Clinical And Basic Research Of Myocardial Injury In Sepsis

Objective(1)To observe the relationship between arterial blood lactate clearance and the prognosis and myocardial injury of patients with septic shock after early goal-directed therapy.(2)To investigate the relationship between mitophagy and sepsis-induced myocardial injury at different stages of sepsis.(3)To investigate the relationship of LAMP2-mediated mitochondrial autophagy lysosomal pathway in autophagy,apoptosis and energy metabolism during sepsis myocardial injury.Methods(1)One hundred fifty-seven Patients with septic shock after early resuscitation and arterial blood lactate(Lac)>2mmoL/L were enrolled from August 2013 to July 2016 in ICU at Peking Union Medical College Hospital,retrospectively.Patients were divided into group A:ScvO2>80%,PI>1.4,group B:ScvO2>80%,PI<1.4,group C:ScvO2<80%,PI>1.4,group D:ScvO2<80%,PI<1.4.baseline data of the patients,APACHE II and SOFA at admission,clinical hemodynamic parameters,tissue perfusion parameters,and arterial blood lactic acid levels at 2h,4h,and 6h after resuscitation were completed.SPSS 19.0 statistical software was used for statistical analysis of the data.(2)Forty SD rats were divided into 4 groups according to the random number table:SAM group,CLP6h group,CLP12h group,CLP18h group.In the SAM group 18 h after surgery,the three CLP groups were anesthetized and opened at 6 h,12 h,and 18 h after operation.Blood samples were collected for preparation of cTnI and heart samples were collected for electron microscopy,immunofluorescence staining;The mRNA levels of Lc3-Ⅱ,Lamp2 were detected by RT-PCR and the protein levels of Lc3-II was detected by Western Blot.Assess autophagy and its role in different stages of sepsis.(3)Forty SD rats were randomly divided into 4 groups:SAM+Baf-A1 group and CLP+Baf-A1 group received bafilomycin A1 by 0.3 mg/kg at 1 hour after the sham operation and the CLP operation.The SAM and CLP groups received the same dose of DMSO.The blood samples of the four groups were collected 18 h later for preparation of cTnI and the heart samples of the four groups were collected 18 h later for preparation of electron microscopy,immunofluorescence staining and TUNEL staining.The mRNA levels of Lamp2 were detected by RT-PCR and the protein levels of Lc3-Ⅱ,Bik and Lamp2 were detected by Western Blot.Assess the roles and relationships of autophagy and apoptosis in the development of sepsis myocardial dysfunction were identified.Results(1)①The rate of lactic acid clearance in the group C were respectively 34.57(21.44,44.20)%,58.33(30.19,70.79)%,and 71.43(53.75,82.79)%at T2、T4、T6.② The incidence of myocardial damage was the highest in group B(85%)and the lowest in group A(45.7%)(P<0.008)on the first day after ICU admission.The incidence of myocardial damage was the lowest(29.3%)in group C and the highest(70%)in group B On the third day after ICU admission(group C vs group B,P<0.008.③The logistic regression analysis showed that the rate of lactic acid clearance at T4 point in group B was related with incidence of myocardial damage at 48h and 72h(OR=0.917,95%CI0.851～0.988;OR=0.920,95%CI 0.854～0.992).④There was no significant difference in the ICU stay time and mortality at 28 days between the 4 groups(P>0.05).(2)〖 The serum cTnI increased gradually after CLP 6h,12h and 18h after operation.There were significantly difference between the four groups(all P=0.000);②LC3-II mRNA expression was higher in CLP6h group than in SAM group and CLP18h group(all P=0.000);LC3-II mRNA was higher in CLP12h group than in SAM group(P=0.028);LC3-II protein level in SAM group was lower than that in CLP6h group(P=0.001),CLP12h(P=0.002),and CLP18h(P=0.000).③Atg5 mRNA level in CLP6h group was higher than that in SAM group(P=0.002),CLP12h group(P=0.012)and CLP18h group(P=0.009).④ Lamp2 mRNA peaked at CLP6h,higher than that of CLP18h group(P=0.037).(3)①The cTnI in the CLP+Baf-Al group was greater than that in the CLP group(P=0.000),and the cTnI in the SAM+Baf-Al group was greater than that in the SAM group(P=0.002).(2)The apoptosis index of CLP+Baf-A1 group was higher than that of CLP group(P=0.001);The apoptosis index of CLP group was higher than that of SAM group(P=0.013).(3)LC3-II protein level in CLP group was higher than that in SAM group and SAM+Baf-A1 group(P=0.000,P=0.007);CLP+ Baf-Al group was higher than SAM+Baf-A1 group(P=0.024);There was no statistically significant difference between the CLP+ Baf-A1 group and the CLP group(P=0.270).(4)Bik protein level in CLP+Baf-A1 group was higher than that in CLP group(P=0.028);CLP group was higher than SAM group(P=0.000)(5)The Lamp2 mRNA level in CLP18h group was the highest in the four groups,higher than that in CLP+ Baf-A1 group((P=0.038)),higher than SAM group(P=0.06);Lamp2 protein level in CLP18h group was higher than SAM group(P=0.000)and CLP+Baf-A1 group(P=0.009).(6)The ATP level in the CLP+Baf-A1 group was lower than that in the CLP group(P=0.032);the ATP level in the CLP group was lower than that in the SAM group(P=0.022).Conclusion(D1)①Unclearance lactic acid is associated with microcirculatory disturbance and cell dysfunction after early resuscitation in patients with septic shock;②Patients with septic shock with slower lactic acid clearance after early resuscitation are prone to myocardial damage;③Early recovery of patients with septic shock After the onset of microcirculatory disturbance and cell dysfunction,the degree of myocardial damage is the most severe,and the incidence of myocardial injury is the highest;④ In patients with septic shock,when the microcirculation is corrected after early resuscitation,cell dysfunction and concomitant myocardial damage.(2)①The level of mitochondrial autophagy is different in different stages of sepsis,increased in early stage and declined in late stage;②The presence of LC3-Ⅱ gene and protein in the late stage of sepsis is inconsistent,which may be related to the inhibition of Lamp-2.③The slow elimination of late autophagosomes in sepsis is associated with myocardial cell mitochondrial damage.④The degree of mitochondrial damage associated with myocardial injury in sepsis;(3)Autophagy and apoptosis are involved in the whole process of development and progression of sepsis-induced myocardial suppression.In the late stage of sepsis,slow autophagy is a factor that induces apoptosis.Lamp2 is a potential factor regulating the balance of autophagy and apoptosis in sepsis-induced myocardial suppression.① Inhibition of lysosomal membrane protein LAMP-2 expression may lead to further elevation of the autophagy marker protein LC3-II,verifying that late mitochondrial LC3-II gene and protein expression mismatches are associated with slow clearance of autophagy proteins;②Inhibiting the expression of LAMP-2 in myocardium of sepsis rats leads to the slow degradation of autophagosome and the expression of apoptotic gene BIK,which up-regulates apoptosis;③LAMP-2 is a potential factor regulating the expression of apoptotic gene BIK in rat with sepsis.