Application of recombinant SPRR2A protein screened based on transcriptome in preparation of high-energy neutron radiation intestinal injury repair agent

Abstract

The application discloses application of recombinant SPRR2A protein based on transcriptome screening in preparation of a high-energy neutron radiation intestinal injury repair agent, identifies, for the first time, a small proline-rich protein family member Sprr2a as a specific sensitive marker for high-energy neutron radiation intestinal injury, and for the first time, applies the recombinant SPRR2A protein to high-energy neutron radiation protection. The recombinant SPRR2A protein can promote intestinal crypt cell proliferation and / or improve intestinal stem cell activity, improve the ability of intestinal crypts to form organoids after high-energy neutron radiation, solve the technical bottleneck that existing protection agents have poor high-LET radiation protection effect, and have important application value in the fields of nuclear accident emergency medical rescue, tumor radiotherapy, space medicine and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of recombinant SPRR2A protein selected based on transcriptome screening in the preparation of a high-energy neutron radiation intestinal injury repair agent. Background Technology

[0002] Neutron radiation, as a typical example of high linear energy transfer (high-LET) ionizing radiation, has significantly stronger biological effects than low-LET radiation such as gamma rays and X-rays. Neutrons can generate recoil protons through elastic scattering or produce charged heavy particles through nuclear reactions, depositing large amounts of energy over short distances within biological tissues, thus causing complex damage such as DNA double-strand breaks, base damage, and chromosomal aberrations. In particular, high-energy neutron radiation (>10 MeV) has stronger penetrating power and a higher tissue-destructive effect, leading to more severe biological damage.

[0003] Among the biological damages caused by neutron radiation, the intestine is one of the most vulnerable and highly sensitive target organs. The intestinal epithelium is characterized by rapid renewal, active proliferation of crypt stem cells, and a delicate mucosal barrier structure. Therefore, after acute radiation exposure, it is highly susceptible to a series of pathological changes, including villous atrophy, crypt structure destruction, impaired intestinal epithelial regeneration, damaged barrier function, and secondary inflammatory reactions.

[0004] Amifostine, currently recognized as the "gold standard" drug for radiation protection, has certain protective effects, but suffers from problems such as a narrow dosing window, significant toxic side effects, and poor tolerability, making it unsuitable for large-scale emergency protection after sudden nuclear accidents or high-energy neutron exposure. CN118512594A discloses a potential drug for treating gastrointestinal syndromes and a possible therapeutic target, which allows for the application of PARP1 inhibitors in the preparation of radiation damage repair drugs. This invention is based on the discovery that the PAR-STING signaling pathway plays a role in DNA damage repair after radiation. By using an appropriate concentration of the PARP1 inhibitor PJ34 to inhibit PARP1 activity, the survival rate of mice after radiation exposure was improved.

[0005] Currently, most existing radiation shielding agents are developed and evaluated based on low-LET radiation models such as gamma rays and X-rays. Their protective efficacy, stability, and tissue specificity against intestinal damage caused by high-energy neutron radiation lack sufficient validation. In particular, there is a lack of safe and effective protective measures against intestinal barrier disruption, crypt damage, and abnormal intestinal stem cell function caused by high-energy neutron radiation. To obtain a novel protective agent that improves intestinal stem cell damage and promotes intestinal mucosal repair, the inventors of this invention, utilizing laboratory technology, conducted extensive research and experimentation to arrive at this invention. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an application of recombinant SPRR2A protein in the preparation of a high-energy neutron radiation intestinal damage repair agent, thereby solving the problems mentioned in the background art.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0008] Application of recombinant SPRR2A protein based on transcriptome screening in the preparation of a high-energy neutron radiation intestinal injury repair agent.

[0009] Furthermore, the recombinant SPRR2A protein can promote the proliferation of intestinal crypt cells and / or enhance the activity of intestinal stem cells.

[0010] Furthermore, the intestinal damage includes one or more of the following: disruption of intestinal crypt structure, decreased activity of intestinal stem cells, and dysfunction of the intestinal mucosal barrier.

[0011] Furthermore, the energy of the high-energy neutron radiation is greater than 10 MeV.

[0012] Furthermore, the repair agent is used to enhance the ability of intestinal crypts to form organoids after exposure to high-energy neutron radiation.

[0013] Furthermore, the repair agent comprises a therapeutically effective amount of recombinant SPRR2A protein and a pharmaceutically acceptable carrier.

[0014] Furthermore, the biological mechanism by which recombinant SPRR2A protein promotes the proliferation of intestinal crypt cells is by increasing the number of Ki67-positive cells in the intestinal crypts; the biological mechanism by which recombinant SPRR2A protein can enhance the activity of intestinal stem cells is by upregulating the expression of Lgr5, Olfm4 and Ascl2 genes.

[0015] The present invention has the following beneficial effects:

[0016] 1. This invention, through RNA-Seq analysis, first identified Sprr2a, a member of the small proline-rich protein family, as a specific and sensitive biomarker for intestinal damage caused by high-energy neutron radiation. It also first applied recombinant SPRR2A protein to high-energy neutron radiation protection, with the mechanism of action focusing on protecting intestinal stem cells and promoting crypt regeneration, thus overcoming the technical bottleneck of poor protective effect of existing protective agents against high LET radiation.

[0017] 2. SPRR2A is an endogenous protein, and its recombinant form has lower immunogenicity and toxicity, and has better safety compared to chemically synthesized protective agents (such as amifostine).

[0018] 3. This invention successfully transforms differentially expressed genes screened from the transcriptome into protein drugs with practical protective efficacy, providing a novel biological agent for the precise prevention and treatment of high-energy neutron radiation damage. It has important application value in fields such as nuclear accident emergency medical rescue, tumor radiotherapy protection, and aerospace medicine. Attached Figure Description

[0019] Figure 1 The dose-dependent transcriptomic changes in mouse intestinal tissue induced by neutron radiation;

[0020] Figure 2 The expression profiles of common downregulated genes induced by neutron radiation and statistical significance analysis were presented.

[0021] Figure 3 rmSPRR2A promotes the proliferation of intestinal crypt tissue after neutron irradiation (Figure A shows Ki-67 staining at 200× and 400×; Figure B shows quantitative analysis of cell count).

[0022] Figure 4 rmSPRR2A promotes the expression of intestinal stem cell markers after neutron irradiation;

[0023] Figure 5 rmSPRR2A promotes the formation and regeneration of intestinal organoids after neutron irradiation. Detailed Implementation

[0024] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims.

[0025] It should be noted that the Ki67, OLFM4, and LGR5 biomarkers and DAPI FISH reagents used in this invention were provided by Servicebio (Wuhan, China). The PCR kit was purchased from Yeasen, Shanghai, China. Other raw materials, unless otherwise specified, are all conventional commercially available products; the methods used in this invention, unless otherwise specified, are conventional methods in the art.

[0026] It should be noted that the C57BL / 6 male mice were obtained from the Chinese Academy of Sciences (Shanghai). All mice were housed in the laboratory animal room under standard conditions. This experiment was approved by the Laboratory Animal Center of the Naval Medical University of China and complied with the "Guidelines for the Care and Use of Laboratory Animals" (NIH Publication No. 85-23, National Academy of Sciences Press, Washington, D.C., revised in 1996).

[0027] Example 1: Investigating biomarkers of intestinal damage caused by ionizing radiation in mice

[0028] To assess the intestinal damage effects of high-energy neutron radiation, age- and weight-matched male C57BL / 6 mice were randomly divided into three groups: 0 Gy, 8 Gy, and 12 Gy. The 8 Gy and 12 Gy groups received whole-body irradiation with 14.1 MeV neutrons, while the 0 Gy group served as a control group without irradiation. Each group contained at least three mice. To assess the protective effect of rmSprr2a, age- and weight-matched male C57BL / 6 mice were also administered rmSprr2a (10 mg / kg / dose) intraperitoneally 2 h and 1 h before irradiation, followed by whole-body irradiation with 8.0 Gy 14.1 MeV neutrons.

[0029] This experiment used the High Intensity DT Fusion Neutron Generator (HINEG) at the Institute of Nuclear Energy Safety Technology, Chinese Academy of Sciences, as the fast neutron radiation source. To meet experimental requirements, mice were exposed to different doses of radiation. Specifically, the mice were placed in a specially designed curved plastic box to ensure that each mouse maintained a consistent distance from the neutron source, thus precisely receiving a single dose of high-energy neutron radiation (HENR).

[0030] Immunohistochemistry and immunofluorescence staining: Mice were sacrificed on day 3.5 after irradiation, and small intestinal tissue was fixed in 4% paraformaldehyde for 24 h. After routine dehydration, embedding, and paraffin sectioning, sections were prepared. For immunohistochemical staining, after dewaxing to water, antigen retrieval was performed, followed by blocking of endogenous peroxidase with peroxidase blocking solution, and then blocking of non-specific binding sites. After removing the blocking solution, Ki67 primary antibody was added, and the sections were incubated overnight at 4 °C. The next day, the sections were washed with PBS, and the corresponding secondary antibodies were added, incubated at room temperature for 1 h, followed by staining, counterstaining, dehydration, and mounting. Ki67 positive expression was observed and evaluated under an optical microscope. For immunofluorescence staining, after dewaxing to water, antigen retrieval was performed, and non-specific binding sites were blocked. OLFM4 and LGR5 primary antibodies were then added, and the sections were incubated overnight at 4 °C. The next day, after washing with PBS, fluorescently labeled secondary antibodies were added, and the sections were incubated at room temperature for 1 h. Cell nuclei were then counterstained with 4',6-diamidino-2-phenylindole (DAPI) in the dark for 5–10 min. After mounting, the slides were observed and images were acquired under a fluorescence microscope. Phosphate-buffered saline (PBS) was used to wash the slides between each step.

[0031] RNA sequencing: Total RNA was extracted from mouse intestines 24 h post-irradiation using Trizol (Invitgen, USA). RNA purity was assessed using NanoVue (GE, USA). Each RNA sample had an A260-A280 ratio greater than 1.8 and an A260-A230 ratio greater than 2.0. Sequencing was performed at OeBiotech (Shanghai, China) using an Illumina HiSeq 2500 system. Raw data was filtered prior to sequencing to produce high-quality, clean data. All subsequent analyses were performed using this clean data.

[0032] RT-PCR: RNA was extracted using the Trizol method, and RNA purity was assessed. A PCR kit (asen, Shanghai, China) was used, and the reaction system was configured according to the manufacturer's protocol. RT-PCR detection was performed on an applied biosystems instrument (ThermoFisher Scienti-Fic, USA).

[0033] Intestinal organoid culture and irradiation: Mouse small intestine was longitudinally cut open and separated from the mesentery, then thoroughly rinsed with pre-cooled PBS. The villi on the intestinal lumen were gently scraped off with forceps, followed by washing with pre-cooled PBS approximately 10 times. The intestinal tissue was then cut into 2–3 mm segments and transferred to a calcium-free culture medium. 2+ / Mg 2+ Incubation was performed for 2 h on ice in 15 mM EDTA / PBS. After incubation, the tissue blocks were vigorously shaken to release crypts, and washed approximately three times (5 min each time) with pre-cooled PBS at 16 × g. The isolated mouse intestinal crypts were seeded into 24-well plates, approximately 200 crypts per well, suspended in matrix gel (Corning, NY, USA), and then cultured in IntestiCult™ organoid growth medium (STEMCELL Technologies, Canada). 24 h before irradiation, the experimental group was pretreated with rmSPRR2A (5 μg / mL), while the control group received an equal volume of PBS. Subsequently, the mouse intestinal crypts in each group were irradiated with 6.0 Gy neutron radiation. After irradiation, organoid culture continued, with the medium changed every 3 days. After 7 days of culture, organoid growth was observed under a microscope and images were acquired. The organoid surface area and budding number were measured using Photoshop software (Adobe, USA).

[0034] Statistical analysis: Data are expressed as mean and standard error. Two-tailed Student's t-test was used to analyze differences between the two groups. Data were analyzed using SPSS 22.0 and GraphPad Prism 7.0. A p-value < 0.05 was considered statistically significant.

[0035] Results analysis:

[0036] This experiment performed sequencing analysis on the gene expression profiles of mouse intestinal tissue 24 hours after neutron irradiation at 8 Gy and 12 Gy. Principal component analysis (PCA) plots showed good intra-group reproducibility and significant inter-group separation in the 0 Gy, 8 Gy, and 12 Gy groups. Figure 1 A). Volcano plots showing grouping based on dose differences. Between the 8 Gy neutron irradiation group and the 0 Gy (control group), 845 genes were upregulated, 1098 genes were downregulated, and 17176 showed no significant difference; in the comparison between the 12 Gy neutron irradiation group and the 8 Gy group, 558 genes were upregulated, 317 genes were downregulated, and 18222 showed no significant difference. Figure 1 B, C).

[0037] Significantly different genes such as Figure 2 As shown in the heatmap, the left heatmap displays the Log2FC value (blue indicates the downregulation fold), and the right heatmap displays the statistical significance p(-log10) (red indicates the significance level). Among the 10 dose-dependent downregulated genes (Sprr2a, Cd2, Cd79a, Fcer2a, Fcrla, Gvin3, Itk, Ms4a1, Pax5, St8sia4), Sprr2a showed the most significant downregulation (after 8 Gy irradiation). ,p(-log 10 (=24.50). Sprr2a belongs to the family of small proline-rich proteins encoded by the epidermal differentiation complex. Its core function is to maintain the integrity of the epithelial barrier and it is a key molecule in protecting against intestinal radiation damage. This gene shows a significant dose-sensitive downregulation in response to high-energy neutron radiation, suggesting that it may serve as a biomarker for neutron radiation-specific damage and a potential radiation protection gene.

[0038] Example 2: Protective effect of rmSPRR2A on intestinal tissue of mice after whole-body neutron irradiation

[0039] 1. rmSPRR2A can promote the proliferation of intestinal crypt tissue in mice. In the experiment, an acute radiation injury animal model was established in mice after whole-body neutron irradiation with 8.0 Gy. Recombinant rmSPRR2A protein was injected intraperitoneally 16 hours and 2 hours before irradiation. Intestinal tissue was collected 84 hours after irradiation for Ki-67 staining. The proliferation of intestinal crypts was analyzed to clarify the promoting effect of Sprr2a on the proliferation of intestinal crypt tissue after neutron radiation.

[0040] Histopathological examination of sections (magnifications of 200× and 400×) showed enhanced proliferative activity in intestinal crypt tissue after rmSPRR2A injection. At 84 hours post-irradiation, the number of Ki67-positive cells in each crypt was significantly increased in the irradiated group. Figure 3 A). Statistical analysis of the number of Ki67-positive cells in each crypt revealed an increase in the number of Ki67-positive cells in each crypt in the drug-treated irradiation group. The adjusted P-value was less than 0.01, indicating a significant difference. Figure 3 B). This indicates that the proliferative activity of intestinal crypt tissue was restored, promoting the survival of intestinal crypts in mice after irradiation.

[0041] 2. Immunofluorescence analysis showed that the expression of intestinal stem cell markers OLFM4 and LGR5 in the intestinal crypts of the drug-treated group was significantly enhanced compared with that of the control group. Figure 4 A, C). Quantitative analysis showed that the mean fluorescence intensity (MFI) of OLFM4 and LGR5 in the IR+rmSPRR2A group was significantly higher than that in the IR+PBS group (P<0.01). Figure 4 (B, D) suggests that rmSPRR2A can effectively maintain the phenotypic characteristics of intestinal stem cells after neutron radiation.

[0042] To further verify this protective effect, RNA was extracted from small intestinal organoids after irradiation and analyzed by RT-PCR. The results showed that, compared with the control group, the mRNA expression levels of stem cell marker genes Lgr5, Olfm4, and Ascl2 in the organoids treated with rmSPRR2A were all significantly upregulated (P<0.01). Figure 4 E). Given that OLFM4, LGR5, and ASCL2 are all key biomarkers for maintaining intestinal stem cell homeostasis, the above results suggest that rmSPRR2A may exert a neutron radiation protection effect by activating the intestinal stem cell regeneration program after injury and promoting crypt repair.

[0043] 3. In the experiment, mouse intestinal crypts were extracted into 24-well plates. Twenty-four hours before irradiation, the mouse intestinal crypts were stimulated with rmSprr2a (5 μg / ml / time), while the control group received PBS. Subsequently, the mouse intestinal crypts were irradiated with a 6.0 Gy neutron radiation source. After irradiation, mouse intestinal crypt organoids were cultured. After 7 days of culture, the intestinal organoids were observed, confirming that rmSprr2a could improve the organoid formation ability of irradiated mice.

[0044] Compared with the control group alone, the drug-treated group had an increased number of crypt-forming organoids, and the number and size of individual organoid buds were also increased. Figure 5 A). Statistical analysis of the percentage of organoid formation revealed a significant increase in the percentage of organoid formation in the drug-treated group, with an adjusted P-value less than 0.01, indicating a highly significant difference. Figure 5 B). Statistical analysis of relative surface area revealed a significant increase in relative surface area in the treatment group, with an adjusted P-value less than 0.01, indicating a highly significant difference. Figure 5C). The above results indicate that rmSPRR2A can significantly promote the regeneration of radiation-damaged intestinal organoids in mice after irradiation.

[0045] In summary, this invention addresses the technical bottleneck of severe intestinal damage caused by high-energy neutron radiation (14.1 MeV) and the lack of tissue specificity and effective protective measures in existing protective agents. It innovatively establishes a protective target discovery strategy based on transcriptome dose gradient screening (0 Gy-8 Gy-12 Gy). RNA-Seq analysis identified Sprr2a, a member of the small proline-rich protein family, as a specific and sensitive biomarker for intestinal damage caused by high-energy neutron radiation, with its expression significantly downregulated after 8 Gy irradiation. The recombinant protein rmSPRR2A was prepared based on the principle of "high energy neutron radiation damage (P<0.001)" and was directly related to the integrity of the epithelial barrier. Through in vitro and in vivo multi-level functional verification, it was confirmed that rmSPRR2A administration significantly reduced villus atrophy, crypt structure destruction, and barrier dysfunction in mice after 8.0 Gy whole-body neutron irradiation, and significantly increased villus length and crypt depth (P<0.01). Mechanistically, rmSPRR2A promotes crypt cell proliferation (increased Ki67⁺ cells) and organoid formation capacity by upregulating the expression of intestinal stem cell markers (LGR5, OLFM4, ASCL2), thereby driving intestinal epithelial regeneration and repair after radiation injury. This invention successfully transformed differentially expressed genes selected from the transcriptome into protein drugs with practical protective efficacy, providing a novel biological agent for the precise prevention and treatment of high-energy neutron radiation injury. It has significant application value in fields such as nuclear accident emergency medical rescue, tumor radiotherapy protection, and aerospace medicine.

[0046] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to what is disclosed in the embodiments and drawings.

Claims

1. The application of recombinant SPRR2A protein in the preparation of a protective agent against intestinal damage from high-energy neutron radiation, characterized in that, The recombinant SPRR2A protein is a recombinant protein encoded by the mouse SPRR2A gene, used to protect against intestinal damage before high-energy neutron radiation. The recombinant SPRR2A protein can promote the proliferation of intestinal crypt cells and / or enhance the activity of intestinal stem cells; The intestinal damage includes one or more of the following: destruction of intestinal crypt structure, decreased activity of intestinal stem cells, and dysfunction of the intestinal mucosal barrier.

2. The application as described in claim 1, characterized in that, The energy of the high-energy neutron radiation is greater than 10 MeV.

3. The application as described in claim 1, characterized in that, The protective agent is used to enhance the ability of intestinal crypts to form organoids after exposure to high-energy neutron radiation.

4. The application as described in claim 1, characterized in that, The protective agent comprises a therapeutically effective amount of recombinant SPRR2A protein and a pharmaceutically acceptable carrier.

5. The application as described in claim 1, characterized in that, The recombinant SPRR2A protein promotes the proliferation of intestinal crypt cells by increasing the number of Ki67-positive cells in the intestinal crypts; the recombinant SPRR2A protein enhances the activity of intestinal stem cells by upregulating the expression of Lgr5, Olfm4 and Ascl2 genes.

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