Scutellarein Inhibits LPS-Induced Inflammation through NF-κB/MAPKs Signaling Pathway in RAW264.7 Cells

Inflammation is a severe topic in the immune system and play a role as pro-inflammatory mediators. In response to such inflammatory substances, immune cells release cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Lipopolysaccharide (LPS) is known as an endotoxin in the outer membrane of Gram-negative bacteria, and it catalyzes inflammation by stimulating the secretion of inflammatory-mediated cytokines such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) by stimulated immune cells. Among the pathways involved in inflammation, nuclear factor kappa (NF-кB) and mitogen-activated protein kinases (MAPKs) are important. NF-kB is a diploid composed of p65 and IkBα and stimulates the pro- gene. MAPKs is a family consisting of the extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38, JNK and p38 play a role as proinflammatory mediators. Thus, we aim to determine the scutellarein (SCU) effect on LPS stimulated RAW264.7 cells. Furthermore, since scutellarein has been shown to inhibit the SARS coronavirus helicase and has been used in Chinese medicine to treat inflammatory disorders like COVID-19, it would be required to examine scutellarein’s anti-inflammatory mechanism. We identified inflammation-inducing substances using western blot with RAW264.7 cells and SCU. And we discovered that was reduced by treatment with SCU in p-p65 and p-IκBα. Also, we found that p-JNK and p-ERK were also decreased but there was no effect in p-p38. In addition, we have confirmed that the iNOS was also decreased after treatment but there is no change in the expression of COX-2. Therefore, this study shows that SCU can be used as a compound to treat inflammation.     

Progress and Prospects of Non-Canonical NF-κB Signaling Pathway in the Regulation of Liver Diseases

Non-canonical nuclear factor kappa B (NF-κB) signaling pathway regulates many physiological and pathological processes, including liver homeostasis and diseases. Recent studies demonstrate that non-canonical NF-κB signaling pathway plays an essential role in hyperglycemia, non-alcoholic fatty liver disease, alcoholic liver disease, liver regeneration, liver injury, autoimmune liver disease, viral hepatitis, and hepatocellular carcinoma. Small-molecule inhibitors targeting to non-canonical NF-κB signaling pathway have been developed and shown promising results in the treatment of liver injuries. Here, the recent advances and future prospects in understanding the roles of the non-canonical NF-κB signaling pathways in the regulation of liver diseases are discussed.     

Luteolin-3′-O-Phosphate Inhibits Lipopolysaccharide-Induced Inflammatory Responses by Regulating NF-κB/MAPK Cascade Signaling in RAW 264.7 Cells

Luteolin (LT), present in most plants, has potent anti-inflammatory properties both in vitro and in vivo. Furthermore, some of its derivatives, such as luteolin-7-O-glucoside, also exhibit anti-inflammatory activity. However, the molecular mechanisms underlying luteolin-3′-O-phosphate (LTP)-mediated immune regulation are not fully understood. In this paper, we compared the anti-inflammatory properties of LT and LTP and analyzed their molecular mechanisms of action; we obtained LTP via the biorenovation of LT. We investigated the anti-inflammatory activities of LT and LTP in macrophage RAW 264.7 cells. We confirmed from previously reported literature that LT inhibits the production of nitric oxide and prostaglandin E₂, as well as the expression of inducible NO synthetase and cyclooxygenase-2. In addition, expressions of inflammatory genes and mediators, such as tumor necrosis factor-α, interleukin-6, and interleukin-1β, were suppressed. LTP showed anti-inflammatory activity similar to LT, but better anti-inflammatory activity in all the experiments, while also inhibiting mitogen-activated protein kinase and nuclear factor-kappa B more effectively than LT. At a concentration of 10 μM, LTP showed differences of 2.1 to 44.5% in the activity compared to LT; it also showed higher anti-inflammatory activity. Our findings suggest that LTP has stronger anti-inflammatory activity than LT.     

Neocinnamomum caudatum Essential Oil Ameliorates Lipopolysaccharide-Induced Inflammation and Oxidative Stress in RAW 264.7 Cells by Inhibiting NF-κB Activation and ROS Production

Neocinnamomum caudatum (Lauraceae) plant is used in the traditional system of medicine and is considered a potential source of edible fruits, spices, flavoring agents and biodiesel. The leaves, bark and roots of the species are used by local communities for the treatment of inflammatory responses, such as allergies, sinusitis and urinary tract infections. However, there is no scientific evidence to support the molecular mechanism through which this plant exerts its anti-inflammatory effect. The aim of the current research was to characterize the chemical constituents of bark (NCB) and leaf (NCL) essential oil of N. caudatum and to elucidate its anti-inflammatory action in lipopolysaccharide (LPS)-treated RAW 264.7 cells. Essential oils extracted by hydrodistillation were further subjected to gas chromatography mass spectrometry (GC-MS) analysis. The major constituents in bark essential oil identified as β-pinene (13.11%), α-cadinol (11.18%) and α-pinene (10.99%), whereas leaf essential oil was found to be rich in β-pinene (45.21%), myrcene (9.97%) and α-pinene (9.27%). Treatment with NCB and NCL at a concentration of 25 µg/mL exerted significant anti-inflammatory activity by significantly reducing LPS-triggered nitric oxide (NO) production to 45.86% and 61.64%, respectively, compared to the LPS-treated group. In the LPS-treated group, the production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β, decreased after treatment with essential oil, alleviating the mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. The essential oil also inhibited the production of intracellular ROS and attenuated the depletion of mitochondrial membrane potential in a concentration-dependent manner. Pretreatment with NCB also reduced nuclear factor kappa-B (NF-κB)/p65 translocation and elevated the levels of endogenous antioxidant enzymes in LPS-induced macrophages. The present findings, for the first time, demonstrate the anti-inflammatory potential of both bark and leaf essential oils of N. caudatum. The bark essential oil exhibited a significantly more important anti-inflammatory effect than the leaf essential oil and could be used as a potential therapeutic agent for the treatment of inflammatory diseases.     

Tabersonine Inhibits the Lipopolysaccharide-Induced Neuroinflammatory Response in BV2 Microglia Cells via the NF-κB Signaling Pathway

The occurrence and development of neurodegenerative diseases is related to a variety of physiological and pathological changes. Neuroinflammation is one of the major factors that induces and aggravates neurodegenerative diseases. The most important manifestation of neuroinflammation is the activation of microglia. Therefore, inhibiting the abnormal activation of microglia is an important way to alleviate the occurrence of neuroinflammatory diseases. In this research, the inhibitory effect of tabersonine (Tab) on neuroinflammation was evaluated by establishing the BV2 neuroinflammation model induced by lipopolysaccharide (LPS). It was found that Tab significantly inhibited the production and expression of nitric oxide (NO), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and reactive oxygen species (ROS) in BV-2 cells stimulated by LPS. In addition, Tab can also inhibit the activation of nuclear factor-κB (NF-κB) induced by LPS, thus regulating inflammatory mediators such as inducible nitric oxide synthase (iNOS). These results indicated that Tab regulated the release of inflammatory mediators such as NO, IL-1β, TNF-α, and IL-6 by inhibiting NF-κB signaling pathway, and exerting its anti-neuroinflammatory effect. This is the first report regarding the inhibition on LPS-induced neuroinflammation in BV2 microglia cells of Tab, which indicated the drug development potential of Tab for the treatment of neurodegenerative diseases.     

Anti-Inflammatory Effects of 6-Methylcoumarin in LPS-Stimulated RAW 264.7 Macrophages via Regulation of MAPK and NF-κB Signaling Pathways

Persistent inflammatory reactions promote mucosal damage and cause dysfunction, such as pain, swelling, seizures, and fever. Therefore, in this study, in order to explore the anti-inflammatory effect of 6-methylcoumarin (6-MC) and suggest its availability, macrophages were stimulated with lipopolysaccharide (LPS) to conduct an in vitro experiment. The effects of 6-MC on the production and levels of pro-inflammatory cytokines (interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α) and inflammatory mediators (nitric oxide (NO), prostaglandin E₂ (PGE₂)) in LPS-stimulated RAW 264.7 cells were examined. The results showed that 6-MC reduced the levels of NO and PGE₂ without being cytotoxic. In addition, it was demonstrated that the increase in the expression of pro-inflammatory cytokines caused by LPS stimulation, was decreased in a concentration-dependent manner with 6-MC treatment. Moreover, Western blot results showed that the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which increased with LPS treatment, were decreased by 6-MC treatment. Mechanistic studies revealed that 6-MC reduced the phosphorylation of the mitogen-activated protein kinase (MAPK) family and IκBα in the MAPK and nuclear factor-kappa B (NF-κB) pathways, respectively. These results suggest that 6-MC is a potential therapeutic agent for inflammatory diseases that inhibits inflammation via the MAPK and NF-κB pathways.     

Isoorientin Inhibits Amyloid β25–35-Induced Neuronal Inflammation in BV2 Cells by Blocking the NF-κB Signaling Pathway

Alzheimer’s disease (AD) is a severe neurodegenerative disorder. AD is pathologically characterized by the formation of intracellular neurofibrillary tangles, and extracellular amyloid plaques which were comprised of amyloid-beta (Aβ) peptides. Aβ induces neurodegeneration by activating microglia, which triggers neurotoxicity by releasing various inflammatory mediators and reactive oxygen species (ROS). Nuclear factor-kappa B (NF-κB) is expressed in human tissues including the brain and plays an important role in Aβ-mediated neuronal inflammation. Thus, the identification of molecules that inhibit the NF-κB pathway is considered an attractive strategy for the treatment and prevention of AD. Isoorientin (3′,4′,5,7-Tetrahydroxy-6-C-glucopyranosyl flavone; ISO), which can be extracted from several plant species, such as Philostachys and Patrinia is known to have various pharmacological activities such as anticancer, antioxidant, and antibacterial activity. However, the effect of ISO on Aβ-mediated inflammation and apoptosis in the brain has yet to be elucidated. In the present study, we investigated whether ISO regulated Aβ-induced neuroinflammation in microglial cells and further explored the underlying mechanisms. Our results showed that ISO inhibited the expression of iNOS and COX-2 induced by Aβ_25–35. And, it inhibited the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, ISO reduced the ROS production in Aβ_25–35-induced BV2 cells and inhibited NF-κB activation. Furthermore, ISO blocked Aβ_25–35-induced apoptosis of BV2 cells. Based on these findings, we suggest that ISO represents a promising therapeutic drug candidate for the treatment and prevention of AD.     

Ginsenoside Rb1 Mitigates Escherichia coli Lipopolysaccharide-Induced Endometritis through TLR4-Mediated NF-κB Pathway

Endometritis is the inflammatory response of the endometrial lining of the uterus and is associated with low conception rates, early embryonic mortality, and prolonged inter-calving intervals, and thus poses huge economic losses to the dairy industry worldwide. Ginsenoside Rb1 (GnRb1) is a natural compound obtained from the roots of Panax ginseng, having several pharmacological and biological properties. However, the anti-inflammatory properties of GnRb1 in lipopolysaccharide (LPS)-challenged endometritis through the TLR4-mediated NF-κB signaling pathway has not yet been researched. This study was planned to evaluate the mechanisms of how GnRb1 rescues LPS-induced endometritis. In the present research, histopathological findings revealed that GnRb1 ameliorated LPS-triggered uterine injury. The ELISA and RT-qPCR assay findings indicated that GnRb1 suppressed the expression level of pro-inflammatory molecules (TNF-α, IL-1β and IL-6) and boosted the level of anti-inflammatory (IL-10) cytokine. Furthermore, the molecular study suggested that GnRb1 attenuated TLR4-mediated NF-κB signaling. The results demonstrated the therapeutic efficacy of GnRb1 in the mouse model of LPS-triggered endometritis via the inhibition of the TLR4-associated NF-κB pathway. Taken together, this study provides a baseline for the protective effect of GnRb1 to treat endometritis in both humans and animals.     

Acetylharpagide Protects Mice from Staphylococcus Aureus-Induced Acute Lung Injury by Inhibiting NF-κB Signaling Pathway

Staphylococcus aureus (S. aureus)-induced acute lung injury (ALI) is a serious disease that has a high risk of death among infants and teenagers. Acetylharpagide, a natural compound of Ajuga decumbens Thunb. (family Labiatae), has been found to have anti-tumor, anti-inflammatory and anti-viral effects. This study investigates the therapeutic effects of acetylharpagide on S. aureus-induced ALI in mice. Here, we found that acetylharpagide alleviated S. aureus-induced lung pathological morphology damage, protected the pulmonary blood-gas barrier and improved the survival of S. aureus-infected mice. Furthermore, S. aureus-induced myeloperoxidase (MPO) activity of lung homogenate and pro-inflammatory factors in bronchoalveolar lavage (BAL) fluid were suppressed by acetylharpagide. Mechanically, acetylharpagide inhibited the interaction between polyubiquitinated receptor interacting protein 1 (RIP1) and NF-κB essential modulator (NEMO), thereby suppressing NF-κB activity. In summary, these results show that acetylharpagide protects mice from S. aureus-induced ALI by suppressing the NF-κB signaling pathway. Acetylharpagide is expected to become a potential treatment for S. aureus-induced ALI.     

β-Myrcene Mitigates Colon Inflammation by Inhibiting MAP Kinase and NF-κB Signaling Pathways

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders that include Crohn’s disease (CD) and ulcerative colitis (UC). The incidence of IBD is rising globally. However, the etiology of IBD is complex and governed by multiple factors. The current clinical treatment for IBD mainly includes steroids, biological agents and need-based surgery, based on the severity of the disease. Current drug therapy is often associated with adverse effects, which limits its use. Therefore, it necessitates the search for new drug candidates. In this pursuit, phytochemicals take the lead in the search for drug candidates to benefit from IBD treatment. β-myrcene is a natural phytochemical compound present in various plant species which possesses potent anti-inflammatory activity. Here we investigated the role of β-myrcene on colon inflammation to explore its molecular targets. We used 2% DSS colitis and TNF-α challenged HT-29 adenocarcinoma cells as in vivo and in vitro models. Our result indicated that the administration of β-myrcene in dextran sodium sulfate (DSS)-treated mice restored colon length, decreased disease activity index (DAI), myeloperoxidase (MPO) enzyme activity and suppressed proinflammatory mediators. β-myrcene administration suppressed mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) pathways to limit inflammation. β-myrcene also suppressed mRNA expression of proinflammatory chemokines in tumor necrosis factor-α (TNF-α) challenged HT-29 adenocarcinoma cells. In conclusion, β-myrcene administration suppresses colon inflammation by inhibiting MAP kinases and NF-κB pathways.     

Polyphenols from the Peels of Punica granatum L. and Their Bioactivity of Suppressing Lipopolysaccharide-Stimulated Inflammatory Cytokines and Mediators in RAW 264.7 Cells via Activating p38 MAPK and NF-κB Signaling Pathways

Punica granatum L. (Punicaceae) is a popular fruit all over the world. Owning to its enriched polyphenols, P. granatum has been widely used in treating inflammation-related diseases, such as cardiovascular diseases and cancer. Twenty polyphenols, containing nine unreported ones, named punicagranins A–I (1–9), along with eleven known isolates (10–20), were obtained from the peels. Their detailed structures were elucidated based on UV, IR, NMR, MS, optical rotation, ECD analyses and chemical evidence. The potential anti-inflammatory activities of all polyphenols were examined on a lipopolysaccharide (LPS)-induced inflammatory macrophages model, which indicated that enhancing nitric oxide (NO) production in response to inflammation stimulated in RAW 264.7 cells was controlled by compounds 1, 3, 5–8, 10, 11, 14 and 16–20 in a concentration-dependent manner. The investigation of structure–activity relationships for tannins 6–8 and 12–20 suggested that HHDP, flavogallonyl and/or gallagyl were key groups for NO production inhibitory activity. Western blotting indicated that compounds 6–8 could down-regulate the phosphorylation levels of proteins p38 MAPK, IKKα/β, IκBα and NF-κB p65 as well as inhibit the levels of inflammation-related cytokines and mediators, such as IL-6, TNF-α, iNOS and COX-2, at the concentration of 30 μM. In conclusion, polyphenols are proposed to be the potential anti-inflammatory active ingredients in P. granatum peels, and their molecular mechanism is likely related to the regulation of the p38 MAPK and NF-κB signaling pathways.     

Pseudognaphalium affine Extract Alleviates COPD by Inhibiting the Inflammatory Response via Downregulation of NF-κB

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease with limited therapeutic options. Pseudognaphalium affine (D. Don) Anderb. is a medicinal and edible plant used to treat cough, asthma, and COPD for a long time in folk medicine. The objective of this study is to evaluate the effect of Pseudognaphalium affine (D. Don) Anderb. extract (GAE) and investigate the possible underlying mechanism in vivo and in vitro. In vivo, the administration of GAE in a rat COPD model could significantly ameliorate lung damage and pulmonary function by inhibiting the production of pro-inflammatory cytokines. Western blot and real-time PCR results showed that GAE could suppress nuclear translocation of nuclear factor-kappa B (NF-κB), which indicated that GAE down-regulated the NF-κB pathway. Moreover, GAE protected against tumor necrosis factor (TNF)-α induced inflammatory response in BEAS-2B and inhibited the NF-κB pathway. All data suggested that GAE exhibited its anti-COPD effect by inhibiting pro-inflammatory cytokines, which may be associated with the inhibition of the NF-κB pathway.     

Diterpenoid Compounds Isolated from Chloranthus oldhamii Solms Exert Anti-Inflammatory Effects by Inhibiting the IKK/NF-κB Pathway

Chloranthus oldhamii Solms (CO) is a folk medicine for treating infection and arthritis pain but its pharmacological activity and bioactive compounds remain mostly uncharacterized. In this study, the anti-inflammatory compounds of C. oldhamii were identified using an LPS-stimulated, NF-κB-responsive RAW 264.7 macrophage reporter line. Three diterpenoid compounds, 3α-hydroxy-ent-abieta-8,11,13-triene (CO-9), 3α, 7β-dihydroxy-ent-abieta-8,11,13-triene (CO-10), and decandrin B (CO-15) were found to inhibit NF-κB activity at nontoxic concentrations. Moreover, CO-9 and CO-10 suppressed the expression of IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells. The inhibitory effect of CO-9 on TNF-α and IL-6 expression was further demonstrated using LPS-treated bone marrow-derived macrophages. Furthermore, CO-9, CO-10, and CO-15 suppressed LPS-triggered COX-2 expression and downstream PGE2 production in RAW 264.7 cells. CO-9 and CO-10 also reduced LPS-triggered iNOS expression and nitrogen oxide production in RAW 264.7 cells. The anti-inflammatory mechanism of the most effective compound, CO-9, was further investigated. CO-9 attenuated LPS-induced NF-κB activation by reducing the phosphorylation of IKKα/β (Ser176/180), IκBα (Ser32), and p65 (Ser534). Conversely, CO-9 did not affect the LPS-induced activation of MAPK signaling pathways. In summary, this study revealed new anti-inflammatory diterpenoid compounds from C. oldhamii and demonstrated that the IKK-mediated NK-κB pathway is the major target of these compounds.     

Crataegus laevigata Suppresses LPS-Induced Oxidative Stress during Inflammatory Response in Human Keratinocytes by Regulating the MAPKs/AP-1, NFκB,and NFAT Signaling Pathways

Crataegus laevigata belongs to the family Rosaceae, which has been widely investigated for pharmacological effects on the circulatory and digestive systems. However, there is limited understanding about its anti-oxidative stress and anti-inflammatory effects on skin. In this study, 70% ethanol C. laevigata berry extract (CLE) was investigated on lipopolysaccharide (LPS)-stimulated keratinocytes. The LPS-induced overproduction of reactive oxygen species (ROS) was suppressed by the treatment with CLE. In response to ROS induction, the overexpression of inflammatory regulating signaling molecules including mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1), nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB), and nuclear factor of activated T-cells (NFAT) were reduced in CLE-treated human keratinocytes. Consequently, CLE significantly suppressed the mRNA levels of pro-inflammatory chemokines and interleukins in LPS-stimulated cells. Our results indicated that CLE has protective effects against LPS-induced injury in an in vitro model and is a potential alternative agent for inflammatory treatment.     

Identification of Potential Anti-Neuroinflammatory Inhibitors from Antarctic Fungal Strain Aspergillus sp. SF-7402 via Regulating the NF-κB Signaling Pathway in Microglia

Microglia play a significant role in immune defense and tissue repair in the central nervous system (CNS). Microglial activation and the resulting neuroinflammation play a key role in the pathogenesis of neurodegenerative disorders. Recently, inflammation reduction strategies in neurodegenerative diseases have attracted increasing attention. Herein, we discovered and evaluated the anti-neuroinflammatory potential of compounds from the Antarctic fungi strain Aspergillus sp. SF-7402 in lipopolysaccharide (LPS)-stimulated BV2 cells. Four metabolites were isolated from the fungi through chemical investigations, namely, 5-methoxysterigmatocystin (1), sterigmatocystin (2), aversin (3), and 6,8-O-dimethylversicolorin A (4). Their chemical structures were elucidated by extensive spectroscopic analysis and HR-ESI-MS, as well as by comparison with those reported in literature. Anti-neuroinflammatory effects of the isolated metabolites were evaluated by measuring the production of nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in LPS-activated microglia at non-cytotoxic concentrations. Sterigmatocystins (1 and 2) displayed significant effects on NO production and mild effects on TNF-α and IL-6 expression inhibition. The molecular mechanisms underlying this activity were investigated using Western blot analysis. Sterigmatocystin treatment inhibited NO production via downregulation of inducible nitric oxide synthase (iNOS) expression in LPS-stimulated BV2 cells. Additionally, sterigmatocystins reduced nuclear translocation of NF-κB. These results suggest that sterigmatocystins present in the fungal strain Aspergillus sp. are promising candidates for the treatment of neuroinflammatory diseases.