Objectives: To investigate the effect of borneol on cutaneous toxicity of gilteritinib and to explore possible compounds that can intervene with the cutaneous toxicity.
Methods: C57BL/6J male mice were given gilteritinib by continuous gavage for 28 d and the damage to keratinocytes in the skin tissues was observed with hematoxylin and eosin (HE) staining, TUNEL assay and immunohistochemistry. Human keratinocytes HaCaT were treated with gilteritinib, and cell death and morphological changes were examined by SRB staining and microscopy; apoptosis of HaCaT cells was examined by Western blotting, flow cytometry with propidium iodide/AnnexinⅤ double staining and immunofluorescence; the accumulation of cellular reactive oxygen species (ROS) was examined by flow cytometry with DCFH-DA. Compounds that can effectively intervene the cutaneous toxicity of gilteritinib were screened from a natural compound library using SRB method, and the intervention effect of borneol on gilteritinib cutaneous toxicity was further investigated in HaCaT cells and C57BL/6J male mice.
Results: In vivo studies showed pathological changes in the skin with apoptosis of keratinocytes in the stratum spinosum and stratum granulosum in the modeling group. Invitro studies showed apoptosis of HaCaT cells, significant up-regulation of cleaved poly (ADP-ribose) polymerase (c-PARP) and gamma-H2A histone family member X (γ-H2AX) levels, and increased accumulation of ROS in gilteritinib-modeled skin keratinocytes compared with controls. Screening of the natural compound library revealed that borneol showed excellent intervention effects on the death of HaCaT cells. In vitro, cell apoptosis was significantly reduced in the borneol+gilteritinib group compared to the gilteritinib control group. The levels of c-PARP, γ-H2AX and ROS in cells were significantly decreased. In vivo, borneol alleviated gilteritinib-induced skin pathological changes and skin cell apoptosis in mice.
Conclusions: Gilteritinib induces keratinocytes apoptosis by causing intracellular ROS accumulation, resulting in cutaneous toxicity. Borneol can ameliorate the cutaneous toxicity of gilteritinib by reducing the accumulation of ROS and apoptosis of keratinocytes in the skin tissue.
目的: 研究吉列替尼皮肤毒性的发生机制并寻找有效干预策略。方法: 对C57BL/6J雄性小鼠连续28 d灌胃给予吉列替尼，通过皮肤形态观察、苏木精-伊红染色、TUNEL检测、免疫组织化学法检测小鼠皮肤组织角质形成细胞损伤情况。对人角质形成细胞HaCaT给予吉列替尼，通过磺酰罗丹明B（SRB）染色法和显微镜观察法考察细胞死亡和形态学变化；蛋白质印迹法、流式细胞术结合碘化丙啶/Annexin Ⅴ双染法、免疫荧光技术考察吉列替尼作用下HaCaT细胞凋亡情况；流式细胞术结合2 ´ ，7 ´-二氯荧光黄双乙酸盐（DCFH-DA）法考察细胞活性氧累积情况。采用SRB染色法筛选天然化合物库中能有效干预吉列替尼皮肤毒性的天然化合物。HaCaT细胞给予吉列替尼和/或冰片联合处理，考察冰片对吉列替尼皮肤毒性的体外干预效果。C57BL/6J雄性小鼠连续灌胃给予吉列替尼和/或冰片28 d，考察冰片对吉列替尼皮肤毒性的体内干预效果。结果: 体内研究中，组织病理学检查结果提示吉列替尼皮肤毒性造模成功，且模型组雄性小鼠皮肤发生病理性改变，棘层和颗粒层角质形成细胞发生凋亡。体外研究中，与正常对照组比较，吉列替尼模型组发生凋亡、切割型多腺苷二磷酸核糖聚合酶（c-PARP）和磷酸化组蛋白H2AX（γ-H2AX）水平显著上调，活性氧累积明显增多。在天然化合物库中筛选发现冰片对HaCaT细胞死亡显示出极好的干预效果。在体外，相较于吉列替尼对照组，冰片+吉列替尼组凋亡明显减少，细胞中c-PARP、γ-H2AX及活性氧水平显著下降。在体内，冰片可缓解吉列替尼诱导的雄性小鼠皮肤病理性改变和皮肤细胞凋亡。结论: 吉列替尼通过引起细胞内活性氧累积诱导角质形成细胞凋亡，导致皮肤毒性损伤。冰片可以通过减少活性氧累积和皮肤角质形成细胞凋亡，缓解吉列替尼的皮肤毒性作用。.
Keywords: Borneol; Cell apoptosis; Cutaneous toxicity; Gilteritinib; Keratinocyte; Mice.