Study On The Lung Injury Induced By Intratracheal Inoculation Of Ricin Aerosol And On The Intervention Of Ricin Intoxication

The Ricinus plants are widely cultivated around the world,and the highly toxic ricin can be easily produced using castor bean seeds.Exposure to ricin via various routes may cause severe tissue damage,where inhalation is considered more lethal than either injection or ingestion.Ricin inhalation may lead to pulmonary edema,acute lung injury,and acute respiratory distress syndrome,among others,eventually leading to death.Currently there is no effective treatment for ricin.In view of the fact that ricin can be used for potential terrorist threat or as a biological warfare agent,and the current research on the mechanism of ricin induced lung injury are not clear enough.In order to improve the understanding for mechanism of ricin induced lung injury,this study conducted a series of studies from both in vivo and in vitro to screen target cells,proteins,genes or other intervention molecules with research value for clinical treatment of ricin poisoning.For in vivo studies,the female C57BL/6 mice were challenged with multiple aerosol concentrations of RT by intratracheal inoculation to calculate the dose of RT LD₅₀.The death pattern of mice challenged with RT at the dose 2×LD₅₀ were recorded,and the lungs or/and serum of mice,who were challenged with 2×LD₅₀doses of RT,were analyzed in the further studies at different time points according to the death pattern of mice.The analyses include histopathological analysis of lungs based on hematoxylin-eosin staining,time-course genome-wide transcriptome analysis of lungs based on RNA sequencing,the determination of important cytokines from serum and bronchoalveolar lavage fluid?BALF?based on high-throughput liquid chip laboratory technique,the dynamic changes of major innate immune cell populations in lungs based on flow cytometry,gene expression characteristics and intercellular communication analysis of major cell populations in lungs based on single-cell RNA sequencing technology.For in vitro studies,the mouse-derived mononuclear/macrophage cell line,RAW264.7,were used to investigate whether the small molecule compound Retro-2can protects macrophages exposed to RT,and try to explored the mechanism?s?underlying the protective effect exerted on macrophages by Retro-2 against RT.The results of in vivo studies showed that the LD₅₀ doses of RT is about 5?g/Kg body weight of mice,and mice will be died largely from the 3rd day post-exposed to RT at the dose of 2×LD₅₀.Histopathological analysis showed that the lung lesions of the mice treated with RT deteriorate gradually.Vascular leakage was observed in the lungs locally at 24 h post-exposure;pulmonary edema was observed in the lungs locally at 48 h post-exposure;extensive vascular leakage and pulmonary edema could be observed in the lungs at 72 h post-exposure.Time-course transcriptome analysis of lungs showed that the expression of genes that participate in acute phase immune and/or inflammatory response increased rapidly,the expression of genes related to hormone transport and secretion were also up-regulated subsequently.The expression of genes related to leukocyte chemotaxis,especially neutrophil chemotaxis,and leukocyte migration were also up-regulated gradually.Conversely,the expression of genes related to antioxidant capacity decreased sharply at the early time point?less than 12 h?,and the expression of genes related to the cellular component of the extracellular matrix and anchored component of the membrane decreased through all time points.Moreover,the first six genes with the most significant change were Jun,Gdf15,Atf3,Osm,Cxcl2?protein names:MIP-2?,and Egr1 at the early time point?4h post-exposure to RT?.Besides,the measurement of cytokines showed that the serum protein levels of GRO-?,MCP-1,GM-CSF,IL-6 and TNF-?were increased,and the BALF protein levels of Eotaxin,MCP-1,MIP-1?,MIP-1?,MCP-3,IP-10,RANTES,GRO-?,MIP-2,IL-6,IL-1?,TNF-?,IL-23,IL-18,and GM-CSF were increased significantly.The analysis of flow cytometry showed that the number of alveolar macrophages decreased significantly at 4 h post-exposure of RT,and the number of neutrophils,interstitial macrophages 1 and interstitial macrophages 3 increased gradually at 8 h post-exposure of RT.Besides,the decreased number of Batf3⁺dendritic cells was detected at 48 and 72 h post-exposure of RT.The single-cell transcriptome analysis showed that non-immune-related cells were at the core of communication between cell populations.The expression of genes related to cell cycle and apoptosis in Clara cells,and genes related to apoptosis and pyroptosis in mesothelial cells were particularly up-regulated post-exposed to RT.The expression of a large number of interested transcription factors and/or pro-inflammatory cytokines/chemokines genes were up-regulated in alveolar epithelial cells and macrophages post-exposed to RT,The interested up-regulated genes in alveolar epithelial cells include Jun,Atf3,Egr1,Cxcl1,Cxcl2,Cxcl10,Csf2 and Il6;the interested up-regulated genes in macrophages include Atf3,Osm,Cxcl2,Ccl2,Ccl3,Ccl4,Ccl7,Il1b and Tnf.In addition,RT also leads to the activation of fibroblasts and monocytes.The results of in vitro studies showed that 20?M Retro-2 could protect cells from death during all time intervals following RT treatment,alleviate the increase of cytokine-related m RNA and the decrease of capacity of protein synthesis that induced by challenging with 200 ng/m L RT.Moreover,the endoplasmic reticulum stress response caused by RT in cells could be alleviated by pretreating with Retro-2.The results of in vivo studies suggest that there are severe inflammatory lung injuries in mice induced by the RT dose of 2×LD₅₀,and macrophages and alveolar epithelial cells may be the most important cells in the initiation of lung inflammation caused by RT.The harmful effect of RT on other non-immune-related cells cannot be ignored,especially RT may cause fatal damage to Clara cells and mesothelial cells,and the role of this damage in the development of lung injury is also necessary to study in the future research.In addition,the expression of genes related to antioxidant capacity and cellular component of the extracellular matrix decreased in lungs caused by RT,and the role in alleviating lung injury is worthy of further study by intervening the down-regulated expression of these genes.The results of in vitro studies suggest that Retro-2 can protect macrophages from death caused by RT,and it is worthwhile to investigate potential of Retro-2 for clinical applications in RT poisoning in the future research.