Use of metrnl to prevent and treat radiation enteropathy

Abstract

The present application relates to the application of Metrnl in preventing and treating radiation enteropathy. The present application first uses Metrnl in preventing and treating radiation enteropathy, and finds that the expression of Metrnl in intestinal epithelial cells is related to radiation enteropathy. By establishing a Metrnl intestinal epithelial cell-specific knockout and overexpression mouse model, it is confirmed that the lack of Metrnl in intestinal epithelial cells leads to more severe damage after irradiation, and Metrnl intestinal-specific overexpression mice can reduce radiation damage. In a cell model, after Metrnl knockdown, ionizing radiation causes more severe DNA damage, and cell proliferation ability is weakened, while Metrnl overexpression can resist ionizing radiation damage, and METRNL recombinant protein has a preventive effect on ionizing radiation damage. The present application first confirms that Metrnl has an effect of preventing and treating radiation enteropathy, and the protection mechanism is related to reducing DNA damage in intestinal cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of Metrnl in the prevention and treatment of radiation enteropathy. Background Technology

[0002] Radiation-induced enteropathy refers to persistent and recurrent radiation-induced intestinal damage that occurs in patients with pelvic and abdominal tumors after radiotherapy. In addition, nuclear radiation injury can also involve the intestines. Common symptoms include diarrhea, constipation, abdominal distension, abdominal pain, anal pain, rectal bleeding, and fecal incontinence. A small number of patients experience persistent symptoms and may even develop serious complications such as intestinal obstruction, perforation, enterocutaneous fistula, and refractory intestinal bleeding. The main pathological changes in radiation-induced enteropathy include abnormal proliferation of mucosal blood vessels, submucosal fibrosis, and obstructive endarteritis. With the widespread use of radiotherapy, the incidence of radiation-induced enteropathy has increased. Therefore, exploring drugs for treating radiation-induced enteropathy is particularly important.

[0003] METRNL is a secreted protein belonging to the same secreted protein family as Meteorin, also known as Meteorin-like, Cometin, Subfatin, Interleukin-41, and Meteorin-β. Its amino acid sequence shows high similarity across different species. METRNL is distributed throughout various tissues and organs, particularly highly expressed in white adipose tissue, barrier tissues such as the intestinal and respiratory epithelium, and vascular endothelium. Since its discovery, METRNL has had numerous reported functions, involving lipid metabolism, atherosclerosis, neurotrophic factors, damage repair, and immune inflammation. The inventors' research group has conducted several studies on METRNL in recent years, including its effects on lowering blood lipids and blood sugar, protecting the endothelium, combating atherosclerosis, and improving cognitive impairment. However, the application of Metrnl in the prevention and treatment of radiation enteropathy has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing an application of Metrnl in the prevention and treatment of radiation enteropathy.

[0005] In a first aspect of the invention, the use of the Metrnl protein or gene or their synergists in the preparation of a medicament for the prevention and treatment of radiation enteropathy is provided.

[0006] The aforementioned prevention and / or treatment include prevention and / or treatment of radiation enteropathy.

[0007] Furthermore, the radiation enteropathy mentioned above refers to either acute radiation enteropathy or chronic radiation enteropathy.

[0008] Furthermore, the drug reduces weight loss caused by radiation enteropathy and mitigates ionizing radiation damage.

[0009] Furthermore, the drug reduces DNA damage caused by ionizing radiation to intestinal cells.

[0010] Furthermore, the synergist is selected from agonists, upregulators, or stabilizers.

[0011] Furthermore, the drug may also contain conventional pharmaceutical carriers or pharmaceutically acceptable excipients, and be prepared into various dosage forms as a pharmaceutical composition. For example, it can be prepared into conventional solid dosage forms such as tablets, powders, or capsules; when used for injection, it can be prepared into an injection solution. In various formulations, the weight content of the active ingredient is 0.01% to 99.9%.

[0012] In a second aspect of the present invention, a method for screening potential substances to improve radiation enteropathy is provided, comprising the following steps: (1) contacting a candidate substance with a system containing Metrnl protein or gene; (2) observing the effect of the candidate substance on the expression and activity of Metrnl protein or gene, wherein if the candidate substance can promote the expression of Metrnl gene or increase the activity of Metrnl protein, then the candidate substance is a potential substance to improve radiation enteropathy.

[0013] Furthermore, the radiation enteropathy mentioned above refers to either acute radiation enteropathy or chronic radiation enteropathy.

[0014] In a third aspect of the invention, the use of Metrnl protein in the preparation of a protective agent against ionizing radiation damage to intestinal epithelial cells is provided.

[0015] Furthermore, the Metrnl protein refers to the recombinant METRNL protein.

[0016] This invention is the first to use Metrnl for the prevention and treatment of radiation-induced enteropathy, discovering that the expression of Metrnl in intestinal epithelial cells is associated with radiation-induced enteropathy. By establishing mouse models of Metrnl specific knockout and overexpression in intestinal epithelial cells, it was confirmed that the lack of Metrnl in intestinal epithelial cells leads to aggravated damage after irradiation, while mice with intestinal-specific overexpression of Metrnl can alleviate radiation damage. In the Caco-2 cell model, Metrnl knockdown exacerbates DNA damage caused by ionizing radiation and weakens cell proliferation, while Metrnl overexpression can resist ionizing radiation damage. In cell experiments, administration of recombinant METRNL protein confirmed its role in mitigating ionizing radiation damage. This invention is the first to demonstrate that Metrnl has a role in preventing and treating radiation-induced enteropathy, and its protective mechanism is related to reducing DNA damage in intestinal cells. Attached Figure Description

[0017] Appendix Figure 1 The changes in body weight over time in KO and WT mice after abdominal irradiation. Statistical method: t-test, *P<0.05, n=7.

[0018] Appendix Figure 2 The changes in body weight over time in OE and WT mice after abdominal irradiation. Statistical method: t-test, *P<0.05, n=7.

[0019] Appendix Figure 3 Metrnl knockdown in Caco-2 cells exacerbates ionizing radiation damage. A. Verification of mRNA levels after Metrnl knockdown; B. Verification of protein levels after Metrnl knockdown; C. Effects of ionizing radiation on the expression levels of c-Myc and γ-H2aX proteins after Metrnl knockdown. Statistical methods used were t-tests, *P<0.05, **P<0.01, n=3.

[0020] Appendix Figure 4 : Overexpression of Metrnl in Caco-2 cells to resist damage caused by ionizing radiation. A. Verification of mRNA levels after Metrnl overexpression; B. Verification of protein levels after Metrnl overexpression; C. Effect of ionizing radiation on the expression levels of c-Myc and γ-H2aX proteins after Metrnl overexpression. Statistical method: t-test, *P<0.05, ***P<0.001, n=3.

[0021] Appendix Figure 5 Recombinant human METRNL protein (rhMETRNL) alleviates ionizing radiation damage. A. Effect of rhMETRNL on the expression levels of γ-H2aX and c-Myc proteins after ionizing irradiation; B. Statistical graph of γ-H2aX and c-Myc proteins. Statistical method: t-test, *P<0.05, n=3. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0023] Unless otherwise specified, the experimental steps or conditions in the following examples can be performed according to the conventional experimental procedures and conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0024] Metrnl protein and gene

[0025] The Metrnl protein used in this article can be naturally occurring, for example, it can be isolated and purified from mammals.

[0026] Furthermore, the Metrnl protein can also be artificially prepared, for example, using conventional genetic engineering techniques. Any suitable Metrnl protein is applicable to this invention. The Metrnl protein includes the full-length Metrnl protein or its bioactive fragment. As a specific embodiment, the Metrnl protein can be a human Metrnl protein, approximately 30 kDa in size, with its amino acid sequence shown in NP_001004431.1.

[0027] The amino acid sequence of the Metrnl protein, formed by the substitution, deletion, or addition of one or more amino acid residues, is also included in this invention. The Metrnl protein or its biologically active fragment includes a sequence of substitutions for a portion of conserved amino acids, the substitutions of which do not affect its activity or retain a portion of its activity. Appropriate amino acid substitutions are well-known techniques in the art, which can be readily implemented and ensure that the biological activity of known molecules is not altered. These techniques have made it clear to those skilled in the art that, generally, changing a single amino acid in a non-essential amino acid region of a polypeptide does not change its biological activity.

[0028] Any bioactive fragment of the Metrnl protein can be used in this invention. Here, a bioactive fragment of the Metrnl protein refers to a polypeptide that retains all or part of the function of the full-length Metrnl protein. Typically, the bioactive fragment retains at least 50%, 60%, and up to 99% or 100% of the activity of the full-length Metrnl protein.

[0029] This invention can also utilize modified or altered Metrnl proteins, comprising all or some of their amino acids. For example, Metrnl proteins can be modified or altered to improve half-life, efficacy, metabolism, and / or protein potency. The modified or altered Metrnl protein can be a conjugate of a Metrnl protein, or a substitute or artificial amino acid thereof. The modified or altered Metrnl protein or gene may differ from the natural Metrnl protein or gene to some extent, but still possess the functions described in this invention without causing other adverse reactions or toxicity. In other words, any variation that does not affect the biological activity of the Metrnl protein or the biological function of the gene can be used in this invention.

[0030] Metrnl enhancer and its uses

[0031] The term "Metrnl enhancer" includes agonists, upregulators, stabilizers, etc., and refers to any substance that can increase the activity and stability of Metrnl, upregulate Metrnl expression, or increase the effective duration of Metrnl action. These substances can all be used in this invention. They can be compounds, small chemical molecules, biomolecules, etc. The biomolecules can be at the nucleic acid level (including DNA and RNA), at the protein level, or can be viral products that upregulate Metrnl expression, etc.

[0032] Example 1: Metrnl intestinal epithelial cell-specific knockout mice exacerbate ionizing radiation damage.

[0033] Metrnl intestinal epithelial cell-specific knockout mice (Metrnl loxP / loxP Villin-Cre was previously constructed by our research group; Metrnl loxP / loxP Villin-Cre and Metrnl loxP / loxP When the pups born from the breeding program reach 4 weeks of age, they are separated from their mothers, housed separately by sex, and genetically identified. At 8 weeks of age, the mice are separated by genotype and sex. loxP / loxP Villin-Cre(KO) and littermate control mice Metrnl loxP / loxP The weight ratio (WT) was set at 7:7. On the day of irradiation, the weight of each mouse was recorded, and 0.8% sodium pentobarbital was injected intraperitoneally at a volume of 0.1 ml / 10 g. After the mice were deeply anesthetized, they were placed in a fixed box and marked. The mice were then transported to the Co-60 γ-ray source at the Radiation Center of Naval Medical University for abdominal irradiation. The total radiation dose for the abdomen was set at 30 Gy according to experimental needs. After irradiation, the mice were returned to their cages, and their weight was measured and recorded at fixed times each day. A growth curve was plotted based on the changes in the mice's weight.

[0034] The results are as follows Figure 1 As shown, Metrnl loxP / loxP Villin-Cre(KO) and littermate control mice Metrnl loxP / loxP Following 30 Gy abdominal irradiation, the body weight of mice initially decreased and then gradually recovered over time. Knockout (KO) mice experienced a 20% weight loss before recovering, while WT mice experienced a 12% weight loss before recovering. A significant difference was observed between the two groups on day 4. Therefore, the weight loss was more pronounced in Metrnl intestinal epithelial cell-specific knockout mice, and Metrnl deficiency exacerbated post-irradiation damage.

[0035] Example 2: Metrnl intestinal epithelial cell-specific overexpression in mice can alleviate ionizing radiation damage.

[0036] Metrnl intestinal epithelial cell-specific overexpression mice (R26-LSL-Metrnl) + / - Villin-Cre was previously constructed by our research group and utilized R26-LSL-Metrnl. + / - Metrnl conditionally overexpressing homozygous mice (R26-LSL-Metrnl) were bred through self-pollination of mice. + / + R26-LSL-Metrnl mice were bred by mating them with Villin-Cre mice. + / - Villin-Cre(OE) and R26-LSL-Metrnl + / - (WT) In irradiation, pups born from breeding mice were separated from their mothers at 4 weeks of age and housed separately by sex for genetic identification. At 8 weeks of age, mice were separated by genotype and sex, with the ratio of OE to littermate WT mice set at 7:7. On the day of irradiation, the weight of each mouse was recorded, and 0.8% sodium pentobarbital was injected intraperitoneally at a volume of 0.1 ml / 10 g. After deep anesthesia, the mice were placed in a fixed box and marked, then transported to the Co-60 gamma-ray source at the Radiation Center of Naval Medical University for abdominal irradiation. A total radiation dose of 30 Gy was set for abdominal irradiation as needed. After irradiation, the mice were returned to their cages, and their weight was measured and recorded daily at fixed times. Growth curves were plotted based on the changes in mouse weight.

[0037] The results are as follows Figure 2 As shown, R26-LSL-Metrnl + / - After 30 Gy abdominal irradiation, Villin-Cre (OE) mice and their littermate control WT mice showed a trend of initial decrease followed by recovery and increase in body weight over time. WT mice experienced a 13% weight loss before starting to recover, while OE mice only experienced a 5% weight loss before starting to recover. Furthermore, a significant difference was observed between the two groups on day 3. Therefore, mice with Metrnl intestinal epithelial cell-specific overexpression experienced less weight loss, and Metrnl intestinal-specific overexpression mice can mitigate radiation damage.

[0038] Example 3: Metrnl knockdown in Caco-2 cells exacerbates ionizing radiation damage.

[0039] Lentiviral transfection: Metrnl knockdown virus was purchased from Hanheng Biotechnology. After the Caco-2 cells in the cell culture flask reached confluence, the digested cells were distributed at 25 cm⁻¹. 2 5×10 culture flasks 5The cells were plated. After good adhesion, the medium was replaced with fresh DMEM complete medium (high glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin-amphotericidal B solution), and transfected according to the virus titer (MOI = 30). The Metrnl knockdown experimental group was labeled KD and the control group was labeled NC, and both were cultured until the culture flasks reached confluence.

[0040] Caco-2 cell Metrnl knockdown validation: RNA and protein were extracted from cells in the KD and NC groups. RNA was reverse-engineered into cDNA, and real-time quantitative PCR was performed using Human Metrnl and Human GAPDH primers to validate the Metrnl knockdown rate in Caco-2 cells. Western blotting experiments were performed on protein samples from cells in the KD and NC groups to validate the knockdown rate at the protein expression level.

[0041] Cell irradiation experiment: After cell counting in the KD and NC groups, the cells were irradiated at a rate of 4 × 10⁻⁶. 5 Cells were seeded into six-well plates and cultured in DMEM complete medium for 36 h, then replaced with FBS-free high-glucose DMEM for another 12 h. The cells were then transferred to the Co-60 γ-ray source at the Radiation Center of Naval Medical University, where a total irradiation dose of 10 Gy was set. After irradiation, the cells were cultured for another 24 h, and cell proteins were extracted. The levels of γ-H2aX and c-Myc were then measured.

[0042] The knockdown verification results are as follows Figure 3 A and Figure 3 As shown in B, Metrnl knockdown was successful in Caco-2 cells, with the KD group achieving an 80% knockdown rate at the mRNA level. Figure 3 A), at the protein level, the KD group showed a knockdown rate of 70% ( Figure 3 B).

[0043] After 10 Gy ionizing radiation, the expression of γ-H2aX protein in the Metrnl knockdown group of Caco-2 cells was significantly increased compared with that in the control group, while the expression level of c-Myc protein was significantly decreased. Figure 3 C). This indicates that Metrnl knockdown in Caco-2 cells exacerbates DNA damage caused by ionizing radiation and weakens cell proliferation.

[0044] Example 4: Overexpression of Metrnl in Caco-2 cells resists damage caused by ionizing radiation.

[0045] Overexpression virus transfection: The Metrnl overexpression virus was purchased from Hanheng Biotechnology. After the Caco-2 cells in the cell culture flasks reached confluence, the digested cells were transfected at 25 cm⁻¹. 2 5×10 culture flasks 5The cells were plated. After good adhesion, the medium was replaced with fresh DMEM complete medium (high glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin-amphoteric acid B solution), and transfected according to the virus titer (MOI = 30). The Metrnl overexpression experimental group was labeled OE and the control group was labeled NC, and both were cultured until the culture flasks reached confluence.

[0046] Validation of Metrnl overexpression in Caco-2 cells: RNA and protein were extracted from cells in the OE and NC groups. RNA was reverse-engineered into cDNA, and real-time quantitative PCR was performed using Human Metrnl and Human GAPDH primers to verify the overexpression rate of Metrnl in Caco-2 cells. Western blotting experiments were performed on protein samples from cells in the OE and NC groups to verify the overexpression rate at the protein expression level.

[0047] Cell irradiation experiment: After cell counting in the OE and NC groups, the cells were irradiated at a rate of 4 × 10⁻⁶. 5 Cells were seeded into six-well plates and cultured in DMEM complete medium for 36 h, then replaced with FBS-free high-glucose DMEM for another 12 h. The cells were then transferred to the Co-60 γ-ray source at the Radiation Center of Naval Medical University, where a total irradiation dose of 10 Gy was set. After irradiation, the cells were cultured for another 24 h, and cell proteins were extracted. The levels of γ-H2aX and c-Myc were then measured.

[0048] Overexpression validation results are as follows Figure 4 A and Figure 4 As shown in B, Metrnl overexpression was successful in Caco-2 cells, with the relative expression level in the OE group being approximately 4000 times that in the NC group at the mRNA level. Figure 4 A), at the protein level, the relative expression level in the OE group was 3 times that in the NC group. Figure 4 B).

[0049] After 10 Gy ionizing radiation, the expression of γ-H2aX protein in the Metrnl overexpression group of Caco-2 cells was significantly reduced compared with the control group, while the expression level of c-Myc protein was significantly increased. Figure 4 C). This indicates that overexpression of Metrnl in Caco-2 cells reduces DNA damage caused by ionizing radiation, enhances cell proliferation, and demonstrates resistance to ionizing radiation damage.

[0050] Example 5: Recombinant human METRNL protein (rhMETRNL) alleviates ionizing radiation damage

[0051] Caco-2 cells were divided into 4×10 5Cells were seeded per well and labeled as the recombinant human METRNL protein-treated group (rhMETRNL) and the control group (NC group). Both groups were cultured in DMEM complete medium for 36 h, then switched to FBS-free high-glucose DMEM for another 12 h. 20 μL of PBS was added to each well of the NC group, and 20 μL of rhMETRNL (939-MN-050) at a concentration of 20 μg / ml (400 ng rhMETRNL per well) was added to each well of the rhMETRNL group. Cells from both groups were then analyzed under normal conditions (non-irrad) and ionizing radiation (irrad) conditions. The non-irrad group was cultured routinely, while the irrad group was sealed with film to prevent contamination and transported to the Co-60 γ-ray source at the Radiation Center of Naval Medical University. The total irradiation dose was set to 10 Gy, and the cells were cultured for another 24 h after irradiation. Proteins were extracted from both the non-irrad and irrad groups, and the expression levels of γ-H2aX and c-Myc proteins were detected.

[0052] The results are as follows Figure 5 As shown, unirradiated Caco-2 cells in the NC and rhMETRNL groups showed almost no expression of γ-H2aX, while there was no significant difference in c-Myc protein expression between the two groups. After irradiation, both the NC and rhMETRNL groups expressed γ-H2aX and c-Myc proteins, with the c-Myc protein expression level in the rhMETRNL group significantly higher than that in the NC group. Conversely, the γ-H2aX expression level in the rhMETRNL group was significantly lower than that in the NC group. Therefore, rhMETRNL has a role in mitigating ionizing radiation damage.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The application of human Metrnl protein or reagents overexpressing the human Metrnl gene in the preparation of drugs for the prevention and treatment of radiation enteropathy, characterized in that, The radiation enteropathy mentioned above is acute radiation enteropathy, and the drug mentioned above reduces DNA damage caused by ionizing radiation to intestinal epithelial cells.

2. The application according to claim 1, characterized in that, The drug also includes a pharmaceutical carrier.

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