Application of lignoceric acid in the treatment of sepsis

Abstract

The present invention discloses the use of lignoceric acid in the treatment of sepsis, relating to the field of pharmaceutical application technology. The invention involves the use of lignoceric acid or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for preventing and / or treating sepsis. The present invention demonstrates that lignoceric acid can inhibit the inflammatory storm caused by sepsis, thereby enabling the preparation of a medicament for treating sepsis.

Description

Technical Field

[0001] The invention relates to application of lignoceric acid in treating sepsis, and belongs to the technical field of drug application. Background Art

[0002] Sepsis is a systemic, critical illness triggered by infection. Its core pathogenic mechanism is an uncontrolled immune and inflammatory response to pathogens. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) activate immune cells, leading to the release of large amounts of proinflammatory cytokines (such as TNF-α, IL-1β, and IL-6), creating an inflammatory storm. This excessive inflammatory response causes increased vascular permeability, tissue edema, hypoperfusion, and organ damage, and is a key driver of the early pathological changes in sepsis. Subsequently, immunosuppression, coagulation abnormalities, and metabolic disorders may develop, further contributing to the development of multiple organ failure. Therefore, the inflammatory response plays a central role in the pathogenesis of sepsis, and anti-inflammatory therapies that target inflammatory factors or modulate the immune response have become an important strategy for controlling the progression of sepsis and reducing mortality.

[0003] Lignoceric acid (CAS: 557-59-5) is a 24-carbon saturated fatty acid (24:0) synthesized in the developing brain. Lignoceric acid is a byproduct of lignin production and is widely present in plant waxes and oils, such as peanut oil and shepherd's purse oil. It also occurs in trace amounts in animal fats and is a component of sphingolipids such as cerebrosides. Studies have shown that lignoceric acid can be used in the study of Zellweger's encephalohepatic-hepatorenal syndrome and adrenoleukodystrophy, but its therapeutic efficacy in sepsis has not been reported. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an application of lignoceric acid in the treatment of sepsis to inhibit the inflammatory storm caused by sepsis, thereby being used to prepare a drug for treating sepsis.

[0005] To achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0006] The present invention provides use of lignoceric acid or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing and / or treating sepsis.

[0007] Furthermore, the sepsis is induced by lipopolysaccharide.

[0008] Furthermore, the sepsis is induced by lipopolysaccharide of Gram-negative bacteria.

[0009] Furthermore, the lignoceric acid or a pharmaceutically acceptable salt thereof reduces the level of inflammatory cytokines, alleviates lung cell infiltration and reduces the expression of inflammatory cytokines in the lung.

[0010] Furthermore, the inflammatory cytokines include TNF-a and IL-6.

[0011] Furthermore, the lung cells include lung macrophages and lung neutrophils.

[0012] Furthermore, the lung inflammatory cytokines include TNF-a and IL-6 mRNA.

[0013] Furthermore, the safe drug concentration of the lignoceric acid or a pharmaceutically acceptable salt thereof is 1 to 400 μM, calculated as lignoceric acid.

[0014] Furthermore, the dosage of the lignoceric acid or a pharmaceutically acceptable salt thereof is 10-50 mg / kg, calculated as lignoceric acid.

[0015] Furthermore, the administration of the lignoceric acid or a pharmaceutically acceptable salt thereof includes: intraperitoneal injection and oral administration.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention uses C57BL / 6J mice as research subjects to establish a lipopolysaccharide (LPS)-induced sepsis mouse model, and administers intraperitoneal injection of lignoceric acid for treatment. By detecting multiple indicators such as the survival rate of mice in the treatment group and the non-treatment group, inflammatory indicators TNF-a and IL-6 in serum and bronchoalveolar lavage fluid, lung pathological damage, and lung inflammatory cell infiltration, it is effectively demonstrated that lignoceric acid has a certain therapeutic effect on LPS-induced sepsis. Lignoceric acid can be used as a potential drug for the clinical treatment of sepsis and has a good development and application background. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the effect of lignoceric acid concentration on the viability of human macrophage cell line THP-1;

[0019] Figure 2 This is a schematic diagram of the test of the effect of lignoceric acid in treating sepsis;

[0020] Figure 3 Schematic diagram of the effect of lignoceric acid on lung injury, where (a) is a schematic diagram of the staining of lung pathological sections of mice in each group after 24 hours of LPS treatment, and (b) is a schematic diagram of lung injury scores;

[0021] Figure 4 Schematic diagram of MPO immunohistochemical staining in mouse lungs in the test of the effect of lignoceric acid on lung immune cell infiltration;

[0022] Figure 5Schematic diagram of F4 / 80 immunohistochemical staining of mouse lungs in the test of the effect of lignoceric acid on lung immune cell infiltration;

[0023] Figure 6 Schematic diagram of the detection of inflammatory factor transcription levels in mouse lung tissue in the test of lignoceric acid inhibiting inflammatory cytokine release, wherein (a) is a schematic diagram of the detection of inflammatory cytokine IL-6 transcription levels, and (b) is a schematic diagram of the detection of inflammatory cytokine TNF-a transcription levels;

[0024] Figure 7 Schematic diagram of the detection of inflammatory factor levels in mouse serum in the test of lignoceric acid inhibiting the release of inflammatory cytokines, wherein (a) is a schematic diagram of the detection of inflammatory cytokine IL-6 levels, and (b) is a schematic diagram of the detection of inflammatory cytokine TNF-a levels;

[0025] Figure 8 Schematic diagram of the detection of inflammatory factor levels in mouse peritoneal lavage fluid in the test of lignoceric acid inhibiting the release of inflammatory cytokines, wherein (a) is a schematic diagram of the detection of inflammatory cytokine IL-6 levels, and (b) is a schematic diagram of the detection of inflammatory cytokine TNF-a levels;

[0026] Figure 9 This is a schematic diagram of the TLR4 signaling pathway activation and inflammatory response after LPS stimulation of primary peritoneal macrophages in vitro in the test of lignoceric acid inhibiting TLR4 signaling pathway activation, wherein (a) is a schematic diagram of the TLR4 signaling pathway activation after LPS stimulation of primary peritoneal macrophages in vitro, (b) is a schematic diagram of the IL-6 inflammatory factor response after LPS stimulation of primary peritoneal macrophages in vitro; (c) is a schematic diagram of the TNF-α inflammatory factor response after LPS stimulation of primary peritoneal macrophages in vitro. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0028] The experimental materials and equipment used in the examples of the present invention are shown in the following table:

[0029] Table 1: Experimental equipment and instruments

[0030]

[0031] Table 2: Experimental materials and reagents

[0032] Experimental reagents and consumables Product number or batch number company 1.5mL EP tube BS-15-M biosharp biotechnology company 5mL EP tube BS-50-M biosharp biotechnology company 15mL centrifuge tube 430791 Corning Company 50mL centrifuge tube BS-500-M biosharp biotechnology company 1mL syringe K20211213 Shanghai Kindly Co., Ltd. 4% paraformaldehyde P0099 Shanghai Biyuntian Biotechnology Co., Ltd. slides GP26013 Shanghai Keyi Co., Ltd. Cover glass GP26011 Shanghai Keyi Co., Ltd. 75% alcohol 20200928 Sinopharm Group Anhydrous ethanol 10009218 Sinopharm Group Xylene 534056-500ML Sigma, USA Neutral resin S30509 Source Leaf Bio EDTA pH 9.0 antigen retrieval solution 221021S425ak Fuzhou Maixin Biotechnology Development Co., Ltd. DAB color development P0202 Shanghai Biyuntian Biotechnology Co., Ltd. Hematoxylin C0107 Shanghai Biyuntian Biotechnology Co., Ltd. Eosin C0109 Shanghai Biyuntian Biotechnology Co., Ltd. Immunohistochemistry pen ADI-950-233-0001 Aimejie Technology RNA extraction kit SB-R001 Shanghai Shenger Biotechnology Co., Ltd. Reverse transcription reagents R212-01 Nanjing Novizan Co., Ltd. 96-well PCR plate PP-96-HS-0100 Beijing Landje Technology Co., Ltd. THP-1 cells ZQ0086 Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Mouse TNF-α ELISA Kit 88-7324-77 Thermo Fisher Scientific Inc. Mouse IL-6 ELISA Kit 88-7064-88 Thermo Fisher Scientific Inc. 96-well plate 9018 Corning Incorporated MPO antibodies CPTC-MPO-1 Nonprofit Developmental Research Hybridoma Bank F4 / 80 antibody 28463-1-AP Wuhan Sanying Biotechnology Co., Ltd. LPS L2630 Sigma Corporation of America broth B2551 Millipore RPMI 1640 medium C11875500BT Gibco Fetal bovine serum A5256701 Gibco Penicillin and Streptomycin P1400 Solebao 20X TBS ST663 Blue Sky skimmed milk powder A600669 Shenggong Bioengineering Co., Ltd. Tween 20 A600560-0500 Shenggong Bioengineering Co., Ltd. BSA A500023-0100 Shenggong Bioengineering Co., Ltd. RIPA P0013B Blue Sky 5X Loading P0015L Blue Sky ECL luminescent liquid SB-WB004 Shenger Biotechnology Co., Ltd. Protein Marker SB-WB129 Shenger Biotechnology Co., Ltd. SDS-PAGE precast gel SB-FP15010 Shenger Biotechnology Co., Ltd. CCK8 SB-CCK8 Shenger Biotechnology Co., Ltd. Lignoceric acid HY-121883 MCE

[0033] The present invention uses 6-8 week old male C57BL / 6 mice weighing 22±2 g, purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd., and housed in an SPF animal room with an indoor relative temperature of 22±2°C, a relative humidity of 50%~60%, a light-dark alternation time of 12 h, and daily ultraviolet disinfection and sterilization for 30 minutes. The cages are ventilated 10-20 times / hour, 5-7 mice are placed in each cage, and all mice have free access to food and water. The bedding is made of processed, crushed, and sterilized corn cobs, and the bedding is changed once a week.

[0034] Quantitative statistical graphics were generated using Graphpad Prism software, version 9.0. All experimental data were quantitative and normally distributed, expressed as mean ± standard error. Statistical analysis was performed using Graphpad Prism software, version 9.0. If the variances of the groups were equal, t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons between multiple groups. Graphs were plotted using Graphpad Prism software, version 9.0. P < 0.05 indicated statistical significance (* P < 0.05, ** P < 0.01, *** P < 0.001).

[0035] The present invention is analyzed below in conjunction with specific embodiments:

[0036] Example 1: Cytotoxicity test of lignoceric acid

[0037] THP-1 cell culture:

[0038] Human macrophage cell line THP-1 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and placed in an incubator at 37°C and 5% CO2. Cells were passaged when the cell confluence reached more than 90%.

[0039] Lignoceric acid-treated cells:

[0040] Collect cells in the logarithmic growth phase and adjust the cell suspension concentration to 5×10 3 Each well of the 96-well plate was seeded with 100 μL of culture medium and cultured for 24 h to allow the cells to adhere.

[0041] After replacing the culture medium, the concentration gradient groups were added with 0μM, 10μM, 20μM, 50μM, 100μM, 200μM, 400μM, 410μM, 420μM, and 450μM lignoceric acid to treat the cells for 48h, and a control group was set up at the same time, with 3 replicates in each group.

[0042] Cell viability assay:

[0043] After the experimental treatment is completed, remove the 96-well plate and add 10 μL of CCK-8 reagent to each well. Continue incubation for 1 hour. After removal, measure the absorbance (OD value) of each well at 450 nm using a microplate reader. Taking the OD value at 0 h as 1, calculate the relative viability of the remaining time points. The cell proliferation ability is evaluated based on the relative cell viability. The calculation expression of cell viability is:

[0044] Cell viability calculation formula: Cell viability change fold = OD value on day n / OD value on day 0

[0045] The assay was repeated three times.

[0046] Test results such as Figure 1 As shown in the data, there was no significant difference in the cell viability of THP-1 cells at a concentration range of 400 μM lignoceric acid, which indicates that the safe dose range of lignoceric acid for THP-1 cells is within 400 μM.

[0047] Example 2: Testing the Effect of Lignoceric Acid on Sepsis

[0048] Mice were given a lethal dose of LPS (10 mg / kg) by intraperitoneal injection. Subsequently, the treatment groups were given lignoceric acid at doses of 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, and 70 mg / kg using the same method. The drugs were given again 12 hours later, and the survival of the two groups of mice was observed for 120 hours.

[0049] The results are as follows Figure 2 As shown, lignoceric acid increased the survival rate of mice with lipopolysaccharide-induced sepsis from zero to 30% (P < 0.05), with lignoceric acid at a dose of 50 mg / kg having the best therapeutic effect. Doses exceeding 50 mg / kg significantly reduced the therapeutic effect of lignoceric acid, indicating that lignoceric acid has a positive effect in treating sepsis, with the optimal dose being 50 mg / kg.

[0050] Example 3: Lignoceric acid improves sepsis-related lung injury test

[0051] Experimental animal model and drug administration:

[0052] Mice were given a lethal dose of LPS (10 mg / kg) by intraperitoneal injection. Then, the treatment group was given low-dose (Low, 20 mg / kg) and high-dose (High, 50 mg / kg) of lignoceric acid using the same method. The drug was administered again 12 hours later. The mice were killed 24 hours later and samples were collected.

[0053] Mouse lung tissue sample collection:

[0054] After blood collection is complete, place the mouse in the supine position. Wet the chest and abdominal fur with a 75% alcohol-disinfected cotton ball. Grasp the mouse skin with toothed forceps and use ophthalmic scissors to cut the skin from the xiphoid process to the neck. Then, use untoothed forceps to gently lift the xiphoid process to separate the lung tissue from the diaphragm. Make a small incision at the junction of the xiphoid process and the diaphragm. Cut the diaphragm along the left and right sides, and cut the thorax from the left and right ends of the diaphragm toward the head to fully expose the entire chest cavity. Excise the lung tissue, remove the left lobe of the lung, and fix it in 4% paraformaldehyde fixative. Place the remaining tissue in a 1.5mL EP tube, quickly plunge it into liquid nitrogen, and finally transfer it to a -80°C freezer for storage.

[0055] Detection of mouse lung tissue damage and immune cell infiltration:

[0056] H&E and immunohistochemical staining were used to detect lung tissue damage, neutrophil and macrophage infiltration in mice.

[0057] Lung injury pathological score:

[0058] Each group of mice had 5 samples. The H&E-stained lung tissue sections were placed under a light microscope to observe the pathological changes. The lung tissue damage was quantitatively scored based on the bleeding in the mouse lung tissue, whether the alveoli were congested, the infiltration of inflammatory cells including neutrophils and macrophages in the alveolar cavity, and the thickness of the alveolar septa. The scoring criteria are as follows:

[0059] 0: Normal lung tissue; 1: Mild lung injury, ≤ 25% lung involvement; 2: Moderate lung injury, >25% to 50% lung involvement; 3: Severe lung injury, >50% to <75% lung involvement; 4: Very severe, almost the entire lung involved. This procedure was performed blindly by a single investigator who was unaware of the slice number.

[0060] The results of the effects of lignoceric acid on lung injury are as follows Figure 3 As shown in the results, compared with septic mice, the lung tissue damage of mice treated with lignoceric acid was significantly reduced. The results showed that lignoceric acid can significantly improve sepsis-related lung injury, and the therapeutic effect is dose-dependent.

[0061] Effects of lignoceric acid on lung immune cell infiltration Figure 4 and Figure 5 As shown in the results, compared with mice with sepsis, the infiltration of neutrophils and macrophages in the lungs of mice treated with lignoceric acid was significantly reduced. This indicates that lignoceric acid can significantly improve the inflammatory response associated with sepsis, and the therapeutic effect is dose-dependent.

[0062] Example 4: Test on the inhibition of inflammatory cytokine release by lignoceric acid

[0063] Mouse blood sample collection:

[0064] Mice were anesthetized with an intraperitoneal injection of 1% pentobarbital, and blood samples were collected by cardiac puncture. The blood was then allowed to stand at room temperature for 1 hour to allow the serum to precipitate. The blood was then centrifuged at 5000 rpm for 10 minutes in a precooled 4°C centrifuge. The serum was collected and stored at -80°C until further use.

[0065] Mouse peritoneal lavage fluid sample collection:

[0066] After killing the mouse by cervical dislocation, use small scissors to cut open the abdominal fur. Use a 5ml syringe to draw up 5mL of PBS and inject it into the mouse's peritoneal cavity. After gently rubbing the mouse, use the syringe to aspirate the PBS from the mouse's peritoneal cavity. Repeat this lavage process one to two times. During this procedure, be sure not to pierce any internal organs in the mouse's peritoneal cavity with the syringe tip. Keep the lavage fluid on ice. After all mice have been collected, centrifuge them together (300xg, 5 minutes), remove the supernatant, and store at -80°C until needed.

[0067] Inflammatory cytokine (TNF-a, IL-6) detection:

[0068] The levels of inflammatory cytokines TNF-α and IL-6 in mouse serum and peritoneal lavage fluid were detected by ELISA, and the expression of inflammatory cytokines TNF-α and IL-6 in mouse lung tissue was detected by real-time fluorescence quantitative PCR.

[0069] The results of the effects of lignoceric acid on the expression of inflammatory cytokines in the lungs are as follows Figure 6 As shown in the results, compared with septic mice, the expression of inflammatory cytokines TNF-α and IL-6 in the lung tissue of mice treated with lignoceric acid was significantly reduced. Therefore, lignoceric acid can significantly inhibit the expression of inflammatory cytokines in the lungs of septic mice, and the therapeutic effect is dose-dependent.

[0070] The results of the effects of lignoceric acid on the levels of inflammatory cytokines are as follows Figure 7 and Figure 8 As shown in the results, compared with mice with sepsis, lignoceric acid treatment significantly reduced TNF-α and IL-6 levels in the serum and peritoneal lavage fluid of mice. Therefore, lignoceric acid significantly inhibited inflammatory cytokine levels in mice with sepsis. The therapeutic effect was dose-dependent.

[0071] Example 5: Test on the inhibition of TLR4 signaling pathway activation by lignoceric acid

[0072] Isolation and in vitro stimulation of mouse peritoneal macrophages:

[0073] Mice were intraperitoneally injected with 3-4 ml of broth (prepared in advance, autoclaved, and stored at room temperature, protected from light). Three days later, the mice were sacrificed by cervical dislocation. The outer layer of peritoneal fur was cut open with a small pair of scissors (do not rupture the peritoneum). 5 ml of PBS was drawn up using a 5 ml syringe and injected into the mouse's peritoneal cavity, gently agitating the surrounding tissue. The PBS in the peritoneal cavity was then aspirated and placed into a 15 ml centrifuge tube. A second 5 ml PBS lavage was performed to obtain more peritoneal macrophages. The cells were centrifuged at 1200 rpm for 10 minutes, resuspended in RPMI 1640 medium containing 10% FBS, counted, and plated in a microplate. After stimulation with LPS (100 ng / ml) and pyrimethicone (10 μM), the cells were harvested and protein samples were prepared using RIPA lysis buffer. Protein samples were then diluted with 5X loading buffer, denatured in a metal bath at 90°C for 10 minutes, and stored at -80°C until further use.

[0074] TLR4 signaling pathway activation test:

[0075] Western blot was used to detect the phosphorylation levels of p65 and IKBα, and the expression level of GAPDH in primary peritoneal macrophages; real-time fluorescence quantitative PCR was used to detect the expression of TNF-α and IL-6 in primary peritoneal macrophages.

[0076] The above test results are as follows Figure 9 As shown, lignoceric acid significantly inhibited the phosphorylation of p65 and IKBα in primary mouse peritoneal macrophages stimulated with LPS, while maintaining no significant changes in p65 and IKB protein expression. Furthermore, lignoceric acid significantly suppressed the expression of inflammatory cytokines TNF-α and IL-6. These results demonstrate that lignoceric acid significantly inhibits TLR4 signaling pathway activation and inflammatory responses.

[0077] In summary, lignoceric acid is an effective treatment for sepsis. In vivo, it significantly improved the survival rate of septic mice, improved lung damage in septic mice, and inhibited the expression and release of inflammatory cytokines. Furthermore, in vitro, it significantly inhibited the activation of the TLR4 signaling pathway and suppressed the inflammatory response. Therefore, lignoceric acid could be used to prepare drugs for the treatment of sepsis and related inflammatory diseases.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of lignoceric acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating sepsis.

2. The use according to claim 1, characterized in that The sepsis was induced by lipopolysaccharide.

3. The use according to claim 2, characterized in that The sepsis is induced by lipopolysaccharide of Gram-negative bacteria.

4. The use according to claim 1, characterized in that The lignoceric acid or a pharmaceutically acceptable salt thereof can reduce the level of inflammatory cytokines, alleviate lung cell infiltration and reduce the expression of lung inflammatory cytokines.

5. The use according to claim 4, characterized in that The inflammatory cytokines include TNF-a and IL-6.

6. The use according to claim 4, characterized in that The lung cells include lung macrophages and lung neutrophils.

7. The use according to claim 4, characterized in that The lung inflammatory cytokines include TNF-a and IL-6 mRNA.

8. The use according to claim 1, characterized in that The safe dosage concentration of lignoceric acid or a pharmaceutically acceptable salt thereof is 1 to 400 μM, where the concentration is calculated based on lignoceric acid.

9. The use according to claim 1, characterized in that The dosage of the lignoceric acid or a pharmaceutically acceptable salt thereof is 10-50 mg / kg, with the concentration being calculated based on lignoceric acid.

10. The use according to claim 1, characterized in that The administration methods of the lignoceric acid or its pharmaceutically acceptable salt include: intraperitoneal injection and oral administration.

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