P38 MAPK Inhibitor SB203580 Research Articles

P38 MAPK inhibitor SB203580 enhances anticancer activity of PARP inhibitor olaparib in a synergistic way on non-small cell lung carcinoma A549 cells

The Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib gives promising results against various types of cancers in clinical trials. The combination of drugs always increases therapeutic efficacy because of targeting multiple pathways of cancer progression. Our objective was to explore the potential synergistic anticancer activities of olaparib combined with p38 MAPK inhibitor (MAPKi) SB203580 on non-small cell lung carcinoma (NSCLC) A549 cells. The effects of the individual compound and their combination on cell survival, DNA damage as detected by γH2AX foci, expression of key proteins in Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ) repair, caspase 3 activation, nuclear fragmentation and telomerase regulation were studied in A549 cells. The results showed that olaparib and SB203580 individually reduced cell viability in a dose-dependent manner but combined treatment synergistically reduced cell viability. Olaparib combined with SB203580 significantly reduced error-free HR repair via reducing MRE11-RAD50 and promoted error-prone NHEJ repair by increasing Ku70-Ku80 leading to increased DNA damage-induced apoptosis. Notably, the alteration of proteins in HR/NHEJ pathways, DNA damage and induction of apoptosis was significant by combined treatment but not by 1 μM olaparib treatment alone. In addition, combined treatment reduced telomerase activity more than single treatment via reducing telomerase subunits. These data implicated that the anticancer potential of olaparib was significantly increased by combining SB203580 through increasing DNA damage-induced apoptosis and inhibiting telomerase activity.

Laminaria japonica polysaccharide attenuates podocyte epithelial-mesenchymal transformation via TGF-β1-mediated Smad3 and p38MAPK pathways

In the present work, we explored the interventional effect and potential mechanism of a purified Laminaria japonica polysaccharide (LJP61A) on podocyte epithelial-mesenchymal transition (EMT) in TGF-β1-induced podocytes and adriamycin-treated mice. Results showed that compared to the model groups, LJP61A significantly up-regulated the levels of epithelial markers (Nephrin, WT-1, podocin) and down-regulated the levels of mesenchymal markers (α-SMA, FN1) in vitro and in vivo, thus preventing EMT-like morphological changes of podocytes, proteinuria and kidney injury. Smad3 and p38MAPK are two central pathways mediating podocyte EMT activated by TGF-β1. We found that LJP61A suppressed TGF-β1-induced activation of Smad3, Smad4 and p38MAPK in vitro and in vivo. Moreover, the inhibitory actions of LJP61A on podocyte EMT were synergistically strengthened by Smad3 inhibitor SIS3 and p38MAPK inhibitor SB203580. Taken together, these findings revealed that LJP61A could prevent podocyte EMT, which might be related to the inhibition of TGF-β1-mediated Smad3 and p38MAPK pathways.

Bone marrow stromal cells-derived exosomes reduce neurological damage in traumatic brain injury through the miR-124-3p/p38 MAPK/GLT-1 axis

BackgroundMounting evidence indicates that stem cell-derived exosomal miRNAs have therapeutic effects on traumatic brain injury (TBI). This research is focused on exploring the molecular processes of miR-124-3p obtained from bone marrow stromal cells-derived exosomes (BMSCs-Exos) in attenuating posttraumatic glutamate-mediated excitotoxicity. MethodsWe created a TBI rat model and analyzed the expression profile of miRNA through miRNA microarray. The miR-124-3p and p38 MAPK levels were analyzed utilizing RT-qPCR and western blotting. Dual-luciferase reporter (DLR) assay showed the targeting relationship between miR-124-3p and p38 MAPK. We subsequently conducted a TUNEL assay and flow cytometry to evaluate the neuronal apoptotic rate in an in vitro glutamate-mediated excitotoxicity model treated with BMSCs-Exos enriched with miR-124-3p (BMSCs-ExosmiR-124-3p). Moreover, the levels of p38 MAPK and glutamate transporter-1 (GLT-1) were measured by western blotting. Furthermore, BMSCs-ExosmiR-124-3p were administered to the TBI rats, and their neuroprotective effects were observed using western blotting, immunohistochemistry, histological staining, magnetic resonance imaging (MRI), and Morris water maze (MWM). ResultsThe results revealed that the brains of TBI rats exhibited lowered miR-124-3p and enhanced p38 MAPK levels. DLR assay demonstrated miR-124-3p's role in targeting p38 MAPK and negatively regulating its expression. In vitro and in vivo studies confirmed that BMSCs-ExosmiR-124-3p attenuated glutamate-mediated excitotoxicity by downregulating p38 MAPK and upregulating GLT-1 expressions via transferring exosomal miR-124-3p. Moreover, histopathological evaluation and MRI results showed that BMSCs-ExosmiR-124-3p remarkably alleviated neuronal cell death and minimized the lesion volumes post-TBI. MWM outcomes illustrated that BMSCs-ExosmiR-124-3p treatment could substantially improve neurological function post-TBI. Furthermore, the effects of treatment with p38 MAPK inhibitor SB203580 were similar to BMSCs-ExosmiR-124-3p. ConclusionOverall, the outcomes of the current report highlighted that BMSCs-ExosmiR-124-3p can lead to the upregulation of GLT-1 in TBI rat models by inhibiting the p38 MAPK signaling pathway, hence alleviating glutamate-mediated excitotoxicity and attenuating neurological damage post-TBI.

Porphyromonas gingivalis-induced periodontitis could contribute to cognitive impairment in Sprague-Dawley rats via the P38 MAPK signaling pathway.

Periodontitis is one of the most common oral diseases and has been shown to be a risk factor for systemic diseases. Our aim was to investigate the relationship between periodontitis and cognitive impairment and to explore the role of the P38 MAPK signaling pathway in this process. We established a periodontitis model by ligating the first molars of SD rats with silk thread and injecting Porphyromonas gingivalis (P. gingivalis) or P. gingivalis plus the P38 MAPK inhibitor SB203580 at the same time for ten weeks. We assessed alveolar bone resorption and spatial learning and memory using microcomputed tomography and the Morris water maze test, respectively. We used transcriptome sequencing to explore the genetic differences between the groups. The gingival tissue, peripheral blood and hippocampal tissue were assessed for the cytokines TNF-α, IL-1β, IL-6, IL-8 and C reactive protein (CRP) with enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). We observed the presence of P. gingivalis in the hippocampus of rats by paraffin-fluorescence in situ hybridization (FISH). We determined the activation of microglia by immunofluorescence. Finally, Western blot analysis was employed to determine the expression of amyloid precursor protein (APP), β-site APP-cleaving enzyme 1 (BACE1) and P38MAPK pathway activation. We demonstrated that silk ligature-induced periodontitis plus injection of P. gingivalis into subgingival tissue could lead to memory and cognitive impairment. Transcriptome sequencing results suggested that there were neurodegenerative diseases in the P. gingivalis group, and the MWM test showed that periodontitis reduced the spatial learning and memory ability of mild cognitive impairment (MCI) model rats. We found high levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) and CRP in the gingiva, peripheral blood and hippocampus, and the expression of APP and BACE1 was upregulated, as was the P38 MAPK pathway activation. Activated microglia and the presence of P. gingivalis were also found in the hippocampus. P38 MAPK inhibitors mitigated all of these changes. Our findings strongly suggest that topical application of P. gingivalis increases the inflammatory burden in the peripheral and central nervous systems (CNS) and that neuroinflammation induced by activation of P38 MAPK leads to impaired learning and memory in SD rats. It can also modulate APP processing. Therefore, P38 MAPK may serve as a linking pathway between periodontitis and cognitive impairment.

OPN up-regulated proliferation and invasion of head and neck squamous cell carcinoma through the p38MAPK signaling pathway

Osteopontin (OPN) is aberrantly expressed in various tumors. However, its role and detailed mechanisms in head and neck squamous cell carcinoma (HNSCC) have not been extensively described. Expression of OPN in HNSCC was examined at the gene and protein levels. The effect of cell proliferation ability was examined by Cell Counting Kit-8, colony formation assay, cell invasiveness by Transwell assay, the effect of OPN on protein expression of Capase-3 and Bcl2 by Western blotting, and the expression of p38MAPK signaling pathway by p38MAPK inhibitor SB203580. We found that OPN expression was higher in human HNSCC tissues than in adjacent tissues. Osteopontin may regulate the proliferation and invasion of HNSCC cells through the p38-MAPK signaling pathway. Our study identifies an important role for OPN in HNSCC and further demonstrates that it may regulate the proliferation and invasion of HNSCC cells by activating the p38-MAPK signaling pathway. Osteopontin may be a promising prognostic and diagnostic indicator and a potential target for cancer therapy.

PSKH1 affects proliferation and invasion of osteosarcoma cells via the p38/MAPK signaling pathway.

Malignant osteosarcoma (OS) is a tumor of bone and soft tissue that metastasizes early and has a high mortality rate. Protein serine kinase H1 (PSKH1), an autophosphorylating human protein serine kinase, controls the trafficking of serine/arginine-rich domain, with downstream effects on mRNA processing. It is also associated with tumor progression. However, how this protein contributes to OS progression and metastasis is unknown. The present study evaluated the potential effect of PSKH1 on proliferation of human OS cells. OS cell lines were used in Cell Counting Kit-8, colony formation, wound-healing and Transwell assays, to investigate cellular processes such as proliferation, migration and invasion and underlying molecular mechanisms. Expression of PSKH1 in OS tissue was significantly greater than in adjacent non-malignant tissue. PSKH1 knockdown inhibited the proliferation, migration and invasion of OS cells. Conversely, PSKH1 overexpression promoted proliferation of OS cells. PSKH1 upregulated phosphorylated-p38 in OS cells. Moreover, the p38 MAPK inhibitor SB203580 effectively blocked the tumor-promoting action of PSKH1. Furthermore, PSKH1 knockdown inhibited tumor growth and metastasis in vivo. In conclusion, these findings suggested that PSKH1 promoted OS proliferation, migration and invasion. Thus, PSKH1 may serve an oncogenic role in the development of human OS.

Sulforaphane attenuates platelet granule secretion through down-regulating glycoprotein VI-mediated p38 MAPK/cPLA2 signaling pathway

ABSTRACT Glycoprotein VI (GPVI) is the major collagen receptor on the platelet surface and is important in mediating granule secretion and atherothrombosis. Sulforaphane (SFN), a dietary isothiocyanate enriched in cruciferous vegetables, exerts multiple biological activities. This study aimed to investigate the efficacy of SFN on GPVI-mediated signaling and platelet granule secretion in vitro. We herein demonstrated that SFN greatly attenuated granule secretion in human gel-filtered platelets in response to collagen, including inhibiting CD62P and CD63 expression and PF4, CCL5, and ATP release. Mechanistically, these inhibitory effects of SFN were mainly mediated by its attenuation of cytosolic phospholipase A2 (cPLA2) phosphorylation and thromboxane A2 (TxA2) generation. Moreover, SFN did not further decrease the inhibitory effects of p38 MAPK inhibitor SB203580 on cPLA2 phosphorylation, TxA2 generation and granule secretion. Thus, SFN attenuates platelet granule secretion via down-regulating GPVI-mediated p38 MAPK/cPLA2/TxA2 signaling, which may play important preventive roles in cardiovascular diseases.

Luteoloside Induces G0/G1 Phase Arrest of Neuroblastoma Cells by Targeting p38 MAPK.

Luteoloside has shown anti-inflammatory, antiviral, and antitumor properties. However, the effect and mechanism of luteoloside on neuroblastoma cells remain unknown. The proliferation of human neuroblastoma cells (SH-SY5Y and SK-N-AS) treated with different concentrations of luteoloside (0, 12.5, 25, and 50 μM) was detected by the MTT assay and colony formation assay. Cell apoptosis and cell cycle were examined by Hoechst staining and flow cytometry. A subcutaneous tumorigenesis model was established in nude mice to evaluate the effect of luteoloside on tumor growth in vivo. Bioinformatics, molecular docking techniques, and cellular thermal shift assays were utilized to predict the potential targets of luteoloside in neuroblastoma. The p38 MAPK inhibitor SB203580 was used to confirm the role of p38 MAPK. Luteoloside inhibited the proliferation of neuroblastoma cells in vitro and in vivo. Luteoloside slightly induced cellular G0/G1 phase arrest and reduced the expression levels of G0/G1 phase-related genes and the proteins cyclin D1, CDK4, and C-myc, which are downregulated by p38 MAPK pathways. Meanwhile, p38 was identified as the target of luteoloside, and inhibition of p38 MAPK reversed the inhibitory effect of luteoloside on neuroblastoma cells. Luteoloside is a potential anticancer drug for treating neuroblastoma by activating p38 MAPK.

Ubiquitin D promotes the progression of rheumatoid arthritis via activation of the p38 MAPK pathway.

Ubiquitin D (UBD), a member of the ubiquitin‑like modifier family, has been reported to be highly expressed in various types of cancer and its overexpression is positively associated with tumor progression. However, the role and mechanism of UBD in rheumatoid arthritis (RA) remain elusive. In the present study, the gene expression profiles of GSE55457 were downloaded from the Gene Expression Omnibus database to assess differentially expressed genes and perform functional enrichment analyses. UBD was overexpressed by lentivirus transfection. The protein level of UBD, p‑p38 and p38 in RA‑fibroblast‑like synoviocytes (FLSs) were examined by western blotting. Cell Counting Kit‑8 and flow cytometry assays were used to detect the functional changes of RA‑FLSs transfected with UBD and MAPK inhibitor SB202190. The concentrations of inflammatory factors (IL‑2, IL‑6, IL‑10 and TNF‑α) were evaluated using ELISA kits. The results revealed that UBD was overexpressed in RA tissues compared with in the healthy control tissues. Functionally, UBD significantly accelerated the viability and proliferation of RA‑FLSs, whereas it inhibited their apoptosis. Furthermore, UBD significantly promoted the secretion of inflammatory factors (IL‑2, IL‑6, IL‑10 and TNF‑α). Mechanistically, elevated UBD activated phospohorylated‑p38 in RA‑FLSs. By contrast, UBD overexpression and treatment with the p38 MAPK inhibitor SB202190 not only partially relieved the UBD‑dependent effects on cell viability and proliferation, but also reversed its inhibitory effects on cell apoptosis. Furthermore, SB202190 partially inhibited the effects of UBD overexpression on the enhanced secretion of inflammatory factors. The present study indicated that UBD may mediate the activation of p38 MAPK, thereby facilitating the proliferation of RA‑FLSs and ultimately promoting the progression of RA. Therefore, UBD may be considered a potential therapeutic target and a promising prognostic biomarker for RA.

A Cyclopentanone Compound Attenuates the Over-Accumulation of Extracellular Matrix and Fibrosis in Diabetic Nephropathy via Downregulating the TGF-β/p38MAPK Axis.

Excessive accumulation of the extracellular matrix (ECM) is a crucial pathological process in chronic kidney diseases, such as diabetic nephropathy, etc. The underlying mechanisms of how to decrease ECM deposition to improve diabetic nephropathy remain elusive. The present study investigated whether cyclopentanone compound H8 alleviated ECM over-deposition and fibrosis to prevent and treat diabetic nephropathy. HK-2 cell viability after treatment with H8 was measured by an MTT assay. ECM alterations and renal fibrosis were identified in vitro and in vivo. A pharmacological antagonist was used to detect associations between H8 and the p38 mitogen-activated protein kinase (p38MAPK) signaling pathway. H8 binding was identified through computer simulation methods. Studies conducted on high glucose and transforming growth factor β1 (TGF-β1)-stimulated HK-2 cells revealed that the p38MAPK inhibitor SB 202190 and H8 had similar pharmacological effects. In addition, excessive ECM accumulation and fibrosis in diabetic nephropathy were remarkably improved after H8 administration in vivo and in vitro. Finally, the two molecular docking models further proved that H8 is a specific p38MAPK inhibitor that forms a hydrogen bond with the LYS-53 residue of p38MAPK. The cyclopentanone compound H8 alleviated the over-deposition of ECM and the development of fibrosis in diabetic nephropathy by suppressing the TGF-β/p38MAPK axis.

Upregulation of dynorphin/kappa opioid receptor system in the dorsal hippocampus contributes to morphine withdrawal-induced place aversion.

Aversive emotion of opioid withdrawal generates motivational state leading to compulsive drug seeking and taking. Kappa opioid receptor (KOR) and its endogenous ligand dynorphin have been shown to participate in the regulation of aversive emotion. In the present study, we investigated the role of dynorphin/KOR system in the aversive emotion following opioid withdrawal in acute morphine-dependent mice. We found that blockade of KORs before pairing by intracerebroventricular injection of KOR antagonist norBNI (20, 40 μg) attenuated the development of morphine withdrawal-induced conditioned place aversion (CPA) behavior. We further found that morphine withdrawal increased dynorphin A expression in the dorsal hippocampus, but not in the amygdala, prefrontal cortex, nucleus accumbens, and thalamus. Microinjection of norBNI (20 μg) into the dorsal hippocampus significantly decreased morphine withdrawal-induced CPA behavior. We further found that p38 MAPK was significantly activated in the dorsal hippocampus after morphine withdrawal, and the activation of p38 MAPK was blocked by pretreatment with norBNI. Accordingly, microinjection of p38 MAPK inhibitor SB203580 (5 μg) into the dorsal hippocampus significantly decreased morphine withdrawal-produced CPA behavior. This study demonstrates that upregulation of dynorphin/KOR system in the dorsal hippocampus plays a critical role in the formation of aversive emotion associated with morphine withdrawal, suggesting that KOR antagonists may have therapeutic value for the treatment of opioid withdrawal-induced mood-related disorders.

Dexmedetomidine protects against acute lung injury in mice via the DUSP1/MAPK/NF-κB axis by inhibiting miR-152-3p.

Acute lung injury (ALI) is a debilitating condition in clinics. Dexmedetomidine (Dex) is known for its anti-apoptotic and anti-inflammatory properties. This study attempted to investigate the protective mechanism of Dex in ALI mice. Mice were pretreated with Dex before model establishment by tracheal injection of lipopolysaccharide (LPS). Pulmonary function indexes and wet-to-dry (W/D) ratio were measured. Pulmonary pathological changes were observed through HE staining, CD31+-positive mouse pulmonary microvascular endothelial cells (MPMVECs) were counted through immunofluorescence staining, and apoptosis was detected through TUNEL staining. miR-152-3p mimic, sh-DUSP1, or p38 MAPK inhibitor was delivered into MPMVECs, followed by combined treatment of Dex and LPS. miR-152-3p expression, apoptosis, levels of apoptosis- and MAPK/NF-κB pathway-associated proteins, and inflammatory factors were measured through RT-qPCR, flow cytometry, Western blot, and ELISA. The binding relationship of miR-152-3p and DUSP1 was verified through bioinformatics software and dual-luciferase assay. ALI mouse model was established after injection of miR-152-3p antagomir. Dex improved ALI mouse pulmonary function and mitigated injury in mice and MPMVECs. miR-125-3p overexpression or sh-DUSP1 partially abolished the protection of Dex on MPMVECs. miR-152-3p targeted DUSP1. sh-DUSP1 partially averted the protection of Dex on MPMVECs. Dex inhibited the activation of the MAPK/NF-κB pathway in MPMVECs mediated by LPS, which was partially reversed by sh-DUSP1. The p38 MAPK inhibitor SB203580 antagonized the protective effect of Dex on MPMVECs mediated by sh-DUSP1. Similarly, downregulation of miR-152-3p mitigated ALI via the DUSP1/MAPK/NF-κB axis in vivo. Dex relieved ALI in mice via the DUSP1/MAPK/NF-κB axis by down-regulating miR-152-3p.

Ox-LDL-Induced Endothelial Progenitor Cell Oxidative Stress via p38/Keap1/Nrf2 Pathway.

Background Nrf2 which was recently reported to regulate the antioxidant genes and cellular redox regulators was highly expressed in EPCs. However, its role in ox-LDL-induced EPC oxidative stress and apoptosis has not been fully illustrated. Methods EPCs isolated from human peripheral blood mononuclear cells were treated with different concentrations of ox-LDL, Keap1 siRNA, and a specific p38 MAPK inhibitor SB203580 and then used to assay the cytoplasmic Nrf2, nuclear Nrf2, NAD(P) H:quinone oxidoreductase 1 (NQO1) and Bax/Bcl-2 levels with Western blot, NQO1 mRNA levels with RT-PCR, ROS levels with H2DCF-DA, loss/disruption of mitochondrial membrane potential with JC-1, apoptosis with Annexin V and PI, migration with transwell chambers, and tube formation with Matrigel. Results ox-LDL decreased the nuclear Nrf2/Histone H3 to cytoplasmic Nrf2/GAPDH ratio, NQO1 mRNA, and protein levels. ox-LDL enhanced ROS production, induced the loss of membrane potential, and increased the cell shrinkage, pyknotic nuclei, and apoptosis of EPCs. Keap1 siRNA increased Nrf2 nuclear translocation, NQO1 mRNA transcription, and protein expression and prevented ROS generation and formation of JC-1 monomers. ox-LDL increased the activation of p38. SB203580 significantly eliminated ox-LDL induced inhibition of Nrf2 nuclear translocation, depression of NQO1 mRNA transcription, generation of ROS, and formation of JC-1 monomers in EPCs. Keap1 siRNA decreased the Bax/Bcl-2 ratio which was increased by ox-LDL in EPCs. ox-LDL decreased EPC migration and tube formation. Keap1 siRNA preserved the migration and tube formation of EPCs. Conclusion ox-LDL activated EPCs p38/Keap1/Nrf2 pathway and induced oxidative stress, dysfunction, and apoptosis of EPCs.

Inhibitor of protein kinase N3 suppresses excessive bone resorption in ovariectomized mice

The long-term inhibition of bone resorption suppresses new bone formation because these processes are coupled during physiological bone remodeling. The development of anti-bone-resorbing agents that do not suppress bone formation is urgently needed. We previously demonstrated that Wnt5a-Ror2 signaling in mature osteoclasts promoted bone-resorbing activity through protein kinase N3 (Pkn3). The p38 MAPK inhibitor SB202190 reportedly inhibited Pkn3 with a low Ki value (0.004μM). We herein examined the effects of SB202190 on osteoclast differentiation and function in vitro and in vivo. Bone marrow cells were cultured in the presence of M-csf and GST-Rankl to differentiate into multinucleated osteoclasts. Osteoclasts were treated with increasing concentrations of SB202190. For in vivo study, 10-week-old female mice were subjected to ovariectomy (OVX). OVX mice were intraperitoneally administered with a Pkn3 inhibitor at 2mg/kg or vehicle for 4weeks, and bone mass was analyzed by micro-CT. SB202190 suppressed the auto-phosphorylation of Pkn3 in osteoclast cultures. SB202190 significantly inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation. SB202190 reduced c-Src activity in osteoclast cultures without affecting the interaction between Pkn3 and c-Src. A treatment with SB202190 attenuated OVX-induced bone loss without affecting the number of osteoclasts or bone formation by osteoblasts. Our results showed that Pkn3 has potential as a therapeutic target for bone loss due to increased bone resorption. SB202190 is promising as a lead compound for the development of novel anti-bone-resorbing agents.

Lidocaine relieves murine allergic rhinitis by regulating the NF-κB and p38 MAPK pathways.

Allergic rhinitis (AR) is one of the most common chronic inflammatory diseases and its main feature is nasal mucositis. It has been recently revealed that lidocaine demonstrates optimal effects in the treatment of various diseases. However, a limited number of studies have examined the association between lidocaine and AR. In the present study, the AR mouse model was established to explore the effects of lidocaine in AR and to further analyze its molecular mechanism. Subsequently, different concentrations of lidocaine were provided to the animals by intranasal administration and a series of indices were assessed. The data indicated that the frequencies of mouse sneezing and nose rubbing were suppressed following an increase in lidocaine concentration. Subsequently, the number of inflammatory cells was measured. Wright's-Giemsa staining results indicated that lidocaine significantly decreased the numbers of leukocytes, eosinophils, neutrophils and lymphocytes in the nasal lavage fluid (NLF) of AR mice. In addition, the expression levels of ovalbumin (OVA)-specific immunoglobulin E (IgE), leukotriene C4 (LTC4) and certain inflammatory factors were assessed by ELISA. Lidocaine reduced OVA-specific IgE and LTC4 expression in NLF and plasma derived from AR mice. It also decreased the expression levels of IL-4, IL-5, IL-13, IL-17 and TNF-α. Lidocaine caused upregulation of IFN-γ and IL-2 expression levels. Subsequently, western blot analysis indicated that lidocaine suppressed phosphorylated (p)-p38 and p-p65 expression levels in AR mice. Collectively, the results indicated that the NF-κB and p38 MAPK signaling pathways were involved in the lidocaine-mediated relief of AR in mice. In order to further verify the association between the NF-κB and p38 MAPK signaling pathways and AR in mice, the effects of the NF-κB inhibitor IMD-0354 and the p38 MAPK inhibitor SB 203580 were assessed on AR mice. The results indicated that these two compounds exhibited similar inhibitory effects on AR mice as those noted with the use of lidocaine. These findings suggested that lidocaine represented a novel therapeutic agent for AR.

Sheep β-Defensin 2 Regulates Escherichia coli F17 Resistance via NF-κB and MAPK Signaling Pathways in Ovine Intestinal Epithelial Cells.

Simple SummaryThis study was conducted to explore the antibacterial ability of sheep β-defensin 2 (SBD-2) against E. coli F17 infection in ovine intestinal epithelial cells (OIECs). Our data revealed that E. coli F17 induces SBD-2 expression in OIECs in vitro, which appears to be mediated through the activation of the signaling pathways NF-κB and MAPK. Our results provide a novel insight for the functionality of SBD-2, which could be useful for developing anti-infective drugs and/or breeding for E. coli diarrhea disease-resistant sheep.Escherichia coli (E. coli) F17 is a member of enterotoxigenic Escherichia coli, which can cause massive diarrhea and high mortality in newborn lambs. β-defensin is mainly produced by the epithelial tissue of the gastrointestinal tract in response to microbial infection. However, the molecular mechanism of sheep β-defensin 2 (SBD-2) against E. coli F17 remains unclear. This study aims to reveal the antibacterial ability of SBD-2 against E. coli F17 infection in sheep. Firstly, we established the culture system of ovine intestinal epithelial cells (OIECs) in vitro, treated with different concentrations of E. coli F17 for an indicated time. Secondly, we performed RNA interference and overexpression to investigate the effect of SBD-2 expression on E. coli F17 adhesion to OIECs. Finally, inhibitors of NF-κB and MAPK pathways were pre-treated to explore the possible relationship involving in E. coli F17 infection regulating SBD-2 expression. The results showed that E. coli F17 markedly (p < 0.01) upregulated the expression levels of SBD-2 mRNA and protein in a concentration- and time-dependent manner. Overexpression of SBD-2 contributed to enhancing E. coli F17 resistance in OIECs, while silencing SBD-2 dramatically improved the adhesion of E. coli F17 to OIECs (p < 0.05 or p < 0.01). Furthermore, E. coli F17 stimulated SBD-2 expression was obviously decreased by pre-treatment with NF-κB inhibitor PDTC, p38 MAPK inhibitor SB202190 and ERK1/2 MAPK inhibitor PD98095 (p < 0.05 or p < 0.01). Interestingly, adhesion of E. coli F17 to OIECs were highly enhanced by pre-treated with PDTC, SB202190 and PD98095. Our data suggested that SBD-2 could inhibit E. coli F17 infection in OIECs, possibly through NF-κB and MAPK signaling pathways. Our results provide useful theoretical basis on developing anti-infective drug and breeding for E. coli diarrhea disease-resistant sheep.

High-Throughput Screening Platform in Postnatal Heart Cells and Chemical Probe Toolbox to Assess Cardiomyocyte Proliferation.

Restoring lost heart muscle is an attractive goal for cardiovascular regenerative medicine. One appealing strategy is the therapeutic stimulation of cardiomyocyte proliferation, which inter alia remains challenging due to available assay technologies capturing the complex biology. Here, a high-throughput-formatted phenotypic assay platform was established using rodent whole heart-derived cells to preserve the cellular environment of cardiomyocytes. Several readouts allowed the quantification of cycling cardiomyocytes, including a transgenic H2B-mCherry system for unequivocal, automated detection of cardiomyocyte nuclei. A chemical genetics approach revealed pronounced species differences and furnished pan-kinase inhibitors 5 and 36 as potent and robust inducers of endoreplication and acytokinetic mitosis. Combined profiling of the commonly used p38 MAPK inhibitors SB203580 (1), SB239063 (2) and a novel set of skepinone-L (6) derivatives pointed to off-target effects beyond p38 that might be critical for effective cardiomyocyte cytokinesis. Kinome-focused screening eventually furnished TG003 (38) as a novel candidate for stimulating cardiomyocyte proliferation.

Enhanced GRP78 protein expression via the IRE1α/ASK1/p38 MAPK pathway during As2O3-induced endoplasmic reticulum stress in BEAS-2B cells

Inorganic arsenic is widely present in the environment. Exposure to moderate to high concentrations of arsenic from drinking water or air can cause various cancers and multisystem dysfunction. Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) stress sensor of unfolded protein response (UPR) under stress conditions and it enhances cell survival. The aim of this study is to investigate molecular mechanisms of arsenic-induced GRP78 expression in BEAS-2B cells model. We found that GRP78 protein expression was enhanced, while the level of GRP78 mRNA expression did not change under arsenic trioxide (As2O3)-induced ER stress condition in BEAS-2B cells. Cycloheximide, a protein synthesis inhibitor, completely inhibited As2O3-induced GRP78 protein expression. GRP78 mRNA expression was inhibited by actinomycin-D (Act-D). However, GRP78 protein expression was upregulated in the presence of Act-D under As2O3-induced ER stress condition. These data indicated that the upregulation of GRP78 protein under As2O3-induced UPR condition was possibly due to the increased biosynthesis of GRP78 protein. Moreover, both inositol-requiring enzyme 1α (IRE1α) RNase and kinase inhibitor KIRA6 and IRE1α kinase inhibitor APY29 completely inhibited As2O3-induced GRP78 protein expression and phosphorylation of JNK, ERK and p38 MAPK. Activation of apoptotic signaling kinase 1 (ASK1) is a downstream effector of IRE1α kinase. ASK1 inhibitor selonsertib and p38 MAPK inhibitor SB203580 partially inhibited As2O3-induced GRP78 protein expression, respectively. Our results suggested that As2O3 enhanced GRP78 protein expression in BEAS-2B cells via IRE1α kinase/ASK1/p38 MAPK signaling pathway. To our knowledge, this is the first report on illuminating the related mechanisms of increased GRP78 protein expression in As2O3-induced ER stress condition through a novel IRE1α pathway.

Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1

Fibroblast growth factor 21 (FGF21) acts as an endocrine factor, playing important roles in the regulation of energy homeostasis, glucose and lipid metabolism. It is induced by diverse metabolic and cellular stresses, such as starvation and cold challenge, which in turn facilitate adaptation to the stress environment. The pharmacological action of FGF21 has received much attention, because the administration of FGF21 or its analogs has been shown to have an anti-obesity effect in rodent models. In the present study, we found that 3-O-acetyloleanolic acid, an active constituent isolated from the fruits of Forsythia suspensa, stimulated FGF21 production concomitant with the up-regulation of a transcription factor, nuclear receptor Nr4a1, in C2C12 myotubes. Additionally, significant increases in mFgf21 promoter activity were observed in C2C12 cells overexpressing TGR5 receptor in response to 3-O-acetyloleanolic acid treatment. Treatment with the p38 MAPK inhibitor SB203580 was effective at suppressing these stimulatory effects of 3-O-acetyloleanolic acid. Pretreatment with SB203580 also significantly repressed FGF21 mRNA abundance and FGF21 secretion in C2C12 myotubes after 3-O-acetyloleanolic acid stimulation, suggesting that p38 activation is required for the induction of FGF21 by ligand-activated TGR5 in C2C12 myotubes. These findings collectively indicated that TGR5 receptor signaling drives FGF21 expression via p38 activation, at least partly, by mediating Nr4a1 expression. Thus, the novel biological function of 3-O-acetyloleanolic acid as an agent having anti-obesity effects is likely to be mediated through the activation of TGR5 receptors.

Gambogenic Acid Enhances Chemosensitivity of Multiple Myeloma to Bortezomib through p53/ROS/p38 MAPK Apoptotic Pathway

Background: Multiple myeloma (MM) is the second most prevalent hematological cancer (10%) with a five-year survival rate of only 49%. Approval of bortezomib, the first in-class proteasome inhibitor, has significantly improved outcomes of MM in the last few years; however, the almost inevitable refractory or resistance has made a cure of MM remains out of reach. Identification and validation of novel effective targeted agents are necessary. As one of the natural compounds isolated from Traditional Chinese Medicine gamboge, gambogenic acid (GNA) is a very promising antitumor agent and has been proved to have more potent anticancer effect and less systemic toxicity according to early investigations. In our previous studies, we have demonstrated that GNA could synergistically potentiate bortezomib-induced apoptosis of MM. In this study, our aim was to further explore and understand the concrete apoptotic action mechanism of GNA and bortezomib combination treatment against MM in pre-clinical models.Methods: We performed pre-clinical studies in myeloma cell lines and mouse models treated by GNA and bortezomib, alone and in combination. Cell counting kit-8 (CCK-8) assay, combination index (CI) isobologram, flow cytometry (FCM), western blot, xenograft tumor models, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immunohistochemistry (IHC) and oxidation-sensitive fluorescent dye carboxy-DCFDA were applied in this study. The thiol antioxidant N-acetylcysteine (NAC) and p38 MAPK inhibitor SB203580 were used to detect the effect of GNA and bortezomib combination therapy on ROS, p53 and p38 MAPK.Results: Both p53 wild type cell line MM.1S and mutant type cell line RPMI 8226 myeloma cell lines were sensitive to GNA therapy. Interestingly, GNA only enhanced the chemosensitivity of bortezomib on p53 wild type cell line MM.1S. Both 0.51μM GNA combined with 2.3nM bortezomib and 0.90μM GNA combined with 4.0nM bortezomib to MM.1S cells showed a more markedly apoptotic rate of MM compared with that of the single agent group of each accordingly (p <0.01; p <0.01), and the apoptosis was presented in a dosage- and time-dependent manner. Increased apoptosis of MM.1S cells was found via the activation of PARP cleavage, p53, Caspase-3 cleavage and Bax and inhibition of Bcl-2 expression. An increased antitumor effect of combination therapy of GNA and bortezomib on MM.1S xenograft models were also observed. A significant generation of ROS was detected after exposure of GNA and bortezomib in MM.1S cells. The expression level of phospho-p38MAPK was upregulated by combination therapy compared with that of single agent (p<0.05; p<0.05). To characterize the mechanisms of action of GNA in combination with bortezomib, we pretreated MM.1S cells with NAC and SB203580. We found that the apoptosis of MM.1S cells could be inhibited by both NAC and SB203580 (p<0.01; p<0.01). However, the generation of ROS could only be inhibited by NAC (p<0.05), and the elevated expression of p53 could be inhibited neither by NAC nor SB203580. These results indicated that p53/ROS/p38 MAPK pathway is a pre-requisite for GNA to accomplish the enhancement of chemosensitivity of MM to bortezomib.Conclusions: We demonstrate that GNA could potentiate apoptosis of MM induced by bortezomib in vivo and in vitro, and p53/ROS/p38 MAPK pathway plays a central role of the apoptotic action. Thus, our data provide a persuasive rationale for the successful utilization of dual GNA and bortezomib for chemotherapy in MM patients in the future. DisclosuresNo relevant conflicts of interest to declare.