“RT-PK”（radiotherapy-pharmacokinetic）现象是指辐射产生的生物学效应改变化疗药物的药代动力学特征。目前，超过一半的癌症患者选择联合放化疗进行恶性肿瘤的治疗，随着该治疗方案的广泛应用，研究者发现部分化疗药物存在“RT-PK”现象，“RT-PK”现象的存在不仅限制了化疗药物的疗效，在一定程度上也增加了毒副作用，因此探究“RT-PK”现象的发生机制是放化疗使用中的重要任务。阿帕替尼作为国内自主研发的口服小分子激酶抑制剂，在临床上联合放疗广泛用于非小细胞肺癌、肝癌、乳腺癌等恶性肿瘤治疗，目前尚未有研究报道阿帕替尼是否存在“RT-PK”现象。

为了探究阿帕替尼与放疗合用是否存在“RT-PK”现象及该现象的发生机制，本研究建立了LC-MS/MS定量方法和UPLC-Q-TOF-MS/MS定性方法，选择SD大鼠和HepG2细胞进行照射，考察腹部X射线辐射后阿帕替尼的PK和代谢酶基因表达的变化。结果显示，我们所建立的LC-MS/MS定量分析方法符合《M10：生物分析方法验证及样品分析》指导原则的要求。在0.5 Gy和2 Gy 时，阿帕替尼在大鼠血浆中的AUC_0-t和C_max明显降低，AUC_0-t分别减少了33.8%和76.3%；翻转肠和肠灌流实验结果显示，辐射组和正常组的肠道吸收无显著差异；在组织分布研究中，我们发现X射线辐射后阿帕替尼在肝脏和小肠组织中分布明显下降；排泄实验结果显示，辐射并未影响阿帕替尼在尿粪中的排泄情况；然而，在对大鼠血浆、尿液和粪便进行代谢物鉴定和差异代谢产物分析时，发现在正常组和辐射组分别检测到53和64个代谢产物，辐射组尿液和粪便中分别发现5个和9个新代谢产物，提示辐射显著改变了阿帕替尼的体内代谢。综上所述，X射线辐射后阿帕替尼的血浆暴露量和组织内暴露量明显降低，药物的体内代谢途径增加，代谢加速，证实阿帕替尼联合放疗存在显著的“RT-PK”现象。同时，上述结果提示辐射后阿帕替尼的体内代谢改变可能是发生“RT-PK”现象的主要因素。

X射线辐射后大鼠肝组织中CYP3A4、CYP2E1、CYP2C9、UGT2B7、UGT1A3的mRNA表达明显增加，CYP2D6在辐射后48 h和72 h的mRNA表达升高却无显著性差异；CYP1A2在辐射组中表达显著减少，CYP2C19除IR-48 h组显著增加外其他与正常组表达量基本一致。进一步的蛋白表达检测发现，X射线辐射增加了HepG2细胞内CYP3A4、CYP2D6、CYP2E1、UGT2B7的蛋白表达量。该结果初步证明X射线辐射诱导代谢酶的表达明显增加，进而促进了阿帕替尼在体内代谢。

为了进一步探究辐射诱导肝组织代谢酶基因表达的分子机制，本研究使用X射线照射SD大鼠和HepG2细胞，考察腹部X射线辐射后大鼠肝功能、氧化应激、核转录因子变化。腹部X射线辐射后进行H&E和血清生化指标检测，发现辐射组大鼠肝组织产生了损伤，H&E染色结果显示部分肝细胞水肿和细胞浸润，ALT、AST、LDH活性相比正常组明显升高，表现为先升高后降低，而ALP活性相比正常组无显著变化。肝组织纤维化检测发现大鼠血清中HA、Hyp、LN的含量在辐射组和正常组之间无显著差异，表明辐射后未出现肝组织纤维化。进一步的氧化应激检测发现，X射线辐射后大鼠肝组织和HepG2细胞中ROS明显升高，而抗氧化酶SOD、CAT活性和GSH含量显著降低，表明X射线辐射后抗氧化系统和氧化系统失衡，产生了氧化应激。WB和IF实验表明，X射线辐射显著增加细胞核中Nrf2蛋白含量，降低细胞质中Nrf2蛋白含量，同时作为RXR-α上游调控因子，Nrf2表达的增加可能进一步促进RXR-α表达。进一步研究表明，X射线辐射后的确增加了PXR、CAR、RXR-α的mRNA表达，尤其在IR-24 h和IR-48 h组的大鼠肝组织中与对照组相比显著增加，PXR、CAR、RXR-α作为药物代谢酶表达的上游调控因子，其表达的增加促进了代谢酶的表达。以上结果表明腹部X射线显著增加代谢酶上游调控因子Nrf2、PXR、CAR、RXR-α的表达，为了验证X射线辐射的确是影响Nrf2蛋白入核和表达介导代谢酶的改变，我们使用Nrf2激活剂（TBHQ）和抑制剂（ML385）进行验证，发现辐射组的Nrf2、CYP3A4、CYP2E1、CYP2D6蛋白表达明显升高；抑制剂组Nrf2蛋白表达和入核被显著抑制后，其下游CYP3A4、CYP2E1、CYP2D6蛋白表达同样显著下降；在辐射组中加入抑制剂后明确抵消了辐射引起的蛋白增加，因此，初步证实X射线辐射激活Nrf2蛋白后促进了代谢酶的表达进而促进阿帕替尼的药物代谢。

总之，腹部X射线照射明显影响阿帕替尼在大鼠体内的PK，发生“RT-PK”现象。进一步的研究发现该现象的发生与代谢酶的表达相关，腹部X射线辐射显著增加代谢酶的表达，加速了阿帕替尼在体内代谢及降低了血浆暴露。另外，代谢酶的表达是由转录因子调控，深入研究发现辐射产生的过量ROS促进了Nrf2蛋白的表达和入核，进一步促进下游核受体和代谢酶的表达，进而改变药物PK。因此，本研究系统的阐述了阿帕替尼“RT-PK”现象的发生机制，本研究模式可为探究其他药物“RT-PK”现象发生机制提供参考，且实验结果可为临床中阿帕替尼联合放疗的治疗方案、调整临床用药剂量以及实现精准治疗提供实验基础和理论依据。

The "radiotherapy-pharmacokinetic"(RT-PK) phenomenon means that the biological effects of radiation change the pharmacokinetic characteristics of chemotherapeutic drugs. At present, more than half of the cancer patients choose the combination of radiotherapy and chemotherapy for the treatment of malignant tumors. With the wide application of this treatment program, researchers have found that there is a "RT-PK" phenomenon in some chemotherapeutic drugs. The existence of "RT-PK" phenomenon not only limits the efficacy of chemotherapeutic drugs, but also increases the side effects to a certain extent. Therefore, to explore the mechanism of "RT-PK" phenomenon is an important task in the use of radiotherapy and chemotherapy. As an oral small molecular kinase inhibitor independently developed in China, apatinib is widely used in combination with radiotherapy in the treatment of non-small cell lung cancer, liver cancer, breast cancer and other malignant tumors. However, there is no report on the existence of "RT-PK" phenomenon in apatinib.

In order to explore the existence and mechanism of "RT-PK" phenomenon in apatinib combined with radiotherapy, LC-MS/MS quantitative analysis and UPLC-Q-TOF-MS/MS qualitative analysis were established. SD rats and HepG2 cells were irradiated to investigate the changes of pharmacokinetics and metabolic enzyme gene expression of apatinib after abdominal X-ray radiation. The results show that the quantitative analysis method of LC-MS/MS established by us meets the requirements of the guiding principle of ICH "M10 Bioanalytical Method Validation and Study Sample Analysis". At 0.5Gy and 2Gy, the AUC_0-t and C_max of apatinib in rat plasma decreased significantly, and the AUC_0-t decreased by 33.8% and 76.3%, respectively. The results of intestinal flip test showed that there was no significant difference in intestinal absorption between radiation group and normal group. In the study of tissue distribution, we found that the distribution of apatinib in liver and small intestine decreased significantly after X-ray radiation. The results of excretion experiment showed that radiation did not affect the excretion of apatinib in urine and feces; however, in the identification of metabolites and analysis of differential metabolites in plasma, urine and feces of rats, it was found that 53 and 64 metabolites were detected in the normal group and radiation group, and 5 and 9 new metabolites were found in urine and feces of the radiation group, respectively, suggesting that radiation significantly changed the metabolism of apatinib in vivo. To sum up, after X-ray radiation, the plasma exposure and tissue exposure of apatinib were significantly decreased, the metabolic pathway of the drug in vivo was increased, and the metabolism was accelerated, which confirmed that there was a significant "RT-PK" phenomenon in apatinib combined with radiotherapy. At the same time, the above results suggest that the metabolic changes of apatinib after radiation may be the main factor for the occurrence of "RT-PK" phenomenon.

The mRNA expression of CYP3A4, CYP2E1, CYP2C9, UGT2B7 and UGT1A3 in rat liver tissue increased significantly after X-ray irradiation, while the mRNA expressions of CYP2D6 were increased at 48 h and 72 h after radiation without significant differences; the expression of CYP1A2 was significantly decreased in the radiation group, and the expression of CYP2C19 was basically the same as that of the normal group except for the significant increase in the IR-48 h group. Further protein expression detection showed that X-ray radiation increased the protein expression of CYP3A4, CYP2D6, CYP2E1 and UGT2B7 in HepG2 cells. The results showed that X-ray radiation induced a significant increase in the expression of metabolic enzymes, which promoted the metabolism of apatinib in vivo.

In order to further explore the molecular mechanism of metabolic enzyme gene expression in liver tissue induced by radiation, SD rats and HepG2 cells were irradiated with X-rays to investigate the changes of liver function, oxidative stress and nuclear transcription factors after abdominal X-ray radiation. After abdominal X-ray radiation, the liver tissue injury was found in the radiation group, and some hepatocyte edema and cell infiltration were found in the radiation group. The activities of ALT, AST and LDH were significantly higher than those in the normal group, while the activity of ALP had no significant change compared with the normal group. The detection of liver fibrosis showed that there was no significant difference in the contents of HA, Hyp and LN in serum between the radiation group and the normal group, indicating that there was no liver fibrosis after radiation. Further oxidative stress detection showed that ROS in rat liver tissue and HepG2 cells increased significantly, while antioxidant enzyme SOD, CAT activity and GSH content decreased significantly after X-ray radiation, indicating that the antioxidant system and oxidative system were out of balance after X-ray radiation, resulting in oxidative stress. WB and IF experiments showed that X-ray irradiation significantly increased the content of Nrf2 protein in the nucleus and decreased the content of Nrf2 protein in the cytoplasm. As an upstream regulatory factor of RXR-α, the increased expression of Nrf2 may further promote the expression of RXR-α. Further studies showed that X-ray irradiation did increase the mRNA expression of PXR, CAR and RXR- α, especially in the liver tissues of rats in IR-24h and IR-48h groups compared with the control group. As upstream regulators of drug metabolic enzyme expression, the increased expression of PXR, CAR and RXR- α promoted the expression of metabolic enzymes. The above results showed that abdominal X-ray significantly increased the expression of upstream regulatory factors Nrf2, PXR, CAR and RXR- α of metabolic enzymes. In order to verify that X-ray radiation does affect the entry and expression of Nrf2 protein into the nucleus to mediate the changes of metabolic enzymes, we used Nrf2 activator (TBHQ) and inhibitor (ML385) to verify that the protein expression of Nrf2, CYP3A4, CYP2E1 and CYP2D6 increased significantly in radiation group. After the expression of Nrf2 protein and its entry into the nucleus were significantly inhibited in the inhibitor group, the expression of downstream CYP3A4, CYP2E1 and CYP2D6 protein also decreased significantly, and the increase of protein induced by radiation was clearly offset by the addition of inhibitor in the radiation group. Therefore, it was preliminarily confirmed that X-ray radiation activated Nrf2 protein promoted the expression of metabolic enzymes and promoted the drug metabolism of apatinib.

The results of this study showed that abdominal X-ray irradiation significantly affected the pharmacokinetics of apatinib in rats, resulting in the phenomenon of "RT-PK". Further studies found that the occurrence of this phenomenon was related to the expression of metabolic enzymes. Abdominal X-ray radiation significantly increased the expression of metabolic enzymes, accelerated the metabolism of apatinib in vivo and reduced plasma exposure. In addition, the expression of metabolic enzymes is regulated by transcription factors. In-depth studies have found that excessive ROS produced by radiation promotes the expression and entry of Nrf2 protein into the nucleus, further promotes the expression of downstream nuclear receptors and metabolic enzymes, and then changes the pharmacokinetics of drugs. In a word, this study systematically expounds the mechanism of apatinib "RT-PK" phenomenon, and the model of this study can provide reference for exploring the mechanism of other drugs "RT-PK" phenomenon, and the experimental results can provide experimental and theoretical basis for clinical treatment of apatinib combined with radiotherapy, adjustment of clinical drug dose and realization of accurate treatment.