In 2016, the third international consensus on sepsis and septic shock (sepsis 3.0) defined it as life-threatening organ dysfunction caused by the imbalance of host response to infection[1]. The mortality rate of sepsis increases from 10% in children to 40% in the elderly, and the average mortality rate is about 30%.The mortality of patients with more severe syndrome and septic shock is 50% or higher[2]. If the treatment is not started early and the pathogen is not eradicated, sepsis can lead to organ dysfunction, hypotension,and circulatory failure, often leading to death.
One of the most significant features of sepsis is coagulation activation, during sepsis, many factors activate coagulation function while inducing an inflammatory response [3].At the same time,the anticoagulation system and fibrinolysis system are down regulated. After the initial trigger event, the inflammatory response is enhanced by several positive feedback circuits, which can activate the coagulation cascade. Therefore, in the process of occurrence and development of many diseases, inflammation and coagulation coexist. Inflammatory factors can promote the production and activation of prothrombin, enhance the procoagulant activity, cause microvascular thrombosis, and promote the occurrence and development of diffuse intravascular coagulation (DIC); whereas coagulation can be widely involved in mediating the inflammatory response[4]. The over activation of coagulation may cause damage to the host body, leading to sepsis-induced coagulopathy (SIC)[5]. which has an important impact on the prognosis of sepsis[6].
The host's innate immune system, which consists of neutrophils, macrophages, dendritic cells, natural killer cells, T-cells, and other immune cells, is the first line of defense against infections[7]. Among various immune cells, macrophages play a key role in immune homeostasis and inflammatory response by secreting a series of key cytokines. Macrophages can directly sense the damage-related pattern molecule(DAMP) released during inflammation-induced tissue injury through pattern recognition receptor (PRR), leading to the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammatory bodies [8]. Therefore, it is of great significance to pay attention to and regulate the immune function of macrophages for the treatment of sepsis.
The RAW264.7 cell line is the most commonly used in vitro research model for screening anti-inflammatory active substances and studying inflammation[9]. Under the action of inducers (such as lipopolysaccharide[LPS]), RAW264.7 cells simulate inflammatory response and release or upregulate a variety of inflammatory mediators, such as nitric oxide (NO),cyclooxygenase-2 (COX-2),tumor necrosis factor-α(TNF-α),and Interleukin 6 (IL-6).In Gram-negative bacterial infections, LPS can activate various cells, such as monocytes and vascular endothelial cells, to release a variety of inflammatory mediators, such as TNF, IL-1β, and IL-6, and produce a series of inflammatory reactions by contacting coagulation factor VII and complement system to resist the invasion of pathogenic microorganisms[10]. While causing inflammation, pathogenic microorganisms and some of their components can also cause coagulation. This in turn up-regulates the expression of tissue factor(TF) in monocytes/macrophages and vascular endothelial cells, leading to coagulation and thrombosis.Studies have indicated that TFs are potential mediators of intracellular signal transduction of established inflammatory pathways[11]. Normal blood cells and vascular endothelial cells do not express TF. The content of TF in normal plasma is very low, whereas the levels of tissue factor pathway inhibitor (TFPI) are about 1000 times higher than that of TF[12]. However, macrophages can be induced to express TF under the action of certain stimuli [13], and are considered important triggers for clotting in diseased states[14]. Many inflammatory substances such as endotoxins and tumor necrosis factors, IL-1, IL-6, IL-8, and C-reactive proteins can cause mononucleocytes/macrophages and vascular endothelial cell expression histokines[15]. TF pathway is not only responsible for the initiation of the coagulation process in physiological hemostasis but also plays a key role in the process of thrombosis under pathological conditions. Therefore, in the treatment of sepsis, it is of great significance to pay attention to and stimulate macrophage function[16].
It has been observed that toll-like receptors TLR (such as TLR2 and TLR4), microbial products, and other inflammatory mediators initiate signaling pathways leading to NF-κB nuclear accumulation and enhanced gene transcription which are responsible for the expression of cytokines, chemokines, adhesion molecules, and other mediators of infection-related inflammatory response[17].Transcription regulator NF-κB is a core participant in the expression of many immunomodulate media involved in acute inflammatory responses [18]. Nuclear factor-kappa B (NF-κB), which is widely expressed in the central nervous system, is a protein transcription factor and a key regulator of inflammation and coagulation response, as well as the expression of many genes, such as iNOS and COX2. It also promotes the secretion of a variety of pro-inflammatory cytokines, such as TNF-α and IL-1β[19].
The combination of NF-κB with the inhibitory protein IκBα in the cytoplasm blocks the nuclear positioning sequence of NF-κB and inhibits its entry into the nucleus[20]. Under physiological conditions, NF-κB and its inhibitor IκBα binds and remain dormant in the cytoplasm. Once activated, NF-κB is transferred to the nucleus to start the transcription of downstream genes[21]. We found that LPS increased the activity of NF-κB, followed by an increase in the expression of iNOS and COX2. BNF downregulates the increased expression of these two pro-inflammatory mediators by inhibiting the activity of NF-κB, and the results show a decrease in pro-inflammatory mediator secretion.
Other examples of immune response regulation by specific inhibitory methods involve kinases (such as p38 or Akt) or transcription factors (such as NF-κB) has shown its pathophysiological importance in experimental models [22].Protein kinase B (PKB) or Akt is a widely expressed serine/threonine kinase with a PH domain that selectively binds inositol 3-phosphate [23]. Studies have shown that AKT1, which is one of three closely related serine/threonine protein kinases, may play a dual role in carcinogenesis not only by inhibiting apoptosis but also by inhibiting invasion and metastasis.AKT signal imbalance is the underlying cause of several life-threatening diseases, including diabetes, neurodegenerative syndrome, and various cancers. In the immune response of sepsis, the inositol phosphate 3-kinase (PI3K) signaling pathway and Akt play an important role because they regulate the release of inflammatory factors and control downstream signaling molecules[24]. PI3K/AKT pathways are known to be activated by a variety of extracellular and intracellular signals. They are important cell signaling molecules and key survival factors involved in proliferation, differentiation, apoptosis, and several other cellular processes.
Heme oxygenase-1 (HO-1), which is a famous phase II detoxification enzyme, plays a powerful role in maintaining cellular homeostasis and protects cells from damage caused by oxidative stress and inflammatory response[25, 26]. It has been observed that During oxidative stress, HO-1 is upregulated during oxidative stress and inflammatory conditions to enhance cellular defense mechanisms against oxidative damage[27]. In normal cells, HO-1 is expressed at a low levels; however, its expression can be stimulated in cells without stimulation and can be rapidly induced by by various oxidation inducers, such as including LPS[28], heme, and hypoxiaa, etc. Under inflammatory conditions, the enhancement of HO-1 expression plays an important role in cell protection[29]. HO-1 catalyzes the first, rate-limiting step in the degradation of heme to carbon monoxide (CO), biliverdin, and ferrous iron[26].Biliverdin is converted to bilirubin which is an effective endogenous antioxidant with recognized anti-inflammatory properties[30].CO has a variety of biological functions, including anti-inflammatory properties[31].In pure inflammatory models of disease, such as endotoxin exposure, HO-1 deficient mice are vulnerable to oxidant-induced tissue damage and death[32].HO-1 reportedly inhibits the excessive production of TNF-α and IL-1β[33] in LPS-induced RAW264.7 cells.In animal models, HO-1 deficient mice exhibit severe inflammation[32],and HO-1 overexpression produces anti-inflammatory effects[27].
The expression of HO-1, like several other phase II detoxifying enzymes, is induced by the binding of upstream activators to the starter region of the antioxidant-response element (ARE) [34].The nuclear factor erythroid 2-related factor 2 (Nrf2) acts as an upstream activator for is an upstream medium for Phase II detoxifying enzymes (including HO-1)[35]. Apart from being a pleiotropic protein, it is a basic leucine zipper redox sensitive transcription factor that regulates the basic and induced expression of a series of antioxidant and other cryoprotective genes by binding with cis-acting genes s[36]. Under normal conditions, Nrf2 is degraded in the cytoplasm after binding to Keap1, resulting in the decreased accumulation of Nrf2 in the nucleus and reduced transcription of a range of cell-protective genes[37], such as HO-1 [37].Under inflammatory conditions, Nrf2 is dissociated from Keap1 and is transported to the nucleus to activate the expression of HO-1, thereby resisting inflammatory responses. Based on these and other studies, it is now widely believed that Nrf2/HO-1 plays a key role in regulating the inflammatory response, enhancing the cell's resistance to inflammation.
Beta-naphthoflavone (BNF) is a synthetic derivative of naturally occurring flavonoids that have antioxidant and anti-inflammatory effects. BNF itself is not toxic or carcinogenic, and as a ligand of aromatic hydrocarbon receptors, BNF is widely used in the pharmaceutical industry. In recent years, BNF has been reported to prevent peritonitis by reducing TNF-α induced endothelial cell activation[38], with antioxidant and anti-inflammatory potential. In addition, numerous studies have shown that BNF therapy reduces neonatal hyperoxic lung injury[39] and acute kidney injury by inhibiting inflammation in surrounding tissues[40]. BNF has also been shown to alleviate colitis induced by sodium dextran sulfate[41].
Previous research has indicated that BNF inhibits the activity of NF-κB and increases the expression of pro-inflammatory mediators, such as iNOS and COX2.However, the role of BNF in sepsis-related inflammatory and coagulation response is still unclear. Therefore, in this study, we utilized LPS-stimulated RAW264.7 cells and cecal ligation and puncture (CLP) animal model to study the anti-inflammatory and anticoagulant activity of BNF and the molecular mechanisms involved.