2',4-Dihydroxy-3',4',6'-trimethoxychalcone from Chromolaena odorata possesses anti-inflammatory effects via inhibition of NF-κB and p38 MAPK in lipopolysaccharide-activated RAW 264.7 macrophages.

CONTEXT: Immune dysregulation has been implicated in the pathogenesis of many diseases. Macrophages play a crucial role contributing to the onset, progression, and resolution of inflammation. Macrophage inflammatory mediators are of considerable interest as potential targets to treat inflammatory diseases. OBJECTIVE: The present study was conducted to elucidate the anti-inflammatory mechanism of 2',4-dihydroxy-3',4',6'-trimethoxychalcone (1), the major chalcone isolated from Chromolaena odorata (L.) R.M.King & H.Rob, against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. MATERIALS AND METHODS: Cell viability, nitric oxide (NO), and proinflammatory cytokines of LPS-activated RAW 264.7 cells were measured by MTT, Griess, and ELISA assays, respectively. Cell lysates were subjected to Western blotting for investigation of protein expression. RESULTS AND DISCUSSION: Treatment with the major chalcone 1 significantly attenuated the production of NO and proinflammatory cytokines, tumor necrosis factor-α, interleukin-1ß, and interleukin-6 in a dose-dependent manner. The chalcone suppressed nuclear factor-κB (NF-κB) stimulation by preventing activation of inhibitor κB kinase (IKK) α/ß, degradation of inhibitor κB (IκB) α, and translocation of p65 NF-κB into the nucleus. Additionally, the chalcone markedly repressed the phosphorylation of p38 mitogen-activated protein kinase (MAPK), but no further inhibition was detected for c-Jun N-terminal activated kinases or extracellular regulated kinases. Thus, suppression of NF-κB and p38 MAPK activation may be the core mechanism underlying the anti-inflammatory activity of 2',4-dihydroxy-3',4',6'-trimethoxychalcone (1). CONCLUSION: These findings provide evidence that 2',4-dihydroxy-3',4',6'-trimethoxychalcone (1) possesses anti-inflammatory activity via targeting proinflammatory macrophages. This anti-inflammatory chalcone is a promising compound for reducing inflammation.

Curcumin pyrazole blocks lipopolysaccharide-induced inflammation via suppression of JNK activation in RAW 264.7 macrophages.

BACKGROUND: Targeting inflammatory macrophages and their products is an effective method for controlling inflammation. The pyrazole analog of curcumin (curcumin pyrazole, PYR) has been reported to possess superior anti-inflammatory activity to curcumin (CUR). However, the role of PYR anti-inflammatory activity in macrophages has not yet been elucidated. OBJECTIVE: To examine the anti-inflammatory effects of PYR and CUR in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages and determine the role of mitogen-activated protein kinases (MAPK) in their activity. METHODS: Nitrite level was investigated by the Griess assay. The expression of inducible nitric oxide (NO) synthase, cyclooxygenase-2 (COX-2), and MAPK proteins were analyzed by western blot analysis. The pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay. RESULTS: LPS-induced NO secretion in RAW 264.7 macrophages was potently inhibited by PYR (IC50 = 3.7 ± 0.16 µM), at a higher efficacy than CUR (IC50 = 11.0 ± 0.59 µM). Treatment with identical concentrations of PYR and CUR demonstrated that PYR drastically inhibited iNOS and COX-2 expression, whereas CUR only blocked COX-2. PYR reduced the LPS-induced secretion of TNF-α to a greater extent than CUR and both similarly reduced IL-1ß and IL-6 levels. Activation of c-Jun N-terminal kinase (JNK) MAPK was significantly decreased in LPS-activated RAW 264.7 macrophages upon PYR but not CUR treatment. CONCLUSION: PYR exhibited a more potent anti-inflammatory activity than CUR. This activity is partly mediated by PYR-depended inhibition of the JNK signaling pathway and underscores the utility of PYR as an anti-inflammatory agent in macrophages.

Phosphodiesterase type 10A inhibitor attenuates lung fibrosis by targeting myofibroblast activation.

Pulmonary fibrosis (PF) is a fatal and irreversible respiratory disease accompanied by excessive fibroblast activation. Previous studies have suggested that cAMP signaling pathway and cGMP-PKG signaling pathway are continuously down-regulated in lung fibrosis, whereas PDE10A has a specifically expression in fibroblasts/myofibroblasts in lung fibrosis. In this study, we demonstrated that overexpression of PDE10A induces myofibroblast differentiation, and papaverine, as a PDE10A inhibitor used for vasodilation, inhibits myofibroblast differentiation in human fibroblasts, Meanwhile, papaverine alleviated bleomycin-induced pulmonary fibrosis and amiodarone-induced oxidative stress, papaverine downregulated VASP/ß-catenin pathway to reduce the myofibroblast differentiation. Our results first demonstrated that papaverine inhibits TGFß1-induced myofibroblast differentiation and lung fibrosis by VASP/ß-catenin pathway.

Time­dependent changes in NLRP3 and Nrf2 levels in lipopolysaccharide­induced acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe clinical conditions with a high mortality rate. Nucleotide­binding oligomerization domain (NOD)­like receptor containing pyrin domain 3 (NLRP3) and nuclear factor E2­related factor 2 (Nrf2) have been reported to be associated with ALI. However, the dynamic changes in the levels of these factors in lipopolysaccharide (LPS)­induced lung injury remain unclear. Thus, the present study aimed to determine the LPS­induced activation of immunological cascades, as well as the NLRP3/Nrf2 signaling pathway at different stages of lung injury. For this purpose, mice were divided into six groups as follows: The control, LPS­4 h, LPS­24 h, LPS­48 h, LPS­96 h and LPS­144 h groups. LPS (4 mg/kg) was administered intratracheally to induce lung injury. Flow cytometry was used to determine the changes in macrophages, neutrophils and T­cell subsets in lung tissue, hematoxylin and eosin staining were used to measure the histopathological changes in lung tissues, ELISA was performed to evaluate the levels of cytokines, western blot analysis was used to measure the levels of inflammatory proteins, and reverse transcription­quantitative PCR used to determine the mRNA level of a target gene. Following LPS administration, evident histopathological damage with neutrophil infiltration was observed which peaked at 48 h. The levels of interleukin­1ß, keratinocyte­derived chemokine, macrophage inflammatory protein 2 and tumor necrosis factor a were markedly increased in bronchoalveolar lavage fluid and serum from the mice, and these levels peaked at 4 h. Moreover, LPS promoted Toll like receptor­4 expression and reactive oxygen species production, thus activating NLRP3/Nrf2 signaling and pyroptosis. Collectively, the present study demonstrates that LPS triggers multiple inflammatory molecules and immune cells during ALI, which may be closely involved in the irregular redox status, NLRP3/Nrf2 pathway and pyroptosis.

Phosphodiesterase 4B is required for NLRP3 inflammasome activation by positive feedback with Nrf2 in the early phase of LPS- induced acute lung injury.

Acute lung injury (ALI) is associated with overproduction of inflammatory mediators in lung tissue. Previous studies have revealed that inflammation induces activation of phosphodiesterase 4B (PDE4B) accompanied by the production of inflammatory mediators, but the detailed mechanism remains unclear. Here, we focused on the NOD-, LRR- and pyrin domain-containing protein 3(NLRP3) inflammasome complexes to study the crosstalk between PDE4B and NF-E2-related factor 2 (Nrf2). We used global knockout PDE4B or Nrf2 mice to prepare LPS induced acute lung injury model by intratracheally administration, and LPS primed bone marrow-derived macrophages (BMDMs), following overexpression of PDE4B or Nrf2, luciferase activity analysis, and chIP-qPCR analyses. We found that deficiency of PDE4B could potently attenuate the lung histopathological changes, suppress the secretion of pro-inflammatory mediators such as tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6, IL-18, and cleaved caspase-1, 8, and GSDMD accompanied with defective activation of the ROS/Nrf2/NLRP3. Meanwhile deficiency of Nrf2 showed the similar results. Furtherly, overexpression by PDE4B or Nrf2 plasmid transfection in MH-S cells could enhance the Nrf2 or PDE4B expression. Luciferase analysis suggested that Nrf2 activated PDE4B promoter activity, while PDE4B could increase Nrf2 substrate ARE activity in MH-S cells in dose dependent manners. ChIP-qPCR analyses showed that Nrf2 bound to the PDE4B promoter region at Ì´ 1532 to Ì´1199 position in macrophages. Altogether, deficiency of PDE4B inhibit the inflammasome activation and pyroptosis in LPS stimulated lung injury model and macrophages by regulating ROS/Nrf2/NLRP3 activation. The study provides new insight that PDE4B is required for NLRP3 inflammasome activation by positive feedback with Nrf2.

PDE9 Inhibitor PF-04447943 Attenuates DSS-Induced Colitis by Suppressing Oxidative Stress, Inflammation, and Regulating T-Cell Polarization.

Ulcerative colitis (UC) is a form of inflammatory bowel disease, which manifests as irritation or swelling and sores in the large intestine in a relapsing and remitting manner. In a dextran sulfate sodium sulfate (DSS)-induced UC model in female mice, we found that the levels of cyclic guanosine monophosphate (cGMP) are reduced, while the expression of phosphodiesterase 9A (PDE9A) is highest among all phosphodiesterase (PDEs). Since PDE9 has the highest affinity toward cGMP, we evaluated the selective PDE9 inhibitor PF-04447943 (PF) as a potential candidate for UC treatment. PF has been extensively studies in cognitive function and in sickle cell disease, but not in models for inflammatory bowel disease (IBD). Therefore, we used female C57BL/6 mice treated with 3% DSS alone or co-treated with PF or sulfasalazine (SASP) to study the body weight, colon length, histopathology, and measure superoxide dismutase (SOD), malondialdehyde (MDA), and cGMP level, as well as cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-12/23 (IL-12/23), interleukin-10 (IL-10), and pathways including nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), and inflammasome activation. In addition, the number of dendritic cells (DC) and regulatory T cells (Treg cell) was assessed in the spleen, lymph node, and colon using flow cytometry. DSS reduced the number of goblet cells, decreased colon lengths and body weights, all of them were attenuated by PF treatment. It also suppressed the elevated level of inflammatory cytokines and increased level the anti-inflammatory cytokine, IL-10. PF treatment also reduced the DSS-induced inflammation by suppressing oxidative stress, NF-κB, STAT3, and inflammasome activation, by upregulating nuclear factor erythroid 2-related factor 2 (Nrf-2) and its downstream proteins via extracellular signal-regulated kinase (ERK) phosphorylation. Importantly, PF reversed imbalance in Treg/T helper 17 cells (Th17) cells ratio, possibly by regulating dendritic cells and Treg developmental process. In summary, this study shows the protective effect of a PDE9A inhibitor in ulcerative colitis by suppressing oxidative stress and inflammation as well as reversing the Treg/Th17 cells imbalance.

Estimating Dynamic Cellular Morphological Properties via the Combination of the RTCA System and a Hough-Transform-Based Algorithm.

The xCELLigence real-time cell analysis (RTCA) system has the potential to detect cellular proliferation, migration, cytotoxicity, adherence, and remodeling. Although the RTCA system is widely recognized as a noninvasive and efficient tool for real-time monitoring of cellular fate, it cannot describe detailed cell morphological parameters, such as length and intensity. Transforming growth factor beta(TGF-ß) induced the epithelial-mesenchymal transition (EMT), which produces significant changes in cellular morphology, so we used TGF-ß to treat A549 epithelial cells in this study. We compared it with lipopolysaccharide (LPS) and cigarette smoke extract (CSE) as stimulators. We developed an efficient algorithm to quantify the morphological cell changes. This algorithm is comprised of three major parts: image preprocessing, Hough transform (HT), and post-processing. We used the RTCA system to record the A549 cell index. Western blot was used to confirm the EMT. The RTCA system showed that different stimulators produce different cell index curves. The algorithm determined the lengths of the detected lines of cells, and the results were similar to the RTCA system in the TGF-ß group. The Western blot results show that TGF-ß changed the EMT markers, but the other stimulator remained unchanged. Optics-based computer vision techniques can supply the requisite information for the RTCA system based on good correspondence between the results.

Electroacupuncture ameliorates cardiopulmonary bypass induced apoptosis in lung via ROS/Nrf2/NLRP3 inflammasome pathway.

AIMS: Electroacupuncture (EAc) has a pulmonary protective effect during cardiopulmonary bypass (CPB), but its molecular mechanisms including inflammasome activation signaling pathways remains unclear. MATERIALS AND METHODS: Male Sprague Dawley rats were divided into control, CPB + EAc and CPB groups. Lung injury model was developed by CPB treatment and EAc (2/100 Hz) was carried out before CPB in the CPB + EAc group. Lung tissues were collected at two time points (0.5 h; 2 h) to determine cytokines release by ELISA kits, and protein expressions by Western blot. Serum collected at two time points (0.5 h; 2 h) from CPB and CPB + EAc treated groups were used in NR8383 cells to confirm the effect of EAc. KEY FINDINGS: CPB significantly increased the inflammatory mediators, histological damage and expression of inflammasome related protein and apoptosis, when compared with control group. The level of tumor necrosis factor-α(TNF-α), interleukin (IL)-18 and IL-1ß in the CPB + EAc treated group was significantly decreased along with histological changes compared to CPB. Moreover, EAc inhibited the activation of Nod like receptor protein-3 (NLRP3) inflammasome complex, caspase-8 and activated NF-E2-related factor 2 (p-Nrf2). In addition, serum from the CPB + EAc group prevented CPB induced activation of inflammasome and related mediators, reducing ROS generation and apoptosis in NR8383 macrophages. SIGNIFICANCE: These findings indicate that EAc had a critical anti-apoptotic role by suppression of ROS/Nrf2/NLRP3 inflammasome pathway. EAc might be a possible therapeutic treatment for CPB-induced acute lung injury.