the culture medium, 100 μL of PL solution was added to each well at a gradient concentration of 0, 1, 2, 4, 8, 16, and 32 μM. On days 2 and 4 after administration, the culture medium was removed and the drug was administered again using the same concentration gradient. On day 6, the culture medium was removed, and 10 μL of CCK-8 solution was added to each well and incubated for 2 hours. The absorbance of the cells at a wavelength of 450 nm was measured using a microplate reader, and the relative cell viability in each well was calculated: cell viability = [A(treated group) - A(blank)] / [A(control group) - A(blank)].

[0041] according to Figure 1 The results showed that PL had no cytotoxicity against RAW264.7 cells at concentrations less than or equal to 2 μM. However, at concentrations greater than 2 μM, cell viability gradually decreased. The IC50 value of PL in RAW264.7 cells was 9.282 μM. Therefore, concentrations less than or equal to 2 μM were selected for subsequent experiments.

[0042] (2) Intracellular antibacterial effect of PL combined with metabolic inhibitors targeting host cells

[0043] RAW264.7 cells cultured to the logarithmic growth phase were cultured at 2×10 5 The cells were seeded at a density of 100 cells / mL in 12-well plates and incubated for 24 hours. H37Ra-GFP cells in the logarithmic growth phase were resuspended in culture medium and added to the 12-well plates at a multiplicity of infection (MOI) of 5:1. The cells were incubated for 4 hours. The bacterial suspension was removed and incubated with 50 ng / mL amikacin solution for 1 hour. The cells were administered on days 0, 2, and 4 after infection. The cells were harvested on day 6, and GFP fluorescence intensity was measured by flow cytometry and observed under a fluorescence microscope.

[0044] Drug concentration setting:

[0045] ①3-BP: set up Mock group, Control group, 1μM PL group, 0.5μM PL group, 0.2μM PL group, 50μM 3-BP group, 5μM 3-BP group, 1μM PL + 50μM 3-BP group, 1μM PL + 5μM 3-BP H group, 0.5μM PL + 50μM 3-BP group, 0.5μM PL + 5μM 3-BP group, 0.2μM PL + 50μM3-BP group, 0.2μM PL + 5μM 3-BP group;

[0046] ②RAPA: set up Mock group, Control group, 1μM PL group, 0.5μM PL group, 0.2μM PL group, 0.05μM RAPA group, 0.01μM RAPA group, 1μM PL + 0.05μM RAPA group, 1μMPL + 0.01μM RAPA group, 0.5μM PL + 0.05μM RAPA group, 0.5μM PL + 0.01μM RAPA group, 0.2μM PL + 0.05μM RAPA group, 0.2μM PL + 0.01μM RAPA group;

[0047] ③MET: set up Mock group, Control group, 1μM PL group, 0.5μM PL group, 0.2μM PL group, 100μM MET group, 10μM MET group, 1μM PL + 100μM MET group, 1μM PL + 10μM MET group, 0.5μM PL + 100μM MET group, 0.5μMPL + 10μM MET, 0.2μM PL + 100μM MET group, 0.2μM PL + 10μM MET group;

[0048] ④ORL: Mock group, Control group, 1μM PL group, 0.5μM PL group, 0.2μM PL group, 50μM ORL group, 5μM ORL group, 1μM PL+50μM ORL group, 1μM PL+5μM ORL group, 0.5μM PL+50μM ORL group, 0.5μM PL+5μM ORL group, 0.2μM PL+50μM ORL group, and 0.2μM PL+5μM ORL group were set.

[0049] The results showed that 0.2μM, 0.5μM, and 1μM PL could not significantly reduce the MFI value and GFP+ cell ratio after the action of 3-BP, RAPA, MET, and ORL.

[0050] (3) Intracellular antibacterial effect of PL combined with metabolic inhibitors targeting pathogens

[0051] The method is the same as (2)

[0052] Drug concentration setting:

[0053] ① INH: Mock group, Control group, 1μM PL group, 0.5μM PL group, 0.2μM PL group, 0.25μg / mL INH group, 0.05μg / mL INH group, 1μM PL + 0.25μg / mL INH group, 1μMPL + 0.05μg / mL INH group, 0.5μM PL + 0.25μg / mL INH group, 0.5μM PL + 0.05μg / mL INH group, 0.2μM PL + 0.25μg / mL INH group, 0.2μM PL + 0.05μg / mL INH group;

[0054] ②ETH: set up Mock group, Control group, 1μM PL group, 0.5μM PL group, 0.2μM PL group, 0.5μM ETH group, 0.1μM ETH group, 1μM PL + 0.5μM ETH group, 1μM PL + 0.1μM ETH group, 0.5μM PL + 0.5μM ETH group, 0.5μM PL + 0.1μM ETH group, 0.2μM PL + 0.5μM ETH group, 0.2μM PL + 0.1μM ETH group;

[0055] ③BDQ: Mock group, Control group, 1μM PL group, 0.5μM PL group, 0.2μM PL group, 4μg / mL BDQ group, 0.4μg / mL BDQ group, 1μM PL+4μg / mL BDQ group, 1μM PL+0.4μg / mL BDQ group, 0.5μM PL+4μg / mL BDQ group, 0.5μM PL+0.4μg / mL BDQ group, 0.2μM PL+4μg / mL BDQ group, and 0.2μM PL+0.4μg / mL BDQ group were set.

[0056] The results showed that 0.2μM, 0.5μM, and 1μM PL could not significantly reduce the MFI value and GFP+ cell ratio after the action of ETH and BDQ. Figure 2 、 3 The results showed that 0.2μM, 0.5μM, and 1μM PL had no significant effect on the MFI and GFP+ cell ratio of Mycobacterium tuberculosis in macrophages when used alone. When used in combination with 1.25μg / mL, 0.25μg / mL, and 0.05μg / mL INH, the MFI and GFP+ cell ratio of INH when used alone were significantly reduced. Figure 4 It was shown that the fluorescence intensity was weakened when 0.5 μM PL was combined with INH.

[0057] The dilution plate method was then used to further verify the effect of 0.5 μM PL combined with INH on the intracellular survival rate of H37Rv. On the 6th day, the supernatant was discarded and washed three times with PBS, lysed with 0.1% Tween 80 for 10 minutes, and diluted to 10 with 7H9 medium. -1 , 10 -2 , 10 -3 times, 60uL of each was spread on 7H10 plates, and colonies were counted after culturing for 21 days.

[0058] according to Figure 5 The results showed that the intracellular survival rates of Mycobacterium tuberculosis were 10.17±0.17%, 14.13±0.52%, and 43.83±1.66% when 1.25μg / mL, 0.25μg / mL, and 0.05μg / mL INH were used alone, respectively. After combined use with 0.5μM PL, the survival rates decreased to 2.87±0.26%, 5.69±0.34%, and 14.13±0.52%, respectively. When 0.5μM PL was used alone, the survival rate was 107.52±11.73%, which showed no significant change compared with the Control group.

[0059] The above results indicate that PL can serve as an antibacterial synergist of INH, and 0.2μM-1μM PL enhances the anti-tuberculosis effect of INH in macrophages.

[0060] 4. PL increases ROS levels in macrophages infected with Mycobacterium tuberculosis

[0061] (1) A H37Ra-infected RAW264.7 cell model was established. Control, 0.2 μM PL, 0.5 μM PL, and 1 μM PL groups were set up. Different concentrations of PL were added on days 0, 2, and 4 after infection, respectively. DCFH-DA probes were incubated at 1 h, 6 h, 12 h, 1 day, 3 days, and 6 days after administration, and fluorescence intensity was measured using a multifunctional microplate reader.

[0062] (2) H37Ra-infected RAW264.7 cell models and uninfected models were constructed. A negative control (DMSO), a positive control (from the Biyuntian ROS detection kit), and a 0.5 μM PL group were administered on days 0, 2, and 4 after infection, respectively. The DCFH-DA probe was incubated on day 6 after infection, and the mean fluorescence intensity was measured using flow cytometry.

[0063] according to Figure 6 Results A showed that after the action of 0.2μM-1μM PL, the intracellular ROS levels increased significantly at different time points. Figure 6Results (B and C) show that after treatment with 0.5 μM PL, ROS levels in H37Ra-infected cells increased significantly on day 6, consistent with the results detected by a multifunctional microplate reader. In uninfected cells, ROS levels did not change significantly after treatment with 0.5 μM PL, indicating that PL specifically increases ROS levels in M. tuberculosis-infected macrophages.

[0064] 4. PL enhances autophagy in macrophages infected with Mycobacterium tuberculosis

[0065] (1) Total protein extraction: A H37Ra-GFP-infected RAW264.7 cell model was constructed, and the control group, 5 mM NAC + 0.5 μM PL + 0.25 μg / mL INH group, 0.25 μg / mL INH group, 0.5 μM PL group, and 0.5 μM PL + 0.25 μg / mL INH group were set up. On the 6th day, RIPA lysis buffer was added to each well and lysed on ice for 20 min. The lysate was collected and ultrasonically disrupted on ice. The supernatant was collected by centrifugation at 12,000 g for 10 min at 4°C, and protein quantification was performed according to the instructions of the BCA protein quantification kit.

[0066] (2) SDS-PAGE electrophoresis: Based on the results of BCA quantification, adjust the concentration of each protein sample with 5× SDS-PAGE protein loading buffer. Boil in water for 10 minutes, then cool on ice for 5 minutes. Load 20 μg / lane of the sample onto the prepared gel. Set the electrophoresis voltage to 80 V. When the bromophenol blue indicator band runs through the stacking gel, adjust the voltage to 120 V.

[0067] (3) Western blot hybridization transfer: Cut the gel of appropriate size near the target band, use 0.45 μm PVDF membrane, and transfer it by electroporation at 300 mA on ice for 90 min.

[0068] (4) Immunoreaction: The PVDF membrane was blocked in 5% skim milk for 1 h, washed 3 times with TBST, incubated with primary antibody at 4°C overnight, washed 3 times with TBST, incubated with secondary antibody at room temperature for 1.5 h, and washed 3 times with TBST.

[0069] (5) Chemiluminescence: Make sure the membrane is in full contact with the color developing solution, place it on a chemiluminescence imager, set the exposure gradient, and adjust the exposure time to achieve the best exposure effect.

[0070] according to Figure 7Results showed that when PL was used in combination with INH, the ratio of LC3BⅠ to LC3BⅡ protein expression was significantly lower than when either drug was used alone, indicating that PL can enhance autophagy in macrophages infected with Mycobacterium tuberculosis after INH treatment. Autophagy levels were significantly increased after treatment with the ROS scavenger NAC, and PL specifically increased ROS levels in macrophages infected with Mycobacterium tuberculosis, suggesting that PL can promote autophagy by increasing ROS levels, thereby inhibiting the growth of Mycobacterium tuberculosis.

[0071] The above experiments show that piperlongamide can specifically increase the level of ROS in macrophages infected with Mycobacterium tuberculosis, and can enhance macrophage autophagy by increasing the level of ROS, thereby achieving the purpose of anti-Mycobacterium tuberculosis; therefore, piperlongamide has the effect of significantly enhancing the effect of isoniazid in inhibiting the growth of Mycobacterium tuberculosis in macrophages, and proposes a new use of piperlongamide to enhance isoniazid's inhibition of intracellular Mycobacterium tuberculosis, as well as the use of piperlongamide combined with isoniazid in the preparation of drugs for the treatment of tuberculosis.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of piperlongum amide to enhance the inhibitory effect of isoniazid on intracellular Mycobacterium tuberculosis.

2. The use according to claim 1, characterized in that: Piperlongamin inhibits Mycobacterium tuberculosis by enhancing the autophagy level of Mycobacterium tuberculosis-infected macrophages after isoniazid treatment.

3. The use according to claim 1, characterized in that: The concentration of piperlongum amide is 0.2 μM to 1 μM; the concentration of isoniazid is 0.05 to 1.25 μg / mL.

4. The use according to claim 3, characterized in that: The concentration of piperlongumamide was 0.5 μM.

5. The application of piperlongum amide combined with isoniazid in the preparation of drugs for the treatment of tuberculosis.

6. The use according to claim 5, characterized in that: The medicine contains therapeutically effective amounts of piperlongum amide, isoniazid and pharmaceutically acceptable carriers and / or excipients.

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