Study On Plasma Metabonomics And Hepatic Genomics In Rats Subjected To Hemorrhagic Shock

Background:Hemorrhagic shock(HS) is generally precipitated by traumatic event that results in an acute loss of blood from the intravascular space. It remains a major cause of death and disability in battlefield injuries, as well as in civilian trauma. With the deeper recognition of mechanism and development of treatment for HS, treatment success rate tend to ascend in recent years. But many of these people with severe HS will reach hospitals in salvageable condition but will still die. One of the reasons is that the syndrome of hemorrhagic shock in humans leads to cardiovascular failure and death by impairing the several important organs including heart, liver, spleen, lung, kidney and brain. Another reason for the continued deaths from injury is an incomplete understanding of the human response to hemorrhagic shock, and new therapeutic approaches need to be explored.The main factors leading HS to refractory are cascade effects and multiple organ dysfunction syndrome, and they are correlated with variations of substance and energy metabolism. The rapid development of HS is accompanied by a series of abnormal metabolism implicated in many different pathways, such as sugar, lipid, amino acid, vitamin, nucleotide and other metabolism. 1H-nuclear magnetic resonance (1H-NMR)-based metabonomic approach is a newly developed technology that can be exploited to be a useful tool for disease diagnosis. And it has been widely used in many fields, but no report is found in HS so far.The liver is responsible for maintaining homeostasis in the body and is the main source of energy to peripheral organs. Hence, it is readily susceptible to the ischemic injury associated with hemorrhagic shock. Disturbances in liver function profoundly affect other body systems leading to multiple organ failure and death. Therefore, the elucidation of hepatic responses to shock is of great importance in the pathogenesis and treatment of HS.Objective and methods:First, to detect the plasma metabonomic profile of rats subjected to HS and Control group by 1H-NMR analyzer. The 1H-NMR spectra of two groups is analyzed and the principal components(PC) are recognized by principal components analysis(PCA). Second, to analysis the PC detailed. Third, to detect differential gene expressions in mouse liver of HS and Control group used Affymetrix Gene Chip Rat 230 2.0 Array. Finally, to evaluate the effect of fat emulsion(FE) on rats subjected to HS.Contents and results:1. Establish rat model in HSAfter rat model being operated and lying quietly for 10 minutes, Hemorrhage was performed through the femoral arterial catheter. The total amount of blood withdrawn and the mean artery blood pressure(MAP) was recorded. The amount of blood withdrawn were 45.31±5.34% and 32.37±3.36% of total blood respectively in model of severe HS(SHS) and moderate HS(MHS); the MAP were 51.25±10.77mmHg and 80.87±7.42mmHg respectively in SHS and MHS.2. Analysis of plasma metabonomics of Rats Subjected to HS based on 1H-NMR.Plasma was collected and its 1H-NMR spectra were acquired. The metabonomic profile of two groups were able to be distinguished after the data being processed by principal components analysis(PCA). The remarkable differenceso?f plasma metabonomic profile between two groups were lactate and lipids,such as very low density lipoprotein(VLDL), low density lipoprotein(LDL) and unsaturated fatty acid.3. Analysis of plasma lipid profile of rats subjected to HS.Following the results indicated by PCA, plasma lipid profile of rat subjected to SHS and MHS were measured, such as free fatty acids(FFA), total cholesterol(TC), triglyceride(TG), high density lipoprotein cholesterol(HDL-C), low density lipoprotein cholesterol(LDL-C), thromboxane B2(TXB2), 6-keto-prostaglandin Fla(6-Keto-PGF1a) and 8-iso-prostaglandin F2a (8-iso-PGF2a).Results of plasma lipid profile: ①Compared with Control, the levels of FFA,TC,HDL-C and the ratio of HDL-C/LDL-C(H/L) decreased, and the levels of TG,LDL-C increased significantly. The severer of the HS was, the more the levels of FFA,TG,TC and H/L decreased. The longer the time of HS lasting, the more the FFA levels and H/L decreased. With the time of HS lasting, the levels of TG increased first, then decreased.②Compared with Control, the levels of TXB2, 8-iso-PGF2a and the ratio of TXB2/6-keto-PGF1a(T/K) increased, and the severer of HS was, the more the items increased. The levels of 6-Keto-PGF1a decreased, and the severer of HS was, the more the items decreased. Compared among three group of MHS-30, MHS-60 and MHS-120, the levels of TXB2, 8-iso-PGF2a and the ratio of T/K decreased first, then increased in MHS-120. The levels of 6-Keto-PGF1a increased first, then decreased in MHS-120.4. Hepatic gene expression of rats subjected to hemorrhagic shock.Hepatic gene expression profiles of each group were detected by Affymetrix Gene Chip Rat 230 2.0 Array and 9 genes were selected to undergo semi-quantitative RT-PCR. The gene chip arrays results showed that 303 genes expression in the HS group was significantly different from the Control group; and of the 303 genes, 256 genes were up-regulated and 47 genes were down-regulated. Their biological function of 107 up-regulated genes and 13 down-regulated genes were known. Differentially expressed genes were categorized based on the best available information regarding their biological functions. Genes with multiple functions were assigned to a single category. These included many genes of transport, transcription, signaling, response to stress, metabolism, biosynthesis, development, cell adhesion, proliferation, differentiation, cell cycle and apoptosis. 10 genes of them were related with lipid metabolism, such as SCD1,FASN,ACLY,ELOVL6,HMGCR,CYP7A1,CYP4A12,EGR1,NR1i3 and LCN2, which occupied 8.33% of genes whose biological function were known.5. Effect of fat emulsion(FE) on rats subjected to HS.60 male Wistar rats were randomly divided into a Control group, HS group, and HS-F group, with 20 rats in each group. The survival time, survival rate and mean artery blood pressure(MAP) were recorded. The bacteria translocation of liver, spleen, kidney and mesenteric lymph nodes were detected. The levels of serum TNF-a, FFA, TG, TC, HDL-C and LDL-C were measured. Result: compared with the HS group, survival time prolonged and elevated survival rate in 120min and 180min increased significantly in the HS-F group; the MAP was increased significantly; bacterial translocation was lower in HS-F rats; serum levels of TNF-a were lower in HS-F rats compared with HS rats, and those of FFA and TG were elevated in HS-F rats compared with HS rats, but no difference of HDL-C and LDL-C was shown between them.Conclusion:1. The remarkable differences of plasma metabonomic profile between two groups were lactate and lipids.2. The serum levels of FFA, TC, TG, HDL-C, LDL-C and the ratio of H/L were closely related to the severity and the lasting time of HS.3. The serum levels of TXB2, 6-Keto-PGF1a, 8-iso-PGF2a and the ratio of T/K were closely related to the severity and the lasting time of HS.4. Multiple hepatic genes differentially expressed in rats subjected to HS, and 10 genes were related to lipid metabolism, which occupied 8.33% of genes whose biological function were known.5. FE intervention on HS rats prolonged survival time and elevated survival rate, decreased bacterial translocation and TNF-a, increased FFA and TG after hemorrhage.6. Our study indicated that lipid metabolism maybe is an important item monitoring the course of HS, and to interfere lipid metabolism may effect on HS.