Application of kynurenase protein inhibitor in preparation of medicine for treating radioactive intestinal injury

Abstract

The invention discloses application of a kynurenase protein inhibitor in preparation of a medicine for treating radioactive intestinal injury, and belongs to the technical field of biological medicine. The invention discloses the interaction between the kynurenase protein KYNU and the radioactive intestinal injury for the first time, and through gene knockout and expression research of an MAPK signal channel, it is clear that the radioactive intestinal injury can be effectively relieved by inhibiting the expression of the KYNU. Based on the research results, the invention emphatically puts forward that carbidopa can be used as a kynureninase protein KYNU inhibitor and plays a key role in radioactive intestinal injury, so that a new medicine application for treating radioactive intestinal injury is provided aiming at KYNU inhibition; the invention also provides a small interfering RNA (siRNA) for inhibiting the expression of kynureninase protein KYNU for the first time, the KYNU is knocked down, so that the cell proliferation after irradiation can be increased, the cell apoptosis and oxidative stress can be reduced, and KYNU knock-down has a protective effect on radioactive intestinal injury.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of a kynurenine protein KYNU inhibitor in the preparation of a medicine for treating radiation-induced intestinal damage. Background Art

[0002] Radiation-induced intestinal injury (RIII) is a common gastrointestinal complication caused by radiotherapy for abdominal, pelvic and retroperitoneal tumors, also known as radiation enteritis. Up to 90% of patients experience acute gastrointestinal symptoms such as abdominal pain, diarrhea and bloating within 3 months, and about 50% of patients develop chronic gastrointestinal symptoms (such as fibrosis, occlusive vasculitis, atrophy, etc.) within 1 year. This seriously affects the patient's quality of life and treatment effect. At present, the pathogenesis of radiation enteritis is not fully understood, but many research reports have shown that epithelial damage, damage to the vascular system, and intestinal immune and microbial disorders are related to radiation intestinal injury. At present, there is no unified and effective method for the prevention and treatment of RIII in clinical practice.

[0003] Kynurenine protein (KYNU) is a hydrolase involved in tryptophan metabolism, which promotes the synthesis of NAD+ cofactors through the kynurenine pathway. KYNU is expressed in almost all organs of the body, with higher expression levels in the liver, bladder, and appendix. In addition to several types of cancer, KYNU is also involved in various inflammatory and cardiovascular diseases through different pathways. The activity of KYNU is closely related to the occurrence and development of many diseases, such as neuropsychiatric diseases, inflammation and immune response. Therefore, drug development targeting kynurenine protein has potential application prospects. Some recent studies have emphasized the key role of KYNU in intestinal inflammatory diseases, including ulcerative colitis (UC). However, its role in radiation-induced intestinal injury is still unclear, so in-depth research on its structure and function will help to more clearly reveal the regulatory mechanism of tryptophan metabolism and provide new ideas and methods for drug development and clinical diagnosis.

[0004] Carbidopa is a peripheral decarboxylase inhibitor, currently commonly used to treat Parkinson's disease and Parkinson's syndrome, and is often used in combination with levodopa to improve the efficacy of levodopa by inhibiting the decarboxylation of peripheral levodopa. Currently, there are no reports on the mechanism of action and physiological functions of carbidopa and KYNU in vivo. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide the use of a kynurenine protein KYNU inhibitor in the preparation of a drug for treating radiation-induced intestinal damage.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The invention discloses application of a kynurenine protein KYNU inhibitor in preparing a medicine for treating radiation intestinal damage.

[0007] Preferably, the drug is a drug that affects radiation-induced intestinal damage via the MAPK pathway.

[0008] Further preferably, the drug is a drug that reduces cell apoptosis and oxidative stress.

[0009] Preferably, the kynurenine protein KYNU inhibitor is carbidopa.

[0010] Further preferably, carbidopa is capable of reducing the effect of radiation on cell viability.

[0011] More preferably, carbidopa can significantly reduce radiation-induced cell apoptosis and promote cell proliferation.

[0012] More preferably, carbidopa is capable of reducing the increase in ROS levels induced by radiation.

[0013] Preferably, the drug is si-KYUN, which is a small interfering RNA that inhibits the expression of kynurenine protein KYNU through RNA interference mechanism, and its sense strand sequence is shown in SEQ ID NO:1, and its antisense strand sequence is shown in SEQ ID NO:2.

[0014] The present invention also discloses the use of carbidopa as a kynurenine protein KYNU inhibitor in the preparation of a drug for treating radiation intestinal damage. The drug is a drug capable of alleviating the inflammatory response associated with radiation enteritis.

[0015] The present invention also discloses a small interfering RNA for inhibiting the expression of kynurenine protein KYNU. The small interfering RNA is named si-KYUN, and its sense chain sequence is shown in SEQ ID NO:1, and its antisense chain sequence is shown in SEQ ID NO:2.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention reveals for the first time the interaction between the kynurenine protein KYNU and radiation-induced intestinal damage. Through gene knockout and expression studies of the MAPK signaling pathway, the association between KYNU and radiation-induced intestinal damage is clarified, that is, inhibiting KYNU expression can effectively reduce radiation-induced intestinal damage. Based on this innovative research result, the present invention further studies and obtains: 1. The present invention studies and proves that carbidopa can be used as a kynurenine protein KYNU inhibitor, plays a key role in radiation intestinal injury, finds that carbidopa can be used as a therapeutic drug for radiation enteritis, and deeply studies its mechanism of action for radiation enteritis. Through experimental verification, the therapeutic drug carbidopa of the present invention has a protective effect on both mice and in vitro cells in acute radiation enteritis. Therefore, a new use of drugs for treating radiation intestinal injury is proposed for KYNU inhibition, which has important scientific significance and clinical value for early treatment and prevention of the occurrence of acute radiation enteritis.

[0017] 2. Experimental verification shows that siRNA inhibits gene expression through RNA interference mechanism and is generally regarded as an inhibitor. This invention also proposes for the first time a small interfering RNA (siRNA) that inhibits the expression of kynurenine protein KYNU, named si-KYUN. The experimental results show that knocking down KYNU can increase cell proliferation after irradiation, reduce cell apoptosis and oxidative stress, suggesting that knocking down KYNU has a protective effect on radiation-induced intestinal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :A PCA plot of transcriptome sequencing of radiation enteritis mice and control mice; B volcano plot; C two data sets of radiation enteritis mice on the GEO database were selected for STEM time series analysis, and genes with an upward trend were selected; D intersection analysis of the above genes with the transcriptome data set submitted by oneself; E GOKEGG analysis of the genes in the intersection; F chord plot of GOKEGG analysis; G heat map of genes with LogFC greater than 1 and Padj less than 0.01; H. The protein expression of KYNU increased after IR; I. Protein quantification; J. PCR results of kynu; K. Histochemical image of kynu on mouse colon tissue.

[0019] Figure 2 :A. sikynu knocked down the protein expression of KYNU; B. Protein quantification; C. PCR results after knocking down KYNU; D. CCK8 results after siKYNU; E. CCK8 results of sikynu after IR; F. Statistics of ROS flow cytometry results; G. Statistics of flow cytometry apoptosis double staining results; H. Flow cytometry of ROS staining; I. Flow cytometry of apoptosis double staining.

[0020] Figure 3 :A. PCA plot of transcriptome sequencing of IR+siKynu and IRsiNC; B. Volcano plot; C. Heat map; D. KEGG map; E. RECTome map; F. GSEA map; G. WB verification of MAPK pathway.

[0021] Figure 4 :A. CCK8 experiment of carbidopa (CBP) concentration gradient; B. CCK8 experiment of carbidopa concentration gradient after IR; C. ROS staining by flow cytometry; D. Apoptosis double staining by flow cytometry; E. ROS flow cytometry result statistics; F. Apoptosis double staining by flow cytometry result statistics; G. MAPK pathway WB verification.

[0022] Figure 5 :A. Flow chart of carbidopa radiation enteritis animal experiment; B. Changes in mouse weight; C. Weight value of mice on the last day; D. Schematic diagram of mouse colon length; E. Statistics of mouse colon length; F. Pathological score; G. Number of glands; H. Intestinal permeability experiment (FITC dextran experiment); I. HE image of mice. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings: In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0026] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings: The present invention provides a drug carbidopa for treating radiation enteritis. The present invention is further described below in conjunction with specific embodiments, which are intended to explain the present invention rather than to limit it.

[0028] The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified. The reagents and materials used in the examples can be purchased conventionally, and the quantitative experiments involved in the examples are repeated at least three times, and the results are averaged.

[0029] The C57 mice used were purchased from the School of Translational Medicine of Yangzhou University (Yangzhou, Jiangsu, China).

[0030] Carbidopa was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA).

[0031] Example 1: KYNU expression is elevated in mice with radiation enteritis To study the key genes in the development of radiation enteritis, transcriptome sequencing was performed on the normal control (hereinafter referred to as the NC group) and the model colon tissue, i.e., the radiation enteritis group (hereinafter referred to as the IR group). Figure 1 The volcano plot showed that 318 genes were significantly up-regulated and 760 genes were significantly down-regulated. Compared with the NC group, 318 genes were significantly up-regulated and 760 genes were significantly down-regulated in the intestine of the IR group mice ( Figure 1 Middle B). STEM time series analysis was performed on two sets of radiation enteritis mouse data sets in the GEO database, and genes with an increasing trend were selected ( Figure 1 KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis and cluster heat map showed that there was a significant difference in the expression of KYNU gene in the colon between the NC group and the IR group ( Figure 1Western blotting and qPCR analysis further confirmed that the KYNU mRNA and protein levels in the colon tissues of mice in the IR group were significantly upregulated ( Figure 1 In addition, colon tissues of mice with radiation enteritis were collected and KYNU histochemical staining was performed. Compared with the control group, the expression of KYNU in the colon of mice with radiation enteritis was significantly increased ( Figure 1 This suggests that KYNU is involved in the occurrence and pathogenesis of radiation enteritis.

[0032] Example 2: KYNU gene knockout has a protective effect on RIII NCM460 cells are widely used in cell experiments to study the mechanisms of colorectal diseases. In order to explore the role of KYNU in intestinal homeostasis and inflammation, this experiment constructed KYNU small interfering RNA (si-KYNU) for in vitro experiments. The results showed that under normal untreated conditions, si-KYNU had no obvious toxic reaction. The effect of siKYNU on cells was measured under simulated conditions. The results showed that si-KYNU can promote the proliferation of NCM460 cells after irradiation and has anti-radiation effects in vitro. Apoptosis is the most important form of IR-induced cell death ( Figure 2 AC). First, flow cytometry was used to detect apoptosis in NCM460 cells treated with si-KYNU 6 hours after irradiation. Compared with the IR group, si-KYNU treatment significantly reduced apoptosis in irradiated cells, confirming the protective effect of si-KYNU against radiation-induced apoptosis ( Figure 2 Reactive oxygen species (ROS) assay showed that oxidative stress in NCM460 cells treated with siKYNU was significantly reduced ( Figure 2 In summary, it was shown that knocking down KYNU increased cell proliferation and reduced apoptosis and oxidative stress after irradiation, suggesting that knocking down KYNU has a protective effect on RIII.

[0033] The si-KYUN sequence used above is named KYNU-Homo-173, which is a small interfering RNA (siRNA) with the following double-stranded sequence: Sense strand: CAUUGCGGCUGAACUCAAATT (SEQ ID NO: 1) Antisense strand: UUUGAGUUCAGCCGCAAUGTT (SEQ ID NO: 2) Example 3: The mechanism of KYNU injury is closely related to the expression of MAPK signaling pathway To further explore the mechanism by which KYNU knockout alleviates RIII damage, RNA-seq analysis was performed on NCM460 cells in the IR+si-NC and IR+si-KYNU groups. DEG analysis showed that compared with the IR+si-NC group, 2067 and 2568 genes were significantly upregulated and downregulated in the IR+si-KYNU group, respectively ( Figure 3 The cluster heat map showed that after KYNU knockout, there were significant differences in gene expression between the IR+si-NC and IR+si-KYNU groups ( Figure 3 In addition, KEGG and Reactome pathway enrichment analysis showed that some degs were enriched in the MAPK signaling pathway ( Figure 3 The heat map showed that genes in the MAPK pathway were significantly downregulated in the IR+si-KYNU group ( Figure 3 In addition, the phosphorylation level of ERK, a key protein in the MAPK pathway, was increased in NCM460 cells. However, KYNU knockout significantly reduced ERK phosphorylation, which was consistent with the results of gene expression analysis ( Figure 3 Collectively, these results suggest that KYNU may affect RIII via the MAPK pathway.

[0034] Example 4: CBP can reduce radiation damage to NCM460 cells CBP and benserazide are KYNU inhibitors used to treat neurodegenerative diseases such as Parkinson's disease. Given the proinflammatory effects of KYNU, this study investigated the radioprotective effects of CBP in vitro. The CCK-8 assay was used to detect the effect of CBP on the viability of NCM460 cells. To determine the optimal concentration for radioprotection, a range of CBP concentrations were tested, and the activity of CCK-8 was measured after a radiation dose of 8 Gy ( Figure 4 The results showed that CBP had the lowest toxicity when the concentration was ≤10µm, and its radiation protection effect was most obvious at this concentration ( Figure 4 CBP treatment promoted the proliferation of NCM460 cells after irradiation, indicating that it has a protective effect against radiation damage in vitro.

[0035] Radiation-induced cell death is caused by multiple factors, among which apoptosis is the most important. To investigate the protective mechanism of CBP, flow cytometry was performed 6 hours after irradiation. ROS detection showed that oxidative stress in NCM460 cells treated with CBP was significantly reduced ( Figure 4 Compared with the IR group, the apoptosis of cells treated with CBP was significantly reduced, thus confirming the mitigating effect of CBP on radiation-induced apoptosis ( Figure 4Similarly, Western blot analysis of the MAPK pathway showed that the expression of related proteins was reduced after CBP treatment ( Figure 4 The above results indicate that CBP can promote the proliferation of NCM460 cells, reduce the increase of ROS level induced by radiation, and reduce cell death after radiation.

[0036] Example 5: Protective effect of CBP on mouse RIII In this study, the potential therapeutic effect of CBP was tested in a mouse model of radiation enteritis. The RIII model was established by TAI with 13 Gy of current, and mice were pretreated and treated with CBP ( Figure 5 The mice in the IR group showed significant weight loss, decreased water and food intake, and decreased activity. The significant weight loss, decreased activity, and decreased water and food intake indicated that the irradiated mice had RIII ( Figure 5 This experiment measured the changes in colon length, pathological scores, and gland number in mice, indicating that CBP treatment alleviated the RIII response ( Figure 5 We also verified the same results using an intestinal permeability test (FITC-dextran test) ( Figure 5 (shown in H).

[0037] H&E staining showed that the colon mucosal tissue structure of the control group was normal, with intact crypts and villi. In contrast, the intestinal crypts of mice in the IR group were significantly reduced and the intestinal villi were shortened, indicating that the mice in the IR model group were severely damaged. Compared with the IR group, the number of relatively intact intestinal crypts in the IR+CBP group increased. This suggests that CBP reduces RIII, which is associated with inflammation and oxidative stress ( Figure 5 (as shown in I).

[0038] Overall, CBP treatment protected mice from radiation-induced colonic injury and alleviated the inflammatory response associated with radiation enteritis. Therefore, CBP has the potential to be developed into a drug for the treatment of radiation-induced intestinal injury.

[0039] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Application of kynurenine protein KYNU inhibitor in the preparation of drugs for the treatment of radiation-induced intestinal damage.

2. The use according to claim 1, characterized in that The drug is a drug that affects radiation-induced intestinal damage via the MAPK pathway.

3. The use according to claim 2, characterized in that The medicine is a medicine for reducing cell apoptosis and oxidative stress.

4. The use according to claim 1, characterized in that The kynurenine protein KYNU inhibitor is carbidopa.

5. The use according to claim 4, characterized in that Carbidopa can reduce the effects of radiation on cell viability.

6. The use according to claim 5, characterized in that Carbidopa can significantly reduce radiation-induced cell apoptosis and promote cell proliferation.

7. The use according to claim 5, characterized in that Carbidopa can reduce the increase in ROS levels induced by radiation.

8. The use according to claim 1, characterized in that The drug is si-KYUN, which is a small interfering RNA that inhibits the expression of kynurenine protein KYNU through RNA interference mechanism. The sense strand sequence is shown in SEQ ID NO:1, and the antisense strand sequence is shown in SEQ ID NO:

2.

9. Use of carbidopa as a kynurenine protein KYNU inhibitor in the preparation of a drug for treating radiation-induced intestinal damage, characterized in that: The medicine is a medicine capable of alleviating the inflammatory response associated with radiation enteritis.

10. A small interfering RNA for inhibiting the expression of kynurenine protein KYNU, characterized in that: The small interfering RNA is named si-KYUN, and its sense strand sequence is shown in SEQ ID NO:1, and its antisense strand sequence is shown in SEQ ID NO:2.

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