Application of a functional lipid in the preparation of drugs for treating systemic lupus erythematosus

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of a functional lipid in the preparation of drugs for treating systemic lupus erythematosus (SLE). Specifically, the functional lipid MLnM exhibits significant therapeutic effects in a pristane-induced SLE mouse model, effectively alleviating SLE-related clinical symptoms, improving intestinal barrier function, reducing oxidative stress levels, regulating the balance of inflammatory cytokine secretion, and reducing systemic inflammatory responses by inhibiting the expression of immune cell migration-related genes. The discovery of MLnM provides a new direction for the treatment of SLE and other autoimmune diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a functional lipid in the preparation of a drug for treating systemic lupus erythematosus. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by multi-organ damage and autoantibody formation. Its core pathological mechanisms involve the disruption of immune tolerance, abnormal recognition of autoantigens, and imbalance in immune cell metabolic function. Current clinical treatment mainly relies on glucocorticoids and immunosuppressants. While these drugs can alleviate symptoms, long-term use often brings serious side effects, such as increased risk of infection, metabolic disorders, and organ toxicity, severely impacting patients' quality of life. In recent years, studies have found that abnormal lipid metabolism is closely related to the pathogenesis, disease activity, and organ damage of SLE. For example, oxidized low-density lipoprotein exacerbates autoimmune responses by activating inflammatory pathways, abnormal lipid raft stability enhances abnormal B cell activation, and dysregulation of the gut microbiota-immune axis further amplifies systemic inflammatory responses. Therefore, intervention strategies targeting lipid metabolism have become a new direction for SLE treatment.

[0003] Current research on functional lipids largely focuses on single functional molecules (such as the anti-inflammatory effects of ω-3 polyunsaturated fatty acids), with no systematic exploration of multidimensional metabolic and immune synergistic regulatory systems. MLnM (decanoic acid-linolenic acid-decanoic acid) is a novel functional lipid synthesized via enzymatic transesterification. Its unique "medium-chain-long-chain-medium-chain" triglyceride design integrates the dual characteristics of rapid energy supply and targeted immune regulation. Decanoic acid, as a medium-chain fatty acid, can be rapidly absorbed by the body and converted into energy, while linolenic acid, as a long-chain polyunsaturated fatty acid, has the potential for anti-inflammatory effects, immune regulation, and promotion of gut microbiota homeostasis. MLnM specifically enriches linolenic acid at the sn-2 position, enabling its specific hydrolysis in the human body and maximizing its nutritional value and health benefits. However, no studies have yet systematically explored the possible link and mechanism of action between structural lipids and SLE treatment. Summary of the Invention

[0004] In order to overcome the limitations of existing technologies in the treatment of systemic lupus erythematosus (SLE) and to discover new functional lipids and establish a link with SLE treatment, this invention provides an application of functional lipids in the preparation of drugs for treating systemic lupus erythematosus.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for preparing a functional lipid MLnM, characterized in that the method comprises the following steps:

[0007] 1) Medium-chain fatty acids react with glycerol in an esterification reaction catalyzed by lipase to form 1,3-glycerol esters;

[0008] 2) The oil containing linolenic acid is reacted with the 1,3-glyceride obtained in step 1) to obtain MLnM.

[0009] Furthermore, in step 1) of the preparation method, the medium-chain fatty acid is decanoic acid, the lipase is Novozym 435 lipase, and step 1) yields 1,3-dicepanoic acid glyceride.

[0010] Furthermore, the esterification reaction conditions are as follows: using vinyl decanoate and glycerol as raw materials, dichloromethane is added, followed by lipase, and the reaction is carried out in an ice bath for 1-5 hours. After adding more lipase, the reaction is continued for 1-3 hours. Dichloromethane is removed by rotary evaporation, and the product is purified by low-temperature crystallization. The product is dissolved in petroleum ether, frozen overnight, and then filtered to obtain 1,3-dicoctanediglycerate.

[0011] Further, in step 2), the 1,3-dicoctanoic acid glyceride obtained in step 1) is reacted with linolenic acid in a mass ratio of 1.3~1.6:1, and the 1,3-dicoctanoic acid glyceride and linolenic acid are reacted under conditions containing 4-dimethylaminopyridine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dichloromethane to obtain MLnM.

[0012] In a second aspect, a functional lipid MLnM is provided, characterized in that it is prepared by the method described in the first aspect.

[0013] Thirdly, a pharmaceutical composition for treating systemic lupus erythematosus is provided, characterized in that the pharmaceutical composition contains the functional lipids described in the second aspect.

[0014] Furthermore, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0015] Furthermore, the pharmaceutical composition is administered orally or by injection.

[0016] Furthermore, the dosage of the functional lipid MLnM in the pharmaceutical composition for an individual is 500-1000 mg / kg body weight.

[0017] Fourthly, the use of the method described in the first aspect or the functional lipid MLnM described in the second aspect in the preparation of a drug for treating systemic lupus erythematosus is provided.

[0018] Fifthly, the present invention provides a kit for evaluating the therapeutic effect of the drug described in the second aspect on systemic lupus erythematosus, the kit comprising reagents for detecting anti-dsDNA antibodies, inflammatory factor levels, and liver and kidney function indicators in an individual's serum.

[0019] Furthermore, the inflammatory factors include, but are not limited to, IL6, TNFα, and IFNγ.

[0020] Preferably, the kit further includes reagents for detecting the levels of oxidative stress markers in individual tissues.

[0021] Furthermore, the oxidative stress markers include, but are not limited to: SOD, T-AOC, GSH-px, MDA, and CAT.

[0022] More preferably, the kit further comprises reagents for detecting gene expression levels associated with immune cell migration in individual tissues.

[0023] Furthermore, the genes associated with immune cell migration include, but are not limited to: ITGA4, ITGB7, MAdCAM-1, CCR9, and CCL25.

[0024] More preferably, the kit further includes reagents for detecting metabolites in individual intestinal tissue, and / or reagents for detecting the expression levels of epithelial tight junction proteins and mucins.

[0025] Furthermore, the metabolites in the intestinal tissue include, but are not limited to: short-chain fatty acids, including but not limited to: lactic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, etc.; and / or,

[0026] The epithelial tight junction proteins include, but are not limited to: ZO-1, Claudin-1, and Occludin; the mucins include, but are not limited to: Muc2.

[0027] Preferably, the reagent includes primers and / or probes for gene expression detection.

[0028] In a sixth aspect, the present invention provides a system for evaluating the treatment effect of MLnM, the system comprising a detection module and an analysis and judgment module; the detection module is used to detect various indicators related to systemic lupus erythematosus in an individual after MLnM treatment, and transmit the data of various indicators related to systemic lupus erythematosus to the analysis and judgment module, the analysis and judgment module evaluating the treatment effect of MLnM based on the indicator data.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] First, this invention utilizes a novel functional lipid, MLnM, obtained through a unique molecular design. MLnM possesses a unique "medium-chain-long-chain-medium-chain" triglyceride structure, specifically enriching linolenic acid (C18:3) at its sn-2 position and binding decanoic acid (C10:0) at its sn-1 / 3 position. This integrates the dual characteristics of rapid energy supply and targeted immune regulation, demonstrating potential for precise nutritional intervention. MLnM also exhibits excellent oxidative stability and biocompatibility, with a peroxide value of only 0.06 g / 100 g, indicating that it is not prone to oxidative degradation during storage and processing, making it suitable for long-term use, long-term storage, and industrial applications.

[0031] Second, this invention, through the construction of an SLE animal model and combined with multi-omics analysis techniques, for the first time elucidated the role of MLnM in regulating the immune-metabolic network and homeostasis. These mechanisms demonstrate that MLnM can precisely intervene in the pathological process of SLE. Specifically, this manifests as metabolic remodeling and immune regulation. Metabolic remodeling includes regulating key pathways such as fatty acid biosynthesis and glycerophospholipid metabolism, promoting the synthesis of short-chain fatty acids (acetic acid, butyric acid, isovaleric acid), and restoring intestinal metabolic homeostasis. Immune regulation includes regulating the secretion of pro-inflammatory factors (IL6, TNFα, IFNγ) and anti-inflammatory factors (IL10, IL22), inhibiting pro-inflammatory signaling pathways such as NF-κB and S1PR1-STAT3, thereby achieving immune homeostasis reconstruction.

[0032] Third, the findings of this invention revealed that MLnM exhibits significant therapeutic effects in a pristane-induced SLE mouse model, effectively alleviating SLE-related clinical symptoms, improving intestinal barrier function, reducing oxidative stress levels, regulating the balance of inflammatory cytokine secretion, and reducing systemic inflammatory responses by inhibiting the expression of immune cell migration-related genes. The discovery of MLnM provides a new direction for the treatment of SLE and other autoimmune diseases.

[0033] Fourth, besides SLE, MLnM holds promise for precision nutritional intervention in other autoimmune diseases (such as rheumatoid arthritis and Sjögren's syndrome) and chronic inflammatory diseases. Its unique molecular structure gives it broad application value in the food, cosmetics, and pharmaceutical fields, for example, developing it into functional foods, dietary supplements, or adjunctive therapies to provide patients with green, efficient, and low-side-effect treatment options. Attached Figure Description

[0034] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0035] Figure 1The results show the levels of double-stranded DNA (A), alanine aminotransferase (B), aspartate aminotransferase (C), total serum protein (D), serum creatinine (E), serum urea (F), high-density lipoprotein (G), low-density lipoprotein (H), and total serum cholesterol (I) in the serum samples of mice in each experimental group in Example 1 of this invention.

[0036] Figure 2 The staining results are shown for the mouse organ samples from each experimental group in Example 1 of this invention.

[0037] Figure 3 The figures show the mRNA expression results and immunofluorescence detection results in the intestinal epithelial tissue of mice in each experimental group in Example 1 of the present invention. Figures A to D show the relevant mRNA expression results in the intestinal epithelial tissue of mice in each experimental group, E and F show the fluorescence quantitative analysis results of DAPI / ZO-1 and DAPI / Claudin-1, respectively, and G shows the immunofluorescence detection results of DAPI / ZO-1 and DAPI / Claudin-1.

[0038] Figure 4 The results show the detection results of five oxidative stress markers in Example 1 of this invention: SOD (superoxide dismutase) (A), T-AOC (total antioxidant capacity) (B), GSH-px (glutathione peroxidase) (C), MDA (malondialdehyde) (D), and CAT (catalase) (E).

[0039] Figure 5 The results show the mRNA expression of antioxidant factors in the colon tissue of mice in each experimental group in Example 1 of this invention, where A~E correspond to the expression results of SOD1, CAT, Gpx1, NFE2L2 and Keap1, respectively.

[0040] Figure 6 The results show the mRNA expression of pro-inflammatory factors IL6 (A), IL10 (B), IL22 (C), TNFα (D), IFNγ (E), ITGA4 (F), ITGB7 (G), MAdCAM-1 (H), CCR9 (I), CCL25 (J), GPR41 (K) and GPR43 (L) in the colon of mice in each experimental group in Example 1 of this invention.

[0041] Figure 7 The figure shows the changes in the content of short-chain fatty acids in the feces of mice in each experimental group in Example 1 of the present invention, including lactic acid (A), acetic acid (B), propionic acid (C), butyric acid (D), valeric acid (E), and isovaleric acid (F). Detailed Implementation

[0042] The MLnM used in this invention is a triglyceride with a "medium-chain-long-chain-medium-chain" triester structure, bound to n-decanoic acid (C10:0) at the sn-1 / 3 position. The peroxide value of MLnM was measured according to the national standard GB5009.227-2023, and the result was 0.06 g / 100 g, indicating that MLnM is not easily oxidized and deteriorated, making it suitable for storage, processing, and long-term use. This invention further utilizes MLnM in the study of its role as a matrix in SLE, innovatively revealing its multidimensional mechanism in regulating systemic lupus erythematosus (SLE), providing a novel strategy for precision nutritional intervention. By deeply exploring the synergistic regulatory effect of MLnM on the immune metabolic network and the immune system, this invention not only provides a new perspective for the treatment of SLE but also opens up new target directions for the treatment of other autoimmune diseases. It is worth emphasizing that any new mechanisms revealed by this invention, and all related applications, are covered within the scope of protection of this invention.

[0043] The detailed steps of the novel mechanism involved in this invention are as follows: First, a pristane-induced SLE mouse model is constructed to simulate the pathological characteristics of human SLE; second, the model mice are administered MLnM solution by gavage, with a high-dose group of 1000 mg / kg and a low-dose group of 500 mg / kg; then, the administration is continued for 21 days, and the therapeutic effect of MLnM is evaluated through a series of experimental methods, including blood biochemistry analysis, histopathological examination, detection of oxidative stress markers, analysis of inflammatory factor expression, and detection of metabolites.

[0044] In the aforementioned new mechanism, "constructing a pristane-induced SLE mouse model" refers to inducing mice to produce autoantibodies and nephritis by intraperitoneal injection of pristane, thus mimicking the pathological characteristics of SLE. The establishment of this model provides a foundation for subsequent evaluation of treatment efficacy.

[0045] In the aforementioned new mechanism, "gavage treatment of model mice with a specific dose of MLnM solution" refers to dissolving MLnM in physiological saline and administering a specific dose of MLnM to mice via gavage to achieve interventional treatment for SLE. The high-dose group and the low-dose group received 1000 mg / kg and 500 mg / kg of MLnM solution, respectively, while the control group received an equal volume of physiological saline.

[0046] In the aforementioned new mechanism, "continuous administration for 21 days" refers to administering oral gavage to mice daily during a 21-day experimental period to ensure the sustained effect of MLnM in mice and thus evaluate its long-term therapeutic efficacy.

[0047] In the aforementioned new mechanism, "evaluating the therapeutic effect of MLnM through a series of experimental methods" refers to employing multiple experimental techniques, including blood biochemistry analysis, histopathological examination, detection of oxidative stress markers, analysis of inflammatory factor expression, and metabolite detection, to comprehensively assess the therapeutic effect of MLnM on SLE. These methods cover multi-dimensional assessments from the molecular to the tissue level, ensuring the comprehensiveness and accuracy of the experimental results.

[0048] In the aforementioned new mechanism, "blood biochemistry analysis" refers to the use of a fully automated biochemistry analyzer to detect the levels of inflammatory factors such as anti-dsDNA antibodies, IL-6, and TNF-α in serum, as well as liver and kidney function indicators, in order to assess the impact of MLnM on SLE-related inflammatory responses and organ function.

[0049] In the aforementioned new mechanism, "histopathological examination" refers to the observation of pathological changes in mouse organs and HE staining and immunofluorescence staining of colon tissue to evaluate the repair effect of MLnM on tissue damage caused by SLE.

[0050] In the aforementioned new mechanism, "oxidative stress marker detection" refers to measuring the levels of SOD, T-AOC, GSH-px, MDA, and CAT to assess the effect of MLnM on oxidative stress levels in SLE mice.

[0051] In the aforementioned new mechanism, "inflammatory factor expression analysis" refers to the use of real-time quantitative PCR to detect the expression of IL6, TNFα, IFNγ, IL10, IL22, and immune cell migration-related genes to assess the regulatory role of MLnM in SLE-related inflammatory responses.

[0052] In the aforementioned new mechanism, "metabolite detection" refers to the use of gas chromatography-mass spectrometry (GC-MS) to detect metabolites in intestinal tissue and analyze the effect of MLnM on the metabolic profile of mice in order to assess its corrective effect on SLE-related metabolic disorders.

[0053] Through the above steps and methods, this invention comprehensively reveals the multidimensional mechanism of MLnM in regulating SLE, providing an innovative solution for precision nutritional intervention.

[0054] Furthermore, in the description of this invention, it should be noted that unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0055] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] Example 1: Treatment of SLE mice with functional lipid MLnM

[0057] The experimental design for this embodiment is as follows:

[0058] I. Preparation scheme of MLnM

[0059] Weigh 21.6 g of vinyl decanoate and 4 g of glycerol, add 2 mL of dichloromethane, then add 1.88 g of Novozym 435 lipase. Stir magnetically in an ice-water bath at 0-10 °C for 3 h, then add 0.6 g of Novozym 435 lipase and continue the reaction for another 2 h. After the reaction is complete, dissolve the lipase in a suitable amount of dichloromethane, filter to separate the lipase, and remove the dichloromethane by rotary evaporation to obtain a white crystalline crude product. Purify the crude product by low-temperature crystallization. Dissolve the white crystals in 500 mL of petroleum ether, freeze overnight at -20 °C, and filter to obtain 1,3-dicoctanediglycerate (purity > 99% (LC), yield 87.65%).

[0060] Weigh 9.6 g of linolenic acid and 14 g of 1,3-didecanoic acid glyceride, and add 4-dimethylaminopyridine (DMAP, 1.66 g), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 8.04 g), and 40 mL of dichloromethane, respectively. Stir at room temperature for 12 h, allow to stand and separate, concentrate the upper layer by rotary evaporation, and purify by silica gel column chromatography to obtain MLnM (purity >99% (LC), yield 93.10%), eluent n-hexane / ethyl acetate (9:1, v / v).

[0061] II. Experimental Animals

[0062] The experimental animals used were 6-week-old SPF-grade female C57BL / 6J mice (Beijing Huafukang Biotechnology Co., Ltd.). This research followed the National Health and Medical Research Council's "Guidelines for the Care and Use of Laboratory Animals" and was approved by the Animal Ethics Committee of China Agricultural University. All experimental mice were housed in a standardized environment with a barrier (temperature 23℃±2℃, humidity 55%±5%, 12h light / dark cycle).

[0063] III. Construction of SLE model mice:

[0064] Sixty C57BL6 / J mice were used. All mice were acclimatized to the environment for 14 days before the experiment. Then, the SLE model was induced by inoculating 0.5 mL of pristane. The significant increase in serum dsDNA content after hair removal on the back of the mice was used as a method to evaluate the success of model establishment.

[0065] IV. Experimental Methods:

[0066] Sixty SLE model mice underwent different interventions, including: a high-dose MLnM group (HCl), a low-dose MLnM group (LCL), an SLE model group, an SLE control group, an Ln (linolenic acid) intervention group, and a PC (hydroxychloroquine) intervention group, with 10 SLE model mice in each group, randomly assigned. The specific steps are as follows:

[0067] 1. Treatment methods for high-dose group, low-dose group, and model group

[0068] (1) Pristane-induced SLE model mice in the high-dose MLnM group, low-dose MLnM group and SLE model group were acclimatized for one week to ensure that their physical health was stable and there was no obvious infection or injury.

[0069] (2) Mice in the high-dose group were given 1000 mg / kg of MLnM solution orally by gavage daily, while mice in the low-dose group were given 500 mg / kg of MLnM solution orally by gavage daily. The 1000 mg and 500 mg refer to the mass of MLnM in the MLnM solution. Mice in the SLE model group were given physiological saline by gavage at a volume of 10 mL / kg body weight.

[0070] (3) Administer the drug continuously for 21 days, closely observe the mice’s daily behavior, hair condition, weight changes and other clinical manifestations during the period, and record any abnormalities.

[0071] (4) After administration, mice were deeply anesthetized, blood samples were collected, and serum was separated to detect the levels of anti-dsDNA antibodies, IL-6, TNF-α and other inflammatory factors, as well as liver and kidney function indicators in the serum.

[0072] (5) Dissect the mice, observe and record the pathological changes of the organs, measure the weight of immune-related organs such as the spleen and liver, and calculate the organ coefficient.

[0073] (6) Take mouse colon tissue, fix it in 4% paraformaldehyde solution, prepare paraffin sections, perform HE staining and immunofluorescence staining, and observe the integrity of the intestinal barrier and the infiltration of inflammatory cells.

[0074] (7) Collect mouse fecal samples and perform 16S rRNA sequencing analysis to analyze the structure and diversity of gut microbiota.

[0075] 2. Treatment of the SLE control group

[0076] Ten pristane-induced SLE model mice were also acclimatized for one week to ensure stable health and absence of obvious infection or injury. For the following 21 days, no specific interventions were administered to the mice; they were simply fed normally. During this period, the mice's daily behavior, coat condition, weight changes, and other clinical manifestations, as well as any possible abnormalities, were closely observed and recorded.

[0077] The subsequent operations are the same as steps (4) to (8) above.

[0078] 3. Treatment methods for the Ln intervention group and the PC intervention group

[0079] Except for the Ln intervention group mice being administered 500 mg / kg of Ln solution orally via gavage daily, and the PC intervention group mice being administered 100 mg / kg of PC solution orally via gavage daily, all other procedures were the same as those in Section 1 above.

[0080] V. Testing Items and Methods

[0081] In the animal experiments of this invention, the following tests were performed on mice in each experimental group:

[0082] 1) Blood biochemistry analysis

[0083] Mouse blood was centrifuged at 1000 g for 15 min at 4 °C. The supernatant was carefully collected and stored at -80 °C. Analysis was performed using a fully automated biochemical analyzer (Mavis BS-430). Low-density lipoprotein (LDL), high-density lipoprotein (HDL), and total cholesterol (TC) levels were examined. Serum total protein (TP), alanine aminotransferase (ALT), and aspartate transaminase (AST) levels were measured to assess hepatotoxicity. Creatinine (CREA) and urea (UREA) levels were examined to assess nephrotoxicity.

[0084] 2) HE staining of tissues and PAS staining of kidneys

[0085] Samples were fixed with 4% paraformaldehyde, embedded in paraffin, and then cut into 3-5 µm thick tissue sections. The sections were dewaxed twice with xylene for 5 min each time, followed by soaking in a series of ethanol solutions (100%, 95%, 90%, 80%, 70%, 50%) and double-distilled water for 3 min each. After rehydration, the tissue sections were stained with hematoxylin for 10 min, rinsed with distilled water, and then stained with eosin for 2 min. Finally, the sections were rinsed with distilled water, soaked in xylene for 3 min, and mounted with neutral resin.

[0086] Mouse kidney tissue was fixed in 4% paraformaldehyde for 7 hours, followed by post-fixation in 10% formalin for 20 hours. After thorough fixation, the tissue was dehydrated with a gradient of alcohols and embedded in paraffin to prepare 2-3 μm sections. The sections were dewaxed in xylene, hydrated with a gradient of alcohols, and then oxidized with 0.5% periodic acid solution for 6-8 minutes to expose the 1,2-ethylene glycol groups of glycogen and glycoproteins. The sections were then thoroughly rinsed with distilled water. Under light-protected conditions, the sections were stained with Schiff's reagent at room temperature for 7-10 minutes, rinsed with running water for 5 minutes to remove unbound dye, differentiated with 0.5% sodium metabisulfite for 40 seconds to reduce non-specific background staining, rinsed with running water, and rapidly counterstained with hematoxylin for 1 minute. The nucleocytoplasmic contrast was clarified by differentiation with hydrochloric acid alcohol and blueing with ammonia. Finally, the sections were dehydrated with a gradient of alcohols, cleared with xylene, and mounted with neutral resin.

[0087] 3) Immunofluorescence of colon tissue

[0088] Paraffin-embedded colon tissue sections were dewaxed in xylene and then soaked in a gradient of ethanol (100%, 95%, 90%, 80%, 70%, 50%) and double-distilled water for 3 min. After rehydrated sections were blotted dry with filter paper, the tissue area was marked with a hydrophobic pen. Antigen retrieval was then performed using sodium citrate buffer, followed by boiling and cooling to 50°C, repeated twice. After retrieval, slides were washed three times with PBS for 5 minutes each time, and then immersed twice in a permeation buffer containing 0.2% gelatin and 0.25% Triton for 10 minutes each time. The slides were then transferred to a humidified chamber, covered with 5% BSA, and blocked at room temperature in the dark for 1 hour. After removing the blocking solution, diluted primary antibody was added and incubated overnight at 4°C. The next day, the slides were washed twice with PBS (10 minutes each time) and re-treated with permeation buffer once. Fluorescent secondary antibody was added and reacted at room temperature in the dark for 1-2 hours. In the final rinsing stage, the slides were washed three times with PBS (10 minutes each time) and treated once with 10 mM CuSO4 / 50 mM NH4Cl solution. After air-drying, the slides were mounted with a DAPI-containing anti-quenching mounting medium. Images of the stained colon tissue from the same area were obtained using a fluorescence microscope.

[0089] 4) Oxidation factor ELISA detection

[0090] After collecting mouse blood samples, centrifuge at 1000g for 10 minutes at 4°C. After centrifugation, separate the supernatant and store it at -80°C for further analysis. To determine the content of oxidative factors in serum, an enzyme-linked immunosorbent assay (ELISA) kit was used, strictly following the instructions in the kit's manual.

[0091] 5) Real-time quantitative PCR analysis

[0092] Total RNA was extracted from colon tissues of mice in different treatment groups using Trizol reagent. The extracted RNA was reverse transcribed using the HiScript IV RTSuperMix for qPCR (+gDNA wiper) (Novizan, R423-01) detection kit. The generated cDNA was used for real-time quantitative PCR according to the instructions of the Taq Pro Universal SYBR qPCR Master Mix (Novizan, Q712-02) detection kit. 1 ng of template RNA, 3 μL of 5×gDNA wiper Mix, and RNase-free ddH2O were added to 15 mL and incubated at 42°C for two minutes. Then, 5 mL of 4×HiScript IV qRTSuperMix was added, and the mixture was incubated at 37°C for 15 minutes, followed by incubation at 85°C for 5 seconds to obtain the reverse transcription product. 2 μL of the reverse transcription product was added to a mixture of 10 μL of 2×Taq Pro Universal SYBR qPCR Master Mix, 0.4 μL of the front primer, 0.4 μL of the back primer, and 7.2 μL of ddH2O. The qPCR reaction system consisted of an initial incubation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds followed by 60°C for 30 seconds. Amplification and melting curves of the PCR products were measured using a Prism VIIA7 real-time PCR system (Thermo Fisher Scientific). The expression level of the target gene was determined using the 2ΔΔCt method.

[0093] Table 1. List of RT-qPCR primers

[0094]

[0095] 6) Determination of short-chain fatty acids in feces

[0096] Weigh an appropriate amount of cecal contents and acidify with concentrated sulfuric acid (98%, v / v). Add the appropriate reagents according to the ratio of 900 μL of diethyl ether and 100 μL of internal standard (1000 μg / mL, 2-ethylbutyric acid) per sample. Vortex the mixture for 5 min, sonicate in an ice-water bath for 30 min, let it stand at -20℃ for 30 min, and centrifuge at 4℃ and 12000g for 15 min. Add anhydrous sodium sulfate to the supernatant after centrifugation, vortex for 5 min, and then centrifuge at 4℃ and 12000g for 15 min. Collect the supernatant and filter it through a 0.22 μm filter membrane. Prepare mixed standard solutions containing lactic acid, acetic acid, propionic acid, butyric acid, valeric acid, and isovaleric acid at concentrations of 5, 50, 100, 200, 300, 400, and 500 μg / mL. Add the same internal standard as in the sample to the mixed standard solutions of different concentrations and filter through a 0.22 μm filter membrane. The sample filtrate and the mixed standard solution filtrate were analyzed using a Shimadzu gas chromatograph equipped with an HP-INNOVAX column (30 m × 0.250 mm, 0.25 μm). Gas chromatographic conditions: injector temperature 230 °C, flame ionization detector temperature 250 °C, column temperature increased from 100 °C to 180 °C at a rate of 5 °C / min and held at 180 °C for 4 min. The short-chain fatty acid content in the cecal contents was analyzed by comparing the retention time with that of the mixed standard solution and by incorporating the peak area of ​​the internal standard 2-ethylbutyric acid.

[0097] VI. Test Results

[0098] (1) The spleen and liver of mice in the SLE control group were significantly enlarged, and the organ coefficients were significantly increased. The significant decrease in the thymus index may be the result of a combination of factors, including autoimmune attack, abnormal T cell activation, hematopoietic differentiation deviation, and metabolic imbalance. These organ coefficients were improved in the treatment groups, and the high-dose MLnM group was more effective than the low-dose MLnM group. Colon length reflects the severity of inflammation to some extent. In contrast, the colon length in the SLE control group was significantly shortened and recovered after treatment. These results indicate that functional lipid MLnM can effectively alleviate and improve the clinical symptoms of pristane-induced SLE mice.

[0099] Table 2. Organ coefficients of spleen, liver, kidney, thymus, and colon length in mice of each experimental group.

[0100]

[0101] (2) Figure 1 The results showed that the level of anti-dsDNA antibodies was significantly increased in the SLE control group. Figure 1In the high-dose MLnM group and PC intervention group, antibody levels were lower than those in the low-dose MLnM group and Ln intervention group, indicating more effective treatment. Among liver function indicators, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were simultaneously significantly elevated. Figure 1 (B and C) indicate parenchymal hepatocellular damage, which may be related to the direct attack on hepatocellular cells by autoimmune hepatitis secondary to SLE or the systemic cytokine storm during disease activity, while a slight increase in total protein suggests increased globulin synthesis due to chronic inflammation. Figure 1 (D). Renal function indicators (serum creatinine CREA, serum urea UREA) were significantly elevated in the SLE control group, possibly due to SLE-induced renal impairment. Figure 1 (E and F). Abnormal lipid metabolism is a more significant early warning indicator; a decrease in high-density lipoprotein (HDL) and a simultaneous increase in low-density lipoprotein (LDL) and total cholesterol (TC) not only reflect lipid metabolism disorders ( Figure 1 The presence of G, H, and I in the blood is more likely to accelerate the progression of atherosclerosis, which is closely related to the high incidence of cardiovascular events in SLE patients. The significant improvement in blood biochemical parameters in the high-dose MLnM group indicates that the novel functional lipid MLnM has a certain regulatory effect in the treatment of SLE.

[0102] (3) Figure 2 The pathological features of the SLE control group highly conformed to the typical organ damage pattern of systemic lupus erythematosus (SLE): abundant lymphocyte infiltration in the thymus suggested disruption of the T cell developmental microenvironment, possibly stemming from autoantibody-mediated thymic epithelial cell damage; splenic red pulp expansion and white pulp lymphocyte aggregation reflected B cell overactivation, consistent with the abnormal proliferation of germinal centers driven by follicular helper T cells commonly seen in lupus models; glomerular mesangial cell proliferation and decreased PAS-positive material in kidney HE staining may be closely related to basement membrane thickening and podocyte damage caused by immune complex deposition in lupus nephritis. In the treatment group, the thymic structural damage in the Ln group was slightly less severe than in the SLE group, with fewer infiltrating cells, possibly due to the anti-inflammatory effect of linolenic acid (ω-3 polyunsaturated fatty acids) inhibiting the inflammatory process. The pathological differences between LCL and HCL revealed a dose-dependent effect; the LCL group showed a more pronounced red-white pulp boundary and less lymphocyte infiltration in the spleen than the SLE group, indicating that low-dose drugs could partially suppress inflammation. The HCL group showed less lymphocyte infiltration and higher glomerular basement membrane integrity, with lower liver vacuolation than the SLE group, indicating that the HCL group had a significant and better therapeutic effect than the LCL group. Hydroxychloroquine, as a positive control drug for SLE, reduced thymic inflammation and glomerular immune complex deposition by inhibiting the TLR signaling pathway.

[0103] (4) Epithelial tight junction proteins play an important role in the intestinal mechanical barrier. Figure 3The results showed that the mRNA expression of tight junction proteins ZO-1, Claudin-1, Occludin, and mucin Muc2 was significantly decreased in the SLE control group. Both the high-dose MLnM group and the PC intervention group showed some repair effects, but the PC intervention group had a better therapeutic effect. Figure 3 (AD). Further validation was performed using immunofluorescence detection of ZO-1 and Claudin1 in the tissue, and the immunofluorescence results were quantitatively analyzed. Figure 3 (Middle EG). These results indicate that MLnM can improve impaired intestinal epithelial integrity by enhancing the expression of tight junction proteins and mucins associated with intestinal epithelial tissue.

[0104] (5) To further investigate the effect on the antioxidant properties of the colonic barrier in mice. Figure 4 Figure A shows that SOD levels in the SLE control group were significantly decreased, while those in the high-dose MLnM group and the PC intervention group were significantly increased. As a core enzyme for scavenging superoxide radicals, the increased activity of SOD may be due to MLnM enhancing primary defense against free radicals by activating SOD gene transcription or stabilizing the enzyme protein structure. Figure 4 Figure B shows that T-AOC reflects the comprehensive capacity including both enzymatic and non-enzymatic antioxidants. The high-dose MLnM group showed a significantly better effect compared to the SLE control group, and was also more effective than the low-dose MLnM group and the Ln intervention group. Glutathione peroxidase (GSH-px), an important peroxidase widely present in the body, showed a significantly decreased expression level in the SLE group, which recovered after MLnM treatment. Figure 4 (C). Malondialdehyde (MDA), as an end product of lipid peroxidation, accumulates rapidly in the SLE control group, while its levels are significantly reduced in both the high-dose MLnM group and the PC intervention group. Figure 4 The significant recovery of catalase levels in the high-dose MLnM group suggests that it may enhance CAT transcription by activating the Nrf2 pathway. Figure 4 (E).

[0105] (6) The expression of antioxidant factor mRNA in colon tissue was also measured. Figure 5 Figure A shows the expression of SOD1 mRNA in the colon (relative to GAPDH). The figure shows a significant decrease in expression in the SLE control group. Figure 5 The expression of β-CAT mRNA showed a similar trend to that of SOD1, with no significant difference between the SLE control group and the high-dose MLnM group. Figure 5 The results showed that the expression levels of Gpx1 mRNA in the high-dose MLnM group and the PC intervention group were significantly higher than those in the SLE control group, and the PC intervention group had the best treatment effect. Figure 5 D NFE2L2 mRNA and Figure 5 Data on Keap1 mRNA levels in the SLE control group showed a significant decrease in NFE2L2 levels and a significant increase in Keap1 expression. MLnM treatment showed the opposite trend, with increased NFE2L2 expression and decreased Keap1 expression.

[0106] (7) Continue to investigate whether functional lipids can modulate the expression of inflammatory factors in mice. For example... Figure 6 As shown, in the colon of SLE control mice, the pro-inflammatory factor IL6 (IL-6) Figure 6 (A), TNFα ( Figure 6 D), IFNγ ( Figure 6 The significantly elevated mRNA expression of IL-10 (E) in the SLE control group indicated a significant inflammatory response in the colon. Meanwhile, the anti-inflammatory factor IL-10 (E) was significantly elevated. Figure 6 IL-22 (B) Figure 6 The mRNA expression of IL-6 (IL-6), TNF-α, and IFNγ was significantly decreased in the SLE control group. Compared with the SLE control group, the mRNA expression of pro-inflammatory factors IL-6, TNF-α, and IFNγ was significantly reduced in the HCL group and the positive control group (PC), while the mRNA expression of anti-inflammatory factors IL-10 and IL-22 was increased, but the differences were not significant. This suggests that high-dose MLnM and PC intervention can effectively inhibit the expression of pro-inflammatory factors in the colon of SLE control mice, reduce the inflammatory response, and may also play a role in increasing the expression of anti-inflammatory factors. In addition, ITGA4 (… Figure 6 (China F), ITGB7 ( Figure 6 (G), MAdCAM-1 ( Figure 6 H), CCR9 ( Figure 6 (I), CCL25 ( Figure 6 Genes related to immune cell migration, such as GPR41 (GPR41), were significantly expressed higher in the SLE control group than in the control group, indicating increased immune cell migration in the SLE control group. After treatment with high-dose MLnM and PC intervention, the expression of these genes was significantly reduced, indicating that high-dose MLnM and PC intervention could inhibit the migration of immune cells to the colon and reduce inflammatory infiltration. Figure 6 (Middle K) and GPR43 ( Figure 6 The expression of MLnM was significantly higher in the SLE control group than in the control group, while its expression decreased after treatment in the high-dose MLnM group and the PC intervention group, suggesting that the high-dose MLnM group and the PC intervention group may affect intestinal immunity and inflammation by regulating short-chain fatty acid receptors.

[0107] (8) Lactic acid, acetic acid, propionic acid, butyric acid, etc., are not only important products of intestinal microbial metabolism, but also play an important role in intestinal barrier function, immune regulation, and energy metabolism. Figure 7The results showed that the levels of short-chain fatty acids in the gut of the PC intervention group were significantly higher than those of the SLE control group. This indicates that the synthesis or secretion of short-chain fatty acids in the gut of the SLE control group may be inhibited, or that the composition of their gut microbiota may have changed, leading to a decrease in short-chain fatty acid levels. The high-dose MLnM group showed significantly higher levels of lactic acid (MLnM). Figure 7 A), acetic acid ( Figure 7 (B), butyric acid ( Figure 7 D) and isovaleric acid ( Figure 7 The levels of propionic acid (MLnM) were significantly higher in the MLnM treatment group than in the SLE control group, indicating that MLnM treatment restored the levels of these short-chain fatty acids to some extent, possibly by regulating gut microbiota composition and increasing the synthesis or secretion of short-chain fatty acids, thereby improving gut function. However, the levels of propionic acid (MLnM) were significantly higher in the MLnM treatment group than in the SLE control group. Figure 7 (C) and valeric acid ( Figure 7 In the SLE control group (CLE), the improvement was not significant. The high-dose MLnM group may act on lactic acid, acetic acid, butyric acid, and isovaleric acid through specific mechanisms (e.g., regulation of certain key microbiota or metabolic pathways), but its effect on the metabolism of other short-chain fatty acids is weak. Meanwhile, although the intestinal short-chain fatty acid content in the high-dose MLnM group was lower than that in the PC intervention group, the improvement effect in the high-dose MLnM group may become more significant with increasing treatment dose. This difference may be related to the different degrees of regulation of gut microbiota by different doses of treatment. In summary, MLnM treatment has a certain improving effect on the intestinal short-chain fatty acid content in the SLE control group. Therefore, MLnM holds promise for extending to precision nutritional intervention for other autoimmune diseases (such as rheumatoid arthritis and Sjögren's syndrome) and chronic inflammatory diseases.

[0108] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementations of the invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the invention, and these all fall within the protection scope of the invention. Therefore, the protection scope of this invention should be determined by the appended claims.

Claims

1. Use of functional lipid MLnM in the preparation of a medicament for treating systemic lupus erythematosus, characterized in that, The functional lipid is prepared by the following method: 1) taking vinyl decanoate and glycerol as raw materials, adding dichloromethane, then adding lipase, and reacting in an ice bath for 1-5 h, then adding lipase and continuing to react for 1-3 h; removing dichloromethane by rotary evaporation, purifying the product by low-temperature crystallization, dissolving in petroleum ether, filtering after being refrigerated overnight to obtain 1,3-didecanoate glycerol; 2) reacting linolenic acid with the 1,3-didecanoate glycerol obtained in step 1) to obtain MLnM; The lipase is Novozym 435 lipase.

2. Use according to claim 1, characterized in that, In step 2), the mass ratio of the 1,3-didecanoate glycerol obtained in step 1) to linolenic acid is 1.3-1.6:1, and the 1,3-didecanoate glycerol and linolenic acid are reacted under the condition of containing 4-dimethylaminopyridine, 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride and dichloromethane to obtain MLnM.

Images