Application of polypeptide in preparation of medicine for treating radioactive intestinal injury

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of polypeptide in preparation of a medicine for treating radioactive intestinal injury. The polypeptide sequence provided by the invention is Thr-Met-Val-Asp-Ala, is called TMVAD small peptide for short, and is subjected to N-terminal acetylation and C-terminal amidation modification. Results of embodiments prove that mouse animal experiments prove that the TMVAD small peptide has a radiation protection effect in an X-ray induced mouse radioactive intestinal injury model, and can significantly improve the survival rate of irradiated mice, reduce the inflammatory factor level of the irradiated mice, improve the intestinal permeability, promote the regeneration of crypt cells and repair the intestinal mucosal barrier. Compared with the existing symptomatic support treatment or non-specific polypeptide control, the TMVAD small peptide has a remarkable radiation protection effect, so that the TMVAD small peptide can be used as a novel and effective radiation protection and radioactive intestinal injury treatment medicine for clinical treatment of radioactive intestinal injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a polypeptide in the preparation of drugs for treating radiation-induced intestinal injury. Background Technology

[0002] Radiotherapy is an important means of cancer treatment. It uses high-energy ionizing radiation to induce DNA damage in tumor cells, thereby inhibiting their proliferation or inducing apoptosis. However, while ionizing radiation kills tumor cells, it inevitably damages surrounding normal tissues as well. As a radiation-sensitive organ, the intestines are often accompanied by radiation-induced intestinal injury during pelvic cancer radiotherapy.

[0003] Radiation-induced intestinal injury can be divided into acute and chronic types. Acute radiation-induced intestinal injury usually occurs within 3 months after radiotherapy, mainly manifesting as nausea, abdominal pain, and diarrhea, and usually resolves spontaneously. Chronic radiation-induced intestinal injury occurs more than 3 months after the end of radiotherapy, presenting as persistent or progressive damage, characterized by severe mucosal epithelial destruction and intestinal wall fibrosis, often irreversible, and seriously affecting the patient's quality of life. Clinical studies show that the incidence of acute radiation-induced intestinal injury can reach 80%, of which about 50% of patients eventually progress to chronic injury.

[0004] Current clinical treatment mainly relies on symptomatic support, including antidiarrheal medications, glucocorticoids, amino acid preparations, and enteral nutritional support. These methods are effective in relieving symptoms, but they cannot fundamentally repair the intestinal barrier or effectively prevent disease recurrence. In recent years, novel therapies such as stem cell transplantation, probiotic preparations, and exosomes have gradually gained attention, but their clinical application remains limited due to their complex preparation, high cost, insufficient safety profile, and lack of large-scale clinical validation.

[0005] Peptide drugs, due to their small molecular weight, good biocompatibility, and low immunogenicity, exhibit unique advantages in anti-inflammation, anti-oxidation, and tissue repair. Studies have shown that peptides have potential applications in intestinal injury repair by regulating the release of inflammatory factors, improving intestinal barrier function, and promoting epithelial cell regeneration. However, the systematic development and application of peptide drugs for radiation-induced intestinal injury still lacks progress.

[0006] Therefore, there is an urgent need for a polypeptide drug to prepare drugs for intestinal radiation protection and treatment of radiation-induced intestinal injury. Summary of the Invention

[0007] The purpose of this invention is to provide an application of a polypeptide in the preparation of a drug for treating radiation-induced intestinal injury. The polypeptide provided by this invention has a radiation-protective effect in an X-ray-induced mouse model of radiation-induced intestinal injury, and can significantly improve the survival rate of irradiated mice, reduce their inflammatory factor levels, improve intestinal permeability, promote crypt cell regeneration, and repair the intestinal mucosal barrier.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the use of a polypeptide in the preparation of a drug for the prevention and / or treatment of radiation-induced intestinal injury, wherein the polypeptide is a TMVAD small peptide; the amino acid sequence of the TMVAD small peptide is Thr-Met-Val-Asp-Ala (SEQ ID NO. 1).

[0009] Preferably, the polypeptide is used to improve the survival rate of subjects with radiation-induced intestinal injury, reduce intestinal barrier damage, inhibit intestinal inflammatory response, and / or promote intestinal tissue repair.

[0010] Preferably, the drug dosage range is 10–50 mg / kg.

[0011] Preferably, the radiation-induced intestinal injury is intestinal injury induced by X-ray abdominal irradiation, and the total dose of the X-ray irradiation is 10 Gy-11.5 Gy, with a dose rate of 2 Gy / min.

[0012] Preferably, the active ingredient of the pharmaceutical composition is a TMVAD peptide, and it also includes a pharmaceutically acceptable carrier.

[0013] Preferably, the drug is formulated as an enteric-coated tablet, enteric-coated capsule, enema, colon-targeted gel, or intraperitoneal injection.

[0014] The present invention also provides the use of the above-described small peptide TMVAD in the preparation of a drug for alleviating the increase of inflammatory factors in radiation-induced intestinal injury.

[0015] The present invention also provides the use of the above-described small peptide TMVAD in the preparation of a drug for reducing intestinal permeability caused by radiation-induced intestinal injury.

[0016] The present invention also provides the application of the above-described small peptide TMVAD in the preparation of a drug for promoting the proliferation of crypt cells after radiation-induced intestinal injury.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides the application of TMVAD small peptides in the prevention and / or treatment of radiation-induced intestinal injury. Through intraperitoneal injection, this invention has shown that TMVAD small peptides can significantly rescue mice from death caused by radiation-induced intestinal injury, while also significantly reducing the levels of inflammatory factors produced by radiation-induced intestinal injury, alleviating symptoms of radiation-induced intestinal injury, and promoting intestinal crypt regeneration. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The results show the survival rate and body weight change curves of the TMVAD small peptide administration group and each control group under the condition of intraperitoneal irradiation of 11.5 Gy in mice. Figure 2 To compare the changes in small intestine length at different time points under 10 Gy irradiation and to evaluate the intervention effect of TMVAD small peptide administration; Figure 3 To assess intestinal permeability in mice on day 3 after 10 Gy irradiation using FITC-dextran and compare the differences between the TMVAD small peptide administration group and the control group; Figure 4 The expression of inflammatory factor mRNA in the small intestine tissue of mice on day 3 after 10 Gy irradiation reflects the regulatory effect of TMVAD small peptide on inflammatory response. Figure 5 HE staining results of small intestinal tissue on day 3 after 10 Gy irradiation; Figure 6 To compare the length of small intestinal villi and histological damage scores on day 3 after 10 Gy irradiation; Figure 7 Ki67 immunostaining results of small intestine sections on day 3 after 10 Gy irradiation; Figure 8 This is a schematic diagram showing the statistical results of surviving crypts in the small intestine on day 3 after 10 Gy irradiation. Detailed Implementation

[0020] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0021] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0022] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0023] The TMVAD small peptide of this invention was synthesized by Tanzhen Biotechnology Co., Ltd.; the FastQuant cDNA first-strand synthesis kit and the fluorescence quantitative detection kit (SuperReal PreMix (SYBR Green)) were purchased from Beijing Tiangen Biotech Co., Ltd.; the primers for qRT-PCR were synthesized by Shanghai Sangon Biotech Co., Ltd., and purified by UNIPAGE; the Ki67 antibody was purchased from Abmart; and the FITC-dextran powder was purchased from Sigma.

[0024] The SPF-grade male C57BL / 6 mice used in this invention were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0025] In the immunohistochemical analysis of this invention, the slides were analyzed using the anti-mouse / rabbit universal immunohistochemical detection kit PK10006, which was purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0026] Example 1: Protective effect of TMVAD small peptide against radiation-induced intestinal injury in mice induced by lethal dose of X-rays. 1.1 Mouse feeding and irradiation This invention uses 64 male SPF-grade C57BL / 6 mice aged 8-9 weeks and weighing approximately 23 g. All mice are placed under 12-hour light-dark cycles and given free access to food and water.

[0027] Sixty-four mice were divided into three groups: a solvent group (n=21), a negative control group (n=21), and an experimental group (n=22).

[0028] The experimental group, negative control group, and solvent group were administered intraperitoneal injections starting 2 days before irradiation at a concentration of 30 mg / kg, once daily, for 7 days post-irradiation. The experimental group received 3 mg / mL TMVAD peptide solution, the negative control group received 3 mg / mL AAAAA peptide solution, the solvent group received an equal volume of ddH2O, and the blank control group received no treatment.

[0029] The AAAAA peptide sequence is Ala-Ala-Ala-Ala-Ala, and all peptide segments have undergone N-terminal acetylation and C-terminal amidation modification.

[0030] 1.2 Construction of a radiation-induced intestinal injury model After anesthetizing, three groups of mice underwent X-ray irradiation of a specific area of ​​their abdomen, 2.5 cm wide, extending from the xiphoid process of the sternum to the pubic symphysis. The total irradiation dose was 11.5 Gy, with a dose rate of 2 Gy / min. During the irradiation, a 5 cm thick lead block was used to shield the rest of the mice's bodies.

[0031] 1.3 Indicator Testing Mouse body weight was monitored daily until it returned to baseline, and survival was observed for 30 days post-treatment. Statistical analysis was performed using GraphPad Prism 8.0 software, and differences between groups were compared using the ANOVA test. P < 0.05 was considered statistically significant.

[0032] Experimental results are as follows Figure 1 As shown, under abdominal irradiation with 11.5 Gy X-rays, irradiation caused weight loss in mice, leading to diarrhea and death on day 4. Weight gradually recovered by day 8. There was no significant difference in weight between groups, indicating that the TMVAD small peptide did not cause abnormal weight gain.

[0033] At this dose, the survival rate of the solvent group (ddH2O) and the control small peptide group (AAAAA) was only 20%-30%, but the survival rate of the TMVAD small peptide experimental group could reach 70%, indicating that TMVAD small peptide can significantly improve the survival of mice.

[0034] Therefore, this peptide drug can significantly alleviate death caused by radiation-induced intestinal injury.

[0035] Example 2: The therapeutic effect of TMVAD small peptide on radiation-induced intestinal injury in mice induced by sublethal doses of X-rays. The selected experimental animals, reagents, and dosages are the same as in Example 1. Fifty-eight mice were selected and divided into four groups: a non-irradiated group (n=4), a solvent group (n=18, 3-5 at each time point), a negative control group (n=18, 3-5 at each time point), and an experimental group (n=18, 3-5 at each time point). The administration method, concentration, and cycle were the same as in Example 1, i.e., intraperitoneal injection starting 2 days before irradiation, once daily until the 7th day after irradiation.

[0036] Model construction: After anesthesia, mice in all three groups were irradiated with 10 Gy X-rays to the abdomen according to the irradiation parameters in Example 1 (dose rate 2 Gy / min, lead blocks to shield other parts).

[0037] The indicators are as follows: 2.1 Intestinal length measurement Three to five mice in each group were sacrificed at 1, 3, 7, and 14 days after irradiation, and the small intestine was dissected and its length was measured.

[0038] Experimental results are as follows Figure 2 As shown, under a sublethal dose of 10 Gy, the shortening of the small intestine was most significant on day 3 of irradiation. The length of the small intestine in the experimental group was significantly increased compared with that in the solvent group and the negative control group, indicating that intestinal damage reached its peak at this time. By day 7, the length of the intestine began to gradually recover, and the recovery speed in the experimental group was faster.

[0039] These results indicate that 10 Gy abdominal irradiation can establish a stable acute intestinal injury model, with day 3 being the period of most significant injury.

[0040] Therefore, subsequent experiments selected the third day after irradiation as the main observation time point to reflect the acute injury state of radiation-induced intestinal injury.

[0041] 2.2 Intestinal permeability testing On the third day after irradiation, mice were fasted for 12 hours and then weighed. They were then administered FITC-Dextran by gavage (50 mg / 100g). Three hours later, blood was collected from the mice's eyeballs, and the serum was centrifuged to determine the fluorescence intensity. The fluorescence intensity was measured using a full-wavelength multi-mode microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The fluorescence intensity was normalized based on the fluorescence index of the non-irradiated group.

[0042] The results are as follows Figure 3 As shown, on day 3 after 10 Gy irradiation, the irradiation significantly increased serum fluorescence signal, indicating impaired intestinal barrier function. Compared with the control group, the permeability of the TMVAD small peptide treatment group was reduced, indicating that the small peptide can alleviate intestinal barrier damage in the early stage after irradiation.

[0043] 2.3 Detection of inflammatory factor expression On day 3 post-irradiation, small intestinal tissue was collected from mice, and total RNA was extracted using the trizol method. The extracted RNA was then quantified using a spectrophotometer. cDNA was synthesized from the RNA samples via reverse transcription, and the expression levels of TNF-β, TNF-α, IL-1β, and SAA in the samples were detected using a SYBR Green PCR kit. The primer sequences are shown in Table 1. The reaction conditions were: 95℃ pre-denaturation for 30 s, 95℃ for 5 s, 55℃ for 30 s, and 72℃ for 1 min, for 40 cycles. The relative levels of TNF-β, TNF-α, IL-1β, and SAA relative to the internal control β-actin were calculated based on Ct values. At least three mice were included in each group, and data were normalized to those from the non-irradiated group.

[0044] Table 1 RT-PCR primer sequences

[0045] The results are as follows Figure 4As shown, qRT-PCR was used to detect the expression of inflammatory factors TNF-β, TNF-α, IL-1β and serum amyloid protein SAA in the small intestine tissue of the experimental group on day 3 after 10 Gy irradiation, compared with the control group. This indicates that TMVAD peptide can alleviate radiation-induced inflammatory response in mice.

[0046] 2.4 Histopathological examination On the third day after irradiation, small intestinal tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Sections were prepared according to standard procedures and stained with hematoxylin and eosin (H&E). The sections were observed under an optical microscope, and the number of crypts was determined using CaseViewer software. The Chiu six-grade method was used to score intestinal epithelial damage. The number of surviving crypts was counted by Ki67 immunohistochemical staining (following the kit instructions: blocking, primary antibody incubation, secondary antibody incubation, DAB staining, hematoxylin counterstaining, and neutral resin mounting).

[0047] The results are as follows Figure 5 As shown, the 10 Gy irradiation solvent group and the negative control group resulted in shortened and broken small intestinal villi, disordered crypt structure, and significant edema of the lamina propria; while after treatment with TMVAD small peptides, the crypt structure was intact and elongated, the villi were longer and wider, and the intestinal tissue structure was significantly improved. Figure 6 Statistical results also showed that the TMVAD small peptide treatment group significantly increased villous length compared to the solvent group, and the Chiu score results further confirmed that the TMVAD small peptide treatment group had the lowest degree of damage.

[0048] Moreover, as Figure 7 , 8 As shown, TMVAD peptides significantly increased the number of surviving crypts on day 3 after 10 Gy irradiation, indicating that they can protect and promote the proliferation of crypt stem cells, alleviate acute inflammatory damage, and promote tissue repair.

[0049] In summary, the TMVAD peptide demonstrated significant protective effects and therapeutic potential in a mouse model of radiation-induced intestinal injury. This peptide improved survival, reduced inflammation, enhanced intestinal barrier function, and promoted tissue regeneration, providing a novel strategy for the prevention and treatment of radiation-induced intestinal injury.

[0050] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a polypeptide in the preparation of drugs for the prevention and / or treatment of radiation-induced intestinal injury, characterized in that, The polypeptide is a TMVAD peptide; the amino acid sequence of the TMVAD peptide is Thr-Met-Val-Asp-Ala.

2. The application according to claim 1, characterized in that, The peptide is used to improve the survival rate of subjects with radiation-induced intestinal injury, reduce intestinal barrier damage, inhibit intestinal inflammatory response, and / or promote intestinal tissue repair.

3. The application according to claim 1, characterized in that, The dosage range of the drug is 10–50 mg / kg.

4. The application according to claim 1, characterized in that, The radiation-induced intestinal injury is intestinal damage induced by X-ray abdominal irradiation, with a total X-ray dose of 10 Gy-11.5 Gy and a dose rate of 2 Gy / min.

5. The application according to claim 1, characterized in that, The active ingredient of the pharmaceutical composition is a TMVAD small peptide, and it also includes a pharmaceutically acceptable carrier.

6. The application according to claim 1, characterized in that, The drug is formulated as enteric-coated tablets, enteric-coated capsules, enemas, colon-targeted gels, or intraperitoneal injections.

7. The use of the small peptide TMVAD of claim 1 in the preparation of a medicament for alleviating elevated inflammatory factors in radiation-induced intestinal injury.

8. The use of the small peptide TMVAD of claim 1 in the preparation of a medicament for reducing intestinal permeability caused by radiation-induced intestinal injury.

9. The use of the small peptide TMVAD of claim 1 in the preparation of a drug for promoting the proliferation of crypt cells after radiation-induced intestinal injury.

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