WO2022150948A1 - Use of nicotinamide mononucleotide in preparation of anti-radiation injury preparation - Google Patents
Use of nicotinamide mononucleotide in preparation of anti-radiation injury preparation Download PDFInfo
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- WO2022150948A1 WO2022150948A1 PCT/CN2021/071192 CN2021071192W WO2022150948A1 WO 2022150948 A1 WO2022150948 A1 WO 2022150948A1 CN 2021071192 W CN2021071192 W CN 2021071192W WO 2022150948 A1 WO2022150948 A1 WO 2022150948A1
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- nicotinamide mononucleotide
- nmn
- irradiation
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/13—Nucleic acids or derivatives thereof
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
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Definitions
- the invention belongs to the field of biomedicine, and particularly relates to the application of nicotinamide mononucleotide in the preparation of anti-radiation damage preparations and preparations containing nicotinamide mononucleotide.
- Ionizing radiation is widely used in industry, medical care, military and other fields. The probability of radiation workers and the public being exposed to radiation is greatly increased. In daily life, the use of electronic products and planes will also suffer from radiation damage to varying degrees. Therefore, radiation damage and protection more academic and public attention.
- Radiotherapy as an important method in current cancer treatment, usually causes severe damage to adjacent tissues and irradiated normal tissues while killing tumor tissues. DNA damage caused by ionizing radiation is the basis of radiation biological effects in organisms, various tissues of the body, cells and even cell sub-levels, and is the main cause of radiation biological effects.
- some supportive therapies are mainly used in clinical practice, mainly symptomatic treatment, including drug antiemetic, analgesic, blood transfusion, body fluids, electrolytes and other nutritional supplements, and antibiotics to treat infection.
- thiol-containing compound amifostine WR-2721
- WR-2721 thiol-containing compound amifostine
- An ideal radioprotectant should have the characteristics of safety to human body, convenient source, easy oral administration, and rapid absorption.
- Nicotinamide ribonucleotide is synthesized from nicotinamide (Nic) and 5'-phosphate pyrophosphate by nicotinamide phosphotransferase (NAMPT) and is a key intermediate of NAD + .
- NAMPT nicotinamide phosphotransferase
- NMN has been shown to enhance NAD + biosynthesis and improve various pathologies in mouse disease models, such as myocardial and cerebral ischemia, neurodegenerative diseases such as Alzheimer's disease, and diabetes.
- NAD + Most of the pharmacological effects of NMN are carried out by promoting the synthesis of NAD + , because direct administration of high doses of NAD + will cause side effects such as insomnia, fatigue, anxiety, and the ability of NAD + to penetrate the plasma membrane is also worse than that of NMN.
- NAD + is an important coenzyme in the body, and several studies have shown that supplementation of NAD + precursors in vitro can delay aging. Nicotinamide mononucleotide (NMN), as the precursor of NAD + , has low toxicity and high safety, and has become a research hotspot in the field of anti-aging and tumor suppression, and NMN's involvement in DNA damage repair has also been widely concerned.
- NAD + Nicotinamide mononucleotide
- the Sirtuins family is an NAD + -dependent family that is widely expressed in mammalian cells.
- the family has 7 members, namely: SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7.
- SIRT2 is located in the cytoplasm
- SIRT3, SIRT4 and SIRT5 are located in the mitochondria
- SIRT6 and SIRT7 are located in the nucleus. Due to their differences in location and structure in cells, they produce different enzymatic activities.
- the Sirtuins family is associated with aging-related diseases, such as neurodegenerative diseases, cardiovascular diseases, etc.
- SIRT1 is the member with the highest homology to the yeast silent information regulator Sir2.
- the gene is located on chromosome 10 and encodes a total of 747 amino acids.
- the relative molecular weight of the protein is about 120kDa, and the main distribution area is the nucleus.
- the NAD + /NADH binding domain is the active center of SIRT1, and such a key structure determines the important role of SIRT1 in mammalian cells. It can remove the acetyl groups of histones H1, H3 and H4. At the same time, it can also act on many non-histone proteins, such as: p53, PGC-1, etc., and can also modify DNA repair proteins such as PARP1 and Ku 70.
- Previous studies have shown that SIRT1 can regulate bone metabolism by regulating the Wnt/-catenin pathway.
- SIRT6 is mainly located on chromosome 19 in the nucleus, and there are two isoforms, one containing 8 exons encoding a protein of 355 amino acids in length, and the other containing 7 exons encoding a protein of 328 amino acids in length.
- SIRT6 protein has 3 enzymatic activities: deacetylase activity, long-chain deacylase activity and single ADP ribosyltransferase activity.
- SIRT6 has a weak deacetylation effect and is a site-specific histone deacetylase that targets the acetylation sites of histone H3 lysines K9, K56 and K18 (H3K9ac, H3K56ac and H3K18ac) and has the ability to regulate DNA damage repair , glucose and lipid metabolism, stress tolerance and inflammatory response and the function of aging.
- SIRT6 acts as a core repressor of several transcription factors NF-kB, HIF1 ⁇ , c-Jun and c-Myc involved in aging, cancer and metabolism, promoting DNA damage-dependent chromatin changes, which are Essential for repair; and maintains telomeric chromatin structure, preventing genomic instability and cellular senescence.
- SIRT7 is located on chromosome 17 and has a sequence of 400 amino acids. It is the most similar (56% identical) to the protein sequence of SIRT6 in the mammalian sirtuins family, and has lysine residues at positions 18, 36, and 37 of histone H3. (H3K18ac, H3K36ac, H3K37ac) have specific deacetylation, participate in tumorigenesis, promote rDNA (ribosomal DNA) heterochromatin silencing and genome stability, transcription elongation or DNA damage repair. SIRT7 promotes cell survival after injury by inhibiting ⁇ H2AX and p53 aggregation at DNA damage sites and MAPK signaling.
- SIRT7 can promote the deacetylation of ATM to initiate DNA damage repair. Furthermore, SIRT7 can be recruited to the injury site in a PARP1-dependent manner and regulate the level of H3K18Ac at the injury site, thereby promoting the recruitment of 53BP1 to DSBs and promoting NHEJ repair. In addition, SIRT7 can also catalyze the desuccinylation of H3K122 at the injury site. , promoting chromatin remodeling during DNA repair.
- the purpose of the present invention is to provide a new medicinal use of nicotinamide mononucleotide and a pharmaceutical composition containing nicotinamide mononucleotide.
- the first aspect of the present invention is to provide the application of nicotinamide mononucleotide in the preparation of anti-radiation damage preparations.
- the preparation includes drugs, health products, food additives or skin care products; the radiation is ionizing radiation.
- the preparation for resisting radiation damage is a preparation for reducing the apoptosis rate of cells after irradiation or alleviating the inhibition of cell proliferation by irradiation.
- the anti-radiation damage preparation is a preparation for reducing the degree of DNA damage after irradiation and promoting the repair of DNA damage. It can be seen from the foregoing that DNA damage is a key link in the occurrence of radiation damage, and reducing the degree of DNA damage helps to fundamentally Protection from radiation damage.
- the drug for reducing the degree of DNA damage after irradiation is a preparation for promoting the up-regulation of PARP and SIRT expression.
- the present invention verifies the protective effect of nicotinamide mononucleotide on radiation damage from two microscopic perspectives of cells and genes.
- NMN neuropeptide
- the drug concentration of 1 mM NMN was selected for treatment, and the drug was added 24 hours before irradiation, and different doses of radiation were used respectively, and the colony formation was observed after 14 days.
- the results showed that NMN treatment significantly improved the survival rate of cells after irradiation, effectively reduced the apoptosis rate caused by ionizing radiation, and at the same time increased the cell survival rate after irradiation; in addition, NMN could significantly reduce the degree of DNA damage, and further studies showed that increasing SIRT1 gene expression level. It shows that the use of nicotinamide mononucleotide before irradiation can reduce the damage of ionizing radiation and reduce the sensitivity of cells and even genes to radiation.
- the anti-radiation injury preparation of the present invention is nicotinamide mononucleotide as the only active ingredient or a pharmaceutical composition comprising nicotinamide mononucleotide.
- the medicine or pharmaceutical composition of the present invention can be made into any dosage form with commonly used pharmacy auxiliary materials, for example, it can be decoction, powder, pill, wine, lozenge, glue, plaster, tea, koji , cake, lotion, stick, thread, strip, nail, moxibustion iron, ointment, elixir, microcapsule, intravenous emulsion, liposome preparation, aerosol, prodrug preparation, injection, Mixtures, oral ampoules, tablets, capsules, drop pills, emulsions, ointments, rubber plasters, films, sponges, iontophoresis, or transdermal absorption agents; health products are oral capsules, tablets or powder; skin care products are lotions, sprays, masks or eye creams.
- the second aspect of the present invention is to provide an anti-radiation injury preparation, which is composed of nicotinamide mononucleotide or its derivatives and pharmaceutically, health-care, food or cosmetic acceptable excipients.
- the nicotinamide mononucleotide of the present invention is used to prevent radiation damage, and is particularly suitable for radiation damage associated with radiation workers, cancer radiotherapy patients, etc., and is administered in advance to people who may suffer from radiation lung damage, and the administration method is not limited to Oral, injection, etc.
- the present invention verifies the protective effect of nicotinamide mononucleotide on radiation damage from two microscopic perspectives of cells and genes: cell experiment results show that NMN treatment can significantly improve the survival rate of cells after irradiation, and effectively reduce cell damage caused by ionizing radiation. Apoptosis rate, while improving cell survival rate after irradiation; in addition, NMN can significantly reduce the degree of DNA damage, while increasing the expression level of SIRT1 gene. It shows that the use of nicotinamide mononucleotide before irradiation can reduce the damage of ionizing radiation and reduce the sensitivity of cells and even genes to radiation. Therefore, the present invention provides a new basis for nicotinamide mononucleotide to protect against radiation damage.
- nicotinamide mononucleotide is a mature commercial product, its pharmacological action is clear, the toxic and side effects are small, and the drug safety has been clinically recognized, and the new indication of nicotinamide mononucleotide provided by the present invention can be faster achieve clinical translation. Therefore, nicotinamide mononucleotide has great potential in the protection of clinical radiation injury.
- Figure 1 is a graph showing the effect of nicotinamide mononucleotide on cell clones after irradiation.
- Figure 2 is the result of the effect of nicotinamide mononucleotide on apoptosis induced by radiation.
- Figure 3 shows the effect of nicotinamide mononucleotide on cell viability after irradiation.
- A is the effect on the survival rate of MODE-K cells after irradiation
- B is the effect on the survival rate of 293T cells after irradiation.
- Figure 4 shows the effect of nicotinamide mononucleotide on DNA damage in A549-NRF2KO cells after irradiation.
- A is the gel electrophoresis result of the "comet” experiment
- B is the comparison result of the tail moment of the "comet” experiment
- C is the cell immunofluorescence experiment result of the formation of the DNA damage early response protein ⁇ H2AX
- D is the DNA damage early response protein of different groups Comparison of ⁇ H2AX foci numbers.
- Figure 5 shows the effect of nicotinamide mononucleotide on DNA damage of human skin fibroblasts HFF-1 after irradiation.
- A is the gel electrophoresis result of the "comet” experiment;
- B is the comparison result of the tail moment of the "comet” experiment;
- C is the cell immunofluorescence experiment result of the formation of the DNA damage early response protein ⁇ H2AX;
- D is the DNA damage early response protein of different groups ⁇ H2AX foci quantity comparison
- Figure 6 shows the effect of nicotinamide mononucleotide on DNA damage in normal human skin fibroblasts HFF-1.
- A is the gel electrophoresis result of the "comet” experiment of NMN combined with different DNN damage inducers
- B is the comparison result of the tail moment of the "comet” experiment
- C is the result of the cellular immunofluorescence experiment of the formation of the DNA damage early response protein ⁇ H2AX
- D Comparison of the number of DNA damage early response protein ⁇ H2AX foci in different groups.
- Figure 7 shows the effect of nicotinamide mononucleotide on the viability and cell cycle of HFF-1 cells after irradiation.
- A is the effect of HFF-1 cell viability after irradiation; -1 cell cycle effect results.
- Figure 8 shows the results of protein expression levels of SIRT1 ⁇ SIRT6 ⁇ SIRT7 and PARP in cells after nicotinamide mononucleotide-promoted irradiation.
- Nicotinamide mononucleotide can improve the survival rate of cells after irradiation
- Literature has proved that NMN can improve the aging of mice, and NRF2 not only plays an important role in DNA damage, and NRF2 knockout mice show the characteristics of premature aging, so we chose the A549-NRF2KO cell line for experiments.
- the cells were divided into two groups, the control group and the NMN-treated group, and the two groups of cells were cultured under the same conditions. 24h before irradiation, the NMN treatment group selected 1mM NMN drug concentration to treat the cells, and the control group was not treated. Then, the two groups of cells were irradiated with 0, 0.5, 1, 2, 4, and 6 Gy respectively, and the colony formation was observed 14 days after irradiation.
- ionizing radiation treatment and NRF2 knockout resulted in increased apoptosis, so apoptosis was examined.
- the cells were divided into control group, NMN group, irradiation (IR) group and NMN+IR group.
- the control group neither added NMN nor irradiated; the NMN group added NMN without irradiation; the irradiation (IR) group Irradiation; NMN+IR group added NMN first and then irradiated.
- NMN irradiation
- NMN+IR group The cells in the irradiation (IR) group and the NMN+IR group were irradiated with 8 Gy, and the samples were collected at 24h, 48h, and 72h after irradiation. The results are shown in Figure 2. After 24h of irradiation, NMN has a certain recovery effect on cell apoptosis. , but with the prolongation of irradiation time, the recovery effect became weaker and weaker.
- MODE-K and 293T cells were selected for experiments. 0, 0.5 and 1 mM NMN were added 24 h before irradiation, and then 0, 0.5, 1, 2, and 4 Gy were irradiated for different treatment conditions, and cultured for 7 days for staining.
- the comet assay can effectively detect and quantify the degree of DNA single-strand or double-strand break nick damage in cells.
- the comet experiments were performed on A549-NRF2KO cells treated with different conditions, and the results are shown in Figure 4: It was found that the "comet" tail moment of the NMN+IR group was significantly shorter than that of the single irradiation (IR) group ( Figures A and B). ), indicating that NMN can reduce DNA damage in cells after irradiation.
- NMN attenuates DNA damage in skin fibroblasts after irradiation
- Human skin fibroblasts HFF-1 were selected for research, and CPT (topoisomerase inhibitor, induced DNA replication fork collapse caused dsDNA damage), HU (induced replication fork arrest caused ssDNA damage), MMS (alkylated four kinds of drugs that induce different forms of DNA damage, and further explore the effect of NMN on different forms of DNA damage. influences.
- NMN By examining the cell cycle, compared with the single irradiation group, NMN could significantly prolong the G1 phase when irradiated for 12h, but the improvement of the cell cycle was not obvious when irradiated for 6h and 24h (B, C, D).
- NMN treatment promotes the up-regulation of SIRT1 ⁇ SIRT6 ⁇ SIRT7 and PARP expression
- the NRF2KO cell line was selected, and samples were collected 1h, 6h, 12h, and 24h after irradiation, and WB experiments were performed. The results are shown in Figures 8A-C. According to the experimental results, we found that the protein levels of SIRT1, SIRT6, SIRT7 and PARP were up-regulated after NMN treatment. With the prolongation of irradiation time, compared with the single irradiation group, NMN promoted the up-regulation of several protein levels to a higher degree, which may be an effective pathway for NMN to play a protective role.
- NMN can significantly promote cell proliferation, improve cell survival after irradiation, reduce the formation of DNA damage protein ⁇ H2AX foci after irradiation, and shorten comet tail distance, significantly improving DNA damage.
- NAD + -dependent sirtuin SIRT family was significantly increased after NMN treatment, and the level of DNA damage repair protein SIRT1 was also significantly increased, providing a new basis for NMN to prevent and treat damage caused by ionizing radiation.
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Abstract
The present invention relates to the technical field of medicines, in particular, provided is the use of a nicotinamide mononucleotide (NMN) in an anti-radiation injury preparation. In vitro cell experiments show that the NMN can significantly promote the proliferation ability of normal cells and senescent cells, and improve the survival rate of irradiated cells, reduce the formation of DNA damage protein γH2AX foci after irradiation, shorten the comet tail distance, significantly improve DNA damage conditions, and enhance the ability of same to perform DNA damage repair. In addition, an NMN-treated PARP protein and NAD +-dependent deacetylase SIRT family are significantly increased, and the levels of DNA damage repair protein SIRT1, telomere-related SIRT6 and transcription-related SIRT7 protein are significantly increased, which provides a new basis for NMN to prevent and treat the damage caused by ionizing radiation.
Description
本发明属于生物医药领域，具体涉及烟酰胺单核苷酸在制备抵抗辐射伤害制剂中的应用，以及含有烟酰胺单核苷酸的制剂。The invention belongs to the field of biomedicine, and particularly relates to the application of nicotinamide mononucleotide in the preparation of anti-radiation damage preparations and preparations containing nicotinamide mononucleotide.
电离辐射在工业、医疗、军事等领域广泛应用，放射工作人员和公众受照的几率大大增加，日常生活中，使用电子产品、乘坐飞机等也会受到不同程度的辐射伤害，因此辐射伤害及防护更加受到学界及公众的关注。放疗作为目前癌症治疗中的重要手段，对肿瘤组织杀伤的同时通常会对癌旁组织以及受照的正常组织造成严重的损伤。电离辐射导致的DNA损伤是生物体、机体各种组织，细胞乃至细胞亚水平上的辐射生物效应基础，是辐射生物效应的主要原因。Ionizing radiation is widely used in industry, medical care, military and other fields. The probability of radiation workers and the public being exposed to radiation is greatly increased. In daily life, the use of electronic products and planes will also suffer from radiation damage to varying degrees. Therefore, radiation damage and protection more academic and public attention. Radiotherapy, as an important method in current cancer treatment, usually causes severe damage to adjacent tissues and irradiated normal tissues while killing tumor tissues. DNA damage caused by ionizing radiation is the basis of radiation biological effects in organisms, various tissues of the body, cells and even cell sub-levels, and is the main cause of radiation biological effects.
目前，对于辐射损伤的治疗，临床上主要采用一些支持疗法，主要为对症治疗，包括药物止吐、止痛，输血、输体液、电解质等营养补充和抗生素治疗感染。此外，已批准上市的辐射防护药物仅有含巯基化合物氨磷汀(amifostine,WR-2721)，用于肿瘤患者放疗前使用，但其副作用较大，包括低血压、恶心、呕吐、嗜睡、过敏性皮疹、发热、休克等缺点限制了其开发和使用。理想的辐射防护剂应该具备对人体安全、来源方便、便于口服、可以迅速吸收等特点，目前临床上仍然缺乏理想的辐射防护和治疗药物，因此，亟需寻找广谱性、高效性、低毒性的新型辐射防护和辐射治疗药物。At present, for the treatment of radiation injury, some supportive therapies are mainly used in clinical practice, mainly symptomatic treatment, including drug antiemetic, analgesic, blood transfusion, body fluids, electrolytes and other nutritional supplements, and antibiotics to treat infection. In addition, only the thiol-containing compound amifostine (WR-2721) is the only approved radioprotective drug for use in cancer patients before radiotherapy, but its side effects include hypotension, nausea, vomiting, drowsiness, and allergies. Disadvantages such as rash, fever, and shock limit its development and use. An ideal radioprotectant should have the characteristics of safety to human body, convenient source, easy oral administration, and rapid absorption. At present, there is still a lack of ideal radioprotective and therapeutic drugs in clinical practice. Therefore, it is urgent to find broad-spectrum, high-efficiency, and low-toxicity drugs. of new radiation protection and radiation therapy drugs.
烟酰胺单核苷酸(nicotinamide ribonucleotide，NMN)由烟酰胺(nicotinamide,Nic)和5’-磷酸基焦磷酸通过烟酰胺磷酸基转移酶(NAMPT)合成，是NAD
+的关键中间体。NMN已被证明可以增强NAD
+的生物合成，改善小鼠疾病模型的各种病理，如心肌和脑缺血、阿尔茨海默病等神经退行性疾病和糖尿病。NMN大部分药理作用是通过促进NAD
+合成进行的，因为直接给药高剂量的NAD
+会出现失眠、疲劳、焦虑等副作用，并且NAD
+对质膜的穿透能力也比NMN差。
Nicotinamide ribonucleotide (NMN) is synthesized from nicotinamide (Nic) and 5'-phosphate pyrophosphate by nicotinamide phosphotransferase (NAMPT) and is a key intermediate of NAD + . NMN has been shown to enhance NAD + biosynthesis and improve various pathologies in mouse disease models, such as myocardial and cerebral ischemia, neurodegenerative diseases such as Alzheimer's disease, and diabetes. Most of the pharmacological effects of NMN are carried out by promoting the synthesis of NAD + , because direct administration of high doses of NAD + will cause side effects such as insomnia, fatigue, anxiety, and the ability of NAD + to penetrate the plasma membrane is also worse than that of NMN.
NAD
+是体内重要的辅酶，已有多项研究表明，体外补充NAD
+前体可以延缓衰老。烟酰胺单核苷酸(NMN)作为NAD
+的前体，毒副作用小，安全性高，在抗衰老和抑癌领域 已成为研究的热点，并且NMN参与DNA损伤修复也被广泛关注。
NAD + is an important coenzyme in the body, and several studies have shown that supplementation of NAD + precursors in vitro can delay aging. Nicotinamide mononucleotide (NMN), as the precursor of NAD + , has low toxicity and high safety, and has become a research hotspot in the field of anti-aging and tumor suppression, and NMN's involvement in DNA damage repair has also been widely concerned.
Sirtuins家族是一个NAD
+依赖的家族，于哺乳动物细胞中广泛表达。目前该家族共有7个成员，分别为：SIRT1、SIRT2、SIRT3、SIRT4、SIRT5、SIRT6、SIRT7。SIRT2存在于细胞质，SIRT3、SIRT4和SIRT5位于线粒体，SIRT6、SIRT7位于细胞核。由于它们在细胞中分布位置和结构上的差异，使其产生了不同的酶活性。Sirtuins家族与衰老相关疾病有关，例如：神经退行性疾病、心血管疾病等。
The Sirtuins family is an NAD + -dependent family that is widely expressed in mammalian cells. At present, the family has 7 members, namely: SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7. SIRT2 is located in the cytoplasm, SIRT3, SIRT4 and SIRT5 are located in the mitochondria, and SIRT6 and SIRT7 are located in the nucleus. Due to their differences in location and structure in cells, they produce different enzymatic activities. The Sirtuins family is associated with aging-related diseases, such as neurodegenerative diseases, cardiovascular diseases, etc.
在Sirtuins家族中，SIRT1是与酵母菌沉默信息调节因子Sir2同源性最高的成员，该基因位于10号染色体，共编码了747个氨基酸，其蛋白质相对分子量约为120kDa，主要分布区域为细胞核。NAD
+/NADH结合区是SIRT1的活性中心，这样的关键结构决定了SIRT1在哺乳动物细胞中的重要作用。它可以脱去组蛋白H1、H3和H4的乙酰基，与此同时，也可以作用于许多非组蛋白，例如：p53、PGC-1等，还可以修饰PARP1和Ku 70等DNA修复蛋白质。已有的研究表明，SIRT1可以通过调节Wnt/-catenin通路来调节骨代谢。
In the Sirtuins family, SIRT1 is the member with the highest homology to the yeast silent information regulator Sir2. The gene is located on chromosome 10 and encodes a total of 747 amino acids. The relative molecular weight of the protein is about 120kDa, and the main distribution area is the nucleus. The NAD + /NADH binding domain is the active center of SIRT1, and such a key structure determines the important role of SIRT1 in mammalian cells. It can remove the acetyl groups of histones H1, H3 and H4. At the same time, it can also act on many non-histone proteins, such as: p53, PGC-1, etc., and can also modify DNA repair proteins such as PARP1 and Ku 70. Previous studies have shown that SIRT1 can regulate bone metabolism by regulating the Wnt/-catenin pathway.
SIRT6主要位于细胞核内19号染色体，存在2种异构体，一种含有8个外显子编码355个氨基酸长度的蛋白，另一种含有7个外显子编码的328个氨基酸长度的蛋白。SIRT6蛋白具有3种酶活性：去乙酰化酶活性、长链脱酰基酶活性和单ADP核糖基转移酶活性。SIRT6去乙酰化作用较弱，是位点特异性组蛋白去乙酰化酶，针对组蛋白H3赖氨酸K9、K56和K18(H3K9ac、H3K56ac和H3K18ac)的乙酰化位点，具有调节DNA损伤修复、糖脂代谢、压力耐受及炎症反应和衰老的功能。以往的研究表明，SIRT6充当了与衰老，癌症和新陈代谢有关的几种转录因子NF-kB，HIF1α，c-Jun和c-Myc的核心抑制剂，促进依赖DNA损伤的染色质变化，这是DNA修复必不可少的；并维持端粒染色质结构，防止基因组不稳定和细胞衰老。SIRT6 is mainly located on chromosome 19 in the nucleus, and there are two isoforms, one containing 8 exons encoding a protein of 355 amino acids in length, and the other containing 7 exons encoding a protein of 328 amino acids in length. SIRT6 protein has 3 enzymatic activities: deacetylase activity, long-chain deacylase activity and single ADP ribosyltransferase activity. SIRT6 has a weak deacetylation effect and is a site-specific histone deacetylase that targets the acetylation sites of histone H3 lysines K9, K56 and K18 (H3K9ac, H3K56ac and H3K18ac) and has the ability to regulate DNA damage repair , glucose and lipid metabolism, stress tolerance and inflammatory response and the function of aging. Previous studies have shown that SIRT6 acts as a core repressor of several transcription factors NF-kB, HIF1α, c-Jun and c-Myc involved in aging, cancer and metabolism, promoting DNA damage-dependent chromatin changes, which are Essential for repair; and maintains telomeric chromatin structure, preventing genomic instability and cellular senescence.
SIRT7位于17号染色体，有400个氨基酸序列，在哺乳动物sirtuins家族种，与SIRT6的蛋白质序列最为相似(56％同一性)，对组蛋白H3的第18、36、37位赖氨酸残基(H3K18ac、H3K36ac、H3K37ac)有特异性去乙酰化作用，参与肿瘤发生，促进rDNA(ribosomal DNA)异染色质沉默和基因组稳定性、转录延伸或DNA损伤修复。SIRT7通过抑制γH2AX和p53在DNA损伤位点的聚集和MAPK信号通路，促进细胞损伤后的存活。SIRT7可以促进ATM的去乙酰化，使其启动DNA损伤修复。并且，SIRT7可以以PARP1依赖的方式募集到损伤位点，调节损伤位点H3K18Ac水平，从而促进53BP1在DSBs的募集，促进 NHEJ修复，此外，SIRT7还可以催化损伤位点上H3K122的去琥珀酰化，促进DNA修复过程的染色质重塑。SIRT7 is located on chromosome 17 and has a sequence of 400 amino acids. It is the most similar (56% identical) to the protein sequence of SIRT6 in the mammalian sirtuins family, and has lysine residues at positions 18, 36, and 37 of histone H3. (H3K18ac, H3K36ac, H3K37ac) have specific deacetylation, participate in tumorigenesis, promote rDNA (ribosomal DNA) heterochromatin silencing and genome stability, transcription elongation or DNA damage repair. SIRT7 promotes cell survival after injury by inhibiting γH2AX and p53 aggregation at DNA damage sites and MAPK signaling. SIRT7 can promote the deacetylation of ATM to initiate DNA damage repair. Furthermore, SIRT7 can be recruited to the injury site in a PARP1-dependent manner and regulate the level of H3K18Ac at the injury site, thereby promoting the recruitment of 53BP1 to DSBs and promoting NHEJ repair. In addition, SIRT7 can also catalyze the desuccinylation of H3K122 at the injury site. , promoting chromatin remodeling during DNA repair.
目前尚未有文献报道烟酰胺单核苷酸在电离辐射损伤中的防治作用，尤其在辐射后DNA损伤修复中的作用机制。At present, there is no literature report on the preventive effect of nicotinamide mononucleotide in ionizing radiation damage, especially the mechanism of action in the repair of DNA damage after radiation.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种烟酰胺单核苷酸新的医药用途，以及含有烟酰胺单核苷酸的药物组合物。The purpose of the present invention is to provide a new medicinal use of nicotinamide mononucleotide and a pharmaceutical composition containing nicotinamide mononucleotide.
本发明的第一方面在于提供烟酰胺单核苷酸在制备抵抗辐射伤害制剂中的应用。优选的，该制剂包括药物、保健品、食品添加剂或护肤品；辐射为电离辐射。The first aspect of the present invention is to provide the application of nicotinamide mononucleotide in the preparation of anti-radiation damage preparations. Preferably, the preparation includes drugs, health products, food additives or skin care products; the radiation is ionizing radiation.
进一步，该抵抗辐射伤害制剂为降低细胞照后凋亡率或减轻照射对细胞增殖抑制的制剂。Further, the preparation for resisting radiation damage is a preparation for reducing the apoptosis rate of cells after irradiation or alleviating the inhibition of cell proliferation by irradiation.
进一步，该抵抗辐射伤害制剂为为降低照后DNA损伤程度和促进DNA损伤修复的制剂，由前所述可知，DNA损伤是辐射损伤发生的关键环节，降低DNA损伤程度有助于从根本上进行辐射损伤的防护。Further, the anti-radiation damage preparation is a preparation for reducing the degree of DNA damage after irradiation and promoting the repair of DNA damage. It can be seen from the foregoing that DNA damage is a key link in the occurrence of radiation damage, and reducing the degree of DNA damage helps to fundamentally Protection from radiation damage.
进一步，该降低照后DNA损伤程度的药物为促进PARP与SIRT表达上调的制剂。Further, the drug for reducing the degree of DNA damage after irradiation is a preparation for promoting the up-regulation of PARP and SIRT expression.
为了验证烟酰胺单核苷酸对于电离辐射损伤的防护效果，本发明从细胞和基因两个微观角度，验证了烟酰胺单核苷酸对辐射损伤的防护作用。In order to verify the protective effect of nicotinamide mononucleotide on ionizing radiation damage, the present invention verifies the protective effect of nicotinamide mononucleotide on radiation damage from two microscopic perspectives of cells and genes.
选择1mM的NMN药物浓度进行处理，照射前提前24h加药，并分别采用不同剂量的射线照射，14天后观察克隆形成情况。结果显示，NMN处理显著提高了细胞照射后的存活率，有效降低电离辐射造成的细胞凋亡率，同时提高辐射后细胞生存率；此外，NMN能够显著降低DNA损伤程度，进一步研究显示，提高SIRT1基因的表达水平。说明照前运用烟酰胺单核苷酸能够减轻电离辐射损伤，降低细胞乃至基因对放射的敏感性。The drug concentration of 1 mM NMN was selected for treatment, and the drug was added 24 hours before irradiation, and different doses of radiation were used respectively, and the colony formation was observed after 14 days. The results showed that NMN treatment significantly improved the survival rate of cells after irradiation, effectively reduced the apoptosis rate caused by ionizing radiation, and at the same time increased the cell survival rate after irradiation; in addition, NMN could significantly reduce the degree of DNA damage, and further studies showed that increasing SIRT1 gene expression level. It shows that the use of nicotinamide mononucleotide before irradiation can reduce the damage of ionizing radiation and reduce the sensitivity of cells and even genes to radiation.
本发明所述的抵抗辐射伤害制剂是烟酰胺单核苷酸作为唯一活性成分或者是包含烟酰胺单核苷酸的药物组合物。The anti-radiation injury preparation of the present invention is nicotinamide mononucleotide as the only active ingredient or a pharmaceutical composition comprising nicotinamide mononucleotide.
本发明所述的药物或药物组合物可以和药学上常用的辅料制成任何剂型，例如其可以是为汤剂、散剂、丸剂、酒剂、锭剂、胶剂、膏药、茶剂、曲剂、糕剂、露剂、棒剂、线剂、条剂、钉剂，灸熨剂，膏剂、丹剂、微型胶囊、静脉乳剂、脂质体制剂、气雾剂、前体药制剂、注射剂、合剂、口服安瓿剂、片剂、胶囊剂、滴丸剂、乳剂、软膏剂、橡胶硬 膏、膜剂、海绵剂、离子透入剂，或透皮吸收剂；保健品为口服式胶囊剂、片剂或粉剂；护肤品为乳液、喷剂、面膜或眼霜。The medicine or pharmaceutical composition of the present invention can be made into any dosage form with commonly used pharmacy auxiliary materials, for example, it can be decoction, powder, pill, wine, lozenge, glue, plaster, tea, koji , cake, lotion, stick, thread, strip, nail, moxibustion iron, ointment, elixir, microcapsule, intravenous emulsion, liposome preparation, aerosol, prodrug preparation, injection, Mixtures, oral ampoules, tablets, capsules, drop pills, emulsions, ointments, rubber plasters, films, sponges, iontophoresis, or transdermal absorption agents; health products are oral capsules, tablets or powder; skin care products are lotions, sprays, masks or eye creams.
本发明的第二方面在于提供一种抵抗辐射伤害制剂，由烟酰胺单核苷酸或其衍生物以及药学、保健品学、食品学或化妆品学上可接受的辅料组成。The second aspect of the present invention is to provide an anti-radiation injury preparation, which is composed of nicotinamide mononucleotide or its derivatives and pharmaceutically, health-care, food or cosmetic acceptable excipients.
本发明的烟酰胺单核苷酸用于预防辐射损伤，特别适用于放射工作人员、癌症放疗治疗患者等伴有的放射性损伤，提前给药于可能遭遇放射性肺损伤的人群，给药方式不限于口服、注射等。The nicotinamide mononucleotide of the present invention is used to prevent radiation damage, and is particularly suitable for radiation damage associated with radiation workers, cancer radiotherapy patients, etc., and is administered in advance to people who may suffer from radiation lung damage, and the administration method is not limited to Oral, injection, etc.
发明的作用与效果The role and effect of the invention
本发明从细胞和基因两个微观角度，验证了烟酰胺单核苷酸对辐射损伤的防护作用：细胞实验结果显示，NMN处理能够显著提高细胞照射后的存活率，有效降低电离辐射造成的细胞凋亡率，同时提高辐射后细胞生存率；此外，NMN能够显著降低DNA损伤程度，同时提高SIRT1基因的表达水平。说明照前运用烟酰胺单核苷酸能够减轻电离辐射损伤，降低细胞乃至基因对放射的敏感性。因此，本发明为烟酰胺单核苷酸防护辐射损伤提供了新的依据。The present invention verifies the protective effect of nicotinamide mononucleotide on radiation damage from two microscopic perspectives of cells and genes: cell experiment results show that NMN treatment can significantly improve the survival rate of cells after irradiation, and effectively reduce cell damage caused by ionizing radiation. Apoptosis rate, while improving cell survival rate after irradiation; in addition, NMN can significantly reduce the degree of DNA damage, while increasing the expression level of SIRT1 gene. It shows that the use of nicotinamide mononucleotide before irradiation can reduce the damage of ionizing radiation and reduce the sensitivity of cells and even genes to radiation. Therefore, the present invention provides a new basis for nicotinamide mononucleotide to protect against radiation damage.
此外，由于烟酰胺单核苷酸是成熟的市售产品，其药理作用明确、毒副作用小，药物安全性已得到临床认可，本发明提供的烟酰胺单核苷酸的新适应症可较快实现临床转化。因此，烟酰胺单核苷酸在临床辐射损伤的防护应用上具有巨大潜力。In addition, because nicotinamide mononucleotide is a mature commercial product, its pharmacological action is clear, the toxic and side effects are small, and the drug safety has been clinically recognized, and the new indication of nicotinamide mononucleotide provided by the present invention can be faster achieve clinical translation. Therefore, nicotinamide mononucleotide has great potential in the protection of clinical radiation injury.
图1是烟酰胺单核苷酸对照射后细胞克隆的影响结果图。Figure 1 is a graph showing the effect of nicotinamide mononucleotide on cell clones after irradiation.
图2是烟酰胺单核苷酸对辐射造成的细胞凋亡的影响结果。Figure 2 is the result of the effect of nicotinamide mononucleotide on apoptosis induced by radiation.
图3为烟酰胺单核苷酸对辐射后细胞生存率的影响结果。其中，A为对辐射后MODE-K细胞生存率的影响结果；B为对辐射后293T细胞生存率的影响结果。Figure 3 shows the effect of nicotinamide mononucleotide on cell viability after irradiation. Among them, A is the effect on the survival rate of MODE-K cells after irradiation; B is the effect on the survival rate of 293T cells after irradiation.
图4为烟酰胺单核苷酸对辐射后A549-NRF2KO细胞的DNA损伤影响结果。其中，A为“彗星”实验凝胶电泳结果；B为“彗星”实验尾矩对比结果；C为DNA损伤早期应答蛋白γH2AX形成情况的细胞免疫荧光实验结果；D为不同组DNA损伤早期应答蛋白γH2AX foci数量对比。Figure 4 shows the effect of nicotinamide mononucleotide on DNA damage in A549-NRF2KO cells after irradiation. Among them, A is the gel electrophoresis result of the "comet" experiment; B is the comparison result of the tail moment of the "comet" experiment; C is the cell immunofluorescence experiment result of the formation of the DNA damage early response protein γH2AX; D is the DNA damage early response protein of different groups Comparison of γH2AX foci numbers.
图5为烟酰胺单核苷酸对辐射后人皮肤成纤维细胞HFF-1的DNA损伤影响结果。其中，A为“彗星”实验凝胶电泳结果；B为“彗星”实验尾矩对比结果；C为DNA损伤早 期应答蛋白γH2AX形成情况的细胞免疫荧光实验结果；D为不同组DNA损伤早期应答蛋白γH2AX foci数量对比Figure 5 shows the effect of nicotinamide mononucleotide on DNA damage of human skin fibroblasts HFF-1 after irradiation. Among them, A is the gel electrophoresis result of the "comet" experiment; B is the comparison result of the tail moment of the "comet" experiment; C is the cell immunofluorescence experiment result of the formation of the DNA damage early response protein γH2AX; D is the DNA damage early response protein of different groups γH2AX foci quantity comparison
图6为烟酰胺单核苷酸对正常人皮肤成纤维细胞HFF-1的DNA损伤的影响结果。其中，A为NMN联合不同DNN损伤诱导剂的“彗星”实验凝胶电泳结果；B为“彗星”实验尾矩对比结果；C为DNA损伤早期应答蛋白γH2AX形成情况的细胞免疫荧光实验结果；D为不同组DNA损伤早期应答蛋白γH2AX foci数量对比。Figure 6 shows the effect of nicotinamide mononucleotide on DNA damage in normal human skin fibroblasts HFF-1. Among them, A is the gel electrophoresis result of the "comet" experiment of NMN combined with different DNN damage inducers; B is the comparison result of the tail moment of the "comet" experiment; C is the result of the cellular immunofluorescence experiment of the formation of the DNA damage early response protein γH2AX; D Comparison of the number of DNA damage early response protein γH2AX foci in different groups.
图7为烟酰胺单核苷酸对辐射后HFF-1细胞生存率以及细胞周期的影响，A为对辐射后HFF-1细胞活力的影响结果；B～D为不同辐照时间点，对HFF-1细胞周期的影响结果。Figure 7 shows the effect of nicotinamide mononucleotide on the viability and cell cycle of HFF-1 cells after irradiation. A is the effect of HFF-1 cell viability after irradiation; -1 cell cycle effect results.
图8为烟酰胺单核苷酸促进辐射后细胞内SIRT1\SIRT6\SIRT7以及PARP的蛋白表达水平结果。Figure 8 shows the results of protein expression levels of SIRT1\SIRT6\SIRT7 and PARP in cells after nicotinamide mononucleotide-promoted irradiation.
下面结合实施例和附图对本发明进行详细描述。但下列实施例不应看作对本发明范围的限制。实施例不包括对传统方法的详细描述，如细胞克隆检测方法、细胞凋亡测试方法、免疫荧光染色方法等。这样的方法对于本领域中具有普通技术的人员是众所周知的或者根据供应商提供的试剂盒步骤进行即可。The present invention will be described in detail below with reference to the embodiments and accompanying drawings. However, the following examples should not be construed as limiting the scope of the present invention. The examples do not include detailed descriptions of traditional methods, such as cell clone detection methods, apoptosis detection methods, immunofluorescence staining methods, and the like. Such methods are well known to those of ordinary skill in the art or can be performed according to the kit procedures provided by the suppliers.
1、烟酰胺单核苷酸能够提高照射后细胞的存活率1. Nicotinamide mononucleotide can improve the survival rate of cells after irradiation
文献证明，NMN可以改善小鼠的衰老情况，而NRF2不仅在DNA损伤中发挥重要作用，并且NRF2敲除的小鼠呈现出早衰的特征，因此我们选择A549-NRF2KO细胞系进行实验。将细胞分成对照组和NMN处理组两个大组，两组细胞的培养条件相同。照射前24h，NMN处理组选择1mM的NMN药物浓度对细胞进行处理，对照组不进行处理。而后分别采用0、0.5、1、2、4、6Gy对两组细胞进行照射，照射后14天观察克隆形成情况。Literature has proved that NMN can improve the aging of mice, and NRF2 not only plays an important role in DNA damage, and NRF2 knockout mice show the characteristics of premature aging, so we chose the A549-NRF2KO cell line for experiments. The cells were divided into two groups, the control group and the NMN-treated group, and the two groups of cells were cultured under the same conditions. 24h before irradiation, the NMN treatment group selected 1mM NMN drug concentration to treat the cells, and the control group was not treated. Then, the two groups of cells were irradiated with 0, 0.5, 1, 2, 4, and 6 Gy respectively, and the colony formation was observed 14 days after irradiation.
结果如图1所示，NMN处理显著提高了细胞照射后的存活率，尤其是在4、6Gy处理的条件下。因此，以下未标明照射条件的实验，我们均采用照射前24h在培养基中加入1mM的NMN，和4Gy照射的实验条件。The results are shown in Figure 1, NMN treatment significantly improved the survival rate of cells after irradiation, especially under the conditions of 4 and 6 Gy treatment. Therefore, in the following experiments that do not indicate irradiation conditions, we all used the experimental conditions of adding 1 mM NMN to the medium 24 hours before irradiation, and irradiating with 4 Gy.
2、NMN处理对电离辐射造成的细胞凋亡具有恢复作用2. NMN treatment has a recovery effect on apoptosis caused by ionizing radiation
电离辐射处理和NRF2基因敲除都会引起细胞凋亡增加，因此对细胞凋亡情况进行了检测。将细胞分为对照组、NMN组、辐照(IR)组以及NMN+IR组，对照组既不添加NMN 也不进行辐照；NMN组添加NMN后不进行辐照；辐照(IR)组进行辐照；NMN+IR组先添加NMN后再进行辐照。Both ionizing radiation treatment and NRF2 knockout resulted in increased apoptosis, so apoptosis was examined. The cells were divided into control group, NMN group, irradiation (IR) group and NMN+IR group. The control group neither added NMN nor irradiated; the NMN group added NMN without irradiation; the irradiation (IR) group Irradiation; NMN+IR group added NMN first and then irradiated.
对辐照(IR)组以及NMN+IR组细胞进行8Gy照射，在照射后24h、48h、72h进行收样，结果如图2所示，照射24h后，NMN对细胞凋亡具有一定的恢复作用，但随着照射时间的延长，该种恢复作用越来越弱。The cells in the irradiation (IR) group and the NMN+IR group were irradiated with 8 Gy, and the samples were collected at 24h, 48h, and 72h after irradiation. The results are shown in Figure 2. After 24h of irradiation, NMN has a certain recovery effect on cell apoptosis. , but with the prolongation of irradiation time, the recovery effect became weaker and weaker.
3、NMN有效影响辐射后细胞生存3. NMN effectively affects cell survival after irradiation
选择MODE-K、293T细胞进行实验。在照射前24h分别加入0、0.5和1mM NMN，而后针对不同处理条件进行0、0.5、1、2、4Gy照射处理，培养7天染色。MODE-K and 293T cells were selected for experiments. 0, 0.5 and 1 mM NMN were added 24 h before irradiation, and then 0, 0.5, 1, 2, and 4 Gy were irradiated for different treatment conditions, and cultured for 7 days for staining.
结果如图3所示，在同一照射条件下，细胞生存率随着NMN浓度的增加而逐渐提高；在同一NMN处理条件下，细胞生存率随着辐照强度的增加而降低。The results are shown in Figure 3. Under the same irradiation condition, the cell viability gradually increased with the increase of NMN concentration; under the same NMN treatment condition, the cell viability decreased with the increase of irradiation intensity.
4、NMN削弱辐射后A549-NRF2KO细胞的DNA损伤4. NMN attenuates DNA damage in A549-NRF2KO cells after irradiation
彗星实验可以有效地检测并定量分析细胞中DNA单链或双链断裂缺口损伤程度。对不同条件处理后的A549-NRF2KO细胞进行了彗星实验分析，结果如图4所示：发现NMN+IR组组的“彗星”尾矩要明显短于单一照射(IR)组(图A、B)，说明NMN可以减少照射后细胞的DNA损伤。The comet assay can effectively detect and quantify the degree of DNA single-strand or double-strand break nick damage in cells. The comet experiments were performed on A549-NRF2KO cells treated with different conditions, and the results are shown in Figure 4: It was found that the "comet" tail moment of the NMN+IR group was significantly shorter than that of the single irradiation (IR) group (Figures A and B). ), indicating that NMN can reduce DNA damage in cells after irradiation.
为了进一步验证NMN的作用效果，检测了照射后6h、12h、24h的DNA损伤早期应答蛋白γH2AX的形成情况。细胞免疫荧光的实验结果(图C)以及的γH2AX foci数量统计结果(图D)表明，在照射后6、12、24h，加药组的γH2AX foci数量均低于单一照射组，表明NMN能够降低辐射后细胞的DNA损伤程度。In order to further verify the effect of NMN, the formation of DNA damage early response protein γH2AX was detected at 6h, 12h and 24h after irradiation. The experimental results of cell immunofluorescence (Figure C) and the statistical results of the number of γH2AX foci (Figure D) showed that the number of γH2AX foci in the dosing group was lower than that in the single irradiation group at 6, 12, and 24 h after irradiation, indicating that NMN can reduce the amount of γH2AX foci. Degree of DNA damage in cells after irradiation.
5、NMN削弱辐射后皮肤成纤维细胞的DNA损伤5. NMN attenuates DNA damage in skin fibroblasts after irradiation
在人皮肤成纤维细胞HFF-1中同样发现NMN预先处理组的“彗星”尾矩要明显短于单一照射组(图5A、B)，说明NMN可以减少照射后细胞的DNA损伤。In human skin fibroblasts HFF-1, it was also found that the "comet" tail moment of the NMN pretreatment group was significantly shorter than that of the single irradiation group (Fig. 5A, B), indicating that NMN could reduce the DNA damage of cells after irradiation.
为了进一步验证NMN的作用效果，检测了照射后6h、12h、24h的DNA损伤早期应答蛋白γH2AX的形成情况。细胞免疫荧光的实验结果(图5C)以及的γH2AX foci数量统计结果(图5D)表明，在照射后6、12、24h，加药组的γH2AX foci数量均低于单一照射组，表明NMN能够降低辐射后细胞的DNA损伤程度。In order to further verify the effect of NMN, the formation of DNA damage early response protein γH2AX was detected at 6h, 12h and 24h after irradiation. The experimental results of cell immunofluorescence (Figure 5C) and the statistical results of the number of γH2AX foci (Figure 5D) showed that the number of γH2AX foci in the dosing group was lower than that in the single irradiation group at 6, 12, and 24 h after irradiation, indicating that NMN can reduce the amount of γH2AX foci. Degree of DNA damage in cells after irradiation.
6、NMN对其他形式的DNA损伤的修复作用6. The repair effect of NMN on other forms of DNA damage
选择人皮肤成纤维细胞HFF-1进行研究，分别选择了CPT(拓扑异构酶抑制剂，诱导DNA复制叉塌陷导致的dsDNA损伤)、HU(诱导复制叉停滞导致ssDNA损伤)、MMS(烷基化试剂，诱导DNA复制叉塌陷导致的dsDNA损伤)、MMC(DNA交联剂，诱导特定位点的DNA损伤)四种诱导不同形式DNA损伤的药物进行处理，进一步探究NMN对不同形式DNA损伤的影响。Human skin fibroblasts HFF-1 were selected for research, and CPT (topoisomerase inhibitor, induced DNA replication fork collapse caused dsDNA damage), HU (induced replication fork arrest caused ssDNA damage), MMS (alkylated four kinds of drugs that induce different forms of DNA damage, and further explore the effect of NMN on different forms of DNA damage. influences.
同样，采用1mM的NMN提前24h进行处理，然后分别加入2mM HU处理2h，200μM MMS处理8h，10nM CPT处理16h，300ng/mlMMC处理12h，并进行“彗星”实验，结果如图6所示。Similarly, 1 mM NMN was used for 24 h treatment in advance, and then 2 mM HU was added for 2 h, 200 μM MMS for 8 h, 10 nM CPT for 16 h, and 300 ng/ml MMC for 12 h, and the "comet" experiment was performed. The results are shown in Figure 6.
通过对彗星尾距分析，发现实验浓度的CPT和HU对DNA损伤的影响较弱，但在NMN+HU处理时，尾距明显短于HU单独处理组，在其他三种药物处理组未发现NMN的保护作用(图A、B)。接下来对CPT处理对γH2AX的影响进行探究，细胞免疫荧光的实验结果表明在CPT处理16h后，NMN+CPT组的γH2AX foci数量低于单加药组(图C、D)，说明NMN对其他形式的DNA损伤也具有一定的修复作用。By analyzing the comet tail distance, it was found that the experimental concentrations of CPT and HU had a weak effect on DNA damage, but in the NMN+HU treatment group, the tail distance was significantly shorter than that in the HU alone treatment group, and NMN was not found in the other three drug treatment groups. protective effect (Figures A and B). Next, the effect of CPT treatment on γH2AX was explored. The experimental results of cellular immunofluorescence showed that the number of γH2AX foci in the NMN+CPT group was lower than that in the single-dosing group after 16 h of CPT treatment (Figures C and D), indicating that NMN has no effect on other Forms of DNA damage also have a certain repair effect.
7、细胞存活实验与细胞周期7. Cell survival experiments and cell cycle
CCK8筛选药物浓度(重复三次)。对照和照射后的HFF-1细胞中，加入1mM的NMN，培养24h、48h、72h收样，加入10μL CCK8试剂，加入2h后，测吸光度。结果如图7所示，NMN显著提高照射后细胞的活性(A)。CCK8 screening for drug concentration (repeated three times). In the control and irradiated HFF-1 cells, 1 mM NMN was added, cultured for 24 h, 48 h, and 72 h, samples were collected, 10 μL of CCK8 reagent was added, and the absorbance was measured after adding 2 h. As a result, as shown in Fig. 7, NMN significantly increased cell viability after irradiation (A).
通过考察细胞周期，相对单一辐照组，照射12h时，NMN能够显著延长G1期，但在照射6h以及24h时，对于细胞周期的改善不明显(B、C、D)。By examining the cell cycle, compared with the single irradiation group, NMN could significantly prolong the G1 phase when irradiated for 12h, but the improvement of the cell cycle was not obvious when irradiated for 6h and 24h (B, C, D).
8、NMN处理促进SIRT1\SIRT6\SIRT7以及PARP表达上调8. NMN treatment promotes the up-regulation of SIRT1\SIRT6\SIRT7 and PARP expression
为了进一步研究NMN发挥保护作用的机制，验证了NMN对辐射后细胞内DNA损伤修复蛋白SIRT1的表达情况影响。In order to further study the mechanism of NMN's protective effect, the effect of NMN on the expression of DNA damage repair protein SIRT1 in cells after irradiation was verified.
选择NRF2KO细胞系，在照射后1h、6h、12h、24h分别收样，进行WB实验，结果如图8A～C所示。根据实验结果，我们发现NMN处理后SIRT1、SIRT6、SIRT7以及PARP的蛋白水平上调。随着照射时间的延长，相对于单一照射组，NMN对于几种蛋白水平的上调促进程度更高，可能是NMN发挥保护作用的有效通路。The NRF2KO cell line was selected, and samples were collected 1h, 6h, 12h, and 24h after irradiation, and WB experiments were performed. The results are shown in Figures 8A-C. According to the experimental results, we found that the protein levels of SIRT1, SIRT6, SIRT7 and PARP were up-regulated after NMN treatment. With the prolongation of irradiation time, compared with the single irradiation group, NMN promoted the up-regulation of several protein levels to a higher degree, which may be an effective pathway for NMN to play a protective role.
通过上述体外细胞实验可知，NMN可以显著促进细胞的增殖能力，并且提高照射后的细胞存活率，减少照射后DNA损伤蛋白γH2AX foci的形成情况，以及缩短彗星尾距，显著改善了DNA损伤情况，并且NMN处理后NAD
+依赖的去乙酰化酶SIRT家族都显著升高，DNA损伤修复蛋白SIRT1的水平也明显上调，为NMN预防和治疗电离辐射带来的损伤提供了新的依据。
It can be seen from the above in vitro cell experiments that NMN can significantly promote cell proliferation, improve cell survival after irradiation, reduce the formation of DNA damage protein γH2AX foci after irradiation, and shorten comet tail distance, significantly improving DNA damage. In addition, the NAD + -dependent sirtuin SIRT family was significantly increased after NMN treatment, and the level of DNA damage repair protein SIRT1 was also significantly increased, providing a new basis for NMN to prevent and treat damage caused by ionizing radiation.
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. The above-mentioned embodiments and descriptions only illustrate the principle of the present invention. Such changes and improvements fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.
Claims (8)
- 烟酰胺单核苷酸在制备抵抗辐射伤害制剂中的应用。The application of nicotinamide mononucleotide in the preparation of anti-radiation damage preparations.
- 根据权利要求1所述的烟酰胺单核苷酸在制备抵抗辐射伤害制剂中的应用，其特征在于：The application of nicotinamide mononucleotide according to claim 1 in the preparation of preparations for resisting radiation damage, characterized in that:其中，所述制剂包括药物、保健品、食品添加剂或护肤品，Wherein, the preparation includes drugs, health products, food additives or skin care products,所述辐射为电离辐射。The radiation is ionizing radiation.
- 根据权利要求1所述的烟酰胺单核苷酸在制备抵抗辐射伤害制剂中的应用，其特征在于：The application of nicotinamide mononucleotide according to claim 1 in the preparation of preparations for resisting radiation damage, characterized in that:其中，所述抵抗辐射伤害制剂为降低细胞照后凋亡率或减轻照射对细胞增殖抑制的制剂。Wherein, the anti-radiation damage preparation is a preparation that reduces the apoptosis rate of cells after irradiation or reduces the inhibition of cell proliferation by irradiation.
- 根据权利要求1所述的烟酰胺单核苷酸在制备抵抗辐射伤害制剂中的应用，其特征在于：The application of nicotinamide mononucleotide according to claim 1 in the preparation of preparations for resisting radiation damage, characterized in that:其中，所述抵抗辐射伤害制剂为降低照后DNA损伤程度和促进DNA损伤修复的制剂。Wherein, the anti-radiation damage preparation is a preparation that reduces the degree of DNA damage after irradiation and promotes DNA damage repair.
- 根据权利要求4所述的烟酰胺单核苷酸在制备抵抗辐射伤害制剂中的应用，其特征在于：The application of nicotinamide mononucleotide according to claim 4 in the preparation of anti-radiation damage preparation, it is characterized in that:其中，所述抵抗辐射伤害制剂为促进PARP与SIRT表达上调的制剂。Wherein, the anti-radiation damage preparation is a preparation that promotes the up-regulation of the expressions of PARP and SIRT.
- 根据权利要求1～5任一项所述的烟酰胺单核苷酸在制备抵抗辐射伤害制剂中的应用，其特征在于：The application of the nicotinamide mononucleotide according to any one of claims 1 to 5 in the preparation of an anti-radiation damage preparation, characterized in that:其中，所述抵抗辐射伤害制剂是烟酰胺单核苷酸作为唯一活性成分或者是包含烟酰胺单核苷酸的制剂。Wherein, the anti-radiation damage preparation is nicotinamide mononucleotide as the only active ingredient or a preparation comprising nicotinamide mononucleotide.
- 根据权利要求2所述的烟酰胺单核苷酸在制备抵抗辐射伤害制剂中的应用，其特征在于：The application of nicotinamide mononucleotide according to claim 2 in the preparation of anti-radiation damage preparation, it is characterized in that:其中，所述药物为汤剂、散剂、丸剂、酒剂、锭剂、胶剂、膏药、茶剂、曲剂、糕剂、露剂、棒剂、线剂、条剂、钉剂，灸熨剂，膏剂、丹剂、微型胶囊、静脉乳剂、脂质体制剂、气雾剂、前体药制剂、注射剂、合剂、口服安瓿剂、片剂、胶囊剂、滴丸剂、乳剂、软膏剂、橡胶硬膏、膜剂、海绵剂、离子透入剂或透皮吸收剂；Wherein, the medicine is decoction, powder, pill, wine, lozenge, glue, plaster, tea, koji, cake, dew, stick, thread, strip, nail, moxibustion iron preparations, ointments, pills, microcapsules, intravenous emulsions, liposome preparations, aerosols, prodrug preparations, injections, mixtures, oral ampoules, tablets, capsules, drop pills, emulsions, ointments, rubber plasters, films, sponges, iontophores or transdermal absorbers;所述保健品为口服式胶囊剂、片剂或粉剂；Described health product is oral capsule, tablet or powder;所述护肤品为乳液、喷剂、面膜或眼霜。The skin care products are lotions, sprays, facial masks or eye creams.
- 一种抵抗辐射伤害的制剂，其特征在于：A preparation for resisting radiation damage, characterized in that:由烟酰胺单核苷酸或其衍生物以及药学、保健品学、食品学或化妆品学上可接受的辅料组成。It is composed of nicotinamide mononucleotide or its derivatives and acceptable excipients in pharmacy, health product, food or cosmetic.
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