Study On The Mechanism Of Susceptibility Of Host Cells To SARS-CoV-2 Sensitized By Ionizing Radiation

Severe acute respiratory syndrome coronavirus type 2,also known as COVID-19(SARS-CoV-2),can cause novel coronavirus(Covid-19),which has rapidly developed into a global pandemic since it appeared at the end of 2019,posing a serious threat to public health and the global economy.Covid-19 is mainly transmitted by air,contact,faeces and aerosol,and its transmission power and pathogenicity are stronger than that of the SARS virus that broke out in 2003.Although Covid-19 has a general population susceptibility,elderly people and patients with basal metabolic diseases are more susceptible to severe pneumonia than healthy individuals.It is worth noting that patients with clinical tumor radiotherapy also have a high susceptibility to COVID-19.Previous studies have suggested that low immunity and organ function are the main reasons that lead to the susceptibility of radiotherapy patients to Covid-19.Because ionizing radiation can produce complex biological effects on irradiated cells and tissues,the susceptibility change of host cells is also a pathogenic factor that can not be ignored.Covid-19 adheres to the virus mainly through the high affinity binding of Spike protein to the host cell membrane receptor ACE2,and enters the cell through endocytosis under the action of host protease.The endocytosis of the virus is related to sphingolipids,cholesterol and endocytosis proteins on the host cell membrane.Studies have shown that Acid sphingomyelinase,ASM)exists in lysosomes,which can be activated and transferred to the cell membrane under the stimulation,and the sphingolipids on the membrane are hydrolyzed to generate hydrophobic product Ceramide,CER),which can merge the lipid rafts into a larger ceramide-rich membrane structure domain platform,which is considered as the site for intracellular ingestion and uptake of pathogens.Recent studies have proved that ASM/CER is related to endocytosis in Covid-19.In addition,caveolin-1(Cav-1)is an essential part of the formation of caverns.It mediates the formation of pits and participates in the endocytosis of many pathogens.The pathogen first binds to the pit area rich in sphingolipids and cholesterol on the cell membrane,and then the pit invades to form endocytosis vesicles to mediate virus entry into the cell.Therefore,it is not clear whether ionizing radiation will cause the abnormal expression of ASM/CER and Cav-1,which will lead to the increase of infection in Covid-19.In this paper,the effect of ionizing radiation on Covid-19’s invasion of host cells was discussed,and the related mechanism was analyzed from two aspects: sphingomyelin metabolism and Cav-1 expression change.Firstly,the model of Covid-19 pseudovirus cell infection in vitro was established,and the effect of ionizing radiation on virus infection was evaluated.Combined with RNA transcriptome sequencing and bioinformatics analysis,it was found that ionizing radiation activated sphingomyelin metabolism in cells.Through molecular biology experiments,the promoting effect of ASM/CER pathway on Covid-19 infection was clarified.On this basis,the infection model of Covid-19 pseudovirus in vivo was established,and it was successfully verified that ionizing radiation increased the infection in vivo by acting on ASM.At the same time,through the comprehensive analysis of transcription sequencing results,it was found that Cav-1 in irradiated cells was significantly up-regulated The pseudovirus experiment confirmed that the enhanced expression of Cav-1 promoted Covid-19 to invade the host cells.The specific research results are as follows:1.Covid-19 pseudovirus cell infection model was successfully established,and it was found that single 1.0Gy,2.0Gy and 4.0Gyγ-ray irradiation could promote pseudovirus infection in a dose-dependent manner.2.Transcriptome sequencing showed that ionizing radiation could significantly change the sphingomyelin metabolism of cells,and different molecular biological experiments revealed that irradiation could up-regulate the expression of ASM in human alveolar epithelial A549 cells and human intestinal epithelial Caco-2 cells.3.It was found that the level of sphingomyelin metabolite CER increased after A549 and Caco-2 cells were exposed to ionizing radiation,and the results of liquid chromatography-mass spectrometry showed that the contents of C16-CER,C18-CER and C24-CER were significantly increased after irradiation.Using ASM interfering RNA and ASM function inhibitor amitriptyline(AMT)can reverse the above abnormalities,suggesting that ionizing radiation activates sphingomyelin metabolism through ASM/CER axis.4.ASM silencing,cholesterol inhibitor and AMT intervention can weaken the formation of lipid rafts in cell membrane induced by ionizing radiation,and significantly reduce the invasion of Covid-19 pseudovirus into host cells in vitro.5.The model of Covid-19 pseudovirus infection in vivo was successfully constructed,and it was confirmed that AMT intervention can weaken the enhancement effect of ionizing radiation on virus infection,suggesting that the sphingomyelin metabolism inhibitor AMT has the application potential to prevent clinical radiotherapy patients from being susceptible to Covid-19.6.It is found that ionizing radiation can up-regulate the expression of Cav-1 in A549 cells.By using Cav-1 silencing and Covid-19 pseudovirus experiment in vitro,it is clear that ionizing radiation can enhance Cav-1-dependent endocytosis and promote Covid-19 infection.To sum up,this study reveals the role and mechanism of ionizing radiation in sensitizing host cells to Covid-19 by enhancing sphingomyelin metabolism and Cav-1-dependent endocytosis.On this basis,it is found that AMT can weaken the effect of ionizing radiation on enhancing virus infection,thus providing new ideas for clinical radiotherapy patients to prevent and treat Covid-19 infection.