Yi-Maun Subeq

Melatonin Ameliorates Hemorrhagic Shock-Induced Organ Damage in Rats

BACKGROUND Hemorrhagic shock (HS) followed by resuscitation can result in production of several inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), leading to multiple organ dysfunction. Melatonin can attenuate organ damage with its anti-inflammation effects. The present study was designed to investigate the effects of melatonin on the physiopathology and cytokine levels after HS in rats. METHODS HS was induced in rats by withdrawing 40% of the total blood volume (6 mL/100 gm body weight) from a femoral artery catheter, immediately followed by intravenous injection of 10mg/kg melatonin. Mean arterial pressure and heart rate were monitored continuously for 48 h after the start of blood withdrawal. Biochemical parameters, including levels of hemoglobulin, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), and lactate, were determined 30 min before and 0, 1, 3, 6, 12, 24, and 48 h after induction of HS while an equal volume of normal saline was replaced as fluid resuscitation. Cytokine levels including TNF-α and IL-6 in the serum were measured at 1, 24, and 48 h after HS. The kidney, liver, lung, and small intestine were removed for pathology assessment at 48 h after HS. RESULTS HS significantly increased the heart rate, blood GOT, GPT, BUN, Cre, LDH, CPK, lactate, TNF-α, and IL-6 levels, and decreased hemoglobulin and mean arterial pressure in rats. Treatment with melatonin preserved the mean arterial pressure, decreased tachycardia, and markers of organ injury, and suppressed the release of TNF-α and IL-6, with no change in hemoglobulin after HS in rats. CONCLUSION Treatment with melatonin suppresses the release of serum TNF-α and IL-6, and decreases the levels of markers of organ injury associated with HS, thus ameliorating HS-induced organ damage in rats.

Low-dose erythropoietin aggravates endotoxin-induced organ damage in conscious rats

Endotoxin shock can induce the production of several inflammatory mediators such as TNF-alpha, IL-6, and IL-1beta, leading to multiple organ dysfunction and death. Erythropoietin (EPO) has been found to interact with its receptor (EPO-R), expressed in a wide variety of non-hematopoietic tissues, to induce a range of pleiotropic cytoprotective actions. We investigated the effects of low doses of EPO (300U/kg, intravenous administration) on the physiopathology and cytokine levels in endotoxin shock in conscious rats. Endotoxin shock was induced by intravenous injection of Escherichia coli lipopolysaccharide (20mg/kg) in conscious rats. Mean arterial pressure (MAP) and heart rate (HR) were continuously monitored for 48h after LPS administration. Levels of biochemical and cytokine parameters, including glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), and creatine phosphokinase (CPK) were measured at 0, 1, 3, 6, 9, 12, 18, 24, and 48h after sepsis. Serum TNF-alpha, IL-6, and IL-1beta level was measured at 1h after sepsis. Endotoxin shock significantly increased blood GOT, GPT, BUN, Cre, LDH, CPK, TNF-alpha, IL-6, IL-1beta levels, and HR, while it decreased MAP. EPO further increased the markers of organ injury (GOT, GPT, BUN, Cre, LDH, and CPK), inflammatory biomarkers (TNF-alpha, IL-6, and IL-1beta) and did not affect MAP and HR after LPS. EPO disserved endotoxin shock-induced liver, kidney, lung, and small intestine damage in conscious rats. In conclusion, pre-treatment with low doses of EPO increased the release of TNF-alpha, IL-6, and IL-1beta, along with aggravating endotoxin shock-induced markers of organ injury in conscious rats.

Fluvastatin attenuates severe hemorrhagic shock-induced organ damage in rats☆

OBJECTIVES Multiple organ dysfunction resulting from hemorrhagic shock (HS) and subsequent resuscitation was mediated by several inflammatory factors such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10). The present study was designed to investigate the protective effects of fluvastatin on these mediators after HS in rats. METHODS The experimental rats were randomly divided into three groups. The vehicle group received only vitamin K without HS, the HS-control group received vitamin K and HS, and the HS-experimental group received both vitamin K and fluvastatin (1mg/kg) before HS. HS was produced by bleeding from a femoral arterial catheter to remove 60% of total blood volume (6ml/100g BW) over 30min. The mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 12h after the start of blood withdrawal. The biochemical parameters, including arterial blood gas, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), and lactate were obtained at 30min before induction of HS and at 0, 1, 3, 6, 9 and 12h after HS. Equal volume of normal saline was given to replace blood volume loss. Cytokine levels including TNF-alpha and IL-10 in serum were measured at 1h after HS. Kidney, liver, lung and small intestine were removed for pathology examination at 48h after HS. RESULTS HS significantly increased HR, blood GOT, GPT, BUN, Cre, LDH, CPK, lactate, TNF-alpha and IL-10 levels, and also induced metabolic acidosis and decreased MAP in rats. Pre-treatment with fluvastatin was found to improve survival rate, preserved MAP, decreased the markers of organ injury, suppressed the release of TNF-alpha and increased IL-10 after HS in rats. CONCLUSION Pre-treatment with fluvastatin can suppress the release of serum TNF-alpha and can also increase serum IL-10 level to protect HS-induced multi-organ damage in rats.

Recombinant human erythropoietin reduces rhabdomyolysis-induced acute renal failure in rats

BACKGROUND Rhabdomyolysis is one of the causes of acute renal failure. Erythropoietin (EPO) has been found to interact with its receptor (EPO-R) expressed in a large variety of non-haematopoietic tissues to induce a range of pleiotropic cytoprotective actions. In this study, we used recombinant human erythropoietin (rhEPO) to study the effects on the glycerol-induced rhabdomyolysis with acute renal failure in rats. METHODS Twenty-four rats were divided into three groups as glycerol group, glycerol+EPO group and normal saline+EPO group. Rhabdomyolysis was induced by intramuscular injection of 10 mlkg(-1) 50% glycerol in rats. Ten minutes later, the rats received an intravenous injection of rhEPO (300 Ukg(-1)). Biochemical substances, including haemoglobin, blood urea nitrogen (BUN), creatinine (Cre), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and creatine phosphokinase (CPK), were measured at 0, 1, 3, 6, 9, 12, 18, 24 and 48 h. Rats were sacrificed 48 h later after glycerol administration and the kidneys were removed immediately for pathology and immunohistochemistry (IHC). RESULTS Intramuscular injection of glycerol significantly increased blood BUN, Cre, GOT, GPT and CPK levels and induced severe histopathologic damage in the kidneys. Nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS) were increased and E-cadherin was decreased after glycerol administration, as detected by IHC in the kidneys. Post-treatment with rhEPO decreased blood BUN, Cre, GOT, GPT and CPK levels, decreased markers of kidney injury and suppressed the release of NF-κB and iNOS after rhabdomyolysis. CONCLUSION Treatment with rhEPO suppressed the activities of NF-κB and iNOS, decreased BUN, Cre, GOT, GPT and CPK levels, and decreased the markers of kidney injury after rhabdomyolysis. These actions ameliorated rhabdomyolysis-induced acute renal failure in rats.

Single dose intravenous thioacetamide administration as a model of acute liver damage in rats

Thioacetamide (TAA) has been used extensively in the development of animal models of acute liver injury. Frequently, TAA is administered intraperitoneally to induce liver damage under anaesthesia. However, it is rarely administered by intravenous injection in conscious rats. The experiments in this study were designed to induce acute liver damage by single intravenous injection of TAA (0, 70 and 280 mg/kg) in unrestrained rats. Biochemical parameters and cytokines measured during the 60‐h period following TAA administration, included white blood cells (WBC), haemoglobulin (Hb), platelet, aspartate transferase (GOT), alanine transferase (GPT), total bilirubin (TBIL), direct bilirubin (DBI), albumin, ammonia (NH3), r‐glutamyl transpeptidase (r‐GT), tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6). Rats were sacrificed by decapitation 60 h after TAA administration and livers were removed immediately for pathology and immunohistochemical (IHC) examination. Another group of rats were sacrificed by decapitation 1, 6 and 24 h after TAA administration and livers were removed immediately for time course change of pathology and IHC examination. TAA significantly increased blood WBC, GOT, GPT, TBIL, DBIL, NH3, r‐GT, TNF‐α and IL‐6 levels but decreased the blood Hb, platelet and albumin level. The levels of histopathological damage in the liver after intravenous TAA administration were also increased with a dose‐dependent trend and more increased at 60 h after TAA administration. The levels of inducible nitric oxide synthase (iNOS) and nuclear factor‐κB (NF‐κB) detected by IHC in the liver after intravenous TAA administration were also increased with a dose‐dependent trend and more increased at 1 h after TAA administration. Single intravenous TAA administration without anaesthesia is a restorable animal model which may be used to investigate acute liver damage.

Erythropoietin protects severe haemorrhagic shock-induced organ damage in conscious rats.

OBJECTIVE Erythropoietin (EPO) has pleiotropic cytoprotective actions. We investigated the effects of EPO on the physiopathology and cytokine levels after haemorrhagic shock (HS) in conscious rats. METHODS Rats received an intravenous injection of 300 U/kg EPO over 10 min followed by HS via withdrawal of 60% of total blood volume from a femoral arterial catheter (6 ml/100 g body weight) over 30 min. Mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 18 h after the start of blood withdrawal. Levels of biochemical parameters, including haemoglobin, GOT, GPT, BUN, creatinine (Cr), LDH, CPK, and lactate were measured at 30 min before the induction of HS and 0, 1, 3, 6, 9, 12, and 18 h after HS. Cytokine levels, including TNF-alpha and IL-6, in serum were measured at 1, 9, and 18 h after HS. The kidneys, liver, lungs, and small intestine were removed for pathology assessment at 48 h after HS. RESULTS HS significantly increased HR, blood GOT, GPT, BUN, Cr, LDH, CPK, lactate, TNF-alpha, and IL-6 levels and decreased haemoglobin and MAP in rats. Pre-treatment with EPO improved survival rate, preserved the MAP, decreased the tachycardia and markers of organ injury, suppressed the release of TNF-alpha and IL-6 after HS in rats. CONCLUSION Pre-treatment with EPO suppresses the release of serum TNF-alpha and IL-6, along with decreasing the levels of markers of organ injury associated with HS, with such actions ameliorating HS-induced organ damage in rats.

Calcitriol decreases TGF-β1 and angiotensin II production and protects against chlorhexide digluconate-induced liver peritoneal fibrosis in rats.

Peritoneal fibrosis is a major complication of peritoneal dialysis that can lead to ultrafiltration failure. This study investigates the protective effects of calcitriol on chlorhexidine digluconate-induced peritoneal fibrosis in rats. Peritoneal fibrosis was induced in Sprague-Dawley rats by daily administration of 0.5mL 0.1% chlorhexidine digluconate in normal saline via peritoneal dialysis for 1week. Rats received daily intravenous injections of calcitriol (low-dose, 10ng/kg; or high-dose, 100ng/kg) for 1week. After 7days, conventional 4.25% Dianeal (30mL) was administered via peritoneal dialysis over 4h. Peritoneal solute transport was calculated from the dialysate concentration relative to its concentration in the initial infused dialysis solution (D4/D0 glucose) for glucose, and the dialysate-to-plasma concentration ratio (D4/P4 urea) at 4h for urea. Rats were then sacrificed and the liver peritoneum was harvested for immunohistochemical analysis via microscopy. After dialysis, the D4/P4 Urea level was reduced; increases were observed in the D4/D0 glucose level and the levels of active transforming growth factor-β1 and angiotensin II in serum and dialysate; the liver peritoneum and muscle peritoneum was markedly thickened, and the expression of α-SMA, fibronectin, collagen, vascular endothelial growth factor, angiotensin II, transforming growth factor-β1, and phosphorylated Smad2/3 (P-Smad2/3)-positive cells in the liver peritoneum was elevated in the peritoneal fibrosis group compared with the vehicle group. Calcitriol decreased the serum and dialysate active transforming growth factor-β1 and angiotensin II level, decreased the thickness of the liver peritoneum and muscle peritoneum, and decreased the expression of α-SMA, fibronectin, collagen, vascular endothelial growth factor, angiotensin II, transforming growth factor-β1, and P-Smad2/3-positive cells in liver peritoneum cells. High-dose calcitriol exhibited better protective effects against peritoneal fibrosis than did the lower dose. Calcitriol protected against chlorhexidine digluconate-induced peritoneal fibrosis in rats by decreasing transforming growth factor-β1 and angiotensin II production.

Valsartan decreases TGF-β1 production and protects against chlorhexidine digluconate-induced liver peritoneal fibrosis in rats

Peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The present study was designed to investigate the protective effects of valsartan on chlorhexidine digluconate-induced PF by decreasing TGF-β1 production in rats. PF was induced in Sprague-Dawley rats by daily administration of 0.5 ml 0.1% chlorhexidine digluconate in normal saline via peritoneal dialysis (PD) tube for 1 week. Rats received daily intravenous injections of low dose valsartan (1 mg/kg) or high dose valsartan (3 mg/kg) for 1 week. After 7 days, conventional 4.25% Dianeal (30 ml) was administered via a PD catheter with a dwell time of 4 h and assessed of peritoneal function. At the end of dialysis, rats were sacrificed and the liver peritoneum was harvested for microscopically and immunohistochemistry. There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D₄/P(4Urea) level was reduced, the D₄/D₀ glucose level, serum and dialysate transforming growth factor-β1 (TGF-β1) level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, collagen, and vascular endothelial growth factor (VEGF) were elevated in the PF group compared with the vehicle group. High dose of valsartan decreased the serum and dialysate TGF-β1 level, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen, and VEGF-positive cells in liver peritoneum. The low dose of valsartan did not protect against chlorhexidine digluconate-induced PF in rat. Valsartan protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-β1 production.

Aliskiren ameliorates chlorhexidine digluconate-induced peritoneal fibrosis in rats.

Eur J Clin Invest 2010; 40 (4): 301–309

Freshwater clam extract ameliorates acute liver injury induced by hemorrhage in rats.

The freshwater clam is a widely-consumed shellfish and is used as a remedy for chronic hepatitis in Asia. However, its contribution to acute liver injury (ALI) remains unclear. The aim of this study is to assess the protective effects of freshwater clam extract (CE) in ALI induced by hemorrhage in rats. Rats were randomly divided into 5 groups, (1) blood loss (BL) 40%, (2) CE 150 mg/kg plus BL 40%, (3) CE 75 mg/kg plus BL 40%, (4) CE 150 mg/kg, and (5) CE 75 mg/kg groups. CE was given by femoral vein catheter in Groups 2 to 5. Initial hemorrhage was induced by withdrawing blood (loss 40% of total blood volume) from a femoral arterial catheter after CE administration in Groups 2 and 3. The levels of blood tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured at several time points during the experimental period. Rats were sacrificed after 48 hours, and the liver was harvested for hematoxylin and eosin (HE) stain to show liver tissue injury. The results indicated that hemorrhage significantly decreased mean arterial pressure (MAP), increased blood AST, ALT and LDH levels and induced liver injury. Pre-treatment with the CE increased MAP and IL-10 levels and decreased AST, ALT, LDH and TNF-alpha levels after hemorrhage. The HE stains showed diminished organ injury in the CE groups. In conclusion, freshwater clam extract is a potential immunomodulating agent and ameliorates acute liver injury.