基于NF-κB与Nrf2信号阐释骨髓辐射旁效应“毒损髓络”病机的生物学机制及“芪归药对”的干预作用

Radiation induced bystander-effects (RIBE) is one reason for myelosuppression caused by radiotherapy. The redox homeostasis imbalance in bone marrow (BM) is the key event. NF-κB and Nrf2 signal pathway might regulate the process by means of crosstalk. Astragalus and Angelica Compatibility (AAC) can prevent and cure radiation induced myelosuppression, but the exact mechanism is unclear. Therefore hypothesis is described that AAC can regulate NF-κB and Nrf2 signal pathway to cope with marrow meridian collaterals damage of RIBE and maintain BM redox homeostasis to promote the recovery of immune and hematopoietic function. Using immunofluorescence, enzyme biochemistry, RNA interference, real-time RT PCR and Western blot techniques, it is observed that ACC affects the proliferation, apoptosis, senescence, the expression of genes relating NF-κB, Nrf2 signal pathway, apoptosis and aging of the bystander cells from the animal and co-culture cell model in order to clarify the target and mechanism of ACC to maintain BM redox homeostasis on the level of gene transcription, protein expression and enzyme activity from two aspects of the hematopoietic stem cells and microenvironment by regulation of NF-κB and Nrf2 signaling pathway. The study can provide theoretical basis and experimental evidence for the clinical application of the traditional Chinese medicine on the basis of ACC preventing myelosuppression for chemotherapy.

辐射旁效应是放疗造成骨髓抑制的原因之一，骨髓的氧化还原稳态失衡是关键事件，NF-κB与Nrf2通路可能存在串话调控着此过程。具有益气补血，滋补排毒功效的“芪归药对”对骨髓抑制有防治作用，但具体机制不清。故提出“芪归药对”通过调控NF-κB与Nrf2信号通路，对抗辐射旁效应“毒损髓络”的作用，维持骨髓的氧化还原稳态，促进骨髓造血免疫功能恢复的假说。复制辐射旁效应的在体离体模型，利用免疫荧光、RNA干扰、realtime RT-PCR及Western blot等技术，观察“芪归药对”对旁细胞增殖凋亡衰老状况、NF-κB与Nrf2通路及凋亡衰老相关基因表达的影响，从HSC及造血微环境两个角度，在基因转录、蛋白表达及酶活性水平，阐明“芪归药对”通过调控NF-κB与Nrf2通路维持骨髓氧化还原稳态的作用靶点及机理，为以“芪归药对”为基础配伍的中药复方防治骨髓抑制的临床应用奠定理论基础，提供实验依据。

辐射旁效应是放疗造成骨髓抑制的原因之一，骨髓的氧化还原稳态失衡是关键事件，NF-κB与Nrf2通路可能存在串话调控着此过程。具有益气补血，滋补排毒功效的“芪归药对”对骨髓抑制有防治作用，但具体机制不清。在“芪归药对”通过调控NF-κB与Nrf2通路，对抗辐射旁效应“毒损髓络”的作用，维持骨髓的氧化还原稳态，促进骨髓造血免疫功能恢复的假说基础上，复制辐射旁效应的离在体模型，观察了“芪归药对”自成复方当归补血汤对旁细胞增殖凋亡衰老状况、NF-κB与Nrf2通路及凋亡衰老相关基因表达的影响，研究发现：辐射旁效应可引起骨髓旁细胞内ROS、RNS的水平升高，模型小鼠血清中IL-6、TNF-α的含量增多，使旁细胞处于炎症氧化应激状态，促使旁细胞凋亡或衰老，参与模型鼠骨髓抑制的发生发展过程。当归补血汤明显改善模型鼠的生存状态，提高骨髓旁细胞生长活性，减轻骨髓旁细胞DNA损伤程度，降低骨髓旁细胞凋亡、衰老的发生率，推动细胞周期的进程，促进旁细胞的损伤修复。当归补血汤能缓解旁细胞的炎症氧化应激状态，可能与复方能激活抗氧化应激系统Nrf2通路及抑制促发炎症反应NF-κB通路的活性有关。当归补血汤通过上调旁细胞Nrf2基因及其下游靶基因SOD、CAT表达，可有效增强辐射小鼠血清中SOD、CAT酶活性；当归补血汤通过抑制NF-κB通路，减少炎症相关分子cox-2、iNOS的表达，促进骨髓旁细胞bcl-2/bax在mRNA、蛋白表达水平都呈上调趋势，caspase3在mRNA、蛋白表达水平都呈下调趋势，降低旁细胞凋亡率；当归补血汤还可影响调控细胞周期的p16Ink4a、CDK4及CyclinD1等基因的表达，推动旁细胞细胞周期进程，促进增殖，降低旁细胞衰老的发生率。本课题利用免疫荧光、realtime RT-PCR及Western blot等技术，在基因转录、蛋白表达及酶活性水平，初步明确了“芪归药对”自成复方当归补血汤能通过调控NF-κB与Nrf2通路，维持骨髓氧化还原稳态，缓解电离辐射旁效应造成的骨髓抑制。在骨髓辐射旁效应损伤过程中，“芪归药对”的作用靶点及机理，为以“芪归药对”为基础配伍的中药复方防治骨髓抑制的临床应用奠定理论基础，提供实验依据。