| BackgroundHemorrhagic shock (HS) remains the most common cause of death in severely injured patients, it is also a common reason for death in some non-injured hemorrhagic patients (e.g. acute hemorrhage due to peptic ulcer, the rupture of esophageal varicose vein and some special gynaecological and obstetric diseases) Although massive blood loss may contribute to early mortality, a significant number of deaths occur indirectly due to the development of multiple organ failure (MOF) days to weeks after the initial injury. The kidney and liver are especially vulnerable to hemorrhagic shock due to their complex microvascular structures and high oxygen dependency, and renal and liver damage has been shown to be independently associated with increased mortality in critically injured patients. Injured patients with renal or liver damage have significant higher mortality when compareing with patients with no organ failure.Persistent mitochondrial dysfunction has been associated with increased MOF and higher mortality in severely injured patients. Because mitochondria utilize over95%of the available oxygen to synthesize the majority of cellular adenosine triphosphate(ATP), impaired perfusion during hemorrhagic shock negatively impacts mitochondrial function and cellular energy stores. Moreover, although reperfusion can restore adequate tissue oxygen levels, it can exacerbate mitochondrial dysfunction by increasing the generation of reactive oxygen species (ROS). Enhanced ROS not only directly damage mitochondrial proteins, they can also lead to the formation of the mitochondrial permeability transition pore, loss of cytochromec, and enhanced apoptosis. Despite resuscitation, persistent mitochondrial dysfunction following hemorrhagic shock may contribute to cellular injury and organ failure. Therefore, improving mitochondrial function and reducing mitochondrial oxidant-induced injury are important steps in mitigating hemorrhage-induced renal damage.Acute insulin resistance often occurs following trauma and hemorrhage. It manistests as hyperinsulinemia and hyperglycemia, both of which have been proved to be independent risk factors for adverse outcomes in severely injured patients. From the researches using models of type II diabetes and obesity, mitochondrial dysfunction contributes to the phenomenon of insulin resistance and hyperglycemia. Mitochondria play a vital role in metabolism and shock-induced mitochondrial dysfunction, resulting in decreased (3-oxidation, impaired ATP production, and increased ROS. Although ROS can directly damage mitochondrial proteins and impair function, ROS can also activate various serine kinases that regulate the insulin signaling cascade. Moreover, post-injury acute insulin resistance and hyperglycemia can be minimized by blocking the increased production of ROS. Therefore, resuscitation strategies that either mitigate oxidative stress or improve acute insulin resistance could prove beneficial for patients with hemorrhagic shock.Resveratrol(RSV), a natural polyphenol produced by several plants (e.g. grapes, polygonum cuspidatum, peanuts and mulberries), has been shown to have multiple salutary effects in a variety of chronic disease states (e.g. diabetes, alzheimer’s disease, coronary artery disease and obesity, etc.) as follows:promoting mitochondrial function and biogenisis, reducing oxidative damage, improving insulin resistance, regulating blood glucose and suppressing inflammation, etc. Sirtuin-1(SIRT1), a direct ligand of RSV, was proposed to be the major effector for many of the biological effects of RSV. SIRT1is NAD+(Nicotinamide adenine dinucleotide+)/NADH dependent deacetylase, it deacetylates and activates peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC1-a), a master regulator of mitochondrial biogenesis that coactivates the nuclear respiratory factors1(NRF1) and NRF2and induces the transcription of different genes involved in mitochondrial protection and anti-oxidant defense system. A number of studies have focused on the protection of RSV on the cardiac insufficient, hepatic and intestinal injury after trauma and hemorrhage, there is, however, little information on the impact of RSV on hemorrhagic shock induced mitochendrial dysfunction and acute insulin resistace following hemorrhagic shock, and it is also unclear with the potential mechanisms involved in these effects of RSV.In this study, we used a well-validated decompensated HS model and animals were resusciated with the supplementation of RSV. Through the usage of a various of mitochondrial measurements as well as techniques of molecular biology, we tried to investigate the salutary effects of RSV on mitochondrail dysfunction and acute insulin resistance following HS and resuscitation, we also tried to disclose the potential mechanisms involved in these benefits. These results will provide a new strategy for early resuscitation of HS and contribute to the usage of RSV in critically illed patients. They are of great academic significance and potential clinical value.Objectives1. To investigate whether the supplementation of RSV during resuscitation would improve liver and kidney mitochondrial dysfunction and oxidative stress damage, as well as to explore the potential mechanism of these benefits on kidney mitochondria and the role of SIRT1-PGC1a pathways.2. To study the improvements of RSV on acute insulin resistance following HS and resuscitation and to determine the role of mitochondrial function druing the above process.Methods1. Animal model:Using a well-validated decompensated HS model, the male Long-Evans rats were anesthetized and underwent placement of femoral vascular catheters. A5-cm midline laparotomy was performed to simulate soft tissue trauma. Mean arterial pressure (MAP) and heart rate (HR) were continuously monitored and recorded. Animals were passively bled via the femoral artery and maintained at a MAP of40mmHg. When the blood pressure could no longer be maintained without fluid infusion, a MAP of40mmHg was sustained by incrementally infusing0.2cc boluses of lactated Ringer’s solution (LR). Animals were considered to be in severe shock when40%of the shed volume had been returned in the form of LR boluses. Animals were then resuscitated with four times the shed volume in LR with or without RSV (30mg/kg, dissolved in DMSO) over60minutes. Animals (n=6per group) were sacrificed prior to hemorrhage (Sham), at Severe Shock (SS), and following LR Resuscitation (LR) or LR+RSV Resuscitation (LR+RSV) Before sacrifice, blood samples were taken and assayed for arterial blood gases, lactate, hemoglobin, glucose, and electrolytes, the plasma was stored at-80℃until other analysis. The animals were then euthanized and tissues removed immediately, liver and kidney mitochondria were isolated using differential centrifugation.2. The measurements of respiratory capacities of individual mitochondrial complexes: At each time point, liver and kidney mitochondria were used to assess respiratory capacities of individual complexes (CI, CII, and CIV) using high-resolution respirometry.3. Fluorometry was used to determine the total ROS production of liver and kidney mitochondria, as well as the NAD+and NADH levels in kidney tissue.4. spectrophotometry was used to determine the electron flow activities of mitochondrial complex IV and the activity of citrate synthase, which is commonly used as a quantitative enzyme marker for the presence of intact mitochondria.5. Western blot was used to determine the expression levels of3-nitrotyrosine (an indicator of nitrosative stress) and4-hydroxynonenal (4-HNE, a measure of lipid peroxidation from reactive oxygen species) in kidney, as well as the expression levels of phosphorylated insulin receptor substrate1(pY612IRS-1), which is the active form of IRS-1.6. A blood biochemistry analyser was used to measure liver funtion as reflected by glutamic-pyruvic transaminase(ALT)and glutamic oxalacetic transaminase(AST), and kidney function as refeclted by blood urea nitrogen (BUN) and serum creatinine (SCr)7. Total RNA was isolated by using the TRIzol reagent extraction kit. Real-time quantitative RT-PCR was used quantify mRNA from SIRT1, PGC1-α, anti-oxidative enzymes [superoxide dismutase2(SOD2), catalase and cyclooxygenase2(COX2)] and mitochondrial biogenesis factors [mitochondrial transcription factor A (TFAM), NRF1and NRF2] in kidney;8. Following resuscitation with LR or LR+RSV, blood glucose levels were measured every15minutes for1,5hours;9. Insulin, corticosterone and proinflammatory cytokines, including Tumor necrosis factor-a (TNF-a) and interlukin-6(IL-6), were determined using commercially available enzyme-linked immuno sorbent assay (ELISA) kits;10. Total glucagon-like peptide-1(GLP-1) and glucagon were determined using commercially available radioimmunoassay kits;11. The HOMA-IR index is used clinically to characterize insulin resistance using the equation:insulin level (μ.U/ml) x glucose (mg/dl)/405.ResultsPart Ⅰ1. Vital signs and Laboratory Parameters:Compared with LR resuscitation, LR+RSV resuscitation was associated with significantly less lactate production (10.2±3.0mmol/L versus6.9±3.3mmol/L,p<0.05), improved HCO3-levels (11.3±2.8mmol/L versus17.2±3.5mmol/L, p<0.05) and less hyponatremia (130.3±6.1mmol/L versus138.0±3.4mmol/L,p<0.05). RSV supplementation, however, did not significantly improve the MAP, arterial pH, BUN, Scr, ALT or AST levels.2. RSV supplementation during resuscitation restored liver and renal mitochondrial function following HS and resuscitation:Hemorrhagic shock resulted in significant decreased respiratory capacity of all complexes in liver (all p<0.05). Compared to LR alone, RSV supplementation during resuscitation significantly restored CⅠ, CⅡ and CIV-dependent respiratory capacities (874.9±223.9pmol/s/mg versus 589.9±62.8pmol/s/mg;2682.9±421.5pmol/s/mg versus1733.4±128.1pmol/s/mg;9080.0±1324.7pmol/s/mg versus5441.7±483.4pmol/s/mg; all p<0.05)Hemorrhagic shock resulted in significant decreased respiratory capacity of all complexes in kidney (all p<0.05). Compared to LR alone, RSV supplementation during resuscitation significantly restored CII and CIV-dependent respiratory capacities (2000.0±234.9pmol/s/mg versus3137.0±349.5pmol/s/mg mitochondria and7857.1±695.8pmol/s/mg versus11615.4±1518.9pmol/s/mg mitochondria, respectively; all p<0.05), as well as elctron flow activity of CIV (1687.3±69.3nMol/min/mg versus1253.8±79.9nMol/min/mg mitochondria; p<0.05)3. RSV treatment during resuscitation ameliorated renal mitochondrial oxidative stress following HS and resuscitation both in liver and kidney:RSV supplementation during resuscitation significantly reduced liver and kidney mitochondrial ROS production following HS and resuscitation (all p<0.05). In kidney, RSV supplementation significantly ameliorated the degree of lipid peroxidation (4-HNE), but did not change the nitrogen oxidative damage following hemorrhagic shock or resuscitation as measured by3-NT.4. RSV supplement increased the mRNA expression of SIRT1and the NAD+-NADH ratio in kidney:Severe shock resulted in a dramatic decline in the mRNA expression of SIRT1which was reversed by RSV administration during resuscitation (p<0.05). Resuscitation with RSV significantly decreased the NADH concentration and nearly doubled the NAD+-to-NADH ratio when compared to LR.5. RSV increased mRNA expression of PGC1-α and anti-oxidant enzymes in kidney, but did not promote mitochondrial biogenesis:In kidney, the mRNA expression of PGC1-α and anti-oxidant enzymes (SOD2and catalase) were significantly elevated in the RSV resuscitation group when compared to LR resuscitation alone, however, RSV did not change the expression of COX2. Furthermore, we did not observe a significant change in mitochondrial abundance as measured by citrate synthase activity, as well as the mRNA expression levels of mitochondrial biogenesis factors (TFAM, NRF1and NRF2) following resuscitation. Part II1. RSV significantly lowered blood glucose, decreased insulin levels and improved insulin resistance following resuscitation:Compared to resuscitation with LR alone, animals treated with RSV had significantly lower blood glucose (116.0±20.2mg/dl vs.359.0±79.5mg/dl, p<0.05) and plasma insulin levels (1.0±0.4ng/ml vs.6.5±3.7ng/ml, p<0.05), as well as lowered Ln HOMA-IR index following resuscitation (1.30±0.42vs.4.18±0.68,p<0.05) following resuscitation. Blood glucose levels continued to be significantly lower in RSV treated animals at45,60and90minutes following resuscitation.2. RSV increased plasma GLP-1levels following resuscitation, but did not influence the levels of glucagon and corticosterone:Our model of decompensated hemorrhagic shock resulted in significantly elevated plasma GLP-1, glucagon and corticosterone levels. Compared to LR alone, RSV supplementation during resuscitation was associated with significantly higher GLP-1levels (385.8±56.6ng/ml vs.187.3±1.1ng/ml, p<0.05), but did not influence glucagon and corticosterone levels.3. RSV did not influence plasma TNF-a and IL-6levels:The inflammatory cytokines TNF-a and IL-6were significantly elevated following hemorrhagic shock and resuscitation. Resuscitation with RSV, however, did not significantly reduce levels of cytokines.4. RSV supplementation preserved the active form of IRS-1in both liver and kidney tissues:Phosphorylated tyrosine612IRS-1(pY612IRS-1) is the active form of IRS-1that activates several insulin signaling pathways. Following resuscitation with LR, the expression level of pY612IRS-1in liver and kidney tissue was significantly reduced. Resuscitation with RSV, however, preserved the expression of phosphorylated IRS-1(p<0.05)5. The respiratory capacities of liver mitochondrail complexes II and IV significantly correlated with insulin resistance index:Using Pearson correaltion analysis, the respiratory capacities of liver mitochondrail complexes II and IV significantly correlated with Ln HOMA-IR index (r=0.552and0.665, respectively, all p< 0.05). The respiratory capacities of liver mitochondrail complex I and kidney mitochondrail complexes I, II and IV did not significantly correlate with Ln HOMA-IR index (all p>0.05)Conclusions1. Resveratrol supplementation led to a restoration in liver and kidney mitochondrial function and alleviated oxidative stress injury following hemorrhagic shock and resuscitation. In kidney, these mitochondrial-protective effects appear to be mediated via stimulation of a SIRT1-PGCl-α-antioxidant pathway rather than via mitochondrial biogenesis.2. RSV supplementation during resuscitation robustly improved acute insulin resistance following hemorrhagic shock and resuscitation. These effects of RSV may be mediated by improved mitochondrial function, decreased mitochondrial ROS and increased GLP-1secretion. |
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