Application of ailanone in inhibiting HK2 regulation glycolysis and treating metabolic inflammation cross disease

Abstract

The invention provides application of ailanone in inhibiting HK2 regulation glycolysis and treating metabolic inflammation cross diseases, and belongs to the technical field of biological medicines. The invention reveals for the first time that ailanone (AIL) can significantly alleviate sepsis-related myocardial damage by targeting HK2, and has good biological safety. After the AIL is used for treating an animal model, the heart blood pumping function can be remarkably recovered; the level of myocardial injury markers is reduced; the tissue damage is obviously relieved. Meanwhile, a staining experiment proves that the AIL can effectively reduce the LPS-induced myocardial cell apoptosis rate and obviously reduce the Ly6G level, and proves that the AIL can improve cell apoptosis and immune injury. The ailanthus altissima ketone inhibits HK2 to regulate glycolysis, breaks the metabolic abnormality loop in the SCM pathogenesis process, and is suitable for treatment of multiple metabolic inflammation cross diseases such as sepsis, myocarditis, inflammatory cardiovascular diseases and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of ailanthone in inhibition of HK2 regulation of glycolysis and treatment of metabolic inflammation cross diseases. BACKGROUND

[0002] Sepsis is a systemic inflammatory response syndrome triggered by infection, with a mortality rate of 30%-70%. Its pathological process begins with an uncontrolled inflammatory storm and eventually progresses to multiple organ dysfunction. Septic cardiomyopathy (SCM), as a critical complication of sepsis, is characterized by myocardial contractile dysfunction and progressive myocardial edema-fibrosis remodeling, significantly worsening the prognosis of patients. The STING pathway triggers the release of type I interferon (IFN-1) and inflammatory factors by recognizing DNA of bacterial / host origin in sepsis, thereby activating endothelial cells. Activated endothelial cells rapidly release angiopoietin-2 (Ang-2) and von Willebrand factor (vWF) from Weibel-Palade bodies, leading to increased vascular permeability, capillary leakage, and microthrombosis, thereby exacerbating myocardial cell edema and causing severe cardiac dysfunction.

[0003] Ailanthone (AIL) is a natural compound derived from the bark of Chinese medicinal tree ailanthus altissima, which has antibacterial, anti-inflammatory, and antitumor activities. Studies have shown that ailanthone can effectively inhibit the proliferation of non-small cell lung cancer cisplatin-resistant A549 / DDP cells and induce apoptosis, while also enhancing the sensitivity of the drug-resistant cells to cisplatin, demonstrating the potential to reverse tumor multidrug resistance. In addition, ailanthone can strongly interfere with the progression of liver and lung fibrosis by inhibiting the core TGF-β / Smad signaling pathway and preventing abnormal tissue scarring. Emerging research has also shown that ailanthone can help clear accumulated abnormal proteins in neurodegenerative diseases by activating autophagy, and alleviate excessive immune response in models of autoimmune diseases such as psoriasis by suppressing the STAT3 / NF-κB pathway. These findings collectively establish ailanthone as a lead compound that can act on multiple core targets, and its broad therapeutic spectrum makes it a valuable asset for future drug development. SUMMARY

[0004] The application provides application of ailanthone in inhibition of HK2 regulation of glycolysis and treatment of metabolic inflammation cross diseases. Ailanthone can intervene in the pathogenesis of metabolic inflammation cross diseases from multiple angles by inhibiting HK2 enzyme activity, improving cardiac function, suppressing inflammatory response, and reducing myocardial cell apoptosis, and has clear clinical application prospects.

[0005] The application provides application of an agent for inhibiting HK2 from regulating glycolysis in preparation of a medicine for treating metabolic inflammation cross diseases.

[0006] In a preferred mode of the application, the agent for inhibiting HK2 from regulating glycolysis comprises an effective component derived from the skin of Ailanthus altissima.

[0007] In a preferred mode of the application, the agent for inhibiting HK2 from regulating glycolysis comprises ailanthone.

[0008] In a preferred mode of the application, the metabolic inflammation cross diseases comprise at least one of the following: sepsis, myocarditis and inflammatory cardiovascular diseases.

[0009] In a preferred mode of the application, the therapeutic effect of the medicine comprises at least one of the following: inhibition of HK2 enzyme activity, improvement of heart function, inhibition of inflammatory response and reduction of myocardial cell apoptosis.

[0010] In a preferred mode of the application, the inhibition of inflammatory response comprises inhibition of at least one of the following inflammatory factors: TNF-alpha, IL-1beta, IL-6, chemotactic factor Cxcl5 and chemotactic factor Ccl2.

[0011] The application further provides a medicine for treating metabolic inflammation cross diseases, wherein the active component comprises ailanthone, and further comprises a pharmaceutically acceptable adjuvant.

[0012] In a preferred mode of the application, the working concentration of ailanthone in the medicine is not less than 5 mg / kg.

[0013] In a preferred mode of the application, the dosage form of the medicine comprises an injection.

[0014] In a preferred mode of the application, the type of the adjuvant comprises a solvent, and the solvent comprises a mixed solution of DMSO, PEG300, Tween-80 and physiological saline.

[0015] Beneficial effects: The application first discloses a small molecule compound of natural origin, ailanthus integrifolia ketone (AIL), which can significantly alleviate sepsis-related myocardial injury by targeting HK2, has good biological safety, does not cause obvious liver and kidney tissue damage, can maintain the integrity of the tissue structure, and does not cause obvious hepatotoxicity and nephrotoxicity. In the embodiments of the application, a LPS-induced sepsis cardiomyopathy mouse model is used for systematic evaluation, and the results show that LPS treatment significantly reduces the ejection fraction (EF) of the mouse heart, and AIL treatment significantly restores the heart pumping function; the myocardial injury markers CK-MB, TnT and LDH are significantly increased in the LPS group, and AIL significantly reduces the levels of these serum markers; LPS induces myocardial cell swelling, and AIL treatment significantly alleviates tissue damage. At the same time, it is proved by staining experiments that AIL can effectively reduce the LPS-induced myocardial cell apoptosis rate and significantly reduce the Ly6G level, proving that AIL can improve cell apoptosis and immune damage.

[0016] The AIL disclosed in the application can significantly reduce the levels of serum inflammatory factors induced by LPS, including TNF-alpha, IL-1beta and IL-6, and simultaneously reduce the corresponding mRNA expression in the heart tissue; and down-regulate the mRNA levels of chemotactic factors Cxcl5 and Ccl2 in the myocardial tissue of sepsis mice. In summary, the ailanthus integrifolia ketone disclosed in the application can intervene the pathogenesis of sepsis cardiomyopathy from multiple angles by inhibiting the HK2 enzyme activity, improving heart function, inhibiting inflammatory response and reducing myocardial cell apoptosis, and has a clear clinical application prospect.

[0017] The application breaks the abnormal metabolic loop in the SCM pathogenesis process by inhibiting HK2 to regulate glycolysis through the high-biological-safety ailanthus integrifolia ketone, simultaneously acts on metabolic regulation, inflammatory factors, cell apoptosis and myocardial function, and it is proved by animal experiments that the AIL can significantly alleviate the progression of SCM, and is suitable for the treatment of various metabolic and inflammatory cross diseases such as sepsis, myocarditis and inflammatory cardiovascular diseases. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The results of AIL can specifically block HK2 and inhibit LPS-induced inflammatory response are shown in the figure, wherein A: three-dimensional structure of HK2 protein and docking frame schematic diagram; B: three-dimensional docking mode of part of small molecule drugs and HK2 protein molecules is shown using YINFO online platform (https: / / cloud.yinfotek.com); C: characteristics of the top three potential HK2 inhibitors; D: Western blotting is used to verify the inhibition ability of part of small molecule drugs on the expression of HK2 protein in AC16 cells at different concentrations; E: levels of TNFalpha, IL1beta and IL6 in AC16 cells, n=6; the data in E is statistically analyzed by two-factor variance analysis and post-hoc test, and the data is represented by mean ± standard error; P <0.001, P <0.0001; Figure 2 Figure 1 shows the biocompatibility test results for AIL. A: Representative HE staining images of mouse livers treated with the vector or AIL (scale bar: 50 μm); B: Representative HE staining images of mouse kidneys treated with the vector or AIL (scale bar: 50 μm); C: Serum ALT and AST level detection results; D: Serum BUN and CREA level detection results. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BUN: Blood urea nitrogen; CREA: Creatinine; Vector group: n=6 mice; AIL group: n=6 mice; All data are expressed as mean ± standard error. P The values ​​were calculated using a two-tailed Student's t-test, and ns: no significant difference; Figure 3 The figures show the results of AIL improving myocardial injury induced by SCM. In the figures, AB: echocardiographic analysis of mouse cardiac function, including left ventricular ejection fraction and left ventricular fractional shortening (n=6); CE: detection of serum myocardial injury markers, including creatine kinase isoenzyme, troponin T, and lactate dehydrogenase (n=6); F: representative image of mouse heart sections stained with hematoxylin and eosin (HE), scale bar: 500 μm (black) or 50 μm (red); G: immunofluorescence staining showing the expression of TnI and Ly6G in cardiac tissue; H: representative image of mouse heart sections stained with TUNEL, scale bar: 100 μm; I: analysis of inflammatory factor levels, detecting changes in TNFα, IL1β, and IL6; J: mRNA levels of inflammatory factors in cardiac tissue (n=6). Data in AE and HJ were statistically analyzed using two-way ANOVA and post-hoc tests. Data are expressed as mean ± standard error. P <0.01, P <0.001, P <0.0001; Figure 4 The graph shows the results of AIL inhibiting Cxcl15 and Ccl2 mRNA levels in SCM. Data were analyzed using two-way ANOVA and post-hoc tests. Data are expressed as mean ± standard error. P <0.0001. Detailed Implementation

[0019] The application provides application of an agent for inhibiting HK2 from regulating glycolysis in preparation of a medicine for treating metabolic inflammation cross diseases.

[0020] In the application, 2-deoxy-D-glucose (2-DG) is a glucose analogue and a hexokinase inhibitor, and has been widely used for inhibiting glycolysis. Hexokinase 2 (HK2, Gene ID: 3099) is a main rate-limiting enzyme in the aerobic glycolytic pathway, and plays a key role in promoting the Warburg effect. However, due to the competition of endogenous glucose for the active site of hexokinase, the efficacy of 2-DG is limited. In the application, the three-dimensional structure (PDB code: 2NZT) of human HK2 is determined by using RCSB protein database (http: / / www.rcsb.org / ), and the structure has been resolved by nuclear magnetic resonance (NMR) technology. Notably, a pocket-like structure is observed inside the HK2 protein. In order to identify natural small molecule compounds targeting HK2, molecular docking and virtual screening are performed on more than 15,000 natural compounds in the L6020 alternative natural compound library. According to the binding affinity, AIL showing the highest affinity to HK2 is screened, and AIL shows a significant inhibitory effect on HK2. It is also proved by Western blot analysis that AIL can effectively reduce the levels of TNF-α, IL-1β and IL-6 in LPS-treated AC16 cells, indicating that the AIL can play a role as an inhibitor of HK2.

[0021] It is found in the embodiments of the application that the AIL can specifically block HK2 and inhibit LPS-induced inflammatory response, and the AIL can effectively inhibit the progression of sepsis cardiomyopathy by targeting HK2, and improve myocardial injury caused by SCM, specifically, including inhibiting HK2 enzyme activity, improving heart function, inhibiting inflammatory response and reducing myocardial cell apoptosis, wherein the inflammatory response includes inflammatory response caused by at least one of the following inflammatory factors: TNF-α, IL-1β, IL-6, chemokine Cxcl5 (Gene ID: 20311) and chemokine Ccl2 (Gene ID: 20296). Therefore, the AIL in the application can be effective for various metabolic inflammation cross diseases, including at least one of the following: sepsis, myocarditis and inflammatory cardiovascular disease.

[0022] The application also provides a medicine for treating metabolic inflammation cross diseases, wherein the active ingredient comprises ailanthoidol, and further comprises a pharmaceutically acceptable adjuvant.

[0023] The working concentration of the anjeroone in the medicine is not less than 5 mg / kg. The dosage form of the medicine is not specially limited in the present application, and the medicine can be prepared by using the conventional medicine dosage form in the art, and the types and amounts of excipients are adjusted according to the dosage form, for example, in the examples, an injection is selected, and in one example, the solvent of the injection includes a mixed solution of DMSO, PEG300, Tween-80 and physiological saline, for example, 100 μL of 20.8 mg / mL clear DMSO stock solution is added into 400 μL of PEG300, and mixed uniformly; then 50 μL of Tween-80 is added into the above system, and mixed uniformly; and then 450 μL of physiological saline is further added to make up to 1 mL.

[0024] In order to further illustrate the present application, the application of anjeroone provided by the present application in inhibiting HK2 to regulate glycolysis and treating metabolic inflammatory cross diseases is described in detail below in combination with examples, but they cannot be understood as limiting the protection scope of the present application.

[0025] Example 1 Molecular docking affinity screening The present application determines the three-dimensional structure of human HK2 (PDB code: 2NZT) by using RCSB protein database (http: / / www.rcsb.org / ), and the structure has been resolved by nuclear magnetic resonance (NMR) technology. It is worth noting that a pocket-like structure is observed inside the HK2 protein (FIG. 1A). In order to identify natural small molecule compounds targeting HK2, molecular docking and virtual screening are performed on more than 15,000 natural compounds in the L6020 alternative natural compound library. According to the binding affinity, the top three small molecules are selected as candidate compounds (FIG. 1B and FIG. 1C). Figure 1 Figure 1

[0026] Among these candidate compounds, AIL shows the highest affinity to HK2 and exhibits a significant inhibitory effect on the enzyme (FIG. 1D). Consistently, Western blot analysis confirms that AIL can effectively reduce the levels of TNF-α, IL-1β and IL-6 in LPS-treated AC16 cells (FIG. 1E), which further indicates that AIL can act as an inhibitor of HK2. Figure 1 Figure 1

[0027] Example 2 2.1 LPS modeling In order to evaluate the effect of AIL on myocardial injury induced by sepsis, the present application adopts a LPS-induced sepsis cardiomyopathy (SCM) mouse model.

[0028] ​​​​To construct a septic cardiomyopathy (SCM) model, we administered intraperitoneal injections of Escherichia coli O111:B4 lipopolysaccharide (LPS, product number S1732, purchased from Shanghai Beyotime Biotechnology Co., Ltd.) to mice at a dose of 10 mg / kg. Simultaneously, a vector control group was set up, in which mice were injected intraperitoneally with an equal volume of sterile saline (0.9% NaCl).

[0029] 2.2 Assessment of potential hepatotoxicity and nephrotoxicity of AIL For interventional studies, ailanthus ketone (5 mg / kg) was administered intraperitoneally one hour before LPS challenge. Twelve hours after LPS or saline administration, heart, liver, and kidney tissues were harvested, immediately flash-frozen in liquid nitrogen, and stored at -80°C for subsequent analysis. Simultaneously, arterial blood samples collected from the abdominal aorta were coagulated at room temperature for 15 min, followed by centrifugation at 3,000 × g at 4°C for 10 min. The resulting serum supernatant was aliquoted and stored at -80°C for ELISA detection.

[0030] This invention involves hematoxylin-eosin (HE) staining of liver and kidney tissues from AIL-treated mice. HE staining results showed normal tissue structure, intact cell morphology, and no histopathological abnormalities. Figure 2 Similarly, kidney sections showed intact glomerular and tubular structures, indicating that the integrity of the kidney was maintained. Figure 2 (Middle B). Consistent with this, serum alanine aminotransferase (ALT) (39.83 ± 3.65 vs 39.00 ± 3.66 U / L) and aspartate aminotransferase (AST) (88.17 ± 11.41 vs 95.0 ± 8.4 U / L), reflecting liver function, and serum blood urea nitrogen (BUN) (9.25 ± 0.88 vs 8.67 ± 0.90 mg / dL) and creatinine (58.72 ± 4.91 vs 61.13 ± 4.56 μM), reflecting kidney function, showed no significant changes. Figure 2 (C and D in the middle).

[0031] 2.3 Effects of AIL on myocardial function and injury markers Mice were assessed for cardiac function using M-mode echocardiography with a Vevo2100 system (VisualSonics, Toronto, Canada). After anesthesia, chest hair was carefully removed, and ultrasound coupling gel was applied to ensure acoustic contact. Two-dimensional M-mode ultrasound tracks were acquired from the parasternal short-axis view (papillary muscle level) and long-axis view. Hemodynamic parameters were analyzed over at least six consecutive cardiac cycles. Key measurements included left ventricular end-systolic diameters (LVIDs) and end-diastolic diameters (LVIDd), as well as left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV). Ejection fraction (EF) and fractional shortening (FS) were calculated using the following formula: , Formula I; Formula II.

[0032] Troponin T (TnT) levels were measured using an ELISA kit from Shanghai Youjing Biotechnology according to the prescribed procedure; serum creatine kinase isoenzyme MB (CK-MB) and lactate dehydrogenase (LDH) concentrations were measured using a fully automated biochemical analyzer from Wuhan Saiwei Biotechnology.

[0033] Frozen sections of cardiac tissue were first washed with PBS, followed by fixation with 4% paraformaldehyde (PFA) for 30 min. After three cycles of PBS washing, the sections were permeabilized with 0.1% Triton X-100 for 10 min. They were then incubated with goat antiserum for 1 hour to block non-specific binding sites. Primary antibody incubation was performed overnight at 4°C. The next day, after the sections were brought to room temperature, they were co-incubated with fluorescently labeled secondary antibody for 1 hour according to the supplier's instructions. After washing again with PBS, the nuclei were stained with DAPI for 5 min under light-protected conditions. Finally, images were acquired using a fluorescence microscope.

[0034] This invention utilizes echocardiography, biochemical assays, and immunofluorescence staining to assess myocardial function and injury markers. Echocardiographic analysis revealed significant heart failure in LPS-treated mice. Notably, AIL treatment restored cardiac ejection function, indicating a protective effect on myocardial function. Figure 3 (A and B). Serum markers of myocardial injury, including CK-MB, TnT, and LDH, were significantly elevated in LPS-treated mice (687.932 ± 36.104 vs 123.30 ± 11.27 U / L; 134.82 ± 26.09 vs 3.8820 ± 1.1061 pg / mL; 1146.084 ± 65.807 vs 109.106 ± 10.524 U / L), but were significantly reduced by AIL ( Figure 3Fig. 5. AIL attenuates LPS-induced myocardial injury. Representative H&E-stained sections of heart tissue from control, LPS-treated and AIL-treated mice (C-E). Consistent with these findings, H&E staining showed that LPS treatment resulted in myocardial swelling, which was alleviated in AIL-treated mice (F). Figure 3 Fig. 6. AIL attenuates LPS-induced myocardial injury. Representative H&E-stained sections of heart tissue from control, LPS-treated and AIL-treated mice (C-E). Consistent with these findings, H&E staining showed that LPS treatment resulted in myocardial swelling, which was alleviated in AIL-treated mice (F).

[0035] Fig. 7. AIL attenuates LPS-induced myocardial injury. Representative H&E-stained sections of heart tissue from control, LPS-treated and AIL-treated mice (C-E). Consistent with these findings, H&E staining showed that LPS treatment resulted in myocardial swelling, which was alleviated in AIL-treated mice (F). Figure 3 Fig. 8. AIL attenuates LPS-induced myocardial injury. Representative H&E-stained sections of heart tissue from control, LPS-treated and AIL-treated mice (C-E). Consistent with these findings, H&E staining showed that LPS treatment resulted in myocardial swelling, which was alleviated in AIL-treated mice (F). Figure 3 Fig. 9. AIL attenuates LPS-induced myocardial injury. Representative H&E-stained sections of heart tissue from control, LPS-treated and AIL-treated mice (C-E). Consistent with these findings, H&E staining showed that LPS treatment resulted in myocardial swelling, which was alleviated in AIL-treated mice (F). Figure 3 Fig. 10. AIL attenuates LPS-induced myocardial injury. Representative H&E-stained sections of heart tissue from control, LPS-treated and AIL-treated mice (C-E). Consistent with these findings, H&E staining showed that LPS treatment resulted in myocardial swelling, which was alleviated in AIL-treated mice (F). Figure 3 Fig. 11. AIL attenuates LPS-induced myocardial injury. Representative H&E-stained sections of heart tissue from control, LPS-treated and AIL-treated mice (C-E). Consistent with these findings, H&E staining showed that LPS treatment resulted in myocardial swelling, which was alleviated in AIL-treated mice (F). Figure 4 Therefore, the AIL described herein significantly attenuated sepsis cardiomyopathy by targeting HK2.

[0036] Although the above embodiments have been described in detail, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the above embodiments without creativity, which are within the protection scope of the present application.

Claims

1. The application of a reagent that inhibits HK2-regulated glycolysis in the preparation of drugs for treating cross-diseases of metabolic inflammation.

2. The application according to claim 1, characterized in that, The reagent that inhibits HK2-regulated glycolysis includes effective components derived from neem.

3. The application according to claim 2, characterized in that, The reagents used to inhibit HK2-regulated glycolysis include ailanthus ketone.

4. The application according to claim 1, characterized in that, The metabolic inflammatory cross-diseases include at least one of the following: sepsis, myocarditis, and inflammatory cardiovascular disease.

5. The application according to claim 4, characterized in that, The therapeutic effects of the drug include at least one of the following: inhibiting HK2 enzyme activity, improving cardiac function, inhibiting inflammatory response, and reducing cardiomyocyte apoptosis.

6. The application according to claim 5, characterized in that, The inhibition of the inflammatory response includes inhibiting at least one of the following inflammatory factors: including TNF-α, IL-1β, IL-6, chemokine Cxcl5, and chemokine Ccl2.

7. A drug for treating cross-diseases of metabolic inflammation, characterized in that, The active ingredients include ailanthus ketone, as well as pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that, The working concentration of ethosone in the drug is not less than 5 mg / kg.

9. The drug according to claim 7, characterized in that, The dosage form of the drug includes injections.

10. The drug according to claim 7, characterized in that, The excipients include solvents, which include a mixture of DMSO, PEG300, Tween-80 and physiological saline.

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