Open Access

Thymoquinone modulates the expression of sepsis‑related microRNAs in a CLP model

  • Authors:
    • Khalid M. Alkharfy
    • Ajaz Ahmad
    • Basit L. Jan
    • Mohammad Raish
    • Muneeb U. Rehman
  • View Affiliations

  • Published online on: April 14, 2022
  • Article Number: 395
  • Copyright: © Alkharfy et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 4.0].

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Sepsis is a clinical syndrome common in critical care settings. In the present study, the therapeutic effect of thymoquinone (TQ) on the expression of sepsis‑related microRNAs (miRNAs/miRs), levels of inflammatory markers, organ dysfunction and mortality were investigated in a cecal ligation and puncture (CLP) rat model. A single dose of TQ (1 mg/kg) was administered to animals 24 h after CLP and the mortality rate was assessed up to 7 days following the induction of sepsis. In addition, blood samples were collected at different time points and the expression levels of miRNAs (i.e. miR‑16, miR‑21, miR‑27a and miR‑34a) were examined, along with the levels of inflammatory cytokines (i.e. TNF‑α, IL‑1α, IL‑2, IL‑6 and IL‑10) and sepsis markers (i.e. C‑reactive protein, endothelial cell‑specific molecule‑1, VEGF, procalcitonin and D‑dimer). Liver, kidney and lung tissues were also collected for further histological examination. Treatment with TQ significantly downregulated the miRNA expression levels, as well as the levels of inflammatory cytokines and early‑stage sepsis biomarkers by 30‑70% at 12‑36 h (P<0.05). Furthermore, CLP model rats treated with TQ exhibited an ~80% increase in survival rate compared with that in the untreated CLP group. In addition, TQ induced the preservation of organ function and structure. In conclusion, the present study demonstrated a promising therapeutic effect of TQ against the sequelae of sepsis.


Sepsis is a potentially lethal condition that is commonly encountered in intensive care units (ICUs) (1-4). Septic shock is a subset of sepsis that is associated with a greater risk of mortality than sepsis alone (5). The usual host reaction is complex, aiming to detect and control pathogen incursion, and to initiate immediate tissue repair. The body activates both cellular and the humoral immunity, which release massive quantities of pro-inflammatory and anti-inflammatory mediators, leading to systemic inflammatory response syndrome (SIRS) (6,7). Aggravation of these mechanisms can cause a series of events that may lead to endothelial injury, tissue hypoperfusion, intravascular coagulation, multiple organ dysfunction syndrome and possibly death (8).

Despite improvements in critical care medicine, sepsis and septic shock continue to be among the leading causes of death and a serious challenge to clinicians and scientists (1,9). Some of the most important approaches in sepsis investigation consist of focusing on agents that may modify systemic inflammation; however, these efforts have not had much success. Another important feature of sepsis is cellular apoptosis, which can lead to organ failure (10-13). Parenchymal cells of the lung and the liver, as well as intestinal epithelial cells have been reported to exhibit higher levels of apoptotic death in animal models of sepsis following microvascular dysfunction and tissue hypoxia compared with non-septic animals (14,15). Unlike genomic DNA, which is static, RNA expression can dynamically change between healthy and diseased states, and thus can provide real-time information regarding cellular function. miRNAs have been recognized as a class of gene expression regulators that serve significant roles in normal cell function and in disease development, including the pathogenesis of cardiovascular disorders, cancer and inflammation (16). miRNAs are 19-24 nucleotide-long, endogenous, non-coding RNAs that function as post-transcriptional suppressors of gene expression by interfering with target mRNA translation or stability; notably, miRNAs affect multiple target genes (17). There is growing evidence that miRNA dysregulation corresponds with the clinical symptoms of sepsis (18). Increases in the levels of circulating miRNAs originating from lipopolysaccharide (LPS)-stimulated monocytes and monocyte-derived dendritic cells during sepsis have also been reported (19,20). In addition, the pathophysiological responses associated with sepsis, such as inflammation, shock or even ileus in the CLP model may contribute to the upregulation of circulating miRNAs. It has also been shown that miRNA dysregulation can correspond to clinical symptoms and severity of sepsis (18,21,22). Therefore, miRNAs could be potential therapeutic targets for sepsis management. In the present study, the expression levels of miRNAs (miR-16, miR-21, miR-27a and miR-34a) were examined, shortlisted from a group of miRNAs which have been shown to upregulate/downregulate during sepsis in multiple studies (16,18,23-25).

Thymoquinone (TQ) has been reported to possess notable immunomodulatory effects, as well as other pharmacological properties, including anti-inflammatory activity, improvement of microvascular function and regulation of endothelial nitric oxide synthase (26,27). In the present study, it was hypothesized that TQ could regulate miRNA expression levels, protect organs and improve survival in sepsis. Furthermore, changes in sepsis markers, such as cytokines, C-reactive protein (CRP), VEGF, endothelial cell-specific molecule-1 (ESM-1 or Endocan-1), procalcitonin (PCT) and D-dimer, were detected in a cecal ligation and puncture (CLP) model (28).

Materials and methods

Animals and CLP model

A total of 60 male Wistar albino rats (Rattus norvegicus), weighing 200-220 g, age, 8-10 weeks, were used in the present study. The animals were housed in filter-top cages in a temperature-controlled environment (23±2˚C, 40-60% humidity with a 12-hour light/dark cycle) and were provided access to standard rat chow and water ad libitum. The animals were allowed to acclimate for 1 week before the experiments were conducted. The present study was approved by the Ethics Committee of the College of Pharmacy, King Saud University (Riyadh, Saudi Arabia; approval no. KSU-SE-19-17).

Under aseptic conditions, the rats (n=10/group) were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). The animal was placed on a surgical tray 5 min after injection, its abdomen was shaved and an ~1-cm cut was made into the abdomen. The cecum was exposed, ligated and punctured at two points with a 21G needle; in the sham group, the cecum was not punctured. Subsequently, the cecum was placed back into the body and the incision was sutured. At 1 h following CLP, each animal received 1 ml sterile saline intraperitoneally for resuscitation, and the animals were allowed ad libitum access to food and water. At 24 h after CLP, a single intramuscular dose of TQ (1 mg/kg; prepared in 10% DMSO) was administered in the thigh muscles of the hind limbs to the animals in the treatment group (CLP + TQ). The sham and CLP control groups were treated only with 10% DMSO. For the survival study, animal mortality was assessed up to 7 days after TQ treatment. In another set of experiments, blood samples (0.5 ml) were collected in sterile tubes from the tail vein prior to TQ administration, and 12, 24 and 36 h post-treatment. The blood was centrifuged (3,000 x g, 4˚C for 10 min) immediately after collection, and plasma and serum were stored at -80˚C. The animals were subsequently euthanized with ketamine (100 mg/kg) and xylazine (10 mg/kg). The death of the animals was confirmed by lack of movement, absence of heartbeat and respiration over a sufficient period of time. The liver, kidney and lung tissues were harvested, dissected and stored in formalin for histopathological examination.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was isolated from the serum samples using miRNeasy kit (Qiagen, Inc.) according to the manufacturer's protocol. The quality and quantity of total RNA were checked using a spectrophotometer. To remove any DNA contamination, the total RNA was treated with DNase enzyme from a DNase 1 kit (Millipore Sigma). Total RNA, including miRNA, was reverse transcribed into cDNA using MystiCq microRNA cDNA Synthesis Mix (Millipore Sigma) according to the manufacturer's protocol. Prior to miRNA quantification by RT-qPCR, conventional PCR was run for all the primers used in the present study to check amplification specificity. Primer details are presented in Table I. In conventional PCR, all the primers produced only one amplification band corresponding to their expected amplicon size (data not shown). qPCR was subsequently performed using SYBR Green (Roche Molecular Diagnostics) according to the manufacturer's protocol; U6 small nuclear ribonucleoprotein was used as a reference gene. For each miRNA, the reaction mixture consisted of 10 µl 2X SYBR Green master mix, 0.25 µl each reverse and forward primer, 1 µl cDNA and 8.5 µl nuclease-free water. The following thermocycling conditions were used: 40 cycles of denaturation at 94˚C for 4 min, annealing at various temperatures as provided in Table I, and extension at 72˚C for 15 sec. To confirm the product specificity, melting curve analysis was performed. The relative expression levels of the different miRNAs were determined using the 2-ΔΔCq method (29). Relative expression levels of the miRNAs detected in the present study are presented as fold change.

Table I

List of primers used for PCR.

Table I

List of primers used for PCR.

GenePrimer sequence (5'-3')Annealing temperature (˚C)Expected size (bp)

[i] F, forward; miR, microRNA; R, reverse.

Biomarkers and biochemical analyses of plasma

ELISA was used to estimate the concentrations of inflammatory cytokines, including TNF-α (cat. no. PRTA00), IL-1α (RRA00), IL-2 (SR2000), IL-6 (SR6000B), and IL-10 (SR1000), according to the manufacturer's protocols (R&D Systems, Inc.). A quantitative sandwich ELISA was used to assess the levels of sepsis biomarkers, including CRP (EK0978), and VEGF (EK0540; all Boster Biological Technology, Pleasanton, CA, USA) and ESM-1 (MBS762527; MyBioSource, Inc. San Diego, CA, USA), according to the manufacturer's protocols. The concentration of PCT (CSB-E13419r) was measured using a specific ELISA kit (CUSABIO, Houston, TX, USA). Similarly, D-dimer (CSB-E12984r-1) levels were determined using an ELISA kit (Cosmo Bio Co., Ltd.); changes in color were measured using a spectrophotometer at 450 nm and were quantified using constructed standard curves. Pertinent biochemical parameters, such as alanine transaminase (ALT, aspartate transaminase, (AST) alkaline phosphatase (ALP), serum creatinine and blood urea nitrogen (BUN), were measured using colorimetric methods (HUMAN Diagnostics Worldwide, Wiesbaden, Germany).

Histopathological examination

Liver, kidney and lung tissues were sliced into small pieces and fixed in 10% formalin for 24 h at room temperature. The fixed tissues were then embedded in paraffin. Subsequently, the tissues were cut into 4-5-µm sections, deparaffinized and rehydrated with methanol as previously described (30). Morphological changes induced by sepsis with or without TQ treatment were assessed by evaluating the organ tissues using H&E staining; tissues were observed under a light microscope as previously described (31).

Statistical analysis

All of the data analysis was carried out using GraphPad Prism version 6 (GraphPad Software, Inc.) and SPSS 20.0 software package (IBM Corp.). Variables are presented as the mean ± SEM. Animal survival was assessed using Kaplan-Meier analysis and log-rank test. For the miRNA expression analysis, the relative expression of the different miRNAs was determined by the 2-ΔΔCq method, normalized with ΔCq=Average CqmiRNA-Average CqU6. The relative expression levels of miRNAs are presented as fold change, calculated from the mean Cq values for each group. Differences in the miRNA expression, biochemical parameters and inflammatory biomarkers between the groups were evaluated using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.


Effect of TQ on animal survival and biochemical estimation

Animals that underwent CLP started displaying signs of infection including lethargy, piloerection, huddling and a decrease in water and food uptake within 12 h of surgery. As shown in Fig. 1, 24 h post-CLP, death started to occur in the CLP groups but significantly decreased when TQ was administered in the CLP+TQ group. The mortality rate reached 100% in the rats in the untreated CLP group at 5 days, whereas animals in the TQ treatment group exhibited an increase in survival rates of up to 80%; no deaths were reported in the sham group during this time. Results from the biochemical analyses used to assess organ function showed a significant increase in liver enzymes (i.e. ALT, AST and ALP) in the CLP group, which were reduced within 12 h of TQ treatment. Similarly, TQ administration significantly improved the kidney function as shown by a reduction in serum creatinine and BUN compared with that in the CLP control (Table II).

Table II

Comparison of biochemical parameters between different experimental groups.

Table II

Comparison of biochemical parameters between different experimental groups.

 Time after TQ, h
ALP, IU/lSham118.79±4.73131.85±5.94129.41±5.37121.32±5.18
 CLP 292.66±6.94b 358.25±12.20b 388.56±18.20b 418.74±10.68b
 CLP + TQ 278.08±5.57b 191.12±4.99b,c 236.01±5.50b,c 260.44±6.10b,c
AST, IU/lSham121.45±3.76129.19±4.65119.79±3.26124.52±4.84
 CLP 238.89±5.13b 275.14±7.64b 289.04±8.86b 320.86±2.96b
 CLP + TQ 224.35±8.38b,c 157.76±6.48b,c 177.35±7.59b,c 187.49±6.86b,c
ALT, IU/lSham29.66±1.9434.87±2.4931.76±2.0927.06±2.78
 CLP 53.37±2.01b 58.15±1.62b 62.42±1.82b 69.32±1.39b
 CLP + TQ 51.31±0.58b 38.12±1.55c 41.02±1.08b,c 46.51±1.27b,c
Creatinine, mg/dlSham1.30±0.121.27±0.221.32±0.431.30±0.59
 CLP2.15±1.22 2.39±0.84b 2.66±1.02b 3.13±0.60b
 CLP + TQ 2.18±0.51b 1.58±0.65c 1.75±.22b,c 1.91±0.78b,c
BUN, mg/dlSham49.07±0.6551.98±0.7753.69±0.5252.85±0.61
 CLP 91.16±0.38b 101.95±0.65b 125.85±0.76b 129.37±0.35b
 CLP + TQ 95.87±0.20b,c67.65 ±0.66b,c 74.51±0.42b,c 81.75±0.91b,c

[i] aTime 0 h represents the time point 24-h post-sepsis induction;

[ii] bP<0.05 vs. Sham;

[iii] cP<0.05 vs. CLP. ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen; CLP, cecal ligation and puncture; TQ, thymoquinone.

Effect of TQ on miRNA and biomarker estimation

The expression levels of miR-16, miR-21, miR-27a and miR-34a were significantly increased at 12 and 24 h after CLP compared with the sham group (Fig. 2). Treatment with TQ significantly downregulated the miRNA expression levels by 40-80%. Similarly, the concentrations of IL-1α, IL-2, IL-6, IL-10 and TNF-α, were significantly increased in the CLP group compared with those in the sham group, and TQ treatment resulted in a significant reduction in the levels of the inflammatory cytokines in a time dependent manner and effect of TQ seems to diminish by the 36 h (Figs. 3 and 4A; P<0.05).

The effect of CLP on sepsis biomarkers CRP, VEGF and ESM-1 was also determined. CLP resulted in a notable increase in the concentrations of the sepsis biomarkers, which was reversed by TQ administration (Fig. 4B-D; P<0.05). TQ treatment also mitigated the increased levels of D-dimer and PCT in septic model animals (Fig. 5A and B), which was consistent with its antiseptic effect.

Histopathology observations

The histological evaluation (Fig. 6) of rat renal tissues from the sham group showed normal architecture of the renal cortex, renal glomeruli and renal tubules (Fig. 6Aa). By contrast, animals having undergone the CLP procedure exhibited deterioration of the renal cortex and medullary tubules, and some interstitial hemorrhages with mononuclear cell infiltration (Fig. 6Ab). In addition, epithelial tubular necrotic areas and cellular atrophy were observed. CLP model animals treated with TQ exhibited reduced deterioration of the renal cortex, renal glomeruli and renal tubules (Fig. 6Ac); in addition, reduced dilatation of renal glomeruli and cortical tubules was observed. No signs of deterioration in cellular organelles and no inferential hemorrhages were found (Fig. 6Ac).

Similarly, the rat liver from the sham group exhibited normal liver composition with a prominent nucleus, well preserved cytoplasm, central vein and a compact arrangement of hepatocytes without any fatty lobulation (Fig. 6Ba). By contrast, animals in the untreated CLP group exhibited acute cellular swelling, congestion of the central vein, medium centrilobular necrosis of hepatocytes and apoptotic bodies with sinusoidal dilatation (Fig. 6Bb). An acute massive focal infiltration of mononuclear cells in the portal area, and sinusoidal infiltration in the central zones with vacuolization and steatosis were also evident (Fig. 6Bb). CLP animals treated with TQ displayed distinct features, such as pentagonal or hexagonal lobules, with central veins and borderline hepatic triads or tetrads, engrained in connective tissues (Fig. 6Bc). A mild degree of centrilobular necrosis of hepatocytes was observed but no apoptotic bodies were seen, whereas slight sinusoidal dilatation and mild congestion of central vein were detected. A small number of mononuclear cells and sinusoidal intrusion in the central zones with less vacuolization and steatosis was detected but without signs of edematous tissue (Fig. 6Bc).

Lung tissues from animals in the sham group displayed normal structure and composition, with a well-preserved alveolar space and pulmonary interstitium, and no inflammatory cell infiltration into the alveolar cavity (Fig. 6Ca). Evaluation of lung tissues of the animals in the CLP group showed acute edema, emphysema and pulmonary interstitial hyperemia, resulting in impaired alveolar architecture with penetrations of mononuclear cells causing alveolar congestion and hyperemia in the pulmonary capillaries (Fig. 6Cb). By contrast, tissues from CLP model animals treated with TQ displayed limited inflammatory cell infiltration and restoration of the normal alveolar structure (Fig. 6Cc).


Sepsis is a detrimental condition with a high mortality rate in ICUs; therefore, scientists and clinicians have focused on identifying new therapeutic modalities to combat its negative outcomes (32,33). The CLP animal model has been extensively used to study new approaches for sepsis management (34). The constant source of bacteria in this model is the punctured cecum and endotoxin, which is the main constituent of the external membrane of bacterial cell walls, activates several pathophysiological events of gram-negative sepsis (35). The overproduction of reactive oxygen species (ROS) produced under these conditions has been implicated in tissue injury (36). Subsequently, CLP triggers pathological changes in the lung, liver and kidney tissues, signifying varying degrees of organ injury. In the present study, it was demonstrated that treatment of CLP model animals with TQ improved animal survival by up to 80% over a period of 7 days. This coincided with a significant reduction in the expression levels of specific miRNAs, levels of circulating inflammatory cytokines and early-stage sepsis biomarkers, as well as preservation of liver, lung and kidney functions.

Among the circulating miRNAs that have been reported to be upregulated following CLP, miR-16 serves a vital role in sepsis. Notably, deletion of miR-16 in myeloid cells has been shown to significantly decrease Escherichia coli-associated mortality in several sepsis models (37). Consistently, miR-16 overexpression can decrease both phagocytosis and production of mitochondrial ROS. Additionally, lack of miR-16 has been reported to enhance secretion of cytokines and chemokines from bone-marrow-derived macrophages at the early stages of infection (38). Furthermore, Gao and Yu (39) identified IκB kinase-β (IKKβ) as a direct target of miR-16, the expression of which was negatively regulated by miR-16 at the mRNA and protein expression levels, indicating that miR-16 may suppress the inflammatory response by inhibiting the IKKβ/NF-κB signaling pathway. Notably, bacterial infection-induced miR-16 upregulation may cause significant organ damage and cell death. Another study demonstrated a positive correlation between circulating miR-16 in serum and death of patients infected with sepsis (23). Therefore, it may be concluded that miR-16 has a crucial role in tissue damage and cell death in sepsis and associated SIRS.

Another miRNA that is highly expressed in different immune cells, including monocytes, macrophages, T and B lymphocytes, and dendritic cells, is miR-21(25). Numerous studies have shown that different inflammatory stimuli, such as LPS, lipids (prostaglandin E2 and resolvin A1) and cytokines, can trigger miR-21 expression (25,40,41). miR-21 has also been shown to be upregulated in acute sepsis and sustained in late sepsis in patients and mice (41-43). McClure et al (44) demonstrated that administration of miR-21 antagomir to BALB/c mice improved animal survival and decreased the bacterial load following CLP. Determination of the functions of miR-21 have been difficult owing to its multiple mRNA target interactions, as well as its complex regulation in response to extracellular signals (25). Furthermore, miR-21 has been associated with numerous key processes of inflammation, such as detection and response to homeostatic disturbances throughout the body as well as coordinating these responses appropriately, thus serving a dynamic function in inflammatory responses (25,45). Differing from other mediators, the presence of miR-21 is not restricted to pro-inflammatory or immunosuppressive states; it can act as a crucial signal to mediate the homeostasis between both states (25,45).

miR-27a is another miRNA that has been reported to serve an important role in the regulation of inflammatory responses in sepsis (24). Inhibition of miR-27a has been shown to downregulate the expression levels of TNF-α and IL-6 by reducing the phosphorylation levels of NF-κB p65 subunit and through the inhibition of its DNA-binding activity (24). Moreover, miR-27a neutralization has been suggested to upregulate peroxisome proliferator-activated receptor γ, possibly inhibiting the production of monocyte inflammatory cytokines and activating macrophages to boost innate defense against pathogens, thereby downregulating TNF-α expression, relieving inflammation and increasing the survival rate of patients with sepsis (46,47).

A previous study has reported that accumulation of ROS and reduced antioxidant enzyme activities increase oxidative stress and serve important roles in the progression of sepsis, leading to mitochondrial dysfunction and organ failure (48). miR-34a regulates oxidative stress and autophagy through the inhibition of silent information regulator T1 (SIRT1) and autophagy gene 4B signaling. Differential miR-34a expression has also been suggested as a potential prognostic biomarker, in addition to providing insight into the mechanisms of endothelial dysfunction of septic shock. Notably, miR-34a can target and inhibit BCL-2 and SIRT1, which are important negative regulators of endothelium apoptosis and cellular senescence (49,50). Therefore, increased circulating miR-34a levels in response to cytokine stimulation may contribute to septic shock-induced endothelial dysfunction through its effects on apoptosis and senescence.

Collectively, targeting these miRNAs, particularly after the initial phase of infection, may provide a novel therapeutic and/or diagnostic tool for sepsis. The present study demonstrated that CLP-induced experimental sepsis resulted in a time-dependent upregulation in the expression levels of these selected miRNAs, which was subsequently mitigated by treatment with TQ.

Multiple cytokine biomarkers have been identified over the past few decades for the diagnosis and treatment of sepsis, of which TNF-α, IL-1, IL-2, IL-6 and IL-10 are some of the most important mediators of inflammation (51,52). TNF-α serves a role in apoptosis, cell survival, inflammation and immunity (53). IL-1 is a prototypical pro-inflammatory cytokine that aids in the stimulation of local and systemic responses. TNF-α and IL-1 have been demonstrated to augment inflammatory cascades through the stimulation of macrophages, which release pro-inflammatory cytokines, such as IL-6 and IL-8, as well as ROSs, reactive nitrogen species and lipid mediators that are vital in sepsis-induced organ failure (54,55). Results from the present study demonstrated that TQ treatment reduced TNF-α and IL-1α levels during sepsis, which may explain its protective effects on CLP-induced sepsis. These results are consistent with our previous findings showing beneficial effects of TQ using a slightly different sepsis model (27,56).

IL-2 has varying and sometimes opposing functions during inflammation, contributing to both the initiation and the termination of the immune response (55). The increased plasma levels of IL-2 in response to CLP infection may act as a prognostic marker for septic shock (55). IL-2 is released from T and B lymphocytes, possibly contributing to the pathogenesis of sepsis (57). In the present study, TQ treatment significantly decreased the levels of IL-2 in septic animals. Another important cytokine mediator that enhances the production of acute phase reactants in the liver is IL-6(58). IL-6 is rapidly produced in response to infections and tissue injuries, contributing to host defense by stimulating the acute phase responses, hematopoiesis and immune reactions. The present study results confirmed that TQ could regulate IL-6 concentration in a CLP model. IL-10 is a key anti-inflammatory cytokine, which is released in response to TNF-α and IL-1α (59-61). Notably, the present study also demonstrated that TQ reduced IL-10 levels at an early time point, which might be a consequence of the initial downregulation of the release of pro-inflammatory cytokines in septic animals.

Precise assessment of sepsis remains a challenge in most ICU settings; therefore, there is a high demand for accurate and early diagnostic biomarkers (62). An early diagnostic biomarker for sepsis must have a rapid turnaround time and be widely available for effective therapeutic potential. CRP, a well-known marker of inflammation, has been suggested to bind the phospholipid components of microorganisms, thereby enabling eradication by macrophages (63,64). During systemic inflammation of a microbial origin, the levels of CRP are increased, as observed in the present study. TQ treatment was able to decrease CRP levels in the CLP model rats, thus indicating its ability to ameliorate the levels of early phase reactant proteins.

VEGF was originally considered to be only a potent stimulator of endothelial permeability, but has since been reported to enhance proliferation and survival of endothelial cells (65,66). Previous studies have shown that a number of features of VEGF make it a strong candidate to control inflammation (66-68). An association between higher circulating levels of VEGF and severe human septic shock has also been reported (69,70). Notably, sepsis or septic shock is associated with a time-dependent surge in the circulating levels of VEGF (71,72). The present findings revealed that the circulating levels of VEGF were significantly increased following CLP, whereas TQ administration substantially decreased these levels. These findings suggested that TQ may also exert its effect on sepsis, at least partially, by controlling the production of VEGF which are in support of our previous finding using a different sepsis model (27,56).

Endotoxins, such as LPS, induce endothelial cell contraction and the development of intercellular gaps, thus increasing permeability of blood vessels (73,74). In addition, it has been demonstrated that ESM-1 can cause vascular responses in in vivo models of inflammation, as well as increase barrier permeability and the passage of leukocytes by upregulating cytokines during sepsis (75). ESM-1 release has been shown to be neutralized by ESM-1 antibodies in a CLP-induced mouse model of sepsis (75). The treatment of rats with TQ in the present study markedly reduced ESM-1 release, providing consistent evidence of its promising effect for the management of sepsis and septic shock (56).

PCT is a peptide precursor of calcitonin hormone that has been identified as a biomarker of bacterial infection (76,77). Moreover, it can be used in evaluating a suitable treatment response, determining severity of sepsis, and estimating morbidity and mortality rates (77-82). Similarly, increased D-dimer levels are another sign of systemic thrombosis, which has been identified as an effective predictor of mortality in severe sepsis (83-86). In the present study, CLP increased PCT and D-dimer levels in a time-dependent manner. Treatment with TQ reduced the levels of both markers, particularly at the 12-h time point, and improved the outcome of sepsis, including animal survival.

From a histopathological perspective, liver, kidney and lung tissue samples of the sham group had normal features, whereas rats subjected to CLP exhibited substantial histopathological alterations. For example, liver sections of the CLP group displayed penetration of inflammatory cells along with necrotic damage; this may be due to increased ROS and lipid peroxidation (12,87). TQ has been shown to attenuate ROS and lipid peroxidation, as well as organ injury caused by various agents (27,88). In the present study, TQ treatment improved both morphological and histological features of the tissue. TQ significantly mitigated kidney and lung injuries in CLP model rats. In particular, renal tissue of septic rats exhibited degeneration in the renal cortex and medullary tubules, which was ameliorated by TQ. Similarly, lung tissue exhibited damage to the alveolar structure with infiltration of mononuclear cells; these pathological changes were restored to normal alveolar architecture with TQ treatment.

In conclusion, the present study demonstrated the effects of TQ in sepsis management by reducing morbidity and mortality in a CLP model. These potential effects are thought to be due to immunomodulation and control of inflammatory status, including effects on the expression of relevant miRNAs, under septic conditions.


The authors extend their sincere appreciation to the Deanship of Scientific Research at King Saud University (Riyadh, Saudi Arabia) for carrying out this work through research group no. RG-1441-337.


Funding: The present study was supported by the Deanship of Scientific Research, KSU (RG-1441-337).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

KMA and BLJ wrote the manuscript, and KMA designed the experiments. AA and MR carried out animal experiments and analyzed the data. MUR and BLJ carried out the microRNA study. KMA, AA and BLJ confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of the College of Pharmacy, King Saud University (Riyadh, Saudi Arabia; approval no. KSU-SE-19-17).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.



Fleischmann C, Scherag A, Adhikari NKJ, Hartog CS, Tsaganos T, Schlattmann P, Angus DC and Reinhart K: International Forum of Acute Care Trialists. Assessment of global incidence and mortality of hospital-treated sepsis. Current Estimates and Limitations. Am J Respir Crit Care Med. 193:259–272. 2016.PubMed/NCBI View Article : Google Scholar


Martin GS, Mannino DM, Eaton S and Moss M: The epidemiology of sepsis in the United States from 1979 through 2000. New Engl J Med. 348:1546–1554. 2003.PubMed/NCBI View Article : Google Scholar


Angus DC and Wax RS: Epidemiology of sepsis: An update. Crit Care Med. 29 (7 Suppl):S109–S116. 2001.PubMed/NCBI View Article : Google Scholar


Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, Schein RM and Sibbald WJ: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest. 101:1644–1655. 1992.PubMed/NCBI View Article : Google Scholar


Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, Bellomo R, Bernard GR, Chiche JD, Coopersmith CM, et al: The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA. 315:801–810. 2016.PubMed/NCBI View Article : Google Scholar


Koyama I, Matsunaga T, Harada T, Hokari S and Komoda T: Alkaline phosphatases reduce toxicity of lipopolysaccharides in vivo and in vitro through dephosphorylation. Clin Biochem. 35:455–461. 2002.PubMed/NCBI View Article : Google Scholar


Männel DN: Advances in sepsis research derived from animal models. Int J Med Microbiol. 297:393–400. 2007.PubMed/NCBI View Article : Google Scholar


Bentala H, Verweij WR, der Vlag AH-V, van Loenen-Weemaes AM, Meijer DKF and Poelstra K: Removal of phosphate from lipid a as a strategy to detoxify lipopolysaccharide. Shock. 18:561–566. 2002.PubMed/NCBI View Article : Google Scholar


Annane D, Buisson CB, Cariou A, Martin C, Misset B, Renault A, Lehmann B, Millul V, Maxime V and Bellissant E: APROCCHSS Investigators for the TRIGGERSEP Network. Design and conduct of the activated protein C and corticosteroids for human septic shock (APROCCHSS) trial. Ann Intensive Care. 6(43)2016.PubMed/NCBI View Article : Google Scholar


Ayala A and Chaudry IH: IMMUNE dysfunction in murine polymicrobial sepsis. Shock. 5 (Suppl 1):S27–S38. 1996.PubMed/NCBI


Lang JD and Matute-Bello G: Lymphocytes, apoptosis and sepsis: Making the jump from mice to humans. Crit Care. 13(109)2009.PubMed/NCBI View Article : Google Scholar


Matsuda H, Ishikado A, Nishida N, Ninomiya K, Fujiwara H, Kobayashi Y and Yoshikawa M: Hepatoprotective, superoxide scavenging, and antioxidative activities of aromatic constituents from the bark of Betula platyphylla var. japonica. Bioorg Med Chem Lett. 8:2939–2944. 1998.PubMed/NCBI View Article : Google Scholar


Wesche DE, Lomas-Neira JL, Perl M, Chung CS and Ayala A: Leukocyte apoptosis and its significance in sepsis and shock. J Leuk Biol. 78:325–337. 2005.PubMed/NCBI View Article : Google Scholar


Coopersmith CM, Chang KC, Swanson PE, Tinsley KW, Stromberg PE, Buchman TG, Karl IE and Hotchkiss RS: Overexpression of Bcl-2 in the intestinal epithelium improves survival in septic mice. Crit Care Med. 30:195–201. 2002.PubMed/NCBI View Article : Google Scholar


Coopersmith CM: Inhibition of intestinal epithelial apoptosis and survival in a murine model of pneumonia-induced sepsis. JAMA. 287:1716–1721. 2002.PubMed/NCBI View Article : Google Scholar


Sonkoly E and Pivarcsi A: MicroRNAs in inflammation. Int Rev Immunol. 28:535–561. 2009.PubMed/NCBI View Article : Google Scholar


Ratti M, Lampis A, Ghidini M, Salati M, Mirchev MB, Valeri N and Hahne JC: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) as new tools for cancer therapy: First steps from bench to bedside. Target Oncol. 15:261–278. 2020.PubMed/NCBI View Article : Google Scholar


Ardekani AM and Naeini MM: The role of microRNAs in human diseases. Avicenna J Med Biotechnol. 2:161–179. 2010.PubMed/NCBI


Taganov KD, Boldin MP, Chang KJ and Baltimore D: NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci. 103:12481–12486. 2006.PubMed/NCBI View Article : Google Scholar


Ceppi M, Pereira PM, Dunand-Sauthier I, Barras E, Reith W, Santos MA and Pierre P: MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells. Proc Natl Acad Sci. 106:2735–2740. 2009.PubMed/NCBI View Article : Google Scholar


Essandoh K and Fan GC: Role of extracellular and intracellular microRNAs in sepsis. Biochim Biophys Acta. 1842:2155–2162. 2014.PubMed/NCBI View Article : Google Scholar


Krol J, Loedige I and Filipowicz W: The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet. 11:597–610. 2010.PubMed/NCBI View Article : Google Scholar


Wang HJ, Zhang PJ, Chen WJ, Feng D, Jia YH and Xie LX: Four serum microRNAs identified as diagnostic biomarkers of sepsis. J Trauma Acute Care Surg. 73:850–854. 2012.PubMed/NCBI View Article : Google Scholar


Wang Z, Ruan Z, Mao Y, Dong W, Zhang Y, Yin N and Jiang L: miR-27a is up regulated and promotes inflammatory response in sepsis. Cell Immunol. 290:190–195. 2014.PubMed/NCBI View Article : Google Scholar


Sheedy FJ: Turning 21: Induction of miR-21 as a key switch in the inflammatory response. Front Immunol. 6(19)2015.PubMed/NCBI View Article : Google Scholar


Woo CC, Kumar AP, Sethi G and Tan KHB: Thymoquinone: Potential cure for inflammatory disorders and cancer. Biochem Pharmacol. 83:443–451. 2012.PubMed/NCBI View Article : Google Scholar


Alkharfy KM, Ahmad A, Raish M and Vanhoutte PM: Thymoquinone modulates nitric oxide production and improves organ dysfunction of sepsis. Life Sci. 143:131–138. 2015.PubMed/NCBI View Article : Google Scholar


Hubbard WJ, Choudhry M, Schwacha MG, Kerby JD, Rue LW III, Bland KI and Chaudry IH: Cecal ligation and puncture. Shock. 24:52–57. 2005.PubMed/NCBI View Article : Google Scholar


Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar


Bancroft J and Gamble M: Theory and practice of histological techniques. Churchill Livingstone Pub, Edinburgh, 2002.


Drury R and Wallington E: Carlton's histological techniques, 4th ed. 1967. Oxford University Press, New York, Toronto, 1967.


Calandra T, Glauser MP, Schellekens J and Verhoef J: Treatment of gram-negative septic shock with human igg antibody to escherichia coli J5: A prospective, double-blind, randomized trial. J Infect Dis. 158:312–319. 1988.PubMed/NCBI View Article : Google Scholar


Frazier WJ and Hall MW: Immunoparalysis and adverse outcomes from critical illness. Pediatr Clin North Am. 55:647–668. 2008.PubMed/NCBI View Article : Google Scholar


Dejager L, Pinheiro I, Dejonckheere E and Libert C: Cecal ligation and puncture: The gold standard model for polymicrobial sepsis? Trends Microbiol. 19:198–208. 2011.PubMed/NCBI View Article : Google Scholar


Menezes G, Amaral S, Alvarenga D and Cara D: Surgical procedures to an experimental polymicrobial sepsis: Cecal Ligation and Puncture. Braz J Vet Pathol. 1:77–80. 2008.


Mittal M, Siddiqui MR, Tran K, Reddy SP and Malik AB: Reactive oxygen species in inflammation and tissue injury. Antiox Redox Signal. 20:1126–1167. 2014.PubMed/NCBI View Article : Google Scholar


Precone V, Stornaiuolo G, Amato A, Brancaccio G, Nardiello S and Gaeta GB: Different changes in mitochondrial apoptotic pathway in lymphocytes and granulocytes in cirrhotic patients with sepsis. Liver Int. 33:834–842. 2013.PubMed/NCBI View Article : Google Scholar


Moon HG, Yang J, Zheng Y and Jin Y: MiR-15a/16 regulates macrophage phagocytosis after bacterial infection. J Immunol. 193:4558–4567. 2014.PubMed/NCBI View Article : Google Scholar


Gao Y and Yu Z: MicroRNA-16 inhibits interleukin-13-induced inflammatory cytokine secretion and mucus production in nasal epithelial cells by suppressing the IκB kinase β/nuclear factor-κB pathway. Mol Med Rep. 18:4042–4050. 2018.PubMed/NCBI View Article : Google Scholar


Löffler D, Brocke-Heidrich K, Pfeifer G, Stocsits C, Hackermüller J, Kretzschmar AK, Burger R, Gramatzki M, Blumert C, Bauer K, et al: Interleukin-6-dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer. Blood. 110:1330–1333. 2007.PubMed/NCBI View Article : Google Scholar


Sheedy FJ, Palsson-McDermott E, Hennessy EJ, Martin C, O'Leary JJ, Ruan Q, Johnson DS, Chen Y and O'Neill LAJ: Negative regulation of TLR4 via targeting of the proinflammatory tumor suppressor PDCD4 by the microRNA miR-21. Nat Immunol. 11:141–147. 2010.PubMed/NCBI View Article : Google Scholar


McClure C, Brudecki L, Ferguson DA, Yao ZQ, Moorman JP, McCall CE and Gazzar ME: MicroRNA 21 (miR-21) and miR-181b couple with nfi-a to generate myeloid-derived suppressor cells and promote immunosuppression in late sepsis. Infect Immun. 82:3816–3825. 2014.PubMed/NCBI View Article : Google Scholar


Goodwin AJ, Guo C, Cook JA, Wolf B, Halushka PV and Fan H: Plasma levels of microRNA are altered with the development of shock in human sepsis: An observational study. Crit Care. 19:2015.PubMed/NCBI View Article : Google Scholar


McClure C, Ali E, Youssef D, Yao ZQ, McCall CE and El Gazzar M: NFI-A disrupts myeloid cell differentiation and maturation in septic mice. J Leukoc Biol. 99:201–211. 2016.PubMed/NCBI View Article : Google Scholar


Lu TX and Rothenberg ME: Diagnostic, functional, and therapeutic roles of microRNA in allergic diseases. J Allergy Clin Immunol. 132:3–13; quiz 14. 2013.PubMed/NCBI View Article : Google Scholar


Jiang C, Ting AT and Seed B: PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines. Nature. 391:82–86. 1998.PubMed/NCBI View Article : Google Scholar


Chawla A: Control of macrophage activation and function by PPARs. Circul Res. 106:1559–1569. 2010.PubMed/NCBI View Article : Google Scholar


Mantzarlis K, Tsolaki V and Zakynthinos E: Role of oxidative stress and mitochondrial dysfunction in sepsis and potential therapies. Oxid Med Cell Longev. 2017(5985209)2017.PubMed/NCBI View Article : Google Scholar


Ackermann EJ, Taylor JK, Narayana R and Bennett CF: The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides. J Biol Chem. 274:11245–11252. 1999.PubMed/NCBI View Article : Google Scholar


Potente M and Dimmeler S: Emerging roles of SIRT1 in vascular endothelial homeostasis. Cell Cycle. 7:2117–2122. 2008.PubMed/NCBI View Article : Google Scholar


Zhang JM and An J: Cytokines, inflammation, and pain. Int Anesthesiol Clin. 45:27–37. 2007.PubMed/NCBI View Article : Google Scholar


Schulte W, Bernhagen J and Bucala R: Cytokines in sepsis: Potent immunoregulators and potential therapeutic targets-an updated view. Mediators Inflamm. 2013(165974)2013.PubMed/NCBI View Article : Google Scholar


Parameswaran N and Patial S: Tumor necrosis factor-α signaling in macrophages. Crit Rev Eukaryot Gene Expr. 20:87–103. 2010.PubMed/NCBI View Article : Google Scholar


Cohen J: The immunopathogenesis of sepsis. Nature. 420:885–891. 2002.PubMed/NCBI View Article : Google Scholar


Fong Y, Tracey KJ, Moldawer LL, Hesse DG, Manogue KB, Kenney JS, Lee AT, Kuo GC, Allison AC and Lowry SF: Antibodies to cachectin/tumor necrosis factor reduce interleukin 1 beta and interleukin 6 appearance during lethal bacteremia. J Exp Med. 170:1627–1633. 1989.PubMed/NCBI View Article : Google Scholar


Alkharfy KM, Ahmad A, Jan BL and Raish M: Thymoquinone reduces mortality and suppresses early acute inflammatory markers of sepsis in a mouse model. Biomed Pharmacother. 98:801–805. 2018.PubMed/NCBI View Article : Google Scholar


Hoyer KK, Dooms H, Barron L and Abbas AK: Interleukin-2 in the development and control of inflammatory disease. Immunol Rev. 226:19–28. 2008.PubMed/NCBI View Article : Google Scholar


Tanaka T, Narazaki M and Kishimoto T: IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol. 6:a016295. 2014.PubMed/NCBI View Article : Google Scholar


Pestka S, Krause CD, Sarkar D, Walter MR, Shi Y and Fisher PB: Interleukin-10andrelatedcytokines andreceptors. Ann Rev Immunol. 22:929–979. 2004.PubMed/NCBI View Article : Google Scholar


Couper KN, Blount DG and Riley EM: IL-10: The master regulator of immunity to infection. J Immunol. 180:5771–5777. 2008.PubMed/NCBI View Article : Google Scholar


Howard M, Muchamuel T, Andrade S and Menon S: Interleukin 10 protects mice from lethal endotoxemia. J Exp Med. 177:1205–1208. 1993.PubMed/NCBI View Article : Google Scholar


Calfee CS and Pugin J: The search for diagnostic markers in sepsis. Am J Respir Crit Care Med. 186:2–4. 2012.PubMed/NCBI View Article : Google Scholar


Benzaquen LR, Yu H and Rifai N: High sensitivity c-reactive protein: An emerging role in cardiovascular risk assessment. Crit Rev Clin Lab Sci. 39:459–497. 2002.PubMed/NCBI View Article : Google Scholar


Gabay C and Kushner I: Acute-phase proteins and other systemic responses to inflammation. N Engl J Med. 340:448–454. 1999.PubMed/NCBI View Article : Google Scholar


Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS and Dvorak HF: Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science. 219:983–985. 1983.PubMed/NCBI View Article : Google Scholar


Leung D, Cachianes G, Kuang W, Goeddel D and Ferrara N: Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 246:1306–1309. 1989.PubMed/NCBI View Article : Google Scholar


Hotchkiss RS and Karl IE: The pathophysiology and treatment of sepsis. N Engl J Med. 348:138–150. 2003.PubMed/NCBI View Article : Google Scholar


Voelkel NF, Cool C, Taraceviene-Stewart L, Geraci MW, Yeager M, Bull T, Kasper M and Tuder RM: Janus face of vascular endothelial growth factor: The obligatory survival factor for lung vascular endothelium controls precapillary artery remodeling in severe pulmonary hypertension. Crit Care Med. 30 (5 Suppl):S251–S256. 2002.PubMed/NCBI View Article : Google Scholar


Pickkers P, Sprong T, Eijk LV, Hoeven HVD, Smits P and Deuren MV: Vascular endothelial growth factor is increased during the first 48 hours of human septic shock and correlates with vascular permeability. Shock. 24:508–512. 2005.PubMed/NCBI View Article : Google Scholar


van der Flier M, van Leeuwen HJ, van Kessel KP, Kimpen JL, Hoepelman AI and Geelen SP: Plasma vascular endothelial growth factor in severe sepsis. Shock. 23:35–38. 2005.PubMed/NCBI View Article : Google Scholar


Yano K, Liaw PC, Mullington JM, Shih SC, Okada H, Bodyak N, Kang PM, Toltl L, Belikoff B, Buras J, et al: Vascular endothelial growth factor is an important determinant of sepsis morbidity and mortality. J Exp Med. 203:1447–1458. 2006.PubMed/NCBI View Article : Google Scholar


Thickett DR, Armstrong L, Christie SJ and Millar AB: Vascular endothelial growth factor may contribute to increased vascular permeability in acute respiratory distress syndrome. Am J Respir Crit Care Med. 164:1601–1605. 2001.PubMed/NCBI View Article : Google Scholar


Marshall JC, Vincent JL, Fink MP, Cook DJ, Rubenfeld G, Foster D, Fisher CJ Jr, Faist E and Reinhart K: Measures, markers, and mediators: Toward a staging system for clinical sepsis. A report of the fifth toronto sepsis roundtable, toronto, ontario, canada, october 25-26, 2000. Crit Care Med. 31:1560–1567. 2003.PubMed/NCBI View Article : Google Scholar


Peters K, Unger RE, Brunner J and Kirkpatrick CJ: Molecular basis of endothelial dysfunction in sepsis. Cardiovasc Res. 60:49–57. 2003.PubMed/NCBI View Article : Google Scholar


Lee W, Ku SK, Kim SW and Bae JS: Endocan elicits severe vascular inflammatory responses in vitro and in vivo. J Cell Physiol. 229:620–630. 2014.PubMed/NCBI View Article : Google Scholar


Becker KL, Snider R and Nylen ES: Procalcitonin assay in systemic inflammation, infection, and sepsis: Clinical utility and limitations. Crit Care Med. 36:941–952. 2008.PubMed/NCBI View Article : Google Scholar


Nakamura A, Wada H, Ikejiri M, Hatada T, Sakurai H, Matsushima Y, Nishioka J, Maruyama K, Isaji S, Takeda T and Nobori T: Efficacy of procalcitonin in the early diagnosis of bacterial infections in a critical care unit. Shock. 31:586–591. 2009.PubMed/NCBI View Article : Google Scholar


Çetinkaya M, Özkan H, Köksal N, Çelebi S and Hacımustafaoğlu M: Comparison of serum amyloid A concentrations with those of C-reactive protein and procalcitonin in diagnosis and follow-up of neonatal sepsis in premature infants. J Perinatol. 29:225–231. 2008.PubMed/NCBI View Article : Google Scholar


Kim KE and Han JY: Evaluation of the clinical performance of an automated procalcitonin assay for the quantitative detection of bloodstream infection. Korean J Lab Med. 30:153–159. 2010.PubMed/NCBI View Article : Google Scholar


Ugarte H, Silva E, Mercan D, De Mendonca A and Vincent JL: Procalcitonin used as a marker of infection in the intensive care unit. Crit Care Med. 27:498–504. 1999.PubMed/NCBI View Article : Google Scholar


Deis JN, Creech CB, Estrada CM and Abramo TJ: Procalcitonin as a marker of severe bacterial infection in children in the emergency department. Pediatr Emerg Care. 26:51–60. 2010.PubMed/NCBI View Article : Google Scholar


Schneider CP, Yilmaz Y, Kleespies A, Jauch KW and Hartl WH: Accuracy of procalcitonin for outcome prediction in unselected postoperative critically ill patients. Shock. 31:568–573. 2009.PubMed/NCBI View Article : Google Scholar


Amaral A, Opal SM and Vincent JL: Coagulation in sepsis. Intensive Care Med. 30:1032–1040. 2004.PubMed/NCBI View Article : Google Scholar


Fu Y, Jiang H, Li LX, Chen J, Niu Q and Li RX: Correlation of coagulation indicators with inflammatory markers for sepsis in the patients with hematological malignancies. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 22:1381–1385. 2014.PubMed/NCBI View Article : Google Scholar : (In Chinese).


Zhan ZG and Li CS: Prognostic value of D-dimer in patients with sepsis in emergency department: A prospective study. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue. 24:135–139. 2012.PubMed/NCBI(In Chinese).


Rodelo JR, De la Rosa G, Valencia ML, Ospina S, Arango CM, Gómez CI, García A, Nuñez E and Jaimes FA: d-dimer is a significant prognostic factor in patients with suspected infection and sepsis. Am J Emerg Med. 30:1991–1999. 2012.PubMed/NCBI View Article : Google Scholar


Kono H, Asakawa M, Fujii H, Maki A, Amemiya H, Yamamoto M, Matsuda M and Matsumoto Y: Edaravone, a novel free radical scavenger, prevents liver injury and mortality in rats administered endotoxin. J Pharmacol Exp Ther. 307:74–82. 2003.PubMed/NCBI View Article : Google Scholar


Nagi MN, Alam K, Badary OA, al-Shabanah OA, al-Sawaf HA and al-Bekairi AM: Thymoquinone protects against carbon tetrachloride hepatotoxicity in mice via an antioxidant mechanism. Biochem Mol Biol Int. 47:153–159. 1999.PubMed/NCBI View Article : Google Scholar

Related Articles

Journal Cover

Volume 23 Issue 6

Print ISSN: 1792-0981
Online ISSN:1792-1015

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
Spandidos Publications style
Alkharfy KM, Ahmad A, Jan BL, Raish M and Rehman MU: Thymoquinone modulates the expression of sepsis‑related microRNAs in a CLP model. Exp Ther Med 23: 395, 2022
Alkharfy, K.M., Ahmad, A., Jan, B.L., Raish, M., & Rehman, M.U. (2022). Thymoquinone modulates the expression of sepsis‑related microRNAs in a CLP model. Experimental and Therapeutic Medicine, 23, 395.
Alkharfy, K. M., Ahmad, A., Jan, B. L., Raish, M., Rehman, M. U."Thymoquinone modulates the expression of sepsis‑related microRNAs in a CLP model". Experimental and Therapeutic Medicine 23.6 (2022): 395.
Alkharfy, K. M., Ahmad, A., Jan, B. L., Raish, M., Rehman, M. U."Thymoquinone modulates the expression of sepsis‑related microRNAs in a CLP model". Experimental and Therapeutic Medicine 23, no. 6 (2022): 395.