Application of indigo in preparation of medicine for treating Crohn disease

Abstract

The invention provides application of indigo in preparation of a medicine for treating Crohn's disease, and relates to medical application of bisindole compounds. Specifically, the invention relates to a medicine / composition prepared from indigo blue for treating the Crohn's disease, and the medicine / composition can inhibit intestinal inflammatory response of the Crohn's disease and promote mucous membrane healing. Mechanism research finds that the therapeutic mechanism of the invention is to inhibit polarization of M1 type macrophages. Indigo can regulate differentiation of macrophages and reduce the number of spleen M1 type macrophages in Crohn's disease, so that the purpose of treating Crohn's disease is achieved.

Description

Technical Field

[0001] The present invention relates to the medical use of bis - indole compounds, in particular to the application of indigo in the preparation of drugs for treating Crohn's disease. Background Art

[0002] Crohn's disease (CD) is one of the main diseases in the spectrum of inflammatory bowel disease (IBD), characterized by chronic transmural inflammation. The lesions often involve the terminal ileum and colon, and the clinical manifestations include abdominal pain, diarrhea, weight loss, fatigue, etc., seriously affecting the quality of life of patients. In recent years, the incidence of CD has been continuously rising globally. According to statistics, the prevalence of CD in Europe and America has exceeded 200 cases per 100,000 people. Crohn's disease is not only closely related to local complications such as intestinal stricture and fistula, but also co - exists with various extra - intestinal manifestations such as anemia, osteoporosis, and arthritis, and is a risk factor for the occurrence of colon cancer. The conventional clinical drugs for CD mainly include aminosalicylate drugs, glucocorticoid drugs, immunosuppressants, and enteral nutrition, etc. Aminosalicylate drugs are relatively safe with fewer adverse reactions, but usually have poor effects on severe patients and are more suitable for mild to moderate CD and maintenance treatment in the remission period. Glucocorticoid drugs such as methylprednisolone, prednisone, budesonide, etc., although they have a rapid onset of action, cannot maintain treatment and need to be gradually tapered off. Traditional treatment drugs have the characteristics of economy, convenience, and clear mechanisms, but there are obvious limitations, such as drug intolerance, a relatively high incidence of adverse reactions, a relatively high disease recurrence rate, and individual differences, etc. Therefore, the development of effective drugs is of great significance for the treatment of CD and the improvement of the prognosis of patients.

[0003] In traditional Chinese medicine theory, the basic pathogenesis of CD is the accumulation of heat - toxin in the intestine, damaging the intestinal collaterals, mostly belonging to the syndrome of deficiency in origin and excess in superficiality. The main pathological factors include dampness, heat, toxin, etc., which often interact with each other. Heat accumulates in the intestine, damaging the intestinal collaterals, and when the collaterals are damaged and blood overflows, it leads to hematochezia or dysentery with purulent bloody stools; when the heat - toxin is extremely severe, the blood and flesh are corrupted, flowing and forming abscesses, resulting in intestinal fistulas and abdominal abscesses. Therefore, traditional Chinese medicine often diagnoses and treats from aspects such as clearing heat and detoxifying, cooling blood and removing stasis. In this context, the traditional Chinese medicine indigo naturalis has become one of the commonly used drugs for treating CD due to its extensive therapeutic effects. In view of this, in - depth exploration of the specific mechanism of action of indigo and its active ingredients in the treatment of CD not only has great scientific value, but also provides important practical significance for the further development and optimization of the traditional Chinese medicine treatment plan for CD. Indigo is a bis - indole compound widely present in traditional Chinese herbs such as indigo naturalis, and has anti - inflammatory, antibacterial, anti - tumor, immunomodulatory, and cell proliferation - inhibiting effects, and can specifically treat the pathogenesis of CD. However, at present, its effects and mechanism of action on CD are not very clear. Therefore, the present invention has carried out research on the effects and mechanism of action of indigo in the treatment of CD, and is expected to provide a new direction for the clinical diagnosis and treatment of CD. Summary of the Invention

[0004] One of the objectives of this invention is to provide the use of indigo in the preparation of drugs / compositions for treating Crohn's disease, in order to solve the technical problems of drug tolerance, high incidence of adverse reactions, high relapse rate, and individual differences in the treatment of Crohn's disease with conventional drugs.

[0005] In the uses provided by this invention, the drug / composition can inhibit the intestinal inflammatory response in Crohn's disease and promote mucosal healing.

[0006] Furthermore, the mechanism by which the drug / composition treats Crohn's disease is by inhibiting M1 macrophage polarization.

[0007] Furthermore, the dosage form of the drug / composition is tablet, capsule, soft capsule, enteric-coated tablet, enteric-coated tablet capsule, enteric-coated soft capsule, suspension, powder for suspension, orally disintegrating tablet, lozenge, tablet, suppository, enema, ointment, cream or gel.

[0008] Furthermore, the indigo is natural indigo or synthetic indigo.

[0009] Furthermore, the natural indigo is purified from Isatis indigotica leaves, Isatis tinctoria root, Polygonum tinctoria, or Isatis tinctoria.

[0010] The use of indigo in the preparation of drugs / compositions for treating Crohn's disease provided by this invention can effectively inhibit the intestinal inflammatory response in Crohn's disease and promote mucosal healing. Mechanistic studies have found that it can regulate the differentiation of M1 macrophages in Crohn's disease and reshape immune homeostasis, thereby achieving the purpose of treating Crohn's disease. Detailed Implementation

[0011] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0012] According to one aspect of the invention, indigo is provided for use in the preparation of medicaments / compositions for treating Crohn's disease.

[0013] The present invention has the following advantages: (1) Indigo, as a bisindole compound, is widely found in Chinese herbal medicines such as Isatis tinctoria leaf, Isatis tinctoria root, and Indigo naturalis. It has anti-inflammatory, antibacterial, antitumor, immunomodulatory and cell proliferation-inhibiting effects, and can specifically target the pathogenesis of Crohn's disease for treatment. (2) Indigo has the advantages of low relapse rate, low incidence of adverse reactions and low price in the treatment of Crohn's disease; (3) Indigo can effectively inhibit intestinal inflammation in Crohn's disease and promote mucosal healing.

[0014] In a preferred embodiment of the invention, the drug / composition is able to inhibit the intestinal inflammatory response in Crohn's disease and promote mucosal healing.

[0015] In a preferred embodiment of the present invention, the mechanism of the drug / composition for treating Crohn's disease is to inhibit M1 macrophage polarization.

[0016] In a preferred embodiment of the present invention, the dosage form of the drug / composition is tablet, capsule, soft capsule, enteric-coated tablet, enteric-coated tablet capsule, enteric-coated soft capsule, suspension, powder for suspension, orally disintegrating tablet, lozenge, tablet, suppository, enema, ointment, cream or gel.

[0017] In a preferred embodiment of the present invention, the indigo is natural indigo or synthetic indigo.

[0018] In a further preferred embodiment of the present invention, natural indigo is purified from Isatis indigotica leaves, Isatis tinctoria root, Polygonum tinctoria, or Isatis tinctoria. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 Line graphs showing the rate of increase in body weight for each group of mice; Figure 2 Line graphs showing the daily DAI scores of mice in each group; Figure 3 The colon lengths of mice in each group are represented by A, where A represents the total colon length of mice in each group, and B represents the colon length of mice in each group. Figure 4 The spleen weight ratio of mice in each group is recorded, where A represents the spleen of mice in each group and B represents the spleen weight ratio of mice in each group. Figure 5 The spleen weight ratio of mice in each group; Figure 6 The study investigated the regulatory effect of indigo on TNBS-induced macrophages in CD mice. In this study, A represents M1 macrophages in CD mice, B represents M2 macrophages in CD mice, C represents the content level analysis of M1 macrophages in CD mice, and D represents the content level analysis of M2 macrophages in CD mice.

[0021] The technical solution provided by the present invention will be further described below in combination with test examples.

[0022] Test Example 1: 1 Materials and Methods 1.1 Experimental animals Fourteen healthy SPF-grade male C57BL / 6 mice, weighing 18 - 22 g, were purchased from Beijing Spepharm Biotechnology Co., Ltd., with the experimental animal license number: SCKK (Beijing) 2019 - 0010. They were housed in the SPF-grade animal breeding room of the Experimental Animal Center of Dongfang Hospital, Beijing University of Chinese Medicine. The breeding room had alternating light and darkness (12 h of daylight and 12 h of night), a temperature of 20 - 24 °C, and a relative humidity of 50% - 60%. During the breeding period, the mice had free movement, sufficient clean drinking water, and were fed with normal mouse food or a high-fat diet.

[0023] 1.2 Experimental drugs: Before daily gavage, indigo was weighed and fully dissolved in sterile water at a concentration of 6 mg / ml.

[0024] 1.3 Main reagents and instruments Trinitrobenzenesulfonic acid (TNBS, Sigma - Aldrich); fecal occult blood (OB) reagent (Zhuhai Biosino Biotechnology Co., Ltd.); 10% neutral formalin fixative (Yili, MKYLFEMA - 10%); FBS (Gibco); bovine serum albumin (Beijing BiolDee Biotechnology Co., Ltd.); Percoll, red blood cell lysate, 1×PBS, double antibodies, bovine serum albumin, neutral resin, sterile and enzyme-free water, red blood cell lysate (all purchased from Solarbio); hematoxylin - eosin (HE) staining solution set, 3 mm grinding beads (Wuhan Sevier Biotechnology Co., Ltd.); 75% alcohol disinfectant solution (Shandong Lilkon Technology Co., Ltd.); absolute ethanol, xylene, isopropanol, chloroform (Sinopharm Chemical Reagent Co., Ltd.); flow cytometry antibodies F4 / 80, CD16 / 32, CD206 (Biolegend), RPMI1640 medium (Invitrogen); DP74 microscope (Olympus); flow cytometer (BD Biosciences).

[0025] 1.4 Animal grouping, model establishment and drug administration After one week of acclimatization, mice were randomly assigned to three groups based on body weight: a control group (n=4), a model group (n=4), and an indigo group (n=6). On day 1, the control group received no intervention, while the other 10 mice underwent TNBS enema to establish the CD model. After anesthetizing the mice, a 2 mm diameter soft catheter was used to administer a single enema of TNBS solution (30 mg / kg, 50% ethanol). Once the CD model was successfully established, the indigo group received 60 mg / kg indigo suspension, starting with enema on day 2. To maintain parallelism, the other groups received the same volume of sterile drinking water enema at the same time. Mice were sacrificed on day 7. During this period, the mice's general condition and symptoms were observed daily, and their weight, fecal occult blood tests, and fecal characteristics were recorded. The Disease Activity Index (DAI) was calculated. After the intervention, the mice were harvested. Colonic tissue was separated from the anus, washed with ice-cold PBS, and the colon length was measured and photographed. The colonic tissue was then cut into segments approximately 0.5 m long. Colonic segments from the same location were placed in 10% neutral formalin for HE staining to observe histopathological conditions. Assessment indicators included the number of crypts, the extent of intestinal epithelial damage, and the degree of damage to the mucosa and submucosa. The remaining segments were placed in sterile EP tubes, transferred to liquid nitrogen, and frozen at -80°C for later use. Splenic tissue samples were obtained and ground using a 70μm cell sieve, subjected to schizochrome pluvialis staining, density gradient centrifugation, and flow cytometry staining. Flow cytometry was used to assess the content of mouse spleen macrophage subsets.

[0026] 1.5 Observation Indicators and Methods 1.5.1 General Observation Observe and record the mice's weight, diet, activity level, mental state, fur condition, frequency and characteristics of defecation, etc.

[0027] 1.5.2 Efficacy Evaluation Criteria Calculate the disease activity index (DAI). DAI = (body weight loss index + fecal characteristics + fecal occult blood) / 3. See Table 1 for specific evaluation criteria.

[0028] Table 1 Scoring criteria for the Disease Activity Index

[0029] 1.5.3 Colon length and spleen-to-body weight ratio When collecting samples, the total length of the mouse colon was measured, the spleen was removed and weighed, and the spleen-to-body weight ratio was calculated.

[0030] 1.5.4 Pathological morphological observation During tissue collection, colonic tissue was transversely cut from the mouse colon 1-1.5 cm from the anus. Feces were removed, and the tissue was washed with PBS and fixed in 10% formalin for 24 hours. After removal, the tissue was dehydrated in a gradient of alcohols, then embedded in paraffin, sectioned at 5 μm thickness, mounted, dewaxed, stained with hematoxylin and eosin (HE), and mounted for observation of the mouse colonic tissue morphology and structure under a light microscope. During the preparation process, it is important to fix the animal tissue rapidly after sacrifice to preserve its original morphological structure. The scalpels and scissors used for cutting tissue blocks must be sharp, and the tissue should not be gripped too tightly to avoid deformation of the tissue and cells due to compression. Tissue should be accurately collected according to the anatomical location, and the material should be kept clean. The histopathological features of the colonic tissue were observed under a light microscope, and a histopathological score was performed. Specific evaluation criteria are shown in Table 2.

[0031] Table 2. Scoring criteria for colonic histopathology

[0032] 1.5.5 Flow cytometry detection of macrophage subsets in mouse spleen The entire procedure was performed at 4°C. Mouse spleens were harvested, and the tissue was gently homogenized using a sterile syringe plunger. The cells were filtered through a 70μm cell sieve, subjected to erythrolysis for 10 min, centrifuged, the supernatant discarded, and the cells resuspended. After blocking for 20 min, the cells were centrifuged again at 4°C, 1600 rpm for 5 min, and then resuspended. Flow cytometry antibodies (F4 / 80, CD16 / 32, CD206) were added, and the cells were incubated at 4°C in the dark for 40 min for cell membrane staining. After washing and centrifugation, the cells were resuspended and analyzed by flow cytometry. The data were then analyzed using Flowjo.

[0033] 2. Data Statistics SPSS 22.0 and GraphPad Prism 8.0.1 software were used for data analysis and graphing. Quantitative data were presented using (…). The data were tested for homogeneity of variance, and one-way ANOVA was used for comparisons between groups. When the variances were homogeneous, Tukey's test was used for comparisons between the two groups, and Dunnett's t3 test was used for comparisons between groups with unequal variances. The overall efficacy rate was a count data point, and the chi-square test was used. A p-value < 0.05 was considered statistically significant.

[0034] 3 Results 3.1 Effects of indigo on the general condition of TNBS-induced CD mice All groups of mice tolerated the modeling and drug intervention well. Behavioral performance was observed in each group. The control group showed no abnormalities, was energetic, had a good appetite and was active, with glossy and smooth fur. Compared to the control group, TNBS-induced mice exhibited significant intestinal symptoms, such as varying degrees of bloody stools, diarrhea, and weight loss, as well as lethargy, slow movement, poor appetite, dull fur, arched backs, and gregarious behavior. After indigo intervention, the mice's mental state, behavioral changes, dull fur, and symptoms of diarrhea and bloody stools were significantly alleviated compared to the model group.

[0035] 3.2 Effects of indigo on body weight in TNBS-induced CD mice Compared with the control group, the body weight of mice in the model group and the indigo group decreased significantly after modeling; compared with the model group, the body weight of mice in the indigo group gradually increased after indigo intervention (comparing the control group and the model group, # P <0.05, ## P <0.01; Compared with the model group, the indigo group, * P < 0.05), see details Figure 1 .

[0036] 3.3 Effects of indigo on DAI scores in TNBS-induced CD mice Compared with the control group, the DAI scores of mice in the model group and the indigo group were significantly increased after modeling. Compared with the model group, the DAI scores of mice in the indigo group gradually decreased after treatment (comparison between control group and model group, #P<0.05, ##P<0.01; comparison between indigo group and model group, *P<0.05), see details. Figure 2 .

[0037] 3.4 Effect of indigo on colon length in TNBS-induced CD mice Compared with the control group, the colon length in the model group was significantly shortened (#P<0.05), and the colon was rigid with obvious intestinal fibrosis. Compared with the model group, the colon length of mice in the indigo group recovered significantly after treatment (*P < 0.05). See details. Figure 3 .

[0038] 3.5 Effects of indigo on spleen and body weight ratio in TNBS-induced CD mice Compared with the control group, the spleen size and length in the model group were significantly increased, and the spleen-to-body weight ratio was significantly higher (#P<0.05). Compared with the model group, the spleen size and length in the indigo group showed a trend of recovery, while the spleen-to-body weight was significantly lower than that in the model group (*P < 0.05). See details. Figure 4 .

[0039] 3.6 Effects of indigo on the histopathology of colonic tissue in TNBS-induced CD mice Under a light microscope, compared with the control group, the model group showed significant edema of the colonic mucosa, intestinal epithelial sloughing, loss of villi, glandular deformation, and local erosion, bleeding, and inflammatory cell infiltration (##P<0.01). Compared with the model group, after indigo treatment, the glandular structure of mice showed significant recovery, the degree of edema was reduced, inflammatory cell infiltration was improved, and no significant erosion or epidermal cell sloughing was observed (**P < 0.01). See details. Figure 5 .

[0040] 3.7 Regulatory effect of indigo on TNBS-induced macrophages in CD mice Mechanistically, mouse spleens were collected, and single-cell suspensions were prepared for staining. Flow cytometry analysis revealed that the levels of M1 macrophages (F4 / 80+, CD16 / 32+) in the spleens of the model group mice were significantly increased (##P<0.01), and the levels of M2 macrophages (F4 / 80+, CD206+) were also increased (#P<0.05). Compared with the model group, the levels of M1 macrophages in the indigo group mice were significantly decreased (*P < 0.05), but the regulatory effect on M2 macrophages was not significant. This suggests that M1 macrophages may be the main target of indigo treatment for Crohn's disease. (See details...) Figure 6 .

[0041] In conclusion, indigo can effectively inhibit the intestinal inflammatory response and promote mucosal healing in Crohn's disease. Its mechanism of action is related to the inhibition of M1 macrophage polarization, which provides new ideas and strategies for the treatment of Crohn's disease.

Claims

1. Use of indigo in the preparation of medicaments / compositions for treating Crohn's disease.

2. The use according to claim 1, characterized in that, The drug / composition can inhibit the intestinal inflammatory response in Crohn's disease and promote mucosal healing.

3. The use according to claim 1, characterized in that, The mechanism of action of the drug / composition in treating Crohn's disease is the inhibition of M1 macrophage polarization.

4. The use according to claim 1, characterized in that, The dosage form of the drug / composition is tablet, capsule, soft capsule, enteric-coated tablet, enteric-coated tablet capsule, enteric-coated soft capsule, suspension, powder for suspension, orally disintegrating tablet, lozenge, tablet, suppository, enema, ointment, cream or gel.

5. The use according to claim 1, characterized in that, The indigo is either natural or synthetic.

6. The use according to claim 5, characterized in that, The natural indigo is purified from Isatis indigotica leaves, Isatis tinctoria root, Polygonum tinctoria, or Isatis tinctoria.

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