Application of type I interferon in preparation of medicine for treating irritable bowel syndrome

Abstract

The invention discloses an application of interferon (IFN-alpha) in preparation of a medicine for treating irritable bowel syndrome. The medicine takes type I interferon (IFN-alpha) as a main component, can inhibit expression of genes related to intestinal smooth muscle contraction, and further remarkably inhibits irritable bowel comprehensive related indexes including visceral sensitivity, colon transfer speed, defecation amount and the like, so that the effect of remarkably relieving the irritable bowel syndrome is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of type I interferon in the preparation of medicines for treating irritable bowel syndrome. Background Art

[0002] Irritable bowel syndrome (IBS) is a chronic functional gastrointestinal disorder with symptoms that are either constant or intermittent. It is estimated to affect up to 20% of the population and is more common in women than in men. Some evidence suggests that middle-aged people are more susceptible to IBS, and familial clustering has also been reported. Symptoms of IBS may vary, but usually include spastic colon, chronic abdominal pain, bloating, and changes in bowel habits including diarrhea, constipation, or both. IBS can be divided into four subtypes: constipation-predominant IBS (IBS-C), diarrhea-predominant IBS (IBS-D), mixed IBS (IBS-M), and unclassified IBS (IBS-U). Among them, diarrhea-predominant IBS is a relatively common digestive disorder characterized by recurrent abdominal discomfort, bloating, and diarrhea. The symptoms of diarrhea-predominant IBS greatly affect the patient's quality of life and social function, and increase the burden on the family and society.

[0003] Because the cause of diarrhea-predominant irritable bowel syndrome is still unclear, current treatment is mainly limited to the use of anti-diarrhea drugs or laxatives to treat symptoms. There is no targeted and precise treatment method and the recurrence rate is high. Most therapies can only improve symptoms in 25-30% of patients. Therefore, it is necessary to deepen the understanding of the pathogenesis of irritable bowel syndrome and discover reliable and valuable biomarkers and therapeutic targets based on its pathogenesis to provide better treatment methods for clinical practice.

[0004] Type I interferon (IFN-α) is an important cytokine. As a key regulatory molecule in the immune system, it plays an important role in regulating immune responses, antiviral defense, and anti-tumor activity. IFN-α is mainly secreted by various types of cells (such as macrophages, dendritic cells, and lymphocytes). It activates a series of signal transduction pathways by binding to specific receptors, ultimately leading to biological effects inside and outside the cells. In clinical applications, IFN-α is widely used in the treatment of various diseases, especially in the field of infectious diseases and tumor treatment, showing significant therapeutic effects. For example, the treatment of chronic hepatitis B and C, and the treatment of malignant tumors such as melanoma and leukemia. In general, as an important immunomodulator, the wide application of IFN-α in clinical treatment not only provides an effective treatment for the treatment of various diseases, but also promotes the research and development of the field of immunotherapy. With the further understanding of its mechanism of action and the continuous advancement of treatment technology, IFN-α is expected to be more widely used in the treatment of more diseases in the future. At present, there is no literature disclosing the role of type I interferon in the treatment of diarrhea-predominant irritable bowel syndrome. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide an application of type I interferon in the preparation of a drug for treating irritable bowel syndrome. The drug has type I interferon as a main component, can inhibit the expression of genes related to intestinal smooth muscle contraction, and thus significantly inhibit the related indicators of irritable bowel syndrome, including visceral sensitivity, colon transit speed and defecation volume, thereby achieving a significant effect of alleviating irritable bowel syndrome.

[0006] To achieve the above objectives, the present invention provides the following technical solutions:

[0007] Application of type I interferon (IFN-α) in the preparation of medicines for treating irritable bowel syndrome.

[0008] Preferably, the irritable bowel syndrome is diarrhea-predominant irritable bowel syndrome.

[0009] Preferably, the type 1 interferon (IFN-α) is used alone as a medicament.

[0010] Preferably, the type I interferon (IFN-α) and other active components and pharmaceutical excipients acceptable to humans are made into a medicament for use.

[0011] More preferably, the dosage form of the medicament includes powder, capsule, pill, sustained-release tablet or injection.

[0012] The beneficial effects of the present invention are:

[0013] The present invention discloses for the first time the role of type I interferon in the preparation of drugs for irritable bowel syndrome, which can significantly alleviate the symptoms of irritable bowel syndrome in mice.

[0014] The present invention provides a new use of type I interferon, which is of great significance for the research and development of diarrhea-type irritable bowel syndrome and can provide a reference for the development of clinical drugs and mechanism research. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a comparison chart of type I interferon content in colon tissue between the NT group and the Xan group.

[0016] Figure 2 These are the results of fluorescent real-time PCR showing the down-regulation of the expression of type I interferon encoding genes Ifna and Ifnb in colon tissues of the NT group, ES group and Xan group.

[0017] Figure 3 The results show that in vitro supplementation of type I interferon inhibits the contraction of smooth muscle cells. Figure 3 A is the collagen gel contraction diagram of smooth muscle cells in the cell-free group (NT), smooth muscle cell PBS-treated group (PBS) and type I interferon-treated group (IFN-α); Figure 3 B is the flow cytometric analysis of calcium flux in smooth muscle cells treated with PBS (PBS) and type I interferon (IFN-α) groups.

[0018] Figure 4 To supplement type I interferon to alleviate the symptoms of irritable bowel syndrome in mice. Figure 4 A, 4B and 4C are the experimental results of visceral sensitivity, defecation volume per unit time and colon propulsion speed of mice, respectively. Figure 4 D is the fluorescence real-time PCR result of down-regulation of smooth muscle contraction-related genes Atp2b4 and Mylk in mouse colon tissue. DETAILED DESCRIPTION

[0019] In order to make the technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. If the conditions are not clearly stated in the embodiments, they are carried out under normal conditions, and the instruments or reagents used are all commercially available.

[0020] Example 1 Changes in the content of type I interferon in irritable bowel syndrome

[0021] Experimental process:

[0022] Wild-type mice aged 6-8 weeks were randomly divided into groups: ①NT control group: normal diet and water; ②Xan group: xanthine-induced irritable bowel syndrome group; ③ES group: electric shock-induced irritable bowel syndrome group. All experimental operations were in accordance with the requirements of the "Guidelines for the Care and Use of Laboratory Animals".

[0023] The Xan group was operated as follows: xanthine (10 mg / mouse) was given by intraperitoneal injection every two days, and the irritable bowel syndrome model was established one week later.

[0024] The ES group was operated as follows: 6-8 week old wild-type C57BL / 6J mice were randomly divided into an experimental group and a control group. All mice were placed in a laboratory room for 1 hour to acclimate to eliminate environmental interference before training. The mice in the experimental group were placed alone in a soundproof cage room with bottom power supply for 2 minutes, and then 5 random 3-second foot shocks (0.6 mA) were given within 120 seconds. The mice in the control group were placed in the same cage without power supply for 120 seconds. This was done once a day, and the model was completed after one week.

[0025] The colon tissue of mice was embedded in paraffin, sliced ​​and stained with IFN-α antibody for immunohistochemistry. At the same time, the colon tissue of mice was ground with a cryogenic grinder, and the colon RNA was extracted and reversed using the Trizol extraction method. The gene expression levels of Ifna and Ifnb were detected using Ifna and Ifnb primers (Ifna forward: 5'-AAGTATTTCCTCACAGCCAGCAG-3', Ifna reverse: 5'-AGTCCATCAGCAGCTCAATGAC-3', Ifnb forward: 5'-CAGCTCCAAGAAAGGACGAAC-3', Ifnbreverse: 5'-GGCAGTGTAACTCTTCTGCAT-3').

[0026] Experimental results:

[0027] Changes of interferon type I levels in irritable bowel syndrome Figure 1 As shown by Figure 1 It can be seen that the content of type I interferon (IFN-α) in the colon tissue of mice with irritable bowel syndrome induced by xanthine (Xan) was significantly reduced ( Figure 1 ), indicating that the level of type I interferon in colon tissue can monitor the condition of irritable bowel syndrome. At the same time, the downregulation of type I interferon encoding genes Ifna and Ifnb in colon tissue in the mouse irritable bowel syndrome model induced by electric shock (ES) and xanthine (CRS) further verified the above results ( Figure 2 ).

[0028] Example 2 In vitro experiment of type I interferon as a drug for treating irritable bowel syndrome Experimental process:

[0029] Colon smooth muscle cells were isolated from mice and treated with PBS (phosphate buffered saline) or IFN-α (1000 U / ml) for 24 h. 6 Cells / ml were resuspended in culture medium. The prepared cell suspension was mixed with collagen gel solution at a ratio of 1:4 to obtain a cell contraction matrix. 0.5 ml of cell contraction matrix was added to each well of a 24-well cell contraction plate and incubated for 1 h. After collagen polymerization, 1.0 ml of culture medium with or without IFN-α was added to each collagen gel lattice to initiate collagen gel contraction. The lattice size was measured at 0, 8, and 24 h to analyze the degree of gel contraction of the cell population.

[0030] Primary colon smooth muscle cells treated with PBS or IFN-α (1000 U / ml) were washed with PBS and 1×10 6 Each cell was added to 1 ml of 1 μM Fluo-4 staining solution and then resuspended to form a single cell suspension. The suspension was then incubated at 37°C in the dark for 30 minutes. NovoCyteTM flow cytometer was used to detect calcium ion basal level, ionomycin (20 μg / ml) and CaCl2 (2 mM) induced calcium influx.

[0031] Experimental results:

[0032] Effects of type I interferon on the contraction of colon smooth muscle cells Figure 3 As shown by Figure 3 A shows that compared with the PBS group, type I interferon (IFN-α) can significantly inhibit the contraction of colon smooth muscle ( Figure 3 A). At the same time, calcium ions play a vital role in the contraction of smooth muscle. Type I interferon (IFN-α) can inhibit calcium influx into smooth muscle cells and thus inhibit the function of actin ( Figure 3 B), the inhibitory effect was obvious at 1000U / ml.

[0033] Example 3

[0034] Application of type I interferon as a drug for the treatment of irritable bowel syndrome in mice

[0035] Experimental groups: ① control group (NT); ② electric shock-induced irritable bowel syndrome group (ES); ③ xanthine-induced irritable bowel syndrome group (Xan); ④ ES model was established and type I interferon (IFN-α1×10 6 U / mouse) treatment; ⑤ Xan modeling and simultaneous administration of type I interferon (IFN-α1×10 6 U / each) treatment.

[0036] The method of inducing irritable bowel syndrome in mice by xanthine and electric shock is the same as that in Example 1.

[0037] Experimental process:

[0038] Wild-type mice aged 6-8 weeks were selected and given the above treatments, and the model was established one week later. After the model was established, the irritable bowel of the mice was detected by using indicators such as the abdominal wall withdrawal reflex test. A significant symptom of irritable bowel syndrome is the increase in visceral sensitivity. Based on this, we tested the visceral sensitivity of the mice by giving the above mice rectal pressure to assess whether the mice had irritable bowel syndrome. The detection device mainly consists of a balloon made of polyethylene material (length: 1 cm; diameter: 0.7 cm) connected to a three-way valve through a catheter, and then the balloon catheter is connected to a pressure gauge and a syringe through a three-way valve. During the experiment, the balloon was inflated by injecting gas with a syringe to apply pressure to give the mouse rectum an expansion pressure, and the pressure value was observed by the pressure gauge. During the experiment, we lubricated the balloon with glycerol and inserted it into the mouse rectum. The proximal end of the balloon was about 2 cm away from the anus. The catheter connected to the balloon was fixed to the tail of the mouse rectum with a rubber strip, and the mouse was allowed to fully adapt for 4-5 minutes. The operator uniformly inflated the balloon with gas, and maintained the pressure at 20, 40, 60, and 80 mmHg for 20 seconds, respectively, and repeated 3 times, with an interval of 4-5 minutes each time. The observer independently and synchronously observed the abdominal withdrawal reflex (AWR) and recorded the score without knowing the inflation pressure and the mouse group. The AWR score is used to assess the visceral sensitivity of mice. (AWR scoring criteria: 0 points: no response to expansion; 1 point: only slight head movement; 2 points: abdominal contraction and trembling; 3 points: abdominal lifting; 4 points: abdominal lifting, pelvic lifting, body bending and arching).

[0039] The colonic transit speed of mice was tested by the phenol red excretion experiment: the mice were fasted for 24 hours but not water, and then gavaged with 200 microliters of 0.05% phenol red solution (phenol red dissolved in 1.5% carboxymethyl cellulose solution). Then the food was given and the feces were collected every 15 minutes and placed in 1 ml of 0.1N sodium hydroxide solution, and the time when phenol red first appeared in the feces of each mouse was recorded.

[0040] At the same time, the colon tissues of the mice were ground using a cryogenic grinder, and colon RNA was extracted and reversed using the Trizol extraction method. The gene expression levels of Atp2b4 and Mylk were detected using Atp2b4 and Mylk primers (Atp2b4 forward: 5'-CACTACGGAGGGGTACAGGAA-3', Atp2b4 reverse: 5'-AAGTCTACGCTTCTCCAAATCTG-3', Mylk forward: 5'-CCATATCCGAGAAATGCTGGG-3', Mylk reverse: 5'-TGGGATTCCAGGTATGTATCACC-3').

[0041] Experimental results:

[0042] The results are as follows Figure 4 As shown in the study, supplementation of type I interferon (IFN-α) in IBS model mice induced by electric shock (ES) and xanthine (CRS) can alleviate the symptoms of IBS, including decreased visceral sensitivity, decreased stool volume per unit time, and slower colon propulsion speed ( Figure 4 AC). At the same time, the down-regulation of smooth muscle contraction-related coding genes Atp2b4 and Mylk in colon tissue further confirmed the above results ( Figure 4 D).

[0043] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of type I interferon (IFN-α) in the preparation of drugs for the treatment of irritable bowel syndrome.

2. The use according to claim 1, characterized in that: The irritable bowel syndrome is diarrhea-type irritable bowel syndrome.

3. The use according to claim 1, characterized in that: The type 1 interferon (IFN-α) is used alone as a pharmaceutical agent.

4. The use according to claim 1, characterized in that: The type I interferon (IFN-α) and other active components and pharmaceutical excipients acceptable to human body are made into medicine for use.

5. The use according to claim 3 or 4, characterized in that: The dosage form of the medicament includes powder, capsule, pill, sustained-release tablet or injection.

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