Hydroxytyrosol ameliorates oxidative challenge and inflammatory response associated with lipopolysaccharide-mediated sepsis in mice

Abstract

Hydroxytyrosol (HT) is among the main bioactive ingredients isolated from olive tree with a variety of biological and pharmacological activities. In the current study, the antioxidative and anti-inflammatory activities of HT were distinguished in the splenic tissue following lipopolysaccharide (LPS)-mediated septic response. Thirty-five Swiss mice were divided into five groups (n = 7): control, HT (40 mg/kg), LPS (10 mg/kg), HT 20 mg+LPS and HT 40 mg+LPS. HT was administered for 10 days, while a single LPS dose was applied. The obtained findings demonstrate that HT administration enhanced the survival rate and decreased lactate dehydrogenase level in LPS-challenged mice. Treatment with HT inhibited the incidence of oxidative damage in splenic tissue through decreasing lipoperoxidation and increasing antioxidant molecules, namely glutathione, superoxide dismutase and catalase. HT also decreased total leukocytes count, C-reactive protein, monocyte chemoattractant protein-1, and myeloperoxidase levels. Additionally, HT suppressed the production levels of tumor necrosis factor-α, interleukin-1β, and interleukin-6. Moreover, mRNA expression of inducible nitric oxide synthase and nitric oxide production were increased after HT administration. Furthermore, HT supplementation resulted in a downregulation of p38 mitogen-activated protein kinase, inhibited the activation of the nuclear factor kappa-B from the nucleus to the cytoplasm, and attenuated infiltration of activated immune cells and tissue injury following LPS injection. Collectively, these findings demonstrate the antioxidative and anti-inflammatory properties of HT against LPS-mediated inflammation and sepsis. Therefore, HT could be applied as an alternative anti-inflammatory agent to minimize or prevent the development of systemic inflammatory response associated with septic shock.

Keywords

Sepsis hydroxytyrosol inflammation oxidative stress spleen

Introduction

Sepsis is defined as a systemic immune response toward the invasion of different pathogens such as fungi, bacteria, viruses and parasites into circulation. Globally, septic shock represents the most leading cause of mortality between patients in intensive care units.¹ The immune response associated with infection includes the activation of various endothelial and innate immune cells including neutrophils, macrophages, lymphocytes, monocytes and natural killer cells.²

In sepsis and following infection, the activated immune cells generate hyper-inflammatory state involving excessive and uncontrolled production of pro-inflammatory cytokines, acute phase proteins and other inflammatory mediators. The inflammatory response at the infected site includes also recruitment and proliferation of leukocytes, robust activity of complement system and increasing level of endothelial adhesion molecules and chemokines.^3,4 The consequential effects include vasodilatation, increased microvascular permeability and hypoperfusion.⁵ During the late septic phase, a state of immunosuppression dominates, resulting in exaggerate multiorgan dysfunction and further clinical consequences throughout the body.⁶

Lipopolysaccharide (LPS) or endotoxin is located in the outer membrane of Gram-negative bacteria, and has been considered as the main pathogenic factor in the development of septic response.⁷ LPS is known to be produced following bacterial lysis or proliferation and crosses the intestinal barrier into circulation. Induction of systemic inflammatory response following LPS injection is widely applied as an established model to understand clinical features associated with sepsis.⁸

Significant therapeutic approaches for treating sepsis have been established, however, mortality rates haven’t significantly reduced.⁹ It has been suggested that agents that able to eliminate endotoxin from the circulation of septic patients would be of considerable clinical benefit.¹⁰ Previous in vivo reports have demonstrated that antioxidant application was found to protect against septic shock and its associated clinical manifestations.²

Hydroxytyrosol (HT) or dihydroxyphenylethanol is a powerful phenolic constituent derived from olive tree and found in its oil. HT is used as a natural food additive especially in meat products preservation.¹¹ Several pharmacological effects have been coupled with HT consumption including antioxidant,¹² anti-bacterial,¹³ anti-tumor,¹⁴ cardioprotective,¹⁵ hepatoprotective,¹⁶ neuroprotective¹² and anti-inflammatory¹⁷ activities. It has been demonstrated that HT is able to attenuate oxidative damage through preventing lipid peroxidation and enhancing the antioxidant defense proteins in different experimental studies.^12,18 HT was found to inhibit the development of inflammatory cascade following LPS and carrageenan injection through downregulating the levels of pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) and other inflammatory mediators, namely inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) along with their products including nitric oxide (NO) and prostaglandin E2 (PGE2), and nuclear factor kappa-B (NF-κB).^16,19

Here, we explored the potential antioxidant and anti-inflammatory properties of HT against LPS-induced septic response through investigating the levels of oxidative stress indices and inflammatory mediators in the splenic tissue of rats.

Materials and methods

3-Hydroxytyrosol (CAS number: 10597-60 -1, 98% purity) and lipopolysaccharide (Escherichia coli serotype 055: B5, EC Number: 297-473-0) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), all other employed chemicals were of analytical grade.

Experimental animals

A total of 35 male Swiss mice (23–27 g, 8 weeks old) were procured from the animal facility of King Fahd for medical research, King Abdul Aziz University (Jeddah, Saudi Arabia). Animals were placed in standard cages at constant environmental conditions (12 h light:12 h dark cycle and at 22 ± 2°C) with water and food supplied ad libitum.

Protocol design

After acclimatization period (10 days), mice were fasted for 12 h and then separated into five equal groups (n = 7) as mentioned below:

Control group: mice in this group received normal saline 0.9% NaCl.

HT 40 mg group: mice in this group were orally administered with HT (40 mg/kg) for 10 consecutive days based on previous studies.^17,20

LPS intoxicated group (septic model): mice in this group were injected intraperitoneally with a single dose of LPS (10 mg/ kg) to induce septic response according to a previous report.²¹

HT 20 mg+LPS: mice in this group were gavaged with HT 20 mg/kg for 10 consecutive days according to an earlier studies,^20,22 then injected intraperitoneally with a single dose of LPS (10 mg/ kg).

HT 40 mg+ LPS: mice in this group were gavaged with HT 40 mg/kg for 10 consecutive days, then injected intraperitoneally with a single dose of LPS (10 mg/ kg).

HT and LPS were prepared in normal saline 0.9% NaCl based on previous researches.^21,23 Mice treated with the highest dose of HT (40 mg/kg) were observed individually for clinical signs of toxicity within the first 6 h after the treatment. 24 h after the last treatment, mice in all experimental groups were fasted for 6 h and decapitated under mild anesthesia (pentobarbital, 300 mg/kg). The blood samples were collected and the spleen was quickly dissected and weighted. For histopathological examination, samples of spleen tissues were kept at 10% neutral formaldehyde. Meanwhile, for biochemical estimations, splenic tissue homogenate 10% (w/v) was prepared by mixing the tissue with ice-cold 50 mM Tris-HCl buffer (pH 7.4) and centrifugation at 3,000 × g for 10 min at 4°C. The developed supernatant was kept at -80°C for biochemical determinations. Additionally, the peritoneal fluid was collected for counting leukocytes.

The experimental design and the employed animals were approved by the Research Ethics Committee, Taif University (Application No.: 41-00153) in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals 8th edition.

LPS-induced mortality

To evaluate the mortality rate elicited following LPS exposure, 30 mice were divided randomly into five groups (6 mice/ group) as illustrated in the protocol design section.

Lactate dehydrogenase activity

Lactate dehydrogenase (LDH) activity was assayed kinetically using commercial kits purchased from Abcam Biochemical Co. (Cambridge, United Kingdom) according to the manufacturers’ protocol.

Oxidative stress markers

Lipoperoxidation level in term of malondialdehyde (MDA in the splenic homogenate was determined using the method demonstrated by Ohkawa et al.²⁴ Meanwhile, the protocol of Ellman²⁵ was employed for the estimation of glutathione (GSH). Splenic superoxide dismutase (SOD) and catalase (CAT) activities were estimated according to the protocols reported by Nishikimi et al.²⁶ and Aebi,²⁷ respectively.

Inflammatory markers

Splenic total nitrite in the term of nitric oxide (NO) was detected colorimetrically using Griess reagent, based on the protocol described by Green et al.²⁸ The levels of CRP (catalog number: MCRP00), TNF-α (catalog number: SMTA00B), L-1β (catalog number: DY401), and IL-6 (catalog number: SM6000B) in the splenic homogenates was measured by commercial ELISA kits (R&D System, Minneapolis, MN, USA) according to the manufacturers’ procedures. Additionally, monocyte chemoattractant protein-1 (MCP-1, catalog number: CSB-E07430 m) and NF-κB (catalog number: CSB-E12108 m) were assayed using ELISA kits purchased from CUSABIO Life Sciences, Wuhan, China following the manufacturer’s procedures. MPO activity was examined spectrophotometrically at 460 nm following the modified experiment demonstrated by Bradley et al.²⁹

Leukocytes count

The peritoneal cavity was lavaged with 3 mL of sterile PBS and the obtained intraperitoneal fluid (IPF) was kept on ice for further analysis. IPF (500 µL) was fixed on slides by cytospin at 500 RCF (relative centrifugal force) for 5 min and stained with Wright’s stain to determine the differential leukocyte count.

Quantitative Real-time PCR

Splenic total RNA was isolated, and first strand cDNA was synthesized according to the manufacturer’s protocol. The mRNA expression of p38MAPK and iNOS in the spleen was determined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) technique using an Applied Biosystems 7500 Instrument. The thermal conditions for qRT-PCR were denaturated initially at 94°C for 2 min, followed by 40 cycles of 94°C for 30 s and 60°C for 30 s, and a final extension at 72°C for 10 min. After PCR amplification, the ΔCt from three repeated experiments was determined by subtracting the Ct value of the standard gene, glyceraldehyde 3-phosphate dehydrogenase (Gapdh) from that of each sample (Ct). The applied primers sequences for GAPDH was 5’-CCCTTAAGAGGGATGCTGCC-3’ (forward) and 5’-ACTGTGCCGTTGAATTTGCC-3’ (reverse), for p38 MPKA was 5’-TGCCCGAACGATACCAGAAC-3’ (forward) and 5’-TGTAGTTTCTTGCCTCATGGCT-3’ (reverse) and for iNOS was 5’-GCGCTCTAGTGAAGCAAAGC-3’ (forward) and 5’-GCACATCAAAGCGGCCATAG-3’ (reverse).

Histological examination

To determine the damage in the spleen following LPS administration, splenic tissue was separated and fixed in 10% neutral formaldehyde overnight at 25°C and embedded in paraffin. Spleen sections with thickness of 4–5 µm were stained with hematoxylin and eosin (H&E) and finally examined using a light microscope at the magnification of 400 ×.

Statistical analysis

The recorded results were analyzed using one-way ANOVA with Duncan’s multiple range tests using the statistical package SPSS, version 17. The statistical difference for all studied parameters was considered significant at P < 0.05. All values are presented as mean ± standard deviation (SD).

Results

HT increased the rate of survive following LPS-mediated septic response

The results illustrated in Figure 1 show the mean survival rate in mice challenged with LPS and pretreated with HT (20 and 40 mg/kg). Mice challenged with LPS started to die after 2 days of LPS injection and 100% died after 7 days. However, in TH (20 mg/kg) pretreated mice, 21% of the total mice were survived, whereas, 37.5% of the experimented mice were survived in TH (40 mg/kg) pretreated mice; demonstrating the protective effect of TH on the survival rate in comparison to the septic mice.

Figure 1.

Effect of 10 days treatment with hydroxytyrosol (HT, 20 and 40 mg/kg) on survive rate following lipopolysaccharide (LPS, 10 mg/kg)-mediated septic response in mice. Results are the mean ± SD (n = 7). Columns with different letters are statistically significant at P < 0.05 as compared to the control and LPS-challenged mice.

HT modulates the LDH level following LPS-mediated septic response

LDH is widely used as a marker for the systemic inflammatory events associated with sepsis. In the current experiment, LPS-injected mice with a single dose (10 mg/kg) showed a significant increase (P < 0.05) in LDH level with respect to its level in the normal group; reflecting tissue hypoxia. However, HT administration at 20 and 40 mg/kg prior LPS injection for 10 days decreased significantly the hyperlactaemia as compared to LPS-mediated septic response (Figure 2).

Figure 2.

Effect of 10 days treatment with hydroxytyrosol (HT, 20 and 40 mg/kg) on lactate dehydrogenase level following lipopolysaccharide (LPS, 10 mg/kg)-mediated septic response in mice. Results are the mean ± SD (n = 7). Columns with different letters are statistically significant at P < 0.05 as compared to the control and LPS-challenged mice.

HT decreases leukocytes count, macrophages, neutrophils and lymphocytes in the peritoneal cavity following LPS-mediated septic response

As illustrated in Figure 3, LPS injection elicited an elevation trend in the total white blood cell population in the intraperitoneal fluid with respect to their number in the control group. However, the pretreatment with HT was found to decrease the increased total blood cell count as compared to LPS injected group. Likewise, LPS injection maintained high macrophages, neutrophils, and lymphocytes count, while HT administration lowered significantly (P < 0.05) the count of these immune cells as compared to LPS-challenged mice.

Figure 3.

Effect of 10 days treatment with hydroxytyrosol (HT, 20 and 40 mg/kg) on total leukocytes, macrophages, neutrophils and lymphocytes count in the peritoneal fluid following lipopolysaccharide (LPS, 10 mg/kg)-mediated septic response in mice. Results are the mean ± SD (n = 7). Columns with different letters are statistically significant at P < 0.05 as compared to the control and LPS-challenged mice.

HT inhibits the production of CRP, MCP-1 and MPO levels following LPS-mediated septic response

CRP, an acute phase protein, MCP-1 is an initiating cytokine, while MPO activity reflects neutrophil sequestration in the spleen, and all these mediators are widely used as inflammatory markers. Septic model induced by LPS showed a marked elevation (P < 0.05) in the levels of CRP, MCP-1 and MPO activity in the splenic tissue as compared to the normal untreated mice. On the other hand, both HT doses decreased significantly these inflammatory proteins with respect to LPS-challenged group (Figure 4).

Figure 4.

Effect of 10 days treatment with hydroxytyrosol (HT, 20 and 40 mg/kg) on C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1), and myeloperoxidase (MPO) levels following lipopolysaccharide (LPS, 10 mg/kg)-mediated septic response in mice. Results are the mean ± SD (n = 7). Columns with different letters are statistically significant at P < 0.05 as compared to the control and LPS-challenged mice.

HT inhibits the overproduction of pro-inflammatory cytokines in the splenic tissue following LPS-mediated septic response

The development of septic response is coupled with a massive production of pro-inflammatory molecules. In the present study, LPS provoked a significant increase (P < 0.05) in the levels of IL-1β, IL-6 and TNF-α in the splenic tissue as compared to the control mice. This inflammatory response was reduced following HT supplementation before LPS with respect to LPS injected mice (Figure 5).

Figure 5.

Effect of 10 days treatment with hydroxytyrosol (HT, 20 and 40 mg/kg) on interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels following lipopolysaccharide (LPS, 10 mg/kg)-mediated septic response in mice. Results are the mean ± SD (n = 7). Columns with different letters are statistically significant at P < 0.05 as compared to the control and LPS-challenged mice.

HT downregulated iNOS expression and decreased NO formation following LPS-mediated septic response

NO is a biological mediator that plays a significant role at the inflamed foci. In the current model, LPS was found to upregulate significantly (P < 0.05) the transcriptional level of iNOS and increase its product, NO in the splenic tissue when compared to the control group. Interestingly, a significant decrease in the mRNA expression of iNOS and NO production were observed in HT+LPS treated groups as compared to LPS exposed mice (Figure 6).

Figure 6.

Effect of 10 days treatment with hydroxytyrosol (HT, 20 and 40 mg/kg) on inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide (NO) level following lipopolysaccharide (LPS, 10 mg/kg)-mediated septic response in mice. Results are the mean ± SD (n = 7). Columns with different letters are statistically significant at P < 0.05 as compared to the control and LPS-challenged mice. PCR data were performed in triplicate using Gapdh as a housekeeping gene.

HT downregulated p38MAPK expression and decreased NF-κB activity following LPS-mediated septic response

To explore the molecular mechanism involved in the septic response and the anti-inflammatory activity of HT, the mRNA expression of p38MAPK and NF-κB level were examined in the splenic tissue. Figure 7 shows that LPS provoked the upregulation of p38MAPK and increased NF-κB level with respect to their expression and level in the control mice. However, these inflammatory regulators were found to be decreased significantly (P < 0.05) in groups treated with HT as compared against LPS treated mice; reflecting the potent anti-inflammatory properties of HT against LPS-mediated septic response in the splenic tissue.

Figure 7.

Effect of 10 days treatment with hydroxytyrosol (HT, 20 and 40 mg/kg) on p38MAPK mRNA expression and nuclear factor kappa B (NF-κB) level following lipopolysaccharide (LPS, 10 mg/kg)-mediated septic response in mice. Results are the mean ± SD (n = 7). Columns with different letters are statistically significant at P < 0.05 as compared to the control and LPS-challenged mice. PCR data were performed in triplicate using Gapdh as a housekeeping gene.

HT suppresses the oxidative challenge in the splenic tissue following LPS-mediated septic response

Sepsis development is coupled with the incidence of oxidative damage. Figure 8 shows the redox status in the splenic tissue. LPS injection disturbed the oxidant/antioxidants balance as represented by the raised lipoperoxidation in term of MDA accompanied by depletion in GSH content and deactivation of CAT as compared to the oxidative status in the control mice. Notably, HT at high dose restored the depleted antioxidants (GSH and CAT) and elevated SOD activity, and decreased the elevated MDA level in the spleen as compared to LPS-exposed group.

Figure 8.

Effect of 10 days treatment with hydroxytyrosol (HT, 20 and 40 mg/kg) on malondialdehyde (MDA, glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) levels following lipopolysaccharide (LPS, 10 mg/kg)-mediated septic response in mice. Results are the mean ± SD (n = 7). Columns with different letters are statistically significant at P < 0.05 as compared to the control and LPS-challenged mice.

HT improved the histopathological deformations in the spleen following LPS-mediated septic response

Splenic tissue stained with H&E showed normal histological architecture in the control and HT treated groups. Meanwhile, LPS-challenged mice exhibited enlarged and fused white pulps demonstrating splenomegaly and active immune response of spleen macrophages around the white pulp. Further, necrotic cells were observed in the examined sections. Additionally, the red pulp showed apoptotic bodies. However, HT pretreated mice showed improvement in the splenic histological structure in a dose-dependent manner (Figure 9).

Figure 9.

Histological alterations in the splenic tissue following the treatment with hydroxytyrosol (HT, 20 and 40 mg/kg) and/or lipopolysaccharide (LPS, 10 mg/kg). (a): the control group, (b): HT treated group, (c): LPS injected group, (d): HT (20 mg/kg)+LPS and (e): HT (40 mg/kg)+LPS, 400x.

Discussion

A growing interest in the application of nutraceuticals in the management of sepsis has increased due to their low adverse effects compared with the traditional pharmacological drugs. HT is a phenolic constituent, extracted from olive trees and has shown numerous biological activates. The current study was designed to distinguish the potential antioxidant and anti-inflammatory potency of HT supplementation against LPS-mediated septic response in mice. LPS is well-known to develop a systemic inflammatory response, resulting in tissue injury and further multiple organ malfunction and death.³⁰

In the present work, LPS-injected mice with a single dose showed a high mortality rate with elevated LDH level. Sepsis is among the leading causes of death between patients in the intensive care units globally. Hyperlactaemia in sepsis has been attributed to the disturbed lactate clearance more than its overproduction. Therefore, enhancing the activity of pyruvate dehydrogenase will led to lower blood lactate concentration.³¹ In parallel, the rate of mortality and LDH activity were reduced after HT administration at doses of 20 and 40 mg/kg. Previous reports demonstrated that HT was found to decrease LDH activity in liver tissue incubated with glucose.³² Authors have related this effect with the antioxidant capacity of HT. Additionally, HT administration showed a neuroprotective effect in hypoxia–reoxygenation model through suppressing LDH efflux in rat brain slices in a dose dependent manner.³³ Moreover, HT decreased significantly the level of LDH following high fat diet-mediated liver injury in rats.¹⁶

The progression of septic response is closely linked with the increased leukocytes population.³⁴ Here, LPS-challenged mice exhibited a marked increase in the total leukocytes number associated with the elevation of neutrophils, macrophages and lymphocytes in the peritoneal fluid. Previous researches recorded the overactivation of leukocytes during the septic response.³⁵ The initial response in septic shock is characterized by the activation and infiltration of innate immunity cells into the infected tissue.³⁶ This response is mainly potentiated by chemokines. Although secretion of chemokines is mandatory for host defense to encounter the bacterial infection, their over secretion has been found to enhance the development of sepsis. Additionally, the activated leukocytes, in combination with the excessive pro-inflammatory cytokines production are suggested to cause multiple organ dysfunction and death.³⁷ Therefore, the decrease in leukocytes number and recruitment is vital for the suppression of inflammatory response associated with sepsis.³⁴

HT administration decreased significantly the total leukocytes population in the peritoneal fluid. This anti-inflammatory effect elicited by HT may inhibit the development of septic response following bacterial infection. Accumulative evidences reported the ability of phenolic compounds to modulate leukocytes proliferation and activity, and cytokines production in order to inhibit the inflammatory response.³⁸

In the current study, LPS-challenged mice showed a marked increase in levels of CRP, MCP-1 and MPO. CRP is acute phase reactant that produced in the liver and used as a valuable sepsis marker.³⁹ During acute inflammation, CRP is believed to bind with the phospholipid constituent of the pathogen, facilitating their phagocytosing and clearance.⁴⁰ MCP-1 is an initiating cytokine that control migration and infiltration of monocytes/macrophages. It is also regulates leukocyte activation and mediate diverse inflammation-enhancing biological activities.⁴¹ Its high extracellular level is associated with systemic inflammatory response and multiple organ failure in septic shock.⁴² MPO is produced mainly by neutrophils and its level is correlated positively with neutrophils population at the infected foci during septic shock.⁴³ In order to attack and phagocytize bacteria, MPO produces highly reactive oxidants including hypochlorous acid. The overproduction of these oxidants was found to cause extracellular collateral damage and multiple organ failure in septic patients resulting in higher mortality.⁴⁴

Sepsis is characterized by a systemic inflammatory response that includes exacerbated pro-inflammatory cytokines production among other active mediators leading to sever cellular impairments. Our findings showed that LPS injection provoked a significant increase in IL-1β, IL-6, TNF-α, iNOS, NO, NF-κB and p38MAPK expression in the splenic tissue. The activated immune and non-immune cells produce IL-1β and TNF-α in a large amount in response to bacterial infection.³ Once secreted, IL-1β and TNF-α augment inflammatory cascade in an autocrine and paracrine manner through enhancing immune cells to produce other pro-inflammatory molecules such as IL-6, reactive oxygen and nitrogenous species and lipid mediators resulting in sepsis-mediated organs malfunction.⁴⁵ Previous reports suggest that the upregulation of iNOS and overproduction of NO may contribute to abnormal homeostatic processes associated with sepsis. The oversecreted cytokines were shown to upregulate iNOS expression and its product, NO in different cells including endothelium, macrophages and other parenchymal cells.⁴⁶ At high concentrations, NO is reacts with superoxide radicals yielding a powerful oxidant, peroxynitrite radicals that prevent the migration of neutrophils to the infected site in severe polymicrobial sepsis.^47,48

Accumulative evidences confirm the role of NF-κB–related pathways in the progression of endotoxin tolerance.⁴⁹ Activation of NF-κB was found to enhance release of pro-inflammatory cytokines and other mediators in different septic models.⁵⁰ NF-κB activation was found to be regulated by p38MAPK in different cells.⁵¹ Additionally, p38MAPK was found to triggers the systemic inflammatory response in septic shock through enhancing the transcriptional levels of different pro-inflammatory cytokines and mediators.⁵²

Attenuation of pro-inflammatory cytokines and mediators associated with the development of systemic inflammation is thought to control the pathogenesis of sepsis. In the present study, HT supplementation remarkably decreased levels of CRP, MCP-1 and MPO, IL-1β, IL-6, TNF-α, iNOS, NO, NF-κB and p38MAPK expression following LPS intoxication. In a clinical trial, HT administration decreased plasma CRP level and isoprostane.⁵³ It is also attenuated significantly chronic inflammation resulted from endoplasmic reticulum stress in insulin resistance murine model via inhibiting serological levels of CRP and its stimulator, IL-6.⁵⁴ Additionally, HT suppressed the inflammatory response produced following LPS intoxication via downregulation of COX-2 expression and TNF-α level in mice.¹⁷ Moreover, HT and procyanidins found in olive and grape seed extract showed anti-inflammatory effects in osteoarthritis mouse model through inhibiting IL-1β-enhanced NO and prostaglandin production. This effect has been explained by the deactivation of p65 NF-kB pathway.⁵⁵

In a previous in vitro study, Richard et al.⁵⁶ stated that HT was able to decrease the secretion of NO, PGE2, pro-inflammatory cytokines including (interleukins-1α, 1β, 6, 12, and TNF-α) and chemokines including MCP-1 and CXCL10 in murine macrophages RAW 264.7 stimulated with LPS. In the same context, HT downregulated iNOS and COX-2 expression and decreased their products namely, NO and PGE2 along with a reduction in the production of IL-1β, IL-6 and TNF-α, and NF-kB activity in RAW 264.7 macrophages treated with LPS.⁵⁷ HT also downregulated the expression of iNOS, PGE2 synthase, matrix metalloproteinase-9 through suppression of NF-kB in RAW 264.7 macrophages activated by LPS. In a another report, HT inhibited neuroinflammation through inhibiting production of IL-1β, IL-6 and TNF-α in LPS-activated microglia and astrocytes as a model for Parkinson disease.⁵⁸ Recently, HT prevented the progression of acute inflammation in the liver tissue through the inhibition of TNF-α, COX-2 and NF-κB expression in a high fat diet-induced metabolic disturbance model.¹⁶ Bedouhene et al.⁵⁹ showed that HT quenched ROS, decreased the elevated MPO and lactoferrin level, and suppressed AKT, p38MAPK, and ERK1/2 phosphorylation in neutrophils stimulated by fMLF (N-formylmethionyl-leucyl-phenylalanine) bacterial peptide. This effect is related to the inhibition of trimeric G-protein activation or suppression of protein tyrosine kinase activity. Collectively, these data suggest that HT, at least in LPS-mediated septic response, exerts its anti-inflammatory activity through the suppression of the production of pro-inflammatory cytokines and mediators.

Crosslink has been previously reported between the progression of inflammatory response and the development of oxidative challenge during septic shock. At the inflamed foci, overactivation of neutrophils elicited a massive ROS generation coupled with excessive secretion of proteases from granules,⁵⁹ that may enhance the induction of oxidative reactions. Oxidative stress has been suggested to play a vital role in the pathogenesis of sepsis.⁶⁰ In the current study, LPS-exposed mice revealed a significant elevation in MDA level accompanied by depletion of GSH and CAT levels in the spleen. During septic response, pro-oxidants including lipoperoxidation was increased, while antioxidant molecules (GSH, SOD and CAT) were observed.⁶¹ The elevated oxidants contribute to endothelial cells disturbances leading to the increase in vascular permeability that play a key role in the progression of sepsis.⁶⁰ The development of oxidative stress during sepsis has been attributed to mitochondrial dysfunction, energy depletion and the decreased vascular tone that led to multiple organ failure.⁶²

Scavenging ROS, decreasing oxidants and enhancing antioxidant and detoxifying proteins are believed to inhibit the oxidative challenge associated with septic response. Here, HT supplementation decreased lipoperoxidation product, MDA and enhanced GSH, SOD and CAT levels in the splenic tissue in response to LPS injection. HT inhibited the induction of oxidative reactions mediated in the splenic tissue following cadmium intoxication through decreasing lipid peroxidation and increasing SOD and CAT activities.⁶³ Additionally, HT decreased the elevated protein carbonyl and lipid peroxidation levels, and increased the depleted hepatic GSH level, glutathione S-transferase, and SOD activity in obese mice.⁶⁴ Earlier in vitro studies reported that HT elevates Cu/SOD expression;⁶⁵ normalizes GSH content, upregulates the expression of glutathione peroxidase, glutathione reductase and glutathione-S-transferase protein,⁶⁶ activates CAT and reduces ROS production.⁶⁷ The antioxidant properties of HT was extended to enhance the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in different experimental designs.^66,68 Previous studies confirmed the ability of HT to scavenge different ROS in different experiments.⁶⁹ This effect has been explained by the existence of o-dihydroxyphenyl moiety in HT that serve as chain breaker by donating a hydrogen atom to peroxyl radicals (ROO*). Reactive ROO* is substituted with the unreactive HT* radical due to the presence of intramolecular hydrogen bond in the phenoxy radical.⁷⁰

Conclusion

Collectively, the recoded results demonstrated that HT administration could exhibit anti-inflammatory and antioxidative effects on LPS-mediated septic response in the spleen. The pretreatment with HT decreased mortality rate, LDH level, leukocytes count, CRP, MCP-1, MPO, IL-1β, IL-6, TNF-α, NO and NF-κB levels. Besides, HT was found to downregulate iNOS and p38MAPK expression. Additionally, HT decreased MDA and increased GSH, SOD and CAT along with the improvement of the histological changes. These findings could give the conclusion that HT could be used to prevent the development of systemic inflammatory response associated with sepsis.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Mohamed A Alblihed

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