Research On Regulatory Mechanism Of Complement C3to Related Pathophysiology Of Hemorrhagic Shock

BACKGROUD AND PURPOSE According to statistics,1/3of the death caused by the trauma is caused by hemorrhagic shock. Although there had been many research on the pathophysiology mechanism of hemorrhagic shock, but it was difficult to avoid cascade successor body injury after acute hemorrhagic shock. So that we awared of that its physiological and pathological mechanisms still needed to be further supplemented and improved in order to find new and effective drug targets and diagnosis and treatment methods. In this study, high-throughput proteomics screening technique was implemented to select specific protein of hemorrhagic shock. The mechanism of specific protein C3in hemorrhagic shock was investigated, in order to further elucidate the pathophysiology of hemorrhagic shock and find an effective indicator to detect the degree of shock and injury progress to hemorrhagic body and explore effective therapeutic target for improving the prognosis.METHODS By bloodletting of Carotid artery, a rat model of hemorrhagic shock was established, with recording blood pressure, heart rate and other hemodynamic parameters. Samples of pasma and liver were gathered. Screening differentially expressed proteins of hemorrhagic shock plasma through two-dimensional electrophoresis and mass spectrometry. Expression differences of plasma protein in rats of hemorrhagic shock was validated by ELISA. By ELISA, Western blot, immunohistochemistry, immunofluorescence and HE staining, expression and distribution of C3in hemorrhagic shock rats was vetified. Detecting level of SOD, MDA, ALT, AST, MT-1and NO in plasma and CX43expression levels of vascular by ELISA, NBT and chemical methods, compared with the expression levels of plasma C3. In order to analysis the effect of C3on vasomotor activity, vascular permeability, oxidative stress, inflammation and liver damage after hemorrhagic shock. Further, under the same conditions, with injection of sCR1to inhibit C3activation, these indicators were detected by ELISA, and liver leukocyte infiltration were assayed by MPO, and the apoptosis of liver and vascular injury were observed by HE staining and TUNEL experiments, so as to assess the impact of C3on vasomotor activity, vascular permeability, oxidative stress, inflammation and tissue damage.RESULT (1) Significant differences were found in10protein spots by screening plasma. Six over-expression protein and two down-expression protein were identified, such as afamin, complement C3, Hemopexin, Fgg Isoform Gamma-A of Fibrinogen gamma chain, F2thrombin, keratin1, Ephrin type-A receptor5isoform1and Type II cytoskeletal6A. It is found that afamin, complement C3, Hemopexin, Fgg Isoform Gamma-A of Fibrinogen gamma chain and F2thrombin were over-expressed in plasma of hemorrhagic rat(P<0.05).(2) Expression level of complement C3was significantly increased in the liver and large blood vessels during hemorrhagic shock; the C3widely distributed in the liver and endothelial cells; it evenly distributed in the cytoplasm of the liver cells and endothelial cells, compared with the nucleus of liver cells in a large number of aggregation. There was clearly visible morphological damage in liver and blood vessels.(3) C3expression levels in plasma was positively correlated with IL-6, TNF-a, MDA, ALT, AST, NO, ET-1SOD, CX43while negatively correlated with SOD and CX43. With NO, ET-1, CX43, ALT and IL-6, C3has a strong correlation; while with SOD, MDA and AST has a moderate correlation.(4) After hemorrhagic shock, by inhibiting activation of C3, with the application of sCRl intervening fluid resuscitation, heart rate and mean arterial blood pressure fastly returned to normal within10min, and could keep relatively stable. The heart rate of HS+sCRl+Saline group was significantly lower than the HS group and HS+Saline group (P<0.05); mean arterial pressure close to the normal control group after resuscitation. And levels of IL-6, TNF-a, MDA, ALT, AST, NO and ET-1were significantly lower than that in normal saline resuscitation group, compared with SOD increased. Leukocyte infiltration, apoptosis and damage in the liver was significantly reduced and vascular morphology also significantly reduced.CONCLUSION We have successfully screened a number of differentially expressed proteins of hemorrhagic shock and defined its content in the peripheral blood. We also clarified the mechanism of C3in hemorrhagic shock as an acute reactive protein and confirmed that C3participate in the hemodynamic hemorrhagic shock, multiple body vasomotor response, vascular permeability, oxidative stress, inflammation, tissue and organ damage and other body reaction during hemorrhagic shock. It is confirmed that sCR1could significantly inhibit the activation of C3in early stage of hemorrhagic shock, so as to stabilize the shock pressure, heart rate and other hemodynamic; improve vascular reactivity after shock, reducing vascular permeability; effective weaken the excessive inflammatory response of body after hemorrhagic shock; reduced oxidative stress; lower damage to the liver and blood vessels. All of them could illustrate that C3may serve as a new hemorrhagic shock indicators to monitor the progress and extent of damage to the body shock. The way of sCRl inhibited C3activation may be a potentially valuable treatment to improve prognosis, providing new ideas and methods for the study of targeting treatment of hemorrhagic shock.