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BACKGROUND Sepsis is a major medical challenge. Magnolol is an active constituent of Houpu that improves tissue function and exerts strong anti-endotoxin and anti-inflammatory effects, but the mechanism by which it reduces intestinal inflammation in sepsis is yet unclear. AIM To assess the protective effect of magnolol on intestinal mucosal epithelial cells in sepsis and elucidate the underlying mechanisms. METHODS Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and regulated on activation, normal T-cell expressed and secreted (RANTES) levels in serum and ileal tissue in animal studies. The histopathological changes of the ileal mucosa in different groups were observed under a microscope. Cell Counting Kit-8 and cell permeability assays were used to determine the concentration of drug-containing serum that did not affect the activity of Caco2 cells but inhibited lipopolysaccharide (LPS)-induced decrease in permeability. Immunofluorescence and Western blot assays were used to detect the levels of RANTES, inhibitor of nuclear factor kappa-B kinase β (IKKβ), phosphorylated IKKβ (p-IKKβ), inhibitor of nuclear factor kappa-B kinase α (IκBα), p65, and p-p65 proteins in different groups in vitro. RESULTS In rats treated with LPS by intravenous tail injection in the presence or absence of magnolol, magnolol inhibited the expression of proinflammatory cytokines, IL-1β, IL-6, and TNF-α in a dose-dependent manner. In addition, magnolol suppressed the production of RANTES in LPS-stimulated sepsis rats. Moreover, in vitro studies suggested that magnolol inhibited the increase of p65 nucleation, thereby markedly downregulating the production of the phosphorylated form of IKKβ in LPS-treated Caco2 cells. Specifically, magnolol inhibited the translocation of the transcription factor nuclear factor-kappa B (NF-κB) from the cytosol into the nucleus and down-regulated the expression level of the chemokine RANTES in LPS-stimulated Caco2 cells. CONCLUSION Magnolol down-regulates RANTES levels by inhibiting the LPS/NF-κB signaling pathways, thereby suppressing IL-1β, IL-6, and TNF-α expression to alleviate the mucosal barrier dysfunction in sepsis.

BACKGROUND Acute lung injury (ALI) is a common and life-threatening complication of severe acute pancreatitis (SAP). There are currently limited effective treatment options for SAP and associated ALI. Calycosin (Cal), a bioactive constituent extracted from the medicinal herb Radix Astragali exhibits potent anti-inflammatory properties, but its effect on SAP and associated ALI has yet to be determined. AIM To identify the roles of Cal in SAP-ALI and the underlying mechanism. METHODS SAP was induced via two intraperitoneal injections of L-arg (4 g/kg) and Cal (25 or 50 mg/kg) were injected 1 h prior to the first L-arg challenge. Mice were sacrificed 72 h after the induction of SAP and associated ALI was examined histologically and biochemically. An in vitro model of lipopolysaccharide (LPS)-induced ALI was established using A549 cells. Immunofluorescence analysis and western blot were evaluated in cells. Molecular docking analyses were conducted to examine the interaction of Cal with HMGB1. RESULTS Cal treatment substantially reduced the serum amylase levels and alleviated histopathological injury associated with SAP and ALI. Neutrophil infiltration and lung tissue levels of neutrophil mediator myeloperoxidase were reduced in line with protective effects of Cal against ALI in SAP. Cal treatment also attenuated the serum levels and mRNA expression of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-6, IL-1β, HMGB1 and chemokine (CXC motif) ligand 1 in lung tissue. Immunofluorescence and western blot analyses showed that Cal treatment markedly suppressed the expression of HMGB1 and phosphorylated nuclear factor-kappa B (NF-κB) p65 in lung tissues and an in vitro model of LPS-induced ALI in A549 cells suggesting a role for HGMB1 in the pathogenesis of ALI. Furthermore, molecular docking analysis provided evidence for the direct interaction of Cal with HGMB1. CONCLUSION Cal protects mice against L-arg-induced SAP and associated ALI by attenuating local and systemic neutrophil infiltration and inflammatory response via inhibition of HGMB1 and the NF-κB signaling pathway.

Objectives We investigated, the effects of aprepitant (APRE) on the lung tissues of rats with an experimental polymicrobial sepsis model (CLP: cecal ligation and puncture) biochemically, molecularly and histopathologically. Methods A total of 40 rats were divided into 5 groups with 8 animals in each group. Group 1 (SHAM), control group; Group 2 (CLP), cecal ligation and puncture; Group 3 (CLP + APRE10), rats were administered CLP + 10 mg/kg aprepitant; Group 4 (CLP + APRE20), rats were administered CLP + 20 mg/kg aprepitant; and Group 5 (CLP + APRE40), rats were administered CLP + 40 mg/kg aprepitant. A polymicrobial sepsis model was induced with CLP. After 16 h, lung tissues were taken for examination. Tumour necrosis factor α (TNF-α) and nuclear factor-kappa b (NFK-b) messenger ribonucleic acid (mRNA) expressions were analysed by real-time PCR (RT-PCR), biochemically antioxidant parameters such as superoxide dismutase (SOD) and glutathione (GSH) and oxidant parameters such as malondialdehyde (MDA) and lung damage histopathologically. Key findings and conclusions The GSH level and SOD activity increased while the MDA level and the expressions of TNF-α and NFK-b were reduced in the groups treated with APRE, especially in the CLP + APRE40 group. The histopathology results supported the molecular and biochemical results.

Objective: We designed an experimental model of sepsis in rats to investigate the effects of agomelatine (AGO) on lung tissues using molecular and histopathological methods. Materials and Methods: In our experimental model, the 32 rats were divided into 4 groups: group 1: control group (HEALTHY); group 2: lipopolysaccharide group (LPS); group 3: LPS plus 50 mg/kg AGO group (LPS + AGO50); and group 4: LPS plus 100 mg/kg AGO group (LPS + AGO100). An LPS-induced sepsis model was performed to replicate the pathology of sepsis. Rats from all 4 groups were killed after 12 hours, and their lungs were quickly collected. To investigate the therapeutic strategy, we evaluated tumor necrosis factoralpha (TNF-alpha) and nuclear factor-kappa B (NF-kappa B) messenger RNA expressions by real-time polymerase chain reaction using molecular methods and lung tissue damage indicators using histopathological methods. Results: The expressions of TNF-alpha and NF-kappa B were reduced in the groups treated with AGO. The histopathology results supported the molecular results. Conclusion: In this experimental study, we demonstrated for the first time the positive effects of AGO on LPS-induced sepsis in lung tissue using molecular and histopathological methods, indicating that it contributes to the prevention of lung damage.

Context Physcion (Phy) exerts several pharmacological effects including anti-inflammatory, antioxidant, and antitumor properties. Objective This study investigates the cytotoxicity and its underlying mechanisms of Phy on breast cancer. Materials and methods Human breast cancer cell MCF-7 was treated with 5–400 µM Phy for 24 h, MCF-7-xenografted BALB/c nude mice and immunosuppressive mice model induced by cyclophosphamide were intraperitoneally injected with 0.1 mL/mouse normal saline (control group) and 30 mg/kg Phy every other day for 14 or 28 days, and pathological examination, ELISA and western blot were employed to investigate the Phy anti-breast cancer property in vitro and in vivo. Results In MCF-7 cells, Phy 24 h treatment significantly reduced the cell viability at dose of 50–400 µM and 24 h, with an IC50 of 203.1 µM, and 200 µM Phy induced 56.9, 46.9, 36.9, and 46.9% increment on LDH and caspase-3, −8 and −9. In MCF-7-xenograft tumour nude mice and immunosuppressive mice, 30 mg/kg Phy treatment inhibited tumour growth from the 8th day, and reduced Bcl-2 and Bcl-xL >50%, HO-1 and SOD-1 > 70% in tumour tissues of immunosuppressive mice. In addition, Phy reduced nuclear factor erythroid 2-related factor 2 > 30% and its downstream proteins, and enhanced the phosphorylation of nuclear factor-kappa B > 110% and inhibitor of NF-кB α > 80% in the tumour tissues of BALB/c mice. Discussion and conclusions This research demonstrated that Phy has an anti-breast cancer property via the modulation of oxidative stress-mediated mitochondrial apoptosis and immune response, which provides a scientific basis for further research on its clinical applications.

BACKGROUND Autoimmune hepatitis is a serious autoimmune liver disease that threatens human health worldwide, which emphasizes the urgent need to identify novel treatments. Stem cells from human exfoliated deciduous teeth (SHED), which are easy to obtain in a non-invasive manner, show pronounced proliferative and immunomodulatory capacities. AIM To investigate the protective effects of SHED on concanavalin A (ConA)-induced hepatitis in mice, and to elucidate the associated regulatory mechanisms. METHODS We used a ConA-induced acute hepatitis mouse model and an in vitro co-culture system to study the protective effects of SHED on ConA-induced autoimmune hepatitis, as well as the associated underlying mechanisms. RESULTS SHED infusion could prevent aberrant histopathological liver architecture caused by ConA-induced infiltration of CD3+, CD4+, tumor necrosis-alpha+, and interferon-gamma+ inflammatory cells. Alanine aminotransferase and aspartate aminotransferase were significantly elevated in hepatitis mice. SHED infusion could therefore block ConA-induced alanine aminotransferase and aspartate aminotransferase elevations. Mechanistically, ConA upregulated tumor necrosis-alpha and interferon-gamma expression, which was activated by the nuclear factor-kappa B pathway to induce hepatocyte apoptosis, resulting in acute liver injury. SHED administration protected hepatocytes from ConA-induced apoptosis. CONCLUSION SHED alleviates ConA-induced acute liver injury via inhibition of hepatocyte apoptosis mediated by the nuclear factor-kappa B pathway. Our findings could provide a potential treatment strategy for hepatitis.

Seomae mugwort, a Korean native variety of Artemisia argyi, exhibits physiological effects against various diseases. However, its effects on osteoarthritis (OA) are unclear. In this study, a Seomae mugwort extract prevented cartilage destruction in an OA mouse model. In vitro and ex vivo analyses revealed that the extract suppressed MMP3, MMP13, ADAMTS4 and ADAMTS5 expression induced by IL‐1β, IL‐6 and TNF‐α and inhibited the loss of extracellular sulphated proteoglycans. In vivo analysis revealed that oral administration of the extract suppressed DMM‐induced cartilage destruction. We identified jaceosidin in Seomae mugwort and showed that this compound decreased MMP3, MMP13, ADAMTS4 and ADAMTS5 expression levels, similar to the action of the Seomae mugwort extract in cultured chondrocytes. Interestingly, jaceosidin and eupatilin combined had similar effects to Seomae mugwort in the DMM‐induced OA model. Induction of IκB degradation by IL‐1β was blocked by the extract and jaceosidin, whereas JNK phosphorylation was only suppressed by the extract. These results suggest that the Seomae mugwort extract and jaceosidin can attenuate cartilage destruction by suppressing MMPs, ADAMTS4/5 and the nuclear factor‐κB signalling pathway by blocking IκB degradation. Thus, the findings support the potential application of Seomae mugwort, and particularly jaceosidin, as natural therapeutics for OA.

Aims Inflammatory response plays a pivotal role in the pathophysiological process of intervertebral disc degeneration (IDD). A20 (also known as tumour necrosis factor alpha-induced protein 3 (TNFAIP3)) is a ubiquitin-editing enzyme that restricts nuclear factor-kappa B (NF-κB) signalling. A20 prevents the occurrence of multiple inflammatory diseases. However, the role of A20 in the initiation of IDD has not been elucidated. The aim of the study was to investigate the effect of A20 in senescence of TNF alpha (TNF-α)-induced nucleus pulposus cells (NPCs). Methods Immunohistochemical staining was performed to observe the expression of A20 in normal and degenerated human intervertebral discs. The NPCs were dissected from the tail vertebrae of healthy male Sprague-Dawley rats and were cultured in the incubator. In the experiment, TNF-α was used to mimic the inflammatory environment of IDD. The cell viability and senescence were examined to investigate the effect of A20 on TNF-α-treated NPCs. The expression of messenger RNA (mRNA)-encoding proteins related to matrix macromolecules (collagen II, aggrecan) and senescence markers (p53, p16). Additionally, NF-κB/p65 activity of NPCs was detected within different test compounds. Results The expression of A20 was upregulated in degenerate human intervertebral discs. The A20 levels of NPCs in TNF-α inflammatory microenvironments were dramatically higher than those of the control group. TNF-α significantly decreased cell proliferation potency but increased senescence-associated beta-galactosidase (SA-β-Gal) activity, the expression of senescence-associated proteins, the synthesis of extracellular matrix, and G1 cycle arrest. The senescence indicators and NF-κB/p65 expression of A20 downregulated group treated with TNF-α were significantly upregulated compared to TNF-α-treated normal NPCs. Conclusion A20 has a self-protective effect on the senescence of NPCs induced by TNF-α. The downregulation of A20 in NPCs exacerbated the senescence of NPCs induced by TNF-α. Cite this article: Bone Joint Res. 2020;9(5):225–235.

Background BMS-470539, a recently introduced selective agonist of the melanocortin 1 receptor, is known to have anti-inflammatory properties. In this study, we investigated the effects of BMS-470539 on lipopolysaccharide (LPS)-induced inflammatory responses and delayed apoptosis with its signaling pathways in human neutrophils. Methods Isolated human neutrophils were incubated with various concentrations of BMS-470539 (1, 10, and 100 µM) in the presence or absence of LPS (100 ng/ml), and the expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha, interleukin (IL)-6, and IL-1β, were assessed. The effects of BMS-470539 on the expression of mitogen-activated protein kinases (MAPKs), such as p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase, and the expression of nuclear factor kappa B (NF-κB) in LPS-stimulated human neutrophils, were evaluated by enzyme-linked immunosorbent assay. Neutrophil apoptosis was also measured by fluorescence-activated cell sorting (annexin V/propidium iodide) in LPS-stimulated neutrophils under treatment with BMS-470539. Results BMS-470539 attenuated LPS-induced expression of pro-inflammatory cytokines, and phosphorylation of MAPKs and NF-κB. LPS stimulation reduced neutrophil apoptosis compared to the controls; however, BMS-470539 significantly inhibited the reduction of neutrophil apoptosis. Conclusions BMS-470539 can suppress the inflammatory responses of LPS-stimulated neutrophils by inhibition of MAPK pathways or NF-κB pathway, and it can also inhibit LPS-delayed neutrophil apoptosis.

Background/Aims: The purpose of this study is to analyze the expression and biological function of lncRNA ANRIL, microRNA-199a, TLR4, and nuclear factor-kappa B (NF-κB) in acute renal injury (AKI) induced by lipopolysaccharide (LPS). Methods: The levels of ANRIL and microRNA-199a in mouse cells and kidneys were detected by quantitative-polymerase chain reaction. Western blot analysis was used for the NF-κB pathway protein. MTT assay was used for cell viability. Enzyme-linked immunosorbent assay was used for the secretion of inflammatory factors in mouse kidney tissue. Apoptosis was measured by flow cytometry and Western blotting. The potential binding region between ANRIL and miR-199a was verified by luciferase reporter assay. Results: The upregulation of ANRIL can reduce the expression of microRNA-199a and increases the number of apoptotic cells. The expression levels of ANRIL in LPS-induced AKI mice and LPS-treated HK2 cells were upregulated compared with the control group. Overexpression of ANRIL increased apoptosis and promoted TLR4 (Toll-like receptor 4), NF-κB phosphorylation, and downstream transcription factor production. Conclusion: ANRIL/NF-κB pathway in LPS-induced apoptosis provided theoretical guidance for ANRIL in the treatment of AKI.

We previously reported that dopamine (DA) attenuated lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines through the formation of DA quinone (DAQ) in murine microglial cell line BV-2 and primary murine microglial cells. To reveal whether DA inhibits the expression of proinflammatory cytokines of microglial cells through the formation of DAQ in the central nervous system (CNS), in this study, we examined the effect of DAQ on LPS-induced mRNA expression of proinflammatory cytokines in C57BL/6 mouse brain under two experimental conditions: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration and l -dopa/carbidopa administration. Acute MPTP administration reduced the number of tyrosine hydroxylase-positive cells in the substantia nigra, and decreased the level of quinoprotein, an indicator of DAQ formation, in the striatum. Real-time RT-PCR analysis revealed that intraperitoneal administration of LPS increased the mRNA levels of proinflammatory cytokines, including tumor-necrosis factor-α and interleukin-1β, in the striatum. These increases were enhanced in MPTP-treated mice. On the other hand, l -dopa/carbidopa administration increased the level of quinoprotein, attenuated the LPS-induced mRNA expression of proinflammatory cytokines, and reduced the LPS-induced increase in the number of microglial cells in the striatum. These results suggest that DA attenuate the expression of proinflammatory cytokines in microglia through the formation of DAQ in the CNS.

Yiping Zhang, Yahua Wu, Xufeng Zhou, Benyi Yi, Lili Wang Department of Biochemistry and Molecular Biology, Basic Medical College of Jiujiang University, Jiujiang City, Jiangxi Province 332000, People’s Republic of ChinaCorrespondence: Yiping ZhangDepartment of Biochemistry and Molecular Biology, Basic Medical College of Jiujiang University, No. 320, Xunyang East Road, Xunyang District, Jiujiang City, Jiangxi Province 332000, People’s Republic of ChinaTel +86-13767275087Email zhangyiping109@163.comPurpose: This study aimed to investigate the regulatory roles of estrogen receptor beta (ERβ) on gastric cancer (GC) cells, and reveal the potential mechanisms relating to nuclear factor-kappa B (NF-κB) signaling.Methods: GC cell lines SGC7901 and MKN45 were transfected with pEGFP-C1-ERβ to overexpress ERβ, and treated with PMA (a NF-κB activator) to activate NF-κB signaling. The cell proliferation and migration, as well as the formation of vessel-like structures in human venous endothelial cells (HUVECs) were detected. The expression of ERβ, NF-κB p65, p-NF-κB p65, Ki67 (a proliferation marker), vascular endothelial growth factor A (VEGF-A) and matrix metalloproteinase 2 (MMP-2), the DNA binding activity of NF-κB p65, the content of VEGF-A, and the activity of MMP-2 were detected in SGC7901 and MKN45 cells.Results: The transfection of pEGFP-C1-ERβ significantly increased the expression of ERβ in SGC7901 and MKN45 cells (P < 0.05). Overexpression of ERβ in SGC7901 and MKN45 cells significantly decreased the cell activity, cell number in G2/M phase, cell migration, the expression of Ki67, VEGF-A and MMP-2, VEGF-A content, MMP-2 activity, as well as the number of vessel-like structures formed by HUVECs (P < 0.05). Overexpression of ERβ also significantly decreased the DNA binding activity and the expression of p-NF-κB p65 in SGC7901 and MKN45 cells (P < 0.05). The anti-tumor effect of ERβ overexpression on GC cells was reversed by the intervention of PMA (P < 0.05).Conclusion: Overexpression of ERβ inhibited the proliferation, migration, and angiogenesis of GC cells through inhibiting NF-κB signaling.Keywords: estrogen receptor beta, gastric cancer, nuclear factor-kappa B, angiogenesis, proliferation

β-amyloid (Aβ) is one of the inducing factors of astrocytes activation and neuroinflammation, and it is also a crucial factor for the development of Alzheimer’s disease (AD). Icariside II (ICS II) is an active component isolated from a traditional Chinese herb Epimedium, which has shown to attnuate lipopolysaccharide (LPS)-induced neuroinflammation through regulation of NF-κB signaling pathway. In this study we investigated the effects of ICS II on LPS-induced astrocytes activation and Aβ accumulation. Primary rat astrocytes were pretreated with ICS II (5, 10, and 20 μM) or dexamethasone (DXMS, 1 μM) for 1 h, thereafter, treated with LPS for another 24 h. We found that ICS II pretreatment dose dependently mitigated the levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) in the astrocytes. Moreover, ICS II not only exerted the inhibitory effect on LPS-induced IκB-α degradation and NF-κB activation, but also decreased the levels of Aβ1–40, Aβ1–42, amyloid precursor protein (APP) and beta secretase 1 (BACE1) in the astrocytes. Interestingly, molecular docking revealed that ICS II might directly bind to BACE1. It is concluded that ICS II has potential value as a new therapeutic agent to treat neuroinflammation-related diseases, such as AD.

The long noncoding RNA X-inactive specific transcript (XIST) plays vital roles in tumor progression. However, the underlying mechanisms remain unclear. This study investigated the effects and mechanisms of targeting XIST on osteosarcoma (OS) cells in vitro and in vivo. We used shRNA to knockdown XIST to evaluate cell growth and apoptosis in U2OS cells in vitro and xenograft formation in vivo. An observed relationship between XIST and the p53 upregulated modulator of apoptosis (PUMA) and nuclear factor-kappa B (NF-kB) pathway was further explored by using small interfering RNA (siRNA). Our results showed that suppression of XIST by short hairpin RNA (shRNA) impeded U2OS cell growth, induced apoptosis and lessened OS xenograft tumor growth. Targeting XIST increased NF-kB-dependent PUMA upregulation in U2OS cells. Upregulation of PUMA is correlated with suppression of XIST-induced apoptosis in U2OS cells. Therefore, inhibition of XIST could promote U2OS cell death via activation of NF-kB/PUMA pathways., Graphical Abstract

Acute pancreatitis (AP) is a digestive disease characterized by pancreatic inflammation. Tetramethylpyrazine (TMP) has been effectively used to ameliorate the damage on intestinal mucosa injury in rats with acute necrotizing pancreatitis (ANP). We aim to study the protective effect of TMP on caerulein-induced AP and to explore the possible mechanism. The mice randomized into control and different experimental groups. AP was induced in mice by 6-hourly intraperitoneal (i.p) injections of caerulein (50 μg/kg at 1 h interval). TMP (i.p, 10 mg/kg, 1 h interval) was administered 3 h before caerulein injection. Administration of TMP attenuated the severity of AP as shown by the histopathology, reduced serum amylase activity and pro-inflammatory cytokines TNF-α and IL-6. Further, TMP enhances the beneficial effect by reducing caerulein-induced NF-κB activation and inducing cell apoptosis in pancreas. Therefore, inhibition of nuclear factor-kappa B(NF-κB) signals by TMP represents a potential therapeutic strategy for the treatment of acute pancreatitis., Graphical Abstract

BACKGROUND Budd-Chiari syndrome (BCS) is an uncommon disorder characterized by obstruction of hepatic venous outflow. To date, the exact mechanism underlying hepatic injury derived from the hepatic venous outflow obstruction in BCS remains largely unknown. AIM To assess the role of NF-κB-mediated inflammation in BCS-induced liver injury in humans and rats. METHODS A total of 180 rats were randomly assigned into nine groups, including four BCS model groups (1, 3, 6 and 12 wk), four sham-operated groups (1, 3, 6 and 12 wk), and a control group. Lipopolysaccharide (LPS) levels in each group were detected by the Tachypleus Amebocyte Lysate assay. The mRNA and protein levels of TLR4, NF-κB, tumor necrosis factor (TNF)-α, interleukin (IL)-2 and interferon (IFN)-γ were quantified. In addition, 60 patients with BCS and 30 healthy controls were enrolled, and their blood samples were analyzed. RESULTS Hepatic and plasma LPS levels were significantly increased in rats. The mRNA and protein expression levels of TLR4, NF-κB and inflammatory cytokines (TNF-α, IL-2 and IFN-γ) in liver tissues were significantly higher in the BCS model groups compared with the other two groups. In addition, the model groups (1, 3, 6 and 12 wk after BCS induction) showed significant differences in the levels of LPS, TLR4, NF-κB, TNF-α, IL-2 and IFN-γ. Notably, there was a significant correlation between the LPS concentrations and mRNA and protein levels of TLR4, NF-κB and inflammatory cytokines. Importantly, it was revealed that the levels of LPS, TLR4, NF-κB and inflammatory cytokines were significantly greater in chronic BCS patients than healthy controls and acute BCS patients. CONCLUSION LPS level is markedly elevated in BCS, in turn activating the TLR4/NF-κB signaling pathway, leading to induction of inflammatory cytokines (TNF-α, IL-2 and IFN-γ) in response to BCS-induced liver injury.

Golgi protein 73 (GP73) is upregulated in a variety of liver diseases, yet the detailed mechanism is poorly characterized. We analyzed GP73 in a retrospective cohort including 4211 patients with chronic liver disease (CLD) or hepatocellular carcinoma (HCC). The effect of deoxycholic acid (DCA) and nuclear factor-kappa B (NF-κB) on expression and release of GP73 in Huh-7 and SMMC7721 cells were studied. A mouse study was used to confirm our findings in vivo. A positive correlation was found between serum GP73 and total bile acid (TBA) in cirrhotic patients (r = 0.540, p &lt, 0.001), higher than that in non-cirrhotic CLD (r = 0.318, p &lt, 0.001) and HCC (r = 0.353, p &lt, 0.001) patients. In Huh-7 and SMMC7721 cells, DCA upregulated the expression and release of GP73 in a dose- and time-dependent manner. After overexpressing NF-κB p65, the promoter activity, GP73 messenger RNA (mRNA) level, and supernatant GP73 level were increased. The promotion effect of DCA on GP73 release was attenuated after inhibiting the NF-κB pathway. Mutating the binding sites of NF-κB in the sequence of the GP73 promoter led to a declined promoting effect of DCA on GP73. The upregulation role of DCA in GP73 expression through the NF-κB pathway was confirmed in vivo. In addition, exposure to DCA caused disassembly of Golgi apparatus. In summary, DCA upregulates the expression and release of GP73 via activating the NF-κB pathway and destroying the Golgi structure.

Background and Objectives: Probiotics have been widely used for host immune system enhancement but with limited knowledge regarding the immunomodulation mechanisms by which they assist the mucosal innate immune response. We investigated the effects of probiotics on the modulation of the innate mucosal immune response particularly in association with Toll-like receptor (TLR)-2, TLR-4 and nuclear factor-kappa B (NF-κB) p65 and p105. Materials and Methods: We randomized 24 male BALB/c mice into four groups. Two groups were administered probiotics for 21 consecutive days; one of these groups was challenged with Lipopolysaccharide (LPS) on day 15. The third group was challenged with only LPS. The fourth group remained untreated. All mice were sacrificed after 21 days. An immunohistochemistry procedure on the ileum was performed and monoclonal antibodies specific for TLR-2, TLR-4 and NF-κB p65 and p105 were used for the analysis of innate lymphoid cells. Results: In the LPS-only treated group, there was a significant decrease in p105, indicating an alternative transcription pathway for the process of pro-inflammatory cytokine production. In the probiotics-only treated group there was significant enhancement of TLR-2 and TLR-4 and NF-κB p65 and p105. When mice treated with probiotics were exposed to LPS, there was a significant decrease in NF-κB p65 and p105, indicating employment of the classical pathway for pro-inflammatory cytokine production. Conclusion: Probiotics can enhance the innate mucosal immune response in healthy mice and can maintain the homeostasis of the gut mucosal immune response against LPS through the activation of the classical NF-κB pathway.

Tripartite motif-containing (TRIM) 52 (TRIM52) is a vital regulator of inflammation. However, the function and mechanisms of TRIM52 in lipopolysaccharide (LPS)-induced inflammatory injury of human periodontal ligament cells (HPDLCs) in periodontitis remain undefined. In the present research, gene expression was determined using a quantitative polymerase chain reaction and Western blot. The effect of TRIM52 on LPS-induced inflammatory injury was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, and enyzme-linked immunosorbent assay (ELISA). We found that TRIM52 expression was up-regulated in LPS-treated HPDLCs. Knockdown of TRIM52 alleviated LPS-induced proliferative inhibition and apoptosis promotion in HPDLCs, as evidenced by a decrease in cleaved caspase-3 expression and caspase-3 activity. Silencing TRIM52 suppressed LPS-induced inflammatory response of HPDLCs, as indicated by the decrease in interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α) levels, and increase in IL-10 levels. TRIM52 knockdown inhibited LPS-induced activation of TLR4/nuclear factor-κ B (NF-κB) signaling pathway. Taken together, knockdown of TRIM52 mitigated LPS-induced inflammatory injury via the TLR4/NF-κB signaling pathway, providing an effective therapeutic target for periodontitis.

The activation of pancreatic stellate cells (PSCs) plays a critical role in the progression of pancreatic fibrosis. Nuclear factor‐kappa B (NF‐κB) is associated with chronic pancreatitis (CP). Previous evidence indicated that NF‐κB in acinar cells played a double‐edged role upon pancreatic injury, whereas NF‐κB in inflammatory cells promoted the progression of CP. However, the effects of NF‐κB in PSCs have not been studied. In the present study, using two CP models and RNAi strategy of p65 in cultured PSCs, we found that the macrophage infiltration and MCP‐1 expression were increased, and the NF‐κBp65 protein level was elevated. NF‐κBp65 was co‐expressed with PSCs. In vitro, TGF‐β1 induced overexpression of the TGF‐β receptor 1, phosphorylated TGF‐β1–activated kinase 1 (p‐TAK1) and NF‐κB in the PSCs. Moreover, the concentration of MCP‐1 in the supernatant of activated PSCs was elevated. The migration of BMDMs was promoted by the supernatant of activated PSCs. Further knockdown of NF‐κBp65 in PSCs resulted in a decline of BMDM migration, accompanied by a lower production of MCP‐1. These findings indicate that TGF‐β1 can induce the activation of NF‐κB pathway in PSCs by regulating p‐TAK1, and the NF‐κB pathway in PSCs may be a target of chronic inflammation and fibrosis.

Berries are a rich source of phytochemicals, especially phenolics well known for protective activity against many chronic diseases. Berries also contain a complex mixture of volatile compounds that are responsible for the unique aromas of berries. However, there is very limited information on the composition and potential health benefits of berry volatiles. In this study, we isolated phenolic and volatile fractions from six common berries and characterized them by HPLC/HPLC-MS and GC/GC-MS, respectively. Berry phenolic and volatile fractions were evaluated for an anti-inflammatory effect using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells by measuring levels of pro-inflammatory cytokines and the nuclear factor-kappa B (NF-&kappa, B) signaling pathway. Results showed that LPS-induced excessive production of nitric oxide (NO), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6) and tumor necrosis factor-&alpha, (TNF-&alpha, ), which were inhibited by berry phenolic and volatile extracts. Moreover, berry phenolic and volatile extracts reduced the nuclear translocation of NF-&kappa, B by blocking the phosphorylation of p65 and degradation of I&kappa, B&alpha, These findings showed that berry volatiles from six berries had comparable anti-inflammatory effects to berry phenolics through the suppression of pro-inflammatory mediators and cytokines expression via NF-&kappa, B down-regulation, despite being present in the fruit at a lower concentration.

Background Preeclampsia (PE) is a frequently occurring pregnancy disorder in the placenta, which results in various maternal and fetal complications. The current study aims to evaluate the role of extracellular vesicles (EVs)-encapsulated microRNA (miR)-101 in biological processes of trophoblasts in PE and its underlying mechanism. Methods Human umbilical cord mesenchymal stem cell (HUCMSC) and HUCMSC-derived EVs were isolated and cultured, after which EV characterization was carried out using PKH67 staining. In silico analyses were adopted to predict the downstream target genes of miR-101, and dual luciferase reporter gene assay was applied to validate the binding affinity. Furthermore, loss- and gain-of-function approaches were adopted to determine the role of miR-101 and bromodomain-containing protein 4 (BRD4) in trophoblast proliferation and invasion using EDU staining and transwell assay. In addition, a rat model of PE was established to verify the function of EV-encapsulated miR-101 in vivo. Results Placental tissues obtained from PE patients presented with downregulated miR-101 expression and upregulated BRD4 and CXCL11 expression. EV-encapsulated miR-101 from HUCMSCs could be delivered into the trophoblast HTR-8/SVneo cells, thus enhancing proliferation and migration of trophoblasts. Mechanically, miR-101 targeted and negatively regulated BRD4 expression. BRD4 knockdown promoted the proliferation and migration of trophoblasts by suppressing NF-κB/CXCL11 axis. EV-encapsulated miR-101 from HUCMSCs also reduced blood pressure and 24 h urine protein in vivo, thereby ameliorating PE. Conclusion In summary, EV-encapsulated miR-101 promoted proliferation and migration of placental trophoblasts through the inhibition of BRD4 expression via NF-κB/CXCL11 inactivation.

Acute lung injury (ALI) is a highly lethal pulmonary disease that causes edema, hypoxemia and respiratory failure. Recent evidence indicates that nuclear factor-kappa B (NF-κB) plays a crucial role in ALI development. However, the regulatory mechanism of NF-κB on ALI remains enigmatic. In this study, we investigated potential molecular mechanism of NF-κB on ALI induced by lipopolysaccharide (LPS). BALB/c mice were subjected to intratracheal spraying of LPS to generate an ALI mode, with the activity of NF-κB in mice tissues being detected by enzyme linked immunosorbent assay (ELISA), and the number of inflammatory cells in bronchoalveolar lavage fluid being counted. Then, the macrophage cell line RAW264.7 exposed to LPS were treated with ammonium pyrrolidinedithiocarbamate (PDTC) (inhibitor of NF-κB), miR-194 mimic, or oe-chemokine receptor type 4 (CXCR4) separately or in combination. After that, ELISA and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to detect the expression level of IL-1β, IL-6, TNF-α, miR-194 and CXCR4, respectively. In addition, the targeting relationship between miR-194 and CXCR4 was verified by dual-luciferase reporter gene assay. The dry/wet ratio of lung and the MPO activity were also measured to assess the inflammatory response in mice. Activation of NF-κB down-regulated the miR-194 expression in LPS-induced ALI. Overexpression of miR-194 alleviated LPS-induced ALI and reduced the expression of inflammatory factors IL-1β, IL-6 and TNF-α via targeting CXCR4. In LPS-induced ALI, NF-κB mediates the CXCR4 expression by inhibiting the expression of miR-194, thus promoting the inflammatory injury of lung.

Rheumatoid arthritis (RA) is among the most prevalent forms of autoimmunity. Gentiopicroside (Gent) is an iridoid glucoside derived from the Gentiana Macrophylla Pall which is used in traditional Chinese medicine to treat RA. The present study was designed to explore the ability of Gent to combat RA and to explore the molecular basis for such anti-RA activity both in vitro using tumor necrosis factor alpha (TNF-α)-stimulated human RA fibroblast-like synoviocytes (RA-FLS) and in vivo using a rat adjuvant-induced arthritis (AIA) model. We found that Gent was able to significantly reduce the swelling of joints and arthritic index scores, with corresponding reductions in synovial inflammatory cell infiltration, synovial hyperplasia, and bone erosion in treated AIA rats. Importantly, Gent 200 mg/kg reduced thymus index in AIA rats, but had no effect on spleen index and body weight, it revealed that Gent was relatively safe at the dose we chose. We further found that Gent was able to suppress the TNF-α-induced proliferation and migration of RA-FLS cells. This suppression was attributed to the ability of Gent to block NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1, thereby disrupting the activation of the NLRP3 inflammasome. Consistent with such suppression, Gent led to a significant decrease in IL-1β secretion by treated cells. Furthermore, this reduction in NLRP3 inflammasome activation was also associated with decreases in the activation of nuclear factor (NF-κB), the production of reactive oxygen species (ROS), and the expression of inflammatory IL-6. Together these findings indicate that Gent can suppress the ROS-NF-κB-NLRP3 axis to alleviate RA symptoms. Chemical compounds studied in this article Gentiopicroside (PubChem CID: 88708).

Alpinetin is a naturally occurring flavonoid from the ginger plants. We previously reported the identification of alpinetin as a ligand of human pregnane X receptor (hPXR). The current study investigated the role of alpinetin as a putative PXR activator in ameliorating chemically induced inflammatory bowel disease (IBD). We found that oral administration of alpinetin significantly alleviated the severity of dextran sulfate sodium (DSS)-induced colitis in mice by decreasing the inflammatory infiltration, the levels of the pro-inflammatory mediators, and the PXR target genes in the colon. In vitro, alpinetin blocked the nuclear translocation of p-p65 in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Further, alpinetin significantly upregulated PXR target genes and inhibited TNF-α-induced NF-κB-luciferase activity in LS174T colorectal cells; however, this regulatory effects were lost when cellular PXR gene was knocked down. In PXR transactivation assays, alpinetin increased both mouse and human PXR transactivation in a dose-dependent manner. Ligand occluding mutants, S247W/C284W and S247W/C284W/S208W, in hPXR-reporter assays, abrogated alpinetin-induced hPXR transactivation. Finally, alpinetin bound to the hPXR-ligand-binding domain (LBD) was confirmed by competitive ligand binding assay. The current study significantly extends prior observations by validating a PXR/NF-κB regulatory mechanism governing alpinetin’s anti-inflammatory effects in a murine model of IBD.

Fibroblasts are the chief secretory cells of the extracellular matrix (ECM) responsible for basal deposition and degradation of the ECM under normal conditions. During stress, fibroblasts undergo continuous activation, which is defined as the differentiation of fibroblasts into myofibroblasts, a cell type with an elevated capacity for secreting ECM proteins. Dipeptidyl peptidase-4 (DPP4) is a ubiquitously expressed transmembrane glycoprotein and exerts effects that are both dependent and independent of its enzymatic activity. DPP4 has been demonstrated to define fibroblast populations in human skin biopsies of systemic sclerosis. Shedding of DPP4 from different tissues into the circulation appears to be involved in the pathogenesis of the diseases. The mechanism underlying soluble DPP4-induced dermal fibrosis has not been clearly determined. The effects of DPP4 on murine 3T3 fibroblasts and human dermal fibroblasts were evaluated by measuring the expression of fibrotic proteins, such as α-SMA and collagen. Soluble DPP4 stimulated the activation of fibroblasts in a dose-dependent manner by activating nuclear factor-kappa B (NF-κB) and suppressor of mothers against decapentaplegic (SMAD) signaling. Blocking proteinase-activated receptor-2 (PAR2) abrogated the DPP4-induced activation of NF-κB and SMAD and expression of fibrosis-associated proteins in fibroblasts. Linagliptin, a clinically available DPP4 inhibitor, was observed to abrogate the soluble DPP4-induced expression of fibrotic proteins. This study demonstrated the mechanism underlying soluble DPP4, which activated NF-κB and SMAD signaling through PAR2, leading to fibroblast activation. Our data extend the current view of soluble DPP4. Elevated levels of circulating soluble DPP4 may contribute to one of the mediators that induce dermal fibrosis in patients.

Background: Ameloblastoma among benign tumors holds a unique position by its locally destructive and invasive nature. Tumors that originate from the odontogenic apparatus or its remnants in the jaws show diverse clinical presentations, behavior and histologic patterns. The differed biological behavior behind follicular and plexiform ameloblastomas has never attained completeness because of the lack of rhythmic correlation regarding the exact mechanism. Nuclear factor-kappa B (NF-κB) pathways play a crucial role in survival, death and differentiation during physiologic and pathologic conditions. With this background, the study has been aimed to investigate the expression of NF-κB in follicular and plexiform ameloblastomas. Objective: The objective of this study was to analyze the immunohistochemical expression pattern of NF-κB in ameloblastoma and to compare the immunohistochemical expression pattern of NF-κB among the histological types of ameloblastoma, follicular and plexiform patterns. Methodology: Total 20 ameloblastomas (10 follicular, 10 plexiform) were immunostained with antihuman NF-κB p65 mouse IgG monoclonal antibody, and the pattern of staining is statistically analyzed using Chi-square test with the level of significance (P < 0.05). Results: Twelve (3 follicular, 9 plexiform) out of 20 ameloblastomas showed immunoreactivity to NF-κB p65. In ameloblastoma, only the peripheral preameloblast-like tall columnar cells showed reactivity, whereas the stellate reticulum-like cells are immunonegative. The staining pattern was membranous in the immunoreactive cells. The results were studied with the associated and inducing pathways from the literature, and a possible mechanism has been proposed. Conclusion: The expression pattern of NF-κB was found to be higher in plexiform ameloblastoma than follicular ameloblastoma.

Low doses of antibiotics are ubiquitous in the marine environment and may exert negative effects on non-target aquatic organisms. Using primary macrophages of common carp, we investigated the mechanisms of action following exposure to several common antibiotics; cefotaxime, enrofloxacin, tetracycline, sulfamonomethoxine, and their mixtures, and explored the immunomodulatory effects associated with the nuclear factor-κB (NF-κB) signaling pathway. A KEGG pathway analysis was conducted using the sixty-six differentially expressed genes found in all treatments, and showed that exposure to 100 μg/L of antibiotics could affect regulation of the NF-κB signaling pathway, suggesting that activation of NF-κB is a common response in all four classes of antibiotics. In addition, the four antibiotics induced nf-κb and NF-κB-associated cytokines expression, as verified by qPCR, however, these induction responses by four antibiotics were minor when compared to the same concentration of LPS treatment (100 μg/L). Antagonists of NF-κB blocked many of the immune effects of the antibiotics, providing evidence that NF-κB pathways mediate the actions of all four antibiotics. Moreover, exposure to environmentally relevant, low levels (0.01–100 μg/L) of antibiotics induced a NF-κB-mediated immune response, including endogenous generation of ROS, activity of antioxidant enzymes, as well as expression of cytokine and apoptosis. Moreover, exposure to mixtures of antibiotics presented greater effects on most tested immunological parameters than exposure to a single antibiotic, suggesting additive effects from multiple antibiotics in the environment. This study demonstrates that exposure of fish primary macrophages to low doses of antibiotics activates the NF-kB pathway. Keywords: Antibiotics, Nuclear factor-κB, Immunomodulatory, Macrophages

Background/Aims: Exercise training (ExT) was associated with cardiovascular diseases including hypertension. The rostral ventrolateral medulla (RVLM) is a key region for central control of blood pressure and sympathetic nerve activity. Therefore, this study aimed to investigate the mechanisms within RVLM that can influence exercise training induced effects in salt-induced hypertension. Methods: Male Wistar rats were fed with a normal salt (0.3%) (NS) or a high salt (8%) (HS) diet for 12 weeks to induce hypertension. Then these rats were given moderate-intensity ExT for a period of 12 weeks. RVLM was used to determine glutamate and gamma-aminobutyric acid (HPLC), phosphorylated IKKβ, Fra-LI, 67-kDa isoform of glutamate decarboxylase (GAD67), proinflammatory cytokines (PIC) and NADPH-oxidase (NOX) subunits expression (Immunohistochemistry and Immunofluorescence, Western blotting). PIC and NF-κB p65 activity in the plasma were evaluated by ELISA studies. Renal sympathetic nerve activity (RSNA) was recorded and analyzed using the PowerLab system. Results: High salt diet resulted in increased mean arterial pressure and cardiac hypertrophy. These high salt diet rats had higher RVLM levels of glutamate, PIC, phosphorylated IKKβ, NF-κB p65 activity, Fra-LI, superoxide, NOX-2 (gp91phox) and 4, and lower RVLM levels of gamma-aminobutyric acid and GAD67, and higher plasma levels of PIC, norepinephrine, and higher RSNA. ExT attenuated these changes in salt-induced hypertensive rats. Conclusions: These findings suggest that high salt diet increases the activity of NF-κB and the levels of PIC and oxidative stress, and induces an imbalance between excitatory and inhibitory neurotransmitters in the RVLM. ExT attenuates hypertension and cardiac hypertrophy partially mediated by attenuating oxidative stress and modulating neurotransmitters in the RVLM.

The effect of NiCl2 on oral squamous cell carcinoma-derived cell line HSC3 was examined. Incubation with 1 mM NiCl2 significantly reduced the expression of MMPs at mRNA and protein levels. The in vivo orthotopic implantation model was established by injecting highly metastatic subcell line HSC3-M3 to nude mouse tongue. After 1 week of injection, mice were fed with or without 1 mM NiCl2-containing water for two to three weeks. Immunohistochamical examination revealed that MMP9 expression was drastically reduced in NiCl2-fed mice. By CT images, cancer mass was observed as a translucent area in control mice. In NiCl2-fed mice, much highly translucent area was observed within the translucent area. Histologically, this area corresponded to the necrotic area in the tumor mass. Real-time PCR analysis revealed the reduced expression of angiogenic factors such as IL-8 and VEGF mRNA in NiCl2-fed mice. To further examine the effect of NiCl2 on metastasis, human β-globin gene expression in regional lymphnodes was compared. The β-globin gene was totaly absent in NiCl2-fed mice. Moreover, various cancer metastasis-related genes were inhibited in NiCl2-fed mice by PCR array analysis. The results indicated that NiCl2 might be a promising new anti-cancer therapeutics for the oral cancer treatment.

Taurochenodeoxycholic acid (TCDCA) is one of main bioactive substances of animals’ bile acid and confers good anti-inflammatory activity. This study aimed to investigate the mechanism of the anti-adjuvant arthritis activity of TCDCA, the actions were observed in vitro on the protein and mRNA expression of cytokines, the activity of nuclear factor-kappa B (NF-κB) and its inhibitory IκBα protein level of peritoneal macrophages (PMs) in adjuvant arthritis (AA) rats. In a definite concentration ranging from 150 μg/mL to 200 μg/mL, the over protein and mRNA expression of TNF-α, IL-1β and IL-6 were remarkably suppressed in the supernatant of PMs treatment with TCDCA. Positive correlations were found between changes of NF-κB activity and expression of TNF-α, IL-1β and IL-6 influenced by TCDCA. The activity of NF-κB was markedly inhibited and IκBα protein level was increased by TCDCA (150 μg/mL,180 μg/mL and 200 μg/mL). TCDCA suppresses the protein and mRNA expression of TNF-α, IL-1β, and IL-6 by inhibiting the NF-κB binding activity, which is mediated through up-regulating the IκBα expression of PMs in AA rats.

Synovial fibroblasts (SFs) play a crucial role in the inflammatory process of rheumatoid arthritis (RA). The highly activated NF‐κB signal in SFs is responsible for most of the synovial inflammation associated with this disease. In this study, we have developed an SF‐targeting liposomal system that encapsulates the NF‐κB‐blocking peptide (NBD peptide) HAP‐lipo/NBD. HAP‐lipo/NBDs demonstrated efficient SF‐specific targeting in vitro and in vivo. Our study also showed a significant inhibitory effect of HAP‐lipo/NBD on NF‐κB activation, inflammatory cytokine release and SF migration capability after zymosan stimulation. Furthermore, the systemic administration of HAP‐lipo/NBDs significantly inhibited synovial inflammation and improved the pathological scores of arthritis induced by zymosan. Thus, these results suggest that an SF‐targeting NF‐κB‐blocking strategy is a potential approach for the development of alternative, targeted anti‐RA therapies.

Nuclear factor‐kappa B (NF‐κB) as a prognostic marker remains unclear in non‐small cell lung cancer (NSCLC). Here, we studied NF‐κB‐p65 (p65) expression and phosphorylated NF‐κB‐p105 (p‐p105) expression in NSCLC and correlated the finding with overall survival (OS) and clinicopathological features. A total of 186 archival samples from patients with surgically resectable NSCLC were probed with p65 and p‐p105 (Ser 932). The p65‐positive expression and p‐p105‐positive expression were defined as distinct nuclear p65 and cytoplasmic p‐p105 labelling in at least 1% of tumour cells, respectively. The positive staining of p65 alone, p‐p105 alone and co‐expression of p65 and p‐p105 were observed in 61 (32.8%), 90 (48.4%) and 35 (18.8%) patients, respectively. Co‐expression of p65 and p‐p105 but not of either p65 or p‐p105 alone was associated with a poor prognosis. Patients with co‐expression of p65 and p‐p105 had a shorter OS than others, median OS 26.5 months versus 64.1 months, HR 1.85 (95% CI: 1.18–2.91), P = 0.007. There was no statistically significant association between clinicopathological characteristics and either p65 or p‐p105 alone or co‐expression of p65 and p‐p105. This indicates that co‐expression of p65 and p‐p105 was a poor prognostic factor, and pathologic studies of NF‐κB expression could include multiple pathway components in NSCLC.

Context: It has been shown that hesperidin induces apoptosis in NALM-6 cells through inhibition of nuclear factor-kappa B (NF-κB) activation. Aims: To investigate the effect of hesperidin on inhibition of NF-κB activation through blocking phosphoinositide 3–kinase (PI3K)/Akt pathway as a main target in cancer treatment, in NALM-6 cells. Materials and Methods: NALM-6 cells were incubated with two concentrations of hesperidin (25, 50 μM) in the presence or absence of insulin (100 nM), as a potent activator of Akt. The cytotoxic activity of hesperidin was determined by 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptotic death was measured by ELISA test using cell death detection ELISA Plus kit. To assay the effect of hesperidin on Akt pathway, the phosphorylation levels of Akt, inhibitor of kappa B alpha (IκBα), and glycogen synthase kinase-3 beta (GSK-3β) and expression level of IκB kinase alpha (IKKα) were determined by Western blot analysis. Results: Hesperidin (both concentrations) significantly reduced cells survival in the presence and absence of insulin compared to untreated cells in a time-dependent manner (P < 0.05). Hesperidin also significantly increased apoptosis in NALM-6 cells even in hyperinsulinemia condition (P < 0.0001). Hesperidin inhibited insulin-induced phosphorylation and activation of Akt, IκBα, and GSK-3β and decreased expression of IKKα. Conclusion: The results of this study demonstrated that cytotoxic and proapoptotic actions of hesperidin are partly mediated through the suppression of PI3K3/Akt/IKK signaling pathway. So, hesperidin might act as a chemotherapeutic agent by targeting cell survival pathways.

In order to investigate the function of toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signal pathways in the pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D), IBS-D animal models were established in wistar rats challenged with acute and chronic stresses (29 days). Wistar rats without stress-challenged were used as controls. IBS-D models were randomly divided into two groups: one was treated with normal saline, another group was treated with TLR4/NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) (50mg/kg/week) for continuous four times. Our results demonstrate that continuous stresses can induce the characteristic symptoms of IBS-D, including high wet stool rate and intestinal flora imbalance. Further examinations of colon tissues show that the protein expression levels of TLR4 and NF-κB in IBS-D groups are higher than that in control group. The secretory levels of interleukin (IL-8), tumor necrosis factor α (TNFα), and myeloid differentiation factor 88 (MyD88) are significantly increased in IBS-D group. Administration with PDTC effectively downregulates levels of these inflammatory factors. In contrast, interleukin-10 (IL-10) is in an opposite alteration with lower levels in IBS-D groups and the PDTC treatment increases it to the levels as in control group. Moreover, inhibition of the TLR4/NF-κB by PDTC improves the microstructure of intestinal mucosa mainly by increasing the height of villi. Our results suggest that TLR4/NF-κB signal pathway plays an important role in the modulation of inflammatory responses in IBS-D, which might be a therapeutic target for the IBS-D. All of these findings also provide the evidence concerning an inherent linkage between the axis of stress/NF-κB/inflammation and IBS-D.

Carotenoids are natural lipophilic pigments with substantial health benefits. Numerous studies have demonstrated the anti-inflammatory activities of carotenoids, especially toward lipopolysaccharide-induced inflammatory responses. As such, there are few reports on the evaluation and comparison of the anti-inflammatory activities of carotenoids against inflammation induced by other stimuli. In this study, we used pathogen-associated molecular patterns, proinflammatory cytokines, degenerated proteins, and chemical irritants as inflammatory inducers to evaluate the anti-inflammatory activities of eight different carotenoids. Each carotenoid showed characteristic anti-inflammatory activities, thus, we conducted a multivariate analysis to clarify the differences among them. Unsubstituted β-ring (i.e., provitamin A) and C8-keto structures of carotenoids were found to be crucial for their inhibitory effects on the activation of nuclear factor-kappa B and interferon regulatory factors, respectively. Furthermore, we found that β-carotene and echinenone treatment increased intracellular retinoid levels in monocytes and that the retinoids showed the similar activities to β-carotene and echinenone. Taken together, the intake of both provitamin A and C8-keto carotenoids (e.g., siphonaxanthin and fucoxanthin) might be effective in improving the inflammatory status of individuals. A multivariate analysis of anti-inflammatory activities is a useful method for characterizing anti-inflammatory compounds.

In this study, the potential of Carissa carandas Linn. as a natural anti-aging, antioxidant, and skin whitening agent was studied. Various parts of C. carandas, including fruit, leaf, seed, and pulp were sequentially extracted by maceration using n-hexane, ethyl acetate, and ethanol, respectively. High-performance liquid chromatography, Folin–Ciocalteu, and Dowd method were used to investigate their chemical compositions. The inhibitory activities of oxidation process, matrix metalloproteinases (MMPs), elastase, hyaluronidase, and tyrosinase were analyzed. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay in a human epidermal keratinocyte line (HaCaT). The results exhibited that ethyl acetate could extract the most ursolic acid from C. carandas, while ethanol could extract the most phenolics and flavonoids. The leaf extract had the highest content of ursolic acid, phenolics, and flavonoids. The leaf extracted with ethyl acetate (AL) had the highest ursolic acid content (411.8 mg/g extract) and inhibited MMP-1, NF-kappa B, and tyrosinase activity the most. Ursolic acid has been proposed as a key component in these biological activities. Although several C. carandas extracts are beneficial to human skin, AL has been proposed for use in cosmetics and cosmeceuticals due to its superior anti-wrinkle, anti-inflammation, and whitening properties.

Aim. This systematic review is aimed at evaluating the literature on the efficacy of naturally available extracts that inhibit cancer. Methods. A literature search was performed to strengthening the reporting of observational studies in epidemiology analysis. Approximately 3000 research articles were initially selected. Of these articles, 200 were included, and 2800 were excluded. On further scrutiny, 150 of the 200 studies were reviews, seminars, and presentations, and 50 were original study articles. Among these articles, 20 studies were selected for the systematic review. Results. The predominant molecular pathways followed by natural extracts were nuclear factor kappa B ligand, suppression of the protein kinase B-Akt/P13K pathway (an intracellular signaling pathway important in regulating cell cycle), vascular endothelial growth factor downregulation, and tumor protein-P53 tumor suppressor upregulation. Conclusions. It is evident that natural extracts have the ability to inhibit cancer progression. Continued research in this field could facilitate the use of natural extracts with currently available anticancer agents.

Pancreatic cancer is often resistant to chemotherapy. We previously showed the efficacy of combination treatment using gemcitabine and nafamostat mesilate (FUT‐175) for patients with unresectable pancreatic cancer. However, the mechanisms that affect the sensitivity of FUT‐175 are not fully understood. The purpose of the present study was to clarify the mechanism of the sensitivity to FUT‐175, with a focus on the activity of glycogen synthase kinase‐3β (GSK‐3β). In vitro, we assessed sensitivity to FUT‐175 in human pancreatic cancer cell lines (PANC‐1 and MIAPaCa‐2) and difference of signaling in these cells by cell proliferation assay, Western blot analysis and microarray. Next, we assessed cell viability, apoptotic signal and nuclear factor‐kappa B (NF‐κB) activity in response to treatment with FUT‐175 alone and in combination with GSK‐3 inhibitor or protein phosphatase 2A (PP2A) by cell proliferation assay, Western blot analysis and enzyme‐linked immunosorbent assay. Phosphorylated GSK‐3β level was significantly higher in MIAPaCa‐2 (high sensitivity cell) than in PANC‐1 (low sensitivity cell). Cell viability and NF‐κB activity were significantly decreased by addition of GSK‐3 inhibitor to FUT‐175, and levels of cleaved caspase‐8 were increased by inhibition of GSK‐3. PP2A inhibitor increased the levels of phosphorylated GSK‐3β and sensitized both cell lines to FUT‐175 as measured by cell viability and apoptotic signal. The results indicate that GSK‐3β activity plays a key role in the antitumor effect of FUT‐175 in pancreatic cancer cells, and regulation of GSK‐3β by PP2A inhibition could be a novel therapeutic approach for pancreatic cancer.

Background/Aims: Aspirin has been demonstrated to possess potent chemopreventive and anticancer effects on prostate cancer. However, the more detailed molecular mechanisms of aspirin to suppress prostate cancer cell invasion have not been clearly elucidated. Methods: Transwell assays were performed to evaluate the effects of aspirin on cell invasion. Matrix metalloproteinases (MMPs) and serine proteinases activities in cell media were examined by gelatin zymography and ELISA. In addition, inhibitor of κB (IκB) kinase-β (IKK-β) phosphorylation and IKK-β kinase activity were measured to assess the effects of aspirin on IKK-β activation. Results: We found that aspirin suppressed the invasion and attachment in human prostate cancer cells. Aspirin treatment significantly resulted in reduction of matrix metalloproteinase-9 (MMP-9) and upregulation of tissue inhibitors of metalloproteinase-1 (TIMP-1) activity, which are the proteolytic enzymes contributing to the degradation of extracellular matrix and basement membrane in cell invasion and metastasis. Our data further showed that aspirin was able to inhibit both urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) expression in the cells. In addition, aspirin treatment caused a strong decrease in nuclear factor-kappa B (NF-κB) activation, inhibitor of κB (IκB)-α phosphorylation together with translocation of NF-κB p65 to nucleus and IκB kinase (IKK)- β activation. Moreover, the inhibitory effects of aspirin on cell invasion were reversed by IKK-β overexpression, while the IKK inhibitor sensitizes the anti-invasive effect of aspirin in prostate cancer cells. Conclusion: The present research concluded that aspirin suppressed prostate cancer cell invasion by reducing MMP-9 activity and uPA expression through decreasing of IKK-β-mediated NF-κB activation, indicating that the ability of aspirin to inhibit cell invasion might be useful in the chemoprevention of metastatic prostate cancer.