Sepsis is a devastating condition characterized by the activation of inflammatory and coagulation pathways in response to microbial systemic infection [
One cell type that is injured during sepsis is the erythrocyte. As red blood cells lyse, hemoglobin is released and oxidized, releasing free heme in the circulation. Free heme can cause tissue damage and programmed cell death through inducing the release of proinflammatory mediators and activation of coagulant system, and thus contributing to multiorgan dysfunction and death [
There are a number of cytoprotective mechanisms against the deleterious effect of sepsis; among them are heme oxygenase-1 (HO-1, formerly heat shock protein 32) and hemopexin (HPx) mediated mechanisms. HO-1 is an inducible enzyme that converts heme into carbon monoxide (CO), biliverdin, and free iron [
HPx is an acute-phase plasma protein expressed mainly in the liver, and its production is enhanced under inflammatory conditions. It has the highest binding affinity to heme, subsequent to heme binding; the heme-HPx complex is internalized in liver cells through LDL receptor-related protein 1- (LRP1-) mediated endocytosis, resulting in cellular heme uptake [
In this context, by using an animal model of sepsis, our study aimed to determine a new role for HO-1 and HPx in the coagulopathy induced by septic inflammation and whether they can affect the host defense mechanism by enhancing the production of the anti-inflammatory cytokine, IL-10, at early stages of sepsis.
The development of experimental sepsis models to elucidate the progression and pathophysiology of clinical sepsis extent over the past decades. The most frequently used sepsis model is cecal ligation puncture (CLP) which was employed in the current study [
The study was conducted on 48 healthy male albino Wistar rats weighing 200–220 g; the animals were housed in an animal facility at the Faculty of Medicine, Alexandria University. A minimum temperature of 10°C in winter and a maximum one of 35°C in the summer were maintained. A period of 12–14 hours of daylight was provided. Food and water were available ad libitum. All experimental procedures were carried out based on the ethical guidelines for care and use of laboratory animals of Alexandria University. The study was approved by the Faculty of Medicine, Alexandria University Ethics Committee.
The animals were divided into 4 experimental groups (12 rats/group):
The rats were fasting for 12 hrs before the procedure but allowed free access to water. Anesthesia was induced by an intraperitoneal injection of pentobarbital (70 mg/kg). A midline laparotomy was performed, and the cecum was identified. Stool contents were milked to the tip of the cecum, which was subsequently ligated 1 cm from the tip with a 2-0 silk tie. The cecum was then perforated four punctures with a 22 G needle to induce severe sepsis and returned into the abdomen. The abdominal wall was closed with a continuous 3-0 silk suture. No antibiotics were used, and the animals had free access to food and water postoperatively. All animals in all experiments received saline to prevent dehydration (60 mL/kg/day subcutaneously) [
48 hrs after CLP, mortality rate was calculated; then animals were deeply anesthetized with 100 mg/kg pentobarbital. The blood was collected via cardiac puncture with EDTA-treated syringe needles immediately mixed with 3.2% sodium citrate at a ratio of 9 : 1 (blood vol/citrate vol). Blood was then centrifuged at 1,500 g for 15 min to separate the plasma that was frozen in aliquots at −70°C until assay. The lungs and livers were excised for histological assessment.
Thawed plasma was transferred into a polypropylene aliquot tube and respinned at 1,500 g for 15 minutes to obtain platelet poor plasma (PPP) where platelet count was <10,000/uL. The top 0.1 mL of plasma was removed and placed in a water bath kept at a 37°C. 0.1 mL of thromboplastin (tissue factor) and 0.1 mL of calcium chloride (CaCl2) were added, and the contents were mixed thoroughly to initiate coagulation [
PPP was obtained as previously mentioned; then 0.1 mL was incubated at 37°C. Phospholipid (cephalin) and contact activator (Kaolin) were added followed by the calcium (all prewarmed to 37°C) and the contents were mixed thoroughly to initiate coagulation [
Both assays were performed in an Amelung KC4 coagulation semiautomated analyzer using electromechanical method of clot detection (Sigma Chemical Co., St. Louis, MO).
PPP was incubated at 37°C with the protein C activator (Protac) for 5 minutes. Then a chromogenic substrate for APC was added. The change in optical density was measured and by comparison against a standard reference curve the protein C level was determined [
Liver homogenate HO-1, plasma, and liver homogenate IL-10 levels were measured by using ELISA kits (Shanghai BlueGene Biotech Co., Ltd., Shanghai, China) according to the manufacturer’s instructions [
Liver tissues were homogenized in homogenization buffer (20 mmol/L potassium phosphate buffer (pH 7.4), 250 mmol/L sucrose, 2 mmol/L ethylene diamine tetra-acetic acid, 2 mmol/L phenylmethylsulfonyl fluoride, and 10 lg/mL leupeptin). The homogenates were centrifuged at 10,000 ×g for 30 min, and the supernatant was further centrifuged at 100,000 ×g for 1 h at 4°C. The pellet was suspended with potassium phosphate buffer followed by sonication for 2 sec at 4°C (microsome fraction) [
Liver and lung specimens were fixed with 10% buffered formalin, embedded in paraffin, sectioned, and examined under light microscopy. Transverse sections were made at five different levels to cover the entire organ, and ten fields were selected randomly from each section and examined for counting the number of thrombi. The mean number of thrombi was calculated for statistical analysis.
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. Quantitative data were described using mean and standard error. The distributions of quantitative variables were tested for normality using Kolmogorov-Smirnov test, Shapiro-Wilk test, and D’Agostino test. Also, histogram and QQ plot were used for vision test. Data were normally distributed, and, thus, comparison between different studied groups was analyzed using
Survival rates were calculated during 48-hour period after the induction of sepsis. Control rats survived the entire period, while rats subjected to CLP showed survival rates of 83.3%, 75%, and 58.3% after 6, 12, and 18 hrs of induction, respectively. Hemin improved the survival rates to 91.6 and 83.3% 6 and 12 hrs after induction of sepsis; also HPx treated rats had the same rates as hemin group after 6 and 12 hrs of sepsis, but survival then decreased to be 75% after 18 hrs and for the rest of the study period (Figure
Survival rates through the study period. Survival rates of CG were 100% through the entire period; CLP showed survival rates of 83.3%, 75%, and 58.3% after 6, 12, and 18 hrs of induction, respectively. CLP + Hm group survival rates were 91.6 and 83.3% 6 and 12 hrs after induction of sepsis. CLP + HPx group survival rates were 91.6%, 83.3%, and 75% after 6, 12, and 18 hrs of sepsis, respectively.
The PT and APTT values were significantly lower in CLP group in comparison to the control one (
Comparison between experimental groups regarding the coagulation parameters.
Control group (CG) | Sepsis group (CLP) | CLP + Hm | CLP + HPx | |
---|---|---|---|---|
Prothrombin time (seconds) | 17.08 ± 0.2 | 13.6 ± 0.2 |
18.6 ± 0.4 |
15.9 ± 0.5 |
Partial thromboplastin time (seconds) | 15.8 ± 0.04 | 9.2 ± 0.3 |
15.6 ± 0.2 |
12.1 ± 0.3 |
APC (IU/dL) | 96.8 ± 2.2 | 59 ± 1.6 |
83 ± 0.6 |
75.5 ± 1.05 |
Data are presented as mean ± SEM (7–12 per group); one-way ANOVA was conducted and the results of post hoc least significance difference comparison were shown. Results are significant at
APC: activated protein C.
Concentration of HO-1 in liver homogenate (ng/mg tissue) among the four groups 48 hours after induction of sepsis. One-way ANOVA test was used to analyze the data presented as mean ± SEM, where
Concentrations of IL-10 (a) in the serum (pg/mL) and (b) in the liver homogenate (pg/mg tissue), measured 48 hours after CLP, using ELISA technique. Data are expressed as mean ± SEM (
The number of thrombi per field. Sections of the livers and lungs from control rats (CG), CLP rats with cecal ligation induced sepsis (CLP), CLP rats pretreated with hemin (CLP + Hm), or CLP rats pretreated with hemopexin (CLP + HPx). Sections were stained with H&E and examined by light microscopy. The numbers of thrombi were counted in 10 randomly selected fields (magnification ×40) per organ and the mean number in the 10 fields was taken. The data were analyzed using one-way ANOVA test,
Histological examination of liver and lung sections showed the appearance of some inflammatory changes that were most prominent in CLP group. These inflammatory changes were moderate in the group CLP + Hm and mild in CLP + HPx group as shown in Figure
Histopathological changes of liver and lung sections of the four experimental groups. Liver and lung sections were stained with H&E and examined by light microscopy (magnification ×40 and ×100 for liver and lung, resp.). (a) Liver tissue—CG: normal liver section of the control group with no pathological changes. CLP group: dense (severe) portal lymphocytic infiltration (arrow) and diffuse macro- and microvesicular fatty changes in hepatocytes (arrow heads). CLP + Hm: focal microvesicular fatty changes in hepatocytes (arrow heads). CLP + HPx: moderate portal lymphocytic infiltration (arrow) and diffuse microvesicular fatty changes in hepatocytes (arrow heads). (b) Lung tissue—CG: lung, thin walled alveoli, no inflammation, and mild congestion. CLP group: thick walled alveoli with dense inflammation, congestion, and numerous thrombi (arrows). CLP + Hm: thin walled alveoli with mild thickness, moderate inflammation, mild congestion, and few thrombi (arrow). CLP + HPx: thin walled alveoli with mild thickness, inflammation, congestion, and few thrombi (arrow).
Liver tissue
Lung tissue
Despite the development of advances in diagnostic and prognostic biomarkers, the presence of promising preclinical animal studies examining the fundamental cellular and biological mechanisms underlying septic physiology, and a number of clinical trials targeting treatment of thromboinflammatory mediators and pathways, there are only few therapeutic agents passing to phase III clinical trials and currently none have witnessed sustained clinical use [
So, currently, more researches continue in this scope to identify and study new therapies that hold promise. So, agents targeting heme oxygenase-1 acting as a cytoprotective enzyme catalyzing the oxidative degradation of toxic heme released during the pathophysiology of sepsis [
Sepsis is associated with impaired hemostasis, with tilt of the balance towards the procoagulant state. In sepsis, the clotting cascade is enhanced through induction of tissue factor as a result of endotoxemia [
The current study, in line with previous research [
Low plasma PC levels can be considered as predictive parameter of early death after CLP as reported by Berg et al. (2006), who showed an enhancement of the survival rate in animals that did not have significant reduction in PC level [
Multiorgan failure is a leading cause of death in sepsis. No specific therapeutic agent, till now, is available to prevent multiorgan failure in sepsis. However, among the promising approaches is using agents that upregulate endogenous intracellular defenses against cellular toxins. One of such agents is hemin, which upregulates cellular levels of HO-1 [
However, there are contradictory studies concerning the role of HO-1 in sepsis physiopathology. One study indicates that the HO-1 pathway leads to increased susceptibility to severe sepsis as it found that treatment of animals with an HO-1 inhibitor increases their survival rate [
The protective role of HO-1 during microbial sepsis has been partly attributed to its ability to suppress the deleterious effect of free heme produced during the course of infection. Therefore, it protects against irreversible tissue damage [
Other reports showed similar results, even though they used a different regimen of hemin administration, being given to rats 12 hrs after the induction of sepsis [
Inflammation and hypercoagulable state are the main features of sepsis [
A recent therapeutic approach against tissue damage of sepsis is the heme scavenger protein, HPx. In the current study, HPx was administered by intraperitoneal route to rats 30 min before the induction of sepsis. The results showed a significant prolongation in the PT and APTT with an increased level of APC. Consequently, control of coagulation was improved; their liver HO-1 and IL-10 (serum and liver) were significantly increased compared to septic rats.
HPx is able to neutralize the rising amounts of free heme. Binding of heme to HPx neutralizes its prooxidant effect. Such binding also inhibits the release of free radicals in hepatocytes due to the presence of heme and thus prevents their apoptosis [
Larsen et al., 2010, found that HPx serum concentration within 48 hrs of presentation with septic shock was positively associated with patient survival time. That is, patients with lower HPx serum concentrations died at earlier time points compared to patients with higher HPx serum concentrations [
Therefore, we consider that administration of exogenous HPx might be used therapeutically to increase tolerance to infection and hence prevent the development of severe sepsis in rats and therefore decrease the mortality rate.
The reported beneficial effect of HPx most probably requires the expression of HO-1 to catabolize HPx-bound heme and to decrease IL-6 and TNF-
Therefore, the anti-inflammatory activity of HPx is attributed to its direct effect on the anti-inflammatory key counterregulatory protein, IL-10. IL-10 is an anti-inflammatory cytokine that was significantly increased after CLP as evidenced in our study. There was a positive correlation between its level in serum and liver in CLP group. This denotes that sepsis induces the production of IL-10 mainly through the stimulation of Kuppfer cells, which are considered to be a primary source of IL-10 during a model of abdominal sepsis as demonstrated by Traegera et al. (2010) [
Kasten et al., 2010 [
Role of IL-10 in sepsis is complex; it could be shown that IL-10 acts posttranscriptionally to downregulate TNF expression as it increases TNF mRNA instability [
However, Song et al., 1999, reported that inhibition of IL-10 12 hrs after CLP can improve survival [
The present study extends these previous reports by demonstrating that hemin and HPx administration upregulate liver HO-1 and reduce CLP induced thrombosis. Hemin or HPx administration was able to prolong the PT and APTT and enhance APC. Also, their administration was able to reduce the inflammatory infiltrate in liver and lung parenchyma and to reduce the number of apoptotic cells. All effects may be partially due to the increased production of the anti-inflammatory cytokine IL-10.
Despite showing the same results, still hemin was superior to HPx in controlling the coagulation and in enhancing the HO-1 production, while HPx was a more potent stimulant for the expression of IL-10.
Activated partial thromboplastin time
Activated protein C
Cecal ligation puncture
Heme oxygenase-1
Hemopexin
Lipopolysaccharide
Phosphate buffered saline
Platelet poor plasma
Prothrombin time
Thrombomodulin.
The authors declare that they have no competing interests.
All the authors contributed to the study design, collection of the data, and writing of the paper. All authors read and approved the final paper. All authors contributed equally to funding this research.
The authors thank Dr. Amany Abdel Bary, the Lecturer of pathology, Faculty of Medicine, Alexandria University, for her contribution to this work by describing all the histologic examined specimens.