Application of nordihydroguaiamic acid and derivatives thereof in preparation of products for treating inflammatory bowel disease

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of nordihydroguaiamic acid and derivatives thereof in preparation of products for treating inflammatory bowel diseases. By exogenous supplementation of nordihydroguaiamic acid (NDGA), DSS-induced mouse enteritis is remarkably relieved, survival prognosis of mice is improved, mucosal barrier dysfunction of UC mice is remarkably improved, the number of inflammatory macrophage subgroups is reduced, transcription generation of colon tissue proinflammatory factors and activated cleavage of pyroptosis-related proteins are inhibited, and the application of the NDGA is developed. Good anti-inflammatory effect and intestinal protection effect are achieved. The NDGA is further found to be capable of remarkably improving the mucosal barrier function disorder of the UC mouse, and the NDGA is prompted to have a good intestinal protection effect; the NDGA can inhibit the transcriptional generation of IL-1beta by regulating and controlling the NR4A1-Serpin1 pathway and reduce the co-localization of NR4A1 and NLRP3 in macrophages, which prompts that the NDGA can regulate and control the interaction relationship of NR4A1 and NLRP3 to inhibit inflammation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of nordihydroguaiaretic acid and its derivatives in the preparation of products for treating inflammatory bowel disease. Background Art

[0002] Ulcerative colitis (UC) is a recurrent nonspecific intestinal inflammatory disease whose pathogenesis has not yet been fully elucidated. With the increasing global incidence, the disease burden of the UC population is rising. Currently, UC treatments mainly include aminosalicylic acid preparations, hormones, immune preparations, small molecule drugs, etc. However, only 30% to 50% of patients achieve clinical remission within 1 year of treatment, and there is an urgent need to explore and tap new treatment methods. Although the mortality rate of UC patients has decreased in recent years, their condition has recurred, which can easily cause intestinal motility disorders, increased risk of colorectal cancer, extraintestinal complications, anxiety and depression, etc., which greatly affect the quality of life of patients and is one of the major global public health issues.

[0003] NDGA is a natural strong antioxidant with antioxidant, anti-tumor, and antibacterial effects. Previous studies have found that it has certain effects on the central nervous system, cardiovascular system, liver, kidneys, and reproductive system. In addition, a foreign authoritative journal reported that the level of NDGA in the metabolites of the intestinal flora of mice fed a high-fat diet was significantly reduced, and NDGA intervention could significantly inhibit the growth of colon cancer organoids, suggesting that NDGA may play an important role in intestinal homeostasis. However, NDGA has not been reported in inflammatory bowel disease, and its effect on enteritis needs to be explained. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide the use of nordihydroguaiaretic acid and its derivatives in the preparation of products for treating inflammatory bowel disease.

[0005] The second aspect of the present invention aims to provide a method.

[0006] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the use of nordihydroguaiaretic acid and its derivatives in the preparation of products for treating inflammatory bowel disease.

[0008] Preferably, the nordihydroguaiaretic acid or its derivative comprises at least one of a pharmaceutically acceptable salt and a pharmaceutically acceptable modification.

[0009] Preferably, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0010] Preferably, the metal salt comprises an alkali metal salt or an alkaline earth metal salt.

[0011] Preferably, the alkali metal salt includes at least one of a sodium salt and a potassium salt.

[0012] Preferably, the alkaline earth metal salt includes at least one of calcium salt, magnesium salt, barium salt and aluminum salt.

[0013] Preferably, the salt formed with the organic base includes a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N,N'-dibenzylethylenediamine.

[0014] Preferably, the salt formed with an inorganic acid includes a salt formed with the following inorganic acids: at least one of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0015] Preferably, the salt formed with the organic acid includes a salt formed with the following organic acids: at least one of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0016] Preferably, the salt formed with a basic amino acid includes a salt formed with the following basic amino acids: at least one of arginine, lysine and ornithine.

[0017] Preferably, the salt formed with the acidic amino acid includes a salt formed with the following acidic amino acids: at least one of aspartic acid and glutamic acid.

[0018] Preferably, the pharmaceutically acceptable modification comprises at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

[0019] Preferably, the inflammatory bowel disease comprises at least one of undefined inflammatory bowel disease, ulcerative colitis, Crohn's disease, undifferentiated colitis, acute colitis, and chronic colitis.

[0020] Preferably, the inflammatory bowel disease is ulcerative colitis.

[0021] Preferably, the product comprises a medicine.

[0022] Preferably, the drug comprises pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

[0023] Preferably, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0024] Furthermore, for the convenience of medication, the active ingredient nordiguaiaretic acid or its derivatives can be processed into a specific dosage form with any one or more pharmaceutically acceptable excipients. These excipients can be diluents (such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol and microcrystalline cellulose, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide and calcium carbonate, etc.), wetting agents (such as water and ethanol, etc.), binders (such as hydroxypropyl methylcellulose, povidone, starch slurry and syrup, etc.), disintegrants (such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants and cross-linked polyvinylpyrrolidone, etc.), lubricants (magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol and micropowder silica gel, etc.), colorants (such as titanium dioxide, sunset yellow, methylene blue and medicinal iron oxide, etc.), coating materials (such as acrylic resin, hydroxypropyl methylcellulose and povidone, etc.), solvents (such as water for injection, ethanol, propylene glycol and glycerol, etc.), acid-base regulators (such as hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid and sodium tartrate, etc.), antioxidants (such as sodium sulfite, sodium pyrosulfite and sodium thiosulfate, etc.), antibacterial agents (such as phenol, benzyl alcohol and thimerosal, etc.), and isotonic regulators (such as sodium chloride and glucose, etc.).

[0025] Preferably, the dosage form of the product includes a dosage form for enteral administration or a dosage form for parenteral administration.

[0026] Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0027] Furthermore, the dosage forms for administration through the gastrointestinal tract include but are not limited to enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, sucking tablets, chewable tablets, effervescent tablets, scratched tablets, sustained-release and controlled-release dosage forms such as sustained-release tablets, sustained-release coated tablets, controlled-release tablets, orally disintegrating tablets, lozenges, oral patches, etc.

[0028] Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

[0029] Furthermore, the injectable dosage forms include but are not limited to injection solutions, injection solutions, intravenous injection solutions, injection suspensions, sterile powders for injection, intravenous injections, water injections, injection emulsions, powder injections, injections, sterile powder injections, freeze-dried powder injections, etc.

[0030] Preferably, the product is administered to a mammal.

[0031] Preferably, the mammal comprises a human.

[0032] The second aspect of the present invention provides any one of the methods 1) to 2), comprising the following steps: treating cells with nordihydroguaiaretic acid or a derivative thereof.

[0033] 1) A method for inhibiting cellular inflammation in vitro for non-therapeutic purposes;

[0034] 2) A method for inhibiting cell pyroptosis in vitro for non-therapeutic purposes.

[0035] The beneficial effects of the present invention are:

[0036] The present invention significantly alleviates DSS-induced enteritis in mice, improves the survival prognosis of mice, significantly improves the mucosal barrier dysfunction of UC mice, reduces the number of inflammatory macrophage subpopulations, inhibits the transcription and generation of proinflammatory factors in colon tissue and the activation and cleavage of pyroptosis-related proteins, and has good anti-inflammatory and intestinal protective effects through exogenous supplementation of nordihydroguaiaretic acid (NDGA). Through evaluation of mucus barrier and mechanical barrier related indicators, it was found that NDGA can significantly improve the mucosal barrier dysfunction of UC mice, indicating that NDGA has a good intestinal protective effect. Through single-cell transcriptome sequencing and in vitro experimental analysis, it was found that NDGA may inhibit the transcription and generation of IL-1β by regulating the NR4A1-Serpine1 pathway, and reduce the co-localization of NR4A1 and NLRP3 in macrophages, suggesting that NDGA may regulate the interaction between the two to inhibit inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0038] Figure 1 This is a diagram showing the mechanism of action of NDGA of the present invention.

[0039] Figure 2The results of Example 1 of the present invention, including: (A) fecal microbiota transplantation experimental design; (B) bloody stool in mice; (C) survival curve of mice; (D) weight changes in mice; (E) DAI scores of mice; (FG) gross appearance of the colon and changes in colon length; (HI) HE staining of the colon and its pathological score; (JK) immunofluorescence detection of tight junction protein (Occludin, ZO-1) expression in colon tissue and its fluorescence quantitative analysis (each group of data is expressed as mean ± SEM, n = 8. *P < 0.05, **P < 0.01; analyzed by two-tailed Students' t-test).

[0040] Figure 3 The results of Example 2 of the present invention, in which: (AB) Shannon and Chao indexes show the alpha diversity of the flora; (C) relative kurtosis of species at the phylum level; (D) principal component analysis shows the differences between the flora groups of group O and group Y; (E) water volcano plot shows the differential metabolites between group O and group Y; (F) principal component analysis shows the differences in metabolites and flora composition at the phylum level between the two groups, and the metabolites are positively correlated with the flora at the phylum level between the two groups; (G) differential metabolites of the flora (top 10 differential metabolites); (H) correlation heat map shows the relationship between differential strains and metabolites; (IJ) evolutionary tree diagram and Sankey diagram show the correlation between differential strains and metabolites; (KL) targeted metabolomics detection of fecal NDGA content and its absorption peak diagram; (each group of data is expressed as mean ± SEM. *P < 0.05, **P < 0.01).

[0041] Figure 4 The results of Example 3 of the present invention are shown in Figure 2, wherein: (A) experimental design of mice; (B) survival curve analysis; (C) changes in mouse body weight; (D) pathological score of mouse colon DAI; (E) blood in stool of mice; (F) gross appearance of colon tissue; (G) colon length; (HI) HE staining of the entire length of colon tissue and its pathological score; (each group of data is expressed as mean ± SEM, n = 3-6. *P < 0.05; analyzed by two-tailed Students' t-test).

[0042] Figure 5The results of Example 4 of the present invention are as follows: (AB) immunofluorescence labeling of colon tissue MUC2 protein and fluorescence quantitative analysis; (CD) PAS staining of mouse colon tissue and analysis of its positive cell number; (EF) immunofluorescence labeling of colon tissue tight junction protein Occludin and fluorescence quantitative analysis; (GH) immunofluorescence labeling of colon tissue tight junction protein ZO-1 and fluorescence quantitative analysis; (each group of data is expressed as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 analyzed by two-tailed Student's t-test).

[0043] Figure 6 The results of Example 5 of the present invention are as follows: (A) Cell stacking diagram of different subpopulations in three groups of samples; (B) tSNE mapping diagram of cell subpopulations (the marked data set is the inflammatory macrophage subpopulation); (C) tSNE distribution diagram of LYZ2 and IL-1β genes; (D) Pathway diagram of differential gene enrichment in the macrophage data set; (E) Gene bubble map distribution of the macrophage data set; (F) Gene expression heat map of each cell subpopulation.

[0044] Figure 7 The results of Example 6 of the present invention are as follows: (A) qRT-PCR detection of pro-inflammatory factor levels in mouse colon tissue; (B) immunofluorescence co-localization of mouse colon tissue; (CE) F4 / 80 positive cells, IL-1β positive cells and CD68 + IL-1β + Double positive cell count; (FG) WB detection of GSDMD protein activation and its quantitative analysis. (Data in each group are expressed as mean ± SEM, n = 8; **P < 0.01, ****P < 0.0001 analyzed by two-tailed Students' t-test).

[0045] Figure 8The results of Example 7 of the present invention, wherein: (A) the effect of different doses of NDGA on inflammatory factors in BMDMs; (B) the effect of NDGA at different times on inflammatory factors in BMDMs; (CD) PI staining of BMDMs and positive cell counts; (E) the level of LDH released from the supernatant of BMDMs; (FI) WB detection of GSDMD, Caspase1 and NLRP3 protein levels in BMDMs and their quantitative analysis; F is the original WB image, G is the quantitative analysis result of GSDMD-NT / FL based on the original WB image, H is the quantitative analysis result of Cleaved-Caspase1 / Pro-Caspase1 based on the original WB image, and I is the quantitative analysis result of Nlrp3 / β-actin based on the original WB image (each group of data is expressed as mean ± SEM, n = 6. *P < 0.05, **P < 0.01, ****P < 0.0001 analyzed by two-tailed Students' t-test).

[0046] Fig. 9 The results of Example 8 of the present invention are as follows: (A) a bar graph of the number of differentially expressed genes between the two groups of BMDMs; (B) a volcano plot of differentially expressed genes; (C) a principal component analysis plot; (D-E) KEGG pathway and GO pathway enrichment analysis of differentially expressed genes in BMDMs; (F) a heat map of differentially expressed genes; (G) GSEA analysis showing pathways inhibited by NDGA; (H) PPI protein interaction analysis; (IJ) immunofluorescence detection of co-localization of NLRP3 and NR4A1 proteins in BMDMs cells and their quantitative analysis; (each group of data is expressed as mean ± SEM, n = 4. *P < 0.05, ****P < 0.0001 analyzed by two-tailed Students' t-test). DETAILED DESCRIPTION

[0047] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0048] The present invention explores the therapeutic effect of NDGA on inflammatory bowel disease from the following perspectives:

[0049] In the previous fecal microbiota transplantation experiment, the applicant found that mice that received fecal flora from old mice (18 months old) showed more severe intestinal inflammation in the experimental enteritis model induced by dextran sulfate sodium (DSS). The changes in the flora were evaluated by 16s rDNA sequencing, and non-targeted metabolomics characterized the differential metabolites of the flora. The results showed that the intestinal flora composition between old mice and adult mice was significantly different, and the α diversity changed; in addition, the flora metabolites were also significantly different, among which the content of nordihydroguaiaretic acid (NDGA) in the feces of old mice was the most significantly reduced. Further association analysis found that the reduction of NDGA may be related to the reduction in the abundance of strains such as Lactbacillus.

[0050] To verify the effect of exogenous supplementation of NDGA on UC, an experimental enteritis model in mice was established using DSS, and different doses of NDGA were given to evaluate the changes in body weight, DAI scores, colon length, and pathological damage in mice. The results showed that NDGA could significantly reduce enteritis in DSS mice and improve the survival prognosis of mice. By evaluating the mucus barrier and mechanical barrier related indicators, it was found that NDGA could significantly improve the mucosal barrier dysfunction of UC mice, indicating that NDGA has a good intestinal protective effect.

[0051] To explore the effect of NDGA on intestinal immune cells, the whole colon tissue of mice was free digested and dispersed into single cells, and all submucosal immune cells were sorted out using CD45 magnetic beads antibody, and single-cell transcriptome sequencing analysis was performed. The results showed that NDGA intervention significantly reduced the number of inflammatory macrophage subpopulations in DSS mice. By immunofluorescence double labeling, it was found that NDGA can significantly reduce the number of inflammatory macrophages in the colon tissue of UC mice. In addition, NDGA intervention can inhibit the transcriptional generation of pro-inflammatory factors in colon tissue and the activation and cleavage of pyroptosis-related proteins, indicating that NDGA has a good anti-inflammatory effect.

[0052] Its potential effects and mechanisms were analyzed by in vitro experiments, primary bone marrow-derived macrophages (BMDMs) were isolated, and a classic macrophage inflammation model was constructed by LPS and ATP stimulation. The results showed that NDGA inhibited the transcription level of macrophage proinflammatory factors in a dose-dependent manner, and reduced the activation cleavage of macrophage pyroptosis protein GSDMD and the activation of NLRP3 inflammasome. The results of transcriptome sequencing analysis suggested that NDGA inhibited the transcription of IL-1β by regulating the NR4A1-Serpine1 pathway. Immunofluorescence experiments verified that NDGA could reduce the co-localization of NR4A1 and NLRP3 in macrophages, suggesting that NDGA may regulate the interaction between the two to inhibit inflammation.

[0053] Figure 1 This is a schematic diagram of the relevant mechanism of NDGA of the present invention, and the specific process and data are as follows.

[0054] Example 1 Experimental animals and model construction

[0055] Eight-week-old male C57BL6J mice were selected and fed with 2.5 wt% DSS aqueous solution for 7 days to establish a mouse ulcerative colitis (UC) model. The specific steps are as follows:

[0056] 1. Select healthy, disease-free mice, making sure they weigh between 20-25 grams.

[0057] 2. The mice were divided into a control group and an experimental group, and the experimental group was further divided into different doses of NDGA intervention groups (0.5 mg / kg, 1 mg / kg and 5 mg / kg).

[0058] 3. Add 2.5wt% DSS to the drinking water of the experimental group mice and feed them for 7 consecutive days with free food intake to induce the formation of UC model.

[0059] Example 2 NDGA intervention

[0060] Different doses of NDGA were administered by gavage for intervention. The specific steps are as follows:

[0061] 1. Prepare NDGA solutions with concentrations of 0.5 mg / kg, 1 mg / kg and 5 mg / kg.

[0062] 2. The mice in the experimental group were gavaged once at a fixed time every day with 0.5 mg / kg, 1 mg / kg and 5 mg / kg respectively for 7 days.

[0063] 3. The mice in the control group were given an equal amount of normal saline.

[0064] Example 3 Observation and Evaluation

[0065] During the modeling and intervention process, the following indicators were observed and evaluated:

[0066] 1. General condition of mice: including mental state, weight changes, blood in stool and survival status.

[0067] 2. Disease activity score: The score is based on the disease symptoms of the mice. The Disease Activity Index (DAI score) is evaluated and scored from three aspects, namely body weight, fecal viscosity, fecal occult blood and other indicators. The DAI score is the sum of the three indicators. The specific scoring rules are as follows:

[0068] Table 1 DAI scoring details

[0069]

[0070] 3. Colon length measurement: After the experiment, the mice were killed, the colon was removed and its length was measured.

[0071] 4. Histopathological evaluation: HE staining was performed on colon tissue to evaluate the pathological damage and inflammatory infiltration of colon epithelial tissue.

[0072] 5. Cell and Molecular Biology Analysis

[0073] 5.1 Immunofluorescence: F4 / 80 (GB11027) and IL-1β (12242) were co-stained to evaluate the infiltration of inflammatory macrophages in the mouse colon.

[0074] 5.2 Western blotting: Detection of the activation and cleavage of macrophage GSDMD (ab209845) and the classic inflammasome pathway proteins Caspase1 (22915-1-AP), Nlrp3 (#15101) and IL-1β (12242).

[0075] 5.3qRT-PCR: Detect the mRNA expression levels of cytokines such as IL-1β, TNF-α, and IL-6 in intestinal mucosal tissue.

[0076] 6. Assessment of mucosal barrier function

[0077] 6.1 Detection of mucosal barrier protein expression: Immunofluorescence was used to detect the expression of tight junction proteins ZO-1 and Occludin.

[0078] 6.2PAS staining: observe the mucus barrier.

[0079] 6.3 Detection of mucus barrier marker protein Mucin2: Immunofluorescence was used to assess the integrity of the mucus barrier.

[0080] 7. Microbiome and metabolomics analysis

[0081] 7.1 16S rDNA sequencing and non-targeted metabolomics (Guangzhou Kidio Biotechnology Co., Ltd.): Analyze the changes in bacterial composition and structure, evaluate the effect of NDGA on bacterial diversity; display the metabolic characteristics of bacterial metabolites, and screen differential metabolites.

[0082] 7.2 Targeted metabolomics (Guangzhou Kidio Biotechnology Co., Ltd.): Verify the NDGA content in the intestinal flora.

[0083] 8. Single-cell transcriptome sequencing

[0084] 8.1 Obtain colon tissue and perform single-cell transcriptome sequencing (Guangzhou Kidio Biotechnology Co., Ltd.) to explore the immune cell population that mediates the NDGA effect.

[0085] 8.2 The potential effects and mechanisms were analyzed through in vitro experiments. Primary bone marrow-derived macrophages (BMDMs) were isolated and a classic macrophage inflammation model was constructed by stimulating lipopolysaccharides (LPS) and adenosine triphosphate (ATP).

[0086] 8.3 Evaluate the effects of NDGA on the transcriptional levels of macrophage proinflammatory factors, the activation and cleavage of the macrophage pyroptosis protein GSDMD, and the activation of the NLRP3 inflammasome.

[0087] 8.4 Transcriptome sequencing (Guangzhou Kidio Biotechnology Co., Ltd.) analysis showed that NDGA may inhibit the transcription of IL-1β by regulating the NR4A1 pathway. Immunofluorescence experiments verified that NDGA can reduce the co-localization of NR4A1 (ab153914) and NLRP3 (#15101) in macrophages.

[0088] Based on the above experimental method, the following experimental results of the effect embodiment are obtained.

[0089] Effect Example 1 Intestinal inflammation and barrier dysfunction in aged UC mice are aggravated

[0090] The intestinal barrier function of the elderly UC population is weakened and the mechanism is not completely clear. This effect example explores the molecules that may mediate this phenomenon from the perspective of the flora. The feces of young mice (2 months old, Young) and old mice (18 months old, Old) were collected respectively, and a sterile PBS solution containing 20% ​​glycerol was used to prepare the suspension supernatant at a concentration of 0.125g / ml for standby use; first, 8-week-old C57bl6 mice were gavaged with quadruple antibiotics (ampicillin 4mg / 20g, neomycin sulfate4mg / 20g, metronidazole 4mg / 20g and vancomycin, 2mg / 20g) for 3 consecutive days to eliminate intestinal flora, and then the collected donor mouse fecal supernatant was gavaged at a dose of 200ul / mouse for 5 consecutive days and transplanted into the recipient mice. After 7 days of modeling with 2.5wt% DSS, the general condition, intestinal inflammation and pathological damage of the two groups of mice were evaluated.

[0091] The results are as follows Figure 2As shown in the figure, compared with young mice, old mice showed more severe intestinal inflammation and pathological damage, which may be related to the interaction between intestinal flora and its metabolites, which needs further explanation. There are few studies on elderly enteritis, which is a relatively blank area in IBD research and deserves further exploration.

[0092] Effect Example 2 Changes in intestinal flora and its metabolites in aged mice

[0093] This effect embodiment uses 2-month-old (Young) and 18-month-old (Old) mice with C57BL6 background, which are divided into Y group and O group. Obtain their feces for 16S rDNA sequencing analysis to describe the changes in their flora composition and structure. By non-targeted metabolomics, characterize the changes in metabolites of the intestinal flora of elderly mice. Finally, the two sequencing data are jointly analyzed to find differential strains and their related metabolites that may mediate the progression of enteritis, and the corresponding metabolites are verified by targeted metabolomics.

[0094] The results are as follows Figure 3 As shown, through the combined analysis of 16s rDNA and non-targeted metabolomics sequencing, it was found that the reduction in the relative abundance of the bacterial metabolites NDGA and Lactbacillus may be related to enteritis in heavier elderly mice. Interestingly, the results of the targeted metabolomics confirmed the hypothesis of the present invention. The content of NDGA in the feces of elderly mice was relatively low; while NDGA was almost undetectable in the feces of mice receiving quadruple antibiotics for bacterial elimination. However, how NDGA and its related strains participate in the regulation of intestinal homeostasis remains to be further explored. Effect Example 3 NDGA alleviates DSS-induced enteritis in UC mice

[0095] To verify the effect of NDGA on UC, this effect example selected 8-week-old male C57BL6J mice and fed them with 2.5% DSS aqueous solution for 7 days to construct a mouse UC model. At the same time, different doses of NDGA (0.5 mg / kg, 1 mg / kg and 5 mg / kg) were administered by gavage. During the modeling process, the general condition of the mice was observed and recorded, and the colon tissue of the mice was obtained on the 7th day for further analysis. For survival analysis, it was observed until the 15th day.

[0096] The results are as follows Figure 4 As shown in the results, UC mice showed weight loss, increased disease activity scores, and shortened colons. Intervention with NDGA significantly improved the above changes, alleviated enteritis in UC mice, and improved the survival prognosis of mice. In addition, after pathological evaluation of the entire length of the colon, it was found that NDGA intervention could improve the destruction of colon villus structure, inflammatory infiltration, and its pathological score. The above results suggest that NDGA can exert an intestinal protective effect in a dose-related manner, with a dose of 5 mg / kg showing good therapeutic effects, but its mechanism of action is unclear.

[0097] Effect Example 4 NDGA intervention improves intestinal barrier dysfunction in UC mice

[0098] Male wild-type C57BL / 6 mice were selected, and the model was established and grouped as before. After model establishment, the colon tissues of the mice were obtained for PAS staining, and immunofluorescence was used to detect MUC2 and tight junction proteins and other indicators to evaluate intestinal mucus and mechanical barrier function.

[0099] The results are as follows Figure 5 As shown in the results, the secretion of mucus substances in the colonic mucosal tissue of UC mice decreased, the thickness became thinner, and the protein levels of tight junction proteins Occludin and ZO-1 decreased, the villus structure was destroyed, shortened, and inflammatory cells aggregated. After NDGA intervention, the weakening of the mucus layer was alleviated in a dose-dependent manner, and colitis was improved. In addition, in the colonic mucosal tissue of UC mice, the epithelial structure was destroyed, the crypts were disordered, and the expression of tight junction proteins was downregulated. NDGA intervention significantly improved the intestinal epithelial structure and promoted the expression of tight junction proteins. The above results suggest that NDGA intervention can effectively improve the mucus barrier and mechanical barrier of the intestine of UC mice.

[0100] Effect Example 5 NDGA reduces the number of inflammatory macrophage subpopulations in the colon of UC mice

[0101] To explore the effect of NDGA on intestinal immune cells, the mice were divided into the following three groups in this effect example: control group, DSS group, and DSS+5 mg / kg NDGA group; after the whole colon tissue of the three groups of mice was free digested and dispersed into single cells, all submucosal immune cells were flow cytometry sorted using CD45 magnetic bead antibodies, and single-cell transcriptome sequencing analysis was performed.

[0102] The results are as follows Figure 6 As shown, the single-cell sequencing results suggest that NDGA can regulate colon immune cells from a macroscopic perspective, among which the effect on macrophages is the most significant. In data sets 0, 1, 2, and 7, the number of macrophage subpopulations expressing inflammatory-related genes was significantly downregulated after NDGA intervention. Therefore, this study will focus on the mechanism of NDGA regulation of macrophages. Effect Example 6 NDGA inhibits pyroptosis of colon macrophages in UC mice

[0103] The results of single-cell sequencing suggest that NDGA can inhibit macrophage inflammation. This effect example first detects the level of pro-inflammatory factors in colon tissue, then performs immunofluorescence detection on colon tissue, uses F4 / 80 and IL-1β double labeling to observe inflammatory macrophages, and observes macrophage infiltration by laser confocal microscopy. WB is also used to detect the activation and cleavage of colon pyroptosis protein GSDMD.

[0104] The results are as follows Figure 7As shown in the results, there was a large number of macrophage infiltration and increased expression of inflammatory cytokines in the colon of UC mice, and NDGA intervention significantly improved the infiltration and activation of colonic macrophages. Detection of the N-terminal of the toxic fragment of GDSMD revealed that NDGA could inhibit the activation cleavage of GSDMD in colon tissue and reduce the level of toxic N-terminal fragments.

[0105] Effect Example 7 NDGA inhibits macrophage pyroptosis and NLRP3 activation

[0106] In order to clarify the specific effect of NDGA on macrophages, this effect example isolated mouse bone marrow-derived macrophages (BMDMs), and after LPS and ATP damage stimulation, different doses of NDGA were given for intervention, and different time periods were set to observe its effect through time-effect and dose-effect experiments. The effect of NDGA on cell pyroptosis was evaluated by observing macrophage death under a microscope, detecting the release of LDH and the level of pyroptosis proteins.

[0107] The results are as follows Figure 8 As shown in the results, NDGA can inhibit the level of inflammatory factors in BMDMs in a dose-dependent manner, and subsequent experiments were performed according to the optimal dose (10 μM). At the same time, through time-effect relationship analysis, it was found that NDGA can significantly reduce the mRNA levels of inflammatory cytokines in the early stage of inflammation in BMDMs. In addition, NDGA can inhibit the activation of pyroptosis protein GSDMD. These results suggest that NDGA has good anti-inflammatory and anti-pyroptosis potential for macrophages. However, the mechanism of action of NDGA on macrophages is not clear.

[0108] Effect Example 8 Orphan nuclear receptor NR4A1 mediates the anti-inflammatory effect of NDGA

[0109] Furthermore, in order to analyze the specific effect of NDGA on regulating macrophage inflammation. This effect example separated and cultured mouse BMDMs, and after LPS (100ng / ml) and ATP (3mM) injury stimulation, NDGA intervention (10μM) was given, that is, divided into two groups: LPS+ATP group, LPS+ATP+NDGA group. Transcriptomic sequencing was used to analyze the genes and signal pathways related to the action of NDGA.

[0110] The results are as follows Fig. 9As shown, this effect example uses transcriptomics to mine NDGA-regulated macrophage-related genes and signaling pathways, and KEGG and GO analysis found that NDGA intervention can significantly inhibit multiple inflammation-related pathways. Further, the difference heat map and PPI protein network interaction analysis suggest that NDGA may inhibit the transcription of IL-1β by regulating the NR4A1-Serpine1 pathway. Through experimental verification, this effect example found that the expression of NR4A1 and NLRP3 in LPS+ATP-stimulated macrophages was increased and the two were significantly co-localized, while NDGA can reduce the co-localization of the two. These results suggest that NR4A1 may mediate the anti-inflammatory effect of NDGA.

Claims

1. Application of nordihydroguaiaretic acid or its derivatives in the preparation of products for treating inflammatory bowel disease.

2. The use according to claim 1, characterized in that: The nordihydroguaiaretic acid or its derivative includes at least one of a pharmaceutically acceptable salt and a pharmaceutically acceptable modification.

3. The use according to claim 2, characterized in that: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid; and / or The pharmaceutically acceptable modification includes at least one of phosphorylation, sulfonation, acylation, glycosylation, ubiquitination, acetylation, methylation, sulfation, phospholipidation, and halogenation.

4. The use according to claim 1, characterized in that: The inflammatory bowel disease includes at least one of undefined inflammatory bowel disease, ulcerative colitis, Crohn's disease, undifferentiated colitis, acute colitis, and chronic colitis; Preferably, the inflammatory bowel disease is ulcerative colitis.

5. The use according to claim 1, characterized in that: The products include pharmaceuticals.

6. The use according to claim 5, characterized in that: The drug includes pharmaceutically acceptable excipients, and / or any one or more other active ingredients.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

8. The use according to claim 5, characterized in that: The dosage form of the product includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration; Preferably, the dosage form for administration via the gastrointestinal tract includes at least one of powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet; Preferably, the non-intestinal administration dosage form includes at least one of an injection dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosal administration dosage form, and a cavity administration dosage form.

9. The use according to claim 8, characterized in that: The product is administered to a mammal; Preferably, the mammal comprises a human.

10. Any of the methods 1) to 2) comprises the following steps: treating cells with nordihydroguaiaretic acid or its derivatives; 1) A method for inhibiting cellular inflammation in vitro for non-therapeutic purposes; 2) A method for inhibiting cell pyroptosis in vitro for non-therapeutic purposes.

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