Fibroblast GSK-3α Promotes Fibrosis via RAF-MEK-ERK Pathway in the Injured Heart.

BACKGROUND: Heart failure is the leading cause of mortality, morbidity, and health care expenditures worldwide. Numerous studies have implicated GSK-3 (glycogen synthase kinase-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms seem to play overlapping, unique and even opposing functions in the heart. Previously, we have shown that of the 2 isoforms of GSK-3, cardiac fibroblast GSK-3ß acts as a negative regulator of myocardial fibrosis in the ischemic heart. However, the role of cardiac fibroblast-GSK-3α in the pathogenesis of cardiac diseases is completely unknown. METHODS: To define the role of cardiac fibroblast-GSK-3α in myocardial fibrosis and heart failure, GSK-3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or Postn-promoter-driven Cre recombinase. Control and GSK-3α KO mice were subjected to cardiac injury and heart parameters were evaluated. The fibroblast kinome mapping was carried out to delineate molecular mechanism followed by in vivo and in vitro analysis. RESULTS: Fibroblast-specific GSK-3α deletion restricted fibrotic remodeling and preserved function of the injured heart. We observed reductions in cell migration, collagen gel contraction, α-SMA protein levels, and expression of ECM genes in TGFß1-treated KO fibroblasts, indicating that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, suggesting the profibrotic role of GSK-3α is SMAD3 independent. The molecular studies confirmed decreased ERK signaling in GSK-3α-KO CFs. Conversely, adenovirus-mediated expression of a constitutively active form of GSK-3α (Ad-GSK-3αS21A) in fibroblasts increased ERK activation and expression of fibrogenic proteins. Importantly, this effect was abolished by ERK inhibition. CONCLUSIONS: GSK-3α-mediated MEK-ERK activation is a critical profibrotic signaling circuit in the injured heart, which operates independently of the canonical TGF-ß1-SMAD3 pathway. Therefore, strategies to inhibit the GSK-3α-MEK-ERK signaling circuit could prevent adverse fibrosis in diseased hearts.

OxLDL-Stimulated Macrophages Transmit Exosomal MicroRNA-320b to Aggravate Viability, Invasion, and Phenotype Switching via Regulating PPARGC1A-Mediated MEK/ERK Pathway in Proatherogenic Vascular Smooth Muscle Cells.

Our previous study revealed oxidized-low density lipoprotein (oxLDL)-stimulated macrophages delivered exosomes to exacerbate vascular smooth muscle cell (VSMC) viability and invasion; and microRNA-320b was enriched in exosomes from oxLDL-stimulated macrophages. This study aimed to further explore molecular mechanisms of exosomal microRNA-320b from oxLDL-stimulated macrophages on cellular functions of VSMCs. Exosomes from oxLDL-stimulated macrophages with microRNA-320b mimic/inhibitor transfection were used to treat VSMCs. Next, microRNA-320b mimic/inhibitor, and microRNA-320b mimic with or without peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) overexpression vector were transfected into VSMCs. Viability, invasion, apoptosis, contractile/synthetic phenotype markers, and MEK/ERK pathway were detected in VSMCs. Exosomes from microRNA-320b mimic-treated macrophages promoted viability, invasion, and synthetic phenotype marker osteopontin, while suppressed apoptosis and contractile phenotype marker α-smooth muscle actin in VSMCs. Importantly, direct microRNA-320b mimic treatment aggravated viability, invasion, and synthetic phenotype transition in VSMCs. However, microRNA-320b inhibitor showed the opposite effects as microRNA-320b mimic. Next, luciferase reporter gene assay showed that microRNA-320b directly bound to PPARGC1A; microRNA-320b also inversely regulated PPARGC1A in VSMCs. Moreover, the effect of microRNA-320b mimic on cellular functions of VSMCs was hampered by PPARGC1A overexpression vector (all P < 0.05). Additionally, microRNA-320b activated MEK/ERKT pathway, which was also suppressed by PPARGC1A overexpression vector (all P < 0.05). OxLDL-stimulated macrophages deliver exosomal microRNA-320b to exacerbate viability, invasion, and synthetic phenotype transition in VSMCs via modulating PPARGC1A-mediated MEK/ERK pathway, thus participating in the progression of atherosclerosis.

BDNF-induced phrenic motor facilitation shifts from PKCθ to ERK dependence with mild systemic inflammation.

Moderate acute intermittent hypoxia (mAIH) elicits a form of phrenic motor plasticity known as phrenic long-term facilitation (pLTF), which requires spinal 5-HT2 receptor activation, ERK/MAP kinase signaling, and new brain-derived neurotrophic factor (BDNF) synthesis. New BDNF protein activates TrkB receptors that normally signal through PKCθ to elicit pLTF. Phrenic motor plasticity elicited by spinal drug administration (e.g., BDNF) is referred to by a more general term: phrenic motor facilitation (pMF). Although mild systemic inflammation elicited by a low lipopolysaccharide (LPS) dose (100 µg/kg; 24 h prior) undermines mAIH-induced pLTF upstream from BDNF protein synthesis, it augments pMF induced by spinal BDNF administration through unknown mechanisms. Here, we tested the hypothesis that mild inflammation shifts BDNF/TrkB signaling from PKCθ to alternative pathways that enhance pMF. We examined the role of three known signaling pathways associated with TrkB (MEK/ERK MAP kinase, PI3 kinase/Akt, and PKCθ) in BDNF-induced pMF in anesthetized, paralyzed, and ventilated Sprague Dawley rats 24 h post-LPS. Spinal PKCθ inhibitor (TIP) attenuated early BDNF-induced pMF (≤30 min), with minimal effect 60-90 min post-BDNF injection. In contrast, MEK inhibition (U0126) abolished BDNF-induced pMF at 60 and 90 min. PI3K/Akt inhibition (PI-828) had no effect on BDNF-induced pMF at any time. Thus, whereas BDNF-induced pMF is exclusively PKCθ-dependent in normal rats, MEK/ERK is recruited by neuroinflammation to sustain, and even augment downstream plasticity. Because AIH is being developed as a therapeutic modality to restore breathing in people living with multiple neurological disorders, it is important to understand how inflammation, a common comorbidity in many traumatic or degenerative central nervous system disorders, impacts phrenic motor plasticity.NEW & NOTEWORTHY We demonstrate that even mild systemic inflammation shifts signaling mechanisms giving rise to BDNF-induced phrenic motor plasticity. This finding has important experimental, biological, and translational implications, particularly since BDNF-dependent spinal plasticity is being translated to restore breathing and nonrespiratory movements in diverse clinical disorders, such as spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS).

Valtrate, an iridoid compound in Valeriana, elicits anti-glioblastoma activity through inhibition of the PDGFRA/MEK/ERK signaling pathway.

BACKGROUND: Valtrate, a natural compound isolated from the root of Valeriana, exhibits antitumor activity in many cancers through different mechanisms. However, its efficacy for the treatment of glioblastoma (GBM), a tumor type with a poor prognosis, has not yet been rigorously investigated. METHODS: GBM cell lines were treated with valtrate and CCK-8, colony formation and EdU assays, flow cytometry, and transwell, 3D tumor spheroid invasion and GBM-brain organoid co-culture invasion assays were performed to assess properties of proliferation, viability, apoptosis and invasion/migration. RNA sequencing analysis on valtrate-treated cells was performed to identify putative target genes underlying the antitumor activity of the drug in GBM cells. Western blot analysis, immunofluorescence and immunohistochemistry were performed to evaluate protein levels in valtrate-treated cell lines and in samples obtained from orthotopic xenografts. A specific activator of extracellular signal-regulated kinase (ERK) was used to identify the pathways mediating the effect. RESULTS: Valtrate significantly inhibited the proliferation of GBM cells in vitro by inducing mitochondrial apoptosis and suppressed invasion and migration of GBM cells by inhibiting levels of proteins associated with epithelial mesenchymal transition (EMT). RNA sequencing analysis of valtrate-treated GBM cells revealed platelet-derived growth factor receptor A (PDGFRA) as a potential target downregulated by the drug. Analysis of PDGFRA protein and downstream mediators demonstrated that valtrate inhibited PDGFRA/MEK/ERK signaling. Finally, treatment of tumor-bearing nude mice with valtrate led to decreased tumor volume (fivefold difference at day 28) and enhanced survival (day 27 vs day 36, control vs valtrate-treated) relative to controls. CONCLUSIONS: Taken together, our study demonstrated that the natural product valtrate elicits antitumor activity in GBM cells through targeting PDGFRA and thus provides a candidate therapeutic compound for the treatment of GBM.

NFIC1 inhibits the migration and invasion of MDA-MB-231 cells through S100A2-mediated inactivation of MEK/ERK pathway.

NFIC is a potent transcriptional factor involved in many physiological and pathological processes, including tumorigenesis. However, the role of NFIC1, the longest isoform of NFIC, in the progression of triple negative breast cancer (TNBC) remains elusive. Our study demonstrates that overexpression of NFIC1 inhibits the migration and invasion of TNBC MDA-MB-231 cells. NFIC1 regulates the expression of S100A2, and knockdown of S100A2 reverses the inhibitive effects of NFIC1 on the migration and invasion of MDA-MB-231 cells. Furthermore, knockdown of S100A2 activates the MEK/ERK signaling transduction pathway that is inhibited by NFIC1 overexperssion. Treatment with MEK/ERK pathway inhibitor, U0126, abolishes the effects of S100A2 knockdown. In addition, overexpression of NFIC1 in MDA-MB-231 cells increases the expression of epithelial markers and decreases the expression of mesenchymal markers, and these effects could also be reversed by knockdown of S100A2. Collectively, these results demonstrate that NFIC1 inhibits the Epithelial-mesenchymal transition (EMT) of MDA-MB-231 cells by regulating S100A2 expression, which suppress the activation of MEK/ERK pathway. Therefore, our study confirms the role of NFIC1 as a tumor repressor in TNBC, and reveals the molecular mechanism through which NFIC1 inhibits the migration and invasion of MDA-MB-231 cells.

Hepatoprotective effect of p-Coumaric acid against KBrO3 -induced apoptosis in HepG2 cells.

In the present study, we investigated the effect of the p-Coumaric acid (PCA), a phenolic acid, on potassium bromate (KBrO3 ) induced oxidative damage, Ras/Raf/MEK signaling, and apoptosis in HepG2 cells. Our findings showed that PCA-treated cells prevented cytotoxicity compared with KBrO3- treated cells. Furthermore, KBrO3 -induced oxidative stress and lipid peroxidation was attenuated by PCA and it also increased the antioxidant levels such as SOD, CAT, and GPX. Additionally, PCA inhibited the KBrO3 -induced DNA damage in HepG2 cells. Moreover, PCA treatment suppressed the activation of Ras/Raf/MEK signaling and increased the expression of PRDX-1. In addition, PCA prevented the KBrO3 -induced apoptosis cascade by altering the expression of proapoptotic, Bax, caspase-3, and antiapoptotic, Bcl-2 proteins. The present study proves that PCA inhibited the KBrO3 -induced oxidative stress, DNA damage, and apoptotic signaling cascade in HepG2 cells.

Cell Division Control Protein 42 Facilitates Diabetic Retinopathy Progression by Activating the MEK/ERK Pathway.

Cell division control protein 42 (CDC42) modulates insulin secretion and angiogenesis to participate in the pathology of diabetic complications and retinal vascular-associated diseases. This study intended to explore the role of CDC42 in the progression of diabetic retinopathy, and the underlying mechanism. Human retinal microvascular endothelial cells (hRMECs) were cultured in 5.5 mM glucose (normal glucose) or 25 mM glucose (high glucose; HG) medium, respectively. CDC42 overexpression plasmid and small interference RNA (oe-CDC42 and si-CDC42) or corresponding negative controls (oe-NC and si-NC) were transfected into hRMECs under HG. Then, platelet-activating factor C-16 (C16-PAF) (MEK/ERK pathway activator) was added to si-CDC42 or si-NC transfected hRMECs under HG. Our study showed that HG increased CDC42 mRNA and protein, cell viability, invasive cell count, branch points, and tube length but reduced cell apoptosis in hRMECs. CDC42 upregulation enhanced cell viability, invasive cell count, branch points, tube length, p-MEK, and p-ERK, but attenuated cell apoptosis. Downregulation of CDC42 exhibited opposite trends. In addition, C16-PAF also increased cell viability, invasive cell count, branch points, and tube length, p-MEK, and p-ERK, but retarded cell apoptosis. Notably, C16-PAF diminished the effect of CDC42 downregulation on the above-mentioned functions in hRMECs under HG. Conclusively, CDC42 promotes HG-induced hRMEC viability and invasion, as well as angiogenesis, but inhibits apoptosis by activating the MEK/ERK pathway, which may be responsible for the progression of diabetic retinopathy.

Novel EGFR-MAPK kinase and ABC transporter inhibitors for HepG2 resistant to Erlotinib.

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer death in the world, with outlook for most patients having a 5-year survivability of less than 5%. In a previous study from our laboratory, novel estrone inspired analogs act as epidermal growth factor receptor (EGFR) inhibitors in HepG2 cells. This study focuses on the effect of these analogs on an HCC cell line resistance to Erlotinib. Lead compounds MMA132 and MMA102 showed 13 and 20 µM IC50 values, respectively against HepG2-R resistant to Erlotinib. These compounds showed cell cycle arrest of the G2 phase up to 54%, and inhibited cell migration of HepG2-R cells up to 48 h. Western blot analysis revealed that MMA132 reduced total EGFR content after 48 h, while MMA102 inhibited MEK kinase by 84% after 48 h. Western blot analysis also revealed that multidrug resistance protein 2 (MRP2) is overexpressed in HepG2-R, suggesting that ABC transporters play a likely cause in drug resistance. MMA102 showed significant inhibition of both P-glycoprotein (83%) and ABCG2 (53%), two additional ABC transporters. Additionally, MMA102 and MMA132 were used in a combination therapy with MK571(MRP1/2 inhibitor) and produced IC50 values of 18 and 10 µM, respectively, better than either MMA102/132 or MK571 alone. To validate our findings, we conducted molecular dynamic simulations with MMA102 and MMA132 in MEK, P-glycoprotein, MRP1, and MRP2. Results coincided with biological findings in which MMA102 orientation is favored in both MEK and P-glycoprotein pockets, whereas MMA132 likely binds with MRP2, as likely suggested by the combinatorial study.

[Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells].

This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.

Suppression of phosphodiesterase IV enzyme by roflumilast ameliorates cognitive dysfunction in aged rats after sevoflurane anaesthesia via PKA-CREB and MEK/ERK pathways.

Postoperative cognitive dysfunction (POCD) is a prevalent disorder after anaesthesia in the elderly patients. Roflumilast (RF), a phosphodiesterase 4 (PDE-4) inhibitor, could improve cognition with no side effects. Here, we sought to explore the efficacy of RF in the improvement of cognitive dysfunction caused by sevoflurane (Sev). Sprague-Dawley rats were anaesthetized, and the hippocampal neurons were treated with Sev to develop in vivo and in vitro POCD models, followed by RF administration. The mechanism of the PKA-CREB and MEK/ERK pathways in the pathogenesis of POCD was explored. Sev impaired the cognitive functions of rats, significantly reduced cyclic adenosine monophosphate (cAMP) concentrations and blocked the PKA-CREB and MEK/ERK pathways. Moreover, the Sev-treated rats and neurons exhibited enhanced apoptosis and reactive oxygen species (ROS). After treatment with RF, rats had better learning and memory function, and the activity of neurons in hippocampus and cortex was improved. Loss-of-function assay indicated that PKA-CREB and MEK/ERK signalling impairment reduced cAMP levels and promoted apoptosis and ROS in rat hippocampus and neurons. Generally, RF promotes neuronal activity in rats after Sev treatment by maintaining cAMP levels and sustaining the activation of PKA-CREB and MEK/ERK pathways. This might offer novel sights for POCD therapy.

3-Nitrobenzanthrone promotes malignant transformation in human lung epithelial cells through the epiregulin-signaling pathway.

Exposure to environmental and occupational contaminants leads to lung cancer. 3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA) is a potential carcinogen in ambient air or diesel particulate matter. Studies have revealed that short-term exposure to 3-NBA induces cell death, reactive oxygen species activation, and DNA adduct formation and damage. However, details of the mechanism by which chronic exposure to 3-NBA influences lung carcinogenesis remain largely unknown. In this study, human lung epithelial BEAS-2B cells were continuously exposed to 0-10-µM 3-NBA for 6 months. NanoString analysis was conducted to evaluate gene expression in the cells, revealing that 3-NBA-mediated transformation results in a distinct gene expression signature including carbon cancer metabolism, metastasis, and angiogenesis. Alterations in tumor-promoting genes such as EREG (epiregulin), SOX9, E-cadherin, TWIST, and IL-6 were involved in epithelial cell aggressiveness. Kaplan-Meier plotter analyses indicated that increased EREG and IL-6 expressions in early-stage lung cancer cells are correlated with poor survival. In vivo xenografts on 3-NBA-transformed cells exhibited prominent tumor formation and metastasis. EREG knockout cells exposed to 3-NBA for a short period exhibited high apoptosis and low colony formation. By contrast, overexpression of EREG in 3-NBA-transformed cells markedly activated the PI3K/AKT and MEK/ERK signaling pathways, resulting in tumorigenicity. Furthermore, elevated IL-6 and EREG expressions synergistically led to STAT3 signaling activation, resulting in clonogenic cell survival and migration. Taken together, chronic exposure of human lung epithelial cells to 3-NBA leads to malignant transformation, in which the EREG signaling pathway plays a pivotal mediating role. • Short-term exposure of lung epithelial cells to 3-NBA can lead to ROS production and cell apoptosis. • Long-term chronic exposure to 3-NBA upregulates the levels of tumor-promoting genes such as EREG and IL-6. • Increased EREG expression in 3-NBA-transformed cells markedly contributes to tumorigenesis through PI3K/AKT and MEK/ERK activation and synergistically enhances the IL-6/STAT3 signaling pathway, which promotes tumorigenicity.

Polyphyllin I improves myocardial damage in coronary artery disease via modulating lipid metabolism and myocardial apoptosis.

Polyphyllin I (PPI) is a famous traditional medicine ingredient, which has been explored in wide range of areas. Nevertheless, whether PPI exerts any functions in coronary artery disease (CAD) is still uncertified. Herein, we probed the effect and mechanism of PPI on lipid metabolism and myocardial dysfunction in myocardial cells and CAD rat model. Hypoxia/reoxygenation (H/R)-treated H9c2 cells model was constructed for the in vitro experiments, and CAD model in vivo was established by high-fat feeding. After management with PPI, the correlated factors of lipid metabolism and myocardial function were investigated. The apoptosis of myocardial cells was assessed by Annexin V-FITC/PI kit and TUNEL staining. The apoptosis-associated factors (caspase 3, cleaved caspase 3, Bax, and Bcl-2) were tested by Western blot analysis. The MEK/ERK inhibitor was applied and the functions of MEK/ERK pathway in myocardial damage were investigated. H/R-treated H9c2 cells model was constructed for the in vitro experiments, and CAD model in vivo was established by high-fat feeding. After management with PPI, the correlated factors of lipid metabolism and myocardial function were investigated. The apoptosis of myocardial cells was assessed by Annexin V-FITC/PI kit and TUNEL staining. The apoptosis-associated factors (caspase 3, cleaved caspase 3, Bax, and Bcl-2) were tested by Western blot analysis. The MEK/ERK inhibitor was applied and the functions of MEK/ERK pathway in myocardial damage were investigated. PPI improved lipid metabolism disorder in H/R-induced H9c2 cells or in CAD rat model. Additionally, PPI attenuated myocardial dysfunction in CAD rats via enhancing left ventricular systolic pressure, maximum rate of change of left ventricular pressure (±dp/dtmax ), and arterial blood flow (CF). The apoptosis of myocardial cells was lessened by PPI management, which was further verified by reducing Bax and cleaved caspase 3 expression. Furthermore, PD0325901 (MEK/ERK inhibitor) weakened the effect of PPI on myocardial dysfunction, lipid metabolism, and myocardial cell apoptosis in CAD rats. The research confirmed the protective effect of PPI on myocardial damage in CAD, which was regulated by MEK/ERK pathway.

Reckoning apigenin and kaempferol as a potential multi-targeted inhibitor of EGFR/HER2-MEK pathway of metastatic colorectal cancer identified using rigorous computational workflow.

In the past two decades, the treatment of metastatic colorectal cancer (mCRC) has been revolutionized as multiple cytotoxic, biological, and targeted drugs are being approved. Unfortunately, tumors treated with single targeted agents or therapeutics usually develop resistance. According to pathway-oriented screens, mCRC cells evade EGFR inhibition by HER2 amplification and/or activating Kras-MEK downstream signaling. Therefore, treating mCRC patients with dual EGFR/HER2 inhibitors, MEK inhibitors, or the combination of the two drugs envisaged to prevent the resistance development which eventually improves the overall survival rate. In the present study, we aimed to screen potential phytochemical lead compounds that could multi-target EGFR, HER2, and MEK1 (Mitogen-activated protein kinase kinase) using a computer-aided drug design approach that includes molecular docking, endpoint binding free energy calculation using MM-GBSA, ADMET, and molecular dynamics (MD) simulations. Docking studies revealed that, unlike all other ligands, apigenin and kaempferol exhibit the highest docking score against all three targets. Details of ADMET analysis, MM/GBSA, and MD simulations helped us to conclusively determine apigenin and kaempferol as potentially an inhibitor of EGFR, HER2, and MEK1 apigenin and kaempferol against mCRC at a systemic level. Additionally, both apigenin and kaempferol elicited antiangiogenic properties in a dose-dependent manner. Collectively, these findings provide the rationale for drug development aimed at preventing CRC rather than intercepting resistance.

Downregulation of lncRNA SNHG16 inhibits vascular smooth muscle cell proliferation and migration in cerebral atherosclerosis by targeting the miR-30c-5p/SDC2 axis.

Atherosclerosis (AS) is the basic lesion underlying the occurrence and development of cerebrovascular diseases. Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in AS. We aimed to explore the role of SNHG16 in AS and the molecular mechanism of VSMC involvement in the regulation of AS. The expression levels of SNHG16, miR-30c-5p and SDC2 were detected by qRT-PCR. CCK-8, wound healing and Transwell assays were used to assess ox-LDL-induced VSMC proliferation, migration, and invasion, respectively. Western blot analysis was used to detect SDC2 and MEK/ERK pathway-related protein levels. A dual-luciferase reporter assay confirmed the binding of SNHG16 with miR-30c-5p and miR-30c-5p with SDC2. SNHG16 and SDC2 expression was upregulated in patients with AS and ox-LDL-induced VSMCs, while miR-30c-5p was downregulated. Ox-LDL-induced VSMC proliferation and migration were increased, and the MEK/ERK signalling pathway was activated. MiR-30c-5p was targeted to SNHG16 and SDC2. Downregulating SNHG16 or upregulating miR-30c-5p inhibited ox-LDL-induced VSMC proliferation and migration and inhibited MEK/ERK signalling pathway activation. In contrast, downregulating miR-30c-5p or upregulating SDC2 reversed the effects of downregulating SNHG16 or upregulating miR-30c-5p. Furthermore, downregulating SDC2 inhibited ox-LDL-induced proliferation and migration of VSMCs and inhibited activation of the MEK/ERK signalling pathway, while upregulating lncRNA SNHG16 reversed the effects of downregulating SDC2. Downregulation of SNHG16 inhibited VSMC proliferation and migration in AS by targeting the miR-30c-5p/SDC2 axis. This study provides a possible therapeutic approach to AS.

PD-L1/PD-L1 signalling promotes colorectal cancer cell migration ability through RAS/MEK/ERK.

Programmed death ligand 1 (PD-L1) is widely known as an immune checkpoint, and immunotherapy through the inhibition of checkpoint molecules has become an important component in the successful treatment of tumours via programmed death 1 (PD-1)/PD-L1 signalling pathways. However, its biological functions and expression profile in colorectal cancer (CRC) are elusive. We previously found that PD-L1 can bind to PD-L1 and cause cell detachment. However, the detailed molecular mechanisms of how PD-L1 binds to PD-L1 and how it transmits signals to the cell remain unclear. In this study, we disclosed that PD-L1 expression was dramatically upregulated in CRC compared to normal tissues. Ectopic expression of PD-L1 inhibits cell adhesive capacity and promotes cell migration in CRC cell lines, while silencing PD-L1 had the opposite effects and suppressed invasion and proliferation. Mechanistically, PD-L1 was found to promote epithelial-mesenchymal transition (EMT) through the ERK signalling molecule pathway and interacted with the 1-86 aa fragment of KRAS to transduce signals. Collectively, our study demonstrated the role of PD-L1 after binding to PD-L1 in CRC, thereby providing a new theoretical basis for further improving immunotherapy with anti-PD-L1 antibodies.

NeiyiKangfu tablets control the progression of endometriosis through inhibiting RAF/MEK/ERK signal pathway by targeting RKIP.

BACKGROUND: NeiyiKangfu tablets (NYKF) are widely used clinically for the treatment of endometriosis (EMS), whose mechanism of action has been extensively studied. Researchers have found that NYKF may control the development of ectopic lesions by inhibiting angiogenesis and inflammatory cytokine secretion. Nevertheless, NYKF's mechanism of action remains unclear. METHODS: In the present study, the function of NYKF in the progression of EMS and the associated underlying mechanism was investigated by in vivo and in vitro experiments. EMS model mice were treated with NYKF and the pro-inflammatory factors and apoptosis of ectopic endometrium as well as RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling activation were assessed. In addition, human endometriosis-derived immortalized entopic stromal (hEM15A) cells transfected with or without RAF kinase inhibitor protein (RKIP)-small-interfering RNA (siRNA) were also treated with NYKF and the proliferation, migration, apoptosis, and RAF/MEK/ERK signaling activation were measured by Cell Counting Kit-8 (CCK-8), flow cytometry, Transwell, and western blot, respectively. RESULTS: Results showed that NYKF increased the expression of RKIP, inhibited RAF/MEK/ERK signaling activation, and induced apoptosis while inhibiting proliferation and migration both in EMS mice and hEM15A cells. RKIP knockdown could inhibit the effect of NYKF treatment, leading to the activation of RAF/MEK/ERK signaling and the proliferation and migration of hEM15A cells. CONCLUSIONS: In conclusion, these results suggest that NYKF treatment promotes apoptosis and inhibits proliferation and migration in EMS by inhibiting the RAF/MEK/ERK signaling pathway by targeting RKIP.

Alnustone promotes megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway.

OBJECTIVES: Thrombocytopenia is a disease in which the number of platelets in the peripheral blood decreases. It can be caused by multiple genetic factors, and numerous challenges are associated with its treatment. In this study, the effects of alnustone on megakaryocytes and platelets were investigated, with the aim of developing a new therapeutic approach for thrombocytopenia. METHODS: Random forest algorithm was used to establish a drug screening model, and alnustone was identified as a natural active compound that could promote megakaryocyte differentiation. The effect of alnustone on megakaryocyte activity was determined using cell counting kit-8. The effect of alnustone on megakaryocyte differentiation was determined using flow cytometry, Giemsa staining, and phalloidin staining. A mouse model of thrombocytopenia was established by exposing mice to X-rays at 4 Gy and was used to test the bioactivity of alnustone in vivo. The effect of alnustone on platelet production was determined using zebrafish. Network pharmacology was used to predict targets and signaling pathways. Western blotting and immunofluorescence staining determined the expression levels of proteins. RESULTS: Alnustone promoted the differentiation and maturation of megakaryocytes in vitro and restored platelet production in thrombocytopenic mice and zebrafish. Network pharmacology and western blotting showed that alnustone promoted the expression of interleukin-17A and enhanced its interaction with its receptor, and thereby regulated downstream MEK/ERK signaling and promoted megakaryocyte differentiation. CONCLUSIONS: Alnustone can promote megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway and thus provides a new therapeutic strategy for the treatment of thrombocytopenia.

Role of MAPKs in TGF-ß1-induced maturation and mineralization in human osteoblast-like cells.

OBJECTIVES: Our study aimed to clarify the role of mitogen-activated protein kinases (MAPKs) in transforming growth factor (TGF)-ß1-stimulated mineralization in the human osteoblast-like MG63 cells. METHODS: The viability of MG63 cells under TGF-ß1 stimulation was assessed by MTS assay. Western blotting determined TGF-ß1-mediated activation of extracellular signal-related protein kinase (ERK), p38, and c-Jun amino-terminal kinase (JNK). Mineralization-related gene expression was examined by quantitative real-time PCR, and mineral deposition levels were evaluated by alizarin red S staining. RESULTS: TGF-ß1 had no effect on MG63 cell proliferation. Activation of p38 was observed at 3 h post TGF-ß1 stimulation. Moreover, JNK phosphorylation was upregulated by TGF-ß1 from 1 to 6 h post stimulation, but had no activation on ERK phosphorylation throughout the experimental period. Treatment with JNK inhibitor diminished the alizarin red S-stained area in a dose-dependent manner. Mineral deposition was unaffected by MEK inhibitor, whereas p38 inhibitor increased the red-stained area. Gene expression levels of ALP and BSP were significantly decreased under treatment with JNK inhibitor and p38 inhibitor. The MEK inhibitor had no effect on the TGF-ß1-mediated upregulation of ALP and BSP. Although all three inhibitors suppressed expression of COL I, none were found to stimulate expression of OCN. CONCLUSIONS: Human osteoblast-like MG63 cells maturation and mineralization are induced through JNK activation of MAPK signaling in response to TGF-ß1.

Therapeutic mechanism of Liangxue-Guyuan-Yishen decoction on intestinal stem cells and tight junction proteins in gastrointestinal acute radiation syndrome rats.

On the basis of the previous research, the Traditional Chinese Medicine theory was used to improve the drug composition for gastrointestinal acute radiation syndrome (GI-ARS). The purpose of this study was to study the therapeutic mechanism of Liangxue-Guyuan-Yishen decoction (LGYD) on GI-ARS and to provide a new scheme for the treatment of radiation injury. Here, we investigated the effects of LGYD on intestinal stem cells (ISCs) in a GI-ARS rat model. Rat health and survival and the protective efficacy of LGYD on the intestines were analyzed. The active principles in LGYD were detected using liquid chromatography-mass spectrometry (LC-MS). ISC proliferation, intestinal epithelial tight junction (TJ) protein expression and regulatory pathways were explored using immunohistochemistry, western blotting (WB) and reverse transcription quantitative polymerase chain reaction (RT-qPCR), respectively. Involvement of the WNT and MEK/ERK pathways in intestinal recovery was screened using network pharmacology analysis and validated by WB and RT-qPCR. LGYD administration significantly improved health and survival in GI-ARS rats. Pathological analysis showed that LGYD ameliorated radiation-induced intestinal injury and significantly promoted LGR5+ stem cell regeneration in the intestinal crypts, upregulated TJ protein, and accelerated crypt reconstruction in the irradiated rats. LC-MS revealed ≥13 constituents that might contribute to LGYD's protective effects. Collectively, LGYD can promote crypt cell proliferation and ISCs after radiation damage, the above effect may be related to WNT and MEK/ERK pathway.

Rice Mitogen-Activated Protein Kinase regulates serotonin accumulation and interacts with cell cycle regulators under prolonged UV-B exposure.

Stress conditions such as UV-B exposure activates MAPKs in Arabidopsis and rice. UV-B radiation is hazardous to plant as it causes photosystem disruption, DNA damage and ROS generation. Here we report its effect on biological pathways by studying the global changes in transcript profile in rice seedling exposed to UV-B radiation for 1 h and 16 h. Short UV-B exposure (1 h) led to moderate changes, while a drastic change in transcript landscape was observed after long term UV-B exposure (16 h) in rice seedlings. Prolonged UV-B exposure negatively impacts the expression of cell cycle regulating genes and several other metabolic pathways in developing seedlings. MAP kinase signaling cascade gets activated upon UV-B exposure similar to reports in Arabidopsis indicating conservation of its function in both dicot and monocot. Expression analysis in inducible overexpression transgenic lines of MPK3 and MPK6 shows higher transcript abundance of phytoalexin biosynthesis gene like Oryzalexin D synthase and Momilactone A synthase, along with serotonin biosynthesis genes. An accumulation of serotonin was observed upon UV-B exposure and its abundance positively correlates with the MPK3 and MPK6 transcript level in the respective over-expression lines. Interestingly, multiple cell cycle inhibitor proteins including WEE1 and SMR1 interact with MPK3 and MPK6 thus, implying a major role of this pathway in cell cycle regulation under stress condition. Overall overexpression of MPK3 and MPK6 found to be detrimental for rice as overexpression lines shows higher cell death and compromised tolerance to UV-B.