GM-0111通过抑制CCL5及内皮-免疫调节发挥放射保护作用的机制探讨

Radiation-induced proctitis (RIP) is a painful inflammatory condition of the rectum affecting up to 20% of cancer patients undergoing pelvic radiation. RIP manifests as a broad spectrum of side effects, which may lead to discontinuation of cancer treatment, and surgical intervention. Current therapeutics are merely symptomatic, highlighting the need for new effective prophylactics. We have demonstrated that synthetic glycosaminoglycan, GM-0111, has promising activity in mitigating the tissue-damaging effects of ionizing radiation when administered prophylactically in a murine model of RIP. We found that immune cell infiltration, and rectal tissue expression of CCL5, a potent chemotactic agent for mast cells and other immune cells, and its associated signaling pathways were significantly reduced with GM-0111 compared to irradiated controls. The mechanisms by which GM-0111 interacts with CCL5 and exerts its prophylactic effects against RIP have yet to be elucidated. We therefore performed a series of in vitro studies using immune and endothelial cells implicated in different stages of RIP. As mast cells hyperplasia and activation have been implicated in RIP development, we hypothesized that GM-0111 inhibits mast cell chemotaxis and degranulation, thereby reducing the immunological response resulting from radiation, including immune cell chemotaxis, activation, and viability and reactive oxygen species (ROS) generation. To test our hypothesis, the effect of GM-0111 treatment on CCL5 secretion and mediated responses was assessed in endothelial cells (HUVECs) and mast cells (P-815 and RBL-2H3). Due to the significant crosstalk between mast cells and macrophages, we also evaluated mast cell degranulation, ROS production, and cell viability using a colorimetric assay, flow cytometry, and quantitative phase imaging. We found that GM-0111 inhibited CCL5-induced chemotaxis with the IC 50 of 82 nM. GM-0111 showed strong binding to CCL5 with K d 3.34 ± 1.21 nM which may play a significant role in inhibiting its chemotactic effect. We also investigated the effect of GM-0111 on CCL5 released from TNF-⍺- and IFN-Ɣ-activated HUVECs, whereby 10 µM GM-0111 caused an 8-fold reduction in CCL5 secretion, suggesting that GM-0111 also interferes with CCL5 production. GM-0111 (10 µM) was found to exert cytoprotective effects on RAW 264.7 cells irradiated with 2.5 Gy and 5 Gy, which may be attributed to decreased ROS production. Collectively, these data demonstrate that GM-0111 exerts coordinated radioprotective effects by attenuating oxidative stress, inhibiting endothelial CCL5 production, and blocking mast cell recruitment. This integrated mechanism positions GM-0111 as a promising prophylactic candidate against radiation-induced injury for supportive cancer care. We would like to acknowledge NIH (5R01CA227225-05) (HG) and NIH (R01CA276653 ) (TAZ) for funding this project.