Study On Neurological Injury And Toxic Mechanism Of Sulfur Mustard Based On Human Induced Pluripotent Stem Cells

Sulfur Mustard(SM),also known as mustard gas,is a vesicant chemical toxicants with strong toxicity and wide damage range.It is called the "king of toxic gases".SM mainly targets the skin,eyes and respiratory tract.After poisoning,it will cause erythema,blisters,pruritus and ulcers.Clinical studies have found that most patients with SM poisoning may present symptoms of nervous system damage,such as anxiety,depression,cognitive decline,headache,muscle weakness,neuralgia and other symptoms.It was previously believed that the effect of SM on the nervous system was mainly due to secondary effects caused by injuries in other parts of the body,rather than a direct effect of SM.However,the relevant clinical data and in vitro and in vivo experimental studies have shown that SM can damage the nervous system and reduce the survival rate of nerve cells in animals.However,the exact toxic effect and toxic mechanism are still unclear,and the research on SM antitoxic drugs is seriously insufficient.Due to the particularity of the nervous system and limited by the source of donor tissue and ethics,it is difficult to directly isolate and culture primary human neurons from neural tissue,so there is a lack of data on the exposure of SM to human neurons.Nerve cells are mostly non-proliferative or terminally differentiated cells,and they have various types.So does SM affect the activity of proliferating inactive human nerve cells?Are the effects the same for different stages or types of nerve cells?Is the neurotoxicity of SM a direct or indirect effect?What is the mechanism of SM neurotoxicity?It is not yet clear.Human induced pluripotent stem cells(hiPSC)are a type of pluripotent stem cells that carry human genetic information and are obtained by reprogramming human somatic cells.The appearance of hiPSC provides an ideal human cell for studying the nerve damage induced by chemical agents.At present,hiPSC can be induced and differentiated into a variety of neural cell subtypes and applied to many aspects such as disease research and drug development process,especially displaying great application potential in the evaluation of drug neurotoxicity and effectiveness.By adding specific induction factors,hiPSC can be directionally induced to differentiate into neural stem cells(NSC)and Neurons,so as to obtain neural cells in different stages.Human neuroblastoma cells(SHSY5Y)is an immortalized nerve cell with high similarity to normal human neurons in morphological,physiological and biochemical characteristics,which has been widely used in the related research on nervous system diseases.Therefore,in this paper,hiPSC,hiPSCderived NSC and Neuron are regarded as different stages of nerve cells,and SH-SY5Y is regarded as another different type of nerve cells,to establish an SM neural cell exposure model and study the toxic effects and toxic mechanism of SM on neurons in different stages and types.In addition,the animal model of exposure was established by subcutaneous injection of SM to dynamically study the effects of SM on the nervous system.This study will provide the basis for the establishment of humanized model of SM nerve injury,indepth understanding of the poisoning mechanism and the research of antitoxic drugs,and has important theoretical and practical significance.I.Study on neurotoxicity of sulfur mustard based on human induced pluripotent stem cellsDifferent concentrations of SM solutions were added to different stages and types of nerve cells(The concentrations of SM to hiPSC:0.001,0.01,0.1,0.5,1.0,10,100,200μmol·L-1;hiPSC-NSC,hiPSC-Neuron and SH-SY5Y:0.1,1.0,10,50,100,200,400,800μmol·L-1),and the cells were treated with 0.2%anhydrous ethanol as the vehicle control,removed after 30 min of interaction with the cells,and then cultured for 24 h to establish a SM neural cell injury model.To study the toxic effects of SM on different stages and types of nerve cells from the aspects of cell confluence and morphology,cell viability and cell proliferation.1.Effects of sulfur mustard on confluence and cell morphology of different stages and types of nerve cellsIn order to study the effect of SM on the cell confluence and morphology of nerve cells,the changes of cell confluence and morphology were monitored by using IncuCyte ZOOM dynamic imaging analyzer 24 h before and after SM exposure.The results showed that the cell confluence of the 800 μmol·L-1 groups of hiPSC-Neuron and SH-SY5Y changed significantly following SM exposure when compared with the vehicle group.Then,with the prolongation of time and the increase of exposure concentration,the cell confluence was gradually reduced,and the original morphology of the cells was gradually changed,with the nuclei shrinking,the morphology becoming round,and the cell debris increasing.The changes in cell confluence and morphology of hiPSC and hiPSC-NSC began about 6-8 h after SM exposure,and then the cell confluence gradually decreased with the prolongation of time and the increase of the exposured concentration.The morphological changes were mainly manifested as cell shrinkage and roundness,gradually blurred morphology,and increased cell debris.The above results indicated that SM could reduce the confluence of nerve cells in a time-and concentration-dependent manner,affecting the normal morphological structure of cells.The different time of SM toxicity in different cells also indicates that different cells have different sensitivities to SM toxicity.2.Effects of sulfur mustard on the activity of different stages and types of nerve cellsIn order to study the effect of SM on neuronal cell viability,the OD450nm values of cells in each group were measured and cell viability was calculated by CCK-8 assay at 24 h after SM exposure.The released of lactate dehydrogenase(LDH)in cells was detected by LDH kits.The percentage of living cells and dead cells was counted using double staining assay of Calcein-AM/PI fluorescence.The results showed that SM reduced the OD450nm values of hiPSC,hiPSC-NSC,hiPSC-Neuron,and SH-SY5Y in a concentration-dependent manner at 24 h after SM exposure,compared to vehicle group.The median inhibitory concentrations of SM on cells were 0.17,11.72,18.78,and 15.92 μmol·L-1,respectively.SM was able to cause the LDH released by cells to gradually increase,and the percentage of dead cells to gradually increase.The above results indicated that SM could reduce the cell viability of hiPSC,hiPSCNSC,hiPSC-Neuron and SH-SY5Y in a concentration-dependent manner.hiPSC was the most sensitive to SM toxicity,with NSC,hiPSC-Neuron and SH-SY5Y showing similar sensitivity to SM toxicity.SM can damage the structure of neuronal cell membrane,significantly reduce the survival rate of nerve cells,and eventually cause a large number of cell deaths.3.Effect of sulfur mustard on proliferation of different stages and types of nerve cellsIn order to study the effect of SM on the proliferation of nerve cells,the percentage of EdU positive cells was detected by EdU fluorescence staining 24 h after exposure to SM.The results showed that the proportion of EdU positive cells in hiPSC,hiPSC-NSC,hiPSCNeuron and SH-SY5Y cells decreased gradually with the increase in SM concentration when compared with the vehicle group.The above results indicated that SM could reduce the proliferation of nerve cells.The research in this part has shown that SM could reduce the cell viability of nerve cells in a concentration-dependent manner.SM damaged hiPSC most seriously,and its damage to hiPSC-NSC and hiPSC-Neuron as well as SH-SY5Y was similar.However,the manifestations of toxic damage are diversity in different cells.SM caused the most severe damage to hiPSC.The main damage effects of SM on hiPSC and hiPSC-NSC were consistent,for example,it reduced cell proliferation ability,leading to the increase of the percentage of dead cells,but had little damage to the cell membrane.The damage of SM to the inactive hiPSC-Neuron was similar to SH-SY5Y,and the damage was mainly manifested as destroying the cell membrane structure,increasing the proportion of dead cells,and reducing cell proliferation ability.Ⅱ.Study on neurotoxicity mechanism of sulfur mustard based on human induced pluripotent stem cellsIn order to further study the toxic mechanism of SM on nerve cells,research was carried out from two aspects:the substances and cytotoxic effect produced by SM.After 24 h,the content of DNA double-strand break marker y-H2A.X in nerve cells and the types of adducts of intracellular SM and DNA,the intracellular contents of NAD+/NADH,ATP,and reactive oxygen species(ROS),the levels of cellular glycolysis and oxidative phosphorylation were detected.To investigate the role of DNA damage,energy metabolism and oxidative stress in the neurotoxicity of SM and to analyze whether changes in these mechanisms are the same in different stages and types of neurons.1.Effect of sulfur mustard on DNA of different stages and types of nerve cellsIn order to study the role of DNA damage in the neurotoxicity of SM,the content of DNA double-strand break marker γ-H2A.X in nerve cells was detected by immunofluorescence assay.SM-DNA adducts in SH-SY5Y cells were detected by high performance liquid chromatography-mass spectrometry at 24 h after SM exposure.The results showed that SM was able to increase the intracellular γ-H2A.X content of hiPSC,hiPSC-NSC and Neuron,and SH-SY5Y in a concentration-dependent manner when compared with controls.Two types of DNA adducts,N7-HETEG and Bis-G,were detected in SH-SY5Y cells treated with SM 10 μmol·L-1.The above results indicated that SM could be added with intracellular DNA after entering into nerve cells,which might lead to increase of DNA double-strand break in nerve cells.2.Effects of sulfur mustard on energy metabolism in different stages and types of nerve cellsIn order to study the role of cellular energy metabolism in the neurotoxicity of SM,the intracellular contents of NAD+/NADH and ATP were detected by NAD+/NADH and ATP kits at 24 h after SM exposure.The levels of glycolysis and oxidative phosphorylation were detected by Seahorse Cell Metabolic Respiratory Dynamics Analyzer.The results showed that,compared to the cell control group,SM was able to reduce the concentration-dependent NAD+/NADH and ATP contents in hiPSC-NSC and SH-SY5Y cells to a greater extent than ATP.The basic glycolysis level,glycolysis capacity maximum and non-glycolysis acidification levels of hiPSC-NSC and SH-SY5Y were gradually decreased with the increase of SM concentration.Proton leak,basal respiratory level,maximal respiratory and ATP production capacity of the cells were also gradually decreased with the increase of SM concentration.hiPSC assay results showed that intracellular NAD+/NADH content decreased and ATP content increased with the increase in SM concentration,compared to the cell control group.The basic glycolytic level,glycolytic maximum and non-glycolytic acidification levels of the cells were increased gradually with the increase of SM concentration.Proton leak,basal respiratory level,maximal respiratory,and ATP production capacity decreased gradually with the increase of SM concentration.The above results indicated that SM could induce gradual decreases in the glycolysis level and mitochondrial oxidative phosphorylation level of hiPSC-NSC and SH-SY5Y cells,as well as the intracellular contents of energy-related substances NAD+/NADH and ATP.SM leads to increased cellular glycolysis,decreased oxidative phosphorylation,decreased intracellular NAD+/NADH content,and increased ATP content in hiPSC.It suggested that SM could cause disorders of energy metabolism in different stages and types of nerve cells.3.Effects of sulfur mustard on ROS content in different stages and types of nerve cellsIn order to study the role of oxidative stress in the neurotoxicity of SM,the changes in the mean fluorescence intensity of ROS in cells were detected by ROS kit at 24 h after SM exposure.The results showed that,compared with the cell control group,with the increase of SM concentration,the average fluorescence intensities of ROS in hiPSC,hiPSC-NSC and Neuron cells were decreased gradually,and the average fluorescence intensity of ROS in SH-SY5Y cells was increased gradually.The above results indicated that SM exposure caused the oxidative stress levels of hiPSC,hiPSC-NSC and Neuron cells to decrease,and the oxidative stress level of SH-SY5Y to increase.This part study showed that SM had significant toxicity to non-proliferation active nerve cells.On the one hand,it might be related to DNA damage caused by the formation of DNA adducts;on the other hand,SM could cause cell damage through other effects,such as cell energy metabolism disorder and oxidative stress.Several mechanisms of SM-induced injury to nerve cells are also present in terminally differentiated neurons,indicating that SM is not only targeted to the proliferation of active cells,but also has toxic damage to the proliferation of inactive cells.In addition,under the same degree of cell damage,the damage mechanism of hiPSC is significantly different from several other nerve cells,which may be related to the characteristics of hiPSC itself.Ⅲ.Effects of sulfur mustard on animal nervous systemIt has been found in clinical studies that the effects of SM on the nervous system can last for many years,with a significant long-term effect.Then,is this long-term effect due to failure to recover from the acute injury of SM or subsequent gradual development?It is not clear yet.In this part,the mice were given 20 mg/kg SM by subcutaneous injection at the back to establish the SM poisoning injury model.The effects of SM on the nervous system of mice after two weeks and two months of poisoning were dynamically observed through new object recognition test,open field test,suspended tail test and rotarod test.The results showed that after two weeks and two months of SM poisoning,compared with the solvent control group,there was no significant difference in the total distance of movement and the time and distance to enter the central area of the open field,the fixed test in the suspended tail test,the preference index for new objects,and the residence time on the rotating rod in the rotating rod test of the SM group.These results indicated that 20 mg/kg SM had no significant effects on anxiety,depressive,cognitive ability and neuromuscular function in mice two weeks and two months after poisoning.It suggested that the clinical neurological symptoms might not be the unrecovered acute injury effect,but rather the gradually developed nerve injury in the late stage.Long-term dynamic detection and observation of SM damage to the nervous system are still needed in the future.In summary:1.In this study,we established a SM-infected model based on human induced pluripotent stem cells and human neuroblastoma cells,which can well reflect the toxic response of nerve cells from the aspects of cell viability,the proportion of dead cells,LDH release and cell proliferation capacity,and provide a good cell model for the follow-up study on the toxicity mechanism and the screening of therapeutic drugs.2.SM has different toxicity on different stages and types of human nerve cells,and the most severe toxicity is on hiPSC.3.SM has a direct toxic effect on slowly proliferating nerve cells,and its toxic mechanism may be related to DNA damage,oxidative stress and energy metabolism disorder.The detailed neurotoxicity mechanism needs further investigation.4.The damage to nervous system caused by SM may not be an unrecovered acute effect,but rather a gradually damaged effect in the late stage.