The Role And Mechanism Of STIM1 Activation In Paraquat Induced Acute Lung Injury

Background: Paraquat(PQ)is a commonly used agent causing death from self poisoning with herbicide,especially in developing countries.There is currently a lack of effective treatment to reverse this process.A large number of studies have reported on the chronic pulmonary fibrosis induced by PQ poisoning,while the underlying mechanisms of the acute lung injury caused by severe PQ poisoning still need further efforts to the exploration.Calcium signaling and oxidative stress are tightly linked to cell cycle and cell death in response to a number of stress conditions.PQ is recognized as a recruiter of oxidative stress,but it is still uncertain whether calcium is involved in the regulatory mechanism.Recent study indicated that stromal interaction molecule 1(STIM1)is a functional calcium sensor in the endoplasmic reticulum(ER),which can effectively sense the concentration changes of the calcium and in order to participate in the regulation of cell functions.However,the regulatory mechanisms and the role of STIM1 in paraquat(PQ)-induced acute lung intoxication remain elusive.The aim of this study was to explore the molecular and cellular mechanisms of PQ induced acute intoxication in the lung,and further determine whether calcium signaling and reactive oxygen species(ROS)participate in the regulatory mechanism.Methods: 1.C57BL/6 mice were treated with 75mg/kg PQ for 48 h by intraperitoneal injection to construct a animal model of acute PQ intoxication in vivo and 16 HBE was used for constructing the model in vitro;2.LDH,MTT assay and trypan blue staining were used to detect cell death in PQ treatment,and TUNEL assay was used to detect apoptosis;3.In order to explore the regulation of STIM1 in the acute lung injury caused by PQ,we constructed the STIM1 overexpression plasmid and we used Crispr-cas9 technique to knock out STIM1 in 16 HBE or inhibit the SOCE activity by 2-APB;4.The flow cytometry detected the cell-ratio in the cell cycle at each phases;5.L-012 was used to detect the production of extracellular ROS,while DHE staining was used for the detection of the intracellular ROS production;6.Fluorescence probe(Fluo-8 AM)was used for detecting the intracellular calcium transients;7.QPCR and Western blot were used to detect the transcription and translation changes in the related genes.Conclusion: Our data demonstrated that PQ(500?M,24 hours)induced intracellular ROS production and enhanced store-operated calcium entry(SOCE)activity which is correlated to STIM1 activation.In addition,PQ(500?M,24-48 hours)caused accelerated cell cycle G1/S transition and then arrested in S phase.While knockouting STIM1 by CRISPR-CAS9 in 16 HBE or inhibiting STIM1 mediated SOCE activation ameliorated cell death caused by acute PQ treatment,which also leaded to alleviating the cell accumulation in S phase through the modulation the expression of cyclinD1,p21,cyclinA2 and CDK2.In conclusion,STIM1 plays an important role in PQ induced cell cycle arrest and cell death in acute lung injury,which may provide us a new potential opportunity to target paraquat induced intoxication.