Use of n-palmitoyl sphingosine in the preparation of anti-inflammatory drugs
By preparing an anti-inflammatory drug containing N-palmitoylsphingosine, the problems of inflammation and gut microbiota imbalance caused by high-salt diet were solved, achieving effective intervention and regulation of gut microbiota in high-salt diet-related inflammation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ARMY MEDICAL UNIV
- Filing Date
- 2023-11-23
- Publication Date
- 2026-07-03
AI Technical Summary
The inflammatory response and gut microbiota imbalance caused by a high-salt diet have not been effectively addressed, the specific mechanisms are unclear, and current technologies lack effective anti-inflammatory drug interventions.
Using N-palmitoyl sphingosine as the active ingredient, an anti-inflammatory drug was prepared to alleviate the inflammatory response caused by a high-salt diet and to intervene in high-salt diet-related inflammation by regulating the composition of gut microbiota metabolites.
It significantly reduces the inflammatory response induced by a high-salt diet, regulates gut microbiota metabolites, and alleviates inflammation promoted by a high-salt diet, providing a new drug intervention strategy.
Smart Images
Figure CN117398371B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology and relates to the application of N-palmitoylsphingosine in the preparation of anti-inflammatory drugs. Background Technology
[0002] With the maturation of salt extraction technology, the liberalization of salt policies, and the improvement of living standards, people have easier access to salt, leading to a higher salt content in food. Studies have shown that in Western countries and China, daily salt intake exceeds 10 grams, which has become a significant contributing factor to chronic non-communicable diseases. High-salt diets are known to induce hypertension, cardiovascular disease, chronic kidney disease, and insulin resistance. Recent research has also found a close relationship between high-salt diets and autoimmune diseases, obesity, neurodegenerative diseases, and asthma.
[0003] One of the key mechanisms by which a high-salt diet promotes disease development lies in its significant pro-inflammatory effect. In vitro and in vivo studies have shown that high salt intake can exert a pro-inflammatory effect by directly regulating the function of inflammatory cells such as macrophages and lymphocytes through sodium ion retention in tissues. However, the body has a good capacity to adapt to a high-salt diet and can regulate sodium ion concentration through various mechanisms. Studies have also found that sodium ion concentrations in most tissues are not high under long-term high-salt diets; therefore, how a high-salt diet promotes inflammatory responses in vivo is not fully understood. Recent studies have shown that a high-salt diet can lead to gut microbiota dysbiosis, and this dysbiosis participates in the pro-inflammatory effect of a high-salt diet, but the specific mechanisms and possible regulatory strategies remain unclear. There are over 1500 species of gut bacteria, and the short-chain fatty acids, bile salts, choline, and other metabolites produced by these bacteria during their metabolic activities further affect subsequent metabolic pathways and other metabolic activities in the host. Therefore, this study explored the effects of a high-salt diet on gut microbiota metabolites and the regulatory effect of intervention on the pro-inflammatory effect of a high-salt diet. Summary of the Invention
[0004] In view of this, the object of the present invention is to provide the use of N-palmitoylsphingosine in the preparation of anti-inflammatory drugs.
[0005] To achieve the above objectives, the present invention provides the following technical solution:
[0006] 1. Application of N-palmitoylsphingosine in the preparation of anti-inflammatory drugs.
[0007] Preferably, the anti-inflammatory effect is against inflammation caused by intestinal flora metabolic disorders.
[0008] Preferably, the anti-inflammatory effect is against high-salt diet-related inflammation.
[0009] More preferably, the high-salt diet is defined as a salt intake exceeding 5 grams per day.
[0010] 2. A pharmaceutical composition comprising N-palmitoylsphingosine.
[0011] 3. Application of the aforementioned pharmaceutical composition in the preparation of anti-inflammatory drugs.
[0012] Preferably, the anti-inflammatory effect is against inflammation caused by intestinal flora metabolic disorders.
[0013] Preferably, the anti-inflammatory effect is against high-salt diet-related inflammation.
[0014] 4. A formulation whose active ingredient is N-palmitoylsphingosine.
[0015] 5. A formulation comprising the aforementioned pharmaceutical composition.
[0016] Preferably, the formulation is an oral formulation.
[0017] The beneficial effects of this invention are as follows:
[0018] The applicant established a high-salt diet model in mice (feed containing 4% sodium chloride and sterilized water containing 1% sodium chloride) for 8 weeks, and induced acute peritonitis in mice by intraperitoneal injection of zymosan-A. The pro-inflammatory effect of the high-salt diet and the alleviating effect of N-palmitoylsphingosine on the inflammatory response in mice with acute peritonitis under a high-salt diet were observed. The total cell count and neutrophil count in the peritoneal fluid of mice were calculated by flow cytometry, and the expression levels of inflammatory factors in the peritoneal fluid were detected.
[0019] The applicant analyzed mouse feces using non-targeted metabolomics to compare the effects of a high-salt diet on gut microbiota metabolites. The study found that a high-salt diet altered the composition of gut microbiota metabolites, which play a crucial role in the progression of inflammation induced by a high-salt diet. This invention discovers that supplementation with N-palmitoyl sphingosine can alleviate inflammation induced by a high-salt diet, providing a novel drug candidate for intervening in high-salt diet-related inflammation. Attached Figure Description
[0020] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following drawings are provided for illustration.
[0021] Figure 1 In vivo, a high-salt diet promotes the inflammatory response of yeast polysaccharide-induced peritonitis; where A is the total cell count, B is the neutrophil count, C is TNF-α, D is MCP-1, and E is IL-6.
[0022] Figure 2 The high-salt diet reduced the level of N-palmitoylsphingosine, a metabolite in the gut microbiota of mice.
[0023] Figure 3 In vivo, N-palmitoylsphingosine improves the inflammatory response induced by a high-salt diet; where A is the total cell count, B is the neutrophil count, C is TNF-α, D is MCP-1, and E is IL-6. Detailed Implementation
[0024] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.
[0025] Example 1
[0026] In vivo, a high-salt diet promotes the inflammatory response of yeast polysaccharides induced by peritonitis.
[0027] After establishing an 8-week high-salt diet model and a normal diet model, mice were induced to develop acute peritonitis by intraperitoneal injection of zymosan-A (Sigma). The total number of cells and neutrophils in the peritoneal fluid of the mice were then measured, and the expression level of inflammatory factors in the peritoneal fluid was also measured to verify that the high-salt diet promotes inflammation.
[0028] 1. Animals
[0029] C57 / BL6 mice, male, 6-8 weeks old, weighing 22-26 grams, were purchased from Speford (Beijing) Biotechnology Co., Ltd., and housed in the SPF-grade animal facility of the Experimental Animal Center of the Third Military Medical University.
[0030] 2. Experimental Grouping
[0031] Normal diet group: Normal feed and sterilized water for 8 weeks
[0032] High-salt diet group: high-salt feed (containing 4% sodium chloride) and sterilized water containing 1% sodium chloride, for 8 weeks.
[0033] 3 Experimental Methods
[0034] After establishing an 8-week high-salt diet model and a normal diet model, mice were induced to develop acute peritonitis by intraperitoneal injection of zymosan-A (0.8 mg / mouse). Six hours after intraperitoneal injection, mice were euthanized by cervical constriction. The peritoneal cavity was first irrigated with 600 ml of pre-chilled HANK's balanced solution (calcium- and magnesium-free) (Procell) (the supernatant was used to detect inflammatory factors), and then further irrigated with 2 ml of pre-chilled HANK's balanced solution (calcium- and magnesium-free). Peritoneal fluid and inflammatory cells were collected. The total cell count and neutrophil count were calculated by cell counting and flow cytometry. Cytokine flow cytometry kits were used to detect the binding of TNF-α, MCP-1, and IL-6 capture beads to their corresponding proteins in the peritoneal fluid supernatant. The expression levels of TNF-α, MCP-1, and IL-6 in the peritoneal fluid were detected by flow cytometry.
[0035] The results are as follows Figure 1 As shown, 6 hours after intraperitoneal injection of yeast polysaccharide, the total number of cells in the peritoneal lavage fluid of the high-salt diet group was significantly higher than that of the normal diet group. Figure 1 (A); Compared with the normal diet group, the high-salt diet group also had a significantly higher neutrophil count than the normal diet group ( Figure 1 (B) Compared with the normal diet group, the expression levels of inflammatory factors TNF-α, MCP-1, and IL-6 in the peritoneal lavage supernatant were significantly increased in the high-salt diet group. Figure 1 (C, D, E) indicates that a high-salt diet can promote the development of inflammation.
[0036] Example 2
[0037] A high-salt diet reduced the level of N-palmitoylsphingosine, a metabolite in the gut microbiota of mice.
[0038] This study investigated the effects of a high-salt diet on the composition of gut microbiota metabolites by performing non-targeted metabolomics analysis on feces from mice in the high-salt diet group and the normal diet group.
[0039] 1. Animals
[0040] C57 / BL6 mice, male, 6-8 weeks old, weighing 22-26 grams, were purchased from Speford (Beijing) Biotechnology Co., Ltd., and housed in the SPF-grade animal facility of the Experimental Animal Center of the Third Military Medical University.
[0041] 2. Experimental Grouping
[0042] Normal diet group: Normal feed and sterilized water for 8 weeks
[0043] High-salt diet group: high-salt feed (containing 4% sodium chloride by mass) and sterilized water containing 1% sodium chloride by mass, for 8 weeks.
[0044] 3 Experimental Methods
[0045] After establishing an 8-week high-salt diet model and a normal diet model, five fecal pellets were collected from each mouse. Samples were then extracted and separated using a HILIC ultra-high performance liquid chromatography (UHPLC) system with a column temperature of 25℃, a flow rate of 0.5 mL / min, and an injection volume of 2 μL. The mobile phase composition was A: water + 25 mM ammonia + 25 mM ammonium acetate, and B: acetonitrile. The gradient elution program was as follows (all percentages are volume percentages): 0-0.5 min, 95% B; 0.5-7 min, B linearly decreasing from 95% to 65%; 7-8 min, B linearly decreasing from 65% to 40%; 8-9 min, B maintained at 40%; 9-9.1 min, B linearly decreasing from 40% to 95%; 9.1-12 min, B maintained at 95%. Throughout the analysis, samples were placed in an autosampler at 4℃ and analyzed sequentially using a randomized order. QC samples were inserted into the sample queue to monitor and evaluate the system stability and the reliability of the experimental data.
[0046] Then, a mass spectrometer was used to collect the primary and secondary spectra of the sample. The ESI source conditions after HILIC chromatographic separation were as follows: Ion Source Gas1 (Gas1): 60, Ion Source Gas2 (Gas2): 60, Curtain gas (CUR): 30, source temperature: 600℃, IonSapary Voltage Floating (ISVF): ±5500 V (both positive and negative modes); TOF MS scan m / z range: 60-1000 Da, product ion scan m / z range: 25-1000 Da, TOF MS scan accumulation time: 0.20 s / spectra, product ion scan accumulation time: 0.05 s / spectra; secondary mass spectrometry was performed using information dependent acquisition (IDA) in high sensitivity mode, with a Declustering potential (DP): ±60 V (both positive and negative modes), Collision Energy: 35 ±15 eV, and IDA settings as follows: Exclude isotopes within 4 Da, Candidate ions to monitor per cycle: 10. Finally, bioinformatics analysis was performed.
[0047] The results showed that significant differentially expressed metabolites were screened out by OPLS-DA with VIP>1 and P<0.05. Compared with the normal diet group, the intestinal flora metabolite N-palmitoylsphingosine was significantly decreased in the high-salt diet group ( Figure 2 ).
[0048] Example 3
[0049] In vivo, N-palmitoylsphingosine alleviates the inflammatory response promoted by high-salt diet
[0050] On the basis of a high-salt diet, N-palmitoylsphingosine was orally administered. Zymosan-A was used to induce peritonitis in mice. By calculating the total number of cells, the number of neutrophils, and the expression levels of inflammatory factors in the peritoneal cavity under inflammatory conditions, the regulatory effect of N-palmitoylsphingosine on the inflammatory response promoted by high-salt diet was examined.
[0051] 1 Animals
[0052] C57 / BL6 male mice, 6-8 weeks old, weighing 22-26 g, were purchased from SPF Biotechnology Co., Ltd. (Beijing). The mice were housed in the SPF animal facility of the Experimental Animal Center of the Third Military Medical University.
[0053] 2 Experimental grouping
[0054] High-salt diet group: High-salt diet (containing 4% sodium chloride by mass concentration) and sterilized water containing 1% sodium chloride by mass concentration for 8 weeks
[0055] N-palmitoylsphingosine treatment: High-salt diet (containing 4% sodium chloride by mass concentration), and sterilized water containing 1% sodium chloride by mass concentration for 8 weeks; N-palmitoylsphingosine was administered by gavage every other day (20 mg / kg body weight) starting from the 7th and 8th weeks of the high-salt diet
[0056] 3 Experimental methods
[0057] In the high-salt diet group and the high-salt diet group treated with N-palmitoylsphingosine, acute peritonitis was induced in mice by intraperitoneal injection of zymosan-A (0.8 mg / mouse). 6 hours after intraperitoneal injection, the mice were sacrificed by cervical dislocation. First, the peritoneal cavity was washed with 600 ml of pre-cooled HANK's balanced salt solution (without calcium and magnesium) (the supernatant was used to detect inflammatory factors), and then the peritoneal cavity was further washed with 2 ml of pre-cooled HANK's balanced salt solution (without calcium and magnesium) to collect peritoneal fluid and inflammatory cells. The total number of cells and the number of neutrophils were calculated by cell counting and flow cytometry. The cytokine flow cytometry kit TNF-α, MCP-1, IL-6 capture beads were combined with the corresponding proteins in the peritoneal fluid supernatant, and the expression levels of TNF-α, MCP-1, and IL-6 in the peritoneal fluid were detected by flow cytometry.
[0058] The results showed that 6 hours after inducing peritonitis, the total number of cells in the peritoneal lavage fluid of mice that had ingested N-palmitoylsphingosine was significantly lower. Figure 3 (A) and neutrophil count ( Figure 3 The expression levels of inflammatory factors TNF-α, MCP-1, and IL-6 were significantly lower in the high-salt diet group compared to the high-salt diet group (B). Figure 3 (C, D, E) indicates that N-palmitoyl sphingosine has a relieving effect on the inflammatory response enhanced by a high-salt diet.
[0059] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. The application of N-palmitoylsphingosine in the preparation of anti-inflammatory drugs, characterized in that, The anti-inflammatory treatment is for acute peritonitis induced by a high-salt diet.
2. The use of a pharmaceutical composition containing N-palmitoylsphingosine in the preparation of an anti-inflammatory drug, characterized in that, The anti-inflammatory treatment is for acute peritonitis induced by a high-salt diet.