Effects And Mechanisms Of Autophagy Induced By X-ray Radiation On The Progression Of Atherosclerosis In ApoE-/- Mice

Objective:Inhibition of autophagy can reduce tumor cell escape of patients with malignant tumors receiving radiotherapy,and is a potentially important means to enhance the efficacy of radiotherapy.This study aimed to investigate the effects and mechanisms of X-ray-induced autophagy on the progression of atherosclerotic plaque in Apo E-/-mice.Methods: 1)Development of radiation-associated atherosclerosis(RAA)mouse model:partial ligation of left common carotid artery followed by a single local X-ray irradiation of the carotid artery(in 5 and 10 Gy dose groups)4 weeks later were performed in 8-week-old Apo E-/-mice.All animals were euthanized and carotid arteries were embedded in OCT for future assessment.The pathological changes of atherosclerotic plaques were evaluated using HE staining.The lipid deposition and collagen content were assessed by Oil Red O and Picric Masson staining.Macrophages(CD68+),Vascular smooth muscle cells(α-actin),and the level of autophagic flux were tested by laser confocal microscopy after immunofluorescence staining.2)Autophagy detection in vitro:The mouse peritoneal macrophages were cultured,stimulated with 7-KC and followed by a single 20-Gy dose of radiation,then the level of macrophage autophagy was assessed by Western blotting,transmission electron microscope(TEM),immunofluorescence and GFP-RFP-LC3 transfection.3)inhibition of autophagy in vivo and vitro: The Apo E-/-mice were intraperitoneally injected with autophagy inhibitor chloroquine(50mg/kg/d)for four weeks to block autophagy;The mouse peritoneal macrophages were stimulated with chloroquine(15ug/ml)to block autophagy in vitro.4)Detection of inflammation levels: the expression of Inflammatory cytokines such as MCP-1,TNF-α and IL-1α and the nucleus translocation of P65 in atherosclerotic lesion were detected by immunofluorescence staining,and the level of ROS in plaque was examined by DHE staining;In vitro,The level of IKBα protein was evaluated by Western blotting,the transcription levels of downstream inflammatory factors MCP-1,TNF-α and IL-1α were detected by RT-PCR,and the ROS level was analyzed by DCF-DA staining.Result: 1)Compared with the non-radiation group,the plaque areas in the 10 Gy dose X-ray irradiation group were increased by 42.7%(P<0.05),the macrophages infiltration and lipid deposition were markedly increased(P <0.05),accompanied with significantly decreased vascular smooth muscle cell numbers and collagen content(P<0.05).2)X-ray radiation prominently reduced the expression levels of P62 and LC3 in blood vessels(P <0.05),and inhibited the expression of P62 and LC3-II induced by 7-KC in vitro(P <0.05).3) Compared with the 10 Gy dose X-ray radiation group,chloroquine obviously increased the plaque area in mice by 26.8% after blocking autophagy(P <0.05).4)After X-ray irradiation,the expression level of MCP-1,TNF-α,IL-1α inflammatory factors in plaque were upregulated,and the ratio of P65 nuclear translocation and ROS levels were increased;In vitro,the m RNA expression level of MCP-1,TNF-α and IL-1α,and intracellular ROS levels were significantly increased after X-ray irradiation(P <0.05);Inhibition of autophagy by chloroquine could further increase the above-mentioned indicators of inflammation and oxidative stress(P <0.05).Conclusion: Irradiation with X-ray promotes the development of atherosclerotic lesions and facilitates autophagic flux in plaque.Autophagy activation negatively regulates Xray-induced atherosclerotic plaque progression by inhibiting inflammatory responses.Inhibition of autophagy exacerbates the progression of radiation-induced atherosclerosis.Therefore,autophagy inhibition may increase the risk of cardiovascular events in tumor patients receiving radiotherapy.