Use of squalene in the preparation of a medicament for the treatment of bacterial pneumonia

Abstract

The application belongs to the technical field of biological medicine, and provides an application of squalene in preparation of a drug for treating bacterial pneumonia, wherein the squalene can be used as an effective active component of the drug, can significantly improve the survival rate of a mouse model of bacterial pneumonia induced by pathogenic bacteria such as Klebsiella pneumoniae or Staphylococcus aureus, and can reduce weight loss and clinical symptoms. Specifically, the squalene can effectively reduce the level of a proinflammatory factor in the lung, increase the level of an anti-inflammatory factor, reduce the bacterial load in blood and main organs, inhibit the polarization of alveolar macrophages to a proinflammatory M1 type, and thus significantly improve the pathological damage of lung tissue. The application provides a safe, effective and natural drug which is not prone to drug resistance for clinical treatment of bacterial pneumonia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of squalene in the preparation of drugs for treating bacterial pneumonia. Background Technology

[0002] Bacterial pneumonia is a major infectious disease that poses a serious threat to human and animal health worldwide, characterized by high morbidity, high mortality, and a high medical burden, particularly affecting infants, the elderly, immunocompromised individuals, and severely ill patients. Klebsiella pneumoniae (Kp), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (PA) are among the main pathogens causing bacterial pneumonia. Kp, as a Gram-negative bacterium, produces LPS, a key virulence factor that triggers the body's immune response and inflammatory reaction. Infection with Kp is characterized by rapid onset, rapid progression, and high mortality, with a significantly increased mortality rate, especially in immunocompromised populations.

[0003] An imbalance between innate and adaptive immunity in the lungs is the core mechanism of bacterial pneumonia pathogenesis. Alveolar macrophages, as the first line of immune defense in the lungs, play a crucial role in pathogen recognition and clearance. When Kp infects the lungs, LPS in its cell wall binds to TLR4 receptors on the macrophage surface, activating inflammatory signaling pathways such as NF-κB, causing macrophages to polarize into the M1 type. M1 macrophages release large amounts of pro-inflammatory factors such as IL-6, TNF-α, and IL-1β, triggering a "cytokine storm" in the lungs, leading to lung congestion, edema, inflammatory cell infiltration, and even alveolar structural damage. At the same time, the overpolarized M1 macrophages have impaired phagocytic and bactericidal functions, failing to effectively clear pathogens, leading to the dissemination of pathogens in the lungs and other organs, exacerbating the infection.

[0004] Currently, the core clinical treatment for bacterial pneumonia is antibiotic therapy, including cephalosporins and carbapenems. However, their mechanism of action is mainly to directly kill or inhibit the reproduction of pathogens, failing to regulate the body's inflammatory response and lacking therapeutic effect on excessive inflammatory damage induced by LPS. Furthermore, long-term and high-dose use of antibiotics has led to the emergence of multidrug-resistant Klebsiella pneumoniae strains, becoming a major challenge in clinical treatment. Antibiotics can also disrupt the balance of intestinal flora, causing side effects such as liver and kidney toxicity and diarrhea, limiting patient tolerance. In addition, existing synthetic anti-inflammatory and anti-pneumonia materials (such as carbon dot nanomaterials) have complex synthesis processes, high costs for large-scale production, and their in vivo metabolic safety has not been fully verified, limiting their clinical application. Existing natural anti-inflammatory substances mostly focus on basic inflammatory models, and there is a lack of research specifically targeting Klebsiella pneumoniae-induced bacterial pneumonia.

[0005] Squalene is a natural unsaturated triterpenoid hydrocarbon compound widely found in deep-sea fish oil, olive oil, shark liver oil, and other animal and plant products. It is a precursor to cholesterol synthesis and possesses excellent biocompatibility, no immune rejection, and is easily metabolized without residue in the body. Current research confirms that squalene has antioxidant, basic anti-inflammatory, and immune-regulating effects, and it has been widely used in health supplements, vaccine adjuvants, and cosmetics. However, there are currently no reports on the use of squalene in treating bacterial pneumonia, particularly in regulating alveolar macrophage polarization and improving Klebsiella pneumoniae-induced lung inflammation. Summary of the Invention

[0006] The purpose of this invention is to provide the application of squalene in the preparation of drugs for treating bacterial pneumonia, in order to solve the problems raised in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The use of squalene in the preparation of a drug for treating bacterial pneumonia, wherein the pathogens of said bacterial pneumonia include Klebsiella pneumoniae and / or Staphylococcus aureus.

[0008] Furthermore, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

[0009] Furthermore, the drug comprises an effective amount of squalene and pharmaceutically acceptable excipients.

[0010] Furthermore, the dosage form of the drug is an injection.

[0011] Furthermore, the dosage of squalene in the drug is 50-100 mg / kg of individual body weight.

[0012] Furthermore, the squalene is used to downregulate pro-inflammatory factors IL-6, TNF-α, and IL-1β, and to upregulate anti-inflammatory factor IL-10.

[0013] Furthermore, the squalene is used to inhibit the M1 polarization of alveolar macrophages.

[0014] Another object of the present invention is to provide the use of squalene in the preparation of a medicament for inhibiting pathogens of bacterial pneumonia, wherein the pathogens of bacterial pneumonia include Klebsiella pneumoniae and / or Staphylococcus aureus.

[0015] This invention provides the application of squalene in the preparation of drugs for treating bacterial pneumonia. Squalene serves as the active ingredient in these drugs, significantly improving the survival rate of mouse models of bacterial pneumonia induced by pathogens such as Klebsiella pneumoniae or Staphylococcus aureus, and reducing weight loss and clinical symptoms. Its mechanism of action involves effectively reducing the levels of pro-inflammatory factors (such as IL-6, TNF-α, and IL-1β) in the lungs, increasing the levels of anti-inflammatory factors (such as IL-10), reducing the bacterial load in the blood and major organs (such as the lungs, spleen, and kidneys), and inhibiting the polarization of alveolar macrophages towards the pro-inflammatory M1 type, thereby significantly improving pathological damage to lung tissue. This invention provides a safe, effective, and less likely to induce drug resistance natural drug for the clinical treatment of bacterial pneumonia, overcoming the technical shortcomings of existing antibiotic therapies, such as drug resistance and the inability to regulate excessive inflammatory responses. Attached Figure Description

[0016] Figure 1 The figures show comparisons of survival rates, weight changes, and clinical scores among the mice in each group. In the figures, A is a comparison of survival rates among the control group, Kp infection group, and 50 mg / kg squalene treatment group; B is a comparison of weight changes among the control group, Kp infection group, and 50 mg / kg squalene treatment group; C is a comparison of clinical scores among the control group, Kp infection group, and 50 mg / kg squalene treatment group; and D is a comparison of survival rates among the control group, MRSA infection group, and 100 mg / kg squalene treatment group.

[0017] Figure 2 The figures show the results of detecting the expression levels of inflammatory factors in the control group, Kp infection group, and 50 mg / kg squalene treatment group. In the figures, A shows the detection results of the mRNA level of pro-inflammatory factor IL-6; B shows the detection results of the mRNA level of pro-inflammatory factor TNF-α; C shows the detection results of the mRNA level of pro-inflammatory factor IL-1β; and D shows the detection results of the mRNA level of anti-inflammatory factor IL-10.

[0018] Figure 3 The image shows a comparison of bacterial load in organs between the Kp infection group and the 50 mg / kg squalene treatment group. In the image, A represents the bacterial load test results in blood; B represents the bacterial load test results in the lungs; C represents the bacterial load test results in the kidneys; and D represents the bacterial load test results in the spleen.

[0019] Figure 4 The graph shows the results of macrophage polarization detection. In the graph, A is the flow cytometry results of the control group, Kp infection group, and 50 mg / kg squalene treatment group; B is the statistical graph of the proportion of M1 macrophages in the control group, Kp infection group, and 50 mg / kg squalene treatment group.

[0020] Figure 5 H&E staining images of lungs from the control group, Kp infection group, and 50 mg / kg squalene treatment group.

[0021] In the figure: * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001; ns indicates P≥0.05. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] In one embodiment of the present invention, the application of squalene in the preparation of a drug for treating bacterial pneumonia is provided. This embodiment of the present invention, based on the pathogenesis of bacterial pneumonia, uses alveolar macrophages as target cells to explore the therapeutic effect of squalene on bacterial pneumonia. The pathogens of bacterial pneumonia include, but are not limited to, Klebsiella pneumoniae (Kp) and / or Staphylococcus aureus; Staphylococcus aureus is preferably methicillin-resistant Staphylococcus aureus (MRSA).

[0024] In a preferred embodiment of the present invention, the medicament for treating bacterial pneumonia includes an effective amount of squalene and pharmaceutically acceptable excipients; specifically, the dosage form of the medicament is an injection, but not limited thereto; the dosage of squalene in the medicament is 50-100 mg / kg of individual body weight.

[0025] This invention demonstrates that squalene can be used as an effective active ingredient in the treatment of bacterial pneumonia, achieving multiple effects such as relieving lung inflammation, clearing pathogens, and protecting against lung tissue damage. In in vivo experiments, intravenous injection of 50 mg / kg squalene significantly reduced the mortality rate of a Kp-induced bacterial pneumonia mouse model, increasing the survival rate from 20% to 90%, and reducing weight loss and alleviating clinical symptoms. Furthermore, intravenous injection of 100 mg / kg squalene significantly reduced the mortality rate of a drug-resistant Staphylococcus aureus (MRSA)-induced bacterial pneumonia mouse model. Squalene can reduce the degree of inflammatory response in the lungs of bacterial pneumonia mouse models, significantly reducing the mRNA levels of IL-6, TNF-α, and IL-1β in the lungs, increasing the mRNA level of IL-10, reducing organ bacterial load, inhibiting macrophage M1 polarization, and significantly improving pathological changes in the lungs. This invention is the first to apply squalene to the treatment of bacterial pneumonia, providing a safe, effective, and non-resistant natural treatment for clinical use, helping to solve the core problems of antibiotic resistance and insufficient anti-inflammatory effects in existing drugs.

[0026] Squalene is natural, safe, and widely available, and is a liquid at room temperature. The squalene used in the embodiments of this invention is a light yellow liquid with a purity of ≥98%, a molecular weight of 410.72, CAS number 111-02-4, and chemical formula: CC(C)=CCCC(C)=CCCC(C)=CCC\C=C( / C)CCC=C(C)CCC=C(C)C, with the following structural formula: ; Since squalene is insoluble in water, a culture medium containing serum was used as a carrier for dissolution in the following cell experiments, and 0.5% of the total solvent in Tween-80 was used for dissolution in the animal experiments.

[0027] It should be noted that, unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, all reagents and materials used are commercially available.

[0028] Example 1: This example verifies the effect of squalene on the mortality rate of mice with Kp and MRSA-induced bacterial pneumonia, as detailed below: Thirty female C57BL / 6 mice, weighing 18-20g, were randomly divided into three groups of 10 mice each: a control group, a Kp infection group, and a 50mg / kg squalene treatment group. Kp was cultured in LB medium until OD=1.5. One mL of bacterial suspension was collected, centrifuged at 7000 rpm for 5 min, and the bacteria were collected, washed twice with physiological saline, and resuspended in 1 mL of physiological saline. The control group was treated with the solvent but not infected with Kp. Mice in the Kp infection group and the 50mg / kg squalene treatment group were anesthetized with 140μL of sodium pentobarbital and then infected with a lethal dose (1×10⁻⁶). 4 A bacterial pneumonia mouse model was established by intranasal infection with *K. pylori* (CFU / mouse). In the squalene treatment group, mice were injected intravenously with 50 mg / kg squalene 2 hours after infection, followed by re-injections at 24 and 36 hours, for a total of three injections. Survival, weight changes, and clinical symptoms were then observed. Results showed that 50 mg / kg squalene increased the survival rate of mice infected with *K. pylori* from 20% to 90%, reduced weight loss, and alleviated clinical symptoms (such as...). Figure 1 (As shown in AC). Furthermore, following the same method, mice were randomly divided into three groups: a control group, an MRSA infection group, and a 50 mg / kg squalene treatment group; the control group was treated with solvent and did not undergo MRSA infection; the MRSA infection group and the 100 mg / kg squalene treatment group were intranasally infected with a lethal dose of MRSA (2 × 10⁻⁶). 8 CFU / mouse); the 100 mg / kg squalene treatment group received a tail vein injection of squalene at a dose of 100 mg / kg 2 hours after infection, followed by injections at 24 hours and 36 hours, for a total of 3 injections. Results showed that 100 mg / kg squalene increased the survival rate of MRSA-infected mice from 17% to 83% (e.g., CFU / mouse). Figure 1 As shown in D).

[0029] Example 2: This example verifies that squalene can alleviate Kp-induced bacterial pneumonia in mice, as detailed below: Following the method in Example 1, 18 female C57BL / 6 mice (18-20g) were randomly divided into three groups of six each: a control group, a Kp infection group, and a 50mg / kg squalene treatment group. Kp was cultured in LB medium until OD=1.5. 1mL of bacterial suspension was collected, centrifuged at 7000rpm for 5min, and after collection, washed twice with physiological saline, then resuspended in 1mL of physiological saline. The control group was treated with the solvent but not infected with Kp. Mice in the Kp infection group and the 50mg / kg squalene treatment group were anesthetized with 140μL of sodium pentobarbital and each mouse was given a sublethal dose (5×10⁻⁶). 330 μL of CFU / mL squalene was administered intranasally to infect Kp. In the 50 mg / kg squalene treatment group, squalene was injected intravenously at a dose of 50 mg / kg 2 hours after infection, and repeated at 24 and 48 hours for a total of three injections. At 60 hours after infection, the heart was anesthetized with 140 μL of sodium pentobarbital, and the mRNA levels of IL-6, TNF-α, IL-1β, and IL-10 in the mouse lungs were detected by RT-qPCR. The results showed that squalene significantly reduced the mRNA levels of pro-inflammatory factors IL-6, TNF-α, and IL-1β in the lungs, and increased the mRNA level of the anti-inflammatory factor IL-10 (e.g., CFU / mL, 30 μL). Figure 2 (As shown in AD) Example 3: This example verifies that squalene can reduce the bacterial load in the organs of mice with Kp-induced bacterial pneumonia, as detailed below: Mice were grouped, Kp infected, and administered the drug as in Example 2. Blood was collected from the tail vein of mice in each group and serially diluted before being plated. For lungs, spleen, and kidneys, 0.1g of the drug was added to 300μL of physiological saline, ground, serially diluted, and then plated. After overnight incubation, bacterial counts were performed. The results showed that 50mg / kg of squalene significantly reduced the bacterial load in mouse blood, lungs, kidneys, and spleen (e.g., ...). Figure 3 (As shown in AD).

[0030] Example 4: This example verifies that squalene can inhibit M1 polarization in macrophages, as detailed below: Mice were grouped, Kp infected, and administered the drugs as in Example 2. After cardiac perfusion, the lungs of mice in each group were ground and digested in digestive fluid at 37°C for 30 min. After washing with PBS, the cells were centrifuged at 4°C for 500g for 5 min. 2 mL of erythrocyte lysis buffer was added, mixed, and lysed for 3-5 min. 6 mL of PBS was added to stop the lysis, and the cells were passed through a 200-mesh sieve and centrifuged at 4°C for 500g for 5 min. The supernatant was discarded. Finally, the cells were resuspended in 300 μL of PBS, filtered through a 300-mesh filter, and centrifuged to remove the supernatant. All procedures were performed on ice. After staining with CD86 antibody by flow cytometry, macrophage polarization was detected by flow cytometry. The results showed that squalene significantly inhibited M1 polarization of macrophages (e.g., ...). Figure 4 (As shown in AB).

[0031] Example 5: This example verifies that squalene can alleviate Kp-induced bacterial pneumonia lung damage in mice, as detailed below: Mice were grouped, Kp infected, and administered the drug as in Example 2. After cardiac perfusion, a small piece of lung tissue was cut from each group of mice, preserved in 4% formaldehyde, and observed under a microscope after H&E staining. The results showed that squalene could significantly reduce inflammatory cell infiltration, alveolar wall collapse, and alveolar wall thickening in the lungs of mice with Kp-induced bacterial pneumonia (e.g., Figure 5 (As shown).

[0032] In summary, the embodiments of the present invention demonstrate for the first time that squalene can be used as an effective active ingredient for the treatment of bacterial pneumonia, and it can achieve the following beneficial technical effects: 1. Squalene can inhibit excessive inflammatory response in the lungs from the immune mechanism level by regulating alveolar macrophage polarization, while enhancing the body's ability to clear pathogens, thus overcoming the shortcomings of traditional antibiotics that can only kill bacteria but not fight inflammation.

[0033] 2. Squalene can downregulate pro-inflammatory factors IL-6, TNF-α, and IL-1β, and upregulate anti-inflammatory factor IL-10, thus blocking the occurrence of "cytokine storm" in the lungs from the source. In vivo experiments have confirmed that it can simultaneously reduce the inflammatory response in the lungs, alleviate systemic inflammatory damage, improve pathological changes in the lungs, and provide comprehensive therapeutic effects.

[0034] 3. Squalene is a naturally extracted active substance with a wide range of sources. It can be extracted in large quantities from plants and animals such as deep-sea fish oil, olive oil, and shark liver oil, or it can be synthesized through microbial fermentation. The raw materials are readily available and inexpensive.

[0035] 4. The mechanism of action of squalene is to regulate the polarization of immune cells and the balance of inflammatory factors in the body. Unlike the mechanism of antibiotics that directly kill bacteria, it does not induce drug resistance in pathogens. It can effectively solve the problem of multidrug-resistant bacteria caused by antibiotic abuse and is suitable for long-term clinical application.

[0036] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. Application of squalene in the preparation of drugs for treating bacterial pneumonia.

2. The application according to claim 1, characterized in that, The pathogens causing the bacterial pneumonia include Klebsiella pneumoniae and / or Staphylococcus aureus.

3. The application according to claim 2, characterized in that, The Staphylococcus aureus mentioned is methicillin-resistant Staphylococcus aureus.

4. The application according to claim 1, characterized in that, The drug comprises an effective amount of squalene and pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The drug is in the form of an injection.

6. The application according to claim 4, characterized in that, The dosage of squalene in the drug is 50-100 mg / kg of individual body weight.

7. The application according to claim 1, characterized in that, The squalene is used to downregulate pro-inflammatory factors IL-6, TNF-α, and IL-1β, and to upregulate anti-inflammatory factor IL-10.

8. The application according to claim 1, characterized in that, The squalene is used to inhibit the M1 polarization of alveolar macrophages.

9. The use of squalene in the preparation of a drug for inhibiting pathogens causing bacterial pneumonia, characterized in that, The pathogens causing the bacterial pneumonia include Klebsiella pneumoniae and / or Staphylococcus aureus.

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