Application of punicalagin in preparation of medicine for treating mycobacterium abscessus infection

Abstract

The invention relates to the field of medicines, and discloses application of punicalagin in preparation of a medicine for treating mycobacterium abscessus infection. The punicalagin has a unique related function of promoting the immune function and antibacterial activity of macrophages on the mycobacterium abscessus in the infected macrophages, so that the punicalagin can be used for treating mycobacterium abscessus infection which is difficult to treat at present, and the possibility of relapse is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to the use of punicalagins that regulate autophagy based on the SIRT1 / FOXO3a axis in preparing a drug for treating Mycobacterium abscessus infection. Background Art

[0002] Mycobacterium abscessus (MAB), a rapidly growing nontuberculous mycobacterium (NTM), has seen a significant increase in global infection rates in recent years. This pathogen poses a serious threat to immunocompromised patients and those with chronic respiratory diseases such as cystic fibrosis, chronic obstructive pulmonary disease (COPD), and bronchiectasis. The 5-, 10-, and 15-year cumulative mortality rates for MAB-related lung infections are as high as 11%, 30%, and 50%, respectively.

[0003] Currently, the treatment of Mycobacterium abscessus faces many severe challenges. On the one hand, the bacteria are multidrug-resistant and are generally resistant to β-lactams, aminoglycosides, and traditional anti-tuberculosis drugs. The resistance spectrum of different subspecies varies significantly. Clinical treatment relies on precise drug sensitivity testing to guide medication, but the testing cycle is long, which often leads to treatment delays. On the other hand, existing core therapeutic drugs, such as clarithromycin and amikacin, have severe hepatotoxicity and kidney toxicity. Long-term combination therapy makes patients poorly tolerated and has a high treatment discontinuation rate. In addition, the biofilm-forming ability of Mycobacterium abscessus further increases the difficulty of treatment, and new therapies such as phage therapy and photodynamic therapy are still in the experimental stage and lack a stable evaluation system. The lag in vaccine development has also led to a lack of preventive measures. Therefore, the development of new targeted drugs is urgent.

[0004] In recent years, host-directed therapy (HDT) has provided a novel approach for the treatment of drug-resistant bacterial infections. HDT modulates host immune mechanisms, such as enhancing phagocytosis, inducing autophagy, and suppressing excessive inflammation, to aid in the clearance of intracellular pathogens. Natural products have garnered significant attention in HDT due to their multi-target regulatory properties. Summary of the Invention

[0005] To address the technical issues of poor efficacy of existing treatments for Mycobacterium abscessus infection, drug resistance, toxic side effects, and other issues, the present invention provides the use of compounds such as punicalagin (the main active ingredient of pomegranate) that regulate autophagy through the SIRT1 / FOXO3a axis signaling pathway in the preparation of drugs for treating Mycobacterium abscessus infection. Punicalagin can effectively inhibit the growth of Mycobacterium abscessus in macrophages by enhancing autophagy and stabilizing mitochondrial membrane structure and function, rather than directly having an antibacterial effect. This is expected to improve the therapeutic effect of Mycobacterium abscessus infection and enhance the therapeutic effect of Mycobacterium abscessus infection by enhancing the clearance ability of macrophages against Mycobacterium abscessus.

[0006] The specific technical solutions of the present invention are:

[0007] The first aspect: providing the use of punicalagin in the preparation of a drug for treating Mycobacterium abscessus infection.

[0008] Furthermore, the therapeutic drug is used to enhance the ability of macrophages to resist Mycobacterium abscessus infection.

[0009] Furthermore, the Mycobacterium abscessus infection is a Mycobacterium abscessus infection of the lungs.

[0010] The second aspect: providing the use of punicalagin in the preparation of a drug for enhancing the ability of macrophages to resist Mycobacterium abscessus infection.

[0011] The third aspect: providing the use of punicalagin in the preparation of drugs that enhance macrophage autophagy. Punicalagin enhances mitochondrial stability by upregulating SIRT1, activates the SIRT1 / FOXO3a axis, promotes autophagy, and inhibits the PI3K / Akt / mTOR signaling pathway, thereby effectively inhibiting the growth of Mycobacterium abscessus in macrophages. In in vivo experiments on mice, treatment with punicalagin significantly reduced the bacterial load in the lungs, alleviated inflammatory cell infiltration, enhanced macrophage autophagy, and especially significantly promoted the clearance of Mycobacterium abscessus infection by interstitial macrophages in the lungs of infected mice. In addition, punicalagin has an inhibitory effect on Mycobacterium abscessus in different states, and can regulate the cell signaling pathways and autophagy-related gene expressions related to Mycobacterium abscessus infection.

[0012] Fourthly, the present invention provides the use of punicalagin in the preparation of a regulator of cell signaling pathways associated with Mycobacterium abscessus infection. Punicalagin can inhibit the activation of the PI3K / Akt / mTOR signaling pathway and the MAPK / ERK signaling pathway, while also inhibiting the activity of the STAT3 signaling pathway, providing a new means for regulating cell signaling pathways associated with Mycobacterium abscessus infection.

[0013] It can be expected that any compound similar to punicalagin that can regulate autophagy based on the SIRT1 / FOXO3a axis is expected to be used in the preparation of drugs for the treatment of Mycobacterium abscessus infection. It can exert related functions by upregulating SIRT1, activating the SIRT1 / FOXO3a axis, and promoting macrophage autophagy.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] Highly effective anti-infection: Punicalagin effectively eliminates intracellular Mycobacterium abscessus by enhancing host autophagy and mitochondrial protection, significantly reducing the bacterial load in the lungs, and reducing the CFU of Mycobacterium abscessus in host interstitial macrophages by >50%.

[0016] Low risk of drug resistance: Punicalagin does not directly kill bacteria, but targets host immune regulation, thus avoiding the extensive drug resistance of Mycobacterium abscessus.

[0017] Anti-inflammation and cell protection: inhibit STAT3 and Akt / mTOR pathways, reduce inflammatory cell infiltration and mitochondrial damage, and improve lung pathological damage.

[0018] Precise targeting: The autophagy activation effect on interstitial macrophages is significantly better than that on alveolar macrophages, improving the specificity of treatment.

[0019] Advantages of natural sources: Based on pomegranate extract, it has high biosafety and can be developed into oral or injectable preparations, suitable for long-term treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 2 shows the effect of punicalagin treatment on MAB-infected macrophages. (A) shows bacterial growth curves in 7H9 culture medium, showing no significant difference after the addition of punicalagin. (B) shows the fluorescence intensity of intracellular Mycobacterium abscessus-green fluorescent protein (MAB-GFP), indicating increased killing ability with increasing punicalagin concentration. (C) shows that punicalagin treatment reduces cell apoptosis. (D) shows that punicalagin reduces mitochondrial damage caused by Mycobacterium abscessus. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns indicates no significant difference.) In the figure, NC indicates uninfected macrophages, MAB indicates infected macrophages, and PUN / PU indicates punicalagin treatment.

[0021] Figure 2This is a verification of the role of punicalagin in specifically regulating the survival of intracellular mycobacteria through SIRT1 expression, where: A shows that punicalagin can promote the protein expression of cellular SIRT1 and SIRT3; B shows that the target of punicalagin is not SIRT3, as demonstrated by the use of 3-TYP (SIRT3 inhibitor); C shows that the target of punicalagin is SIRT1, as demonstrated by the use of ex-527 (SIRT1 inhibitor); D shows that ex-527 inhibited the function of punicalagin in inhibiting intracellular Mycobacterium abscessus; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns indicates no significant difference.

[0022] Figure 3 The results of validation of the key functions of SIRT1 are shown, among which: A indicates that punicalagin treatment failed after si-SIRT1 knockout; B indicates that the promoting effect of punicalagin on autophagy was inhibited after si-SIRT1 knockout, and the expression of function-related proteins was changed; C indicates that si-SIRT1 reversed the function of punicalagin to stabilize the mitochondrial membrane after treatment; D indicates that si-FOXO3 inhibited the function of punicalagin to stabilize the mitochondrial membrane; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns indicates no significant difference.

[0023] Figure 4 Figure 3 The therapeutic effect of punicalagin in the mouse lung MAB infection model, where: A indicates that lung damage in mice was alleviated after punicalagin treatment; B indicates that Mycobacterium pulmonary abscess infection in mice was alleviated after punicalagin treatment; C indicates that autophagy-related indicators in the mouse lung were restored after punicalagin treatment; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns indicates no significant difference.

[0024] Figure 5 The following are the effects of punicalagin treatment on different macrophages and transcriptome sequencing results, among which: A shows the GFP infection of mouse interstitial macrophages after punicalagin treatment, which was significantly alleviated after treatment; B shows the GFP infection of mouse alveolar macrophages after punicalagin treatment, which showed certain differences after treatment; C shows that transcriptome sequencing of mice after punicalagin treatment found that autophagy-related signaling pathways were enhanced; D shows that transcriptome sequencing of mice after punicalagin treatment found that autophagosome formation function was improved; E shows that transcriptome sequencing of mice after punicalagin treatment found that changes in lysosomal function and autophagy-related modules were mainly in interstitial macrophages with higher enrichment indexes; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns indicates no significant difference. Specific implementation plan

[0025] The present invention is described below by way of specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, any changes and advantages that can be imagined by those skilled in the art are included in the present invention, and the scope of protection of the present invention is defined by the appended claims and any equivalents thereof.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this disclosure pertains. Unless otherwise specified, the raw materials and equipment used herein are conventional in the art and can be obtained from conventional commercial sources. The methods used herein are conventional in the art, unless otherwise specified.

[0027] Cell culture:

[0028] The THP-1 cell line was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in RPMI1640 medium supplemented with 1% penicillin-streptomycin. All cells were cultured at 37°C in a 5% CO2 environment. When conducting mycobacterial infection experiments, the culture medium did not contain antibiotics to avoid affecting the viability of the pathogen. THP-1 monocytes were treated with 20 ng / mL Phorbol 12-myristate 13-acetate (PMA) for 16 hours to induce their differentiation into macrophages. In the examples of the present application, THP-1 cells differentiated by PMA are referred to as THP-1 macrophages.

[0029] Bacterial culture:

[0030] Mycobacterium abscessus ATCC 19977 standard strain was purchased from the American Type Culture Collection (ATCC) and cultured with shaking at 37°C for 3 days until stationary phase in Middlebrook 7H9 broth supplemented with 0.05% Tween-80, 0.2% glycerol, and 10% OADC enrichment. The culture was harvested, resuspended in phosphate-buffered saline (PBS) containing 0.05% Tween-20 and 25% glycerol, and then stored at -80°C. Colony-forming units (CFUs) per milliliter were quantified from thawed storage tubes.

[0031] Drug or inhibitor preparation:

[0032] Punicalagin (Aladdin, P117962), 3-TYP (MedChemExpress, HY-108331, 40 μM), and chloroquine (MedChemExpress, HY-17589A, 100 μM) were dissolved in dimethyl sulfoxide (DMSO), while ex-527 (MedChemExpress, HY-15452, 10 μM) and bafilomycin A1 (MedChemExpress, HY-100558, 20 μM) were dissolved in double-distilled water. All solutions were aliquoted and stored at -20°C or -80°C.

[0033] Apoptosis detection:

[0034] Cell apoptosis was assessed using the Annexin V-FITC / PI apoptosis kit (Lianke Biotechnology, AT101). 5 μL of FITC Annexin V and 10 μL of PI were added to THP-1 macrophages and stained for 5 minutes. Apoptosis rates were analyzed using a FACSCanto II flow cytometry system (BD Biosciences, USA).

[0035] MAB infection of THP-1 macrophages and intracellular survival assay:

[0036] THP-1 macrophages were cultured at 5 × 10 5 The cells were seeded in 12-well plates at a density of 10 cells and pretreated with MAB at a multiplicity of infection (MOI) of 10. After incubation for 3 hours, the infected cells were replaced with a culture medium, washed with 50 μg / mL amikacin to remove the MAB in the supernatant, replaced with fresh culture medium and co-cultured with punicalagin (40 μM) for 24 hours. After incubation, the cells were washed three times with preheated sterile PBS to remove extracellular bacteria. Subsequently, the cells were lysed using PBS containing 0.1% Triton X-100, and the lysates were serially diluted and plated on Middlebrook 7H10 agar plates supplemented with 10% oleic acid, albumin, glucose and catalase (OADC) and 0.5% glycerol. The plates were incubated at 37°C for 3 days, the bacterial colonies were counted and the colony forming units (CFUs) were calculated.

[0037] Flow cytometric analysis of MAB-GFP infected cells treated with punicalagin:

[0038] THP-1 cells were pretreated with PMA for 24 h to polarize THP-1 cells into macrophage-like THP-1 cells. THP-1 macrophages were cultured at a density of 5 × 10 5Cells were seeded at a density of 10 cells / well in 12-well plates. Green fluorescent protein (GFP)-labeled MAB-GFP was infected as described above. Punicalagin-treated and untreated groups were also established. Flow cytometry was used to assess the survival of GFP-labeled MAB in THP-1 macrophages in the different treatment groups to analyze the inhibitory effect of punicalagin on intracellular MAB infection.

[0039] Western Blotting:

[0040] Cells from different treatment groups were collected, lysed with RIPA lysis buffer, and total protein was extracted. After the protein concentration was determined by the BCA method, the protein samples were subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane. The PVDF membrane was blocked with 5% skim milk powder for 1 hour, and the primary antibody against the target protein (such as SIRT1, SIRT3, FoxO3a, LC3, p62, ATG7, etc.) was added and incubated overnight at 4°C. The next day, the membrane was washed 3 times with TBST for 10 minutes each time, and then the corresponding secondary antibody was added and incubated at room temperature for 1 hour. After washing the membrane with TBST 3 times again, it was developed using a chemiluminescence reagent, the protein band image was obtained using a gel imaging system, and the protein expression level was analyzed using relevant software.

[0041] Immunofluorescence experiments:

[0042] Cells were seeded in 24-well plates with coverslips placed in advance. After the corresponding treatment, the cells were fixed with 4% paraformaldehyde for 15 minutes and washed three times with PBS. The cells were permeabilized with 0.1% Triton X-100 for 10 minutes, washed with PBS, and blocked with 5% BSA for 30 minutes. Primary antibodies against proteins such as LC3B and LAMP2 were added, and the cells were incubated overnight at 4°C. The next day, the cells were washed three times with PBS, and fluorescently labeled secondary antibodies were added and incubated at room temperature for 1 hour. After washing with PBS, the nuclei were stained with DAPI for 5 minutes, washed again, and mounted with anti-fluorescence quenching mounting medium. Laser confocal microscopy was used to observe and collect images, and the co-localization of proteins such as LC3B and LAMP2 was analyzed to assess the degree of fusion between autophagosomes and lysosomes.

[0043] Real-time quantitative PCR (qRT-PCR):

[0044] Total cellular RNA was extracted using TRIzol reagent and reverse transcribed into cDNA according to the reverse transcription kit instructions. Using cDNA as a template, SYBR Green Master Mix was used for real-time quantitative PCR amplification. Primer sequences were designed based on relevant genes, with β-actin as an internal reference gene. Reaction conditions included 40 cycles of pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, and annealing and extension at 60°C for 30 seconds. The relative expression levels of target genes were calculated using the 2(-ΔΔCt) method to analyze the effects of punicalagin on the expression of autophagy-related genes, SIRT1, and FoxO3a.

[0045] Mitochondrial membrane potential detection:

[0046] JC-1 dye was used to detect mitochondrial membrane potential. Cells from different treatment groups were collected, added with appropriate amounts of JC-1 working solution, and incubated at 37°C for 20 minutes. After washing the cells twice with JC-1 buffer, the intensity ratio of red fluorescence (aggregates) to green fluorescence (monomers) was measured by flow cytometry to assess changes in mitochondrial membrane potential and reflect the effects of punicalagin on mitochondrial function.

[0047] Transmission electron microscopy observation:

[0048] Cell samples were collected, fixed with 2.5% glutaraldehyde, dehydrated with graded ethanol, and embedded in epoxy resin. Ultrathin sections were prepared and double-stained for observation of mitochondrial ultrastructure using transmission electron microscopy to assess the effects of punicalagin on mitochondrial cristae structure and outer membrane integrity in MAB-infected cells.

[0049] Transcriptome sequencing and analysis:

[0050] RNA extraction, library construction, and transcriptome sequencing were performed on cells from the MAB-infected group and the MAB-infected control group treated with punicalagin. After quality control, the sequencing data were aligned with the reference genome for quantitative gene expression analysis. Differentially expressed genes (DEGs) were identified through differential expression analysis, and KEGG pathway enrichment analysis, gene set enrichment analysis (GSEA), and weighted gene co-expression network analysis (WGCNA) were performed. KEGG pathway enrichment analysis was used to identify signaling pathways with significant enrichment in differentially expressed genes; GSEA was used to identify the enrichment of specific gene sets between the two groups; and WGCNA clustered gene expression patterns to construct a co-expression network and identify key modules and genes associated with punicalagin treatment, thereby comprehensively analyzing the mechanism of action of punicalagin in gene expression regulation.

[0051] Experimental results

[0052] 1. Effects of MAB infection on THP-1 macrophages

[0053] 1) Transcriptome changes:

[0054] THP-1 macrophages were infected with MAB at an MOI of 10 for 4 hours. After extracellular bacteria were removed, the cells were cultured for an additional 24 hours, followed by transcriptome sequencing. The results showed significant gene expression differences in MAB-infected macrophages compared to uninfected THP-1 cells. Among these, the oxidative stress-related genes HIF1A (hypoxia-inducible factor 1-α) and NCF1 (NADPH oxidase subunit) were upregulated, indicating elevated intracellular oxidative stress. Concomitantly, the autophagy regulators ATG16L1 and ATG9 were downregulated, leading to impaired autophagosome formation. KEGG pathway analysis revealed significant activation of the TNF and NF-κB signaling pathways, while the Hippo signaling pathway, calcium signaling pathway, and arginine biosynthesis pathway were inhibited. GSEA further confirmed widespread alterations in autophagy-related pathways, as well as HIF-1, Rap1, Wnt, and necroptosis signaling cascades.

[0055] 2) Mitochondrial damage:

[0056] Given that dysregulation of the HIF-1, Wnt, and Hippo pathways is associated with mitochondrial dysfunction, we assessed mitochondrial membrane potential using JC-1 staining. The results showed a time-dependent decrease in mitochondrial membrane potential in MAB-infected cells, starting 4 hours after infection compared with uninfected controls. Furthermore, DCFH-DA assays revealed a time-dependent increase in intracellular reactive oxygen species (ROS) levels during infection. This suggests that mitochondrial dysfunction and ROS generation are prominent features of MAB infection.

[0057] 2. Effect of Punicalagin on MAB Infection in Macrophages

[0058] 1) Antibacterial effect:

[0059] Given that mitochondrial dysfunction and excessive ROS production triggered by MAB infection are key factors in impaired macrophage function, we treated infected macrophages with punicalagin. Figure 1 The results showed that compared with the untreated infection control group, punicalagin treatment significantly reduced the activity of MAB in cells. However, direct addition of punicalagin to 7H9 liquid culture had no significant effect on the growth of MAB (see Figure 1 Middle A), indicating that punicalagin inhibits MAB not through direct antibacterial effect.

[0060] 2) Mitochondrial protection:

[0061] Mitochondrial membrane potential was assessed by JC-1 staining, and it was found that punicalagin treatment could restore the mitochondrial membrane potential of MAB-infected macrophages, indicating that punicalagin enhanced the antibacterial activity of macrophages against MAB while maintaining the stability of mitochondria. At the same time, we used scanning electron microscopy to observe the mitochondria of THP-1 cells. The results showed that the mitochondria in THP-1 cells infected with Mycobacterium abscessus were swollen, the membrane ruptured, the membrane protruded outward to form vesicles or bud-like protrusions, and the cristae disappeared. After the intervention of punicalagin, the swelling of mitochondria in THP-1 cells infected with Mycobacterium abscessus was reduced, the cristae structure could still be observed, and the fragmentation was reduced (see Figure 1 Middle D).

[0062] 3) Concentration dependence and autophagy association:

[0063] MAB-infected THP-1 macrophages were treated with punicalagin at different concentrations (0, 20, 40, and 80 μM) and autophagy and apoptosis markers were measured. Western blotting results showed an increase in the LC3-II / LC3-I ratio in punicalagin-treated cells, accompanied by a dose-dependent degradation of p62, confirming that punicalagin specifically activates autophagic flux. Flow cytometry was used to assess the survival of GFP-labeled MAB in THP-1 macrophages and to quantify the number of MAB in the cells. Figure 1 Figure B shows that punicalagin has a dose-dependent inhibitory effect on intracellular MAB infection. Given that 80 μM punicalagin induces a high rate of cell apoptosis, 40 μM was selected as the treatment concentration for subsequent experiments. At 4 hours post-infection, there was no significant difference in apoptosis levels between the MAB-infected and punicalagin-treated groups. However, at 24 hours, apoptosis levels were lower in the punicalagin-treated group than in the MAB-infected group. Furthermore, compared with MAB-infected THP-1 macrophages not treated with punicalagin, 40 μM punicalagin upregulated the expression of the autophagy-related protein ATG14 at 24 hours post-infection. These results suggest that punicalagin alleviates MAB-induced mitochondrial dysfunction and oxidative stress, enhancing the antimicrobial activity of macrophages by activating autophagy. 40 μM represents the optimal dose, effectively reducing the intracellular MAB burden while maintaining mitochondrial stability and reducing apoptosis.

[0064] 3. Punicalagin induces autophagy and enhances MAB clearance via mTOR and MAPK / ERK signaling pathways 1) Effects on related proteins and signaling pathways:

[0065] Further investigation into the mechanism by which punicalagin protects against MAB infection revealed that treatment with punicalagin had no significant effect on pyroptosis-related proteins. However, it significantly inhibited the activation of mTOR, a key negative regulator of autophagy. Immunofluorescence studies revealed that punicalagin treatment promoted the colocalization of LC3B and LAMP2 in MAB-infected cells, indicating enhanced fusion of autophagosomes with lysosomes and a boost in autophagy. Furthermore, punicalagin-treated THP-1 macrophages significantly reduced the bacterial load, suggesting that autophagy activation contributes to MAB clearance. Furthermore, punicalagin inhibited activation of the MAPK / ERK signaling pathway in MAB-infected cells, with significant downregulation of p-P38 and p-Erk levels. MAPK / ERK signaling is known to negatively regulate autophagy, and its inhibition by punicalagin may further promote the autophagic process. Furthermore, punicalagin treatment also inhibited AKT phosphorylation in MAB-infected cells. AKT phosphorylation plays a key role in regulating key cellular processes such as cell survival, proliferation, and autophagy. Punicalagin inhibition may alleviate AKT-mediated autophagy inhibition, thereby promoting the autophagic process and enhancing bacterial clearance. Taken together, punicalagin promotes autophagy by synergistically regulating multiple signaling pathways. This multifaceted mechanism may contribute to its therapeutic efficacy against MAB infection.

[0066] 2) Verification of key autophagy mechanisms:

[0067] Blocking autophagosome acidification with bafilomycin A1 abolished punicalagin-mediated MAB clearance, confirming the necessity of functional lysosomal degradation. Conversely, chloroquine (CQ), an inhibitor of autophagosome-lysosome fusion, led to LC3-II accumulation in punicalagin-treated macrophages, indicating that punicalagin enhances autophagosome formation without impairing lysosomal processing. Application of the autophagy inhibitor bafilomycin A1 effectively blocked the autophagic process and reversed the effect of punicalagin on the reduction of intracellular MAB. CQ blocked punicalagin-induced autophagy by inhibiting autophagosome-lysosome fusion, inhibiting autophagic flux and leading to increased LC3-II accumulation in punicalagin-treated macrophages, indicating that punicalagin-induced LC3-II accumulation is not due to downstream inhibition of autophagic flux. Transmission electron microscopy showed that punicalagin treatment ameliorated MAB-induced mitochondrial cristae disorder and outer membrane rupture, maintaining structural integrity comparable to that of uninfected controls, which is mechanistically consistent with the ability of punicalagin to stabilize bioenergetic homeostasis, as intact mitochondrial structure is essential for maintaining ATP synthesis and regulating redox balance.

[0068] 3) Transcriptome and key target analysis:

[0069] RNA-seq analysis of punicalagin-treated and untreated MAB-infected cells revealed 1,762 upregulated and 854 downregulated genes in the punicalagin-treated MAB-infected group. KEGG enrichment analysis revealed that multiple key pathways were significantly upregulated in punicalagin-treated MAB-infected THP-1 macrophages compared with the untreated control group, including the p53 signaling pathway, cytokine-cytokine receptor interaction, FoxO signaling pathway, Rap1 signaling pathway, and PPAR signaling pathway (p < 0.05). Of particular note was the concurrent activation of the FoxO pathway, a known regulator of autophagy initiation and lysosomal biogenesis, consistent with our hypothesis that punicalagin may exert its anti-mycobacterial effects by enhancing autophagic clearance. GSEA revealed a significant positive correlation with the FoxO signaling pathway in the punicalagin-treated MAB-infected group compared with the untreated MAB-infected group. Through comprehensive bioinformatics analysis integrating GeneCards, Swiss Target, and RNA-seq data, 16 core differentially expressed genes, including SIRT1, NFKB1, and GPR35, were identified as common targets in the MAB-infected group treated with punicalagin. Further investigation of Sirtuin 1 (SIRT1) revealed upregulation of Sirt1 and FoxO3a expression in punicalagin-treated MAB-infected macrophages compared with MAB-infected macrophages. We found that punicalagin inhibited AKT / mTOR and MAPK signaling, promoted autophagy-lysosomal clearance of MAB, and maintained mitochondrial structure. Furthermore, the therapeutic effects of punicalagin may also involve activation of the SIRT1-FoxO axis, suggesting a multi-pathway, host-directed therapeutic strategy.

[0070] 4. Punicalagin treatment upregulates Sirt1 and FoxO3a in MAB-infected macrophages to promote autophagy 1) Verification of the role of SIRT1 and SIRT3:

[0071] SIRT1 and SIRT3, as important members of the Sirtuin family, play a key role in regulating a variety of biological processes such as metabolism, cell survival, stress response, inflammation and aging through deacetylation activity. Punicalagin treatment upregulated the protein expression levels of SIRT1 and SIRT3 in MAB-infected THP-1 macrophages. To clarify whether punicalagin specifically regulates the survival of intracellular mycobacteria through SIRT1 expression, we used the SIRT3 inhibitor 3-TYP and the SIRT1 inhibitor ex 527 to conduct experiments. Figure 2As shown, flow cytometry analysis revealed that SIRT3 blockade did not attenuate the antibacterial effect of punicalagin, with bacterial load reduction comparable to that of the punicalagin-treated control. Immunofluorescence results also revealed no significant changes in autophagy in 3-TYP-treated cells following MAB infection and punicalagin treatment. However, treatment with the SIRT1 inhibitor ex-527 reduced the expression of autophagy-related proteins in punicalagin-treated MAB-infected THP-1 macrophages, accompanied by a significant decrease in SIRT1 and FOXO3a expression levels. Flow cytometry analysis demonstrated that treatment with the SIRT1 inhibitor ex-527 reduced the phagocytic activity of punicalagin-treated THP-1 cells toward MAB. Quantitative assessment of intracellular MAB confirmed that ex-527 impaired the ability of SIRT1-treated macrophages to effectively fight infection. These results indicate that the SIRT1 inhibitor ex-527 reduces the ability of punicalagin to reduce MAB load in macrophages, while SIRT3 inhibitors have no such effect. This functionally excludes the possibility that SIRT3 is involved in the mechanism of action of punicalagin and strengthens the position of SIRT1 as the main target for coordinating autophagy-mediated pathogen clearance. 2) Validation of key functions of SIRT1:

[0072] Before punicalagin administration, SIRT1 was knocked down in macrophages by siRNA. Figure 3Results showed that SIRT1 knockdown in macrophages significantly impaired the ability of punicalagin-treated THP-1 macrophages to clear intracellular MAB. Given the limitations of qRT-PCR for validating knockdown efficiency, SIRT1 expression was assessed by Western blot analysis, confirming that SIRT1 protein levels were reduced after siRNA treatment. We also observed that SIRT1 downregulation altered punicalagin-induced autophagy-related functions in macrophages. Studying the effect of siRNA-mediated SIRT1 knockdown on LC3B colocalization in MAB-GFP-infected THP-1 cells revealed that MAB infection significantly impaired macrophage autophagy, ultimately leading to cell death and exacerbated infection. However, punicalagin treatment effectively confined GFP-labeled MAB to autophagosomes in infected THP-1 cells. Notably, SIRT1 knockdown via si-SIRT1 further exacerbated MAB infection, as evidenced by the inability of THP-1 macrophages to restrict intracellular MAB proliferation, an increase in the number of GFP-labeled MAB, and a significant impairment of macrophage autophagy, ultimately leading to cell death, exacerbated infection, and increased nuclear fragmentation. Furthermore, punicalagin treatment attenuated the si-SIRT1 knockdown-mediated enhancement of MAB proliferation within THP-1 cells, indicating that the SIRT1-mediated pathway plays a key role in the mechanism of punicalagin-enhanced autophagy. These findings suggest that the protective effect of punicalagin against MAB-induced cell damage depends on SIRT1 activation, which subsequently drives autophagy to promote MAB clearance. This highlights SIRT1 as a key therapeutic target for mitigating MAB infection and the potential of punicalagin as a SIRT1 activator to enhance cellular defense mechanisms against intracellular MAB infection. 3) Role of FoxO3a:

[0073] Based on evidence of FoxO3a upregulation and pathway enrichment in punicalagin-treated macrophages, we investigated the role of FoxO3a in MAB-infected macrophages. Prior to punicalagin administration, FoxO3a expression was silenced by siRNA in MAB-infected THP-1 macrophages. We found that FoxO3a knockdown abolished punicalagin-induced autophagic flux and reduced the expression of ATG7, a key autophagic effector transcriptionally regulated by FoxO3a. Imaging analysis of GFP-tagged MAB colocalization with LC3B demonstrated that punicalagin significantly inhibited MAB infection in THP-1 macrophages. However, si-FOXO3 treatment exacerbated the spread of GFP-tagged MAB within THP-1 macrophages and abolished the anti-infective effect of punicalagin. Furthermore, FOXO3 knockdown impaired autophagosome formation. Finally, flow cytometry was used to assess mitochondrial membrane potential generation in cells treated with si-SIRT1 and si-FoxO3a. The results showed that the mitochondrial membrane potential damage was aggravated in MAB-infected THP-1 macrophages treated with si-SIRT1 and si-FoxO3a. In addition, the protective effect of punicalagin on the mitochondrial membrane potential of infected cells was significantly weakened in si-SIRT1 or si-FoxO3a knockout cells (see Figure 3 These results suggest that punicalagins positively influence autophagic flux by upregulating SIRT1 or FoxO3a to inhibit intracellular MAB growth. These findings mechanistically establish the SIRT1-FoxO3a axis as a key signaling hub through which punicalagins coordinate autophagy to achieve autophagic pathogen clearance.

[0074] 5. Punicalagin treatment enhances host defense against MAB infection in mice

[0075] 1) Lung infection model:

[0076] After confirming that the SIRT1-FoxO3a axis is the key mechanism by which punicalagin induces autophagy-dependent anti-MAB activity in macrophages, we evaluated its therapeutic efficacy in a mouse lung MAB infection model. 7 Mice were inoculated with CFU of MAB and then intraperitoneally injected with punicalagin. Figure 4 As shown, compared with untreated MAB-infected mice, the punicalagin-treated group had milder inflammatory cell infiltration in the lung tissue during infection, reduced alveolar damage, and a significant decrease in the MABCFU count in the bronchoalveolar lavage fluid (BALF). 2) Regulation of autophagy and apoptosis:

[0077] The regulation of autophagy and apoptosis in lung tissues of MAB-infected mice and punicalagin-treated MAB-infected mice was evaluated. Figure 4In Figure C, punicalagin significantly inhibited STAT3 activation, suppressed AKT levels, and reduced cleaved-caspase-3 levels, indicating that it increased autophagy activity while attenuating cell apoptosis, a phenotype consistent with the cytoprotective effects observed in vitro.

[0078] 3) Macrophage Subpopulation Analysis:

[0079] To further investigate the effects of punicalagin on autophagy and related cell signaling pathways in different macrophage subsets, alveolar macrophages (SiglecF) were isolated from the lungs of MAB-infected mice (untreated and punicalagin-treated) using cell sorting technology. + ) and interstitial macrophages (CD11b+). Western blot analysis of autophagy-related proteins as well as SIRT1 and FoxO3a showed that SIRT1 and FoxO3a expression was upregulated in interstitial macrophages of MAB-infected mice lungs treated with punicalagin, while PI3K / mTOR levels were reduced, effectively counteracting autophagy inhibition. Interestingly, similar trends were also observed in alveolar macrophages, further supporting the broad applicability of punicalagin’s host-directed mechanism in regulating autophagy and combating MAB infection. Figure 5 As shown in Figures A and B, the infection status of GFP-MAB in lung macrophages was assessed by flow cytometry. The results showed that punicalagin treatment had a stronger protective effect on interstitial macrophages than on alveolar macrophages, as evidenced by a significant reduction in bacterial load. Although punicalagin also reduced the bacterial load in alveolar macrophages, the severity of infection in alveolar macrophages was still significantly higher than that in interstitial macrophages. This observation highlights the differences in the susceptibility of different macrophage populations to MAB infection and the selective effectiveness of punicalagin in targeting lung macrophages.

[0080] 4) Transcriptome analysis:

[0081] Transcriptome sequencing was performed on alveolar macrophages and interstitial macrophages in the MAB-infected group and the MAB-infected group treated with punicalagin. Figure 5CE results showed that compared with the MAB-infected control group, multiple key signaling pathways and biological processes were significantly enriched in MAB-infected lung macrophages treated with punicalagin, including the PI3K-Akt signaling pathway, MAPK signaling pathway, calcium signaling pathway, FoxO signaling pathway, HIF-1 signaling pathway, phagosome formation, Fcγ receptor-mediated phagocytosis, and endocytosis. Considering that the effect of punicalagin treatment was more significant in interstitial macrophages, we performed gene set enrichment analysis (GSEA) and found that autophagosome maturation was significantly enriched in interstitial macrophages treated with punicalagin, indicating enhanced autophagic activity. This finding is consistent with our previous observation in THP-1 macrophages that SIRT1-mediated FoxO3a activation promotes autophagy to fight intracellular pathogens. The upregulation of autophagosome maturation indicates that punicalagin promotes the clearance of MAB through autophagy, thereby enhancing macrophage effector function and contributing to its therapeutic effect. Weighted gene co-expression network analysis (WGCNA) was used to construct a co-expression network, and modules associated with punicalagin treatment were clustered based on gene expression patterns. Endocytosis, proteolysis, and autophagy processes were significantly enriched in the interstitial macrophage-treated group, whereas only autophagy-related processes were enriched in the alveolar macrophage-treated group. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that autophagy- and lysosome-related functions were significantly more enriched in interstitial macrophages than in alveolar macrophages in punicalagin-treated MAB-infected mice. This result highlights the differential effects of punicalagin on alveolar and interstitial macrophages in MAB-infected mice. Our results suggest that punicalagin protects against MAB infection in the lungs by activating the SIRT1-FoxO3a axis and inhibiting the PI3K / mTOR and STAT3 signaling pathways, thereby enhancing autophagy and reducing apoptosis. This host-directed mechanism leads to reduced lung inflammation and bacterial burden, with interstitial macrophages exhibiting a preferential response through autophagy-lysosome interactions. Transcriptome analysis further highlighted the specific involvement of the autophagy-lysosomal pathway in macrophage subsets, consistent with the therapeutic selectivity of punicalagins. By modulating macrophage intrinsic defenses rather than directly targeting MABs, punicalagins may be a promising candidate for the treatment of persistent mycobacterial infections.

[0082] Through the above detailed experimental methods and specific examples, the mechanism of action of punicalagin in regulating autophagy through the SIRT1 / FOXO3a axis and enhancing the ability of macrophages to resist Mycobacterium abscessus infection was further verified, providing a solid experimental basis for its application in the treatment of Mycobacterium abscessus infection.

[0083] In summary, it can be seen that punicalagin has unique functions related to promoting macrophage immune function and antibacterial activity against Mycobacterium abscessus in infected macrophages, and therefore can be used to treat currently difficult-to-treat Mycobacterium abscessus infections and reduce the possibility of recurrence. Compounds similar to punicalagin or compounds that regulate autophagy based on the SIRT1 / FOXO3a axis can exert related functions by upregulating SIRT1, activating the SIRT1 / FOXO3a axis, and promoting macrophage autophagy, and are expected to be used in the preparation of drugs for treating Mycobacterium abscessus infections.

[0084] The embodiments described above are merely some preferred embodiments of the present invention and are not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. Application of punicalagin in the preparation of drugs for treating Mycobacterium abscessus infection.

2. The use according to claim 1, characterized in that The Mycobacterium abscessus infection is Mycobacterium abscessus lung infection.

3. Application of punicalagin in the preparation of drugs for enhancing the ability of macrophages to resist Mycobacterium abscessus infection.

4. Application of punicalagin in the preparation of drugs for enhancing macrophage autophagy.

5. The use according to claim 4, characterized in that The drug enhances mitochondrial stability by upregulating SIRT1, activates the SIRT1 / FOXO3a axis, and promotes macrophage autophagy.

6. Application of punicalagin in the preparation of inhibitors of cell signaling pathways infected with Mycobacterium abscessus.

7. The use according to claim 6, characterized in that The Mycobacterium abscessus infected cell signaling pathway includes one or more of the PI3K / Akt / mTOR signaling pathway, the MAPK / ERK signaling pathway, and the STAT3 signaling pathway.

8. Application of compounds that regulate autophagy based on the SIRT1 / FOXO3a axis in the preparation of drugs for the treatment of Mycobacterium abscessus infection.

9. The use according to claim 8, characterized in that The drug enhances mitochondrial stability by upregulating SIRT1, activates the SIRT1 / FOXO3a axis, and promotes macrophage autophagy.

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