A Preliminary Study On The Protective Effect Of Estrogen On Vascular Smooth Muscle In Rats With Traumatic Hemorrhagic Shock

Objective:1.Optimize the method of anesthesia to establish rats with severe traumatic hemorrhagic shock;2.To determine the pathophysiological effects of estrogen on rats with severe traumatic hemorrhagic shock without resuscitation;3.To explore the role of GPER-mediated MYCOD in the regulation of E₂in vascular smooth muscle of rats with severe traumatic hemorrhagic shock.Methods:1.The rat model of severe traumatic hemorrhagic shock was randomly divided into four groups.After catheterization of left,right femoral artery and left femoral vein under pentobarbital sodium intraperitoneal anesthesia,the rats were divided into conscious physiological control group（CC,n=10）and continuous pentobarbital sodium intravenous physiological anesthesia group（AC,n=10）.After intubation of left and right femoral artery and left femoral vein under pentobarbital sodium anesthesia,the open wound of abdominal white line 5cm was made.The patients were divided into two groups:50%bloodletting in conscious state as experimental control group（CTHS,n=10）and 50%blood-letting in continuous intravenous anesthesia with pentobarbital sodium as empirical anesthesia group（ATHS,n=10）to observe the changes of physiological conditions,hemodynamics,arterial blood gas analysis,inflammatory factors and survival in each group.2.Therapeutic effect of17-β-estradiol（E₂）on rats with severe traumatic hemorrhagic shock without resuscitation,divided into five groups,sham operation group（Sham,）.Trauma shock treatment group:control group（Vehicle,0.4ml/kg,n=20）,E₂intervention group:low dose E₂group（L-E₂1mg/kg,n=6）,medium dose E₂group（M-E₂5mg/kg,n=20）,high dose E₂group（H-E₂:10mg/kg,n=6）.The physiological conditions,hemodynamics,arterial blood gas analysis,pathological injury of various organs and survival changes were observed in each group.An appropriate amount of blood fluid was extracted at Baseline（BL）,Shock,at the end of the experiment（End）,to detect the changes of inflammatory cytokines in STHS rats without resuscitation treated with E₂by ELISA method.3.GPER-mediated effects of 17β-estradiol on vascular smooth muscle structure and expression of MYCOD,contractile protein SM22-αandα-SMA in rats with severe traumatic hemorrhagic shock were randomly divided into five groups:Sham operation group（Sham group,n=10）,control group（Vehicle,DMSO:0.4ml/kg,n=10）and experimental group:medium dose estradiol group（M-E₂:5mg/kg,n=10）.;Medium dose estrogen+GPER agonist G₁group（M-E₂-G₁:E₂5mg/kg+G₁mg/kg,n=10）;Medium dose estrogen+GPER blocker G₁₅(M-E₂-G₁₅:E₂5mg/kg+G₁₅5mg/kg,n=10)extracted E₂to interfere with the thoracic aorta of unresuscitated STHS rats at the end of the experiment.The ultrastructure and pathological changes of smooth muscle were observed（H-E staining,Masson staining）,Western blot staining to detect the expression of GPER,MYOCD,specific contractile protein SM22-αandα-SMA in thoracic aortic smooth muscle of unresuscitated STHS rat model,and the effects of E₂on the expression of GPER,MYOCD,specific contractile protein SM22-αandα-SMA were observed.Immunohistochemical staining showed that E₂interfered with the expression of GPER,MYCOD,specific contractile protein SM22-αandα-SMA in aortic smooth muscle of unresuscitated STHS rats.Results:1.Compared with CC and AC groups,MAP,HR,+dp/dtmax,Pa CO₂,hemoglobin concentration（ct Hb）,bicarbonate concentration（c HCO₃^-）,base excess（BE）decreased significantly,-dp/dt max,Pa O₂,lactate（Lac）,blood glucose（Glu）,IL-6,TNF-αand SAA increased in ATHS and CTHS groups at the Shock.The level of interleukin-10（IL-10）in ATHS and AC groups was significantly higher than that in CTHS and CC groups.Hemodynamic changes after shock:compared with CTHS group,HR and-dp/dt max decreased significantly in ATHS group(MAP and±dp/dt max increased gradually in ATHS group and decreased gradually in CTHS group.Until the end of the experiment（End）,compared with ATHS group,p H,Pa O₂,ct Hb,Glu and c HCO₃^-in CTHS group continued to decrease,Pa CO₂,Lac,SAA and IL-6 continued to increase except TNF-α,and the increasing trend of IL-10 level was opposite.The survival rate in ATHS group was significantly higher than that in CTHS group.2.The 6 h mortality in L-E₂,M-E₂and H-E₂intervention group was significantly higher than that in Vehicle group（P<0.01）.There was no significant difference in 6 h mortality among M-E₂L-E₂and H-E₂groups（P>0.05）,but the cumulative fatality rate in M-E₂group was the lowest.The blood pressure in Sham group had no obvious change during the whole experiment,but the levels of MAP,HR and+dp/dt max in T_S,each group decreased significantly after the completion of traumatic shock model,and there was no significant difference between the two groups（P>0.05）,which was significantly lower than that in Sham group（P<0.05）,indicating that the degree of shock in traumatic shock model group was the same.After severe traumatic shock,MAP decreased rapidly to below 35mm Hg.After treatment with E₂without resuscitation,it was found that the blood pressure in 20min was slightly higher than that in Vehicle group,and there was no significant difference between E₂intervention group and Vehicle group at 20min.After 20 minutes,the blood pressure in M-E₂group reached stable hypotension level（50-60mm Hg）,while that in Vehicle group decreased continuously,which was significantly lower than that in M-E₂group（p<0.05）.The ventricular contractility was relatively stable in the E₂intervention group,especially in the M-E₂group,and at the end of the experiment(T_S0-1h-T_{S4-6 h}),there was no significant difference in p H,ct Hb,Glu,Lac,c HCO₃^-and BE between the E₂intervention group and the Vehicle group except Pa O₂,Pa CO₂and Glu（P>0.05）,indicating that the metabolic disorder of the E₂intervention group did not worsen with the prolongation of survival time.3.At the end of the experiment,compared with Sham group,the ultrastructural function of aortic vascular smooth muscle cells in severe traumatic shock group was damaged,dense plaque,dense body and intermediate filament were damaged,and the elastic fibers between smooth muscle cells were increased,especially in Vehicle group（P<0.01）.Compared with Vehicle group,M-E₂and M-E₂-G₁groups relied on GPER agonists and could be blocked by GPER blockers.Compared with Sham group,the expressions of GPER,MYOCD,specific contractile protein SM22-αandα-SMA in aortic smooth muscle of STHS rats were significantly increased.Compared with Vehicle group,the expressions of GPER,MYOCD,SM22-αandα-SMA in aortic smooth muscle of M-E₂and M-E₂-G₁groups were significantly higher than those in Vehicle group（P<0.05）.GPER agonist G₁promoted the expression mediated by GPER,whereas GPER antagonist inhibited its expression.Conclusion:1.The conscious STHS rat model fits well with the clinical practice,which is suitable for the study of pathophysiological mechanisms such as drug efficacy,hemodynamics,immune inflammation and body metabolism,and provides a model reference for the next part to study the pathophysiological effects of estrogen on traumatic hemorrhagic shock rats;2.Estrogen attenuates organ injury in early unresuscitated rats with severe traumatic hemorrhagic shock by regulating hemodynamics and internal environment,which may be related to the non-gene rapid regulatory response mediated by GPER;3.GPER fast signal pathway may be involved in regulating the expression of MYOCD and its related contractile proteins in vascular smooth muscle of rats with severe traumatic hemorrhagic shock,but the specific mechanism remains to be further studied.