Suppressing P38 Mitogen-activated Protein Kinase Research Articles

The protective mechanism of human umbilical cord mesenchymal stem cell-derived exosomes against neutrophil extracellular trap-induced placental damage

IntroductionPreeclampsia (PE) is a pregnancy-specific complication. Its etiology and pathogenesis remain unclear. Previous studies have shown that neutrophil extracellular traps (NETs) cause placental dysfunction and lead to PE. Human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) have been widely used to treat different diseases. We investigated whether hUCMSC-EXOs can protect against NET-induced placental damage. MethodsNETs were detected in the placenta by immunofluorescence. The impact of NETs on cellular function and the effect of hUCMSC-EXOs on NET-induced placental damage were evaluated by 5-ethynyl-20-deoxyuridine (EdU) cell proliferation, lactate dehydrogenase (LDH), reactive oxygen species (ROS), and cell migration, invasion and tube formation assays; flow cytometry; and Western blotting. ResultsThe number of placental NETs was increased in PE patients compared with control individuals. NETs impaired the function of endothelial cells and trophoblasts. These effects were partially reversed after N-acetyl-L-cysteine (NAC; ROS inhibitor) or DNase I (NET lysing agent) pretreatment. HUCMSC-EXOs ameliorated NET-induced functional impairment of endothelial cells and trophoblasts in vitro, partially reversed NET-induced inhibition of endothelial cell and trophoblast proliferation, and partially restored trophoblast migration and invasion and endothelial cell tube formation. Exosomes inhibited ROS production in these two cell types, suppressed p38 mitogen-activated protein kinase (p38 MAPK) signaling activation, activated extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, and modulated the Bax, Bim, Bcl-2 and cleaved caspase-3 levels to inhibit apoptosis. DiscussionHUCMSC-EXOs can reverse NET-induced placental endothelial cell and trophoblast damage, possibly constituting a theoretical basis for the treatment of PE with exosomes.

Selenium nanoparticles improve nickel-induced testosterone synthesis disturbance by down-regulating miR-708-5p/p38 MAPK pathway in Leydig cells.

The present study was designed to investigate the role of miR-708-5p/p38 mitogen-activated protein kinase (MAPK) pathway during the mechanism of selenium nanoparticles (Nano-Se) against nickel (Ni)-induced testosterone synthesis disorder in rat Leydig cells. We conducted all procedures based on in vitro culture of rat primary Leydig cells. After treating Leydig cells with Nano-Se and NiSO4 alone or in combination for 24 h, we determined the cell viability, reactive oxygen species (ROS) levels, testosterone production, and the protein expression of key enzymes involved in testosterone biosynthesis: steroidogenic acute regulatory (StAR) and cytochrome P450 cholesterol side chain cleavage enzyme (CYP11A1). The results indicated that Nano-Se antagonized cytotoxicity and eliminated ROS generation induced by NiSO4 , suppressed p38 MAPK protein phosphorylation and reduced miR-708-5p expression. Importantly, we found that Nano-Se upregulated the expression of testosterone synthase and increased testosterone production in Leydig cells. Furthermore, we investigated the effects of p38 MAPK and miR-708-5p using their specific inhibitor during Nano-Se against Ni-induced testosterone synthesis disorder. The results showed that Ni-inhibited testosterone secretion was alleviated by Nano-Se co-treatment with p38 MAPK specific inhibitor SB203580 and miR-708-5p inhibitor, respectively. In conclusion, these findings suggested Nano-Se could inhibit miR-708-5p/p38 MAPK pathway, and up-regulate the key enzymes protein expression for testosterone synthesis, thereby antagonizing Ni-induced disorder of testosterone synthesis in Leydig cells.

Osteocrin ameliorates adriamycin nephropathy via p38 mitogen-activated protein kinase inhibition

Natriuretic peptides exert multiple effects by binding to natriuretic peptide receptors (NPRs). Osteocrin (OSTN) binds with high affinity to NPR-C, a clearance receptor for natriuretic peptides, and inhibits degradation of natriuretic peptides and consequently enhances guanylyl cyclase-A (GC-A/NPR1) signaling. However, the roles of OSTN in the kidney have not been well clarified. Adriamycin (ADR) nephropathy in wild-type mice showed albuminuria, glomerular basement membrane changes, increased podocyte injuries, infiltration of macrophages, and p38 mitogen-activated protein kinase (MAPK) activation. All these phenotypes were improved in OSTN- transgenic (Tg) mice and NPR3 knockout (KO) mice, with no further improvement in OSTN-Tg/NPR3 KO double mutant mice, indicating that OSTN works through NPR3. On the contrary, OSTN KO mice increased urinary albumin levels, and pharmacological blockade of p38 MAPK in OSTN KO mice ameliorated ADR nephropathy. In vitro, combination treatment with ANP and OSTN, or FR167653, p38 MAPK inhibitor, reduced Ccl2 and Des mRNA expression in murine podocytes (MPC5). OSTN increased intracellular cyclic guanosine monophosphate (cGMP) in MPC5 through GC-A. We have elucidated that circulating OSTN improves ADR nephropathy by enhancing GC-A signaling and consequently suppressing p38 MAPK activation. These results suggest that OSTN could be a promising therapeutic agent for podocyte injury.

S-deoxydihydroglyparvin from Glycosmis parva inhibits lipopolysaccharide induced murine macrophage activation through inactivating p38 mitogen activated protein kinase

Macrophages play major roles to produce several pro-inflammatory and inflammatory mediators in chronic inflammatory diseases. All current anti-inflammatory drugs target these mediators to alleviate inflammation. Searching for new anti-inflammatory agents is always needed due to problems from the clinical use of current anti-inflammatory drugs. We intended to evaluate the anti-inflammatory potential of three main compounds, arborinine, methylatalaphylline, and S-deoxydihydroglyparvin (DDGP), from Glycosmis parva leaves and branches on macrophage stimulated by lipopolysaccharide (LPS). Only DDGP demonstrated a potent inhibitor of LPS-activated macrophages. Results indicated that the mRNA level of inducible nitric oxide synthase (iNOS) was inhibited by the treatment in accompany with the decreased nitric oxide (IC50 at 3.47 ± 0.1 μM). DDGP was shown to suppress tumor necrosis factor-α, interleukin (IL)-1, and IL-6 at the mRNA expression and at the released protein levels. In addition, DDGP inhibited the several chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory proteins-1α, and enzymes for prostaglandin (PG) synthesis. It also inhibited PGE2 production. On LPS signaling pathways, DDGP profoundly decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK) in the LPS-treated cells. It had little or no effect on the activation of JNK, ERK and nuclear factor kappa B. In conclusion, results suggested that DDGP from G. parva inhibited expression and production of inflammatory molecules in LPS-activated macrophages through suppressing p38 MAPK activation. DDGP should be a good candidate anti-inflammatory agent in the future.

Resveratrol protects human bronchial epithelial cells against nickel-induced toxicity via suppressing p38 MAPK, NF-κB signaling, and NLRP3 inflammasome activation.

Nickel is a common environmental pollutant that can impair the lung, but the underlying mechanisms have not yet been fully elucidated. Furthermore, natural products are generally used to inhibit cell damage induced by heavy metal. Resveratrol possesses wide biological activities, including anti-inflammation and antioxidative stress. This study was conducted to explore the toxicity of nickel on human bronchial epithelial (BEAS-2B) cells and evaluate the protective effect of resveratrol. The results showed that nickel could induce cell apoptosis, increase oxidative stress, and promote the expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-8, C-reaction protein. Western blot analysis showed that nickel activated p38 mitogen-activated protein kinase (MAPK), nuclear factor-kappa B, and nucleotide-binding oligomerization domain-like receptor pyrin-domain-containing protein 3 pathways, while resveratrol could reverse these effects. Our results suggested that resveratrol could protect BEAS-2B cells from nickel-induced cytotoxicity. Therefore, resveratrol is a potential chemopreventive agent against nickel-induced lung disease.

Ferrostatin-1 mitigates cognitive impairment of epileptic rats by inhibiting P38 MAPK activation

Evidence indicates that ferrostain-1 (Fer-1), a specific inhibitor of ferroptosis, could ameliorate cognitive dysfunction of rats with kainic acid (KA)-induced temporal lobe epilepsy (TLE) by suppressing ferroptosis processes. Recent studies suggest that P38 mitogen-activated protein kinase (MAPK) pathway could be mediated by ferroptosis processes. The activation of P38 MAPK results in cognitive impairment by suppressing the expression of synaptic plasticity-related proteins. However, it is unclear whether Fer-1 can mitigate cognitive impairment of rats with KA-induced TLE by inhibiting P38 MAPK activation. In the present study, treatment with Fer-1 blocked the activation of P38 MAPK, which resulted in an increased expression of synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95) in the hippocampus of rats with KA-induced TLE, hence, ameliorating their cognitive impairment. Also, P38 MAPK activation in the hippocampus of the rats reduced the expression of both PSD-95 and SYP proteins. Treatment of the rats with SB203580, a P38 MAPK-specific inhibitor, prevented the activation of P38 MAPK, which resulted in an increase in SYP and PSD95 protein levels in the hippocampus. These results suggest that Fer-1 could mitigate the cognitive impairment by suppressing P38 MAPK activation thus restoring the expression of synaptic proteins. Ferroptosis processes might be involved in suppressing synaptic protein expression.

Low-intensity pulsed ultrasound attenuates cardiac inflammation of CVB3-induced viral myocarditis via regulation of caveolin-1 and MAPK pathways.

The aggressive immunological activity elicited by acute viral myocarditis contributes to a large amount of cardiomyocytes loss and poor prognosis of patients in clinic. Low‐intensity pulsed ultrasound (LIPUS), which is an effective treatment modality for osteoarthropathy, has been recently illustrated regulating the overactive inflammatory response in various diseases. Here, we aimed to investigate whether LIPUS could attenuate coxsackievirus B3 (CVB3) infection‐induced injury by coordinating the inflammatory response. Male BALB/c mice were inoculated intraperitoneally with CVB3 to establish the model of acute viral myocarditis. LIPUS treatment was given on Day 1, Day 1, 3 and Day 1, 3, 5 post‐inoculation, respectively. All mice were followed up for 14 days. Day 1, 3, 5 LIPUS treatment significantly improved the survival rate, attenuated the ventricular dysfunction and ameliorated the cardiac histopathological injury of CVB3‐infected mice. Western blotting analysis showed Day 1, 3, 5 LIPUS treatment decreased pro‐inflammatory cytokines, increased the activation of caveolin‐1 and suppressed p38 mitogen‐activated protein kinase (MAPK) and extracellular signal‐regulated kinase (ERK) signallings in heart tissue. RAW264.7 cells were treated with lipopolysaccharides (LPS) to simulate the augmented inflammatory response in vivo. LIPUS treatment on RAW264.7 inhibited the expression of pro‐inflammatory cytokines, activated caveolin‐1 and suppressed p38 MAPK and ERK signallings. Transfecting RAW264.7 with caveolin‐1 siRNA blunted the suppression of pro‐inflammatory cytokines and MAPK signallings by LIPUS treatment. Taken together, we demonstrated for the first time that LIPUS treatment attenuated the aggressive inflammatory response during acute viral myocarditis. The underlying mechanism may be activating caveolin‐1 and suppressing MAPK signallings.

Glucocorticoids induce apoptosis and matrix metalloproteinase-13 expression in chondrocytes through the NOX4/ROS/p38 MAPK pathway

Based on the results from our previous study, dexamethasone (Dex) increases reactive oxygen species (ROS) levels and subsequently induces cell death and matrix catabolism in chondrocytes. Nevertheless, the mechanism underlying this phenomenon remains unclear. Nicotinamide adenine dinucleotide (phosphate) (NADPH) oxidase 4 (NOX4) is one of the major enzymes responsible for intracellular ROS production during the inflammatory process. The objective of the current study was to investigate the role of NOX4 in Dex-induced ROS over-production. Healthy chondrocytes were harvested from the cartilage debris from 6 female patients. NOX4 and p38 mitogen-activated protein kinase (MAPK) expression levels in these cells were evaluated in the presence of Dex. Changes in the number of apoptotic and viable Dex-treated chondrocytes were recorded after the cells were treated with NOX and p38 MAPK inhibitors. Changes in matrix metalloproteinase 13 (MMP-13) expression levels in Dex-treated chondrocytes were also investigated. The Dex treatment increased NOX4 expression via the glucocorticoid receptor (GR). Treatment of cells with apocynin, a NOX inhibitor, decreased intracellular ROS levels and inhibited p38 MAPK activation. Treatment of cells with a ROS scavenger also reduced p38 MAPK expression. Treatment of cells with a NOX inhibitor, ROS scavenger and p38 MAPK inhibitor rescued chondrocytes from Dex-induced apoptosis. Moreover, treatment of cells with these agents blocked MMP-13 expression in Dex-treated chondrocytes. NOX4 silencing also suppressed p38 MAPK and MMP-13 expression. Dex triggered apoptosis and MMP-13 expression through the NOX4/ROS/p38 MAPK signaling pathway. NOX4 may be a therapeutic target in the management of Dex-induced complications.

Low-level laser therapy prevents endothelial cells from TNF-α/cycloheximide-induced apoptosis.

Low-level laser therapy (LLLT), widely used in physiotherapy, has been known to enhance wound healing and stimulate cell proliferation, including fibroblast and endothelial cells. Applying LLLT can increase cell proliferation in many kinds of cells including fibroblasts and endothelial cells. However, the protective mechanisms of LLLT on endothelial apoptosis remain unclear. We hypothesized LLLT can protect endothelial cells from inflammation-induced apoptosis. Human endothelial cell line, EA.hy926 cells, and TNF-α/cycloheximide (TNF/CHX) were used to explore the protective effects of LLLT (660nm) on inflammation-induced endothelial apoptosis. Cell viability, apoptosis, caspase-3/7/8/9 activity, MAPKs signaling, NF-κB activity, and inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression were measured. Our results showed that LLLT increased EA.hy926 cell proliferation, attenuated the TNF/CHX-induced apoptosis, and reduced the TNF/CHX-mediated caspase-3/7/8/9 activation. In addition, LLLT increased ERK MAPK phosphorylation and suppressed the TNF/CHX-increased p38 MAPK, JNK, IKK phosphorylation, NF-κB translocation, and iNOS expression. The caspases-3 cleavage and cell death were not increased in cells treating with ERK inhibitor U0126, which implicated that ERK is not to be responsible for the protective effects of LLLT. After treating with p38 mitogen-activated protein kinase (MAPK) activator, the protection of LLLT in cell apoptosis was no longer existed, showing that LLLT protected the endothelial cells by suppressing p38 MAPK signaling. Our results provide a new insight into the possible molecular mechanisms in which LLLT protects against inflammatory-induced endothelial dysfunction.

Surfactant protein D attenuates nitric oxide-stimulated apoptosis in rat chondrocyte by suppressing p38 MAPK signaling

Innate immune molecule surfactant protein D (SP-D), a member of the C-type lectin protein family, plays an indispensable role in host defense and the regulation of inflammation in the lung and other tissues. Osteoarthritis (OA) is a degenerative disease of cartilage, with inflammation that causes pathologic changes and tissue damage. However, it is unknown whether there exist SP-D expression and its potential role in the pathogenesis of OA. In this study, we examined SP-D expression and explored its biological function in a sodium nitroprusside (SNP)-stimulated rat chondrocytes and surgically-induced rat OA model. We found SP-D expression in both human and rat articular chondrocytes, with higher level in normal chondrocytes compared to in OA chondrocytes. Furthermore, In vivo study demonstrated that recombinant human SP-D (rhSP-D) ameliorated cartilage degeneration in surgically-induced rat OA model. In vitro cell culture study showed that rhSP-D markedly inhibited the expression of caspase-3 as an apoptosis biomarker, and decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which resulted in maintaining normal nuclear morphology and increasing mitochondrial membrane potential in SNP-stimulated rat chondrocytes. Collectively, these findings indicate that SP-D expresses in articular chondrocytes and suppresses SNP-stimulated chondrocyte apoptosis and ameliorates cartilage degeneration via suppressing p38 MAPK activity.

Gingerol Inhibits Serum-Induced Vascular Smooth Muscle Cell Proliferation and Injury-Induced Neointimal Hyperplasia by Suppressing p38 MAPK Activation.

Gingerol inhibits growth of cancerous cells; however, its role in vascular smooth muscle cell (VSMC) proliferation is not known. The present study investigated the effect of gingerol on VSMC proliferation in cell culture and during neointima formation after balloon injury. Rat VSMCs or carotid arteries were harvested at 15 minutes, 30 minutes, 1, 6, 12, and 24 hours of fetal bovine serum (FBS; 10%) stimulation or balloon injury, respectively. Gingerol prevented FBS (10%)-induced proliferation of VSMCs in a dose-dependent manner (50 μmol/L-400 μmol/L). The FBS-induced proliferating cell nuclear antigen (PCNA) upregulation and p27(Kip1) downregulation were also attenuated in gingerol (200 μmol/L) pretreated cells. Fetal bovine serum-induced p38 mitogen-activated protein kinase (MAPK) activation, PCNA upregulation, and p27(Kip1) downregulation were abrogated in gingerol (200 μmol/L) and p38 MAPK inhibitor (SB203580, 10 μmol/L) pretreated cells. Balloon injury induced time-dependent p38 MAPK activation in the carotid artery. Pretreatment with gingerol (200 μmol/L) significantly attenuated injury-induced p38 MAPK activation, PCNA upregulation, and p27(Kip1) downregulation. After 14 days of balloon injury, intimal thickening, neointimal proliferation, and endothelial dysfunction were significantly prevented in gingerol pretreated arteries. In isolated organ bath studies, gingerol (30 nmol/L-300 μmol/L) inhibited phenylephrine-induced contractions and induced dose-dependent relaxation of rat thoracic aortic rings in a partially endothelium-dependent manner. Gingerol prevented FBS-induced VSMC proliferation and balloon injury-induced neointima formation by regulating p38 MAPK. Vasodilator effect of gingerol observed in the thoracic aorta was partially endothelium dependent. Gingerol is thus proposed as an attractive agent for modulating VSMC proliferation, vascular reactivity, and progression of vascular proliferative diseases.

Berberine prevents nitric oxide-induced rat chondrocyte apoptosis and cartilage degeneration in a rat osteoarthritis model via AMPK and p38 MAPK signaling.

Chondrocyte apoptosis is an important mechanism involved in osteoarthritis (OA). Berberine (BBR), a plant alkaloid derived from Chinese medicine, is characterized by multiple pharmacological effects, such as anti-inflammatory and anti-apoptotic activities. This study aimed to evaluate the chondroprotective effect and underlying mechanisms of BBR on sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis and surgically-induced rat OA model. The in vitro results revealed that BBR suppressed SNP-stimulated chondrocyte apoptosis as well as cytoskeletal remodeling, down-regulated expressions of inducible nitric oxide synthase (iNOS) and caspase-3, and up-regulated Bcl-2/Bax ratio and Type II collagen (Col II) at protein levels, which were accompanied by increased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and decreased phosphorylation of p38 mitogen-activated protein kinase (MAPK). Furthermore, the anti-apoptotic effect of BBR was blocked by AMPK inhibitor Compound C (CC) and adenosine-9-β-D-arabino-furanoside (Ara A), and enhanced by p38 MAPK inhibitor SB203580. In vivo experiment suggested that BBR ameliorated cartilage degeneration and exhibited an anti-apoptotic effect on articular cartilage in a rat OA model, as demonstrated by histological analyses, TUNEL assay and immunohistochemical analyses of caspase-3, Bcl-2 and Bax expressions. These findings suggest that BBR suppresses SNP-stimulated chondrocyte apoptosis and ameliorates cartilage degeneration via activating AMPK signaling and suppressing p38 MAPK activity.

Piperine inhibits IL-1β-induced IL-6 expression by suppressing p38 MAPK and STAT3 activation in gastric cancer cells.

Piperine, a kind of natural alkaloid found in peppers, has been reported to exhibit anti-oxidative and anti-tumor activities, both in vitro and in vivo. Interleukin-6 (IL-6) is an important cytokine that activates the signal transduction, promotes tumor cell metastasis, and induces malignancy, including in gastric cancer. However, the effects of piperine on IL-6 expression in gastric cancer cells have not yet been well defined. In this study, we investigated the effects of piperine on the IL-6 expression, and examined the underlying signaling pathways via RT-PCR, promoter studies and Western blotting in human gastric cancer TMK-1 cells. Our results showed that piperine inhibited interleukin-1β (IL-1β)-induced IL-6 expression in a dose-dependent manner. In addition, piperine also inhibited IL-6 promoter activity. Experiments with mitogen-activated protein kinase (MAPK) inhibitors and dominant negative mutant p38 MAPK indicated that p38 MAPK was essential for IL-6 expression in the TMK-1 cells. Additionally, signal transducer and activator of transcription 3 (STAT3) was also involved in the IL-1β-induced IL-6 expression in gastric cancer cells. Piperine inhibited IL-1β-induced p38 MAPK and STAT3 activation and, in turn, blocked the IL-1β-induced IL-6 expression. Furthermore, gastric cancer cells pretreated with IL-1β showed markedly enhanced invasiveness, which was partially abrogated by treatment with IL-6 siRNA, piperine, and inhibitors of p38 MAPK and STAT3. These results suggest that piperine may exert at least part of its anti-cancer effect by controlling IL-6 expression through the suppression of p38 MAPK and STAT3.

Propofol selectively inhibits nuclear factor-κB activity by suppressing p38 mitogen-activated protein kinase signaling in human EA.hy926 endothelial cells during intermittent hypoxia/reoxygenation

Intermittent hypoxia/reoxygenation (IHR) induces proinflammatory cytokines, contributing to the pathogenic process of atherosclerosis associated with obstructive sleep apnea (OSA). Two transcription factors, nuclear factor-κB (NF-κB) and hypoxia-inducible factor-1 (HIF-1), have been indicated to mediate proinflammatory cytokines during IHR. The anti-inflammatory effects of propofol have attracted increasing attention in regard to the treament of multiple diseases associated with inflammation. The present study examined whether propofol inhibits NF-κB and HIF-1 activity in vascular endothelial cells during IHR. EA.hy926 endothelial cells were exposed to IHR for 64 cycles with or without propofol treatment. Gene knockdown by transfection of siRNA against p38 mitogen-activated protein kinase (MAPK) was also used to investigate the molecular mechanisms. Compared with the control group, IHR exposure significantly induced the activation of NF-κB and HIF-1, enhanced the mRNA expression of proinflammatory cytokines and increased the activation of p38 MAPK. Propofol dose-dependently inhibited the IHR‑induced activation of NF-κB, but did not change the activation of HIF-1, which was accompanied by decreased levels of proinflammatory cytokines. In addition, IHR‑induced p38 MAPK activity was attenuated by propofol in a similar manner to the reduction in NF-κB activity. Furthermore, knockdown of p38 MAPK with siRNA significantly reduced the IHR‑induced activation of NF-κB, while not affecting HIF-1. These data demonstrate that propofol selectively attenuates the IHR‑induced activation of NF-κB, but not HIF-1, in vascular endothelial cells, and these beneficial effects are likely to be based on the inhibition of the p38 MAPK signaling pathway. Propofol may have the potential to prevent atherosclerosis in patients with OSA by inhibiting NF-κB‑mediated inflammation in the vascular endothelium.

Water-soluble andrographolide sulfonate exerts anti-sepsis action in mice through down-regulating p38 MAPK, STAT3 and NF-κB pathways

Andrographolide is a prescribed drug used for preventing and treating the common cold, influenza, viral infections or allergies. However, its poor water solubility enormously limits its bioavailability. In the present study, we aimed at examining and comparing the effect of andrographolide sulfonate (trade name: Xi-Yan-Ping Injection), a water-soluble form made from andrographolide through sulfonating reaction, on the treatment of murine sepsis model induced by lipopolysaccharide (LPS). Pretreatment with andrographolide sulfonate significantly decreased the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and transaminase activities in serum, attenuated liver and lung damage, and improved the survival of mice with experimental sepsis. Andrographolide sulfonate also remarkably reduced the expression levels of TNF-α, IL-1β, IL-6 and inducible nitric oxide synthase in the injured liver from septic mice. Moreover, andrographolide sulfonate time-dependently suppressed the activation of p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase (ERK1/2) or c-Jun NH2-terminal kinase (JNK). Furthermore, pretreatment with andrographolide sulfonate markedly inhibited the activation of p65 subunit of nuclear factor-κB (NF-κB) as well as signal transducers and activators of transcription 3 (STAT3) in the injured liver from mice with endotoxic shock. Notably, andrographolide sulfonate showed a much stronger alleviation of LPS-induced sepsis in mice compared with andrographolide. Taken together, these results reveal that andrographolide sulfonate ameliorates sepsis in mice through suppressing p38 MAPK, STAT3 and NF-κB pathways and suggest that andrographolide sulfonate has an advantage of andrographolide for the treatment of endotoxin shock.

Histamine differentially regulates the production of Th1 and Th2 chemokines by keratinocytes through histamine H1 receptor

Histamine is a biological amine that plays an important role in allergic responses. However, the involvement of histamine signaling in late allergic responses in the skin is poorly understood. Therefore, we attempted to investigate the involvement of histamine signaling in late allergic responses, especially in keratinocytes (KCs). HaCaT KCs and normal human KCs (NHKs) predominantly expressed histamine H1 receptor (H1R) and H2 receptor (H2R). Histamine suppressed tumor necrosis factor α (TNF-α)- and interferon-γ (IFN-γ)-induced production of CC chemokine ligand 17(CCL17), a type 2 T-helper (Th2) chemokine, by HaCaT KCs. It suppressed the phosphorylation of p38 mitogen-activated protein (MAP) kinase, but not that of extracellular signal-regulated kinases (ERKs), and TNF-α- and IFN-γ-induced nuclear factor κB (NFκB) activity. In contrast, histamine enhanced the production of CXC chemokine ligand 10 (CXCL10), a Th1 chemokine, by TNF-α- and IFN-γ-stimulated HaCaT KCs and NHKs. TNF-α- and IFN-γ-induced CXCL10 production was upregulated by suppression of p38 MAP kinase or NF-κB activity, which could explain histamine involvement. We concluded that histamine suppresses CCL17 production by KCs by suppressing p38 MAP kinase and NF-κB activity through H1R and may act as a negative-feedback signal for existing Th2-dominant inflammation by suppressing CCL17 and enhancing CXCL10 production.

Bone morphogenetic protein 6 (BMP6) and BMP7 inhibit estrogen-induced proliferation of breast cancer cells by suppressing p38 mitogen-activated protein kinase activation

Estrogen is involved in the development and progression of breast cancer. Here, we investigated the effects of bone morphogenetic proteins (BMPs) on breast cancer cell proliferation caused by estrogen using human breast cancer MCF-7 cells. MCF-7 cells express estrogen receptors (ESR1 and ESR2), BMP receptors, and SMAD signaling molecules. Estradiol and membrane-impermeable estradiol stimulated MCF-7 cell proliferation. Estradiol also reduced mRNA levels of ESR1, aromatase, and steroid sulfatase. Treatment with BMPs and activin had no effects on MCF-7 cell proliferation. However, BMP2, BMP4, BMP6, BMP7, and activin suppressed estradiol-induced cell mitosis, with the effects of BMP6, BMP7, and activin being more prominent than those of BMP2 and BMP4. Activin decreased ESR1 mRNA expression, while BMP6 and BMP7 impaired steroid sulfatase expression in MCF-7 cells. Interestingly, SMAD1,5,8 activation elicited by BMP6 and BMP7, but not by BMP2 and BMP4, was preserved even under the exposure of a high concentration of estradiol. The difference of BMP responsiveness was likely due to the differential modulation of BMP receptor expression induced by estradiol. In this regard, estradiol decreased the expression levels of BMPR1A, BMPR1B, ACVR2A, and ACVR2B but did not affect ACVR1 and BMPRII, leading to the sustained effects of BMP6 and BMP7 in estrogen-treated MCF-7 cells. Estradiol rapidly activated MAPK phosphorylation including extracellular signal-regulated kinase 1/2, p38, and stress-activated protein kinase/c-Jun NH2-terminal kinase pathways and BMP6, BMP7, and activin preferentially inhibited estradiol-induced p38 phosphorylation. SB203580, a selective p38 MAPK inhibitor effectively suppressed estradiol-induced cell mitosis, suggesting that p38 MAPK plays a key role in estrogen-sensitive breast cancer cell proliferation. Thus, a novel interrelationship between estrogen and the breast cancer BMP system was uncovered, in which inhibitory effects of BMP6 and BMP7 on p38 signaling and steroid sulfatase expression were functionally involved in the suppression of estrogen-induced mitosis of breast cancer cells.

Apigenin inhibits the production of NO and PGE 2 in microglia and inhibits neuronal cell death in a middle cerebral artery occlusion-induced focal ischemia mice model

Flavonoids have been intensively studied on their pharmacological activities such as anti-cancer, anti-oxidant and anti-inflammation. However, little is known about their neuroprotective effects. Recent studies suggest that inflammation mediated by microglia may play a role in neurodegenerative diseases. In this study, we evaluated the anti-inflammatory effect of various flavonoid compounds by using BV-2, a murine microglia cell line. Of the compounds that were evaluated, apigenin inhibited the production of nitric oxide and prostaglandin E 2 by suppressing the expression of inducible nitric oxide synthase and cyclooxygenase-2 protein, respectively. Moreover, apigenin suppressed p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) phosphorylation without affecting the activity of extracellular signal-regulated kinase (ERK). Apigenin was also found to protect neuronal cells from injury in middle cerebral artery occlusion.

Cytokine activation of p38 mitogen-activated protein kinase and apoptosis is opposed by alpha-4 targeting of protein phosphatase 2A for site-specific dephosphorylation of MEK3.

alpha-4 is an essential gene and is a dominant antiapoptotic factor in various tissues that is a regulatory subunit for type 2A protein phosphatases. A multiplexed phosphorylation site screen revealed that knockdown of alpha-4 by small interfering RNA (siRNA) increased p38 mitogen-activated protein kinase (MAPK) and c-Jun phosphorylation without changes in JNK or ERK. FLAG-alpha-4 coprecipitated hemagglutinin-MEK3 plus endogenous protein phosphatase 2A (PP2A) and selectively enhanced dephosphorylation of Thr193, but not Ser189, in the activation loop of MEK3. Overexpression of alpha-4 suppressed p38 MAPK activation in response to tumor necrosis factor alpha (TNF-alpha). The alpha-4 dominant-negative domain (DND) (residues 220 to 340) associated with MEK3, but not PP2A, and its overexpression sensitized cells to activation of p38 MAPK by TNF-alpha and interleukin-1beta, but not by ansiomycin or sorbitol. The response was diminished by nocodazole or by siRNA knockdown of the Opitz syndrome protein Mid1 that binds alpha-4 to microtubules. Interference by alpha-4 DND or alpha-4 siRNA increased caspase 3/7 activation in response to TNF-alpha. Growth of transformed cells in soft agar was enhanced by alpha-4 and suppressed by alpha-4 DND. The results show that alpha-4 targets PP2A activity to MEK3 to suppress p38 MAPK activation by cytokines, thereby inhibiting apoptosis and anoikis.

Genes regulated by hepatocyte growth factor as targets to sensitize ovarian cancer cells to cisplatin

Advanced ovarian cancers are initially responsive to chemotherapy with platinum drugs but develop drug resistance in most cases. We showed recently that hepatocyte growth factor (HGF) enhances death of human ovarian cancer cell lines treated with cisplatin (CDDP) and that this effect is mediated by the p38 mitogen-activated protein kinase. In this work, we integrated genome-wide expression profiling, in silico data survey, and functional assays to identify transcripts regulated in SK-OV-3 ovarian cancer cells made more responsive to CDDP by HGF. Using oligonucleotide microarrays, we found that HGF pretreatment changes the transcriptional response to CDDP. Quantitative reverse transcription-PCR not only validated all the 15 most differentially expressed genes but also confirmed that they were primarily modulated by the combined treatment with HGF and CDDP and reversed by suppressing p38 mitogen-activated protein kinase activity. Among the differentially expressed genes, we focused functional analysis on two regulatory subunits of the protein phosphatase 2A, which were down-modulated by HGF plus CDDP. Decrease of each subunit by RNA interference made ovarian cancer cells more responsive to CDDP, mimicking the effect of HGF. In conclusion, we show that HGF and CDDP modulate transcription in ovarian cancer cells and that this transcriptional response is involved in apoptosis regulation. We also provide the proof-of-concept that the identified genes might be targeted to either increase the efficacy of chemotherapeutics or revert chemotherapy resistance.