Role And Mechanism Of Dexmedetomidine Regulate The Mitochondrial Fission In The Protection Of Vascular Endothelial Barrier Function In Septic Rats

Sepsis is a life-threatening organ dysfunction due to the host's imbalance in response to infection.The morbidity and mortality of sepsis are very high.In China,more than 3 million people suffer from sepsis every year,and more than 1/3 of them die of sepsis.Although the prevention and treatment measures for sepsis are updated constantly,the effect is not significant.The damage of vascular endothelial barrier function is the key to the multiple organ dysfunction and a critical pathological process of sepsis.Dexmedetomidine is a new sedative drug with high selectivity for the?₂-adrenergic receptor.It can excite the?₂-adrenergic receptor in locus coeruleus,reduce sympathetic activity,and produce dose-dependent sedative and hypnotic effects.In recent years,dexmedetomidine has been found to have some protective effects on the function of septic organs,which may be related to the inhibition of oxidative stress injury,the reduction of the inflammatory response,the reduction of theinflammatory mediators release and the inhibition of apoptosis.However,whether dexmedetomidine has a protective effect of the endothelial barrier function in sepsis is not clear at present.Previous studies have reported that the protective effect of dexmedetomidine on organ function may be closely related to regulating mitochondrial function.Mitochondria are organelles with a variety of critical physiological functions.They are in a dynamic balance of division and fusion in the cell.Mitochondria over-division under pathological stimuli seriously affects the normal function of mitochondria and further causes cell dysfunction and structural changes.Studies have found that dynamin related protein 1,drp1,and endoplasmic reticulum-mitochondrial contact have significant regulatory effects on mitochondrial division.Therefore,it is unclear whether dexmedetomidine can regulate the mitochondrial division of vascular endothelial cells by affecting drp1 and endoplasmic reticulum-mitochondrial contact.Therefore,in this study,We used the cecal ligation and puncture operation,and LPS stimulated vascular endothelial cells to investigate the protective effect and mechanism of dexmedetomidine on the endothelial barrier function of sepsis.The specific content includes three parts:?1?the effect of dexmedetomidine on the endothelial barrier function and organ functions of sepsis;?2?the effect of dexmedetomidine on the morphology and function of mitochondria;?3?the mechanism of dexmedetomidine regulating the mitochondrial division.Experimental methods:?1?The rat model of sepsis was made by cecal ligation and puncture;?2?the vascular permeability of lung was measured by Evans Blue;?3?the mesenteric microvascular permeability was measured by fluorescein isothiocyanate serum albumin?FITC-BSA?;?4?the vascular endothelial cells of rats were cultured to measure the transmembrane resistance?TER?and the permeability of FITC-BSA;?5?the expression of ZO-1 was detected by Western blotting and immunofluorescence;?6?the morphological changes of mitochondria and the translocation of drp1 were observed by laser confocal microscopy.Research contents:Part ?:Effect of dexmedetomidine on vascular endothelial barrier function and organ functions in septic rats1.The results of dexmedetomidine on the permeability of pulmonary and mesenteric microvessels and the expression of related regulatory proteins were observed in septic rats.We used the endothelial cells that were stimulated with LPS to measure the penetration rate of FITC-BSA,TER,and the expression ofZO-1.2.We found the effects of different doses of dexmedetomidine on the liver function,the renal function,myocardial injury markers,and the lactate in septic rats.We used the technique of laser speckle imaging to detect the outcome of dexmedetomidine on the liver and renal blood flow in septic rats.3.We observed the effects of different doses of dexmedetomidine on the survival rate and time in septic rats.Part?:Effect of dexmedetomidine on the morphology and function of mitochondria1.Mitochondria of heart,liver,kidney,and intestine of septic rats were extracted to detect the change of mitochondrial oxygen consumption rate.LPS was used to stimulate endothelial cells,andROS,ATP,and mitochondrial membrane potential are measured.2.The morphological changes of mitochondria were observed by transmission electron microscopy and confocal laser scanning microscope,respectively.Part III:The mechanism of dexmedetomidine regulating mitochondrial division.1.We used Western blotting to measure the effect of dexmedetomidine on the expression and activity of drp1.We Observed the effect of dexmedetomidine on the translocation of drp1by using immunofluorescence and Western blotting.Western blotting was used to detect the effect of dexmedetomidine on the expression and activity of ERK1/2.2.We found the results of dexmedetomidine and?₂ receptor inhibitorson F-actin expression and endoplasmic reticulum mitochondrial contact by using immunofluorescence.Results:Part ?:Effect of dexmedetomidine on vascular endothelial barrier function and organ functions in septic rats1.Dexmedetomidine can significantly decrease the vascular permeability of lungs and mesenteric microvessels,and increase the expression of related regulatory proteins.Dexmedetomidine can dramatically increase the transmembrane resistance of vascular endothelial cells after LPS treated,reduce the permeability of FITC-BSA,and significantly increase the expression of ZO-1.2.Dexmedetomidine can improve the liver and kidney function substantially,protect myocardium,reduce the content of lactate in arterial blood,and increase the blood flow of liver and kidney in septic rats.3.The survival rate and time of septic rats were significantly reduced.Dexmedetomidine can dramatically improve the survival rate and survival time.Part?:Effect of dexmedetomidine on the morphology and function of mitochondria1.Dexmedetomidine can substantially enhance mitochondrial function,which is manifested in inhibition of ROS production,an increase of ATP,mitochondrial membrane potential,and respiratory control ratio.2.In septic rats,mitochondrial division of vascular endothelial cells was increased,dexmedetomidine can significantly improve the mitochondrial morphology.Part ?:The mechanism of dexmedetomidine regulating mitochondrial division.1.In the LPS group,the expression of p-ERK1/2 was obviously increased,and the expression of drp1ser616 was enhanced considerably,and the expression of ser637 was decreased,the translocation of drp1was obviously increased.In the Dex group,the expression of p-ERK1/2 was significantly reduced,and the expression of drp1ser616 was considerably reduced,the expression of drp1ser637 was increased,the translocation of drp1 was decreased significantly.2.In the LPS group,the expression of F-actin was enhanced considerably,and it polymerized around the endoplasmic reticulum to form bundle stress fibers.The contact sites between endoplasmic reticulum and mitochondrion were increased.In the Dex group,the expression of F-actin was significantly decreased,and the bundle stress fibers were not polymerized around the endoplasmic reticulum,and the contact sites between endoplasmic reticulum and mitochondrion were obviously decreased.And use atipamezole could antagonize this effect of dexmedetomidine.Conclusion:1.Dexmedetomidine has a significant protective effect on vascular endothelial barrier function and organ functions of septic rats,and the survival rate and survival time of septic rats were significantly reduced,dexmedetomidine can improve the survival rate and survival time.2.The mitochondrial function of vascular endothelial cells in septic rats is significantly impaired,the mitochondrial division is increased,and mitochondria are significantly fragmented.Dexmedetomidine can dramatically inhibit the mitochondrial division of vascular endothelial cells in septic rats and reduce mitochondrial division.Improve mitochondrial morphology and structure to protect mitochondrial function.3.Dexmedetomidine may regulate mitochondrial division of vascular endothelial cells in septic rats through the following pathways:?1?Dexmedetomidine can control the activation and translocation of drp1 by inhibiting ERK1/2 phosphorylation.?2?Dexmedetomidine inhibits F-actin polymerization and endoplasmic reticulum-mitochondrialcontact?ER-Mito?through the?₂ receptor,which leads to a reduction of mitochondria division.