Inhibition Of P38 Phosphorylation Research Articles

AMP-activated protein kinase contributes to cisplatin-induced renal epithelial cell apoptosis and acute kidney injury.

Cisplatin, a commonly used anticancer drug, has been shown to induce acute kidney injury, which limits its clinical use in cancer treatment. Emerging evidence has suggested that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, is activated by various cellular stresses that deplete cellular ATP. However, the potential role of AMPK in cisplatin-induced apoptosis of renal tubular epithelial cells has not been studied. In this study, we demonstrated that cisplatin activates AMPK (Thr172 phosphorylation) in cultured renal tubular epithelial cells in a time-dependent manner, which was associated with p53 phosphorylation. Compound C, a selective AMPK inhibitor, suppressed cisplatin-induced AMPK activation, p53 phosphorylation, Bax induction, and caspase 3 activation. Furthermore, silencing AMPK expression by siRNA attenuated cisplatin-induced p53 phosphorylation, Bax induction, and caspase 3 activation. In a mouse model of cisplatin-induced kidney injury, compound C inhibited p53 phosphorylation, Bax expression, caspase 3 activation, and apoptosis, protecting the kidney from injury and dysfunction. Taken together, these results suggest that the AMPK-p53-Bax signaling pathway plays a crucial role in cisplatin-induced tubular epithelial cell apoptosis.

Daphnetin inhibits proliferation and inflammatory response in human HaCaT keratinocytes and ameliorates imiquimod-induced psoriasis-like skin lesion in mice

BackgroundPsoriasis is a common chronic inflammatory skin disease. Keratinocytes hyperproliferation and excessive inflammatory response contribute to psoriasis pathogenesis. The agents able to attenuate keratinocytes hyperproliferation and excessive inflammatory response are considered to be potentially useful for psoriasis treatment. Daphnetin exhibits broad bioactivities including anti-proliferation and anti-inflammatory. This study aims to evaluate the anti-psoriatic potential of daphnetin in vitro and in vivo, and explore underlying mechanisms.MethodsHaCaT keratinocytes was stimulated with the mixture of IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α (M5) to establish psoriatic keratinocyte model in vitro. Cell viability was measured using Cell Counting Kit-8 (CCK-8). Quantitative Real-Time PCR (qRT-PCR) was performed to measure the mRNA levels of hyperproliferative marker gene keratin 6 (KRT6), differentiation marker gene keratin 1 (KRT1) and inflammatory factors IL-1β, IL-6, IL-8, TNF-α, IL-23A and MCP-1. Western blotting was used to detect the protein levels of p65 and p-p65. Indirect immunofluorescence assay (IFA) was carried out to detect p65 nuclear translocation. Imiquimod (IMQ) was used to construct psoriasis-like mouse model. Psoriasis severity (erythema, scaling) was scored based on Psoriasis Area Severity Index (PASI). Hematoxylin and eosin (H&E) staining was performed to examine histological change in skin lesion. The expression of inflammatory factors including IL-6, TNF-α, IL-23A and IL-17A in skin lesion was measured by qRT-PCR.ResultsDaphnetin attenuated M5-induced hyperproliferation in HaCaT keratinocytes. M5 stimulation significantly upregulated mRNA levels of IL-1β, IL-6, IL-8, TNF-α, IL-23A and MCP-1. However, daphnetin treatment partially attenuated the upregulation of those inflammatory cytokines. Daphnetin was found to be able to inhibit p65 phosphorylation and nuclear translocation in HaCaT keratinocytes. In addition, daphnetin significantly ameliorate the severity of skin lesion (erythema, scaling and epidermal thickness, inflammatory cell infiltration) in IMQ-induced psoriasis-like mouse model. Daphnetin treatment attenuated IMQ-induced upregulation of inflammatory cytokines including IL-6, IL-23A and IL-17A in skin lesion of mice.ConclusionsDaphnetin was able to attenuate proliferation and inflammatory response induced by M5 in HaCaT keratinocytes through suppression of NF-κB signaling pathway. Daphnetin could ameliorate the severity of skin lesion and improve inflammation status in IMQ-induced psoriasis-like mouse model. Daphnetin could be an attractive candidate for future development as an anti-psoriatic agent.

Preventive Effect of Muscone against Cisplatin Nephrotoxicity in LLC-PK1 Cells.

Cisplatin, one of the most common antitumor agents, is widely applied to treat various cancerous diseases and is included in the World Health Organization Model List of Essential Medicines. Cisplatin therapy is used to treat 10–20% of all cancerous cases, and its cure rate is especially high in testicular cancer (over 90%). However, a major side effect of this anticancer drug is nephrotoxicity, limiting treatment effect and reducing the quality of life in cancer patients. Muscone, an odoriferous constituent of musk, was confirmed to inhibit cisplatin-induced LLC-PK1 kidney proximal tubule cell death in a dose-dependent manner. In term of renal protective mechanism, muscone inhibited cisplatin oxidative toxicity by decreasing reactive oxygen species (ROS) level and stimulating HO-1 expression. Muscone also exerted anti-inflammation effect through inhibition of p38 phosphorylation. Furthermore, muscone mitigated cisplatin-induced apoptosis in LLC-PK1 cells via both intrinsic and extrinsic pathways by inhibiting pro-apoptotic protein Bax expression, and cleaved caspase-3, 7, and 8; and increase of anti-apoptotic protein Bcl-2 level. In addition, the anti-apoptotic effect of muscone also was enhanced by preventing p53 expression and its phosphorylation. Our study showed that muscone may be a potential protective agent against cisplatin-induced nephrotoxicity.

The Natural Flavonoid Naringenin Inhibits the Cell Growth of Wilms Tumor in Children by Suppressing TLR4/NF-κB Signaling.

Nuclear Factor-kappa B (NF-κB) is usually activated in Wilms Tumor (WT) cells and plays a critical role in WT development. The study's purpose was to screen for a NF-κB inhibitor from the natural product library and explore its effects on WT development. Luciferase assay was employed to assess the effects of natural chemicals on NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Naringenin displayed a significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SKNEP- 1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α- induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in doseand time-dependent manner, whereas Toll-Like Receptor 4 (TLR4) overexpression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in a dose- and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Naringenin inhibits WT development via suppressing TLR4/NF-κB signaling.

Targeting steroid receptor RNA activator (SRA), a long non-coding RNA, enhances melanogenesis through activation of TRP1 and inhibition of p38 phosphorylation.

Abnormal skin melanin homeostasis results in refractory pigmentary diseases. Melanogenesis is influenced by gene regulation, ultraviolet radiation, and host epigenetic responses. Steroid receptor RNA activator (SRA), a long noncoding RNA, is known to regulate steroidogenesis and tumorigenesis. However, how SRA contributes to melanogenesis remains unknown. Using RNA interference against SRA in B16 and A375 melanoma cells, we observed increased pigmentation and increased expression of TRP1 and TRP2 at transcriptional and translational levels only in B16 cells. The constitutive phosphorylation of p38 in B16-shCtrl cells was inhibited in cells with knocked down SRAi. Moreover, the melanin content of control B16 cells was increased by SB202190, a p38 inhibitor. Furthermore, reduced p38 phosphorylation, enhanced TRP1 expression, and hypermelanosis were observed in A375 cells with RNA interference. These results indicate that SRA-p38-TRP1 axis has a regulatory role in melanin homeostasis and that SRA might be a potential therapeutic target for treating pigmentary diseases.

Erythronium japonicum Alleviates Inflammatory Pain by Inhibiting MAPK Activation and by Suppressing NF-κB Activation via ERK/Nrf2/HO-1 Signaling Pathway.

Microglial activation-mediated neuroinflammation influences the development of inflammatory pain. The aim of this study was to investigate the anti-inflammatory effects and mechanisms of aqueous Erythronium japonicum extract (EJE) in microglia activation-mediated inflammatory pain. EJE was found to suppress lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), ionized calcium-binding adapter molecule 1 (IBA-1), and pro-inflammatory cytokines in BV2 microglial cells. In addition, LPS-induced c-Jun NH2 terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation were inhibited by EJE. Intriguingly, EJE also inhibited p65 phosphorylation by activating extracellular signal-regulated kinase-1/2 (ERK)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling. Furthermore, the effects of EJE treatment, such as HO-1 induction and the reduction of NF-ĸB activation, were reversed by ERK1/2 inhibition. In an inflammatory pain mouse model, Complete Freund’s Adjuvant (CFA)-induced mechanical allodynia and foot swelling were alleviated by the oral administration of EJE. Consistent with in vitro results, EJE increased HO-1, while decreasing CFA-induced COX-2, IBA-1, and pro-inflammatory cytokines in the spinal cord. Among the components of EJE, butanol most heavily suppressed LPS-induced microglial activation and increased HO-1 expression. These findings indicate that EJE can alleviate inflammatory pain by inhibiting p38 and JNK and by suppressing NF-ĸB via ERK/Nrf2/HO-1 signaling.

Over-expression of human PP5 gene in mice induces corneal hyperplasia and leads to ocular surface squamous neoplasia

Protein phosphatase 5 (PP5) plays an important role in cell proliferation, differentiation, and development. Transgenic PP5 mice (Tg-hPP5 mice) overexpressing human PP5 gene were successfully generated by embryo injection. Tg-hPP5 mice spontaneously developed corneal hyperplasia and ocular surface squamous neoplasia (OSSN). To investigate the mechanism behind PP5-induced corneal hyperplasia, we performed immunohistochemistry, quantitative real-time PCR, and Western Blotting analyses on the corneas of Tg-hPP5 mice at 2 months and 9 months of age. We provide the first demonstration that Tg-hPP5 mice develop corneal hyperplasia at 9-months of age demonstrated via histological analysis and in vitro co-transfection investigation. We also present data that the expression of p53 is significantly reduced while the expression of FGF-7 is significantly increased in Tg-hPP5 mice with corneal hyperplasia. Co-transfection of PP5, p53, and FGF-7-promoter-driven luciferase revealed that PP5 promotes while p53 inhibits FGF-7 expression, which indicates PP5 overexpression inhibits p53 phosphorylation, thereby reducing its tumor suppressor function and increasing FGF-7 expression. In conclusion, PP5 plays a pivotal role in corneal hyperplasia development and its downregulation is a potential target for corneal hyperplasia and OSSN treatment.

Effect of Angelica gigas Nakai Ethanol Extract and Decursin on Human Pancreatic Cancer Cells

Pancreatic cancer (PC) is one of the most severe cancers, and its incidence and mortality rates have steadily increased in the past decade. In this study, we demonstrate the effect of Angelica gigas Nakai extract on pancreatic ductal adenocarcinoma cells. We prepared A. gigas Nakai ethanol extract (AGE) using roots of A. gigas Nakai and detected its active compound decursin from AGE by ultra-performance liquid chromatography analysis. AGE and decursin significantly decreased viability and colony formation of PANC-1 and MIA PaCa-2 cells. AGE and decursin induced G0/G1 phase arrest through downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). Caspase-3-dependent apoptosis of PANC-1 cells was promoted by AGE and decursin. Additionally, nontoxic concentrations of AGE and decursin treatment could suppress matrix metalloproteinase (MMP)-2 and MMP-9 expression and activity by inhibiting p38 phosphorylation. Taken together, this study demonstrates that AGE and decursin have potential properties to be considered in PC treatment.

Glycyrrhizin Inhibits PEDV Infection and Proinflammatory Cytokine Secretion via the HMGB1/TLR4-MAPK p38 Pathway.

Our previous study showed that glycyrrhizin (GLY) inhibited porcine epidemic diarrhea virus (PEDV) infection, but the mechanisms of GLY anti-PEDV action remain unclear. In this study, we focused on the anti-PEDV and anti-proinflammatory cytokine secretion mechanisms of GLY. We found that PEDV infection had no effect on toll-like receptor 4 (TLR4) protein and mRNA levels, but that TLR4 regulated PEDV infection and the mRNA levels of proinflammatory cytokines. In addition, we demonstrated that TLR4 regulated p38 phosphorylation but not extracellular regulated protein kinases1/2 (Erk1/2) and c-Jun N-terminal kinases (JNK) phosphorylation, and that GLY inhibited p38 phosphorylation but not Erk1/2 and JNK phosphorylation. Therefore, we further explored the relationship between high mobility group box-1 (HMGB1) and p38. We demonstrated that inhibition of HMGB1 using an antibody, mutation, or knockdown decreased p38 phosphorylation. Thus, HMGB1 participated in activation of p38 through TLR4. Collectively, our data indicated that GLY inhibited PEDV infection and decreased proinflammatory cytokine secretion via the HMGB1/TLR4-mitogen-activated protein kinase (MAPK) p38 pathway.

Protective role of endothelial calpain knockout in lipopolysaccharide-induced acute kidney injury via attenuation of the p38-iNOS pathway and NO/ROS production

To explore the role of calpain and its signaling pathway in lipopolysaccharide (LPS)-induced acute kidney injury (AKI), animal models of endotoxemia were established by administration of LPS to mice with endothelial-specific Capn4 knockout (TEK/Capn4−/−), mice with calpastatin (an endogenous calpain inhibitor) overexpression (Tg-CAST) and mice with myeloid-specific Capn4 knockout (LYZ/Capn4−/−). Mouse pulmonary microvascular endothelial cells (PMECs) were used as a model of the microvascular endothelium and were stimulated with LPS. Renal function, renal inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) expression, cellular apoptosis, plasma and renal levels of NO and reactive oxygen species (ROS), and phosphorylation of mitogen-activated protein kinase (MAPK) family members (p38, ERK1/2, and JNK1/2) were examined. Moreover, a calpain inhibitor, calpastatin overexpression adenoviruses and MAPK inhibitors were used. Significant renal dysfunction was induced by LPS stimulation, and recovery was observed in TEK/Capn4−/− and Tg-CAST mice but not in LYZ/Capn4−/− mice. Endothelial Capn4 knockout also abrogated the LPS-induced increases in renal iNOS expression, caspase-3 activity and apoptosis and plasma and renal NO and ROS levels but did not obviously affect renal eNOS expression. Moreover, LPS increased both calpain and caspase-3 activity, and only the expression of iNOS in PMECs was accompanied by increased phosphorylation of p38 and JNK. Inhibiting calpain activity or p38 phosphorylation alleviated the increased iNOS expression, NO/ROS production, and cellular apoptosis induced by LPS. These results suggest that endothelial calpain plays a protective role in LPS-induced AKI by inhibiting p38 phosphorylation, thus attenuating iNOS expression and further decreasing NO and ROS overproduction-induced endothelial apoptosis.

Antifibrotic Effects of the Thiazolidinediones in Eosinophilic Esophagitis Pathologic Remodeling: A Preclinical Evaluation.

INTRODUCTION:Eosinophilic esophagitis (EoE) is a T-helper 2 (Th2), eosinophilic disease associated with pathologic tissue remodeling that leads to end-organ dysfunction. During early-stage disease, inflammation and subepithelial fibrosis are coupled and reversible, but in late-stage or therapy-resistant disease, there can be uncoupling of these features with progressive esophageal rigidity and strictures contributing to clinical dysphagia and food impactions. No current pharmacotherapeutic interventions directly target esophageal fibrosis. Based on the ability of the thiazolidinediones (TZD) to regulate intestinal and hepatic fibrosis, we tested the antifibrotic effects of the TZDs, rosiglitazone and pioglitazone, in preclinical studies using primary human esophageal fibroblasts.METHODS:Primary fibroblasts isolated from normal or EoE esophagi were treated with transforming growth factor (TGF)-β1 in the absence or presence of TZDs and, in some experiments, without or with budesonide and analyzed by quantitative real-time PCR and immunoblotting. Immunohistochemical analysis of human esophageal biopsies was performed.RESULTS:EoE esophageal biopsies and esophageal fibroblasts expressed higher levels of the TZD receptor, peroxisome proliferator-activated receptor-γ (PPAR-γ), than normal controls. PPAR-γ was inducible by the Th2 cytokine, interleukin 4 (IL-4). TZD significantly reduced TGF-β1-induced myofibroblast and fibrotic gene and protein expression preferentially in EoE, but not normal esophageal fibroblasts. In esophageal fibroblasts, TGF-β1 increased phosphorylated Smad2/3 and p38, but TZDs preferentially inhibited p38 phosphorylation, suggesting signaling pathway-specific effects. The TZDs were more potent than budesonide at decreasing collagen-1α1 expression.DISCUSSION:The TZDs preferentially exert antifibrotic effects in TGF-β1-activated EoE fibroblasts and provide a preclinical foundation for further investigation of the potential of the TZDs in EoE pathologic remodeling.

Amelioration of LPS‐induced inflammatory response and oxidative stress by astaxanthin in Channa argus lymphocyte via activating glucocorticoid receptor signalling pathways

The present study was conducted to evaluate the effects of astaxanthin (AST) against lipopolysaccharide (LPS)‐induced lymphocyte viability, ultrastructural lesions, apoptosis, oxidative stress and inflammatory responses in Channa argus. Lymphocytes exposed to more than 10 μg/ml LPS alone for 24 hr showed significantly decreased cell viability, elevated nitric oxide (NO) and malondialdehyde (MDA), lactate dehydrogenase (LDH) contents, and increased nuclear factor κB p65 (NF‐κB p65), myeloid differential protein‐88 (MyD88), tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐8 (IL‐8), caspase‐3, caspase‐8 and caspase‐9 gene expression. LPS at a concentration of 10 μg/ml could induce oxidative stress and inflammatory responses in lymphocytes. The activities of antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD)) were significantly decreased after exposure to 10 μg/ml LPS. Besides, AST strikingly antagonized the LPS‐induced negative effects. AST significantly increased the expression of HSP70, HSP90, IκB‐α, and glucocorticoid receptor (GR) and decreased inflammatory responses. Further study showed that AST can activate GR signalling pathway and inhibit p65 phosphorylation. In addition, AST attenuated LPS‐induced apoptosis, mitochondrial swelling, degeneration and vacuolization. Collectively, these findings suggest that AST has protective roles in LPS‐induced cell damage via modulating GR activation in C. argus lymphocytes.

CXCL16 silencing alleviates hepatic ischemia reperfusion injury during liver transplantation by inhibiting p38 phosphorylation

CXC chemokine ligand 16 (CXCL16) has been reported to exacerbate acute kidney injury induced by ischemia-reperfusion (IR). This study aimed to investigate the probable role of CXCL16 in hepatic IR injury during liver transplantation. The expression patterns of CXCL16 and its receptor CXC chemokine receptor 6 (CXCR6) were detected in mouse models of IR injury during liver transplantation and cell models of oxygen-glucose deprivation and reoxygenation (OGD/R)-induced hepatocyte injury using RT-qPCR, Western blot analysis and ELISA. CXCL16 expression was silenced in AML12 cells exposed to OGD/R conditions to determine the role of CXCL16 in cell apoptosis and injury. After treatment with CXCL16 and a p38 phosphorylation inhibitor, SB203580, we examined whether CXCL16 regulated p38 phosphorylation to impact hepatocyte injury. To verify the effects of CXCL16 and p38 phosphorylation in vivo, CXCL16 was silenced and p38 phosphorylation was activated in IR-treated mice. CXCL16 and CXCR6 were highly expressed in mouse models of IR injury and cell models of OGD/R-induced hepatocyte injury. Silencing of CXCL16 in AML12 cells resulted in diminished CXCR6 expression and alleviated OGD/R-exposed cell injury. CXCL16 treatment in AML12 cells brought about increased protein expressions of CXCR6 and p38 phosphorylation and elevated apoptosis rate of hepatocytes. Inhibition of p38 phosphorylation neutralized CXCL16-induced apoptosis of AML12 cells. Furthermore, CXCL16 knockdown in vivo relieved hepatic injury, which was reversed by activation of p38 phosphorylation. Taken together, silencing of CXCL16 might protect against IR injury during liver transplantation by reducing p38 phosphorylation, highlighting the potential of CXCL16 as a promising target for treatment against hepatic IR injury.

Glaucocalyxin A inhibits hydrogen peroxide‐induced oxidative stress and inflammatory response in coronary artery smooth muscle cells

Atherosclerosis is a complex chronic inflammatory disease that remains one of the leading causes of death and disability worldwide. A previous study reported that glaucocalyxin A (GLA), a natural ent-Kaurane diterpenoid triptolide, exhibits anti-atherosclerotic activity. However, the underlying molecular mechanism has not yet been explored. In the present study, we evaluated the anti-atherosclerotic effect of GLA and the underlying mechanism in vitro. Human coronary artery smooth muscle cells (HCASMCs) were stimulated by hydrogen peroxide (H2 O2 ) to induce oxidative stress and inflammation. The results showed that GLA pretreatment improved the viability of H2 O2 -induced HCASMCs. The increased reactive oxygen species production and decreased superoxide dismutase and glutathione peroxidase activities in H2 O2 -induced HCASMCs were reversed by GLA pretreatment. In addition, GLA treatment suppressed the H2 O2 -induced expression of inducible nitric oxide synthase, NADPH oxidase (NOX) 2, and NOX4 in HCASMCs. Moreover, treatment with GLA reduced the production of several inflammatory cytokines, including tumour necrosis factor-alpha, interleukin (IL)-6, and IL-1β in H2 O2 -induced HCASMCs. Furthermore, GLA treatment suppressed the phosphorylation of p38, as well as inactivating the NF-κB signalling pathway. These findings suggested that GLA protected against H2 O2 -induced oxidative stress and inflammation via inhibition of p38 phosphorylation and NF-κB activation in HCASMCs.

Resveratrol attenuates IL‑33‑induced mast cell inflammation associated with inhibition of NF‑κB activation and the P38 signaling pathway.

Resveratrol (RSV), a natural polyphenol found in grapes and other herbal plants, has been reported to possess anti‑inflammatory, anti‑oxidative and anti‑proliferative activities. The aim of the present study was to investigate the effect of RSV on interleukin (IL)‑33‑induced inflammatory responses in mast cells and identify the underlying molecular mechanisms. Rat basophilic leukemia (RBL‑2H3)cells were stimulated with IL‑33 in the presence or absence of RSV. MTT, ELISA, reverse transcription‑quantitative PCR and western blot analyses were then performed in order to assess cytotoxicity, inflammatory cytokine production, suppression of tumorigenicity2receptor expression, protein expression involved in mitogen‑activated protein kinase (MAPK) and nuclear factor (NF)‑κB signaling, respectively. Finally, rats were used to determine the biological effect of RSV invivo. The results revealed that RSV inhibited cell viability and increased cytotoxicity in a dose‑dependent manner. Medium concentration of RSV (10µM) treatment attenuated inflammatory cytokine production, such as IL‑6, IL‑13, tumor necrosis factor‑α and monocyte chemotactic protein‑1, and curbed IL‑33‑induced enhancement of immunoglobulin E‑mediated responses in RBL‑2H3cells, which were associated with the suppression of NF‑κB‑mediated transcription and inhibition of P38 phosphorylation in response to IL‑33 stimulation, but not extracellular signal regulated kinase or JNK. Notably, RSV application also decreased the levels of inflammatory cytokines in rats induced by IL‑33 injection, which was similar to the anti‑inflammatory effect invitro. The data from the present study demonstrated that RSV played a regulatory role in antagonizing the effects of IL‑33 on mast cells both invitro and invivo, suggesting that it has therapeutic potential in IL‑33‑mediated inflammatory diseases that are associated with mast cells.

Anti-inflammatory activity of the extracts from Rodgersia podophylla leaves through activation of Nrf2/HO-1 pathway, and inhibition of NF-κB and MAPKs pathway in mouse macrophage cells.

Recently, Rodgersia podophylla has been reported to exhibit anti-inflammatory activity. However, little is known about the potential mechanisms about its anti-inflammatory activity. We elucidated the anti-inflammatory mechanisms of leaves extracts from Rodgersia podophylla (RP-L) in RAW264.7 cells. LPS-induced NO was measured by Griess and mRNA of pro-inflammatory mediators was analyzed by RT-PCR. Cell viability was measured using MTT assay. The protein level was analyzed by Western blot. RP-L significantly inhibited the production of the pro-inflammatory mediators such as NO, iNOS, IL-1β and IL-6 in LPS-stimulated RAW264.7 cells. RP-L increased HO-1 expression in RAW264.7 cells, and the inhibition of HO-1 by ZnPP reduced the inhibitory effect of RP-L against LPS-induced NO production in RAW264.7 cells. Inhibition of p38, ROS and GSK3β attenuated RP-L-mediated HO-1 expression. Inhibition of ROS inhibited p38 phosphorylation and GSK3β expression induced by RP-L. In addition, inhibition of GSK3β blocked RP-L-mediated p38 phosphorylation. RP-L induced nuclear accumulation of Nrf2, and inhibition of p38, ROS and GSK3β abolished RP-L-mediated nuclear accumulation of Nrf2. Furthermore, RP-L blocked LPS-induced degradation of IκB-α and nuclear accumulation of p65. RP-L also attenuated LPS-induced phosphorylation of ERK1/2 and p38. In GC/MS analysis of RP-L, pyrogallol was detected as bioactive compound for anti-inflammatory activity of RP-L. Pyrogallol was observed to activate HO-1 expression through ROS/GSK3β/p38/Nrf2/HO-1 signaling. Our results suggest that RP-L exerts potential anti-inflammatory activity by activating ROS/GSK3β/p38/Nrf2/HO-1 signaling and inhibiting NF-κB and MAPK signaling in RAW264.7 cells. These findings suggest that RP-L may have great potential for the development of anti-inflammatory drug.

Photoaging Protective Effects of Ranunculus bulumei Methanol Extract.

Ultraviolet B (UVB) radiation is the main cause of photoaging processes including cellular senescence, skin drying, collagen degradation, melanogenesis, and inflammation. These responses occur because UVB induces a change in expression of aging-related genes through regulation of signal pathways such as that of mitogen-activated protein kinases- (MAPKs-) activator protein 1 (AP-1). Ranunculus bulumei, which is used as an herb in Indonesia, belongs to the Ranunculaceae family, which has been reported to perform various physiological effects including antioxidant and anti-inflammation. However, data on the pharmaceutical and cosmeceutical utility of Ranunculus bulumei have not been reported. Therefore, we evaluated the antiaging efficacy of RB-ME, a methanol extract of Ranunculus bulumei. Rb-ME attenuated MMP9 and COX-2 gene expression but enhanced SIRT1 and type-1 collagen in UVB-irradiated HaCaT cells. Rb-ME regulated these gene expressions through inhibition of p38 phosphorylation and inactivation of AP-1. In addition, mRNA expression of HAS-2 and -3, which are involved in skin hydration, was elevated in Rb-ME-treated HaCaT cells. Rb-ME also inhibited melanogenesis by suppression of tyrosinase, MITF, and TYRP-1 mRNA in B16F10 cells under α-MSH treatment. Taken together, these results indicate that Rb-ME has a protective effect on some UVB-induced skin photoaging events such as inflammation, collagen degradation, cellular senescence, skin drying, and melanin production through inhibition of the p38-AP-1 signal cascade, indicating that Rb-ME can be used as an active ingredient for antiaging cosmetics.

Wild-type p53-induced phosphatase 1 (WIP1) regulates the proliferation of swine Sertoli cells through P53.

Wild-type p53-induced phosphatase 1 (WIP1) plays an oncogenic function by increasing cell proliferation in various cancer types. Deficiency in WIP1 expression leads to male infertility, possibly by impairing the blood-testis barrier and spermatogenesis. However, how WIP1 functions in the Sertoli cells to affect male reproduction remains unclear. Thus, in the present study we used a swine Sertoli cell line to investigate whether WIP1 regulated the proliferation of Sertoli cells to participate in male reproduction. The WIP1 inhibitor GSK2830371, WIP1-short interference (si) RNAs and an upstream microRNA (miR-16) were used to inhibit the expression of WIP1, after which the proliferation of swine Sertoli cells, P53 expression and the levels of P53 phosphorylation were determined. Inhibiting WIP1 expression suppressed swine Sertoli cell proliferation, increased P53 expression and increased levels of P53 phosphorylation. In addition, overexpression of miR-16 in swine Sertoli cells resulted in a decrease in WIP1 expression and increases in both P53 expression and P53 phosphorylation. Together, these findings suggest that WIP1 positively regulates the proliferation of swine Sertoli cells by inhibiting P53 phosphorylation, and the miR-16 is likely also involved by targeting WIP1.

Wogonin ameliorate complete Freund's adjuvant induced rheumatoid arthritis via targeting NF-κB/MAPK signaling pathway.

Rheumatoid arthritis (RA) is a chronic and accelerated autoimmune illness with proliferative and damaging synovitis, resulting in joint death and cartilage and bone erosion. This study focused on the potential therapeutic effect of wogonin on complete Freund's adjuvant (CFA) induced RA in rats and the underlying mechanisms. Arthritis was experimentally caused in rats by subcutaneously injecting 0.1 mL of CFA into the subplantar area of the left hind paw under moderate anesthesia on day zero. The regular oral doses of indomethacin/wogonin began on day zero and proceeded after injection to day 35. Wogonin reduced arthritic score considerably, enhanced body weight, and reduced paw thickness. Wogonin also boosted red blood cell considerably along with hemoglobin and reduced white blood cell count and erythrocyte sedimentation rate. Wogonin substantially improved an altered level of oxidative stress markers, antioxidant proteins, and inflammatory cytokines in a dose-dependent way. Wogonin inhibited p38 phosphorylation triggered by CFA and p65 nuclear translocation.

CBMT-32. EGFR SUPPRESSES p53 FUNCTION THROUGH DNA-PKcs BINDING TO p53: NOVEL CROSSTALK BETWEEN EGFR AND TP53 IN GLIOBLASTOMA

Abstract Comprehensive analysis of the TCGA Glioblastoma database has identified 3 prognostically relevant subgroups (Proneural, Classical and Mesenchymal). Interestingly, it has also revealed a near mutual exclusivity of EGFR amplification and TP53 mutations. There are only a few tumors with both amplified EGFR and mutant TP53. We decided to investigate the functional relationship between EGFR and TP53 relative to tumorigenesis and tumor proliferation. We selected from our molecularly characterized GSC bank a series of EGFR amplified+TP53wt/TP53mut lines for this study. Here we report that EGFR amplification overrides the effect of p53wt but co-regulates tumorigenesis with TP53R175H mutation. We show that CRISPR EGFR knockout in TP53wt cells leads to complete loss of cell growth suggesting that EGFR is essential for cell survival, providing growth signal and suppression of p53wt function. We further show that EGFR induces physical binding between p53wt and DNA-PKcs, which suppresses p53wt by inhibiting p53 phosphorylation at S15. The knockdown of DNA-PKcs restores p53wt anti-tumor function. In contrast, GSC262 harboring TP53R175H mutation is the only GSC that survives after EGFR knockout, suggesting that this specific mutation has a gain of function (GOF) to sustain growth without EGFR. This is confirmed by dual knockout of EGFR and TP53R175H showing no surviving cells. Further investigation shows both EGFR and p53R175H induce tumor glycolysis, the major energy metabolism and heightened growth signal for this group. We further show that EGFR mediated suppression of p53R175H is through DNA-PKcs binding to p53R175H and reduced S15 phosphorylation. Taken together, this study reveals 1) EGFR amplification is essential for tumor growth in TP53wt GSCs; 2) novel crosstalk between EGFR and p53 through DNA-PKcs. 3) EGFR suppresses p53wt function by providing the energy required for tumor growth through inducing glycolysis, and 4) TP53R175H is a GOF mutation that sustains life without EGFR by inducing glycolysis.