Application of TNKS1BP1 in preparation of medicine for preventing, relieving and / or treating cognitive hypofunction after radiotherapy
By overexpressing the TNKS1BP1 gene in the hippocampus of mice, a whole-brain radiation model was constructed, which solved the problem of cognitive impairment after radiotherapy, significantly improved the DNA damage and cognitive function of neural stem cells, and provided a new way of cognitive protection after radiotherapy.
Patent Information
- Application Number
- CN202510414220.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-07-11
AI Technical Summary
In the prior art, the problem of cognitive impairment after radiotherapy has not been effectively solved, especially in the radiation therapy of brain tumors in children and adults, impaired hippocampal nerves lead to cognitive dysfunction and lack of effective prevention and treatment methods.
By overexpressing or increasing the Tank Enzyme 1 binding protein 1 (TNKS1BP1) gene or its encoded recombinant vector/virus, especially recombinant lentivirus, it is injected into the hippocampus of the mouse, and building a whole-brain radiation model to protect neural stem cells from DNA damage and improve cognitive function.
Overexpression of TNKS1BP1 significantly inhibits DNA damage to neural stem cells in the hippocampus region of mice, protects cognitive function, reduces cognitive impairment after radiotherapy, improves new object recognition ability and neural stem cell growth rate, and reduces DNA damage.
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Figure CN120285164A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and specifically to the application of TNKS1BP1 in the preparation of a drug for preventing, alleviating and / or treating cognitive decline after radiotherapy. Background Art
[0002] Radiotherapy, as a first-line therapy for almost all types of pediatric and adult brain tumors, plays an important role in curbing the growth of central nervous system malignancies. Cranial irradiation significantly improves the survival rate of patients during the process of preventing the progression of brain cancer, but also increases the risk of severe neurocognitive sequelae in long-surviving patients (Gorbunov NV, Kiang JG. 2021. Brain Damage and Patterns of Neurovascular Disorder after Ionizing Irradiation. Complications in Radiotherapy and Radiation Combined Injury. Radiat Res 196:1-16.). The hippocampus plays a crucial role in short-term memory and learning (Brown PD, Ahluwalia MS, Khan OH, Asher AL, Wefel JS, Gondi V. 2018. Whole-Brain Radiotherapy for Brain Metastases: Evolution or Revolution? J Clin Oncol 36:483-491. Wang QQ, Yin G, Huang JR, Xi SJ, Qian F, Lee RX, Peng XC, Tang FR. 2021. Ionizing Radiation-Induced Brain Cell Aging and the Potential Underlying Molecular Mechanisms. Cells 10.). Under physiological conditions, the dentate gyrus (DG) of the hippocampus is an important region for hippocampal neurogenesis, and neural stem cells therein continuously self-renew through asymmetric division and differentiate into neurons and glial cells (Toda T, Gage FH. 2018. Review: adult neurogenesis contributes to hippocampal plasticity. Cell Tissue Res 373:693-709.). IR exposure reduces or ablates hippocampal neurogenesis.
[0003] Efficient DNA repair mechanisms contribute significantly to maintaining genomic stability by counteracting the accumulation of DNA damage, which is one of the key cellular functions. The accumulation of DNA damage and mutations increases the risk of cancer and is associated with aging (Friedberg EC, Aguilera A, Gellert M, Hanawalt PC, Hays JB, Lehmann AR, Lindahl T, Lowndes N, Sarasin A, Wood RD. 2006. DNA repair: from molecular mechanism to human disease. DNA Repair (Amst) 5:986-96. Hoeijmakers JH. 2009. DNA damage, aging, and cancer. N Engl J Med 361:1475-85. Vijg J, Suh Y. 2013. Genome instability and aging. Annu Rev Physiol 75:645-68.).
[0004] TNKS1BP1 was initially identified as an interacting protein of tankyrase 1 and belongs to the poly(ADP-ribose) polymerase (PARP) superfamily. PARP members play important roles in DNA repair, telomere stability, and mitotic regulation, etc.
[0005] However, there is currently no report on the application of TNKS1BP1 in the preparation of drugs for preventing, alleviating, and / or treating cognitive decline after radiotherapy. Summary of the Invention
[0006] The object of the present invention is to provide the role of TNKS1BP1 in protecting cognitive function after radiotherapy, specifically its application in the preparation of drugs for preventing, alleviating, and / or treating cognitive decline after radiotherapy.
[0007] TNKS1BP1 significantly sensitizes cancer cells to radiation through ionizing radiation (IR) and TNKS1BP1 depletion. After 10 Gy of ionizing radiation, DNA damage is significant in neural stem cells in the mouse brain, but there is no significant increase in TNKS1BP1. The present invention attempts to explore whether increasing TNKS1BP1 can reduce DNA damage in neural stem cells after radiation and improve cognitive function, providing new ideas for the treatment of protecting cognitive function after radiotherapy.
[0008] In the first aspect of the present invention, there is provided the application of TNKS1BP1 in the preparation of drugs for preventing, alleviating, and / or treating cognitive decline after radiotherapy.
[0009] The gene sequence number of the described Tankyrase 1 binding protein 1 (TNKS1BP1) is NM_001081260.2, and the protein sequence number is NP_001074729.1.
[0010] Furthermore, the application is the application of TNKS1BP1 in the preparation of a drug for inhibiting DNA damage of neural stem cells after radiation, reversing the reduction in the growth rate of neural stem cells caused by radiation, and protecting cognitive function.
[0011] The present invention determines through experiments the relationship between TNKS1BP1 in protecting neural stem cells from damage and cognitive function after radiotherapy:
[0012] Overexpression of the TNKS1BP1 gene significantly inhibits DNA damage of mouse neural stem cells and improves cognitive function: The present invention uses mice as experimental subjects. By stereotaxically injecting TNKS1BP1 into the hippocampal region and then constructing a whole-brain radiation model, the role of TNKS1BP1 in protecting cognitive function and reducing hippocampal neural stem cell damage is studied. The results show that overexpression of the TNKS1BP1 gene significantly inhibits DNA damage of neural stem cells in the hippocampal region of mice and protects cognitive function.
[0013] From the above results, it can be seen that overexpression of the TNKS1BP1 gene significantly reduces DNA damage of mouse neural stem cells after radiation and protects cognitive function. Therefore, TNKS1BP1 can inhibit the occurrence of cognitive function decline caused by radiation.
[0014] In the second aspect of the present invention, there is provided the application of a reagent for increasing the content or expression level of TNKS1BP1 in the preparation of a drug for preventing, alleviating, and / or treating cognitive function decline after radiotherapy.
[0015] Furthermore, the reagent for increasing the content or expression level of TNKS1BP1 includes any one of the following:
[0016] A) TNKS1BP1;
[0017] B) A recombinant vector containing the TNKS1BP1 coding gene;
[0018] C) A recombinant virus containing the TNKS1BP1 coding gene.
[0019] Furthermore, in the above C), the recombinant virus containing the TNKS1BP1 coding gene can be a recombinant lentivirus containing the TNKS1BP1 coding gene, a recombinant adenovirus containing the TNKS1BP1 coding gene, a recombinant adeno-associated virus containing the TNKS1BP1 coding gene, etc.
[0020] In one embodiment of the present invention, the reagent for increasing the content or expression level of TNKS1BP1 is a recombinant lentivirus carrying the TNKS1BP1 encoding gene (sequence number NM_001081260.2).
[0021] In the third aspect of the present invention, there is provided a pharmaceutical composition for preventing, alleviating and / or treating post-radiotherapy cognitive decline, the active ingredient of which is a reagent for increasing the content or expression level of TNKS1BP1.
[0022] Furthermore, the pharmaceutical composition for preventing, alleviating and / or treating post-radiotherapy cognitive decline further comprises a pharmaceutically acceptable carrier or excipient.
[0023] The advantages of the present invention are as follows:
[0024] Taking mice as experimental subjects, the present invention studies the role of TNKS1BP1 in protecting cognitive function and reducing hippocampal neural stem cell damage by using stereotaxic injection of TNKS1BP1 into the hippocampal region and then constructing a whole-brain radiation model. The present invention discovers the new function of the TNKS1BP1 gene, and the TNKS1BP1 gene has the effect of improving post-radiotherapy cognitive decline. Based on the function of the TNKS1BP1 gene, TNKS1PB1 can be used to prepare drugs for protecting cognitive function after radiation, providing an effective new approach for protecting cognitive function after radiotherapy. Brief Description of the Drawings
[0025] Figure 1 It is a detection graph of Y-maze and novel object recognition experiments performed on the hippocampal tissue of mice overexpressing TNKS1BP1 one week after radiation, and the results show that TNKS1BP1 protects cognitive function after radiotherapy (*: P < 0.05).
[0026] Figure 2 It is an observation of the neural stem cells of mice overexpressing TNKS1BP1 one week after irradiation, and the results show that overexpression of TNKS1BP1 reverses the decrease in the growth rate of neural stem cells caused by radiation (*: P < 0.05).
[0027] Figure 3 It is a comet assay of the hippocampal tissue of mice overexpressing TNKS1BP1 one day after radiation, and the results show that overexpression of TNKS1BP1 reduces the DNA damage of neural stem cells caused by radiation (*: P < 0.05). Detailed Embodiments
[0028] The following detailed description of the specific embodiments provided by the present invention will be given in conjunction with the examples.
[0029] Experimental animals used in the experiments and their feeding:
[0030] Experimental animals: Male mice aged 8 - 10 weeks with a body weight of 25 g ± 3 g were selected as experimental subjects.
[0031] Feeding environment:
[0032] All experimental animals were housed in the SPF - level animal center of the Naval Medical University. The SPF - level mouse feed was purchased from Shanghai Jihui Biotechnology Co., Ltd. Feeding conditions: room temperature 22 - 24 °C, humidity between 40% - 70%, alternating light and dark illumination time of 12 h, free access to food and water.
[0033] Example 1: Obtaining a mouse model of cranial radiotherapy with over - expressed TNKS1BP1
[0034] 1. Animal grouping: C57BL / 6J male mice were randomly divided into four groups: the group transfected with control virus without irradiation, the group transfected with control virus with irradiation, the group transfected with TNKS1BP1 without irradiation, and the group transfected with TNKS1BP1 with irradiation.
[0035] 2. Obtaining mice with over - expressed TNKS1BP1 in the hippocampal region, model preparation process:
[0036] (1) Anesthesia: Weigh the mice, calculate the amount of anesthetic (3% sodium pentobarbital) required according to 90 mg / kg body weight, inject it intraperitoneally, and record the injection time point. The standard for the success of anesthesia is that the limb tension is small and the mouse is in good condition.
[0037] (2) Surgical area preparation: Shave the hair on the head skin, and then wipe the surgical area with a wet gauze to remove the sebaceous glands, so as not to affect the surgical field of view.
[0038] (3) Intracerebral gene injection:
[0039] The lentivirus carrying TNKS1BP1 (sequence number XM_004869787.2) was provided by Shanghai Genechem Co., Ltd. The virus was injected into the hippocampal regions of the two cerebral hemispheres of mice by stereotaxic injection (Yang W, Wu W, Zhao Y, Li Y, Zhang C, Zhang J, Chen C, Cui S. 2022. Caveolin-1 suppresses hippocampal neuron apoptosis via the regulation of HIF1α in hypoxia in naked mole-rats. Cell Biol Int 46:2060-2074.). According to the stereotaxic atlas of Franklin and Paxinos, the injection position parameters were: AP: -2 mm from bregma; L: -1.5 mm; DV: +1.75 mm. The control group was injected with control lentivirus, and the experimental group was injected with TNKS1BP1 lentivirus.
[0040] (4) Postoperative care
[0041] After stereotaxic injection of the brain, the animals were placed in a breeding cage containing autoclaved bedding, feed, and drinking water, and continued to be bred and observed in the breeding room.
[0042] (5) Preparation of an animal model for cranial radiotherapy
[0043] The animals were fixed in a transparent plastic fixing frame in a conscious state to maintain body position fixation, and were irradiated with 10 Gy 60 Co-ray (dose rate: 1.16 Gy / min).
[0044] Example 2: Detection of cognitive function in mice overexpressing TNKS1BP1 after radiation
[0045] 1. Behavioral detection
[0046] The novel object recognition experiment detects short-term memory function. The experiment includes three phases: adaptation, training, and testing. In the adaptation phase, the animals are allowed to adapt to a black and white box (50 cm × 40 cm × 30 cm) for 30 minutes. The training phase starts 24 hours later, during which the mouse is placed in a box containing two identical objects and allowed to freely explore for 10 minutes, and then the animal is returned to the cage. The feces in the experimental chamber are removed and wiped with 75% ethanol to eliminate any olfactory cues that may affect the animals. In the testing phase, one object is replaced with a new object, and then the animal is allowed to explore for another 10 minutes. The movement trajectories of the animals are recorded and analyzed using customized software to quantify the exploration time of different objects, and the cognitive function of the animals is evaluated using the novel object recognition test index. The calculation formula for the recognition index (DI) is as follows: DI = (new object exploration time - familiar object exploration time) / (new object exploration time + familiar object exploration time), where DI represents the recognition ability of the mouse (Chen CA, Pal R, Yin J, Tao H, Amawi A, Sabo A, Bainbridge MN, Gibbs RA, Zoghbi HY, Schaaf CP. 2020. Combination of whole exome sequencing and animal modeling identifies TMPRSS9 as a candidate gene for autism spectrum disorder. Hum Mol Genet 29:459-470.).
[0047] Example 3: Detection of the growth status of neural stem cells in mice overexpressing TNKS1BP1 after irradiation
[0048] 1. Obtaining mouse neural stem cells overexpressing TNKS1BP1
[0049] Primary mouse neural stem cells were cultured in vitro. After 7 days, they were transfected with a control virus or a TNKS1BP1 lentivirus. After 24 hours, the growth medium without virus was replaced, and an irradiation experiment was carried out 72 hours later.
[0050] 2. Obtaining a neural stem cell irradiation model
[0051] The neural stem cells were irradiated with 10 Gy 60 Co γ-rays in a single dose.
[0052] 3. Light microscopy results
[0053] After 7 days of irradiation, the neurospheres transfected with the control virus and the TNKS1BP group were observed by optical fiber microscopy. The cross-section of the neurosphere was selected as the quantitative basis to evaluate the growth size of the neurosphere.
[0054] 4. Comet assay
[0055] After 1 day of irradiation, the neural stem cells were digested into single cells with Accutase digestion solution, and then the damage degree of neural stem cells was measured by comet assay under neutral conditions (Trevigen, Gaithersburg, MD, USA). CometScore software (TriTek, Sumerduck, VA, USA) was used to analyze the comet (Xiong X, Du Z, Wang Y, Feng Z, Fan P, Yan C, Willers H, Zhang J. 2015. 53BP1 promotes microhomology-mediated end-joining in G1-phase cells. Nucleic Acids Res 43:1659-70.).
[0056] Figure 1 These are the results of mice transfected with the control and TNKS1BP1 viruses in the hippocampal region with or without irradiation. The results showed that overexpression of TNKS1BP1 in the hippocampal region significantly improved the ability of mice to recognize new objects after radiotherapy, that is, it improved the cognitive function of mice. Figure 2 These are the results of hippocampal neural stem cell spheres transfected with the control and TNKS1BP1 viruses after 7 days of irradiation or without irradiation. The results showed that after 7 days of irradiation, TNKS1BP1 significantly alleviated the decrease in the growth rate of neurospheres after irradiation. Figure 3 These are the comet staining results of hippocampal neural stem cells transfected with the control and TNKS1BP1 viruses after 1 day of irradiation or without irradiation. The results showed that overexpression of TNKS1BP1 alleviated the DNA damage of neural stem cells caused by radiation.
[0057] From the above results, it can be seen that in the cellular and animal models of cranial radiotherapy, overexpression of the TNKS1BP1 gene inhibited DNA damage of neural stem cells after radiation and protected cognitive function. Therefore, the TNKS1BP1 gene has the function of protecting neural stem cells from DNA damage caused by radiation and inhibiting the decline of cognitive function after radiotherapy.
[0058] The preferred embodiments of the present invention have been specifically described above. However, the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.
Claims
1. Use of TNKS1BP1 in the preparation of a drug for preventing, alleviating, and / or treating cognitive decline after radiotherapy.
2. The application according to claim 1, wherein, Use of TNKS1BP1 in the preparation of a drug for inhibiting DNA damage of neural stem cells after radiation, reversing the reduction in the growth rate of neural stem cells caused by radiation, and protecting cognitive function.
3. Use of a reagent for increasing the content or expression level of TNKS1BP1 in the preparation of a drug for preventing, alleviating, and / or treating cognitive decline after radiotherapy.
4. The application according to claim 3, characterized in that, The reagent for increasing the content or expression level of TNKS1BP1 includes any one of the following: A) TNKS1BP1; B) A recombinant vector containing the TNKS1BP1-encoding gene; C) A recombinant virus containing the TNKS1BP1-encoding gene.
5. The application according to claim 4, wherein In the above C), the recombinant virus containing the TNKS1BP1-encoding gene can be a recombinant lentivirus containing the TNKS1BP1-encoding gene, a recombinant adenovirus containing the TNKS1BP1-encoding gene, a recombinant adeno-associated virus containing the TNKS1BP1-encoding gene, etc.
6. The application according to claim 4, wherein, The reagent for increasing the content or expression level of TNKS1BP1 is a recombinant lentivirus carrying the TNKS1BP1-encoding gene.
7. A pharmaceutical composition for preventing, alleviating and / or treating cognitive decline after radiotherapy, characterized in that, Its active ingredient is a reagent for increasing the content or expression level of TNKS1BP1.
8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition for preventing, alleviating, and / or treating cognitive decline after radiotherapy further includes a pharmaceutically acceptable carrier or excipient.