Target spot for photodynamic treatment of ulcerative colitis and application thereof
By using EPHX2 protein as a biomarker and therapeutic target for ulcerative colitis, the problem of difficulty in early warning and diagnosis of ulcerative colitis in the prior art has been solved, and more accurate diagnosis and treatment effects have been achieved.
Patent Information
- Application Number
- CN202311570435.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-22
- Publication Date
- 2025-05-23
AI Technical Summary
The prior art is difficult to early warning of the onset and development trend of ulcerative colitis, and diagnosis depends on symptom evaluation and lacks reliable biological markers.
EPHX2 protein is used as a biomarker and therapeutic target for ulcerative colitis, and the expression of EPHX2 protein is reduced by expression inhibitors to improve diagnostic and therapeutic effects.
It improves the early diagnosis ability of ulcerative colitis, provides new targets for the treatment of ulcerative colitis, and enhances the accuracy and therapeutic effect of the drug.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a target for photodynamic therapy of ulcerative colitis and an application thereof. Background Art
[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.
[0003] Ulcerative colitis (UC), also known as nonspecific ulcerative colitis, is a chronic disease that causes inflammation and ulcers in the colon and rectum. The main symptoms during an attack include abdominal pain and diarrhea with blood in the stool, weight loss, fever, and anemia. Usually, the symptoms develop slowly and vary in severity. The symptoms often appear intermittently, and there is often a symptom-free period between two attacks. UC has the characteristics of a wide range of lesions, complex pathogenesis, frequent recurrences, and easy canceration. It has been listed in the list of modern intractable diseases by the World Health Organization (WHO).
[0004] In terms of treatment methods for UC, there are mainly diet therapy, drug therapy and surgical treatment. Diet therapy is effective for mild ulcerative colitis, while drug therapy has a long cycle, is prone to recurrence, and is prone to drug resistance. Surgical treatment is painful for patients, has great mental stress, and causes certain damage to the body.
[0005] With the development of pharmaceutical biotechnology, many new treatment methods have emerged. Among them, photodynamic therapy (PDT) is a promising physical and chemical treatment method with the advantages of rapid onset, strong targeting, low toxicity and side effects, and repeatable treatment. In the early stage, the new photosensitizer LD4 developed by independent synthesis was used to treat UC rat models, which achieved good therapeutic effects.
[0006] In recent years, the main detection methods for UC are colonoscopy and histological examination. However, the current diagnosis of UC still relies on symptom evaluation, and there is no reliable biological marker. In addition, the existing diagnostic criteria cannot predict the onset, efficacy and prognosis at an early stage. Summary of the invention
[0007] In view of the deficiencies in the prior art, the present invention provides a new target for photodynamic therapy of ulcerative colitis and its application, which can improve the deficiencies of the existing diagnosis of ulcerative colitis such as the inability to provide early warning and the inability to predict the onset and development trend, and can assist in the pathological typing of the disease and the research on drug action targets, precision medication, and the research on the pathogenesis, etc.
[0008] To achieve the above objectives and other related objectives, the present invention provides the use of EPHX2 protein as a biomarker for ulcerative colitis.
[0009] The inventors found that the EPHX2 protein was highly expressed in the colorectal tissue of the ulcerative colitis rat model, and in vitro cell experiments demonstrated that the expression of EPHX2 in human inflammatory colorectal epithelial cells was higher than that in normal cell groups.
[0010] The present invention also provides the use of EPHX2 protein as a therapeutic target for ulcerative colitis.
[0011] Furthermore, the present invention provides the use of an EPHX2 protein expression inhibitor in the preparation of a drug for treating ulcerative colitis.
[0012] Preferably, the EPHX2 protein expression inhibitor can reduce the expression level of the EPHX2 protein.
[0013] Preferably, the EPHX2 protein expression inhibitor can inhibit the expression of EPHX2 protein or prevent the expression of EPHX2 protein.
[0014] The present invention provides a medicine for treating ulcerative colitis. The medicine contains a biological agent or a chemical agent which targets EPHX2 and can reduce the expression amount of EPHX2.
[0015] The present invention provides a drug for treating ulcerative colitis, wherein the drug contains a biological agent or a chemical agent which targets EPHX2 and can inhibit the expression of EPHX2.
[0016] The present invention provides the use of EPHX2 protein as a biomarker for ulcerative colitis.
[0017] Those skilled in the art will appreciate that a kit for detecting ulcerative colitis containing a reagent for detecting the expression level of EPHX2 protein also falls within the scope of protection of the present invention.
[0018] Furthermore, the kit is used to diagnose whether a subject suffers from ulcerative colitis or a related disease or to predict the risk of a subject suffering from ulcerative colitis or a related disease.
[0019] The present invention provides an application of a method for detecting the expression amount of EPHX2 protein in preparing a diagnostic system for ulcerative colitis.
[0020] The beneficial effects of the present invention are:
[0021] Compared with the prior art, the present invention found that the expression of EPHX2 protein was correlated with ulcerative colitis, and found that the expression level of EPHX2 protein was significantly increased in ulcerative colitis. The present invention provides a new target for studying new drugs for treating ulcerative colitis, and also provides a detection target for the diagnosis of ulcerative colitis, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 To screen key pathogenic genes of ulcerative colitis and new targets for the treatment of ulcerative colitis based on proteomics data using the Lasso regression model.
[0023] Figure 2 To analyze the amount of immune cell infiltration in normal rats, rats with ulcerative colitis, and rats with ulcerative colitis after photodynamic therapy based on the Cibersort algorithm.
[0024] Figure 3 The Pearson correlation calculation principle was used to analyze the correlation between EPHX2 protein expression and the intestinal infiltration of various immune cells.
[0025] Figure 4 The clinical sensitivity and specificity of EPHX2 protein levels were analyzed by receiver operating characteristic curve.
[0026] Figure 5 The expression levels of EPHX2 protein in the colon tissues of rats in the normal group, ulcerative colitis group and photodynamic therapy group.
[0027] Figure 6 is the expression level of EPHX2 gene in the control group and disease group in in vitro cell experiments. Specific implementation plan
[0028] The following specific implementation methods are combined to better illustrate the present invention. It is worth emphasizing that the embodiments are only for better illustrating the present invention; however, the present invention can be presented in many different forms and is by no means limited to the embodiments described below. If any form of adjustment, modification, deletion and addition is made to the present invention by the staff in this field, it is deemed to be within the scope specified by the claims of this application.
[0029] Example 1 Synthesis of a Lysine-Modified Aminotetraphenylporphyrin Photodynamic Therapeutic Drug (LD4)
[0030] In a 100mL reaction bottle, add 0.90mmol of Boc-Lys(BOC)-OH, i.e. 311.8mg, then add 25mL of THF that has been dried in advance in a nitrogen atmosphere, and stir it with a magnetic force to react. Then cool it to -17°C, then add 0.95mol of triethylamine, i.e. 131.4μL, and then add 0.918mol of ethyl chloroformate, i.e. 87.5μL, and let it react fully for one hour. Filter and discard the precipitate. Dissolve 134.90mg of 5,10,15,20-tetrakis-(4-aminoporphyrin) in 20mL of THF, add the resulting filtrate, and stir it fully at room temperature to complete the reaction. Set up a silicagel, CH2Cl2 / MeOH / NH3·H20 (25%), 30:1:0.15 column for chromatographic separation of the obtained sample. Finally, about 368.9 mg of 5,10,15,20-tetrakis{4-[(S)-2,6-di-tert-butyloxycarbonylaminohexanamide]phenyl}porphyrin with a purity of 93% was obtained.
[0031] In a 100mL reaction bottle, add 0.90mmol of Boc-Lys(BOC)-OH, i.e. 311.8mg, and then add 25mL of THF that has been dried in advance to the bottle in a nitrogen atmosphere, and stir it with magnetic force to react. Then cool it to -17℃, then add 0.95mol of triethylamine, i.e. 131.4μL, and then add 0.918mol of ethyl chloroformate, i.e. 87.5μL, and let it react fully for one hour. When a white precipitate is formed, filter it and discard the precipitate. Dissolve 134.90mg of 5,10,15,20-tetra-(4-aminoporphyrin) in 20mL of THF in advance, add the filtrate obtained above, and stir it fully at room temperature to make it react completely. Set up a silica gel, CH2Cl2 / MeOH / NH3·H20 (25%), 30:1:0.15 column for chromatographic separation of the obtained sample. Finally, 5,10,15,20-tetrakis{4-[(S)-2,6-di-tert-butyloxycarbonylaminohexanamide]phenyl}porphyrin with a purity of 93% was obtained.
[0032] Example 2 Photodynamic therapy for ulcerative colitis in rats
[0033] Rat husbandry
[0034] The rats used for ulcerative colitis modeling were all male SD rats, about 7 weeks old. The rats were kept in a specific pathogen-free (SPF) environment with an ambient temperature of about 22°C, and were fed food and sterile water as required. The animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Laboratory Animal Management Committee / Laboratory Animal Welfare Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences. After 7 days of animal adaptation observation, the animals were weighed for the experiment.
[0035] Establishment of ulcerative colitis rat model
[0036] The model was established by using the trinitrobenzene sulfonic acid (TNBS)-induced ulcerative colitis method. Before formal modeling, the SD rats were fasted for a day and a night, and then anesthetized with 10% chloral hydrate. After that, a 50% ethanol solution containing 30 mg of TNBS prepared in advance, about 0.25 mL, was inserted into the proximal anorectum of the rat 8 cm through a polyethylene rubber catheter with a diameter of about 3 mm, and finally the TNBS solution was injected into the rat colon tissue. Then the rats were fixed in a posture with their heads low and their tails high for about 1 minute. The feces were tested with occult blood test strips every day to observe the condition of the colon tissue. The day of injection of the TNBS solution was recorded as day 0, and the ulcerative colitis model was formally established on the seventh day.
[0037] Animal grouping and treatment
[0038] Thirty-six healthy male SD rats were selected and randomly divided into 6 groups. Group 1: Control group; Group 2: TNBS group (Model group); Group 3: LD4-PDTL group (TNBS + low-dose LD4 drug photodynamic therapy group); Group 4: LD4-PDTM group (TNBS + medium-dose LD4 drug photodynamic therapy group); Group 5: LD4-PDTH group (TNBS + high-dose LD4 drug photodynamic therapy group); Group 6: SASP group (TNBS + positive control drug treatment group).
[0039] Rats treated with LD4 were given enema, and rats treated with SASP were given oral gavage.
[0040] The rats in the low-dose LD4 PDT group were all given 60 μg / kg by enema, the rats in the medium-dose LD4 PDT group were all given 120 μg / kg by enema, and the rats in the high-dose LD4 PDT group were all given 240 μg / kg by enema. As for the rats in the positive control SASP group, they were all given 500 mg / kg by gavage.
[0041] After successful modeling on the seventh day, except for the Control group and the TNBS group, the other groups began treatment on the ninth day, with LD4 administered by enema and SASP administered by gavage, with enema / gavage administered every other day, i.e., drug treatment was given on the ninth, eleventh, thirteenth and fifteenth days, for a total of four times.
[0042] 30 minutes after each administration of the LD4 photodynamic therapy drug group, the colon was irradiated with a 650nm laser photodynamic therapy system at an energy density of 25J / cm2, while the SASP group was not irradiated. During the entire animal experiment period, the body weight and food intake of the rats were measured every day.
[0043] On the 18th day, we fasted the rats. After fasting for 24 hours, on the 19th day, all rats were killed and all colon tissues and blood were taken out. The colon tissues were weighed, quickly frozen in liquid nitrogen, and then placed in a -80℃ refrigerator for subsequent proteomics sequencing, qRT-PCR, western blotting and other experiments.
[0044] Example 3 Screening of key therapeutic targets for ulcerative colitis
[0045] Proteomic Sequencing
[0046] After the rats were killed, the colon tissues of the rats in the Control group, TNBS group and LD4-PDTH group with the best photodynamic therapy effect were collected, quickly frozen in liquid nitrogen, and sent for proteomic sequencing.
[0047] 600 μL of 8 mol / L urea with protease inhibitors (lysate: protease inhibitor = 50:1) was added to the collected colon tissue, and ultrasonicated for 1 second, then left for 2 seconds, and this cycle lasted for 120 seconds. Then, the sample was centrifuged at 14,000 g for 20 minutes at 4°C, and the supernatant was taken for protein quantification.
[0048] 100 μg of protein was enzymatically desalted and eluted with 70% ACN. After elution, the protein was lyophilized. Then, 10 μL of A solution (100% water, 0.1% formic acid) was used to dissolve the lyophilized powder and centrifuged at 14,000 g for about 20 min at 4°C.
[0049] 1 μg of sample was injected from the supernatant and liquid chromatography elution was officially started with the help of Q Exactive mass spectrometer. Subsequent mass spectrometry analysis was processed using MaxQuant software. The qualitative results of proteins need to refer to the database, and the selection of the database is based on the required species, completeness of database annotation and sequence reliability.
[0050] Lasso machine learning algorithm screens key therapeutic targets
[0051] The proteomic data of the three groups of Control-Model-LD4 were classified. In view of the photodynamic therapeutic mechanism of LD4, we selected all proteins that showed an increasing and then decreasing trend in expression level and regarded these proteins as potential therapeutic targets.
[0052] With the help of R software LASSO.R, based on the Control-Model two-group proteomics data of these genes, the Lasso regression model was used to further screen key pathogenic proteins.
[0053] Example 4: Mining the diagnostic value of EPHX2 protein diseases
[0054] The clinical sensitivity and specificity of EPHX2 protein level were analyzed by receiver operating characteristic curve (ROC curve), and the AUC value was 100%. Based on the Cibersort algorithm, the intestinal immune cell infiltration amount of normal rats, ulcerative colitis rats and rats after photodynamic therapy was analyzed.
[0055] Based on the proteomic data of Control, Model and LD4, the correlation between EPHX2 protein expression and intestinal infiltration of various immune cells was analyzed using the Pearson correlation calculation principle.
[0056] Experimental Results
[0057] Based on the proteomic data of LD4 photodynamic therapy for ulcerative colitis, the Lasso machine learning algorithm was used to screen key targets, and it was found that the expression of EPHX2 protein was significantly correlated with ulcerative colitis and could be used as a biomarker for ulcerative colitis. The results of in vitro cell experiments with qPCR showed that the expression level of EPHX2 in the disease group was significantly upregulated compared with that in the control group.
[0058] EPHX2 protein is significantly associated with memory B cells. By inhibiting the expression of EPHX2 protein, the infiltration of various immune cells can be controlled, especially the abnormal increase of memory B cells during intestinal inflammation can be curbed, which can gradually reduce inflammation. The receiver operating characteristic curve analysis showed that it has excellent clinical sensitivity and specificity.
[0059] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. Application of EPHX2 protein as a therapeutic target for ulcerative colitis.
2. Application of EPHX2 protein expression inhibitor in the preparation of drugs for the treatment of ulcerative colitis.
3. Use of the EPHX2 protein expression inhibitor according to claim 2 in the preparation of a drug for treating ulcerative colitis, Features: The EPHX2 protein expression inhibitor can reduce the expression level of the EPHX2 protein or inhibit the expression of the EPHX2 protein or prevent the expression of the EPHX2 protein.
4. The drug for treating ulcerative colitis according to claim 2, Features: The drug contains a biological agent or a chemical agent that targets EPHX2 and can reduce the expression amount of EPHX2 or inhibit the expression of EPHX2.
5. A detection kit for ulcerative colitis, It is characterized in that Contains reagents for detecting EPHX2 protein expression.
6. Application of the method for detecting EPHX2 protein expression in the preparation of a diagnostic system for ulcerative colitis.
7. Application of EPHX2 protein as a biomarker for ulcerative colitis.
8. Application of EPHX2 protein in the preparation of ulcerative colitis detection kit.
9. Use of the biomarker according to claim 7 in preparing a kit for diagnosing whether a subject suffers from ulcerative colitis or a related disease or predicting the risk of a subject suffering from ulcerative colitis or a related disease.
10. Use of EPHX2 protein or EPHX2 protein expression promoter in preparing an ulcerative colitis animal model.
11. A target for photodynamic therapy of ulcerative colitis and its application, Features: The method for screening targets includes the following steps: (1) Proteomic sequencing of colonic tissues of rats with ulcerative colitis after photodynamic therapy; (2) Lasso machine learning algorithm to screen key therapeutic targets; (3) Using the Cibersort algorithm, the intestinal immune cell infiltration in normal rats, rats with ulcerative colitis, and rats after photodynamic therapy was analyzed; (4) Pearson correlation calculation principle was used to analyze the correlation between EPHX2 protein expression and intestinal infiltration of various immune cells; (5) Verify the expression level of EPHX2 protein in the colon tissue of normal rats, ulcerative colitis rats and rats after photodynamic therapy.