Study On The Corrlation Between Serum Cytokines And Liver Function And Mitochondrial Injury With Sepsis

ObjectiveSepsis is the systemic inflammatory response syndrome (SIRS) caused by infection, of which essence is excess cytokines and inflammatory mediators in the host due to autoimmune injury, and further development may lead to septic shock, multiple organ dysfunction syndrome (MODS) and other serious complications. Although widely used antibiotics, the mortality of MODS remains high, sepsis is a crucial task in critical care medicine field. With the rapid development of molecular biology, it has been gradually recognized that the body's response to infection while the final performance of the cells, tissues, organs and overall level. The liver is the vital organs for material and energy metabolism in the body, mitochondrion is the main organelle to produce energy, which may be injured In the early stage of sepsis, liver function and mitochondrion are vulnerable to injury. Therefore, this study detect the septic serum TNF-α, IL-6 and IL-10 levels and liver function, morphological and mitochondrion injury, research the corrlation and possible mechanism between serum cytokines and liver function and mitochondrial injury in Sepsis. Through the research, we try to provide experimental evidence to prevent and treat for septic liver injury.Methods1. Animal preparationThe septic model was set up by receiving lipopolysaccharide(LPS) 10mg/kg by intraperitoneal injection. Eighty SPF SD male rats were randomly divided into eight groups: normal control group (n=10): intraperitoneal injected with 0.9% saline only; LPS group (n=70): intraperitoneal injected with LPS 10mg/kg. LPS group was divided into 7 time points: 3, 6, 12, 24, 36, 48, 72 hours points after LPS intraperitoneal injection2. Measurements and methods2.1 General state of animals Observed the reaction of the rats, including psychosis, diarrhea, vertical hair, skin and mucous bleeding, rectal temperature(T), heart rate(HR) and respiratory rate(RR) and the levels of LPS in the plasm.2.2 Markers of inflammatory reaction The levels of tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6),interleukin-10 (IL-10) and Mannose-binding lectin (MBL) in serum were determined by enzyme-linked immunosorbent assay (ELISA).2.3 Markers of liver biochemistry Liver function: Serum ALT, AST, TG, HDL and LDL were detected by automatic biochemistry analyzer.2.4 Histopathological observation of liver slices2.5 Detection of apoptosis in rat liver slices2.6 Markers of liver mitochondrial injuryMitochondrion of liver were isolated by differential centrifugation. Swelling and membrane potential of liver mitochondrion in rats were analyzed by Flow cytometry (FCM). Liver mitochondrion were observed through electronic microscopy and the degree of injury in liver mitochondrion were analyzed by Flameng semiquantitative evalution of ultrastructure.3. Statistical analysisThe datas were analyzed by SPSS13.0. All results were reported as mean±standard deviation(±s). Differences between groups were determined by ANOVA. If there was statistical significance and homogeneity of variance between groups, Least-significant difference (LSD) was used. If there was statistical significance and heterogeneity of variance, we used Games-Howell. Pearson correlation test was used when analyzing the correlation between membrane potential of mitochondrion and other datas. P values<0.05 was considered significant. Results1. General state of animals All animals in normal control group and LPS group showed septic symptoms and signs.Compared with normal control group, temperature(T), heart rate(HR) and respiratory rate(RR) increased significantly in 3hLPS group,6hLPS group,12hLPS group and 24hLPS group (P<0.05). Compared with normal control group, No statistically differences in HR and RR in 36hLPS group,48hLPS group and 72hLPS group(P<0.05).Compared with normal control group, plasma LPS levels increased significantly in LPS group (P<0.05).2. Markers of inflammatory reaction5.1 Serum TNF-αCompared with normal control group, serum levels TNF-αincreased significantly in 3hLPS group,6hLPS group,12hLPS group and 72hLPS group (P<0.05), and in 3hLPS group, serum levels TNF-αincreased the most significantly. Compared with normal control group, no statistically differences in 24hLPS group and 36hLPS group (P >0.05).5.2 Serum IL-6Compared with normal control group, serum levels IL-6 increased significantly in 3hLPS group,6hLPS group,12hLPS group and 24hLPS group (P<0.05), and in 3hLPS group, serum levels IL-6 increased the most significantly. Compared with normal control group, no statistically differences in 36hLPS group,48hLPS group and 72hLPS group (P >0.05).5.3 Serum IL-10Compared with normal control group, serum levels IL-10 increased significantly in 3hLPS group,6hLPS group,12hLPS group,24hLPS group,36hLPS group and 48hLPS group (P<0.05), and in 6hLPS group, serum levels IL-10 increased the most significantly. There was no statistically differences in 72hLPS group and normal control group (P >0.05). 3. Markers of liver biochemistry3.1 Serum ALT,AST and LDHSerum ALT,AST and LDH were significantly higher in 3hLPS group,6hLPS group than that in normal control group (P<0.05). Compared with normal control group, serum AST and LDH increased significantly in 72hLPS group (P<0.05).4. Detection of apoptosis in rat liver slices Apoptotic rates were significantly higher in 6hLPS group,12hLPS group,24hLPS group and 36hLPS group than that in normal control group (P<0.05), and in 12hLPS group, apoptotic rates increased the most significantly. Compared with normal control group, no statistically differences in 24hLPS group and 72hLPS group (P >0.05).5. Markers of liver mitochondrial injury5.1 Swelling and membrane potential of liver mitochondrionThe difference wasn't significant in swelling of liver mitochondrion among three groups (P>0.05).Compared with normal control group, membrane potential of liver mitochondrion decreased significantly in 3hLPS group,6hLPS group,12hLPS group,24hLPS group,36hLPS group and 48hLPS group (P<0.05), and in 24hLPS group, membrane potential of liver mitochondrion decreased the most significantly. There was no statistically differences in 72hLPS group and normal control group (P >0.05).5.2 Morphology of liver mitochondrionCompared with normal control group, semiquantitative evaluation scores of liver mitochondrion ultrastructure increased significantly in 12hLPS group,24hLPS group,36hLPS group and 48hLPS group (P<0.05), and in 24hLPS group, semiquantitative evaluation scores of liver mitochondrion ultrastructure increased the most significantly. There was no statistically differences in 72hLPS group and normal control group (P >0.05).6. Correlation between this datasSerum TNF-αlevels was positively correlated with serum ALT,AST and LDH levels(r=0.475,P<0.05;r=-0.721,P<0.05;r=0.533,P<0.05;r=0.633,P<0.05). Serum IL-6 levels was positively correlated with serum ALT,AST and LDH levels(r=0.42,P<0.05;r=0.52,P<0.05;r=0.394,P<0.05). Serum IL-10 levels was positively correlated with serum ALT and AST levels(r=0.301,P<0.05;r=0.278,P<0.05);no significant correlated relationship between Serum IL-10 and serum LDH levels(r=0.099,P>0.05). There was no significant correlated relationship between membrane potential of liver mitochondrion and serum ALT,AST and LDH level(sr=0.042,P>0.05;r=0.007,P>0.05;r=0.182,P>0.05).Membrane potential of liver mitochondrion was negatively correlated with serum IL-6 and IL-10 levels(r=-0.451,P<0.05;r=-0.721,P<0.05);no significant correlated relationship between membrane potential of liver mitochondrion and serum TNF-αlevels (r=-0.1,P>0.05).Conclusions1. Septic serum TNF-α, IL-6 and IL-10 levels showed a phase of change, the peak of the serum TNF-α, IL-6 and IL-10 levels earlier than liver function, morphology and liver mitochondrial injury time.2. Septic serum TNF-αlevel showed two peaks of changes, indicating there may be exist a secondary strike.3. Septic serum TNF-α, IL-6 and IL-10 levels were positively correlated with liver dysfunction, indicating serum TNF-α, IL-6 and IL-10 levels may be the factors of liver injury.4. Liver mitochondrial injury in of septic rats is reversible. The membrane potential was negatively correlated with serum IL-6 and IL-10 levels, indicating serum TNF-α, IL-6 and IL-10 levels may be the factors of liver mitochondrial injury.