[摘 要] 目的：探究高迁移率族蛋白B1（HMGB1）中和抗体对肺癌LLC细胞移植瘤模型小鼠放疗联合PD-1抑制剂致放射性 肺损伤的影响。方法: 实验用 25 只雄性 C57BL/6 小鼠，随机分为对照组、PD-1 抑制剂组、放疗组、放疗 + PD-1 抑制剂组及 HMGB1中和抗体 + PD-1抑制剂 + 放疗组，每组5只。向每只动物的右腿皮下移植肺癌LLC细胞作为原发肿瘤，间日左腿皮下 移植LLC细胞作为继发肿瘤。实验第28天处死动物取肺组织和移植瘤，用H-E、Masson染色观察肺组织病理变化情况，免疫组 织化学法检测肺组织和移植瘤组织中 CD4+和 CD8+ T 淋巴细胞浸润情况，酶联免疫吸附实验（ELISA）检测肺组织中TNF‐α、 TGF-β和IL-6的表达。结果: 与对照或PD-1抑制剂组比较，其他3组小鼠移植瘤体积明显缩小（P < 0.01或P < 0.001），HMGB1 中和抗体不能使移植瘤体积进一步缩小；与对照或PD-1抑制剂组比较，其他3组小鼠肺组织的炎症评分和胶原容积分数（CVF） 均明显增加（P < 0.05、P < 0.01、P < 0.001或P < 0.000 1），HMGB1中和抗体可部分抑制放疗的不良反应（P < 0.01或P < 0.001）； 与对照或PD-1抑制剂组比较，其他3组肺组织中CD4+ T淋巴细胞浸润明显增多（均P < 0.001），HMGB1中和抗体可部分抑制肺 组织中CD4+ T淋巴细胞浸润（P < 0.01）；与对照或PD-1抑制剂组比较，其他3组移植瘤组织中CD4+ 、CD8+ T淋巴细胞浸润明显 增加（均P < 0.001），HMGB1中和抗体不能抑制移植瘤组织中T细胞的浸润。与对照或PD-1抑制剂组比较，其他3组肺组织中 IL-6 表达升高，TGF-β 表达下降（均 P < 0.05），HMGB1 中和抗体对肺组织中 TNF-α、IL-6 和 TGF-β 表达无明显影响。结论: HMGB1中和抗体不影响对移植瘤生长抑制的前提下，可能通过减少肺部炎性细胞的浸润，进而减轻肺部炎性损伤及纤维化。

[Abstract] Objective: To investigate the effect of high mobility group box-1 protein (HMGB1) neutralizing antibodies on radiationinduced lung injury in transplanted tumor-bearing mice. Methods: Twenty-five male C57BL/6 mice were randomly divided into five groups: control group, PD-1 inhibitor group, radiotherapy group, radiotherapy + PD-1 inhibitor group, and HMGB1 neutralizing antibody + PD-1 inhibitor + radiotherapy group, with five mice in each group. Subcutaneous transplantation of lung carcinoma LLC cells was performed on the right hind limb of each animal to establish the primary tumor, whilst subcutaneous transplantation of LLC cells on the left hind limb served as the secondary tumor. On day 28 of the experiment, animals were euthanized, and lung tissue and transplanted tumors were collected. Hematoxylin and eosin (H-E) staining and Masson's trichrome staining were performed to observe the pathological changes in the lung tissues of experimental mice. Immunohistochemistry (IHC) was used to detect the infiltration of CD4+ and CD8+ T lymphocytes in lung and transplanted tumor tissues. Enzyme-linked immunosorbent assay (ELISA) was used to examine the expression of TNF-α, TGF-β, and IL-6 in lung tissues. Results: Compared with the control and PD-1 inhibitor groups, the other three groups showed significantly reduced transplanted tumor volumes (P < 0.01 or P < 0.001), with HMGB1 neutralizing antibodies not further decreasing tumor volume. Similarly, compared with the control and PD-1 inhibitor groups, the inflammation scores and collagen volume fraction (CVF) in lung tissues were notably increased in the other three groups (P < 0.05, P < 0.01, P < 0.001, or P < 0.0001), and HMGB1 neutralizing antibodies partially inhibited the adverse effects of radiotherapy (P < 0.01 or P < 0.001). Compared to the control or PD-1 inhibitor groups, CD4+ T lymphocyte infiltration was significantly increased in the lung tissues of the other three groups, and HMGB1 neutralizing antibodies partially inhibited CD4+ T lymphocyte infiltration in lung tissue (P < 0.01). Moreover, in the transplanted tumor tissues, compared with the control or PD-1 inhibitor groups, the other three groups demonstrated significantly increased CD4+ and CD8+ T lymphocyte infiltration (all P < 0.001); however, HMGB1 neutralizing antibodies did not inhibit T cell infiltration in the transplanted tumor tissues. Compared with the control or PD-1 inhibitor groups, IL-6 expression in lung tissue was elevated and TGF-β expression was decreased in the other three groups (P < 0.05), and HMGB1 neutralizing antibodies had no significant effect on the expression of TNF-α, IL-6, or TGF-β in lung tissue. Conclusion: HMGB1 neutralizing antibodies may reduce the infiltration of inflammatory cells in the lungs, thereby alleviating lung inflammatory injury and fibrosis, without affecting the inhibition of transplanted tumor growth effects.

[基金项目] 贵州省自然科学基金项目[No. ZK(2022)key040]