Application of locust tachykinin I acetate in preparation of medicine used as Ddx39b inhibitor

Abstract

The invention provides an application of locust tachykinin I acetate in preparation of a medicine used as a Ddx39b inhibitor, and the locust tachykinin I acetate is proved to have a specific inhibition effect on Ddx39b, and the locust tachykinin I acetate has good safety. Meanwhile, the invention also verifies that the locust tachykinin I acetate has a good curative effect on relieving the acute kidney injury, so that the locust tachykinin I acetate has a good application prospect in relieving and treating AKI (acute kidney injury).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, in particular to the use of T22928L as a Ddx39b inhibitor. BACKGROUND

[0002] Compound Locustatachykinin I acetat

CC(C)[C@@H](C(N[C@@H](CCCNC(N)=N)C(N)=O)=O)NC(CNC([C@H](Cc(cc1)ccc1O)NC([C@H](Cc1ccccc1)NC(CNC([C@H](CO)NC([C@H](CCC1)N1C(CN)=O)=O)=O)=O)=O)=O)=O

[0003] The product number is T22928L. T22928L is a potent, rapid and sustained secretory stimulant in the Malpighian tubules of locusts. It is mainly involved in controlling the formation of primary urine, Malpighian tubule peristalsis and local sphincter function in locusts. In terms of mechanism of action, it may activate intracellular signaling pathways by binding to specific receptors on the surface of Malpighian tubule cells. Studies have speculated that there is sequence homology with mammalian tachykinins such as substance P, and possible related receptors such as Drosophila tachykinin receptor (DTKR) and Neurokinin receptor from Drosophila (NK0) have been identified. Although the specific binding mode and activation mechanism of T22928L with these receptors are not fully clear, it is speculated that it may play a role through similar receptor-mediated mechanisms. After binding to the receptor, it may trigger a series of biochemical reactions within the cell, such as the regulation of ion channels and the production of second messengers, ultimately leading to changes in the secretory activity of Malpighian tubule cells, affecting urine formation and related physiological processes. In terms of research progress, some understanding of its function in locusts has been gained, but further research is needed to understand the detailed molecular mechanisms of action, such as the downstream signaling cascade after receptor activation, the relationship with other neuropeptides or regulatory factors, etc. In addition, the expression regulation and functional changes of T22928L under different environmental conditions or physiological states of locusts also need to be explored.

[0004] Ddx39b (DEAD box helicase 39B) is a key enzyme in RNA metabolism, involved in splicing, translation and regulation of viral replication. It is highly expressed in cancer (such as colorectal cancer, breast cancer), promoting tumor proliferation and metastasis; at the same time, as a host factor of RNA viruses such as new coronavirus, it is involved in viral genome replication. Therefore, Ddx39b inhibitors have become a hot spot for the research and development of anti-cancer and anti-viral drugs.

[0005] Existing inhibitors include small molecule compounds (such as BMS-986165, targeting helicase domain inhibition activity), nucleic acid aptamers and gene silencing technology. The development strategy focuses on structure-guided design (based on crystal structure to screen highly selective molecules) and functional screening. However, there are challenges in selectivity (avoiding off-target of the DEAD family) and pharmacokinetic optimization. SUMMARY

[0006] The present application found in the study that locustatachykinin I acetate has a specific inhibitory effect on Ddx39b and has good safety. Further, the use of locustatachykinin I acetate in the preparation of a medicament for use as a Ddx39b inhibitor is provided.

[0007] In an embodiment according to the present application, the medicament is used to alleviate or treat a disease associated with Ddx39b activation; preferably the disease is selected from one or more of hepatitis D virus (HDV) infection, colorectal cancer, breast cancer, lung cancer, acute kidney injury.

[0008] In an embodiment according to the present application, the medicament is used to alleviate or treat acute kidney injury (AKI).

[0009] In an embodiment according to the present application, the medicament is used to alleviate or treat acute kidney injury caused by ischemia-reperfusion (IRI-AKI).

[0010] In an embodiment according to the present application, the medicament is used to alleviate or block the transition of acute kidney injury (AKI) to chronic kidney disease (CKD).

[0011] The present application first found that locustatachykinin I acetate has a specific inhibitory effect on Ddx39b and has good safety. At the same time, the present application also verified that locustatachykinin I acetate has a good therapeutic effect on alleviating acute kidney injury, and thus has a good application prospect in alleviating and treating AKI. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Schematic diagram for molecular docking analysis of the interaction between Ddx39b protein and small molecule compound T22928L;

[0013] Figure 2 Figure for the detection results of observing the interaction between T22928L and Ddx39b by cell thermal shift assay (CETSA); wherein, (A) is the cell thermal shift assay (CETSA) for observing the interaction between T22928L and Ddx39b and its effect on the thermal stability of Ddx39b protein. (B) is a gray scale analysis chart;

[0014] Figure 3 Figure for the experimental results of HK-2 cell viability treated with small molecule compound T22928L (concentration 1, 5, 10, 20 μM) under H / R model. Wherein, ns: no statistical difference, ***P<0.001, **P<0.01, *P<0.05;

[0015] Figure 4 Figure for the results of flow cytometry detection of the effect of Locustatachykinin I acetate on the DNA content of HK2 cells. Wherein, ns: no statistical difference, ***P<0.001.

[0016] Figure 5 Figure for the results of mRNA stability experiment for detecting the effect of Locustatachykinin I acetate on the mRNA half-life of Cdc20, a key downstream target gene of Ddx39b. **P<0.01;

[0017] Figure 6 Figure for the results of RIP experiment for detecting the effect of Locustatachykinin I acetate treatment on the mRNA binding of Ddx39b and Cdc20;

[0018] Figure 7 Figure for the HE staining results of paraffin sections of mouse heart, liver, spleen, lung and kidney tissues in different concentrations of Locustatachykinin I acetate treatment groups; wherein, scale: 50 μm;

[0019] Figure 8 Figure for the blood routine results of mice in different concentrations of Locustatachykinin I acetate treatment groups; wherein, ns: no statistical difference;

[0020] Figure 9Figure for detecting the liver function of mice in different concentrations of Locustatachykinin I acetate treatment groups; wherein, ns: no statistical difference;

[0021] Figure 10 Figure for detecting the kidney function of mice in different concentrations of Locustatachykinin I acetate treatment groups; wherein, ns: no statistical difference;

[0022] Figure 11 Figure for detecting the effect of Locustatachykinin I acetate on the kidney function of the bilateral IRI-AKI model mice by serum creatinine, wherein, ***P<0.001;

[0023] Figure 12 Figure for analyzing the effect of Locustatachykinin I acetate on the DNA content of renal tubular epithelial cells 7, 14 and 28 days after IRI by flow cytometry; wherein, ***P<0.001;

[0024] Figure 13 Figure for analyzing the effect of Locustatachykinin I acetate on the proportion of pH3+CDK4+ renal tubular epithelial cells 7, 14 and 28 days after IRI by immunofluorescence, wherein, ***P<0.001;

[0025] Figure 14 Figure for detecting the effect of Locustatachykinin I acetate on the renal fibrosis of IRI-induced AKI-to-CKD model; wherein, (A) is the effect of Locustatachykinin I acetate on the renal fibrosis of IRI-induced AKI-to-CKD model detected by HE staining, Masson staining, immunohistochemical α-SMA staining and immunofluorescence Fn staining. Scale bar: 50 μm. (B) is the effect of Locustatachykinin I acetate on the fibrosis area of IRI-induced AKI-to-CKD model. (C) is the effect of Locustatachykinin I acetate on the expression of α-SMA and Fn of IRI-induced AKI-to-CKD model; ns: no statistical difference, ***P<0.001. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and advantages of the present application more clear, the following will be described in detail with reference to the drawings and specific embodiments.

[0027] Unless otherwise specified, the reagents used in the examples are analytical pure, and the progress of all chemical reactions is monitored by thin layer chromatography.

[0028] Example 1 Molecular docking analysis

[0029] The docked compound library is from the T001 compound library of Tsingbiotech (https: / / www.tsbiochem.com / libraries?bd_vid=16597645510441608233). The crystal structure of DDX39B_HUMAN protein (PDB ID: 1XTI) is downloaded from the pdb database, and the conformation of the double-stranded DNA complex with the DDX39B protein (PDB ID: 1XTI) is predicted by Alphafold3. The receptor molecule is hydrogenated and charged. The compound library is processed using the LigPrep module in the software, and the Epik method is used to protonate under the condition of pH 7.0±2.0, desalt, generate tautomers, and maintain the original atomic chirality. Drug molecules with docking scores lower than -7 kcal / mol are selected for further screening.

[0030] Further, by visual inspection of the binding conformation of the compound and the protein, the suitable compounds are screened by removing unreasonable structures, and the compounds capable of forming direct interaction with the 1XTI structure are obtained.

[0031] Through molecular docking analysis, it is found that T22928L can bind to specific amino acid residues of Ddx39b through hydrogen bonding and hydrophobic interaction. Figure 1 CETSA experiment shows that T22928L can bind to Ddx39b. Figure 2 mRNA stability detection shows that T22928L can reduce the stability of Cdc20 mRNA. The above experimental results show that T22928L can bind to Ddx39b and reduce the mRNA stability of Cdc20, a downstream molecule of Ddx39b.

[0032] Example 2 In vitro experiment

[0033] CETSA is an experiment for detecting the binding efficiency of drugs and target proteins in cells. The principle is that when the target protein binds to the drug molecule, it will usually become stable. Referring to the methodological literature [PMID: 25101824], the present application uses DMSO or the screened small molecule compound to treat HK2 cells (derived from the ATCC cell library, purchased from Sangon Biotech) for 24 h, and then extracts the cell protein. Then, it is uniformly distributed to different PCR tubes, and the samples are denatured at different temperatures for 8 min on a PCR instrument, and the samples are freeze-thawed 3 times using liquid nitrogen. The sample is centrifuged, and the protein supernatant is analyzed by western blot.​

[0034] HK-2 cells were subjected to hypoxia for 24 hours in a 1% hypoxic incubator in the absence of glucose and serum, and then returned to normoxia at 37°C in the presence of normal medium. The HK-2 cells were treated with different concentrations of T22928L compound (0, 1, 5, 10, 20 μM) for 24 hours, and cell viability was detected by CCK-8 method. Figure 3 )。

[0035] HK-2 cells were subjected to hypoxia for 24 hours in a 1% hypoxic incubator in the absence of glucose and serum, and then returned to normoxia at 37°C in the presence of normal medium. The HK-2 cells were treated with different concentrations of T22928L compound (0, 1, 5, 10, 20 μM) for 24 hours, and cell viability was detected by CCK-8 method.

[0036] Example 3 Effect of small molecule compound T22928L on inhibition of Ddx39b on the generation of polyploidy tubular cells under H / R conditions

[0037] In previous studies, it was found that Ddx39b can aggravate the generation of polyploidy tubular cells by promoting the expression of Cdc20.

[0038] 1) In the present application, HK-2 cells were subjected to hypoxia for 24 hours in a 1% hypoxic incubator in the absence of glucose and serum, and then returned to normoxia at 37°C in the presence of normal medium. Locustatachykinin I acetate was added to the conventional culture medium and cultured for 24 hours. The cells were collected by centrifugation, fixed with ethanol overnight, stained with DAPI nuclear dye for 30 minutes, and the DNA content was detected by flow cytometry. The experiment found that H / R induced HK2 cells to become polyploid, and the proportion of polyploid HK2 cells decreased after the addition of Locustatachykinin I acetate, which was consistent with the effect of knocking down Ddx39b Figure 4 )。

[0039] to determine whether the small molecule compound T22928L (Locustatachykinin I acetate) affects the generation of polyploidy tubular cells through Ddx39b.

[0040] 2) The Locustatachykinin I acetate was added to the HK-2 cells after 24 hours of hypoxia and reoxygenation treatment, and actinomycin D was added to inhibit transcription after 24 hours. The cells were collected at 1, 2, 4, 6, 8, and 10 hours, respectively, and the mRNA was extracted to detect the effect of Locustatachykinin I acetate on the stability of Cdc20 mRNA. In addition, the HK-2 cells treated with Locustatachykinin I acetate after 24 hours of hypoxia and reoxygenation treatment and the control cells treated with DMSO were resuspended with an equal volume of RIPA lysis buffer (Bi Yun Tian, P0013B, Shanghai, China) and the bound proteins in the cell lysate were precipitated using A / G magnetic beads and Ddx39b antibody. The unbound materials were washed away by a magnetic stand, and the RNA was extracted. The enrichment of Cdc20 was analyzed by q-RTPCR to analyze the effect of Locustatachykinin I acetate on the binding of Cdc20 and Ddx39b. The effects of Locustatachykinin I acetate on the interaction of Ddx39b and Cdc20 mRNA were explored by mRNA half-life experiment and RIP experiment.

[0041] The mRNA stability detection showed that Locustatachykinin I acetate could shorten the half-life of Cdc20 mRNA ( Figure 5 ). The RIP experiment found that the interaction of Ddx39b and Cdc20 mRNA in HK2 cells was blocked after treatment with Locustatachykinin I acetate ( Figure 6 ).

[0042] Biological safety of small molecule T22928L

[0043] Toxicity evaluation was performed by intraperitoneal injection of different concentrations of T22928L (Locustatachykinin I acetate) into mice. Locustatachykinin I acetate was dissolved in normal saline. Different concentrations (0, 5 mg / kg, 10 mg / kg, and 20 mg / kg) of T22928L (Locustatachykinin I acetate) were injected intraperitoneally into mice, and the injection was continued for 28 days. The main organs such as heart, liver, spleen, lung, and kidney were taken for pathological examination, and microvenous blood was extracted for serological detection. The results of HE staining and serological detection showed that compared with the control group, intraperitoneal injection of Locustatachykinin I acetate did not have pathological effects on the main internal organs of mice ( Figure 7 ), and did not affect the blood routine ( Figure 8), liver function ( Figure 9 ) and kidney function ( Figure 10 ), which shows that Locustatachykinin I acetate has good biological safety.

[0044] Example 5 Small molecule T22928L can significantly improve the polyploidy formation of renal tubular epithelial cells in the IRI-induced AKI-to-CKD model

[0045] The present application evaluates the therapeutic effect of T22928L (Locustatachykinin I acetate) on AKI by intraperitoneally injecting Locustatachykinin I acetate (concentration 5 mg / kg) into model mice 4 days after IRI-AKI (ischemia-reperfusion injury-acute kidney injury) surgery.

[0046] First, an ischemia-reperfusion injury-acute kidney injury model is established.

[0047] (1) Intraperitoneally inject about 150 μL of 2% sodium pentobarbital into the mouse, and after the mouse is anesthetized and stabilized, place the mouse prone and fix the limbs with tape.

[0048] (2) Use a shaver to remove the hair in the surgical area (right subcostal margin kidney area) and the surrounding area, and disinfect with iodophor 3 times. Use ophthalmic scissors to successively cut open the mouse skin, subcutaneous tissue, fascia, muscle, and medial peritoneum, respectively, to expose the left and right kidneys.

[0049] (3) Then, use an artery clamp to occlude the renal artery of the IRI group, and cover the surface with a wet gauze soaked in normal saline, and place it in a 37℃ constant temperature incubator. The Sham group is treated in the same way except that the renal artery is not occluded.

[0050] (4) After 30 minutes, remove the artery clamp to restore blood supply to the kidney.

[0051] (5) Intraperitoneally inject Locustatachykinin I acetate (concentration 5 mg / kg) into the AKI model mouse 4 days after surgery.

[0052] On the 7th, 14th, 28th, and 42nd days after surgery, take tail vein blood to evaluate serum creatinine levels (Scr) ( Figure 11 ) by using a creatinine detection kit, to evaluate the kidney function of each group of mice.

[0053] Meanwhile, the mice are sacrificed, and single renal tubular epithelial cells are extracted from the renal cortex, fixed with alcohol for 30 minutes, then stained with DAPI nuclear staining solution for 30 minutes, and the DNA content is detected by flow cytometry ( Figure 12 ).

[0054] In addition, take the 7th, 14th, 28th, 42nd day paraffin section:

[0055] (1) Dehydration, oven drying kidney tissue sections at 65°C for 30 minutes.

[0056] (2) After baking, immerse the tissue sections in a xylene solution for 10 minutes, then sequentially immerse in ethanol solutions with concentrations of 100%, 95%, 80%, and 70% for two minutes each, and finally rinse the sections with ddH2O for 2 minutes.

[0057] (3) Citric acid high temperature and high pressure repair for 15 minutes. Cool to room temperature. Wash for 2 minutes.

[0058] (4) Block at 37°C for 30 minutes.

[0059] (5) Prepare pH3 (Abeam, ab14955) and CDK4 antibodies (Abeam, ab108357) according to 1:100 dilution of the primary antibody.

[0060] (6) Place the tissue sections in a wet box, add an appropriate amount of antibody, and incubate the antibody at 4°C overnight.

[0061] (7) The next day, rewarm at 37°C for 1 hour.

[0062] (8) Wash 3 times for 5 minutes each. All subsequent steps need to be carried out in the dark.

[0063] (9) Prepare 555 wavelength anti-mouse fluorescent secondary antibody (CST, 4409S) and 647 anti-rabbit fluorescent secondary antibody (CST, 4414) in the dark according to 1:400 dilution of the secondary antibody, and add LTL straight marker antibody according to 1:100 dilution.

[0064] (10) Incubate each tissue section at 37°C for 1 hour in the dark. Wash for 10 minutes.

[0065] (11) Add an appropriate amount of DAPI staining solution to each tissue section and stain for 5 minutes. Wash for 10 minutes.

[0066] (12) Add an appropriate amount of anti-fluorescence quencher to the tissue sections, observe the pictures under the confocal microscope, and collect the expression of cell cycle markers pH3 and CDK4 in the kidney tissue paraffin sections (Figure 6) to identify polyploid cells. It was found that during the transformation of AKI to CKD, Locustatachykinin I acetate can significantly inhibit the continuous generation of polyploids after IRI injury. Figure 13

[0067] Example 6 Small molecule 1031873-45-6 can delay the transformation of AKI to CKD​

[0068] Polyploidy generation is one of the key mechanisms of the progression of acute kidney injury (AKI) to chronic kidney disease (CKD). Previous studies have found that Ddx39b promotes polyploidy of renal tubular epithelial cells after AKI, thereby promoting the progression of fibrosis. The present application observes the effect of Locustatachykinin I acetate on IRI-induced renal fibrosis through HE staining, Mason staining and fibrosis marker Fibronectin and a-SMA staining, and explores the therapeutic effect of T22928L (Locustatachykinin I acetate) in the transition from AKI to CKD.

[0069] First, paraffin sample sections of model mice at 28 and 42 days after operation were collected.

[0070] 1. Perform hematoxylin-eosin (HE) staining:

[0071] (1) Take the paraffin tissue section and dehydrate, and bake the kidney tissue section in an oven at 65°C for 30 minutes.

[0072] (2) After baking, immerse the tissue section in a xylene solution for 10 minutes, and then immerse in ethanol solutions with concentrations of 100%, 95%, 80%, and 70% for two minutes each in turn from high to low, and finally rinse the section with ddH2O for 2 minutes.

[0073] (3) Try to spin off the water on the tissue section as much as possible.

[0074] (4) Stain with hematoxylin for 3 minutes, and remove the oxidation film formed on the surface of hematoxylin before staining.

[0075] (5) Quickly rinse with 1% hydrochloric acid alcohol solution for a few seconds to de-differentiate.

[0076] (6) After hematoxylin staining, immerse the section in hot water for 15 minutes to return blue, and under the microscope, the cell nucleus can be observed to change from purple to blue.

[0077] (7) Place the tissue section in the eosin solution for staining for 2 minutes, and then immerse in 95% and 100% anhydrous ethanol solutions in turn to rinse the eosin.

[0078] (8) Place the stained tissue section in a 65°C oven to bake and remove the liquid remaining on the tissue section.

[0079] (9) Immerse the tissue section in a xylene solution, and after transparency for 20 minutes, dry and mount the section.

[0080] (10) Observe the HE staining results under a microscope and collect images.

[0081] 2. MASSON staining:

[0082] 1) Dewaxing and rehydrating, see the method section in chapter 2 for details.

[0083] 2) Staining with Weigert iron hematoxylin staining solution for 5 min.

[0084] 3) Differentiating with acid ethanol differentiation solution for several seconds, and rinsing with tap water for 10 min.

[0085] 4) Rebluing with Masson blue solution for 5 min, and rinsing with tap water for 10 min.

[0086] 5) Washing with distilled water for 1 min.

[0087] 6) Staining with ponceau red staining solution, and rinsing with tap water for 10 min.

[0088] 7) During the above operation, weak acid working solution was prepared according to the ratio of distilled water: weak acid solution = 2: 1, and the weak acid working solution was used for washing for 1 min.

[0089] 8) Washing with phosphomolybdic acid solution for 2 min.

[0090] 9) Washing with weak acid working solution for 1 min.

[0091] 10) Directly placing into aniline blue staining solution for staining for 2 min.

[0092] 11) Washing with prepared weak acid working solution for 1 min.

[0093] 12) Rapid dehydration with 95% ethanol.

[0094] 13) Short-term dehydration with anhydrous ethanol for 3 times.

[0095] 14) Xylene transparency for 2 min each time for 3 times.

[0096] 15) Neutral gum sealing.

[0097] 3. Immunohistochemical staining of fibrosis marker Fibronectin

[0098] (1) The paraffin tissue section was dehydrated, and the kidney tissue section was baked in an oven at 65°C for 30 min.

[0099] (2) After baking, the tissue section was soaked in xylene solution for 10 min, and then sequentially soaked in ethanol solutions with concentrations of 100%, 95%, 80%, and 70% for 2 min each, and finally washed with ddH2O for 2 min.

[0100] (3) The water on the tissue section was shaken as much as possible to prevent residual water from affecting the subsequent experiment.

[0101] (4) High temperature and high pressure repair with citric acid for 15 minutes.

[0102] (5) After water cooling to room temperature, pour off the citric acid repair solution.

[0103] (6) Wash the section for 2 minutes to remove the citric acid repair solution.

[0104] (7) 1% Triton punching for 10 minutes.

[0105] (8) Wash for 2 minutes.

[0106] (9) Peroxidase blocker blocking for 10 minutes.

[0107] (10) Wash for 2 minutes.

[0108] (11) Blocking at 37°C for 30 minutes.

[0109] (12) Fibronectin antibody is prepared according to 1:100 dilution of immune primary antibody.

[0110] (13) Put the tissue section in a wet box, add an appropriate amount of antibody, and incubate the antibody at 4°C overnight.

[0111] (14) The next day, rewarm at 37°C for 1 hour.

[0112] (15) Wash for 10 minutes.

[0113] (16) Add anti-rabbit immunohistochemical secondary antibody.

[0114] (17) Incubate the secondary antibody at 37°C for 1 hour.

[0115] (18) Wash for 10 minutes.

[0116] (19) DAB color developing agent A liquid: B liquid is prepared according to 50:1. Color developing under a microscope.

[0117] (20) After color development is complete, wash with running water for 10 minutes.

[0118] (21) Hematoxylin staining for 3 minutes, remove the oxidation film formed on the surface of hematoxylin before staining.

[0119] (22) Quickly rinse with 1% hydrochloric acid alcohol solution for a few seconds to de-differentiate.

[0120] (23) After hematoxylin staining, immerse the section in hot water for 15 minutes to return blue. Under the microscope, the cell nucleus changes from purple to blue.

[0121] (24) Place the stained tissue section in a 65°C oven to bake and remove the residual liquid on the tissue section.

[0122] (25) After transparent for 20 minutes, the tissue section was dried and mounted.

[0123] 4. Immunofluorescence staining of fibrosis marker a-SMA

[0124] (1) Dehydration, oven-dried kidney tissue section at 65°C for 30 minutes.

[0125] (2) After baking, the tissue section was soaked in xylene solution for 10 minutes, then soaked in 100%, 95%, 80%, 70% ethanol solution for two minutes respectively from high to low, and finally washed with ddH2O for 2 minutes.

[0126] (3) Citric acid high temperature and high pressure repair for 15 minutes. Cool to room temperature. Wash for 2 minutes.

[0127] (4) Blocking at 37°C for 30 minutes.

[0128] (5) a-SMA antibody was prepared according to 1:100 dilution of immune primary antibody.

[0129] (6) The tissue section was placed in a wet box, and an appropriate amount of antibody was added, and the antibody was incubated at 4°C overnight.

[0130] (7) The next day, 37°C rewarming for 1 hour.

[0131] (8) Wash 3 times, 5 minutes each time. After that, all steps need to be carried out in the dark.

[0132] (9) In the dark, 555 wavelength anti-rabbit fluorescent secondary antibody was prepared according to 1:400 dilution of immune secondary antibody.

[0133] (10) Each tissue section was incubated at 37°C for 1 hour in the dark. Wash for 10 minutes.

[0134] (11) Each tissue section was stained with an appropriate amount of DAPI staining solution for 5 minutes. Wash for 10 minutes.

[0135] (12) An appropriate amount of anti-fluorescence quencher was added to the tissue section, and the picture was observed under the confocal microscope.

[0136] The results are shown in Figure 14 The results showed that during the transformation of AKI to CKD, Locustatachykinin I acetate significantly inhibited the deposition of collagen fibers after IRI injury and inhibited the expression of fibrosis markers Fn and a-SMA. This indicates that Locustatachykinin I acetate significantly inhibits the degree of fibrosis after AKI and delays its progression to CKD.

[0137] The above describes the preferred embodiments of the present application. It should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the scope of the present application.

Claims

1. Use of locust tachykinin I acetate in the preparation of drugs used as Ddx39b inhibitors.

2. The use as described in claim 1, wherein, The drug is used to relieve or treat diseases associated with Ddx39b activation; preferably, the disease is selected from one or more of hepatitis D virus (HDV) infection, colorectal cancer, breast cancer, lung cancer, and acute kidney injury.

3. The use as described in claim 1 or 2, wherein, The drug is used to relieve or treat acute kidney injury (AKI).

4. The use as described in any one of claims 1-3, wherein, The drug is used to relieve or treat acute kidney injury (IRI-AKI) caused by ischemia-reperfusion.

5. The use as described in any one of claims 1-4, wherein, The drug is used to alleviate or block the progression of acute kidney injury (AKI) to chronic kidney disease (CKD).

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