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.

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.

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.

[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.

Simvastatin Preferentially Targets FLT3/ITD Acute Myeloid Leukemia by Inhibiting MEK/ERK and p38-MAPK Signaling Pathways.

BACKGROUND: The FLT3/ITD mutation exists in many acute myeloid leukemia (AML) patients and is related to the poor prognosis of patients. In this study, we attempted to evaluate the antitumor activity of simvastatin, a member of the statin class of drugs, in vitro and in vivo models of FLT3/ITD AML and to identify the potential mechanisms. METHODS: Cell Counting Kit-8 (CCK-8) and Annexin V/propidium iodide (PI) staining kits were used to detect cell viability and apoptosis, respectively. Subsequently, Western blot and rescue experiment were applied to explore the potential molecular mechanism. In vivo anti-leukemia activity of simvastatin was evaluated in xenograft mouse models. RESULTS: In vitro experiments revealed that simvastatin inhibited AML progression in a dose- and time-dependent manner, while in vivo experiments showed that simvastatin significantly reduced tumor burden in FLT3/ITD xenograft mouse models. After simvastatin treatment of FLT3/ITD AML cells, intracellular Rap1 was downregulated and the phosphorylation levels of its downstream targets MEK, ERK and p38 were significantly inhibited. The rescue experiment showed that mevalonate, an intermediate product of the metabolic pathway of mevalonate, and its downstream geranylgeranyl pyrophosphate (GGPP) played a key role in this process. Finally, we demonstrate that simvastatin can induce apoptosis of primary AML cells, while having no effect on peripheral blood mononuclear cells from normal donors. CONCLUSIONS: Simvastatin can selectively and effectively eradicate FLT3/ITD AML cells in vitro and in vivo, and its mechanism may be related to the disruption of the HMG-CoA reductase pathway and the downregulation of the MEK/ERK and p38-MAPK signaling pathways.

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.

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.

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.

Functionalized human umbilical cord mesenchymal stem cells and injectable HA/Gel hydrogel synergy in endometrial repair and fertility recovery.

Intrauterine adhesions (IUA) caused by endometrial injury are one of the main causes of female infertility. The current treatments for endometrial injury offer limited clinical benefits and cannot improve endometrial receptivity and pregnancy outcomes. Tissue engineering and regenerative medicine are considered potential solutions to address this concern and may offer effective treatment methods for the regeneration of injured human endometrium. Herein, we prepared an injectable hydrogel based on oxidized hyaluronic acid (HA-CHO) and hydrazide-grafted gelatin (Gel-ADH). The injectable hydrogel showed satisfactory biocompatibility when mixed with human umbilical cord mesenchymal stem cells (hUCMSCs). In an endometrial injury rat model, the treatment with hUCMSCs-loaded injectable hydrogel significantly enhanced the thickness of the endometrium and increased the abundance of blood vessels and glands in the injured endometrium compared to the control group. The hUCMSCs-loaded injectable hydrogel treatment significantly reduced endometrial fibrosis, decreased the expression of the pro-inflammatory factors (IL-1ß and IL-6) and increased the expression of the anti-inflammatory factor (IL-10). This treatment induced endometrial VEGF expression by activating the MEK/ERK1/2 signaling pathway. Moreover, this treatment improved endometrial receptivity to the embryo and restored the embryo implantation rate similar to the sham group (48% in the sham group vs 46% in the treatment group), and this treatment achieved pregnancy and live birth in rats with endometrial injury. In addition, we also preliminarily validated the safety of this treatment in the maternal rats and fetuses. Collectively, our study showed that the hUCMSCs-loaded injectable hydrogel hold potential as an effective treatment strategy promoting rapid recovery of endometrial injury, and this hydrogel is a promising biomaterial for regenerative medicine applications. STATEMENT OF SIGNIFICANCE: 1. Oxidized hyaluronic acid (HA-CHO)/hydrazide-grafted gelatin (Gel-ADH) hydrogel combined with human umbilical cord mesenchymal stem cells (hUCMSCs) are effective in improving the regeneration of endometrium in the endometrial injury rat model. 2. The hUCMSCs-loaded hydrogel treatment promotes the expression of endometrial VEGF through MEK/ERK1/2 signaling pathway and regulates the balance of inflammatory factors. 3. The embryo implantation and live birth rates restore to normal level in the endometrial injury rat model, and the hydrogel has no adverse effects on maternal rats, fetuses, and offspring development after the treatments.

Combination of the LARS1 Inhibitor, BC-LI-0186 with a MEK1/2 Inhibitor Enhances the Anti-Tumor Effect in Non-Small Cell Lung Cancer.

PURPOSE: The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth and proliferation by growth factor coordination and amino acid availability. Leucyl-tRNA synthetase 1 (LARS1) senses the intracellular leucine concentration and mediates amino acid-induced activation of mTORC1. Thus, LARS1 inhibition could be useful in cancer treatment. However, the fact that mTORC1 can be stimulated by various growth factors and amino acids suggests that LARS1 inhibition alone has limitations in inhibiting cell growth and proliferation. We investigated the combined effects of BC-LI-0186, a LARS1 inhibitor, and trametinib, an MEK inhibitor, on non-small cell lung cancer (NSCLC). Materials and Methods: Protein expression and phosphorylation were observed by immunoblotting, and genes differentially expressed between BC-LI-0186-sensitive and -resistant cells were identified by RNA sequencing. The combined effect of the two drugs was inferred from the combination index values and a xenograft model. RESULTS: LARS1 expression was positively correlated with mTORC1 in NSCLC cell lines. BC-LI-0186 treatment of A549 and H460 cells maintained in media supplemented with fetal bovine serum revealed paradoxical phosphorylation of S6 and activation of mitogen- activated protein kinase (MAPK) signaling. Compared with BC-LI-0186-sensitive cells, -resistant cells showed enrichment of the MAPK gene set. The combination of trametinib and BC-LI-0186 inhibited the phosphorylation of S6, MEK, and extracellular signal-regulated kinase and their synergistic effects were confirmed in a mouse xenograft model. CONCLUSION: The combination of BC-LI-0186 and trametinib inhibited the non-canonical mTORC1-activating function of LARS1. Our study demonstrated a new therapeutic approach for NSCLC without targetable driver mutations.

The Tpl2-MEK pathway plays a critical role in spheroid-cultured endometriotic stromal cells.

PROBLEM: Endometriosis is a proliferative disease characterized by cytokine-induced inflammation. The objective of this study was to assess cell growth and PGE2 production induced by TNF-α in endometriotic stromal cells (ESCs) in spheroid cell culture and to identify the signaling pathway involved with a view to finding new therapeutic targets for endometriosis. METHOD OF STUDY: Tissue samples were collected from patients with and without endometriosis. ESCs were isolated from ovarian endometrioma (OE). Gene expression was evaluated by real-time PCR and DNA microarray analysis, the proliferative effect on ESCs by WST-8 assay, and PGE2 production by ELISA. Protein phosphorylation was detected using western blotting. RESULTS: COX-2, aromatase and VEGFA mRNA expression and PGE2 production were significantly elevated in spheroid cell cultures compared to monolayer cell cultures. TNF-α receptor (TNFR) 1 and TNFR2 mRNA was also significantly increased. TNF-α promoted the proliferation and PGE2 production of ESCs in spheroid cell cultures significantly more than in monolayer cell cultures. TNF-α increased the expression of several genes related to the pathophysiology of endometriosis in spheroid ESCs. DNA microarray analysis revealed that the Tpl2 gene, which codes for a MAPK upstream of MEK, was upregulated in OE and endometrium with endometriosis compared to normal endometrium. TNF-α increased the phosphorylation and expression of Tpl2 and MEK, and Tpl2 and MEK inhibitors inhibited TNF-α-induced proliferation and PGE2 production in spheroid ESCs. CONCLUSION: The Tpl2-MEK signaling pathway may play a critical role in the cell growth and PGE2 production induced by TNF-α in spheroid ESCs.

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.

PLX8394, a RAF inhibitor, inhibits enterovirus 71 replication by blocking RAF/MEK/ERK signaling.

Enterovirus 71 (EV71) poses a serious threat to human health, with scattered outbreaks worldwide. There are several vaccines against a few EV71 strains but no efficient drug for the treatment of EV71 infection. Therefore, it is urgent and of significance to develop anti-EV71 drugs. Here, we found that PLX8394, a RAF inhibitor, possesses high antiviral activity against EV71 in vitro, being superior to the traditional clinical drug ribavirin. Moreover, PLX8394 exhibits broad-spectrum antiviral activity against enteroviruses. Notably, in a suckling mouse model, PLX8394 provided a 70% protection rate for EV71-infected mice, reduced the viral load in liver and heart tissues, and relieved the inflammatory response. A mechanistic study showed that PLX8394 inhibited EV71 by suppressing the RAF/MEK/ERK signaling pathway. Thus, PLX8394 lays a foundation for the development of new drugs against EV71.

Curcumin Alleviates Hepatic Ischemia-Reperfusion Injury by Inhibiting Neutrophil Extracellular Traps Formation.

BACKGROUND: Hepatic ischemia-reperfusion injury (IRI) is a common innate immune-mediated sterile inflammatory response in liver transplantation and liver tumor resection. Neutrophil extracellular traps (NETs) can aggravate liver injury and activates innate immune response in the process of liver IRI. However, Curcumin (Cur) can reverse this damage and reduce NETs formation. Nevertheless, the specific regulatory mechanism is still unclear in liver IRI. This study aimed to explore the potential mechanisms that how does Cur alleviate hepatic IRI by inhibits NETs production and develop novel treatment regimens. METHODS: We established a hepatic IRI model by subjecting C57BL/6J mice to 60 min of ischemia, followed by reperfusion for 2 h, 6 h, 12 h, and 24 h respectively. Subsequently, we were separated into 5 groups, namely the I/R group, Cur group, DNase-1 group, Cur + DNase1 group and sham operation group. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST), Hematoxylin-eosin staining, immunofluorescence, and TUNEL analysis were applied to assess liver injury degree and NETs levels. Western blot assay was used to detect the protein levels of apoptosis-related proteins and MEK pathway proteins. RESULTS: Cur could alleviate hepatic IRI by inhibiting the generation of NETs via suppressing the MEK/ERK pathway. In addition, this study also revealed that DNase-1 is vital for alleviating hepatic IRI by reducing the generation of NETs. CONCLUSIONS: Cur combined with DNase-1 was more effective than the two drugs administered alone in alleviating hepatic IRI by inhibiting the generation of NETs. These results also suggested that curcumin combined with DNase-1 was a potential therapeutic strategy to mitigate hepatic IRI.

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).

Design and biological characterization of a series of dual mechanism ERK1/2 inhibitors with a Triazolopyridinone core.

Oncology clinical development programs have targeted the RAS/RAF/MEK/ERK signaling pathway with small molecule inhibitors for a variety of cancers during the past decades, and most therapies have shown limited or minimal success. Specific BRAF and MEK inhibitors have shown clinical efficacy in patients for the treatment of BRAF-mutant melanoma. However, most cancers have shown treatment resistance after several months of inhibitor usage, and reports indicate resistance is often associated with the reactivation of the MAPK signaling pathway. It is widely accepted that an effective MAPK therapy will have a significant impact on curtailing cancer growth and improving patient survival. However, despite more than three decades of intense research and pharmaceutical industry efforts, an FDA-approved, effective anti-cancer ERK inhibitor has yet to be developed. Here, we present the design, optimization, and biological characterization of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate the phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. Our series of dual mechanism ERK1/2 inhibitors, in which we incorporated a triazolopyridinone core, may present potential benefits for enhancing efficacy and addressing the emergence of treatment resistance.

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.

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.

Radiation combined with KRAS-MEK inhibitors enhances anticancer immunity in KRAS-mutated tumor models.

KRAS mutation is a common driver in solid tumors, and KRAS-mutated tumors are relatively resistant to radiotherapy. Therefore, we investigated the combined effect of radiation and KRAS-MEK inhibitors (AMG510 and trametinib) in KRAS-mutated tumors. The expression of programmed death-ligand 1 (PD-L1), major histocompatibility complex (MHC) class I molecules, and cytokines in KRAS-mutated cell lines was assessed using flow cytometry, western blot analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. In vivo, tumor growth, T cell infiltration, and gene sequencing analyses were conducted in 2 murine KRAS-mutated models. Both AMG510 and trametinib decreased the radiation-induced increase in PD-L1 expression. Radiation and trametinib additively induced the expression of CXCL10 and CXCL11 cytokines and MHC class I in murine CT26 and LLC cell lines. The combination of trametinib and radiation controlled tumor growth and induced more infiltration of CD4+ and CD8+ T cells in vivo, wherein tumor inhibition function and the survival period of mice could be reduced by CD8+ and/or CD4+ T cell depletion. The expression levels of immune-related genes also increased in the combination therapy group. Our results indicate that KRAS-MEK inhibitors in combination with radiotherapy can enhance antitumor immunity, providing new therapeutic strategies for KRAS-mutated tumors.