Use of guaiacol in the preparation of a medicament for the treatment of ulcerative colitis

Abstract

The application discloses application of guaiacol in preparation of a drug for treating ulcerative colitis. Cell experiment results show that the guaiacol can enhance the ability of macrophages to remove apoptotic cells; animal experiment results show that the guaiacol can significantly relieve the disease of a dextran sodium sulfate (DSS)-induced ulcerative colitis mouse, and the specific performance is that the guaiacol can slow down the weight loss degree of the colitis mouse, effectively reduce the disease activity index (DAI) of the colitis mouse, significantly inhibit the shortening of the colon of the colitis mouse, the infiltration of inflammatory cells and the mucosal damage, obviously reduce the level of a pro-inflammatory factor in serum of the mouse, and increase the level of a pro-inflammatory repair factor. In conclusion, the guaiacol has definite curative effect on ulcerative colitis, has no obvious side effect, and has a good prospect of being developed into a drug for treating ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical applications of guaiacol, specifically relating to the application of guaiacol in the preparation of drugs for treating ulcerative colitis. Background Technology

[0002] Ulcerative colitis is a chronic, nonspecific inflammatory bowel disease with an unclear etiology. The lesions primarily affect the rectal and colonic mucosa, clinically manifesting as persistent or recurrent diarrhea, bloody and mucous stools, and abdominal pain. The disease is protracted, prone to relapse, and carries a risk of malignant transformation, severely impacting patients' quality of life. Currently, first-line treatments include aminosalicylic acid derivatives, glucocorticoids, and immunosuppressants; however, a significant proportion of patients experience poor response, intolerance to drug side effects, or eventually develop hormone dependence / resistance. For moderate to severe cases, biologics offer more treatment options, but they also suffer from insufficient response rates and secondary failure. Therefore, there is an urgent clinical need to develop novel, highly effective, and safe therapeutic agents.

[0003] During the pathogenesis of ulcerative colitis, a large number of apoptotic cells are produced at the lesion site. If these apoptotic cells are not cleared in time, they will accumulate in large quantities and undergo secondary necrosis, releasing a large number of pro-inflammatory factors, thereby inducing or aggravating the inflammatory response. The clearance of apoptotic cells is mainly mediated by cytotoxicity. Previous published studies have confirmed that cytotoxicity is involved in the pathological response and tissue repair process of ulcerative colitis.

[0004] To date, there are no research reports, either domestically or internationally, on the use of guaiacol for the treatment of ulcerative colitis. Summary of the Invention

[0005] The present invention aims to provide the application of guaiacol in the preparation of drugs for treating ulcerative colitis, in order to overcome the above-mentioned defects of existing drugs for treating ulcerative colitis.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] Application of guaiacol in the preparation of drugs for the treatment of ulcerative colitis.

[0008] A drug for treating ulcerative colitis, the active ingredient of which is guaiacol.

[0009] The above-mentioned medications for treating ulcerative colitis are in the form of injections.

[0010] The injection is an intraperitoneal injection solution.

[0011] During in-depth research into the medicinal uses of guaiacol, the inventors discovered that this substance has significant therapeutic effects on ulcerative colitis. Cellular experiments showed that guaiacol can enhance the ability of macrophages to clear apoptotic cells; animal experiments showed that guaiacol can significantly alleviate the symptoms of ulcerative colitis induced by sodium dextran sulfate (DSS) in mice, specifically by: slowing down weight loss in colitis mice, effectively reducing the disease activity index (DAI) in colitis mice, significantly inhibiting colonic shortening, inflammatory cell infiltration, and mucosal damage in colitis mice, while significantly reducing the levels of pro-inflammatory factors in mouse serum and increasing the levels of pro-inflammatory repair factors. In summary, guaiacol is effective in treating ulcerative colitis with no significant side effects, and has a promising future as a drug for treating ulcerative colitis. Attached Figure Description

[0012] Figure 1 The images show the experimental results and statistical graphs of guaiacol (enhancing cell closure); where a is a high-content image of the cell closure effect, and b is a statistical graph of the cell closure effect.

[0013] Figure 2 This is a schematic diagram of the treatment process for mice with ulcerative colitis in an example of the present invention.

[0014] Figure 3 The figures shown are experimental results of mice in each group in the examples of this invention; where a is a physical image of mouse colon, b is a statistical chart of mouse colon length measurement results, c is a statistical chart of mouse weight change results, and d is a statistical chart of mouse DAI change results.

[0015] Figure 4 The images show the H&E staining results and pathological score statistics of colon sections from mice in each group in this invention example; where: a is the H&E staining result image, and b is the pathological score statistics chart.

[0016] Figure 5 The figures show the TUNEL staining results and apoptotic cell index statistics of colon sections from mice in each group in this invention example. In the figures, a is the TUNEL staining result and b is the apoptotic cell index statistics.

[0017] Figure 6 This is a statistical chart showing the expression levels of inflammation-related factors in the serum of mice in each group in the examples of this invention.

[0018] Figure 7 The images show the H&E staining results of sections of the five internal organs (heart, liver, spleen, lung, and kidney) of mice in each group in the examples of this invention. Detailed Implementation

[0019] I. Experimental Materials

[0020] Main experimental reagents and chemicals: guaiacol, DSS.

[0021] Experimental cells and animals: human leukemia lymphocytes (Jurkat cells), primary macrophages derived from mouse bone marrow; 6-8 week old SPF grade C57BL / 6J male mice.

[0022] II. Experimental Methods

[0023] 1. Effects of guaiacol on macrophage cytotoxicity

[0024] Primary macrophages (BMDM) from mouse bone marrow cultured for 3 days were divided into groups of 5 × 10⁻⁶ cells. 4 The concentration of the cells was seeded into 96-well plates at a concentration of [missing value], and cultured for 3 days. The cells were used for experiments on day 7. Jurkat cells were irradiated with UV for 30 minutes and then incubated for 3 hours to induce apoptosis. BMDM was added to guaiacol (GUA) and tetrandrine (COL, used as a positive control), and incubated for 3 hours. The apoptotic Jurkat cells were stained with pHrodo-red and then seeded at 5 × 10⁻⁶ wells. 5 The concentration of the sample was added to the macrophage culture system, washed twice with PBS after three hours, and then detected using a high-content cell imaging system. The cell burial index was calculated based on the imaging of macrophages phagocytizing apoptotic cells.

[0025] 2. Establishment of a mouse model of ulcerative colitis

[0026] After 1 week of acclimatization feeding, 6-8 week old male C57BL / 6J mice were given free access to drinking water containing 3% DSS for 7 days, and then switched to regular drinking water for another 3 days to complete the modeling process.

[0027] 3. Treatment of mice with ulcerative colitis

[0028] An appropriate amount of guaiacol was dissolved in phosphate-buffered saline (PBS) to prepare a drug delivery solution. At the onset of mouse modeling, the treatment group received the drug daily via intraperitoneal injection at a dose of 125 mg / kg; the normal and model groups received an equal volume of phosphate-buffered saline solution intraperitoneally daily. Daily changes in mouse body weight, fecal blood, and fecal consistency were observed and recorded. Disease Activity Index (DAI) scores were also calculated. DAI scores were based on previous study standards.

[0029] Weight loss: 0 points (no weight loss), 1 point (1-5% weight loss), 2 points (5-10% weight loss), 3 points (10-20% weight loss), 4 points (>20% weight loss); Rectal bleeding: 0 points (no blood), 1 point (positive occult blood test), 2 points (positive occult blood test with visible stool bleeding), 4 points (visible bleeding); Stool condition: 0 points (normal), 2 points (soft stool), 4 points (diarrhea).

[0030] At the end of the experiment, blood was collected from the mice via orbital puncture, and the mice were euthanized. After being left at room temperature for 30 minutes, the blood samples were centrifuged at 3000g for 10 minutes at 4°C, and the supernatant serum was collected and stored at -80°C for enzyme-linked immunosorbent assay (ELISA). Simultaneously, the colon and internal organs (heart, liver, spleen, lungs, and kidneys) of the mice were dissected and collected. After measuring the length of the colon, the colon and internal organs were fixed in 4% paraformaldehyde solution for subsequent immunohistochemical and immunofluorescence staining experiments. The H&E staining pathological scoring of mouse colon tissue was performed according to the scoring criteria in the literature "A Simplified Geboes Score for Ulcerative Colitis" (Source: Jauregui-Amezaga A, Geerits A, Das Y, et al. ASimplified Geboes Score for Ulcerative Colitis. J Crohns Colitis. 2017;11(3):305-313. doi:10.1093 / ecco-jcc / jjw154), as shown in Table 1:

[0031] Table 1 H&E staining pathology scores

[0032]

[0033] 4. ELISA detection of inflammatory-related factors in mouse serum

[0034] Before detection, standard wells, sample wells, and blank wells were prepared on the ELISA plate. Standards were serially diluted and added to the standard wells, serum samples to be tested were added to the sample wells, and diluent was added to the blank wells. Horseradish peroxidase-labeled detection antibody was then added to each well, the plate was capped, and incubated at 37°C for 45 minutes. After incubation, the supernatant was discarded, and the plate was washed five times with washing buffer. Substrate solution was added to each well, and the plate was incubated at 37°C in the dark for 15 minutes. The reaction was then terminated by adding stop solution, and the absorbance (OD value) of each well was measured at 450 nm. A standard curve was plotted based on the OD values ​​of the standards, and the concentration of the target inflammatory factor in the serum samples was calculated based on the standard curve.

[0035] 5. Data and Statistics

[0036] All statistical analyses were performed using GraphPad Prism 10 software. All data are expressed as mean ± standard error (SEM). *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant, while ns indicated no statistically significant difference.

[0037] III. Experimental Results and Analysis

[0038] 1. For example Figure 1 As shown, guaiacol can significantly enhance the cytotoxicity of macrophages and promote the clearance of apoptotic cells by macrophages, providing a cellular basis for the treatment of ulcerative colitis.

[0039] 2. For example Figures 2 to 5 As shown, guaiacol can effectively alleviate the symptoms of ulcerative colitis in mice. Compared with the normal group (Ctrl), the model group (Model) mice showed significant weight loss, severe diarrhea and bloody stools, significantly shortened colon length, and severe damage to colonic tissue structure, specifically manifested as loss of colonic epithelial integrity, abnormal crypt structure, reduced or absent goblet cells, and massive infiltration of inflammatory cells, with a large number of apoptotic cells present in the colonic tissue. Compared with the model group, the weight loss trend of mice in the guaiacol treatment group was effectively inhibited, the colonic shortening phenomenon was significantly improved, the increase of DAI was significantly suppressed, and the integrity of the colonic epithelial barrier was protected, while the infiltration of inflammatory cells and the number of apoptotic cells were significantly reduced.

[0040] 3. For example Figure 6 As shown, the analysis of serum inflammation-related factors in mice revealed that, compared with the normal group, the levels of pro-inflammatory factors (TNF-α, IL-6, IL-1β) and pro-inflammatory repair factors (TGF-β, IL-10) were significantly increased and decreased in the serum of the model group mice. Compared with the model group, the levels of pro-inflammatory factors (TNF-α, IL-6, IL-1β) and pro-inflammatory repair factors (TGF-β, IL-10) were significantly decreased and increased in the serum of the guaiacol treatment group mice, indicating that guaiacol can exert a therapeutic effect by regulating the balance of inflammatory factors.

[0041] 4. For example Figure 7 As shown, the H&E staining results of the sections of the five internal organs (heart, liver, spleen, lung, and kidney) of mice in each group showed that no obvious histopathological abnormalities were found in the five internal organs of mice in the guaiacol treatment group, indicating that guaiacol has good biosafety at the dose used in this experiment.

[0042] In conclusion, guaiacol, a cell closure enhancer, has a significant therapeutic effect on ulcerative colitis and is a rational and effective treatment option with broad prospects for development and application.

Claims

1. Application of guaiacol in the preparation of drugs for the treatment of ulcerative colitis.

2. A treatment for ulcerative colitis, characterized in that, The active ingredient is guaiacol.

3. The medicament for treating ulcerative colitis according to claim 2, characterized in that... This includes guaiacol and pharmaceutically acceptable excipients.

4. The ulcerative colitis treatment drug according to claim 3, characterized in that, The drug is in the form of an injection.

5. The medicament for treating ulcerative colitis according to claim 4, characterized in that, The injection is an intraperitoneal injection solution.

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