The Study of Apoptosis, Inflammation, Autophagy and Organ Functions in Rat Liver Following Hemorrhagic Shock and Protective Mechanism of Ligustrazine.

Injury continues to be the leading cause of death in the first four decades of life in Taiwan. Hemorrhagic shock commonly resulted in early trauma death and immediate tissue ischemia and reperfusion (I/R) injury. Regardless of tremendous clinical efforts, the mortality of post-injury multiple organ failure (MOF) remains high about 40-50%. The MOF was described as uncontrolled infection before, and is hypothesized as a result of a dysfunctional inflammatory response now. But the exact pathogenesis of post-injury MOF remains elusive.Liver is a complex metabolically active organ, and liver failure carries a high mortality rate. Their inflammatory response after ischemia and reperfusion injury is characterized by early production of proinflammatory cytokines, such as TNF-α, which propagate the inflammatory response by upregulating vascular endothelial cell adhesion molecules and increasing the production of neutrophil chemoattractants, including CXC chemokines. These activated neutrophils then damage liver parenchymal cells through their release of reactive oxygen species and proteases. In experimental models, blockade of inflammatory mediators has proven effective against liver injury after hepatic I/R, suggesting that interventional therapies designed to suppress the inflammatory response may have significant therapeutic benefits.However, the inflammatory response of hepatic parenchymal, non- parenchymal cells and recruited neutrophils are not well coordinated after hepatic I/R injury. The dichotomy of NF-κB in promoting or protecting against hepatic I/R injury depended on the different hepatic cell populations. Therefore, the other mediators may also contribute to the divergent role in parenchmal and non-parenchymal cell. Recent studies indicate that apoptosis significantly contributes to I/R-induced inflammation, as well as subsequent tissue damage. Many anti-apoptotic molecules, including bcl-2, bcl-x, and Akt, exert part of their protective functions through maintenance of mitochondrial integrity. Autophagy is a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. It presents an adaptive response to hypoxia and starvation. However, the exact role of autophagy in hepatic I/R injury is not well elucidated in literature.Ligustrazine (tetramethypyrazine), a bioactive component contained in Chuanxiong (Ligusticum chuanxiong Hort), is widely used in the treatment of cardiovascular diseases in China. It has been reported that ligustrazine can increase coronary blood flow and reduce myocardial ischemia in animals through Ca2+ and ATP-dependent vascular relaxation. It also reduces the bioactivity of platelets and platelet aggregation, and inhibits free radicals formation. In addition, ligustrazine has been demonstrated to play a protective role in I/R injury in rats kidney. The exact pathogenesis in liver is rarely reported, but we had observed the anti-inflammatory and anti-apoptotic effect during our previous 150 herbal drugs screening study. In this three-year study, first-year, the hepatocyte and Kuppfer cell isolation techniques will be used to study the change of apoptosis, inflammation, and autophagy effect between shock and resuscitation groups after standardized hemorrhagic shock model in rats. The second year will use three time points of drug administration, as: pre-shock, during and after resuscitation to compare the protection effects of Ligustrazine on the hepatocytes and liver tissue. The third-year will use the optimal administration time-point to study the signal transduction in hepatocytes, Kuppffer cells, and neutrophils before and after the shock and the drug administration.Taken together, we hypothesize that (1) apoptosis and autophagy are involved in hepatic dysfunction in I/R injury, (2) autophagy can be protective, detrimental, or both in response to hepatic dysfunction in I/R injury, (3) Ligustrazine may ameliorate the hepatic I/R injury after hemorrhagic shock. Through the rodent models of hemorrhagic shock state, this study hopes to elucidate the miscellaneous transcriptional signals and pathways on hepatic cell-specific functions in apoptosis, inflammation, autophagy and immunological, & histological responses of liver tissue. Besides, we intend to apply the potential herbal drug to decrease the hepatic I/R injury and investigate the specific roles and interactions between hepatocytes, Kupffer cells and neutrophil before and after drug administration.