The Analysis On The Expression Of Poly(ADP-ribose) Polymerase And Mechanism Of Methylene Blue In Sepsis

Objectives: Main cause of septic shock is Systemic inflammatory response syndrome and the result of the shock is cell dysfunction and necrosis. It always leads to increasement of superoxide anion and nitric oxide. Peroxynitrite is synthesized of superoxide anion and nitric oxide, which will cause DNA cleavage. When Poly(ADP-ribose)polymerase(PARP) is activated in sepsis, it will decrease the concentration of Nicotinamide adenine Dinucleotide(NAD+) and slow down the rate of glucolysis and electron tranport. The activation of PARP can result in decreasement of Adenosine Triphosphate(ATP) synthesis. The reaction will lead to cell dysfuntion and necrosis.Monitoring and rectification of tissue and cell Hypoxia have already been received considerable attention by doctors. Moreover, evidence has already been founded to support that early increasement of oxygen delivery can improve prognosis for septic patients. However, it is found in some researches that strategy of early oxygen delivery has difficulty in improving prognosis of patients when organ function has been seriously damaged in the late stage of septic shock. Therefore, we are urgent to know what had happened in tissue and cell under the"normalization"of hemodynamic parameters. Trzeciak and Rivers generalized this change of septic shock to four issues: 1.General tissue hypoxia 2. Endothelial injury 3. Activation of coagulation system 4. Mircrocirculation and mitochondrial disress syndrome(MMDS). Among those, the two issues that attract most of all: one is the dysfuntion of oxygen utilization of which the central part is the abnormal mitochondrial function, the other is microcirculation dysfunction. The excessive activation of PARP is a key ending mechanism which induces tissue injury and organ dysfunctions and plays an important role in the mechanism of mitochondrial dysfunction. PARP plays a vital role both in early organ dysfunction and in multiple organ failure caused by SIRS(Systemic Inflammatory Response Syndrome). A large amount of evidences support that PARP is an important goal of treatment interventions.There are such mechanisms of methylene blue in septic shock or severe sepsis as follows. Firstly, methylene blue inhibits the production of Nitric Oxide[NO] by inhibiting the activation of iNOS. And it can decrease cGMP by inhibiting effect of sGC caused by NO to reverse shock and cardiovascular function disorder. Secondly, in clinical application, it has been found that methylene blue scavenges free radical directly to provide various ways to the protection of tissue and cell.The mechanism function of methylene blue is to reduce the nitric oxide synthesis and superoxide content. Therefore, methylene blue cuts off the way of PARP from the source of mechanism to improve the necrosis of tissue and cell.In my study, in order to elucidate the expression of Poly(ADP-ribose) polymerase and mechanism of methylene blue in sepsis, I duplicated the model of sepsis by cecal ligation and puncture(CLP).My intention is : firstly, to investigate the role of PARP in sepsis, I examined the expression of PARP in sepsis rats. Secondly, I applied methylene blue before sepsis in rats to elucidate effect of methylene blue on expression and activation of PARP. My aim is to explore whether or not methylene blue inhibits the expression of PARP to improve the necrosis of cell by the way of PARP. As a clinical multiple target point medicine which is cheap, methylene blue has made a breakthrough on the therapy of Sepsis, but the exact mechanism need to be further clarified.Method: Cecal ligation and puncture(CLP) was used to duplicate severe sepsis model. Thirty healthy male Sprague-Dawley rats(250g-300g) were randomly divided into three groups:1.Sham group(n=10): in which the rats'abdomen just was incised and then sutured , administered with saline (0.8ml) through vena dorsalis penis. 2. CLP model group(n=10): in which rats were administered with saline(0.8ml)through vena dorsalis penis before CLP. 3. Methelene blue group(n=10): in which rats were administered with methylene blue(15mg/kg ) through vena dorsalis penis before CLP. At 18h after CLP, we observed the inflammatory reaction of peritoneal cavity after laparotomy. Their lobi inferior, small intestine and kidney were resected. Empirical method: 1.Detection the expression of PARP of each tissue by immumohistochemical method.2. Detection the ration of PARP of small intestine.Statistical analysis was performed by using SPSS 13.0 software package. Datas of Western blot were expressed as mean±SEM(X±s), and the means of each group were analysed with one-way ANOVA. A level 0f P<0.05 was considered statistically to be significant. Datas of immumohistochemical method were analysed with rank transformation. A level of P<0.05 was considered statistically to be significant.Results:1 The general situation of rats in the experiment was abserved: The rats of sham group acted as usual which regained consciousness after anaesthesia. No abdominal dropsy and evil smelling were found when their abdomens were opened. The rats of CLP model group regained consciousness later, and their behavior showed as chills, dyspnoea, pilo-erection, and depression. There were bloody ascites, evil smelling, and edema of intestine after laparotomy. Compared with CLP model group, the manifestations in the rats of methylene blue group were better.2 Results of immumohistochemical: Sham group, CLP model group and methylene blue group were analysed with Parametric test to compare the expression of PARP-1. There were significant difference was observed among three groups(P<0.05). The difference of expression of PARP-1 between the sham group and CLP model group was extremely significent(P<0.05). The significant difference of expression of PARP-1 was found between CLP model group and methylene blue group.3 Result of Western blot: Sham group, CLP model group and methylene blue group were analysed with one-wat ANOVA to compare the expression of PARP-1. There were significant difference was observed among three groups(P<0.05). The difference of expression of PARP-1 between the sham group and CLP model group was extremely significent(P<0.05). The significant difference of expression of PARP-1 was found between CLP model group and methylene blue group.Conclusions:The sepsis model could be duplicated by cecal ligation and puncture(CLP) successfully.1 Compared with rats of sham group, the strained intensity of PARP-1 in the lobi inferior, small intestine and kidney of CLP model group and methylene blue group is stronger by immumohistochemical method (P<0.05). It shows that the expression of PARP-1 of vital organs is strengthened in the rats of sepsis. Same to data available in the literature of overseas research, PARP play a key role on the course of injury and necrosis of tissue in sepsis. The expression of PARP-1 in the lobi inferior, apex of heart, small intestine and kidney of methylene blue group is weaker than the CLP model group. It shows that methylene blue can decrease the expression of PARP to improve the injury and necrosis of multiple organs.2 Compared with rats of CLP model group, ration of PARP-1 is declined based on the Western blot analysis, P<0.05, which has statistical meaning. Methylene blue decreases the production of PARP and further improve the necrosis of cell by mechanism of decreased synthesis of superoxide anion and nitric oxide and level of peroxynitrite. The study clarifed the mechanism of function of methylene blue in sepsis further deeply.