Effect And Mechanism Of ERMC On Endotoxin-induced Acute Lung Injury

OBJECTIVE:Previous studies have found that organelle dysfunction,including mitochondrial dysfunction and endoplasmic reticulum stress,exists in endotoxin-induced acute lung injury.As an important structure between endoplasmic reticulum and mitochondria,ERMC plays an important role in the occurrence and development of many diseases,but it is not clear whether ERMC abnormalities exist in the process of endotoxin-induced acute lung injury.In this study,we mainly discussed the role of ERMC in the pathogenesis of endotoxin-induced acute lung injury.METHODS:In order to verify the role of ERMC in endotoxin-induced acute lung injury and screen the protein molecules that play a major role in it,we evaluated ERMC from animal model and cell model levels,which are divided into three parts.The first part is to construct a mouse model of endotoxin-induced acute lung injury.Firstly,we compared the survival rate and lung injury of BALB/c and C57BL/6 mice within 12 hours after tail vein injection with different doses of LPS（5,10 and 20 mg/kg）.In addition,we observed the effects of different administration routes on the survival rate of BALB/c mice.Through the analysis of the above results,C57BL/6 mice were selected as the research object to construct endotoxin-induced acute lung injury.C57BL/6 mice were injected with LPS 15 mg/kg via tail vein.The control mice were given normal saline of equal volume as control.The lung injury score,W/D,albumin content in BALF,BALF and TNF-a,IL-6,MDA content and SOD activity were observed at four different time points 1,4,8 and 12 hours after LPS injection.By comparing the changes at the above different time points,we come to the conclusion.To determine the best time point for LPS to act on mice.The second part mainly observed the changes of ERMC in endotoxin-induced acute lung injury.The model of endotoxin-induced acute lung injury was established in C57BL/6 mice.The changes of ERMC in lung tissue of endotoxin-induced acute lung injury mice were observed by transmission electron microscopy,and the expression of ERMC components was compared to screen the abnormal protein molecules.In addition,the changes of Ca²⁺and ROS in mitochondria,mitochondrial membrane potential and mitochondrial RCR were detected to reflect the abnormal lead of ERMC.Functional changes.In the third part,we constructed the endotoxin-attacking cell model,and observed whether down-regulating the expression level of IP3R-1 could improve mitochondrial function and inhibit ERMC formation in endotoxin-attacking cell model.The main indicators were the same as animal experiments.In addition,we detected the expression level of apoptosis-related proteins in mitochondrial pathway.RESULTS:In the first experiment,we found that the 12-hour survival rate of BALB/c mice was lower than that of C57BL/6 mice under the same LPS effect,and the difference was statistically significant,but the lung injury degree of the two mice was similar.There was no significant effect of different LPS administration routes on the 12-hour survival rate of BALB/c mice,so we established C57BL/6 mice as the substance of acute lung injury induced by endotoxin.Subsequently,by comparing the changes of each index at different time points after tail vein injection of 15mg/kg LPS,we found that the semi-quantitative score of lung injury,W/D and the albumin in the BALF were the highest 12 hours after LPS injection.In addition,the levels of TNF-a,IL-6 and MDA were the highest in serum and BALF,while the activity of SOD was the lowest.Therefore,we determined that the dosage of LPS in the model was 15mg/kg and the action time was 12 hours.In experiment 2,we found that the formation of ERMC increased,mitochondria swelled and vacuolated,the number of mitochondria decreased in a single field of vision and the cross-sectional area increased significantly;the expression of IP3R-1in lung homogenate and ERMC increased significantly,while the expression of Mfn-2 decreased significantly.The expression of other ERMC components such as IP3R-2,PACS-2 and Sig-1R did not change significantly.In addition,mitochondrial RCR and mitochondrial membrane potential were significantly decreased,while Ca²⁺and ROS contents in mitochondria were significantly increased.In experiment 3,we also found that ERMC formation increased and mitochondrial dysfunction were still observed in endotoxin-attacking cell model.However,silencing IP3R-1 by siRNA gene could reduce mitochondrial swelling,reduce ERMC formation and increase mitochondrial membrane potential and mitochondrial RCR level,decreasing the concentration of mitochondrial Ca²⁺and ROS production in mitochondria in endotoxin-attacking cell model.Moreover,decreasing the expression of IP3R-1 could significantly down-regulate the expression of caspase-9,caspase-3 and Bax proteins related to mitochondrial apoptosis in endotoxin-attacked cells,and significantly up-regulate the expression of Bcl-2.CONCLUSION:In the process of endotoxin-induced acute lung injury,the formation of ERMC in lung tissue cells of mice is increased,and the expression of IP3R-1 involved in Ca²⁺transport is significantly up-regulated,which leads to calcium overload and mitochondrial dysfunction.Down-regulation of the expression of IP3R-1 in cells can inhibit the formation of ERMC in endotoxin-attacked cell model,thereby improving mitochondrial function.The role of Mfn-2 in ERMC in endotoxin-induced acute lung injury is still unclear and needs further study.