The Study Of Tetramethylpyrazine Improving Mitochondrial Dysfunction In Septic Rat Hepatocytes And Its Molecular Mechanism

BackgroundSepsis is a clinical syndrome caused by severe infections that always trigger excessive inflammatory responses.Sepsis,septic shock and multiple organ dysfunction syndrome(MODS)represent the sequential stages of the disease process.The end stage of MODS is organ failure,which is commonly associated with poor clinical outcome and a high mortality rate.In sepsis,the liver plays the role of immune organswith ability to eliminatebacteria and produce inflammatory factors.In addition,sepsis-induced multiple organ injury and failure may be crucially caused by the deterioration of mitochondrial function and consequent failure of cellular energy metabolism.Mitochondria are required for normal cellular function by providing energy,utilizing approximately 98%of total body oxygen.Therefore,mitochondria play an important role in the maintenance of normal organ function..On the other hand,mitochondrial structural integrity is essential for preserving normal mitochondrial function and energy metabolism.During sepsis,the main pathological features are cytopathic hypoxia,disorder of oxygen utilization,which leads to the decrease of energy production,and mitochondrial dysfunction is the fundamental cause of oxygen utilization disorder in cells.Mitochondrial damage and dysfunction may occur in sepsis,and mitochondrial injury may induce proinflammatory mediators release and aggravate inflammatory response.While mitochondrial dysfunction leads to the lack of energy in the tissue cells,causing serious microcirculatory disturbance,and multiple organ failure.Mitochondrial dysfunction is often associated with multiple organ failure caused by an imbalance of systemic inflammatory response.However,the exact mechanism of mitochondrial dysfunction is not fully understood.Tetramethylpyrazine(TMP),an active extract from the Chinese herb"Ligusticum chuanxiong",is widely andvery safely used for the treatment of inflammatory and cardiovascular diseases in traditional Chinese medicine.TMP can act as an antioxidant to quench reactive oxygen species,inhibit lipid peroxidation,and protect enzymatic antioxidants.TMP also has anti-inflammatory and organ protective effects and improves mitochondrial function at the cellular level.However,the molecular mechanisms responsible for these effects of TMP remain elusive.Aquaporin-8(AQP8)is located in the inner mitochondrial membrane of liver cells and is essential for mitochondrial structure becauseit mediate sthe transport of water and other metabolic substrates.AQP8 also contributes to inflammation by mediating mature IL-1? release through the induction of rapid cell swelling.On the other hand,knocking down mitochondrial AQP8 can cause oxidant-induced mitochondrial dysfunction and induce necrotic cell death in human neoplastic hepatic cells..Previous study showed that sepsis induces the tumor necrosis factor-a(TNF-a)-mediated post transcriptional downregulation of AQP8 expression in hepatocytes.Recent studies show thatAQP8 regulates the transport of water.In the process of active oxidative phosphorylation and following apoptotic signal,AQP8 promotes the fast increase of mitochondrial volume.Overall,data have suggested that AQP8 might play an important role in maintaining normal mitochondrial function.Previous research also suggested that TMP could protect the structure of hepatocyte mitochondria in septic rats by increasing AQP8 expression.However,whether TMP protects liver cells from inflammation by improving mitochondrial function,whether a strong correlation exists between AQP8 and the inflammatory response in the septic rat model remain unclear.In the present study,we sought tofurther investigate the effect of TMP on mitochondrial function and elucidate the potential mechanisms underlying the protective effects of TMP in sepsis-induced acute liver injury.Methods96 rats were divided into four groups in a random mode.?:control group(C)accepting sham procedureoperation(CLP);a sham(control)group accepted sham procedureoperation(CLP)?:septic group(S)accepting CLP operation;a septic group accepted CLP operation?:a therapeutic group(T)accepting accepted 60 mg TMP/kg by intravenous injection in caudal vein immediately after CLP;?:a preventive group(P)accepting accepted 60 mg TMP/kgintravenous injection from caudal vein for 7 days before CLPAt the 18h after the operation,12 rats were randomly selected from the four groups and anesthetized with pentobarbital sodium(50 mg/kg body wt ip).,The livers and serum samples were taken for examination.Some of the liver tissues were fixed and embedded,and the remaining liver tissues and serum samples were stored in the refrigerator at-80?.The time to ocurring bvious sepsis symptoms and to death were observed among the remaining 12 rats in each group,and the survival curve was drawn.The levels of serum ALT,AST and m-AST were directly detected by biochemical analyzer.The levels of serum IL-1?,IL-6,HGMB1,TNF-alpha and NO were detected by ELISA.ATP content in liver tissue was detected by ATP kit.Liver sections of 50 nm in size were prepared from the livers of rats,and staining with uranyl acetate and lead citrate.The ultrastructure of mitochondria was observed under electron microscope and liver mitochondria were semi quantitatively analyzed according to the Flameng classification method.The mitochondria of hepatocytes were extracted by differential centrifugation,and the mitochondria were stained by JC-1.The fluorescence intensity was detected by confocal laser scanning microscope and flow cytometry.The ratio of green fluorescence to red fluorescence was used to reflect the change of mitochondrial membrane potential(MMP)indirectly.The activities of Na+-K+-ATPase,Ca2+-ATPase,Mg+-ATPase and Ca2+-Mg2+-ATPase in mitochondria membrane were measured by visible spectrophotometryThe mitochondrial membrane proteins were isolated from hepatic mitochondria,and AQP8 protein was detected by Western blot method.The expression of AQP8mRNA in liver tissues was detected by Real-time PCR.Data were recorded and assessed using SPSS 14.0(IL,USA).Results of the survival time of rats,and taking to develop the symptoms of sepsis,the levels of serum ALT,AST,m-AST levels,serum IL-1?,IL-6,HGMB1,TNF-alpha and NO levels,ATP enzyme activity,AQP8 mRNA expression and AQP8 protein expression were expressed as mean±standard deviation,and The comparison between the four groups was performed using the analysis of variance(ANOVA).The relationship between the optic density of AQP8 band and the levels of serum ALT,AST,m-AST,ATP content in liver cells,and the serum levels of IL-1 beta,IL-6,HGMB1,TNF-a and NO was analyzed.using a Pearson correlation.Results1.The time of developing the symptoms of sepsis and survival time in ratsThe time of developing the symptoms of sepsis in the group S was shorter than that in the group T and group P(P<0.01)and this reveals TMP can postpone the time of developing the symptoms of sepsis.The survival time of rats in the group T and group S was longer than that in the group S(P<0.01).there were no significant differences in the time of sepsis symptoms and survival time between the treatment group T and group P(P>0.05)2.The content of ATP in liver tissue and the levels of serum ALT,AST and m-ASTCompared with the group C,the content of ATP in liver tissue of the group S was significantly decreased(P<0.01);compared with the group S,the ATP content in liver tissue of the group T and group P was increased(P<0.01).The serum levels of ALT,AST and m-AST were increased significantly in the group S than that in the group C(P<0.01);compared with group S,the levels of serum ALT,AST and m-AST were decreased in the group T and group P(P<0.01).3.Changes of mitochondria structure in liver cellsIn the sepsis group,the mitochondrial volume increased,cristae obvious swelling,and vacuolar degeneration,even rupture of the outer membrane and adipose degeneration in mitochondria were observed in rat hepatocytes via electron microscope.In the treatment group and prevention group,the above changes of liver cell mitochondria,such as swelling and vacuolar degeneration,were become better than those in the sepsis group.The semi quantitative score of liver mitochondria in the sepsis group was significantly higher than that in group C(P<0.01);the semi quantitative score of liver mitochondria in the group T and group P was significantly lower than that in group S(P<0.01)4.Hepatic cell mitochondrial membrane potentialThe hepatic mitochondrial membrane potential(MMP)was detected by confocal laser scanning microscopy and flow cytometry.The results showed that the MMP of the sepsis group obviously decreased than that of the control group(P<0.01),while the MMP in the group T and the group P was higher than that in the group S(P<0.01)5.Liver cell mitochondrial ATP enzyme activityCompared with the group C,the activity of Na+-K+-ATPase,Mg+-ATPase,Ca2+-ATPase and Ca2+-Mg2+-ATPase of hepatocytic mitochondrial in the group S was significantly decreased(P<0.01).Compared with the group S,the activity of Na+-K+-ATPase,Mg+-ATPase,Ca2+-ATPase and Ca2+-Mg2+-ATPase of hepatic mitochondrial in the group T and group P were increased significantly(P<0.01).There was no obvious difference between the group T and the group P(P>0.05).6.Expression of mitochondrial AQP8 mRNA in liver cellsCompared with the control group,the expression of AQP8 mRNA of hepatocytic mitochondrial was decreased in the sepsis group(P<0.01);the expression of mitochondrial AQP8 mRNA were higher in the treatment group and prevention group than that in the sepsis group(P<0.01);but there is no significant difference between the treatment group and prevention group(P>0.05).7.Expression of mitochondrial AQP8 protein in liver cellsCompared with the group C,the relative expression of hepatocyte mitochondrial AQP8 was decreased in group S(P<0.01);the relative expression of AQP8 were increased in group T and group P than that in the group S(P<0.01),no significant difference between the group T and group P(P>0.05)8.Serum inflammatory factor levelsThe levels of serum IL-1?,IL-6,HGMB1,TNF-a and NO in the group S were increased significantly than that in the group C(P<0.01),while the levels of serum IL-1?,IL-6,HGMB1,TNF-a and NO in the group T and group P were lower than that in the group S(P<0.01)9.correlation analysisThe expression of mitochondrial AQP8 protein was negatively correlated with the levels of serum ALT,AST and m-AST in rats(P<0.01),and the expression of mitochondrial AQP8 protein was positively related to the ATP content and the mitochondrial membrane potential in liver cells(P<0.01).There was a negatively correlation between the expression of mitochondrial AQP8 in rat hepatocytes and the serum levels of NO and TNF-a(P<0.01).Conclusion1.Tetramethylpyrazine can obviously improve the survival state and reduce the mortality of septic mice.2.There are Increased the release of inflammatory factors and decreased the expression of mitochondrial AQP8,AQP8 mRNA in sepsis,the mitochondrial ATP enzyme activity and mitochondrial membrane potential are also decreased,and mitochondrial structure changes and all of that are marker of mitochondrial dysfunction The expression of mitochondrial AQP8 protein was negatively correlated with liver enzyme parameters,positively correlated with ATP content and mitochondrial membrane potential of liver cells,and negatively correlated with serum TNF-a and NO levels.3.TMP can protect the liver mitochondrial function in septic rats,and the possible mechanism may include:(1)TMP can protect the mitochondrial damage of sepsis by up regulating the expression of mitochondrial AQP8 protein,protecting mitochondrial ATP enzyme activity and stabilizing the mitochondrial membrane potential.(2)TMP can inhibit the excessive release of TNF-a,IL-1?,IL-6 and other inflammatory mediators,thereby inhibiting the uncontrolled inflammatory reaction when sepsis occursOur data demonstrated that TMP could ameliorate hepatocellular mitochondrial dysfunction by decreasing theinflammatory response and increasing AQP8 protein expression.