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EFFECT OF DEXMEDETOMIDINE ON THE SURVIVAL RATE AND LIVER PROTECTION IN SEPTIC MICEObjective: To observe the effects of dexmedetomidine(DEX)on the survival,inflammatory response and acute liver injury of septic mice induced by cecal ligation puncture(CLP).Methods: 105 male C57 BL / 6 mice were randomly divided into the following groups:(I)Sham operation group(CON);(II)CLP induced liver injury + normal saline group(CLP);(III)CLP induced liver injury +DEX group(CLP + DEX).CLP was used to establish model of septic liver injury in mice.DEX 20 ?g / kg or the same amount of normal saline was injected intraperitoneally at 0,2 and 4 hours after operation.The postoperative state of the mice was observed and the mortality was calculated.Liver samples and blood samples of mice were collected at 6,12 and 24 hours after CLP for further study.Hematoxylin eosin staining(HE)was used to detect the morphological changes of liver tissue.Serum ALT,AST,TNF-?,IL-1 ?,IL-6 and IL-10 were measured at 6,12 and 24 hours after CLP.Results:1.After CLP operation,the survival rate of CON group was100%;the survival rates of CLP Group and CLP + DEX group were 81%and 100% at 12 h,58.1% and 83.8% at 24 h,respectively.The 5-day survival rate was 3.2% in CLP group and 12.9% in CLP + DEX group.The 5-day survival rate was significantly different between CLP group and CLP + DEX group.(P < 0.05).2.The results of HE staining showed that the hepatocytes in CON group were orderly arranged,without edema and necrosis;compared with CON group,CLP group had obvious pathological changes,such as inflammatory cell infiltration,hepatocyte swelling and disordered arrangement;the pathological changes in CLP +DEX group were better than CLP group.3.Detection results of liver injury markers: ALT and AST in CLP group increased significantly at 6 h,decreased significantly at 12 h and increased significantly at 24 h,while ALT and AST in CLP + DEX group were significantly lower than those in CLP group at 6 h and 24 h(P < 0.05).There was no significant difference between the two groups at 12 h.4.ELISA results showed that CLP could induce the increase of inflammatory cytokines in mice.Compared with CLP + DEX group,the levels of TNF-?,IL-1? and IL-10 in CLP group were significantly increased at 6 h after CLP operation.After 12 hours of sepsis induction,the levels of TNF-?,IL-1? and IL-10 decreased in CLP group.There was no significant difference between CLP Group and DEX + CLP group at 12 hours.At 24 hours,the levels of the above three inflammatory factors in CLP group were significantly higher than those in CLP + DEX group.The results of IL-6 were different from those of the above-mentioned inflammatory cytokines.IL-6increased in both CLP group and CLP+DEX group at 6-12 h,and there was no statistical difference between the two groups.IL-6 in CLP + DEX group decreased significantly at 24 h,and was still at a high level in CLP group(P < 0.05).Conclusion: DEX has protective effect on acute liver injury induced by CLP,and the 24 h survival rate of mice has increased.At 24 h after the CLP operation,the expression of inflammatory cytokines and markers of liver injury in mice decreased.PART ? THE RELATIONSHIP AND MECHANISM BETWEEN THE PROTECTIVE EFFECT OF DEXMEDETOMIDINE ON LIVER INJURY AND AUTOPHAGY LEVEL IN SEPTIC MICEObjective: To explore the relationship between the anti-inflammatory effect of DEX in sepsis and autophagy,and to study the molecular mechanism of DEX inhibiting inflammatory response and alleviating liver injury in septic mice from the perspective of autophagy.Methods: The same group and CLP procedure as part I.The mice were divided into(I)Sham operation group(CON);(II)CLP induced liver injury + normal saline group(CLP);(III)CLP induced liver injury +DEX group(CLP + DEX).The ultrastructure of liver was observed by transmission electron microscope.The expression of LC3?was detected by immunofluorescence.The expressions of autophagy related proteins LC3?,Beclin-1,p62,LAMP-2,AMPK,p-AMPK and SIRT1 were detected by Western blot.The expression of SIRT1 was detected by immunohistochemistry.Results: 1.Transmission electron microscopy showed that autophagosome structure could be clearly observed at different time points in CLP + DEX group.In CLP Group,typical autophagosome structure was observed in hepatocytes at 6 h and 12 h,and no obvious autophagosome was found at 24 h.2.Western blot results showed that the levels of LC3?and Beclin-1 in liver tissue of mice decreased at 6 h after CLP,reached the peak at 12 h,and then decreased at 24 h in CLP group.Compared with CLP + DEX group,the levels of LC3?and Beclin-1 in CLP group were lower at 6 and 24 hours(P < 0.05),and no significant difference was observed at 12 hours.At 24 h after CLP,the content of p62 in CLP Group was significantly higher than that in CLP +DEX group(P < 0.05).There was no difference in the level of LAMP-2between CLP Group and CLP + DEX group at each time point.The level of p-AMPK in CLP Group increased at 12 h and decreased at 24 h after CLP.In contrast,the level of p-AMPK in CLP + DEX group increased significantly from 12 h to 24 h,and the difference of p-AMPK level between 24 h CLP Group and CLP + DEX group was statistically significant(P < 0.05).Compared with DEX group,SIRT1 level in CLP +DEX group increased significantly at 24 h(P < 0.05).3.The semi quantitative results of immunofluorescence showed that the positive signal of LC3?was strong at 12 h in CLP Group and weakened at 24 h,while the positive signal was enhanced at 12-24 h in CLP + DEX group.The difference of semi quantitative results between CLP Group and CLP+ DEX group at 24 h was statistically significant(P < 0.05).Conclusion: 24 hours after CLP operation,autophagy activity in liver tissue of septic mice treated with DEX is enhanced,AMPK / SIRT1 level is up-regulated,and the expression of inflammatory cytokines decreased.PART ? PROTECTIVE EFFECT OF DEXMEDETOMIDINE ON LO2 HEPATOCYTE INJURY AND DETERMINATION OF AUTOPHAGY FLUXObjective:To investigate the regulatory role of SIRT1 in dexmedetomidine(DEX)induced autophagy and the intensity of autophagy flux after SIRT1 was inhibited.Methods: L02 hepatocyte injury model was established by stimulation of D-GalN/LPS in vitro.1.In order to determine the optimal concentration of D-GalN/LPS to establish liver injury model,the cells were divided into two groups:(1)Control group(CON);(2)Hepatocyte injury group(D-GalN/LPS).Cell viability on the cells treated with LPS(10 ng/mL)and different concentrations of D-GalN(0.1,1,10,50,100 mM)were detected by cell counting kit-8(CCK-8).2.In order to determine the optimal concentration of DEX in hepatocyte injury model,L02 cells were divided into three groups:(1)CON;(2)D-GalN/LPS;(3)DEX treatment group(DEX+D-GalN/LPS).DEX treatment group was divided into four sub-groups according to the dose of DEX(1,10,20,100 ?M),and each sub-group was treated with D-GalN(10 mM)/LPS(10ng/mL).Cell viability were detected at 6 h,12 h,24 h and 48 h by CCK-8.3.L02 cells were divided into six groups:(1)CON;(2)D-GalN/LPS;(3)DEX+D-GalN/LPS(DEX divided into 1 and 10 ?M concentration sub-groups);(4)SIRT1 inhibitor group(DEX+EX527+D-GalN/LPS,DEX divided into 1 and 10 ?M concentration sub-groups).The concentrations of D-GalN,LPS and EX527 were 10 mM,10 ng/mL and10 ?M,respectively.The expression of SIRT1 was detected by Western blot.4.L02 cells were divided into four groups:(1)CON;(2)D-GalN/LPS;(3)DEX+D-GalN/LPS;(4)DEX+EX527+D-GalN/LPS.The concentrations of D-GalN,LPS,DEX and EX527 were 10 mM,10ng/mL,1 ?M and 10 ?M,respectively.Intracellular ROS(reactive oxygen species)levels were measured by 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)method.5.Double labeled adenovirus m RFP-GFP-LC3 was transfected into cells,and the infected conditions of adenovirus were determined by pre-experiment.The groups of cells and the concentration of drugs were the same as that of step(3).The formation of autophagosome and lysosome were observed by fluorescence microscope after 24 h and 48 h of cell culture.Results: 1.Under the co-stimulation of D-GalN/LPS,compared with the CON group,the survival rate of L02 cells decreased gradually with the increase of D-GalN concentration in a dose-dependent manner.Cell viability decreased significantly when the treatment concentration of D-GalN/LPS was equal to or more than 20 mM.In the presence of10 mM D-GalN,the cell viability demonstrated a moderate decrease.The concentrations of D-GalN and LPS were determined as 10 mM and 10ng/mL.2.The results showed that 1,10,20 ?M DEX had protective effect on hepatocyte injury,and 1,10 ?M DEX had better protective effect.There were significant differences between D-GalN/LPS group and 1,10?M DEX group at 6,12,24,48 h(P < 0.05).High concentration of 100 ?M DEX had no protective effect on hepatocytes,and there was no significant difference compared with D-GalN/LPS group.Therefore,the concentration of 1,10 ?M DEX was used for subsequent experiments.3.Western blotting showed that EX527 significantly down regulated the SIRT1 level of L02 cells co-cultured with DEX(1,10 ?M)and D-GalN/LPS.4.Compared with D-GalN/LPS group and DEX+EX527+D-GalN/LPS group,the content of ROS in DEX+D-GalN/LPS group decreased at 24~48 h(P < 0.05).5.When MOI = 100,the fluorescence intensity of the cells was moderate,and the cells grew well,so the concentration(MOI = 100)of the virus was selected for subsequent experiments.After 24 hours of co-culture,there were less yellow autophagosomes and more red lysosomes in D-GalN/LPS group,more yellow autophagosomes in DEX+D-GalN/LPS group,and more autophagosomes in L02 in DEX+EX527+D-GalN/LPS group.The difference of autophagosomes formation between DEX+D-GalN/LPS group and D-GalN/LPS group,DEX+EX527+D-GalN/LPS group was statistically significant(P < 0.01).After 48 hours,red autolysosomes increased in DEX+ D-GalN/LPS group,whileautolysosomes were less in D-GalN/LPS group and DEX+EX527+D-GalN/LPS group.Compared with D-GalN/LPS group and DEX+EX527+D-GalN/LPS group,the difference of autolysosomes formation in DEX+D-GalN/LPS group was statistically increased(P < 0.01).Conclusion: SIRT1 inhibitor can significantly increase intracellular ROS level and reverse the effect of DEX on autophagy flux. |
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