Use of the compound indole propenoic acid in the preparation of a medicament for regulating intestinal epithelial necroptosis for preventing and treating necrotizing enterocolitis in newborns

Abstract

This invention discloses the application of the compound indoleacrylic acid in the preparation of a drug for regulating intestinal epithelial necrotizing apoptosis to prevent neonatal necrotizing enterocolitis (NEC), belonging to the field of pharmaceutical technology. This invention utilizes the compound indoleacrylic acid to alleviate related pathological features in NEC mice, including improving the survival rate of NEC mice, increasing the body weight of NEC mice, reducing small intestinal shortening and disease activity index scores, and reducing histological damage. Furthermore, it can inhibit intestinal epithelial necrotizing apoptosis in NEC mice to alleviate and relieve disease symptoms. The invention also utilizes the compound indoleacrylic acid to prepare a drug that regulates intestinal epithelial necrotizing apoptosis to prevent intestinal inflammation and intestinal necrosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of the compound indoleacrylic acid in the preparation of a drug for regulating intestinal epithelial necrotizing apoptosis and preventing neonatal necrotizing enterocolitis. Background Technology

[0002] Necrotizing enterocolitis (NEC) is a disease characterized by intestinal inflammation and necrosis, commonly occurring in newborns, particularly premature infants. It is one of the most common and severe inflammatory bowel diseases in newborns. In extremely low birth weight premature infants, the mortality rate of NEC can be as high as 30-50%, making the prognosis exceptionally challenging. Clinically, it mainly manifests as abdominal distension, internal inflammation, hematochezia, and intestinal necrosis. NEC progresses rapidly and often quickly impacts the health of newborns. The intestinal epithelium is a crucial barrier against pathogens and toxins in the intestinal lumen; epithelial damage is a hallmark of NEC. The pathogenesis involves localized necrosis of intestinal tissues caused by various factors. When the intestinal flora is disrupted, endotoxins released by bacteria can induce the production of inflammatory factors, further mediating the death of intestinal epithelial cells, ultimately leading to intestinal inflammation and necrosis. Necrotizing apoptosis is a key mechanism driving intestinal epithelial cell death and damage, involving receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Furthermore, short bowel syndrome, intestinal stenosis, and neurodevelopmental disorders are common sequelae in surviving infants after recovery, severely impacting their quality of life and psychological development. Therefore, regulating necrotizing apoptosis in the intestinal epithelium will provide a novel approach to the prevention and treatment of neonatal necrotizing enterocolitis.

[0003] Recent research and clinical data on NEC show that traditional drug therapy accounts for less than 15% of treatment options for NEC. NEC, a severe gastrointestinal emergency in the neonatal period, has a high morbidity and mortality rate among premature infants; however, drug development has been extremely slow. Most drugs currently used clinically for NEC have significant side effects, such as intestinal flora imbalance, immunosuppression, impaired neonatal growth and development, and electrolyte disturbances, greatly limiting their widespread clinical application. For infants with severe NEC, surgical treatment is primarily relied upon, such as intestinal resection and anastomosis, and enterostomy. These surgeries are not only highly invasive but also carry a high risk of postoperative complications, such as anastomotic leakage and short bowel syndrome. Furthermore, the high cost of treatment places a heavy financial burden on families. Therefore, finding new drugs and innovative treatment strategies that can effectively suppress excessive intestinal inflammation, promote the repair of damaged intestinal mucosa, and have high safety remains a critical neonatal health issue that urgently needs to be addressed. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a pharmaceutical application of indoleacrylic acid, a compound that inhibits necrotizing apoptosis of intestinal epithelium and is used to prevent and treat necrotizing enterocolitis in newborns. Indoleacrylic acid is an indole compound produced by the metabolism of tryptophan by intestinal microorganisms; its therapeutic effect in necrotizing enterocolitis has not yet been reported.

[0005] This invention provides the use of the compound indoleacrylic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and treatment of neonatal necrotizing enterocolitis.

[0006] This invention provides the use of the compound indoleacrylic acid or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and treatment of intestinal-related diseases mediated by intestinal epithelial cell necrosis.

[0007] The present invention also provides the use of the compound indoleacrylic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and alleviating related clinical symptoms following necrotizing enterocolitis.

[0008] In one embodiment of the present invention, the relevant clinical symptoms include short bowel syndrome, intestinal stenosis, and intestinal epithelial necrosis.

[0009] In one embodiment of the present invention, during the application process, the compound indoleacrylic acid exhibits inhibition of necrotizing apoptosis of intestinal epithelium. This demonstrates that the compound plays a crucial role in the development and progression of necrotizing enterocolitis and in intestinal epithelial cell necrosis. Therefore, the compound indoleacrylic acid of the present invention can be used to prepare medicaments for treating and alleviating necrotizing enterocolitis in newborns, and can also be used to prepare medicaments for alleviating intestinal diseases associated with intestinal epithelial necrosis.

[0010] This invention provides the use of the compound indoleacrylic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and treatment of neonatal necrotizing enterocolitis by regulating necrotizing apoptosis of intestinal epithelium.

[0011] In one embodiment of the present invention, the structural formula of the compound indoleacrylic acid is shown below:

[0012]

[0013] In one embodiment of the present invention, the pharmaceutically acceptable salt is an inorganic salt or an organic salt; wherein the inorganic salt is selected from hydrochloride, hydrobromide, hydroiodide, perchlorate, sulfate, hydrogen sulfate, nitrate, phosphate, acid phosphate, sodium salt, calcium salt, potassium salt, magnesium salt, silver salt, and lithium salt; and the organic salt is selected from formate, acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, glutarate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, salicylate, p-toluenesulfonate, ascorbate, meglumine salt, tromethamine salt, diethylamine salt, lysine salt, choline salt, arginine salt, tert-butylamine salt, and N,N-dibenzylethylenediamine salt.

[0014] In one embodiment of the present invention, the compound indoleacrylic acid is used to prepare a drug that inhibits necrotizing apoptosis.

[0015] In one embodiment of the present invention, the compound indoleacrylic acid is used to prepare a drug that inhibits necrotizing apoptosis of intestinal epithelium.

[0016] The indoleacrylic acid compound derived from intestinal microorganisms in this invention has the use of inhibiting intestinal epithelial necrosis and apoptosis, and its application in the preparation of drugs for regulating intestinal epithelial necrosis and apoptosis to prevent neonatal necrotizing enterocolitis.

[0017] This invention demonstrates that indoleacrylic acid, at the animal level, dose-dependently alleviates NEC-related pathological features, including improving the survival rate of NEC mice, increasing their body weight, reducing small intestinal shortening and disease activity index scores, and mitigating histological damage. Indoleacrylic acid significantly inhibits the expression of necrosis-related proteins in the ileum tissue of NEC mice. Western blot results show that indoleacrylic acid significantly inhibits the phosphorylation levels of RIPK3 and MLKL in the ileum tissue of NEC mice; immunofluorescence experiments show that indoleacrylic acid reduces the number of p-RIPK3-positive cells in intestinal epithelial cells. Therefore, the compound indoleacrylic acid of this invention has promising applications and can be used to prepare drugs for regulating intestinal epithelial necrosis and apoptosis to treat necrotizing enterocolitis.

[0018] In one embodiment of the present invention, the dosage form of the drug includes decoction, pill, powder, paste, elixir, wine, syrup, extract, lozenge, stick, suppository, medicated paste, and roasted paste.

[0019] In one embodiment of the present invention, the dosage form of the drug includes tablets, granules, blister packs, oral liquids, capsules, pills, mixtures, tinctures, aerosols, films, injections, and injectables.

[0020] In one embodiment of the present invention, the drug further contains medically acceptable excipients, including binders, fillers, disintegrants, lubricants, antioxidants, flavoring agents, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, and colorants.

[0021] In one embodiment of the present invention, the drug further comprises a pharmaceutical carrier.

[0022] In one embodiment of the present invention, the pharmaceutical carrier includes microcapsules, microspheres, nanoparticles, and liposomes.

[0023] The beneficial effects of this invention are as follows:

[0024] (1) The compound indoleacrylic acid has a strong effect in alleviating the pathological features related to NEC;

[0025] (2) The compound indoleacrylic acid can inhibit necrotizing apoptosis of intestinal epithelium and can be used to prepare drugs for the prevention and treatment of necrotizing enterocolitis;

[0026] (3) The compound indoleacrylic acid has shown the effect of alleviating the pathological features related to NEC, proving that it plays a key role in the occurrence and development of intestinal inflammation and short bowel syndrome. Therefore, the compound indoleacrylic acid of the present invention can be used to prepare drugs to alleviate intestinal inflammation and drugs to treat and alleviate the short bowel syndrome that occurs after NEC. Attached Figure Description

[0027] Figure 1 The following graph shows the effects of the compound indoleacrylic acid on the survival rate, weight change, and disease index of NEC mice. (A) Survival rate of NEC mice treated with indoleacrylic acid, recorded every 6 hours; (B) Weight change of NEC mice treated with indoleacrylic acid, recorded every 6 hours; (C) Disease index score of NEC mice treated with indoleacrylic acid. The results show that indoleacrylic acid significantly improved the survival rate, weight loss, and disease index score of NEC mice, indicating that indoleacrylic acid can alleviate the disease symptoms of NEC mice. *P<0.05, **P<0.01, ***P<0.001.

[0028] Figure 2 The results show the effects of the compound indoleacrylic acid on intestinal pathological damage in NEC mice. Figure A compares the improvement in small intestinal length in NEC mice among the blank group, NEC model group, and indoleacrylic acid treatment group; Figure B shows the improvement in ileal villus shortening and crypt loss in NEC mice, i.e., the alleviation of intestinal pathological damage in NEC mice. *P<0.05, **P<0.01, ***P<0.001.

[0029] Figure 3 The figure shows the effect of the compound indoleacrylic acid on necrosis-related apoptosis proteins in the ileum tissue of NEC mice.

[0030] Western blotting was used to detect the phosphorylation levels of MLKL and RIPK3, markers of necroptosis and apoptosis. IA treatment significantly reduced these levels.

[0031] Phosphorylation levels of RIPK3 and MLKL in the ileum tissue of NEC mice. The results indicate that indoleacrylic acid can inhibit necrotizing apoptosis in the ileum tissue of NEC mice. *P<0.05, **P<0.01, ***P<0.001.

[0032] Figure 4 The figure shows the effect of the compound indoleacrylic acid on proteins related to necrosis and apoptosis in intestinal epithelial cells of NEC mice. Immunofluorescence staining of p-RIPK3 and FABP2 (intestinal epithelial cell marker proteins) showed that p-RIPK3 was mainly present in the intestinal epithelial cells of NEC mice, and the number of positive cells decreased after treatment with indoleacrylic acid. The results indicate that indoleacrylic acid inhibits necrosis and apoptosis in intestinal epithelial cells to alleviate NEC. *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0033] The present invention will be further described below with reference to the embodiments. Due to space limitations, the description of the experimental process cannot be very detailed. All parts not described in detail in the experiment are conventional operations well known to those skilled in the art. However, the embodiments are by no means any limitation on the present invention.

[0034] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents required in the following implementations are all commercially available unless otherwise specified.

[0035] The data analysis in the examples all used Graphpad Prism 10.0 statistical analysis software for One-Way ANOVA multiple group comparisons.

[0036] Example 1: Effects of the compound on survival rate, weight change, and disease index in NEC mice

[0037] Newborn mice were randomly divided into five groups: control group, NEC group, and IA group (25, 50, and 75 mg / kg). Mice were administered low, medium, and high doses of IA from day 7 to day 10, and on day 10, TNBS (50 mg / kg) was administered to induce the NEC model. After model establishment, the survival status, weight changes, and DAI scores of the five groups of mice were observed and recorded every 3 hours.

[0038] The results are as follows Figure 1 The results showed that indoleacrylic acid significantly improved the survival rate (A), reduced weight loss (B), and improved disease index score (C) in NEC mice, indicating that indoleacrylic acid could alleviate the disease symptoms in NEC mice. *P<0.05, **P<0.01, ***P<0.001.

[0039] Example 2: Effect of the compound indoleacrylic acid on intestinal pathological damage in NEC mice

[0040] Twelve hours after TNBS-induced NEC model, changes in small intestinal length were examined in five groups of mice. The results showed that the small intestinal length of NEC mice was significantly shorter than that of the control group. Treatment with indoleacrylic acid significantly improved the small intestinal shortening in NEC mice. Paraffin sections of the terminal ileum were prepared and stained with H&E.

[0041] The results are as follows Figure 2 As shown, (A) indoleacrylic acid treatment significantly improved the shortening of the small intestine in NEC mice; (B) indoleacrylic acid significantly improved the shortening of ileal villi and loss of crypts in NEC mice, i.e., indoleacrylic acid alleviated the pathological damage to the intestines of NEC mice. *P<0.05, **P<0.01, ***P<0.001. The results showed that the small intestine length of NEC mice was significantly shorter than that of the control group mice, and indoleacrylic acid treatment significantly improved the shortening of the small intestine in NEC mice; H&E staining results showed that the ileal villi of the NEC group mice were disordered and significantly shortened or even detached, the muscle layer was ruptured, and the crypt structure was missing and significantly destroyed; the ileal villi of the IA (25mg / kg) + NEC group mice were more disordered and shorter, and the crypt structure was less destroyed; the ileal villi of the IA (50 and 75mg / kg) + NEC group mice were neatly arranged and longer, the muscle layer was intact with slight damage, and the crypt structure was more intact. H&E staining of ileal tissues from each group of mice was used to score pathological damage. The results showed that the NEC group had significantly higher scores than the control group, while the scores after IA treatment were significantly lower in the NEC group. Morphological changes in mouse ileal tissue indicate that IA can alleviate NEC-induced ileal pathological damage.

[0042] Example 3: Effects of the compound indoleacrylic acid on necrosis-associated proteins in the ileum of NEC mice

[0043] Twelve hours after TNBS-induced NEC model, the distal ileum tissue of mice in each group was removed, and Western blot was used to detect the expression of phosphorylated proteins of receptor-interacting serine / threonine kinase (RIPK3) and mixed lineage kinase domain-like protein (MLKL). The Western blot method is briefly described as follows: total protein was extracted from ileum tissue and quantified. 30 μg of the total protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane, blocked with 5% skim milk, and incubated with the antibody at 4°C overnight. After incubation with secondary antibody for 2 hours, chemiluminescence reagent was used for detection.

[0044] The results are as follows Figure 3 As shown, indoleacrylic acid treatment significantly reduced the phosphorylation levels of RIPK3 and MLKL in the ileum tissue of NEC mice. The results indicate that indoleacrylic acid can inhibit necrotizing apoptosis in the ileum tissue of NEC mice. *P<0.05, **P<0.01, ***P<0.001.

[0045] Example 4: Effects of the compound indoleacrylic acid on necrosis-associated proteins in NEC mouse intestinal epithelial cells

[0046] Twelve hours after TNBS-induced NEC model, the distal ileum tissue of mice from each group was removed, fixed, embedded, and sectioned in paraffin to detect the effect of the compound indoleacrylic acid on necrosis- and apoptosis-related proteins in NEC mouse intestinal epithelial cells.

[0047] The results are as follows Figure 4 As shown, immunofluorescence staining of p-RIPK3 and FABP2 (intestinal epithelial cell marker proteins) revealed that p-RIPK3 was mainly present in the intestinal epithelial cells of NEC mice, and the number of positive cells decreased after treatment with indoleacrylic acid. These results indicate that indoleacrylic acid inhibits necrotizing apoptosis in intestinal epithelial cells to alleviate NEC. *P<0.05, **P<0.01, ***P<0.001.

Claims

1. The use of the compound indoleacrylic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and treatment of neonatal necrotizing enterocolitis; wherein the prevention and treatment of neonatal necrotizing enterocolitis is achieved by inhibiting necrotizing apoptosis of intestinal epithelial cells.

2. The use of the compound indoleacrylic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and alleviating clinical symptoms associated with necrotizing enterocolitis in newborns; said associated clinical symptoms include short bowel syndrome, intestinal stenosis, and intestinal epithelial necrosis.

3. Use according to claim 1 or 2, characterized in that, The dosage forms of the medicine include decoctions, pills, powders, ointments, elixirs, medicated wines, syrups, tablets, sticks, and suppositories.

4. Use according to claim 1 or 2, characterized in that, The dosage forms of the drug include tablets, granules, oral liquids, capsules, pills, tinctures, aerosols, films, and injections.

5. Use according to claim 1 or 2, characterized in that, The drug also contains medically acceptable excipients, including binders, fillers, disintegrants, lubricants, antioxidants, flavoring agents, emulsifiers, osmotic pressure regulators, and colorants.

6. Use according to claim 1 or 2, characterized in that, The drug also contains a pharmaceutical carrier.

7. Use according to claim 6, characterized in that, The pharmaceutical carriers include microcapsules, microspheres, and liposomes.

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