Application of xylinic acid in treatment of sepsis disease

Abstract

The invention discloses application of xylinic acid in treatment of sepsis, relates to the technical field of medicine application, and comprises application of xylinic acid or pharmaceutically acceptable salt thereof in preparation of a medicine composition for preventing and / or treating sepsis. The application proves that the xylinic acid can inhibit inflammation storm caused by sepsis, so that the xylinic acid is used for preparing the medicine for treating sepsis.

Description

Technical Field

[0001] The present invention relates to the application of lignoceric acid in the treatment of sepsis, belonging to the technical field of drug application. Background Art

[0002] Sepsis is a systemic critical illness caused by infection, and its core pathogenic mechanism is the uncontrolled immune and inflammatory response of the body to pathogens. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) activate immune cells, leading to the release of a large number of pro-inflammatory factors (such as TNF-α, IL-1β, IL-6), forming an inflammatory storm. This excessive inflammatory response causes increased vascular permeability, tissue edema, hypoperfusion, and organ damage, which is the key driving factor for the early pathological changes of sepsis. Subsequently, immunosuppression, coagulation abnormalities, and metabolic disorders may occur, further promoting the development of multiple organ failure. Therefore, the inflammatory response plays a core role in the pathogenesis of sepsis, and anti-inflammatory treatment targeting inflammatory factors or regulating the immune response has become one of the important strategies to control the progression of sepsis and reduce the mortality rate.

[0003] Lignoceric acid (CAS: 557-59-5) is a 24-carbon saturated fatty acid (24:0) synthesized in the developing brain. Lignoceric acid is also a by-product of lignin production and is widely present in plant waxes and oils, such as peanut oil and shepherd's purse oil, and is present in trace amounts in animal fats. It is a component of sphingolipids such as cerebrosides. Existing studies have shown that lignoceric acid can be used in the research of Zellweger cerebrohepatorenal syndrome and adrenoleukodystrophy, but the therapeutic effect of lignoceric acid 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, inhibiting the inflammatory storm caused by sepsis, so as to be used in the preparation of drugs for the treatment of sepsis.

[0005] To achieve the above purpose, the present invention is implemented by the following technical solutions: The present invention provides an application of lignoceric acid or a pharmaceutically acceptable salt thereof in the preparation of drugs for preventing and / or treating sepsis.

[0006] Further, the sepsis is induced by lipopolysaccharide.

[0007] Further, the sepsis is induced by the lipopolysaccharide of Gram-negative bacteria.

[0008] Further, the lignoceric acid or a pharmaceutically acceptable salt thereof reduces the level of inflammatory cytokines, alleviates pulmonary cell infiltration, and reduces the expression of pulmonary inflammatory cytokines.

[0009] Furthermore, the inflammatory cytokines include TNF-a and IL-6.

[0010] Furthermore, the lung cells include lung macrophages and lung neutrophils.

[0011] Furthermore, the lung inflammatory cytokines include TNF-a and IL-6 mRNA.

[0012] Furthermore, the safe dosage concentration of ximenic acid or its pharmaceutically acceptable salt is 1 - 400 µM, calculated as ximenic acid.

[0013] Furthermore, the dosage of ximenic acid or its pharmaceutically acceptable salt is 10 - 50 mg / kg, calculated as ximenic acid.

[0014] Furthermore, the administration methods of ximenic acid or its pharmaceutically acceptable salt include: intraperitoneal injection and oral administration.

[0015] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: The present invention uses C57BL / 6J mice as the research object, establishes a lipopolysaccharide (LPS)-induced sepsis mouse model, and treats it by intraperitoneal injection of ximenic acid. By detecting multiple indicators such as the survival rate of mice in the administration group and the non-administration group, the inflammatory indicators TNF-a and IL-6 in serum and bronchoalveolar lavage fluid, lung pathological damage, and lung inflammatory cell infiltration, it effectively proves that ximenic acid has a certain therapeutic effect on LPS-induced sepsis. Ximenic 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

[0016] Figure 1 It is a schematic diagram showing the effect of ximenic acid concentration on the viability of human macrophage cell line THP-1; Figure 2 It is a schematic diagram showing the test of the effect of ximenic acid in treating sepsis; Figure 3 It is a schematic diagram showing the effect of ximenic acid on lung injury. Among them, (a) is a schematic diagram of the staining of lung pathological sections of mice in each group after LPS treatment for 24 h, and (b) is a schematic diagram of lung injury score; Figure 4 It is a schematic diagram of MPO immunohistochemical staining of mouse lungs in the test of the effect of ximenic acid on pulmonary immune cell infiltration; Figure 5 It is a schematic diagram of F4 / 80 immunohistochemical staining of mouse lungs in the test of the effect of ximenic acid on pulmonary immune cell infiltration; Figure 6Schematic diagram for detecting the transcriptional levels of inflammatory factors in mouse lung tissue in the test of the inhibition of inflammatory cytokine release by montanic acid. Among them, (a) is the schematic diagram for detecting the transcriptional level of inflammatory cytokine IL-6, and (b) is the schematic diagram for detecting the transcriptional level of inflammatory cytokine TNF-a; Figure 7 Schematic diagram for detecting the levels of inflammatory factors in the serum of mice in the test of the inhibition of inflammatory cytokine release by montanic acid. Among them, (a) is the schematic diagram for detecting the level of inflammatory cytokine IL-6, and (b) is the schematic diagram for detecting the level of inflammatory cytokine TNF-a; Figure 8 Schematic diagram for detecting the levels of inflammatory factors in the peritoneal lavage fluid of mice in the test of the inhibition of inflammatory cytokine release by montanic acid. Among them, (a) is the schematic diagram for detecting the level of inflammatory cytokine IL-6, and (b) is the schematic diagram for detecting the level of inflammatory cytokine TNF-a; Figure 9 Schematic diagram for the activation of TLR4 signaling pathway and inflammatory response after stimulating primary peritoneal macrophages with LPS in vitro in the test of the inhibition of TLR4 signaling pathway activation by montanic acid. Among them, (a) is the schematic diagram for the activation of TLR4 signaling pathway after stimulating primary peritoneal macrophages with LPS in vitro, (b) is the schematic diagram for the response of IL-6 inflammatory factor after stimulating primary peritoneal macrophages with LPS in vitro; (c) is the schematic diagram for the response of TNF-α inflammatory factor after stimulating primary peritoneal macrophages with LPS in vitro. Detailed implementation mode

[0017] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and cannot be used to limit the protection scope of the present invention.

[0018] The experimental materials, equipment and instruments used in the embodiments of the present invention are shown in the following table: Table 1: Experimental equipment and instruments

[0019] Table 2: Experimental materials and reagents Experimental reagents and consumables Article number or batch number Company 1.5 mL EP tube BS-15-M biosharp Biotechnology Co., Ltd. 5 mL EP tube BS-50-M biosharp Biotechnology Co., Ltd. 15 mL centrifuge tube 430791 Corning Incorporated, USA 50 mL centrifuge tube BS-500-M biosharp Biotechnology Co., Ltd. 1 mL syringe K20211213 Shanghai Kantele Co., Ltd. 4% Paraformaldehyde P0099 Shanghai Beyotime Biotechnology Co., Ltd. Microscope slide GP26013 Shanghai Keyi Co., Ltd. Coverslip GP26011 Shanghai Keyi Co., Ltd. 75% Alcohol 20200928 Sinopharm Group Absolute ethanol 10009218 Sinopharm Group Xylene 534056-500ML Sigma-Aldrich Co., LLC, USA Neutral resin S30509 Yuanye Bio-Technology Co., Ltd. EDTA pH9.0 Antigen retrieval solution 221021S425ak Fuzhou Maixin Biotechnology Development Co., Ltd. DAB Chromogenic P0202 Shanghai Beyotime Biotechnology Co., Ltd. Hematoxylin C0107 Shanghai Beyotime Biotechnology Co., Ltd. Eosin C0109 Shanghai Beyotime Biotechnology Co., Ltd. Immunohistochemistry pen ADI-950-233-0001 Aimix Group Inc. RNA extraction kit SB-R001 Shanghai Shener Biotechnology Co., Ltd. Reverse transcription reagent R212-01 Nanjing Novizan Biotech Co., Ltd. 96-well PCR plate PP-96-HS-0100 Beijing Landztech Co., Ltd. THP-1 cells ZQ0086 Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Mouse TNF-α ELISA Kit 88-7324-77 Thermo Fisher Scientific Inc., USA Mouse IL-6 ELISA Kit 88-7064-88 Thermo Fisher Scientific Inc., USA 96-well plate 9018 Corning Incorporated, USA MPO antibody CPTC-MPO-1 The Developmental Studies Hybridoma Bank (non-profit) F4 / 80 antibody 28463-1-AP Wuhan Sanying Biotechnology Co., Ltd. LPS L2630 Sigma-Aldrich Co., LLC, USA Broth B2551 Millipore RPMI 1640 Medium C11875500BT Gibco Fetal bovine serum A5256701 Gibco Penicillin-streptomycin P1400 Solarbio 20X TBS ST663 Beyotime Skim milk powder A600669 Sangon Biotech (Shanghai) Co., Ltd. Tween 20 A600560-0500 Sangon Biotech (Shanghai) Co., Ltd. BSA A500023-0100 Sangon Biotech (Shanghai) Co., Ltd. RIPA P0013B Beyotime 5X Loading P0015L Beyotime ECL Luminescence solution SB-WB004 Shener Biotechnology Co., Ltd. Protein Marker SB-WB129 Shener Biotechnology Co., Ltd. SDS-PAGE precast gel SB-FP15010 Shener Biotechnology Co., Ltd. CCK8 SB-CCK8 Shener Biotechnology Co., Ltd. Xylic acid HY-121883 MCE In the embodiments of the present invention, male C57BL / 6 mice aged 6 - 8 weeks with a body weight of 22 ± 2 g were purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd. and raised in a SPF - level animal room. The indoor relative temperature was 22 ± 2 °C, the relative humidity was 50% - 60%, the light - dark cycle was 12 h, and the indoor was disinfected with ultraviolet light for 30 minutes every day; the air change rate of the mouse cages was 10 - 20 times per hour, 5 - 7 mice were placed in each cage, all mice could freely eat and drink, and the bedding was processed, broken, disinfected and sterilized corncob, which was replaced once a week.

[0020] Graphpad Prism software version 9.0 was used to make quantitative statistical pictures. All experimental data were measurement data. The normal distribution was expressed as mean ± standard error. Statistical analysis was performed using Graphpad Prism software version 9.0. If the variances of each group were homogeneous, the t - test was used for comparison between two groups, and one - way ANOVA (one - factor analysis of variance) was used for comparison among multiple groups. Graphpad Prism software version 9.0 was used for drawing, and P < 0.05 indicated statistical significance (* P < 0.05, ** P < 0.01, *** P < 0.001).

[0021] Next, the present invention will be analyzed in combination with specific embodiments: Example 1: Cytotoxicity test of ximenic acid Cultivation of THP - 1 cells: 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. Passage was carried out when the cell confluence reached more than 90%.

[0022] Treatment of cells with ximenic acid: Cells in the logarithmic growth phase were collected, and the cell suspension concentration was adjusted to 5×10 3 cells / well and seeded into 96 - well plates. 100 μL of medium was added to each well, and the cells were cultured for 24 h to allow cell attachment.

[0023] After changing the culture medium, cells were treated with ximenic acid at concentrations of 0 μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 400 μM, 410 μM, 420 μM, 450 μM for 48 h in concentration - gradient groups respectively, and a control group was set up, with 3 replicates in each group.

[0024] Detection of cell viability: After the treatment according to the experimental method was completed, the 96-well plate was taken out, 10 μL of CCK-8 reagent was added to each well, and the culture was continued for 1 hour. After taking out, the absorbance value (OD value) of each well was measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Taking the OD value at 0 h as 1, the relative viability at the remaining time points was calculated respectively, and the cell proliferation ability was evaluated according to the relative cell viability. The calculation formula for cell viability is: Cell viability calculation formula: Fold change in cell viability = OD value on the nth day / OD value on the 0th day The detection was repeated three times.

[0025] The detection results are as Figure 1 shown. There was no significant difference in the cell viability of THP-1 cells under the condition of lignoceric acid within the concentration range of 400 μM, indicating that the safe dose range of lignoceric acid for THP-1 cells is within 400 μM.

[0026] Example 2: Test on the effect of lignoceric acid in treating sepsis Mice were given a lethal dose of LPS (10 mg / kg) by intraperitoneal injection, and then the treatment group was given lignoceric acid at doses of 20 mg / kg, 30 mg / kg, 50 mg / kg, 60 mg / kg, and 70 mg / kg by the same method. After 12 h, the drug was administered again, and finally the survival of the two groups of mice was observed for 120 h.

[0027] The results are as Figure 2 shown. Lignoceric acid can increase the survival rate of lipopolysaccharide-induced septic mice from zero to 30% (P < 0.05), and the lignoceric acid at a dose of 50 mg / kg has the best therapeutic effect. When the dose exceeds 50 mg / kg, the therapeutic effect of lignoceric acid is significantly reduced, indicating that lignoceric acid has a certain positive effect on the treatment of sepsis, and the optimal dose is 50 mg / kg.

[0028] Example 3: Test on the improvement of sepsis-related lung injury by lignoceric acid Experimental animal modeling and drug administration method: Mice were given a lethal dose of LPS (10 mg / kg) by intraperitoneal injection, and then the treatment group was given low-dose (Low, 20 mg / kg) and high-dose (High, 50 mg / kg) lignoceric acid by the same method. After 12 h, the drug was administered again, and the mice were sacrificed after 24 h to collect samples.

[0029] Collection of mouse lung tissue samples: After blood collection, the mice were fixed in the supine position. The fur on the chest and abdomen of the mice was moistened with a cotton ball soaked in 75% alcohol. The skin of the mice was picked up with forceps with teeth, and the skin from the xiphoid process to the neck of the mice was cut open with an ophthalmic scissors. Then, the xiphoid process was gently lifted with forceps without teeth to separate the lung tissue from the diaphragm. A small incision was made at the connection between the xiphoid process and the diaphragm, and the diaphragm was cut along the left and right sides, and the thoracic cavity was cut from the left and right ends of the diaphragm to the cephalic end to fully expose the entire thoracic cavity. The lung tissue was cut off, and a part of the left lobe of the lung was taken and placed in 4% paraformaldehyde fixative for tissue fixation, and the rest were placed in 1.5 mL EP tubes, quickly put into liquid nitrogen, and finally transferred to an -80 °C refrigerator for storage for later use.

[0030] Detection of lung tissue injury and immune cell infiltration in mice: H&E and immunohistochemical staining methods were used to detect lung tissue injury, neutrophil and macrophage infiltration in mice.

[0031] Pathological scoring of lung injury: The sample size of each group of mice was 5. The lung tissue sections stained with H&E were placed under a light microscope to observe pathological changes. According to the bleeding condition of the lung tissue in mice, whether there was congestion in the alveoli, the infiltration of inflammatory cells including neutrophils and macrophages in the alveolar cavity, and the thickness of the alveolar septum, the lung tissue injury was quantitatively scored. The scoring criteria are as follows: 0 points: normal lung tissue; 1 point: mild lung injury, ≤ 25% lung involvement; 2 points: moderate lung injury, > 25% - 50% lung involvement; 3 points: severe lung injury, > 50% - < 75% lung involvement; 4 points: very severe, almost the whole lung involved. This operation was completed by a researcher who was unaware of the section numbers in a single-blind manner.

[0032] The results of the effect of ximenic acid on lung injury are as Figure 3 shown. Compared with septic mice, the lung tissue injury in mice treated with ximenic acid was significantly reduced. The results showed that ximenic acid could significantly improve sepsis-related lung injury, and the therapeutic effect was dose-dependent.

[0033] The effect of ximenic acid on the infiltration of immune cells in the lungs is as Figure 4 and Figure 5 shown. Compared with septic mice, after treatment with ximenic acid, the infiltration of neutrophils and macrophages in the lungs of mice was significantly reduced. It can be seen from this that ximenic acid can significantly improve the inflammatory response related to sepsis, and the therapeutic effect is dose-dependent.

[0034] Example 4: Test for inhibiting the release of inflammatory cytokines by ximenic acid Collection of blood specimens from mice: Mice were anesthetized by intraperitoneal injection of 1% pentobarbital, and blood samples were collected by cardiac puncture. Subsequently, the blood was allowed to stand at room temperature for 1 h to precipitate the serum, and then placed in a centrifuge pre-cooled to 4 °C at a speed of 5000 rpm for 10 min. The serum was collected and stored in a -80 °C refrigerator for later use.

[0035] Collection of mouse peritoneal lavage fluid samples: After the mice were sacrificed by cervical dislocation, the abdominal fur was cut open with small scissors. A 5 ml syringe was used to aspirate 5 mL of PBS, which was then injected into the abdominal cavity of the mice. After gently kneading, the PBS was aspirated from the abdominal cavity of the mice with the syringe. This lavage process could be repeated 1 - 2 times. During the operation, the tip of the syringe should not puncture the organs in the abdominal cavity of the mice. The lavage fluid should be placed on ice and centrifuged (300 xg, 5 min) to remove the supernatant after all the mice had been sampled, and then stored in a -80 °C refrigerator for later use.

[0036] Detection of inflammatory cytokines (TNF-a, IL-6): 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.

[0037] The results of the effect of ximenic acid on the expression of pulmonary inflammatory cytokines are as Figure 6 shown. Compared with septic mice, after treatment with ximenic acid, the expression of inflammatory cytokines TNF-α and IL-6 in the lung tissue of mice was significantly decreased. Therefore, ximenic acid can significantly inhibit the expression of pulmonary inflammatory cytokines in septic mice, and the therapeutic effect is dose-dependent.

[0038] The results of the effect of ximenic acid on the levels of inflammatory cytokines are as Figure 7 and Figure 8 shown. Compared with septic mice, after treatment with ximenic acid, the levels of TNF-α and IL-6 in the serum and peritoneal lavage fluid of mice were significantly decreased. Therefore, ximenic acid can significantly inhibit the levels of inflammatory cytokines in septic mice. The therapeutic effect is dose-dependent.

[0039] Example 5: Test for the inhibition of TLR4 signaling pathway activation by ximenic acid Isolation and in vitro stimulation of mouse peritoneal macrophages: Inject 3 - 4 ml of broth (prepared in advance, autoclaved and stored at room temperature in the dark) into the peritoneal cavity of mice. After 3 days, decapitate the mice to sacrifice them. Use small scissors to cut open the outer peritoneal fur (do not cut the peritoneum). Aspirate 5 ml of PBS with a 5 ml syringe, inject it into the peritoneal cavity of the mice and gently knead. Then aspirate the PBS in the peritoneal cavity and put it into a 15 ml centrifuge tube. You can aspirate another 5 ml of PBS to perform a secondary lavage of the peritoneal cavity of the mice to obtain more peritoneal macrophages. Centrifuge the cells at 1200 rpm for 10 min, resuspend them with RPMI 1640 medium containing 10% FBS, count the cells, and seed them in the well plate. After stimulating the cells with LPS (100 ng / ml) and xylonic acid (10 μM), collect the cells and use RIPA lysis buffer to prepare protein samples. Then dilute the 5X loading buffer, denature it in a metal bath at 90 °C for 10 min, and store it at -80 °C for later use.

[0040] Test for the activation of TLR4 signaling pathway: Use the western blot method to detect the phosphorylation levels of p65 and IKBα and the expression level of GAPDH in primary peritoneal macrophages; use real-time fluorescence quantitative PCR to detect the expression of TNF-α and IL-6 in primary peritoneal macrophages.

[0041] As shown in the above test results Figure 9 After the primary peritoneal macrophages of mice were stimulated with LPS, xylonic acid could significantly inhibit the phosphorylation levels of p65 and IKBα, while the expressions of p65 and IKB proteins did not change significantly. In addition, xylonic acid could significantly inhibit the expressions of inflammatory cytokines TNF-α and IL-6. The above results indicate that xylonic acid can significantly inhibit the activation of TLR4 signaling pathway and inflammatory response.

[0042] In summary, xylonic acid can effectively treat sepsis, significantly improve the survival rate of septic mice in vivo, improve the lung injury of septic mice, inhibit the expression and release of inflammatory cytokines; in vitro, it can significantly inhibit the activation of TLR4 signaling pathway and inflammatory response. Therefore, xylonic acid can be used to prepare drugs for the treatment of sepsis and related inflammatory diseases.

[0043] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

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

2. The application according to claim 1, wherein The sepsis is induced by lipopolysaccharide.

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

4. The application according to claim 1, characterized in that, The lignoceric acid or a pharmaceutically acceptable salt thereof reduces the level of inflammatory cytokines, alleviates pulmonary cell infiltration and reduces the expression of pulmonary inflammatory cytokines.

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

6. The application according to claim 4, wherein The pulmonary cells include pulmonary macrophages and pulmonary neutrophils.

7. The application according to claim 4, characterized in that The pulmonary inflammatory cytokines include TNF-α and IL-6 mRNA.

8. The application according to claim 1, wherein The safe drug concentration of lignoceric acid or a pharmaceutically acceptable salt thereof is 1-400 µM, with the concentration calculated based on lignoceric acid.

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

10. The application according to claim 1, characterized in that The administration methods of lignoceric acid or a pharmaceutically acceptable salt thereof include: intraperitoneal injection, oral administration.

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