Rosmanol and Carnosol Synergistically Alleviate Rheumatoid Arthritis through Inhibiting TLR4/NF-κB/MAPK Pathway

Callicarpalongissima has been used as a Yao folk medicine to treat arthritis for years in China, although its active anti-arthritic moieties have not been clarified so far. In this study, two natural phenolic diterpenoids with anti-rheumatoid arthritis (RA) effects, rosmanol and carnosol, isolated from the medicinal plant were reported on for the first time. In type II collagen-induced arthritis DBA/1 mice, both rosmanol (40 mg/kg/d) and carnosol (40 mg/kg/d) alone alleviated the RA symptoms, such as swelling, redness, and synovitis; decreased the arthritis index score; and downregulated the serum pro-inflammatory cytokine levels of interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor α (TNF-α). Additionally, they blocked the activation of the Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways. Of particular interest was that when they were used in combination (20 mg/kg/d each), the anti-RA effect and inhibitory activity on the TLR4/NF-κB/MAPK pathway were significantly enhanced. The results demonstrated that rosmanol and carnosol synergistically alleviated RA by inhibiting inflammation through regulating the TLR4/NF-κB/MAPK pathway, meaning they have the potential to be developed into novel, safe natural combinations for the treatment of RA.     

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.     

Caragana rosea Turcz Methanol Extract Inhibits Lipopolysaccharide-Induced Inflammatory Responses by Suppressing the TLR4/NF-κB/IRF3 Signaling Pathways

Caragana rosea Turcz, which belongs to the Leguminosae family, is a small shrub found in Northern and Eastern China that is known to possess anti-inflammatory properties and is used to treat fever, asthma, and cough. However, the underlying molecular mechanisms of its anti-inflammatory effects are unknown. Therefore, we used lipopolysaccharide (LPS) in RAW264.7 macrophages to investigate the molecular mechanisms that underlie the anti-inflammatory activities of a methanol extract of Caragana rosea (Cr-ME). We showed that Cr-ME reduced the production of nitric oxide (NO) and mRNA levels of iNOS, TNF-α, and IL-6 in a concentration-dependent manner. We also found that Cr-ME blocked MyD88- and TBK1-induced NF-κB and IRF3 promoter activity, suggesting that it affects multiple targets. Moreover, Cr-ME reduced the phosphorylation levels of IκBα, IKKα/β and IRF3 in a time-dependent manner and regulated the upstream NF-κB proteins Syk and Src, and the IRF3 protein TBK1. Upon overexpression of Src and TBK1, Cr-ME stimulation attenuated the phosphorylation of the NF-κB subunits p50 and p65 and IRF3 signaling. Together, our results suggest that the anti-inflammatory activity of Cr-ME occurs by inhibiting the NF-κB and IRF3 signaling pathways.     

Polyphenols isolated from Broussonetia kazinoki prevent cytokine-induced β-cell damage and the development of type 1 diabetes

The axis of nuclear factor κB (NF-κB)-inducible NO synthase (iNOS)-nitric oxide plays a key role in cytokine- and streptozotocin-mediated pancreatic β-cell damage. In this study, we investigated the effects of kazinol C and isokazinol D isolated from Broussonetia kazinoki on the β-cell viability and function. RINm5F cells and primary islets were used for in vitro and ex vivo cytokine toxicity experiments, respectively. For type 1 diabetes induction, mice were injected with multiple low-dose streptozotocin (MLDS). Cytokine-induced toxicity was completely abolished in both RINm5F cells and islets that were pretreated with either kazinol C or isokazinol D. Both kazinols inhibited the NF-κB signaling pathway, thereby inhibiting cytokine-mediated iNOS induction, nitric oxide production, apoptotic cell death and defects in insulin secretion. Moreover, the occurrence of diabetes in MLDS-treated mice was efficiently attenuated in kazinol-pretreated mice. Immunohistochemical analysis revealed that the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive apoptotic cells and nuclear p65-positive cells were significantly decreased in kazinol-pretreated mice. Our results suggest that kazinol C and isokazinol D block the NF-κB pathway, thus reducing the extent of β-cell damage. Therefore, kazinol C and isokazinol D may have therapeutic value in delaying pancreatic β-cell damage in type 1 diabetes.     

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.     

Berberine,A Phytoalkaloid,Inhibits Inflammatory Response Induced by LPS through NF-Kappaβ Pathway: Possible Involvement of the IKKα

Berberine (BBR), a plant alkaloid, is known for its therapeutic properties of anticancer, cardioprotective, antidiabetic, hypolipidemic, neuroprotective, and hepatoprotective activities. The present study was to determine the molecular mechanism of BBR’s pharmacological activity in human monocytic (THP-1) cells induced by arachidonic acid (AA) or lipopolysaccharide (LPS). The effect of BBR on AA/LPS activated proinflammatory markers including TNF-α, MCP-1, IL-8 and COX-2 was measured by ELISA or quantitative real-time PCR. Furthermore, the effect of BBR on LPS-induced NF-κB translocation was determined by immunoblotting and confocal microscopy. AA/ LPS-induced TNF-α, MCP-1, IL-6, IL-8, and COX-2 markers were markedly attenuated by BBR treatment in THP-1 cells by inhibiting NF-κB translocation into the nucleus. Molecular modeling studies suggested the direct interaction of BBR to IKKα at its ligand binding site, which led to the inhibition of the LPS-induced NF-κB translocation to the nucleus. Thus, the present study demonstrated the anti-inflammatory potential of BBR via NF-κB in activated monocytes, whose interplay is key in health and in the pathophysiology of atherosclerotic development in blood vessel walls. The present study findings suggest that BBR has the potential for treating various chronic inflammatory disorders.     

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.     

Anthocyanins Derived from Vitis coignetiae Pulliat Contributes Anti-Cancer Effects by Suppressing NF-κB Pathways in Hep3B Human Hepatocellular Carcinoma Cells and In Vivo

We previously demonstrated that anthocyanins from the fruits of Vitis coignetiae Pulliat (AIMs) induced the apoptosis of hepatocellular carcinoma cells. However, many researchers argued that the concentrations of AIMs were too high for in vivo experiments. Therefore, we performed in vitro at lower concentrations and in vivo experiments for the anti-cancer effects of AIMs. AIMs inhibited the cell proliferation of Hep3B cells in a dose-dependent manner with a maximum concentration of 100 µg/mL. AIMs also inhibited the invasion and migration at 100 µg/mL concentration with or without the presence of TNF-α. To establish the relevance between the in vitro and in vivo results, we validated their effects in a Xenograft model of Hep3B human hepatocellular carcinoma cells. In the in vivo test, AIMs inhibited the tumorigenicity of Hep3B cells in the xenograft mouse model without showing any clinical signs of toxicity or any changes in the body weight of mice. AIMs inhibited the activation NF-κB and suppressed the NF-κB-regulated proteins, intra-tumoral microvessel density (IMVD) and the Ki67 activity of Hep3B xenograft tumors in athymic nude mice. In conclusion, this study indicates that AIMs have anti-cancer effects (inhibition of proliferation, invasion, and angiogenesis) on human hepatocellular carcinoma xenograft through the inhibition of NF-κB and its target protein.     

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.     

Decreased Human Platelet Activation and Mouse Pulmonary Thrombosis by Rutaecarpine and Comparison of the Relative Effectiveness with BAY11-7082: Crucial Signals of p38-NF-κB

Platelets play a critical role in arterial thrombosis. Rutaecarpine (RUT) was purified from Tetradium ruticarpum, a well-known Chinese medicine. This study examined the relative activity of RUT with NF-κB inhibitors in human platelets. BAY11-7082 (an inhibitor of IκB kinase [IKK]), Ro106-9920 (an inhibitor of proteasomes), and RUT concentration-dependently (1–6 μM) inhibited platelet aggregation and P-selectin expression. RUT was found to have a similar effect to that of BAY11-7082; however, it exhibits more effectiveness than Ro106-9920. RUT suppresses the NF-κB pathway as it inhibits IKK, IκBα, and p65 phosphorylation and reverses IκBα degradation in activated platelets. This study also investigated the role of p38 and NF-κB in cell signaling events and found that SB203580 (an inhibitor of p38) markedly reduced p38, IKK, and p65 phosphorylation and reversed IκBα degradation as well as p65 activation in a confocal microscope, whereas BAY11-7082 had no effects in p38 phosphorylation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay shows that RUT and BAY11-7082 did not exhibit free radical scavenging activity. In the in vivo study, compared with BAY11-7082, RUT more effectively reduced mortality in adenosine diphosphate (ADP)-induced acute pulmonary thromboembolism without affecting the bleeding time. In conclusion, a distinctive pathway of p38-mediated NF-κB activation may involve RUT-mediated antiplatelet activation, and RUT could act as a strong prophylactic or therapeutic drug for cardiovascular diseases.     

The Wnt/β-catenin signaling pathway regulates the development of airway remodeling in patients with asthma

Airway remodeling is a key characteristic of chronic asthma, particularly in patients with a fixed airflow limitation. The mechanisms underlying airway remodeling are poorly understood, and no therapeutic option is available. The Wnt/β-catenin signaling pathway is involved in various physiological and pathological processes, including fibrosis and smooth muscle hypertrophy. In this study, we investigated the roles of Wnt/β-catenin signaling in airway remodeling in patients with asthma. Wnt7a mRNA expression was prominent in induced sputum from patients with asthma compared with that from healthy controls. Next, we induced a chronic asthma mouse model with airway remodeling features, including subepithelial fibrosis and airway smooth muscle hyperplasia. Higher expression of Wnt family proteins and β-catenin was detected in the lung tissue of mice with chronic asthma compared to control mice. Blocking β-catenin expression with a specific siRNA attenuated airway inflammation and airway remodeling. Decreased subepithelial fibrosis and collagen accumulation in the β-catenin siRNA-treated mice was accompanied by reduced expression of transforming growth factor-β. We further showed that suppressing β-catenin in the chronic asthma model inhibited smooth muscle hyperplasia by downregulating the tenascin C/platelet-derived growth factor receptor pathway. Taken together, these findings demonstrate that the Wnt/β-catenin signaling pathway is highly expressed and regulates the development of airway remodeling in chronic asthma.     

Design and Synthesis of New Benzo[d]oxazole-Based Derivatives and Their Neuroprotective Effects on β-Amyloid-Induced PC12 Cells

A series of novel synthetic substituted benzo[d]oxazole-based derivatives (5a–5v) exerted neuroprotective effects on β-amyloid (Aβ)-induced PC12 cells as a potential approach for the treatment of Alzheimer’s disease (AD). In vitro studies show that most of the synthesized compounds were potent in reducing the neurotoxicity of Aβ_25-35-induced PC12 cells at 5 μg/mL. We found that compound 5c was non-neurotoxic at 30 μg/mL and significantly increased the viability of Aβ_25-35-induced PC12 cells at 1.25, 2.5 and 5 μg/mL. Western blot analysis showed that compound 5c promoted the phosphorylation of Akt and glycogen synthase kinase (GSK-3β) and decreased the expression of nuclear factor-κB (NF-κB) in Aβ_25-35-induced PC12 cells. In addition, our findings demonstrated that compound 5c protected PC12 cells from Aβ_25-35-induced apoptosis and reduced the hyperphosphorylation of tau protein, and decreased the expression of receptor for AGE (RAGE), β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), inducible nitric oxide synthase (iNOS) and Bcl-2-associated X protein/B-cell lymphoma 2 (Bax/Bcl-2) via Akt/GSK-3β/NF-κB signaling pathway. In vivo studies suggest that compound 5c shows less toxicity than donepezil in the heart and nervous system of zebrafish.     

Topical Nano Clove/Thyme Gel against Genetically Identified Clinical Skin Isolates: In Vivo Targeting Behavioral Alteration and IGF-1/pFOXO-1/PPAR γ Cues

Antimicrobial resistance is a dramatic global threat; however, the slow progress of new antibiotic development has impeded the identification of viable alternative strategies. Natural antioxidant-based antibacterial approaches may provide potent therapeutic abilities to effectively block resistance microbes’ pathways. While essential oils (EOs) have been reported as antimicrobial agents, its application is still limited ascribed to its low solubility and stability characters; additionally, the related biomolecular mechanisms are not fully understood. Hence, the study aimed to develop a nano-gel natural preparation with multiple molecular mechanisms that could combat bacterial resistance in an acne vulgaris model. A nano-emulgel of thyme/clove EOs (NEG8) was designed, standardized, and its antimicrobial activity was screened in vitro and in vivo against genetically identified skin bacterial clinical isolates (Pseudomonas stutzeri, Enterococcus faecium and Bacillus thuringiensis). As per our findings, NEG8 exhibited bacteriostatic and potent biofilm inhibition activities. An in vivo model was also established using the commercially available therapeutic, adapalene in contra genetically identified microorganism. Improvement in rat behavior was reported for the first time and NEG8 abated the dermal contents/protein expression of IGF-1, TGF-β/collagen, Wnt/β-catenin, JAK2/STAT-3, NE, 5-HT, and the inflammatory markers; p(Ser536) NF-κBp65, TLR-2, and IL-6. Moreover, the level of dopamine, protective anti-inflammatory cytokine, IL-10 and PPAR-γ protein were enhanced, also the skin histological structures were improved. Thus, NEG8 could be a future potential topical clinical alternate to synthetic agents, with dual merit mechanism as bacteriostatic antibiotic action and non-antibiotic microbial pathway inhibitor.     

β-Sitosterol Circumvents Obesity Induced Inflammation and Insulin Resistance by down-Regulating IKKβ/NF-κB and JNK Signaling Pathway in Adipocytes of Type 2 Diabetic Rats

β-sitosterol (SIT), the most abundant bioactive component of vegetable oil and other plants, is a highly potent antidiabetic drug. Our previous studies show that SIT controls hyperglycemia and insulin resistance by activating insulin receptor and glucose transporter 4 (GLUT-4) in the adipocytes of obesity induced type 2 diabetic rats. The current research was undertaken to investigate if SIT could also exert its antidiabetic effects by circumventing adipocyte induced inflammation, a key driving factor for insulin resistance in obese individuals. Effective dose of SIT (20 mg/kg b.wt) was administered orally for 30 days to high fat diet and sucrose induced type-2 diabetic rats. Metformin, the conventionally used antidiabetic drug was used as a positive control. Interestingly, SIT treatment restores the elevated serum levels of proinflammatory cytokines including leptin, resistin, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to normalcy and increases anti-inflammatory adipocytokines including adiponectin in type 2 diabetic rats. Furthermore, SIT decreases sterol regulatory element binding protein-1c (SREBP-1c) and enhances Peroxisome Proliferator–activated receptor-γ (PPAR-γ) gene expression in adipocytes of diabetic rats. The gene and protein expression of c-Jun-N-terminal kinase-1 (JNK1), inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) and nuclear factor kappa B (NF-κB) were also significantly attenuated in SIT treated groups. More importantly, SIT acts very effectively as metformin to circumvent inflammation and insulin resistance in diabetic rats. Our results clearly show that SIT inhibits obesity induced insulin resistance by ameliorating the inflammatory events in the adipose tissue through the downregulation of IKKβ/NF-κB and c-Jun-N-terminal kinase (JNK) signaling pathway.     

Gomisin M2 Ameliorates Atopic Dermatitis-like Skin Lesions via Inhibition of STAT1 and NF-κB Activation in 2,4-Dinitrochlorobenzene/Dermatophagoides farinae Extract-Induced BALB/c Mice

The extracts of Schisandra chinensis (Turcz.) Baill. (Schisandraceae) have various therapeutic effects, including inflammation and allergy. In this study, gomisin M2 (GM2) was isolated from S. chinensis and its beneficial effects were assessed against atopic dermatitis (AD). We evaluated the therapeutic effects of GM2 on 2,4-dinitrochlorobenzene (DNCB) and Dermatophagoides farinae extract (DFE)-induced AD-like skin lesions with BALB/c mice ears and within the tumor necrosis factor (TNF)-α and interferon (IFN)-γ-stimulated keratinocytes. The oral administration of GM2 resulted in reduced epidermal and dermal thickness, infiltration of tissue eosinophils, mast cells, and helper T cells in AD-like lesions. GM2 suppressed the expression of IL-1β, IL-4, IL-5, IL-6, IL-12a, and TSLP in ear tissue and the expression of IFN-γ, IL-4, and IL-17A in auricular lymph nodes. GM2 also inhibited STAT1 and NF-κB phosphorylation in DNCB/DFE-induced AD-like lesions. The oral administration of GM2 reduced levels of IgE (DFE-specific and total) and IgG2a in the mice sera, as well as protein levels of IL-4, IL-6, and TSLP in ear tissues. In TNF-α/IFN-γ-stimulated keratinocytes, GM2 significantly inhibited IL-1β, IL-6, CXCL8, and CCL22 through the suppression of STAT1 phosphorylation and the nuclear translocation of NF-κB. Taken together, these results indicate that GM2 is a biologically active compound that exhibits inhibitory effects on skin inflammation and suggests that GM2 might serve as a remedy in inflammatory skin diseases, specifically on AD.