| With the development of nuclear energy in the military and civilian field, although nuclear energy can improve human being s live, it will bring about many challenges and endanger people healthy. China is a nuclear power. There are nuclear bombs, nuclear-powered ships and other strategic nuclear weapons in the military. Moreover, nuclear power in the civilian is rapidly developing in our country. 1991, Qinshan nuclear power station have been established and is working. So for, 11 power plants, involved in QinShan nuclear power station in ZheJiang Province,Daya Bay nuclear power station in Guangdong Province and Tian Bay nuclear power station in Jiangsu Province have put into commercial operation. The total installed capacity is 9.12 million Kilowatts. In future, the total installed capacity will rise to 40 million and 150 million kilowatts in 2020 and 2035 respectively. This indicated that radiation worker and nuclear power station will influence more people healthy. Therefore, strengthen the study in treating radiation damage and invented an effective and safe drug as a strategic technology to cure radiation damage is very urgent, very important.Recently, foreign scholars found some incredible discoveries, when they researched Toll-like receptors (TLRs) and TLR s agonist. 2008, Burdely LG et al in Science reported that TLR s agonist (bacterial flagellin) have a strong reducing effect of radiation damage, and the article pointed out that TLR5 s agonist administered 2h after radiation can reduced radiation damage. The mechanism may be activation of NF-κB. This means that Toll-like receptor family members (TLRs) and their agonist will in the treatment of radiation damage has a wide range of research prospects. TLRs are evolutionary conservative family members of Pattern Recognition Receptor (PRR), which are able to recognize pathogen associated molecular patterns (PAMPs) of microorganisms. 1988, Hashimoto et al first reported that they found Toll protein in Drosophila. Subsequence, Jules A Hoffmann found that Toll protein mediated innate immunity in Drosophila, especially, inhibited fungal infections, recognized the invading pathogens and secreted a variety of anti-microbial peptides to clear the pathogen infection. 1997, Medzhitov et al first isolated Drosophila Toll protein homologues which known as the human Toll protein by homology analysis of the human body, which induced many scholars interesting. Today, immunologists have been found 11 different TLRs named TLR111, all TLRs are typeâ… transmembrane protein, which have common structure with extracellular leucine-rich repeats and a structurally conserved region in the membrane. This structure is the intracellular signal transduction starting position. PAMPs recognized by TLRs are highly conserved molecular structure, which presented in pathogens. An interaction between TLRs and PAMPs initiate the immune response and activate the natural immune system. Different TLRs recognize different PAMPs, such as TLR1,2,4 and 6 recognize lipid part of the pathogen;TLR5 recognizes pathogen proteins;TLR3,7,8 and 9 recognize nucleic acid molecules of pathogens. In view of the common similarities in the structure of family members TLRs, signal transduction pathways and biological activity, we believe that the other family members of the TLRs also can reduce radiation damage.TLR9 is an important member of the family of TLRs; PAMP agonist is CpG-ODN. CpG-ODN enter into cells by endocytosis, interact with TLR9 in the cytoplasm and active the immune response through MyD88 dependent signaling pathway.MyD88 and TRAF6 induce activation of NF-κB kinase and IκB kinase, which lead to IκB phosphorylation. At last, NF-κB is released into the nucleus to activate target genes and initiate gene transcription of cytokines. Meanwhile, CpG-ODN can induce some cytokines high level of transcription and expression involved in G-CSF,TNF-α,IL-6 through other signaling pathways. Like other TLRs agonists, CpG-ODN play an important role in immune regulation and anti-pathogens, in addition, CpG-ODN also have very important effect in treating cancer, such as enhancement tumor cell sensitivity to radiotherapy and chemotherapy. However, the effect of treating radiation damage still is less reported, it is worth of further study. At present, some articles have indicated that CpG-ODN has a range of biological activity, we speculated that there activity could reduce radiation damage, it is based thatâ‘ TLR9 and TLR5 have high homology (chart.1).TLR9 and TLR5 have similar signal transduction pathway to activate NF-κB;â‘¡TLR9 agonist CpG-ODN can stimulate immune cells to secrete G-CSF, TNF-α, IL-6 etc, which are able to reduce radiation damage. Meantime, TLR9 agonist CpG-ODN has lower toxicity and easier obtaining compared with TLR5 agonist flagellin. Some reports have showed that CpG-ODN has been used as immunomodulator in clinic. In future, if we demonstrated that CpG-ODN can reduce radiation damage, beyond all question, this brings a new direction of radiation damage treating. The study found that CpG-ODN given after radiation significantly reduce radiation damage, including bone marrow hematopoietic system and small intestine. There results indicate that CpG-ODN would have a good prospect.Contents:1. Synthesis of CpG-ODN2395, and determination of the acute toxicity on cells and animals;2. To determine whether CpG-ODN2395 can reduce the radiation damage of bone marrow hematopoietic system;3. To determine whether CpG-ODN2395 can reduce radiation damage of small intestinal tissue.4 To study the mechanism of CpG-ODN2395 reducing the radiation damge.Method:1. Synthesis and administration of CpG-ODN, determination the acute toxicity:(1) Synthesis and administration of CpG-ODN: CpG-ODN sequences were synthesized by Sangon Biotech (Shanghai) Co.Ltd.(2) Cytotoxicity test: HIEC cells were treated with different doses of CpG-ODN (0.02,0.04,0.08,0.15,0.30,0.60,1.25,2.50,5.00μM), MTT assay detect cell survival rate after 48h;(3) Acute toxicity test: 2022g BALB/c mice were administered different doses of CpG-ODN, and continuous were observated for 14d;2. Radiation and administration Mice were divided randomly into several groups. Mice were placed in sepecially designed, well-ventilated acrylic container and subjected toγ-radiation from 60Co source. Mice were administered 50μg CpG-ODN within 30 min after radiation via intraperitoneal injection (i.p) and continued administered at 24h and 48h after radiation;3. To determine that CpG-ODN can reduce the radiation damage of bone marrow hematopoietic system:(1) Survival, LD50/30 and DRF analysis: Mice were exposed to different doses of radiation (6.0,6.5,8 and 10Gy). We determined survival rates for 30d and calculate DRF by dividing the LD50/30 of the radiated and administrated CpG-ODN group and by LD50/30of the radiation alone group;(2) Peripheral blood white blood cell (WBC) and bone marrow cell count: white blood cell and bone marrow cell of mice were collected at 1d (initial phase), 9d (critical phase) and 28d (recovery phase) after 6.0Gy radiation, the number of cells were was determined by using of the cell counter analyser system;(3) Bone marrow histological: Femurs of mice were gained at 1d, 9d and 28d after 6Gy radiation. Femurs were treated by fixation, decalcification and ultimately made into H&E preparation;(4) Exogenous (CFU-S) colony forming unit-spleen assay: CFU-S formation was assayed by the method of Till and McCulloch (1961);4. To determine that CpG-ODN can reduce radiation damage of small intestinal tissue:(1) Intestinal tissue H&E histological: Intestinal tissues of mice were dissected at 5d after 6Gy radiation. Intestinal tissues were treated by fixation and ultimately made into H&E preparation;(2) Intestinal tissue TUNEL staining of apoptotic cell: Intestinal tissues of mice were dissected at 5d after 6Gy radiation. Intestinal tissues were treated by fixation and ultimately made into TUNEL preparation;(3) The viability of crypts in the small intestine by BrdU labeling: S phase cell were labeled in vivo by administering BrdU (2.5mg/mice) 2h before euthanasia. Mice were euthanized 3d after 6Gy radiation; intestinal tissues were dissected, fix and ultimately made into BrdU preparation;(4) Micronuclei cell rate of the human intestinal crypt epithelia cell (HIEC): HIEC cells were exposed to cytochalasin B (CB) for 36h after different doses of radiation, cells were fixed and strained by DAPI. Micronuclei in binucleated cells were scored; 5. To study the mechanism of CpG-ODN2395 reducing the radiation damge.(1)Activation of NF-κB: Western blot assay detected the change IκB-αprotein and p65 protein at 10 and 30min after 8Gy radiation. Quantitative of protein were analyzed by Gene Tools software.(2) The indicators of Oxidative stress in plasma: Plasma of mice was collected at 2d, 4d and 7d after radiation to determine MDA, GSH and SOD;(3) Intestinal tissue SOD expression: Intestinal tissues of mice were dissected at 5h after 6Gy radiation. Intestinal tissues were treated by fixation and ultimately made into SOD preparation;(4) The level of cytokines in plasma: Plasma of mice was collected at 0.5, 1, 2, 4, 8, 24, 48h after 6Gy radiation to determine G-CSF, TNF-αand IL-6 in plasma by ELASA assay.6. The experimental data was analyzed by SPSS13.0 software.Results1. Synthesis of CpG-ODN, determination the acute toxicity:(1) Synthesis of CpG-ODN: CpG-ODN sequence used in this study was CpG-ODN 2395. The sequences were 5'-TCG TCG TTT TCG GCG CGC CG-3' (regular letters represent PS linkage and italic letters represent CpG-ODN dinucleotides);(2) Cytotoxicity test: Survival rate of HIEC was measured at 48h after administration of various doses of CpG-ODN. We found CpG-ODN did not have significant adverse effects on the cells at concentrations less than 2.50μM by MTT assay. 0.1μM CpG-ODN was used in this experiment; therefore, CpG-ODN was safe and did not have any toxicity in cell;(3) Acute toxicity test: The administration of different doses (450, 900, 1350μg per mice) of CpG-ODN did not induce adverse effects on the body weights and general health of the animals or mortality during 14 days observation period. In the following experiment, it was not possible to administer higher dose. Therefore, it was concluded that CpG-ODN was safe and did not any adverse effects.2. To determine that CpG-ODN can reduce the radiation damage of bone marrow hematopoietic system:(1) Survival, LD50/30 and DRF analysis: Mice were exposed to 6.0, 6.5 8.0 and 10.0Gyradiation, the radiation alone group displayed survival rates of 95%, 58%, 35% and 0% respectively; the radiation and administration CpG-ODN group yielded rates of 100%, 89%, 81% and 25% at 6.0, 6.5, 8.0 and 10.0Gy, respectively;(2) WBC and bone marrow cell count: The number of white blood cell and bone marrow cell were detected at 1d (initial phase), 9d (critical phase) and 28d (recovery phase) after 6.0Gy radiation, The data showed that administration of CpG-ODN significantly increased the number of WBC and bone marrow cell on 1d, 9d and 28d after radiation, especially, at 9d and 28d;(3) Bone marrow histological: The bone marrow in the radiation and administration CpG-ODN group is less damage than in the radiation alone group, meantime, bone marrow histological results consistent with bone marrow cell count;(4) Exogenous (CFU-S) colony forming unit-spleen count: CFU-S exhibited many characteristics of primitive hematopoietic stem cells. The result showed that the number of CFU-S per femur was 2.1 fold greater the radiation and administration CpG-ODN group than the radiation alone group;3. To determine that CpG-ODN can reduce radiation damage of small intestinal tissue:(1) Intestinal tissue H&E histological: H&E histological of intestinal tissue exposed to 6Gy radiation after 5d was observed. The results showed that CpG-ODN treatment preserve better the morphology of the small intestinal in the radiation and administration CpG-ODN group than in the radiation alone group.(2) Intestinal tissue TUNEL staining of apoptotic cell: TUNEL preparation of intestinal tissue exposed to 6Gy radiation after 5h was observed and quantitated. The results showed that apoptotic cell rate in the radiation and administration CpG-ODN group was lower than in the radiation alone group.(3) The viability of crypts in the small intestine by BrdU labeling: BrdU preparation of intestinal tissue exposed to 6Gy radiation after 3d was observed and quantitated. The results showed that the viability of crypts rate in the radiation and administration CpG-ODN group was higher than in the radiation alone group.(4) Micronuclei cell rate of HIEC: MNCF was scored after different doses of radiation. The result showed that CpG-ODN treatment can significantly decrease MNCF.4. To study the mechanism of CpG-ODN2395 reducing the radiation damge.(1) Activation of NF-κB: CpG-ODN exhibited degradation of IκB-αand upgradation of p65 at 10 and 30min after radiation, especially at 30min.(2) The indicators of oxidative stress in plasma: The indicators of oxidative stress, including MDA, GSH and SOD, were detected at 2d, 4d and 7d after 6Gy radiation. The results showed that the level of oxidative stress in the radiation and administration CpG-ODN group is lower than in the radiation alone group;(3) Intestinal tissue SOD expression: SOD preparation of intestinal tissue exposed to 6Gy radiation after 3d was observed and quantitated. The results showed that the content of SOD in the radiation and administration CpG-ODN group was higher than in the radiation alone group.(4) The level of cytokines in plasma: CpG-ODN injection can let to induction of some cytokines in plasma, including G-CSF, TNF-αand IL-6.DiscussionTreatment of radiation damage has been a very difficult problem in biological and medical. Many countries have invested much money to study new radiation protection drugs. However, so far, only a kind of radiation protection drug (WR2721) approved by FBA was used in clinic for treating radiation damage from radiotherapy. In recent years, immunologists found that Toll-like receptors family is an important part of innate immune system. The results showed that all the TLRs, except for TLR3, can activate NF-κB through MyD88 dependent signaling pathway and stimulate immune cells to release some cytokines, which inhibited apoptosis, promote cell proliferation and free radical scavenging. 2008, Burdelya LG first reported in Science that TLR5 agonist has a very strong protective effect of radiation, and even TLR5 agonist administrated after radiation also enhanced effect on reduction of radiation damage. There suggests that the family members of TLRs will be widely studied in further.CpG-ODN is TLR9 agonist, which was synthesized by imitating bacterial DNA. CpG-ODN with activation of bacterial DNA can stimulate the mammalian immune response. In 1990s, people found that cancer patients became better by injection of bacterial extracts. Later, the scholar found that bacterial DNA mediated anti-tumor. The results further confirmed that bacterial DNA has Immunostimulatory and anti-tumor effect. Currently, CpG-ODN has been researched in many aspects, including non-Hodgkin s lymphoma, melanoma and sepsis.According to different of structure and immune activation, CpG-ODN was divided into 3 types: A type CpG-ODN, such as CpG-ODN2216, they are characterized by poly G motifs with phosphorotioate (PS) linkages at the 5 and 3 ends and a phosphodister (PO) palindromic. A type CpG-ODN induced large amounts of IFN-αin pDC (dendritic cell), resulting in strong activation NK cell and T cell, but fails to activate B cells; B type CpG-ODN, such as CpG-ODN 2006, they are characterized by a full phosphorothioate backbone with one or more CpG motifs without poly G motifs. B type CpG-ODN are week inducers of IFN-αbut very potent Th1 adjuvants and strong B cell stimulators;C type CpG-ODN, such as CpG-ODN 2395, they combine the immune effects of A type CpG-ODN and B type CpG-ODN. Moreover, C type CpG-ODN is more stability in vivo. So C type CpG-ODN, CpG-ODN2395 is used in this study.In this study, we found that irradiated mice administered CpG-ODN significantly improved the survival rate. Meantime, CpG-ODN can reduce radiation damage of bone marrow and small intestinal tissue. There results showed that CpG-ODN have therapeutic effect on radiation damage. At present although the therapeutic effect of radiation damage mechanism is still not entirely clear, the main reason may be activation of NF-κB. Previous reports have showed that NF-κB was a critical factor in enhancing cell survival after radiation. The reason might be that Activation of NF-κB induces multiple factors that contribute to cell protection and promote tissue regeneration, including apoptosis inhibitors, reactive oxygen species scavengers. Moreover, other reason is that CpG-ODN stimulated immune cells to release some cytokines which reduced radiation damage, such as G-CSF, TNF-αand IL-6. These factors collaborated in reducing bone marrow damage, promoting recovery of bone marrow hematopoietic function, inhibiting of small intestinal crypt epithelial cell apoptosis and maintaining the integrity of the intestinal bacterial.
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