目的 基于蛋白质组学和生物信息学探讨莪术油注射液（ZTO）联合热疗（HT）对非小细胞肺癌放疗（RT）增敏的作用机制。方法 裸鼠sc肺癌A549细胞成瘤后，随机分成模型组（不做处理）、RT（6 Gy X线照射处理，每周1次）组、RT+HT（41～43℃加热处理10 min,2 d 1次）组、RT+ZTO(2 d 1次，每次200 mg·kg^-1)组、RT+HT+ZTO组，每组5只，治疗21 d。记录各组裸鼠肿瘤体积、质量变化，苏木素-伊红（HE）染色观察肿瘤病理改变。体外培养人肺癌A549细胞，分为对照组（不做处理）、RT（6 Gy X线照射处理）组、RT+HT（43℃加热处理30 min）组、RT+ZTO(100μg·m L^-1)组、RT+HT+ZTO组，采用MTT法检测细胞存活率；克隆形成实验、EdU检测细胞增殖能力；流式细胞术检测细胞周期变化；定量蛋白质组学技术筛选各组差异表达蛋白（DEPs）并进行生物信息学分析；流式细胞术检测细胞周期；实时荧光定量PCR（qRT-PCR）和Western blotting检测细胞周期蛋白依赖性激酶4（CDK4）、细胞周期素D3（CCND3）、周期素依赖性激酶抑制因子1A（CDKN1A）表达。结果 与模型组相比，RT组瘤体积和瘤质量显著缩小（P<0.001）；与RT组比较，RT+HT、RT+ZTO和RT+HT+ZTO组瘤体积呈下降趋势，瘤质量显著减小（P<0.01、0.001）,RT+HT+ZTO组效果最显著。HE染色显示，模型组肿瘤细胞密集，核异型性明显；各处理组，肿瘤细胞空泡化，间质增生，细胞密度降低，细胞核染色质加深，RT+HT+ZTO组最显著。与对照组相比，RT组A549细胞相对存活率、克隆形成率、EdU阳性细胞数量显著降低（P<0.01、0.001）；与RT组相比，RT+HT、RT+ZTO和RT+HT+ZTO组相对存活率、克隆形成率、EdU阳性细胞数量显著降低（P<0.01、0.001）,RT+HT+ZTO组治疗效果更明显。RT+HT+ZTO处理后DEPs经生物信息学分析发现与细胞周期、细胞增殖等有关，主要富集在细胞周期、细胞凋亡、代谢途径等信号通路。与对照组相比，RT组G₁期细胞比例升高、G₂期细胞比例降低（P<0.01、0.001）；与RT组相比，RT+ZTO和RT+HT+ZTO组G₁期细胞比例升高（P<0.05、0.001）,RT+HT、RT+ZTO和RT+HT+ZTO组S期细胞比例降低（P<0.01、0.001）。与对照组相比，RT组CDKN1A mRNA和蛋白显著上调（P<0.001）,CDK4、CCND3蛋白表达显著下调（P<0.001）；与RT组比较，RT+HT、RT+ZTO和RT+HT+ZTO组CDKN1A mRNA和蛋白显著上调（P<0.01、0.001）,CCND3 mRNA和蛋白显著下调（P<0.05、0.01、0.001）、CDK4蛋白表达显著下调（P<0.001）;RT+ZTO和RT+HT+ZTO组CDK4 mRNA显著下调（P<0.05、0.01）。结论 ZTO联合HT可通过调控CDK4、CCND3和CDKN1A表达进而调控细胞周期，抑制肺癌A549细胞增殖；还可能通过DNA复制、细胞凋亡、代谢途径等信号通路增强肺癌的RT敏感性。

Objective The mechanism of the sensitization effect of Zedoary Turmeric oil Injection（ZTO） combined with hyperthermia（HT） on radiotherapy（RT） for non-small cell lung cancer（NSCLC） was explored based on proteomics and bioinformatics. Methods After subcutaneous inoculation of A549 lung cancer cells in nude mice, the mice were randomly divided into the model group（no treatment）, the RT group（6 Gy X-ray irradiation once a week）, the RT + HT group（heating at 41—43 ℃ for 10 min, twice a day）, the RT + ZTO group(200 mg·kg^-1 twice a day), and the RT + HT + ZTO group, with 5 mice in each group. The treatment lasted for 21 days. The tumor volume and mass of each group of nude mice were recorded, and the pathological changes of the tumors were observed by hematoxylin-eosin（HE） staining. Human lung cancer A549 cells were cultured in vitro and divided into the control group（no treatment）, the RT group（6 Gy X-ray irradiation）, the RT + HT group（heating at 43 ℃ for 30 min）, the RT + ZTO group(100 μg·mL^-1), and the RT + HT + ZTO group. Cell survival rate was detected by MTT assay; cell proliferation ability was detected by colony formation assay and EdU assay; cell cycle changes were detected by flow cytometry; differentially expressed proteins（DEPs） in each group were screened by quantitative proteomics technology and analyzed by bioinformatics; cell cycle was detected by flow cytometry; the expression of cyclin-dependent kinase 4（CDK4）, cyclin D3（CCND3）, and cyclin-dependent kinase inhibitor 1A（CDKN1A） was detected by real-time fluorescence quantitative PCR（qRT-PCR） and Western blotting. Results Compared with the model group, the tumor volume and mass in the RT group were significantly reduced（P ＜0.001）; compared with the RT group, the tumor volume in the RT + HT, RT + ZTO, and RT + HT + ZTO groups showed a downward trend, and the tumor mass was significantly reduced（P ＜0.01, 0.001）, with the most significant effect in the RT + HT + ZTO group. HE staining showed that the tumor cells in the model group were dense, with obvious nuclear atypia; in each treatment group, the tumor cells were vacuolated, with interstitial hyperplasia, decreased cell density, and deepened nuclear chromatin staining, with the most significant effect in the RT + HT + ZTO group. Compared with the control group, the relative survival rate, colony formation rate, and the number of EdU-positive cells in the A549 cells in the RT group were significantly decreased（P ＜0.01, 0.001）; compared with the RT group, the relative survival rate, colony formation rate, and the number of EdU-positive cells in the RT + HT, RT + ZTO, and RT + HT + ZTO groups were significantly decreased（P ＜0.01, 0.001）, with the most significant effect in the RT + HT + ZTO group. After RT + HT + ZTO treatment, the DEPs were analyzed by bioinformatics and found to be related to the cell cycle, cell proliferation, etc., mainly enriched in the cell cycle, apoptosis, and metabolic pathways. Compared with the control group, the proportion of G₁ phase cells in the RT group increased, and the proportion of G₂ phase cells decreased（P ＜0.01, 0.001）; compared with the RT group, the proportion of G₁ phase cells in the RT + ZTO and RT + HT + ZTO groups increased（P ＜0.05, 0.001）, and the proportion of S phase cells in the RT + HT, RT + ZTO, and RT + HT + ZTO groups decreased（P ＜0.01, 0.001）. Compared with the control group, the mRNA and protein expression of CDKN1A in the RT group were significantly upregulated（P ＜0.001）, and the protein expression of CDK4 and CCND3 was significantly downregulated（P ＜0.001）. Compared with the RT group, the mRNA and protein levels of CDKN1A were significantly upregulated（P ＜0.01 and 0.001）, while the mRNA and protein levels of CCND3 were significantly downregulated（P ＜0.05, 0.01, 0.001）, and the protein expression of CDK4 was significantly downregulated（P ＜0.001） in the RT + HT, RT + ZTO, and RT + HT + ZTO groups. In the RT + ZTO and RT + HT + ZTO groups, the mRNA level of CDK4 was significantly downregulated（P ＜0.05, 0.01）. Conclusion ZTO combined with hyperthermia enhances NSCLC radiosensitivity by modulating CDK4, CCND3, and CDKN1A, thereby disrupting cell cycle progression, potentially via DNA replication, apoptosis, and metabolic pathways.

R285.5

黑龙江省自然科学基金资助项目(LH2023H104); 黑龙江省中医药科研课题项目(ZHY2025-299)