Clinical And Basic Research On Sepsis-related Vascular Injury

Background:Peripheral vascular disorders leading to tissue hypoperfusion play a central role in the pathophysiology of organ failure in septic shock.The Doppler snuffbox resistive index(SBRI)can be an accurate parameter to evaluate the status of peripheral vasculature at the bedside.We evaluated whether the SBRI is related to lactate levels or the peripheral perfusion index(PI)and its ability to predict lactate clearance in septic patients.Uncoupling protein 2(UCP2)is important in maintaining mitochondrial morphology and function in cells.Studies suggest that UCP2 may have a protective effect in sepsis vascular injury,but the specific mechanism is still unclear.Our research is intended to investigate the role of UCP2 in the pathogenesis of sepsis related vascular smooth muscle injury at the cellular level,based on in vitro and in vivo tests.Methods:Part 1:We conducted a prospective observational study in Intensive Care Unit(ICU)of Peking Union Medical College Hospital in China.From July 2019 to December 2019,all consecutive adult patients with septic shock who required ICU admission were included.At the same time,twenty stable postoperative patients were studied as a control group.We recorded the haemodynamic parameters,including the PI and arterial blood lactate,which were measured simultaneously after patient recruitment.The lactic acid clearance rate was calculated in the first 6 hours after admission to ICU.SBRI,velocity time integral(VTI)and left ventricular outflow tract area were measured by bedside ultrasound,and cardiac index(CI)and systemic vascular resistance index(S VRI)were calculated according to the formula.The correlation between SBRI and PI and arterial blood lactic acid was compared.Based on lactate clearance in the first 6 h,the septic shock patients were divided into a group with those with lactate clearance-20%and a group with those with lactate clearance<20%.The ability of SBRI and PI to predict 6h lactic acid clearance was compared.Part 2:The model of sepsis vascular smooth muscle(HA-VSMC)injury induced by Lipopolysaccharides(LPS)was established.In cell experiments,UCP2 overexpressed plasmids and UCP2 knockdown siRNA were used to transfect VSMC to increase or inhibit UCP2 expression.The experiment was divided into six groups:vector group,vector+LPS group,UCP2+LPS group,SiControl group,siControl+LPS group,UCP2 siRNA+LPS group.Mitochondrial morphology was observed by transmission electron microscopy.mRNA levels of mitochondrial fission-related molecules Drp1,fusion related molecules Mfn2 and Opal were detected,and protein levels of Drpl were detected by Western Blot,and mitochondrial fission/fusion status of vascular smooth muscle cells was evaluated.Markers of and VSMC mitochondrial damage were determined respectively:apoptosis of VSMC cells(TUMEL staining),mitochondrial membrane potential,mitochondrial mtDNA detection,reactive oxygen species(ROS)and ATP production.Part 3:Wild-type mice of C57BL/6C strain were selected to establish a sepsis mouse model through cecal ligation and puncture(CLP).The expression of UCP2 was inhibited by intravenous injection of genipin into inner canthus.Blank control group and Sham group were set up.To evaluate the status of mitochondrial dynamics,the mitochondrial morphology of vascular smooth muscle cells of mesenteric artery in each group was observed by electron microscopy,and the expression of fission and fusion-related proteins was detected.Vascular smooth muscle cells and mitochondrial damage markers in mice mesenteric artery were determined in groups:mitochondrial membrane potential,mitochondrial DNA damage,ROS,ATP production,etc.HE staining was used to observe the changes of mesenteric artery microstructure,and TUNEL staining was used to evaluate the apoptosis of vascular smooth muscle cells.Results:Part 1:We evaluated 44 patients with septic shock in the study group and 20 stable postoperative patients in the control group.Patients with septic shock had higher Sequential Organ Failure Assessment scores,procalcitonin levels,cardiac index(CI)and lactate levels than patients in the control group.The SBRI was correlated with the PI and lactate level.The CI was not correlated with lactate level in the patients examined.Based on lactate clearance in the first 6 hours,the septic shock patients were divided into two groups:one with lactate clearance?20%(n=28)and the other with lactate clearance<20%(n=16).The CI was not significantly different between the two groups.The SBRI of the lactate clearance<20%group was higher than that of the lactate clearance?20%group and the control group.The PI of the lactate clearance<20%group was lower than that of the lactate clearance?20%group and the control group.The SBRI cutoff value for predicting 6-h lactate clearance after resuscitation was?1.09,with a sensitivity of 68.8%and a specificity of 85.7%.The PI cutoff value for predicting 6-h lactate clearance after resuscitation was<0.99,with a sensitivity of 64.3%and a specificity of 81.2%.The SBRI was significantly better than the PI for predicting 6-h lactate clearance after resuscitation[area under the curve(AUC):0.805 vs.0.703,P<0.05].Part 2:After LPS intervention,the vector+LPS group had shorter mitochondrial length,increased expression of DrplmRNA and protein,imbalance of division and fusion,increased cell apoptosis,and mitochondrial damage,including:decreased mitochondrial mDNA,decreased membrane potential,increased ROS production,and further decreased ATP synthesis.After inhibition of UCP2 expression,the mitochondrial length and diameter of VSMC in the UCP2 siRNA+LPS group were further shortened compared with that in the siControl+LPS group,the expression of Drp1mRNA and protein were increased,the cell apoptosis was increased,and the mitochondrial damage was aggravated.With the increase of UCP2 expression,the mitochondrial length and diameter of cells in the UCP2+LPS group increased,the expression of Drp1mRNA and protein decreased,the cell apoptosis decreased,and the mitochondrial damage decreased compared with that in the vector+LPS group.Part 3:After CLP build mould,electron microscopy showed the CLP group than the Control group of mesenteric artery VSMC mitochondria length to diameter shortening,mitochondrial division Drp1mRNA related molecules and protein expression increased,mitochondrial mDNA decline,membrane potential,tip mitochondria damage,increased ROS,ATP synthesis decrease,affect the cell energy metabolism,cell apoptosis increased,HE dyeing prompt mesenteric artery pathological damage.However,the inhibition of UCP2 expression by genipin aggravated the excessive mitochondrial division of the mesenteric artery VSMC,and the degree of cell and vascular smooth muscle damage was aggravated.Conclusions:Part 1:The Doppler SBRI is correlated with tissue perfusion parameters in critically ill patients.An abnormal SBRI may be better than the PI for predicting poor lactate clearance in septic patients.Further investigations are required to determine whether correcting an abnormal SBRI and PI may improve the success rate of septic shock resuscitation.Part 2:LPS can cause excessive mitochondrial division of VSMC.UCP2 overexpression can regulate the imbalance of mitochondrial fission and fusion in vascular smooth muscle cells caused by LPS,thus reversing the vascular smooth muscle injury caused by sepsis.Inhibition of UCP2 expression aggravates the imbalance of mitochondrial fission and fusion in VSMC induced by LPS.Part 3:Excessive mitochondrial division of VSMC is present in sepsis-associated mesenteric artery vascular smooth muscle injury.Inhibition of UCP2 expression aggravates the degree of sepsis related vascular injury,leading to irreversible injury of mesenteric artery vascular smooth muscle,It suggests that UCP2 is a key protein for vascular protection in sepsis.UCP2 protects the vascular smooth muscle function of the mesenteric artery in sepsis by maintaining mitochondrial homeostasis.