Application of N-arachinoylglycine in preparation of medicine for treating inflammation-related diseases

Abstract

The invention discloses application of N-arachinoylglycine in preparation of a medicine for treating inflammation-related diseases and belongs to the technical field of medicines, the N-arachinoylglycine is NAGLY, belongs to an acyl glycine compound and can participate in various signal transduction pathways including activation of a G protein coupled receptor, the receptor is related to an inflammation pathway, and the N-arachinoylglycine can be used for preparing a medicine for treating inflammation-related diseases. The invention further discloses application of NAGLY in preparation of drugs for treating ulcerative inflammation related diseases, and experiments prove that after an immune inflammatory reaction inducer lipopolysaccharide LPS is used for inducing an inflammatory reaction in mouse mononuclear macrophages RAW264.7, the N-arachinoylglycine can effectively inhibit expression of inflammatory factors, so that the NAGLY can be used for preparing drugs for treating ulcerative inflammation related diseases, and can be used for preparing drugs for treating ulcerative inflammation related diseases. And a new direction is provided for research and development of medicines for treating diseases related to ulcerative inflammation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases. Background Technology

[0002] From a molecular perspective, N-arachidonoylglycine is a natural compound formed by the linkage of arachidonic acid to glycine via an amide bond, and it is basally expressed in mammalian intestinal tissue. Its polyunsaturated fatty acid chains and polar functional groups (carboxyl and amide bonds) give it the potential to bind to various receptors in the gut (such as G protein-coupled receptors), providing a structural basis for its participation in the regulation of intestinal physiological and pathological processes. Early studies have confirmed that N-arachidonoylglycine plays a role in regulating pain perception and inflammatory responses in the central nervous system and peripheral tissues. The gut, as a complex organ rich in immune cells and neuroendocrine cells, provides a potential environment for N-arachidonoylglycine to exert its effects.

[0003] At the level of inflammatory regulation mechanisms, the core pathological feature of ulcerative colitis (UC) is excessive activation of the intestinal mucosal immune system, accompanied by the massive release of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6, and the continuous activation of signaling pathways such as NF-κB and MAPK. In vitro experiments have shown that N-arachidonoylglycine can exhibit significant anti-inflammatory activity by inhibiting the secretion of these inflammatory factors in macrophages (such as RAW 264.7 cells) and downregulating the phosphorylation level of NF-κB p65. This characteristic is highly consistent with the pathological mechanism of UC—UC patients show increased infiltration of macrophages and neutrophils in the intestinal mucosa, and their excessive activation is a key link in the amplification of inflammation. The regulatory ability of N-arachidonoylglycine on these cells suggests that it may alleviate intestinal mucosal damage by blocking the inflammatory cascade.

[0004] Regarding the shortcomings of current UC treatments, existing drugs suffer from poor specificity and significant side effects: aminosalicylic acid preparations have limited efficacy in severe UC, long-term use of glucocorticoids can lead to further dysbiosis of the gut microbiota, and biologics may cause excessive local immunosuppression in the gut. N-arachidonoylglycine, as an endogenous molecule, has the potential advantages of high biocompatibility and fewer side effects.

[0005] However, research on N-arachidonoylglycine in UC is still in its early stages: its expression levels in the intestinal tissues of UC patients are not yet clear, its specific targets (such as whether it regulates intestinal immunity through specific receptors) and dose-dependent effects still need to be verified, and its interaction mechanisms with other inflammatory mediators (such as prostaglandins and leukotrienes) in the complex intestinal microenvironment remain unclear. These gaps provide research space for further exploring the value of N-arachidonoylglycine as a new candidate molecule for UC treatment, and also highlight the importance of clarifying its mechanism of action for promoting clinical translation. Summary of the Invention

[0006] Technical Problem Solved: This invention addresses the core technical problems in existing treatments for ulcerative colitis (UC), such as significant systemic side effects, difficulty in mucosal repair, difficulty in restoring barrier function, and imprecise immune regulation. It provides an application of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases. N-arachidonoylglycine works through mechanisms such as targeted local intestinal anti-inflammation, promotion of mucosal repair, stabilization of the intestinal barrier, precise immune regulation, and physiological analgesia. Current research is largely at the animal and cellular levels. Future research needs to address translational medicine issues such as in vivo stability and administration routes. However, its unique mechanism of action provides an important direction for novel treatment strategies for UC.

[0007] To achieve the above objectives, this application provides the following technical solution: The use of N-arachidonoylglycine in the preparation of a medicament for treating inflammation-related diseases, the medicament comprising N-arachidonoylglycine or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0008] Furthermore, the molecular formula of the N-arachidonoylglycine is C2 22 H 35 NO3, with a molecular weight of 361.5, has a purity of ≥98% as determined by TLC or HPLC. It appears as a yellow waxy solid. This N-arachidonicoglycine is soluble in DMSO or 100% ethanol and achieves a solubility of 2 mg / mL in PBS at pH 7.2.

[0009] Furthermore, the medication for treating inflammation-related diseases is in the form of tablets, capsules, gels, creams, solutions, patches, powders, pills, granules, suspensions, syrups, injections, suppositories, inhalers, or sprays.

[0010] Furthermore, the excipients include one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, and lubricants.

[0011] Furthermore, the inflammation-related disease is a chronic inflammatory disease or an autoimmune inflammatory disease.

[0012] Furthermore, the chronic inflammatory disease is arthritis or inflammatory bowel disease; the autoimmune inflammatory disease is systemic lupus erythematosus or rheumatoid arthritis.

[0013] Furthermore, the drug for treating inflammation-related diseases inhibits the release of inflammatory factors, wherein the inflammatory factors are one or more of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6).

[0014] Furthermore, the drug for treating inflammation-related diseases modulates inflammatory signaling pathways, wherein the inflammatory signaling pathway is the NF-κB signaling pathway or the MAPK signaling pathway.

[0015] Furthermore, the drug for treating inflammation-related diseases treats inflammation-related cells, which are one or more of macrophages, monocytes, and microglia.

[0016] Furthermore, the macrophages were RAW 264.7 macrophages, used to study the effects of N-arachidonoylglycine on macrophage inflammatory cytokine secretion and activation of inflammation-related signaling pathways.

[0017] Mechanism of action: N-arachidonoylglycine is a type of acylglycine with anti-inflammatory, analgesic, and vasodilatory effects. Acylglycine participates in multiple signal transduction pathways, including the activation of G protein-coupled receptors, which are involved in inflammatory pathways. When an inflammatory response was induced in mouse RAW264.7 monocytes and macrophages using LPS, an inducer of immune inflammation, the various indicators of the inflammatory response were inhibited after the use of N-arachidonoylglycine.

[0018] This application provides the use of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases, which has the following advantages compared with the prior art: 1. From the perspective of the compound itself, N-arachidonoylglycine has a clear chemical structure and specific physicochemical properties. It has high purity and good solubility, and is soluble in DMSO and 100% ethanol. This provides a good material basis for its subsequent experimental research, drug formulation development, etc., and facilitates various in vitro and in vivo experiments, as well as the preparation of drugs in different dosage forms. 2. A series of in vivo and in vitro experiments were conducted using N-arachidonoylglycine. Inflammatory response was induced in mouse monocyte-macrophage RAW264.7 cells using lipopolysaccharide (LPS), an immune inflammatory response inducer. After treatment with N-arachidonoylglycine, all indicators of the inflammatory response were inhibited. 3. In in vivo experiments, N-arachidonoylglycine effectively alleviated DSS-induced colitis in a B6 mouse model. Attached Figure Description Figure 1 The graphs show the effects of N-arachidonoylglycine on inhibiting lipopolysaccharide (LPS)-induced immune inflammatory markers, as detected by qPCR in this application. The left graph shows the dose-dependent inhibitory effect of N-arachidonoylglycine on LPS-induced IL-1B expression; the right graph shows the dose-dependent inhibitory effect of N-arachidonoylglycine on LPS-induced TNF-α expression. Figure 2 The graph shows a comparison of body weight changes in mice in the control group, the DSS-induced colitis group, and the group treated with two different doses of N-arachidonoylglycine. It shows that N-arachidonoylglycine effectively alleviated the weight loss in mice with DSS-induced colitis. Figure 3 The graph shows a comparison of colon length in mice compared to the control group, the DSS-induced colitis group, and the group treated with two different doses of N-arachidonoylglycine. The results show that N-arachidonoylglycine effectively alleviates colonic atrophy and bleeding caused by colitis. Figure 4 The graph shows the changes in colon morphology and structure in mice by HE staining in the control group, DSS-induced colitis group, and two groups treated with two different doses of N-arachidonoylglycine. The graph shows that N-arachidonoylglycine effectively alleviated the damage to cell morphology and structure of mouse colon tissue caused by colitis, and the effect was dose-dependent. Figure 5The graphs show the indicators used in this application to detect the effectiveness of N-arachidonoylglycine in inhibiting inflammation in colitis mice via qPCR. The left graph shows that N-arachidonoylglycine inhibits the DSS-induced increase in IL-6 expression; the middle graph shows that N-arachidonoylglycine inhibits the DSS-induced increase in IL-1β expression; and the right graph shows that N-arachidonoylglycine inhibits the DSS-induced increase in TNF-α expression. Figure 6 This is a graph showing the indicators of N-arachidonoylglycine's effectiveness in alleviating colitis in mice, as detected by Western blot in this application. From top to bottom, the first graph shows that N-arachidonoylglycine inhibits the decrease in Claudin-3 expression induced by DSS; the second graph shows that N-arachidonoylglycine inhibits the decrease in ZO-1 expression induced by DSS; and the third graph is the internal control, indicating the uniformity of protein content. Figure 7 This application uses HE-stained sections to observe the histopathological scores of mouse colon tissue in the control group, DSS-induced colitis group, and the group treated with two different doses of N-arachidonoylglycine. This demonstrates that N-arachidonoylglycine effectively alleviates the damage to cell morphology and structure of mouse colon tissue caused by colitis, and the effect is dose-dependent. Figure 8 This graph shows the quantitative assessment results of weight loss, fecal characteristics, and bloody stools on the last day of observation in mice in the control group, DSS-induced colitis group, and groups treated with two different doses of N-arachidonoylglycine. It visually reflects the effect of N-arachidonoylglycine on the overall disease activity, confirming that it effectively alleviates the disease activity index in colitis mice in a dose-dependent manner. Detailed Implementation

[0019] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0020] Example 1: This example provides the application of N-arachidonoylglycine in the preparation of a drug for treating ulcerative colitis.

[0021] Inflammation was induced in mouse RAW264.7 monocytes / macrophages using the immune-inflammatory inducer LPS (1 μg / ml), followed by treatment with N-arachidonoylglycine (40 mM, 60 mM, 80 mM, 100 mM, 120 mM, 150 mM). Electron microscopy and qPCR analysis revealed that N-arachidonoylglycine reversed the LPS-induced inflammatory response, with the effect becoming more pronounced with increasing concentration. The optimal concentration of N-arachidonoylglycine was 150 mM.

[0022] Ulcerative colitis was induced in B6 mice by adding 3% DSS to their daily drinking water. Treatment groups were administered N-arachidonoylglycine (0.2 mg / kg and 0.4 mg / kg) via gavage. N-arachidonoylglycine treatment alleviated weight loss and rectal bleeding in the mice. Colon length measurement, HE staining, DAI score, histological score, qPCR detection, and Western blot analysis further confirmed that N-arachidonoylglycine effectively alleviated ulcerative colitis. The optimal therapeutic concentration of N-arachidonoylglycine was 0.4 mg / kg.

[0023] On day 0, mice were treated with N-arachidonoylglycine via gavage. The control group and DSS group received the same dose of DMSO + corn oil via gavage. On day 1, 3% dextran sulfate (DSS) was added to the daily drinking water of B6 mice. The water was changed on days 3, 5, and 7. Mice were sacrificed on day 9 (the DSS group mice's body weight decreased to approximately 70% of their original weight). After sacrifice, colonic tissue was harvested, and the colonic length and colonic bleeding were compared among the four groups. The colonic tissue was divided into three segments: 1. Fix with paraformaldehyde, embed and section, and then perform HE staining and histological scoring.

[0024] 2. Place in trizol for subsequent RNA extraction.

[0025] 3. Freeze in a -80℃ freezer for subsequent Western blot analysis.

[0026] Example 2: This example provides the application of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases. N-arachidonoylglycine inhibits LPS-induced immune inflammatory responses at the RNA level.

[0027] 1. Experimental procedure: (1) Mouse mononuclear macrophages RAW264.7 were seeded in 6-well plates, with 5*10 cells per well. 5 Four hours later, the control group was given an equal dose of DMSO, while the treatment group was given the corresponding dose of N-arachidonoylglycine for pre-stimulation for half an hour. Subsequently, the immune-inducing inflammation group and the treatment group were given 1 mg / ml LPS for stimulation for 6 hours. (2) RNA extraction: RNA was extracted using the Yishan Bio RNA extraction kit. After aspirating the culture medium, the cells were washed once with PBS, and 500 mL of Lysis Buffer was added. The cells were then vigorously pipetted 10 times and transferred to an EP tube. The cells were vortexed for 10 seconds to fully lyse them. 500 mL of anhydrous ethanol was added to the lysed cells, and the mixture was thoroughly mixed before being transferred to a centrifuge column. The column was centrifuged at 4000 × g for 1 minute, and the waste liquid was discarded. 500 mL of Wash Buffer was added to the RNA column, and the column was centrifuged at 12000 × g for 1 minute, and the waste liquid was discarded. After centrifuging the empty tube at 12000 × g for 1 minute, the column was transferred to a clean RNase-free 1.5 mL centrifuge tube and allowed to air dry for 2 minutes. 20–50 mL of Elution Buffer was added to the center of the membrane of the RNA column and allowed to stand at room temperature for 2 minutes. The column was centrifuged at 12000 × g for 1 minute, and the eluted RNA solution was added back to the column. The column was allowed to stand on ice for 5 minutes and then centrifuged again to determine the concentration of eluted RNA. (3) Reverse transcription: Reverse transcription was performed using the Novizan reagent kit. 1 mg RNA, 4 mL 4 × gDNA wiper Mix, enzyme-free water added to 16 mL, 42℃ for 2 minutes; then 4 mL 5 × qRT SuperMix II added, 37℃ for 15 minutes, 85℃ for 5 seconds; 2. Experimental Results: qPCR was performed on RAW264.7 monocytes / macrophages treated with LPS and N-arachidonoylglycine to detect inflammatory markers IL-1β and TNF-α. Elevated levels of IL-1β and TNF-α indicated that LPS successfully induced an immune inflammatory response in RAW264.7 cells, while N-arachidonoylglycine inhibited inflammation in a dose-dependent manner.

[0028] like Figure 1 As shown, this indicates that LPS can induce cellular inflammatory responses, but N-arachidonoylglycine can effectively inhibit the occurrence of inflammation.

[0029] Example 3: This example provides the application of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases. N-arachidonoylglycine alleviates weight loss in mice with DSS colitis.

[0030] 1. Experimental procedure: On day 0, mice were treated with N-arachidonoylglycine via gavage. The control and DSS groups were treated with the same dose of DMSO + corn oil via gavage. On day 1, 3% dextran sulfate (DSS) was added to the daily drinking water of B6 mice. The water was changed on days 3, 5, and 7. Mice were sacrificed on day 9 (the DSS group mice's body weight decreased to approximately 70% of their original weight). Mice were weighed daily. 2. Experimental Results: DSS induced colitis in mice, leading to weight loss. Treatment with N-arachidonoylglycine alleviated the weight loss.

[0031] like Figure 2 As shown, this indicates that N-arachidonoylglycine can alleviate DSS-induced colitis.

[0032] Example 4: This example provides the application of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases. N-arachidonoylglycine alleviates colonic bleeding and atrophy in DSS colitis mice.

[0033] 1. Experimental procedure: On day 0, mice were treated with N-arachidonoylglycine by gavage, while the control and DSS groups received the same dose of DMSO + corn oil by gavage. On day 1, 3% dextran sulfate (DSS) was added to the daily drinking water of B6 mice. The water was changed on days 3, 5, and 7. Mice were sacrificed on day 9 (the DSS group mice's body weight decreased to approximately 70% of their original weight). After sacrifice, the colon length of mice in the control group, DSS group, and N-arachidonoylglycine treatment group was compared. 2. Experimental Results: DSS induced colitis in mice, leading to colonic bleeding and atrophy. Treatment with N-arachidonoylglycine alleviated the bleeding and atrophy in the mouse colon.

[0034] like Figure 3 As shown, this indicates that N-arachidonoylglycine can alleviate DSS-induced colitis.

[0035] Example 5: This example provides the application of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases. N-arachidonoylglycine rescued the morphological changes of the colon in DSS colitis mice.

[0036] 1. Experimental procedure: (1) N-arachidonoylglycine was administered by gavage on day 0, while the control group and DSS group were administered the same dose of DMSO + corn oil by gavage. 3% dextran sulfate (DSS) was added to the daily drinking water of B6 mice on day 1. The water was changed on days 3, 5, and 7. Mice were sacrificed on day 9 (the weight of mice in the DSS group decreased to approximately 70% of their original weight). After sacrifice, the colons of all four groups of mice were sectioned and stained with hematoxylin and eosin (HE). (2) HE staining: 1) Place the dewaxed slices into a hematoxylin solution for staining for several minutes.

[0037] 2) Separate the acid and ammonia water for several seconds each.

[0038] 3) Rinse with running water for 1 hour, then immerse in distilled water for a short time.

[0039] 4) Dehydrate in 70% and 90% alcohol for 10 min each.

[0040] 5) Stain with alcohol-eosin staining solution for 2-3 minutes.

[0041] 6) The stained sections are dehydrated with pure alcohol and then transparent with xylene.

[0042] 7) Drip Canada balsam onto the transparent slice and seal it with a coverslip.

[0043] 2. Experimental Results: DSS induced colitis in mice, disrupting the structure and morphology of the mouse colon. Treatment with N-arachidonoylglycine alleviated the structural damage and morphological abnormalities in the mouse colon.

[0044] like Figure 4 As shown, this indicates that N-arachidonoylglycine can alleviate DSS-induced colitis.

[0045] Example 6: This example provides the application of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases. N-arachidonoylglycine inhibits inflammation in the colonic tissue of DSS colitis mice at the RNA level.

[0046] 1. Experimental procedure: (1) N-arachidonoylglycine was administered by gavage on day 0, while the control group and DSS group were administered the same dose of DMSO + corn oil by gavage. 3% dextran sulfate (DSS) was added to the daily drinking water of B6 mice on day 1. The water was changed on days 3, 5, and 7. Mice were sacrificed on day 9 (the weight of mice in the DSS group decreased to approximately 70% of their original weight). Colons were collected from all four groups of mice after sacrifice for RNA extraction. (2) RNA extraction: RNA was extracted using the Qiager RNA extraction kit. 350 mL of lysis buffer was added, and the colon tissue was homogenized into a cell suspension using a tissue homogenizer. The mixture was centrifuged at 12000 × rpm for 10 minutes, and the supernatant was transferred to a clean 1.5 mL centrifuge tube. 350 mL of RLT buffer was added and mixed. 700 mL of 70% ethanol was added and mixed. The liquid was transferred twice to RNeasy collection tubes, centrifuged at 8000 × g for 15 seconds, and the waste liquid was discarded. 700 mL of RW1 buffer was added, centrifuged at 8000 × g for 15 seconds, and the waste liquid was discarded. 500 mL of RPE buffer was added, centrifuged at 8000 × g for 15 seconds, and the waste liquid was discarded. 500 mL of RPE buffer was added, centrifuged at 8000 × g for 2 minutes, and the waste liquid was discarded. The empty tube was centrifuged at full speed for 1 minute. The collection tube was transferred to a new 1.5 mL centrifuge tube, 30-50 mL of enzyme-free water was added, and the tube was centrifuged at 8000 × g for 1 minute. Add the centrifuged solution back into the collection tube, centrifuge at 8000 × g for 1 minute, and determine the concentration of eluted RNA; (3) Reverse transcription: Reverse transcription was performed using the Novizan reagent kit. 1 mg RNA, 4 mL 4 × gDNA wiper Mix, enzyme-free water added to 16 mL, 42℃ for 2 minutes; then 4 mL 5 × qRT SuperMix II added, 37℃ for 15 minutes, 85℃ for 5 seconds.

[0047] 2. Experimental Results: DSS induced colonic inflammation in mice, leading to increased expression of inflammatory cytokines IL-1β, IL-6, and TNF-α. Treatment with N-arachidonoylglycine reduced the expression of these inflammatory cytokines.

[0048] like Figure 5 As shown, this indicates that N-arachidonoylglycine can alleviate DSS-induced colitis in mice.

[0049] Example 7: This example provides the application of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases. N-arachidonoylglycine inhibits barrier damage in the colonic tissue of DSS colitis mice at the protein level.

[0050] 1. Experimental procedure: (1) N-arachidonoylglycine was administered by gavage on day 0, while the control group and DSS group were administered the same dose of DMSO + corn oil by gavage. 3% dextran sulfate (DSS) was added to the daily drinking water of B6 mice on day 1. The water was changed on days 3, 5, and 7. Mice were sacrificed on day 9 (the weight of mice in the DSS group decreased to approximately 70% of their original weight). After sacrifice, the colons of the four groups of mice were collected for protein extraction. (2) Protein extraction: Take 2-3 mm of colon tissue, add 300 mL of protein lysis buffer, and grind the tissue into a cell suspension using a tissue homogenizer. Vortex for 10 seconds to fully lyse the cells, and let stand on ice for 15 minutes. Centrifuge at 13000 rpm for 10 minutes at 4℃, take 40 mL of supernatant and mix with 40 mL of 2 × loading buffer, and boil at 100℃ for 10 minutes to denature the protein.

[0051] 2. Experimental Results: DSS can induce colitis in mice. ZO-1 and Claudin-3 are key proteins in tight junctions, jointly maintaining the integrity of the epithelial cell barrier. Abnormal expression or function of these proteins is closely related to barrier damage in various diseases (such as inflammatory bowel disease and cancer). After treatment with N-arachidonoylglycine, the disruption of the colonic epithelial cell barrier in mice was alleviated, and the expression of ZO-1 and Claudin-3 was upregulated.

[0052] like Figure 6 As shown, this indicates that N-arachidonoylglycine can alleviate the disruption of the colonic epithelial cell barrier in mice and effectively improve colitis in mice.

[0053] Example 8: This example provides the application of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases. N-arachidonoylglycine reduces the pathological histological score of DSS colitis mice.

[0054] 1. Experimental procedure: (1) N-arachidonoylglycine was administered by gavage on day 0, while the control group and DSS group were administered the same dose of DMSO + corn oil by gavage. 3% dextran sulfate (DSS) was added to the daily drinking water of B6 mice on day 1. The water was changed on days 3, 5, and 7. Mice were sacrificed on day 9 (the weight of mice in the DSS group decreased to approximately 70% of their original weight). After sacrifice, the colons of all four groups of mice were sectioned and stained with hematoxylin and eosin (HE). (2) HE staining: 1) Place the dewaxed slices into a hematoxylin solution for staining for several minutes.

[0055] 2) Separate the acid and ammonia water for several seconds each.

[0056] 3) Rinse with running water for 1 hour, then immerse in distilled water for a short time.

[0057] 4) Dehydrate in 70% and 90% alcohol for 10 min each.

[0058] 5) Stain with alcohol-eosin staining solution for 2-3 minutes.

[0059] 6) The stained sections are dehydrated with pure alcohol and then transparent with xylene.

[0060] 7) Drip Canada balsam onto the transparent slice and seal it with a coverslip.

[0061] 8) The four indicators were scored for histopathology under an optical microscope (usually 10x and 40x objectives).

[0062] A: Degree of inflammatory infiltration: 0 points: no inflammatory cell infiltration; 1 point: a small number of inflammatory cells in the mucosal layer (mainly lymphocytes); 2 points: a moderate number of inflammatory cells in the mucosal layer; 3 points: a large number of inflammatory cells in the mucosal layer, including the submucosa; 4 points: severe inflammatory infiltration of the entire layer (mucosal layer + submucosa + muscularis propria), accompanied by neutrophil aggregation.

[0063] B: Mucosal damage 0 points: Mucosa intact, epithelial cells neatly arranged; 1 point: Epithelial cell shedding <50%, no ulceration; 2 points: Epithelial cell shedding >50%, or small area of ​​shallow ulceration (<1 / 2 of the mucosal layer); 3 points: Large area of ​​deep ulceration (involving the submucosa or deeper).

[0064] C: Destruction of crypt structure 0 points: crypt structure is intact and regularly arranged; 1 point: some crypts are twisted or shortened by 1 / 3; 2 points: crypts are reduced by 2 / 3; 3 points: crypts are almost completely disappeared.

[0065] D: Pathological range: 0 points: 0%; 1 point: 1-25%; 2 points: 26-50%; 3 points: 51-75%; 4 points: 76-100%; 2. Experimental Results: DSS induced colitis in mice, damaging the structure and morphology of the mouse colon. N-arachidonoylglycine treatment alleviated the damage to the colonic structure and morphological abnormalities, effectively reducing the histopathological score of the mouse colon in a dose-dependent manner.

[0066] like Figure 7As shown, this indicates that N-arachidonoylglycine can treat DSS-induced colitis.

[0067] Example 9: This example provides the application of N-arachidonoylglycine in the preparation of drugs for treating inflammation-related diseases. N-arachidonoylglycine reduces the DAI score in DSS colitis mice.

[0068] 1. Experimental procedure: (1) N-arachidonoylglycine was administered by gavage on day 0. The control group and DSS group were administered the same dose of DMSO + corn oil by gavage. 3% dextran sulfate (DSS) was added to the daily drinking water of B6 mice on day 1. The water was changed on days 3, 5, and 7. Mice were sacrificed on day 9 (the weight of mice in the DSS group decreased to about 70% of their original weight). Before sacrifice, the weight loss, fecal morphology, and blood in the stool were observed and recorded. (2) The DAI score is a classic macroscopic indicator for assessing the disease activity of experimental colitis (such as mouse models). It quantifies the severity of the disease by integrating three core indicators: changes in animal weight, fecal characteristics, and fecal bleeding.

[0069] The DAI (Disease Activity Index) score typically uses a 0-4 point scale. The total score (0-12 points) is the sum of the scores for the three indicators. A higher score indicates higher disease activity. The specific criteria are as follows: A. Percentage weight loss: 0 points: None; 1 point: 1-5%; 2 points: 5-10%; 3 points: 10-20%; 4 points: >20% B. Stool Appearance: 0 points: Normal; 1 point: Slightly loose; 2 points: Loose stool; 3 points: Watery stool; 4 points: Severe diarrhea C. Fecal occult blood test: 0 points: None; 1 point: Positive fecal occult blood test; 2 points: Small amount of visible blood in stool; 3 points: Large amount of blood in stool; 4 points: Total blood in stool; 2. Experimental Results: DSS induced colitis in mice, leading to weight loss, abnormal fecal morphology, and bloody stools. Treatment with N-arachidonoylglycine alleviated these symptoms, significantly reduced DAI scores in a dose-dependent manner.

[0070] like Figure 8 As shown, this indicates that N-arachidonoylglycine can reduce the DAI score in DSS colitis mice and alleviate colitis symptoms.

[0071] The embodiments selected in the above materials are for ease of understanding and not for limiting the process method. Those skilled in the art can easily modify the process flow or transfer it to other cases without inventive change. If these modifications also fall under the category of similar claims or similar technology of this invention, then the intent of this invention also includes these modifications.

Claims

1. The use of N-arachidonoylglycine in the preparation of medicaments for treating inflammation-related diseases, characterized in that, The medication for treating inflammation-related diseases includes N-arachidonoylglycine or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

2. The application according to claim 1, characterized in that: The molecular formula of N-arachidonoylglycine is C 22 H 35 NO3, with a molecular weight of 361.5, has a purity of ≥98% as determined by TLC or HPLC. It appears as a yellow waxy solid. This N-arachidonicoglycine is soluble in DMSO or 100% ethanol and achieves a solubility of 2 mg / mL in PBS at pH 7.

2.

3. The application according to claim 1, characterized in that: The medications for treating inflammation-related diseases are tablets, capsules, gels, creams, solutions, patches, powders, pills, granules, suspensions, syrups, injections, suppositories, inhalers, or sprays.

4. The application according to claim 1, characterized in that: The excipients include one or more of the following: diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, adsorbent carrier, and lubricant.

5. The application according to claim 1, characterized in that: The inflammation-related diseases mentioned are chronic inflammatory diseases or autoimmune inflammatory diseases.

6. The application according to claim 5, characterized in that: The chronic inflammatory disease is arthritis or inflammatory bowel disease; the autoimmune inflammatory disease is systemic lupus erythematosus or rheumatoid arthritis.

7. The application according to claim 1, characterized in that: The drug for treating inflammation-related diseases inhibits the release of inflammatory factors, wherein the inflammatory factors are one or more of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6).

8. The application according to claim 1, characterized in that: The drug for treating inflammation-related diseases modulates inflammatory signaling pathways, wherein the inflammatory signaling pathway is the NF-κB signaling pathway or the MAPK signaling pathway.

9. The application according to claim 1, characterized in that: The drug for treating inflammation-related diseases treats inflammation-related cells, which are one or more of macrophages, monocytes, and microglia.

10. The application according to claim 9, characterized in that: The macrophages used were RAW 264.7 macrophages, which were used to study the effects of N-arachidonoylglycine on macrophage inflammatory cytokine secretion and activation of inflammation-related signaling pathways.

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