| Inflammation is a defensive reaction of the body tissues against to injury stimulus; it is a complicated process that involves various cell types (such as lymphocyte, macrophage, granulocyte, and endothelial cell, etc.) and multi-components (cytokines, vascular active substances, chemokines, adhesion molecules, inflammation-related enzymes). Inflammation is a unified process of injury and damage resistance. Appropriate inflammatory response is helpful for the human body to resist damage, clear the infection and promote wound healing; however, the excessive inflammatory response will lead to persistent tissue damage, thereby affecting our health.Macrophages are differentiated from blood monocytes after monocytes moving into the tissues. They are widely distributed in different organs, for example:alveolar macrophages in lungs, Kupffer cells in liver, glial cells in nervous system and osteoclast in bone tissue. Macrophages are important immune cells in the body; they have anti-infection, anti-tumor and immunomodulatory effects. But they are also the center cell to produce inflammatory mediators. Macrophages can be activated by various inflammatory stimuli; including bacterial lipopolysaccharide (also known as endotoxin).which is a pathogenic factor of gram-negative bacteria. When LPS released from the death or reproduction gram-negative bacteria which had been entered into the host body, it can induce monocyte-macrophage synthesis and to release various inflammatory mediators, such as NO, prostaglandins, and pro-inflammatory cytokine TNF-alpha, IL-1β, and IL-6etc., causing the body to a series of inflammatory reactions, which seriously can lead to severe toxic shock, systemic inflammatory response syndrome and multiple organ failure. Macrophage and its secreted inflammatory mediators play important roles in inflammatory process. The current studies suggested that macrophages were involved in the occurrence and development of a variety of inflammatory diseases such as atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, sepsis, and so on.NO is a toxic gas free radicals, generated from L-arginine catalyzed by nitric oxide synthase (NOS).it possesses a wide range of biological functions. For example, NO is involved in processes of neural signaling transmission, function regulation of heart and lung, apoptosis and immune defense. Currently known of NOS include nerves nitric oxide synthase (nNOS), endothelial NOS (eNOS) and inducible nitric oxide synthase (iNOS). The first two are also known as the constitutively nitric oxide synthase (cNOS). Low concentrations of NO generated by cNOS catalytic mainly involved in physiological processes; maintain the normal function of cells. High concentrations of NO produced by macrophages are mainly catalyzed by inducible nitric oxide synthase. Short-term, large amount of NO released by macrophage are benefit for killing microbes and tumor cells, however, long-term, excessive NO produced by macrophage mediates organizations injury through DNA damage, inhibition of mitochondrial respiration, reactive nitrogen and other cytotoxic effect, participate the occurrence and development of certain inflammatory diseases. Therefore, inhibition of NO over-production has became a hotspot for the prevention of inflammatory diseases.As a group of important tissue hormones, prostaglandins (PGS) are widely present in animals and humans.They are not only play a strong role in regulating the function of the cardiovascular, gastrointestinal, respiratory and reproductive systems, but also critical in development of fever and pain in inflammation. Cyclooxygenases (COX)(including COX-1, COX-2and COX-3subtypes) are key enzyme catalyzing arachidonic acid to generate prostaglandins. Structure cyclooxygenase COX-1and COX-3are mainly present in blood vessels, stomach, kidney and other tissues. They are involved in vasomotion, platelet aggregation, and regulation of gastric mucosal blood flow, gastric mucus secretion and renal function. The inducible cyclooxygenase COX-2is low expression in normal tissues but large expression in activated macrophages. Traditional non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, rofecoxib act as antipyretic analgesic and anti-inflammatory drug by inhibiting the activity of cyclooxygenase and reducing the production of prostaglandins. They have been widely used in the treatment of rheumatoid arthritis and other inflammatory diseases.TNF-alpha, IL-1β and IL-6are proinflammatory cytokines, which can directly activate immune effector cells (such as T cells, B cells, macrophages, NK cells, etc.). Furthermore, they also promote the production of other inflammatory mediators in a paracrine or autocrine system, and thus amplify the inflammatory response. Normal levels of TNF-alpha, IL-1β, and IL-6is essential to modulate the immune response, anti-infection, promote tissue repair, and cause tumor cell apoptosis. However, excessive production and release of these pro-inflammatory cytokines will destroy the immune balance and cause excessive inflammatory response. For example, certain proinflammatory cytokines have been found increasing in the blood circulation of patients with inflammatory diseases such as rheumatoid arthritis, and the increased amount of these proinflammatory cytokines are correlation with the severity of diseases. Blocking the proinflammatory cytokine-mediated effects by using monoclonal antibodies or receptor antagonists has achieved good results in the treatment of many inflammatory diseases.Macrophages produce the above-mentioned inflammatory mediators via activation of several complex intracellular signal transduction pathways. One of the most important is transcription factors NF-κB (nuclear factor-kappa B) signaling pathway. NF-κB is widely present in mammalian cells, and it participates in inflammation response, immune response as well as cell differentiation and apoptosis. In mammalian cells, NF-κB is usually existed as p50/p65heterodimer. In resting state, NF-κB exists as an inactive form in the cytoplasm by binding to its inhibitory kappa B (IκB) protein. Once cells are activated by external stimuli, IκB protein is degraded; NF-κB rapidly translocates into the nucleus, binds to the promoter elements of its targeted genes, and then regulates transcription of various genes. NF-kB signaling pathway activation is mainly regulated by the degradation of IκB; however, numerous studies have shown that NF-kB activity can also be regulated by direct modification of NF-kB protein by phosphorylation and acetylation. For example, casein kinase Ⅱ can enhance the transcriptional activity of NF-kB through phosphorylation of serine residue of p65subunit.NF-κB signaling pathway is the most important downstream in the LPS-mediated signal transduction pathway. When the NF-κB signaling pathway in macrophage is activated, the expression of inflammatory cytokines, adhesion molecules, chemoattractant molecules and inflammation-related enzymes can be rapidly induced. Therefore, blocking the activation of NF-κB signaling pathway can reduce the production of various inflammatory mediators.The mitogen activated protein kinases (MAPKs) are cellular serine and (or) threonine protein kinases. They play important roles in cell stress, damage responses, inflammatory signal transduction, cell apoptosis, tumor cell proliferation and material metabolism. The MAPKs family has three classic pathway of the subfamily, they are extracellular regulated protein kinase1/2(ERK1/2), c-Jun N-terminal kinase (JNK), p38mitogen-activated protein kinase (p38-MApK). The activation of MAPKs is one of the early events in the cells after LPS treatment in monocytes and macrophages. Furthmore, many important substrates for MAPKs are transcription factors such as NF-κB and AP-1.thus MAPKs are closely related to the synthesis and secretion of inflammatory mediators.Excessive release of inflammatory mediators by activated macrophages is critical involved in inflammatory diseases. Therefore, inhibition of inflammatory mediators’production in activated macrophages has became a good target for anti-inflammatory drug research. Furthermore, drugs targeted at inhibition of macrophages activation and mediators release have their own unique advantages in theory compared with the traditional anti-inflammatory drugs.In China, anti-inflammatory drugs are the second largest category of drugs followed anti-infective drugs in a certain peroid. The anti-inflammatory-immunity drugs are divided into three categories, steroidal anti-inflammatory drugs non-steroidal anti-inflammatory drugs (NSAIDs) and Disease-Modifying Drugs (DMDs). These three types of anti-inflammatory-immunity drugs have been widely applied in clinical practice. Steroidal anti-inflammatory drugs such as dexamethasone have a wide range of physiological and pharmacological effects, but long-term applications of steroidal anti-inflammatory drugs caused the disorder of metabolism, induced or exacerbated infection, lead to osteoporosis, etc. Non-steroidal anti-inflammatory drugs exert anti-inflammatory effect by inhibition of COX and thus inhibit the production of prostaglandins (PGs). They can be divided into non-selective COX inhibitors (such as aspirin) and selective COX-2inhibitor (such as celecoxib). COX inhibitors will cause a lot of side effects compared with the selective COX-2inhibitors. However, further studies found that COX-2is also structural expression in brain, seminal vesicle, and kidneys. The negative impacts by inhibition of COX-2activity in these tissues should pay more attention. Disease-Modifying Drugs contained immunosuppressants, immunostimulants and immunomodulatory agents, For example, rapamycin and leflunomide suppressed the immune response, which mainly be used as anti-rejection agent in organ transplantation; levamisole as an immunostimulants is used for the treatment of autoimmune diseases or as an adjuvant in cancer treatment.With the continuous deepening of the anti-inflammatory immunopharmacological research, we are urgently to find more drugs with good anti-inflammatory activity and fewer side effects. Targeting at the inhibition of macrophage activation and its mediators release may be a good choice.Naphthol derivatives are widely used in medical and industrial applications. They have a naphthol nucleus as a chemical backbone. Some naphthol derivatives have been reported to display a variety of biological activities. For instance,2-substituted-l-naphthol derivatives were shown to inhibit the activities of cyclooxygenase and5-lipooxygenase. A metabolic naphthol derivative,1,4-dihydroxy-2-naphthoic acid, from fermentation by Propionibacterium freudenreichii was reported to suppress bone resorption and dextran sodium sulphate (DSS)-induced colitis. Given the diversity of chemical structure and biological activity of naphthol derivatives, their anti-inflammatory activity is particularly attracted us. Thus, in this study, the LPS-induced macrophage inflammatory model and zymosan-induced mouse peritonitis model are carried out to explore the anti-inflammatory activity of a novel naphthol derivatives Methyl-1-hydroxy-2-naphthoate in vitro and in vivo, and its possible mechanism are also investigated.The major research methods and results are divided into three parts:part I:MHNA inhibited LPS-induced macrophage inflammatory response1, MHNA had no significant effect on cell viability of macrophages.MTT assay was firstly used to analyze the effect of MHNA (0-40μg/ml) on RAW264.7cell viability. MHNA at dose of40μg/ml cocultured with macrophage cell line RAW264.7for24h had no significant effect on macrophage viability (no significant difference in statistically). Thus the maximum dose10μg/ml of MHNA is safe for RAW264.7macrophages.2, MHNA inhibited NO, IL-1β, and IL-6production in LPS-activated macrophagesRAW264.7cells were cocultured with2.5,5.0,10.0μg/ml MHNA and0.1μg/ml LPS for24h. NO and inflammatory cytokine TNF-a, IL-1β and IL-6in cell culture medium were detected by Griess reagent and ELISA methods, respectively. The basal level of NO, TNF-a, IL-1βp and IL-6in cell culture medium is very low. After macrophages being stimulated with100μg/ml LPS for24h, NO, TNF-a, IL-1β and IL-6were increased obviously. MHNA at dose of2.5-10μg/ml significantly dose-dependently inhibited LPS-induced macrophage cells to produce NO, IL-1β and IL-6. However, TNF-a produced by LPS-activated macrophages was not significantly affected by MHNA. 3, MHNA inhibited iNOS and COX-2protein expression in LPS-induced macrophagesRAW264.7cells were cocultured with2.5,5.0,10.0μg/ml MHNA and0.1μg/ml LPS for24h. Total protein was extracted, and then iNOS and COX-2protein expression were investigated by western blot. The results showed that, MHNA significantly inhibited LPS-induced macrophage iNOS and COX-2protein expression in a dose dependent manner.4, MHNA inhibited inflammatory genes expression in LPS-activated macrophagesRAW264.7cells were cocultured with2.5,5.0,10.0μg/ml MHNA and0.1μg/ml LPS for6h. Total cellular RNA from the treated cells was extracted by using TPIzol reagent. Then, inflammatory gene iNOS, COX-2, IL-1β and IL-6mRNA level was quantitated by reverse transcription and realtime-PCR methods. The results show that MHNA inhibited macrophage inflammatory gene iNOS, COX-2, IL-1β and IL-6mRNA expression.part II:anti-inflammatory activity of MHNA in vivo.1MHNA inhibited xylene-induced mouse ear swellingIn order to explore the impact of MHNA in acute inflammatory exudation, a xylene-induced mouse ear swelling test was used. The results show that, MHNA at dose of20,40,80mg/kg orally administered, significantly inhibited mouse ear swelling caused by xylene in a dose dependent manner (P<0.05). The inhibition rates were17.8%,29.5%,45.0%, respectively.2MHNA reduced protein exudation and leukocytes infiltration in mouse peritoneal with peritonitis induced by zymosan A.To further investigate the anti-inflammatory activity of the MHNA in vivo, we constructed zymosan induced murine peritonitis model. In this study, zymosan rapidly induced inflammatory response in mice, causing a lot of protein exudation and leukocytes infiltration in the mouse peritoneal. Dexamethasone10mg/kg and MHNA40mg/kg, MHNA80mg/kg significantly inhibited protein exudation in the abdominal cavity (inhibition rate:45.9%,19.3%and29.4%), they also inhibited leukocyte infiltration (The inhibition rates were:57.4%,22.5%and38.6%).3MHNA inhibited inflammatory cytokine IL-6production in mouse peritoneal with peritonitis.In order to investigate the impact of MHNA on inflammatory cytokines production in zymosan A-induced peritonitis in the abdominal cavity of mice, we examined the cytokine IL-6level in the mouse peritoneal lavage fluid. The results show that IL-6almost can not be detected in peritoneal of normal mouse; but6h after intraperitoneal injection of zymosan, the abdominal cavity of IL-6levels were significantly increased. Dexamethasone10mg/kg, MHNA40mg/kg and MHNA80mg/kg significantly inhibited IL-6production in the abdominal cavity (inhibition rate were48.7%,19.5%and41.2%, respectively).Part III anti-inflammatory mechanism of MHNA1, MHNA inhibited NF-κB signaling pathway activation in macrophagesIn order to explore the mechanism of MHNA inhibiting macrophage inflammatory response, NF-κB signaling pathway activation was detected by western blot, reporter gene assay and electrophoretic mobility shift assay. Pretreated RAW264.7cells with2.5,5.0,10.0μg/ml MHNA for lh, then cells were cocultured with0.1μg/ml LPS for30min (detection iκB degradation and MAPKs phosphorylation by Western blot) or1h (extracted cytosolic and nuclear proteins, then detected NF-κB distribution in the cytoplasm and nucleus by Western blot and investigated NF-κB DNA binding ability by electrophoretic mobility shift assay.Western blot results showed that in LPS-stimulated macrophages, iκB protein degradated significantly, and the cytoplasm of NF-κB obviously reduced while NF-κB within the nucleus increased by compared with unstimulated control cells. This indicated that LPS stimulation induced NF-κB translocated from cytoplasm into the nucleus. Pretreatment with MHNA for1h, significantly attenuated the LPS-induced iκB degradation and NF-κB translocation.Reporter gene results showed that0.1μg/ml LPS caused transcriptional activity of macrophages increased5.3-fold compared to control cells while MHNA significantly inhibited the increasing transcriptional activity of LPS-stimulated macrophagesThe results of electrophoretic mobility shift assay showed that LPS increase NF-κB binding activity to its specific DNA sequences in macrophages. Pretreatment macrophages with MHNA and NF-κB specific inhibitor BAY11-7082for1h inhibited the increase of LPS-induced NF-κB and DNA binding activity.2, MHNA inhibited MAPKs signaling pathway activation in macrophagesRAW264.7cells were pretreated with2.5,5.0,10.0μg/ml MHNA for1h, then cells were cocultured with0.1μg/ml LPS for30min. Total protein was extracted, and MAPKs (p38MAPK, ERK1/2and JNK) phosphorylation were detected by Western blot. The results showed that LPS increased p38MAPK, ERK1/2and JNK phosphorylation in macrophages.2.5-10.Oμg/ml MHNA dose-dependently inhibited p38MAPK and JNK phosphorylation, but had no significant effects on ERK1/2phosphorylation.Conclusion:1, MHNA inhibited LPS-induced macrophage inflammatory response.A, MHNA inhibited inflammatory mediators NO, IL-1β and IL-6production in LPS activated macrophages;B, MHNA inhibited iNOS and COX-2protein expression in LPS activated macrophages;C, MHNA inhibited inflammatory genes (iNOS, COX-2, IL-1β and IL-6) expression in LPS activated macrophages.2, MHNA showed a good anti-inflammatory activity in vivo.A, MHNA inhibited mouse ear swelling caused by xyleneB, MHNA reduced protein exudation and leukocytes infiltration in mouse peritoneal with peritonitis induced by zymosan A.C, MHNA inhibited inflammatory cytokine IL-6production in mouse peritoneal with peritonitis. 3, MHNA inhibited macrophage activation by inhibiting NF-kappa B, p38MAPK and JNK signaling pathway activation.In summary, MHNA inhibited inflammatory mediators’production in LPS-activated macrophages by blocking NF-κB, p38MAPK and JNK signaling pathway activation. Further studies found that MHNA also showed good anti-inflammtroy activity in xylene-induced mouse ear swelling test and zymosan-induced mouse peritonitis. These studies provide a new experimental basis to investigate the anti-inflammatory activity and mechanism of naphthol derivatives in vitro and in vivo. |
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