Un‐fractionated heparin counteracts the systemic inflammatory responses and multiple organ damages caused by endotoxaemia in sheep

Abstract Background Endotoxaemia is believed to be a major cause of mortality and there are several therapeutic regimens for the treatment of this situation. Objectives The present experimental study was conducted to evaluate acute phase response, cardiovascular and hepatorenal damages following the treatment of Ovine experimental endotoxaemia model employing unfractionated heparin (UFH). Materials and Methods Twenty clinically healthy 1‐year‐old fat‐tailed ewes were randomly divided into four equal groups, comprising UFH 200, UFH 400, Ctrl+ and Ctrl‐. Lipopolysaccharide (LPS) from Escherichia coli serotype O55:B5 at 0.4 μg/kg was administered intravenously to the ewes. UFH (at 200 and 400 IU/kg) was administrated to the UFH 200 and UFH 400 groups, respectively. All the ewes were evaluated clinically before and 1.5, 3, 4.5, 6 and 24 hours after LPS injection. Blood samplings were also performed at those hours. We measured serum concentrations of haptoglobin, interferon‐gamma, total antioxidant status, malondialdehyde, cardiac lactate dehydrogenase, cardiac troponin‐I, total bilirubin, alanine transaminase and creatinine. Serum concentrations of acute phase response, cardiovascular, hepatic and renal biomarkers and clinical parameters increased significantly following the induction of endotoxaemia in the groups receiving LPS. Results The significantly lowest concentrations of these parameters at hours 4.5 and 6 among the treatment groups belonged to the UFH 400 sheep. Conclusions UFH could act as an anti‐inflammatory mediator by decreasing inflammatory cytokines and acute phase proteins, modulating oxidative stress biomarkers and reducing multiple organ dysfunction following endotoxaemia in a dose–dependent manner. Furthermore, the anti‐inflammatory effects of UFH at 400 IU/kg were significantly higher than another dose. This research examined the effect of two doses of UFH and higher doses may have more anti‐inflammatory effects that require further studies.


INTRODUCTION
Endotoxaemia is an acute inflammatory condition caused by lipopolysaccharide (LPS) of Gram-negative bacteria in the bloodstream. This condition triggers a series of reactions in the host body called the acute phase response (APR) (Munford, 2016). APR reduces subsequent tissue damages by preventing further invasion of the pathogen, allowing the host body's haemostatic mechanisms to reestablish their condition (Rodrigues et al., 2018). APR increases the production of a number of inflammatory mediators which are associated with local and systemic effects. Following APR, the production of inflammatory mediators, such as acute phase proteins and inflammatory cytokines, increases (Christoffersen et al., 2010). Therefore, by evaluating the circulating values of these parameters, APR could also be evaluated . Oxidative stress is another event that occurs following APR, which could be assessed by measuring the circulating oxidative stress biomarkers (Rodrigues et al., 2018).
Increased production of inflammatory mediators following APR leads to secondary dysfunction of internal organs. Under these conditions, the components of the immune system, which are responsible for defending the host against invasive pathogens, could damage the host tissues and organs. This results in the failure of several organs, which is one of the main symptoms of endotoxaemia and is called multiple organ dysfunction (MOD) (Anderson et al., 2010). Accordingly, through evaluating the circulating biomarkers of each organ, such as liver, kidney and cardiovascular and respiratory system, it is possible to assess the health of the function of each of them (Chalmeh et al., 2014).
To prevent the exacerbation of acute inflammatory responses as well as disorders of internal organs, effective treatments should be taken into consideration for patients with endotoxaemia and inflammation. To date, several studies have been performed on different therapeutic regimens for patients with endotoxaemia in both human and animal models. Heparin is one of the drugs with the potential effects to treat inflammatory conditions (Poterucha et al., 2017).
This drug could be classified into unfractionated heparin (UFH) and low molecular weight heparin (LMWH). UFH is the most widely used anti-coagulant in clinical practice, which is recommended for the prevention of thromboembolism in septic patients; there is also information regarding its anti-inflammatory effects (Li and Ma, 2017).
There are several researches about anti-inflammatory effects of UFH in asthma, cardiopulmonary bypass, inflammatory bowel diseases, acute coronary syndrome, cataract surgery and endotoxaemia at different aspects (Mousavi et al., 2015). Therefore, we hypothesized that UFH might be effective in reducing acute inflammatory reactions and reversing internal organ changes following endotoxaemia. Hence, the present study aimed to investigate the role of UFH in two different doses in the treatment of Ovine experimental endotoxaemia model by evaluating the inflammatory, oxidative stress, hepatic and renal and cardiovascular circulating biomarkers. The obtained results herein may also help to generalize the therapeutic effects of UFH to other inflammatory conditions.

Animals
Twenty clinically healthy 1-year-old fat-tailed ewes (50 ± 5 kg bodyweight) were randomly chosen for the project. Every sheep was given

Chemicals and drugs
The ewes were given 0.

Induction and treatment of endotoxaemia
A Schematic illustration of the study design is presented in Figure 1. Clinical evaluations were performed on all the ewes before and 1.5, 3, 4.5, 6 and 24 hours after LPS injection. Rectal temperature, heart rate (HR) and respiratory rate (

Blood sampling and serological assays
The blood samples were collected from all the ewes through the fixed catheter prior and 1.5, 3, 4.5, 6 and 24 hours after LPS injection in serum collection tubes. After the collections, sera were separated through centrifugation (for 10 min at 3000 × g) and stored at −20 • C until assayed. The haemolysed specimens were not analysed by the laboratory.

Statistical analyses
The data were expressed as mean and pooled standard error of mean

RESULTS
After inducing endotoxaemia, serum concentrations of Hp increased significantly in the groups receiving endotoxin and the rising trend continued until 6 hours in the Ctrl+ group (Table 1; p < 0.05). The Hps in the Ctrl-group were significantly lower than those in the other studied groups at all the hours (except hour 0). After beginning the drug administrations, the levels of Hp in the treatment groups (at hour 4.5) decreased and were substantially lower than those in the Ctrl+ group ( Different letters indicate significant differences among different groups at each study time about each parameter (p < 0.05). *Effect of group, time and group × time following induction and treatment of endotoxaemia by different drugs about each studied parameter. The significance level is lesser than 0.05.
lower than those in the other studied groups at all the hours (except hour 0). The levels of cTnI and LDH in the treatment groups decreased after the beginning of drug administrations (at hour 4.5), which were significantly lower than those in the Ctrl+ group (Table 2; p < 0.05). At hours 4.5, 6 and 24, the UFH 400-receiving sheep had the least significant concentrations of cTnI, and LDH.
In all the endotoxic animals, serum levels of hepatic biomarkers (ALT and total bilirubin) increased significantly after intravenous endotoxin infusion (Table 2; p < 0.05). UFH significantly reduced the levels of these hepatic biomarkers. At hours 4.5, 6 and 24, the least significant concentrations of ALT and total bilirubin were detected in the UFH 400. After endotoxin injection, serum renal biomarker (creatinine) levels of all the studied groups increased substantially and all the endotoxic sheep's levels increased up to hour 3. At hours 4.5, 6 and 24, serum creatinine concentrations in the UFH 400 group were significantly lower than those in the UFH 200 (Table 2; p < 0.05).
Following the induction of endotoxaemia, HR and RR and rectal temperature increased significantly. The increasing pattern of the Ctrl+ group persisted until hour 4.5 (Table 3; p < 0.05). These clinical parameters were significantly lower in the Ctrl-group than in the other studied groups at all the hours (except for hour 0). The quantities of HR, RR and rectal temperature in the treatment groups decreased after beginning drug administration (at hour 4.5), which were significantly lower compared with those in the Ctrl+ group (Table 3; p < 0.05). HR, RR and rectal temperature were found to be significantly lower in the UFH 400 sheep at hours 4.5 and 6. At hour 24, there were no significant differences concerning the amounts of these parameters among the studied groups (p > 0.05).

DISCUSSION
Endotoxaemia is known to be a leading cause of morbidity around the world. As a result, implementing adequate treatment methods that are efficient, reliable, easy and inexpensive is critical in treating this situation. These therapies should have the fewest possible side-effects and be widely available (Mousavi et al., 2015). Therefore, the current research sought to investigate the anti-inflammatory effects of UFH in an Ovine experimental endotoxaemia model by analysing the APR and MOD. Several animal and human studies have suggested that heparin not only effectively prevents the coagulation mechanism in endotoxaemia, but also causes a variety of anti-inflammatory responses against infection. Human endotoxaemia experimental models have indicated that heparin can treat endotoxaemia by inducing coagulation activities (Jaimes et al., 2006;Li et al., 2009;Li et al., 2013;Miranda et al., 2014;Schiffer et al., 2002;Wyns et al., 2015;Zhao et al., 2017). However, in human models, the inhibition of inflammatory pathways following endotoxaemia has not been as clearly demonstrated as in animal Heparin is the most commonly available and least expensive anticoagulant utilized to treat DIC. Haneberg et al. (1983) (Derhaschnig et al., 2003). Heparin has been shown in several studies to reduce the inflammatory response following cardiopulmonary bypass. The use of heparin-treated surfaces in cardiopul-monary bypass circuits has been shown to reduce leukocyte activation and complement cascade activation (Ludwig, 2009). As a result, the need for inotropic support, post-operative mechanical ventilation time and incidence of acute lung injury, on top of the length of hospital stay, decrease, reflecting heparin's beneficial impact in cardiopulmonary bypass circuits (Redmond et al., 1993;Svenmarker et al., 1997).
In these trials, the heparin-coated circuit significantly reduced the levels of cytokines, such as TNF-α, complement complex, neutrophils and elastase, when compared to the non-heparin-coated circuit. The current study found that UFH at 400 IU/kg significantly decreased the inflammatory process and combated MOD after endotoxaemia induction in the studied sheep (Tables 1-3; p < 0.05). According to our results, UFH at 400 IU/kg had anti-inflammatory properties superior to 200 IU/kg.

IFN-γ is the only member of class II IFNs and is important for both innate and adaptive immunity against viral and intracellular bacterial
infections. The role of IFN-γ in the immune system is owing to its ability to directly inhibit virus replication as well as its immune-stimulating and regulating effects (Parameswaran & Patial, 2010;Schroder et al., 2004). IFN-γ reduced in the UFH 200 and UFH 400 after the drugs were administered to these groups. Following the therapy, the concentrations of this cytokine in the UFH 400 were significantly lower than those in the UFH 200 (Table 1; p < 0.05). Since IFN-γ is a marker of inflammation and APR, its substantial reduction following UFH administration at 400 IU/kg, when compared to other drugreceiving ewes, may suggest that UFH has the potential to reduce inflammation following endotoxaemia in this Ovine endotoxaemia model.
Hp is an acute phase protein that has been proposed as a stress biomarker in animals (Eckersall and Bell, 2010). Hp is synthesized in the liver and is a key acute phase protein in several animal species. The amount of Hp in the bloodstream of ruminants is usually negligible, but it increases more than 100-fold once the immune system is stimulated (Eckersall and Bell, 2010;George & Sack, 2018). Several studies have highlighted the significance of Hp as a useful para-clinical parameter for assessing the occurrence and severity of inflammatory diseases in sheep (Chalmeh et al., 2014). Serum Hp determination has shown that this protein can be useful in the diagnosis of infection and inflammatory conditions. According to Chalmeh et al. (2013) The pro-and antioxidants could be used to measure oxidative stress.
MDA is a pro-oxidant derived from lipid peroxidation; however, it is a weak indicator of oxidative damage (Tsikas, 2017). Nonetheless, we employed this parameter to assess lipid peroxidation. Endogenous antioxidants are a network of substances in the body that can deal with an oxidative assault. Antioxidant enzymes are a form of endogenous antioxidant. Since other forms of antioxidants are not taken into account, assessing the enzymes alone cannot decide the status of antioxidants throughout the body (Valko et al., 2007). As a result, by assessing TAS, it would be possible to determine the combined action of all the antioxidants present in the target samples (Erel, 2004). In the current research, circulating levels of TAS significantly reduced after endotoxin infusion with the lowest levels observed 24 hours after endotoxaemia induction in the Ctrl+ ewes (Table 1; p < 0.05).
These findings indicated that the major evolving patterns of TAS are consumptive during APR after endotoxaemia induction. Following the therapies, TAS levels increased and at hour 24, these levels were similar to those in the baseline in the UFH 200 and UFH 400 groups.
The current study found that the serum concentration of oxidative stress biomarkers in the UFH 400 ewes was not significantly different from that in the Ctrl-sheep at hour 24 ( accordingly, cardiovascular isoenzymes and biomarkers alter (Chalmeh et al., 2014). As a result, these diagnostic biomarkers are useful parameters in the early detection of cardiovascular complications caused by ischaemia, injury, or inflammation. The literature describes the changes in cardiac injury biomarkers following endotoxaemia induction in farm animals (Chalmeh et al., 2014;Peek et al., 2008). Serum concentrations of cardiovascular biomarkers increased rapidly and significantly at hour 1.5 after endotoxaemia induction (Table 2; p < 0.05) and remained elevated in the Ctrl+ ewes until hour 24. The emergence of cTnIs as the gold standard, as well as responsive and precise biochemical markers of myocardial injuries, has helped the diagnosis and management of myocardial injuries over the recent years (Aldous, 2013). The rise in cTnI immediately after LPS administration in the current study could indicate myocardial injuries during endotoxaemia. Increased cTnI have been linked to a variety of animal cardiac disorders. Serum cardiac LDH levels significantly increased 1.5 hours after endotoxin infusion in all the endotoxin-receiving sheep (Table 2; p < 0.05). In the Ctrl+ animals, high concentrations of these enzymes were observed at all the hours after endotoxaemia induction and remained elevated up to hour 24 after endotoxin administration. LDH is a cytoplasmic enzyme found in the heart, skeletal muscle, liver, kidney and red blood cells. This enzyme is a marker of increased cellular damage and its increased activity is a result of its increased release from damaged cells, as well as a reflection of metabolic changes in inflamed tissues, specifically the heart (Aldous, 2013). Damage to the skeletal or heart musculature causes a significant rise in serum LDH levels since the majority of the vessels in the body can be thought of as a sufficient reservoir of enzymes liable to be released and identified during pathological situations. As a result, any damage to the vasculature could trigger enzyme leakage, making it a valuable tool in the early detection of pathological conditions .
The findings in the current study indicated that circulating cardiovascular biomarkers significantly increased after endotoxaemia induction in the sheep receiving endotoxin, reflecting the severe cardiovascular damage in this acute inflammatory state. UFH reduced cardiovascular biomarkers and UFH at 400 IU/kg was significantly more effective than UFH at 200 IU/kg. Hence, it is possible to state that UFH is of anti-inflammatory effects on reducing cardiovascular damage and dysfunction in endotoxic sheep.
Liver plays an important role in protective responses to scavenge bacteria and the development of inflammatory mediators during the endotoxaemia (Yao et al., 2013). Endotoxaemia-related liver dysfunction has been historically thought to be a late feature of serious illness, manifesting as jaundice and hyperbilirubinaemia (Lida et al., 2009).
Recent studies, however, have identified liver dysfunction as an early event in endotoxaemia (Marshall, 2012). As a result, it is critical to comprehend the pathophysiological changes leading to endotoxaemiarelated liver dysfunction, which has been characterized as a combination of cellular injury and increased inflammation. Endotoxaemia may cause damage to hepatocytes. This damage is caused by hypoperfusion in the presence of hypovolaemia and insufficient cardiac performance and could be repaired quickly with supportive therapy (Rivera et al., 2003). This injury is distinguished through the leak of transaminase enzymes, which represents acute cellular and mitochondrial damages. According to the literature, ALT is rapidly elevated following an episode of hypotension or shock (Kobashi et al., 2013). When the underlying path was reversed, ALT levels dropped dramatically in a matter of days. Septic shock combined with hypoxic hepatitis can lead to fulminant hepatic failure, which often results in disseminated intravascular coagulation and bleeding. Lactate and amino acid clearance, on top of protein synthesis, reduces after hepatic injuries caused by endotoxaemia. Gluconeogenesis and glycogenolysis are also reduced and hypoglycaemia may occur. In endotoxaemia, jaundice is much more prevalent than hypoxic hepatitis. Jaundice is usually a late complication of serious endotoxaemia. However, even in the absence of fever or leukocytosis, jaundice may occur at an early stage of endotoxaemia (Famularo et al., 2003); hence, serum bilirubin is the most commonly used biomarker for detecting hepatic dysfunction during sepsis (Patel et al., 2013). Bilirubin levels, on the other hand, lack precision in reflecting the broad range of liver dysfunction and distinguishing an acute reaction from a pre-existing organ chronic disease.
The obtained results herein revealed that serum levels of ALT and total bilirubin increased significantly after endotoxaemia induction ( Creatinine, a waste product of phosphocreatine catabolism, is primarily filtered by the kidney though a limited amount is deliberately secreted. While there is some tubular reabsorption of creatinine, this is offset by nearly equal amounts of tubular secretion. Any increase in creatinine levels in the blood is related to excretion and, thus, indicates kidney dysfunction. However, in case of extreme renal impairment, creatinine clearance may be overestimated due to active creatinine secretion, which accounts for a greater fraction of overall creatinine cleared. Higher levels of it than normal could indicate dehydration (Uchino, 2010). Understanding the effects of endotoxaemia on renal blood flow, glomerular filtration rate, renal vasculature and tubular function is critical for identifying potential intervention targets to reduce the complications of endotoxaemia-induced acute renal failure (Wang et al., 2010). In the current study, all the endotoxic sheep had significantly higher circulating creatinine levels after endotoxin infusion (Table 2; p < 0.05). The creatinine rise in this work was most likely due to endotoxin damage to renal tissue and its inability to expel creatinine from the body. Since all the animals in our study received intravenous fluids, an increase in blood creatinine after endotoxaemia could not be attributed to dehydration in the endotoxin-receiving ewes. 4.5 and 6 hours after endotoxaemia induction, serum creatinine concentrations in the UFH 400 group were significantly lower than those in the UFH 200 (Table 2; p < 0.05). As a result, UFH at 400 IU/kg was able to minimize renal failures following endotoxaemia in the sheep and this capacity may be on account of its anti-inflammatory properties.
The body temperature increases after endotoxin administration and it is a right pyrogenic reaction (Centanni, 1984). Small doses of endotoxin induce a monophasic fever response in common laboratory animals whereas moderate to large doses can induce a biphasic fever response. The initial fever response, which is caused by a direct impact on the thermoregulatory centre in the hypothalamus, is thought to have a 1-hour latency period. If a second high occurs, it appears 4 hours after the endotoxin is administered (Coskun et al., 2020). Endotoxaemia causes fever and in the current study, the rectal temperature of the endotoxic sheep increased significantly after endotoxin infusion ( demonstrated that HR analysis is a sensitive method for studying the autonomic nervous system in animals and detecting discrete changes in sympathetic-parasympathetic balance, particularly, vagal function. The inflammatory response and the autonomic nervous system are inextricably related. The acute response to endotoxaemia involves improvements in the autonomic nervous function as well as activation of innate immune mechanisms (Chalmeh et al., 2014). Measuring HR has been suggested to provide insight into the acute effect of sympathetic and cholinergic anti-inflammatory pathways. In the current research, HRs increased substantially after the induction of endotoxaemia. Nonetheless, at hours 4.5 and 6 after endotoxin infusion, this parameter was significantly lower in the UFH 400 group than that in the UFH 200 ewes (Table 3; p < 0.05). The decreasing trend of HR in this group might be linked to lowering APR and balancing the autonomic nervous system.
Endotoxin has a significant impact on the structure and function of the lungs. Endotoxin causes both noticeable and subtle effects on the workings of the airways and the pulmonary circulation. These side-effects include diffuse lung inflammation and pulmonary vascular endothelial damage. Endotoxin can also cause endothelial cell damage in vitro (Rojas et al., 2013). In this study, RR increased significantly during endotoxaemia and decreased following the therapeutic regimens.
The lowest RR was observed in the UFH 400 at hours 4.5 and 6 following endotoxaemia induction (Table 3; p < 0.05), indicating that UFH has anti-inflammatory effects on endotoxaemia in sheep.
Heparin-induced thrombocytopenia (HIT) may occur following heparin administration, which can be fatal if left untreated. Platelet depletion following thrombocytopenia may be rapid or delayed, but platelet counts usually increase after discontinuation of heparin. Therefore, the decision to re-administer heparin should be accompanied by platelet counting and monitoring (Ahmed et al., 2007). There are few studies on HIT in sheep (Connell et al., 2006). The sheep in this study had not previously received UFH and they did not receive UFH again after treatment in this research, therefore, they were less likely to develop complications from HIT. Platelet count was not performed in this study but clinical observations after this experiment did not show any signs of HIT in these sheep. Based on the present study, it can be stated that according to the method used in this research, UFH can be used due to its anti-inflammatory properties in endotoxic sheep.

CONCLUSIONS
In conclusions, we shed light on the fact that UFH acts as an antiinflammatory mediator in sheep by decreasing inflammatory cytokines and acute phase proteins, modulating oxidative stress biomarkers and lowering MOD after E. coli serotype O55:B5-induced endotoxaemia.
Furthermore, the anti-inflammatory effects of UFH at 400 IU/kg were substantially higher than those of UFH at 200 IU/kg; this exhibited the dose-dependent action of this drug. The current study found that UFH has therapeutic effects in the treatment of Ovine endotoxaemia as an inflammatory model. It is possible that the findings be applied to the treatment of other inflammatory conditions and further studies are needed. This research examined the effect of two doses of UFH and higher doses may have more anti-inflammatory effects that require further studies.

ACKNOWLEDGMENTS
We sincerely thank the farmers who allowed this project to be done on their sheep. The authors also thank Shiraz University for financial support.