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Vasopressin controls water permeability in the renal collecting duct by regulating the water channel protein, aquaporin-2 (AQP2). Phosphoproteomic studies have identified multiple proteins that undergo phosphorylation changes in response to vasopressin. The kinases responsible for the phosphorylation of most of these sites have not been identified. Here, we use large-scale Bayesian data integration to predict the responsible kinases for 51 phosphoproteomically identified vasopressin-regulated phosphorylation sites in the renal collecting duct. To do this, we applied Bayes' rule to rank the 515 known mammalian protein kinases for each site. Bayes' rule was applied recursively to integrate each of the seven independent datasets, each time using the posterior probability vector of a given step as the prior probability vector of the next step. In total, 30 of the 33 phosphorylation sites that increase with vasopressin were predicted to be phosphorylated by protein kinase A (PKA) catalytic subunit-α, consistent with prior studies implicating PKA in vasopressin signaling. Eighteen of the vasopressin-regulated phosphorylation sites were decreased in response to vasopressin and all but three of these sites were predicted to be targets of extracellular signal-regulated kinases, ERK1 and ERK2. This result implies that ERK1 and ERK2 are inhibited in response to vasopressin V2 receptor occupation, secondary to PKA activation. The six phosphorylation sites not predicted to be phosphorylated by PKA or ERK1/2 are potential targets of other protein kinases previously implicated in aquaporin-2 regulation, including cyclin-dependent kinase 18 (CDK18), calmodulin-dependent kinase 2δ (CAMK2D), AMP-activated kinase catalytic subunit-α-1 (PRKAA1) and CDC42 binding protein kinase β (CDC42BPB). NEW & NOTEWORTHY: Vasopressin regulates water transport in the renal collecting duct in part through phosphorylation or dephosphorylation of proteins that regulate aquaporin-2. Prior studies have identified 51 vasopressin-regulated phosphorylation sites in 45 proteins. This study uses Bayesian data integration techniques to combine information from multiple prior proteomics and transcriptomics studies to predict the protein kinases that phosphorylate the 51 sites. Most of the regulated sites were predicted to be phosphorylated by protein kinase A or ERK1/ERK2. [ABSTRACT FROM AUTHOR]

Background: Schisandra extract has therapeutic and preventive effects on Alzheimer's disease (AD). Therefore, this study evaluated the anti-AD potential of Schisandrin A (SCH A) using an in vitro cell model. SH-SY5Y and SK-N-SH cells were treated with 20 μM amyloid β-protein (Aβ)25-35. The Aβ25-35-induced cells were then exposed to different concentrations of SCH A (1, 5, 10, 15 μg/mL). Moreover, to further explore the role of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway in the anti-AD effects of SHC A, SH-SY5Y cells were treated with SCH A following incubation with ERK activator LM22B-10. The impact of SCH A on cell viability and apoptosis was evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and flow cytometry. Furthermore, the oxidative stress markers and inflammatory cytokine levels were also assessed. The reactive oxygen species (ROS) levels were examined using 2',7'-Dichlorodihydrofluorescein Diacetate (DCFH-DA) method. Finally, Western blot analysis was employed to evaluate the phospho-ERK1/2 (p-ERK1/2) and ERK1/2. Results: We observed that SCH A treatment (5, 10, 15 μg/mL) substantially increased the cell viability (p < 0.05), and reduced the apoptosis rate (10 and 15 μg/mL) in SHSY5Y and SK-N-SH cells (p < 0.05). SCH A significantly ameliorated oxidative stress and reduced inflammatory cytokine levels in Aβ25-35-induced cells (p < 0.05). Furthermore, SCH A up-regulated the p-ERK1/2 to ERK1/2 ratio in Aβ25-35-induced cells. However, LM22B-10 treatment was found to exacerbate this effect of SCH A (p < 0.05). Conclusion: SCH A reduces the Aβ25-35-induced inflammatory response and oxidative stress in SH-SY5Y and SK-N-SH cells, and the activation of the ERK/MAPK signaling pathway was related to its potential mechanism. [ABSTRACT FROM AUTHOR]

Filaggrin (FLG) is an essential structural protein expressed in differentiated keratinocytes. Insufficient FLG expression contributes to the pathogenesis of chronic inflammatory skin diseases. Saikosaponin A (SSA), a bioactive oleanane-type triterpenoid, exerts anti-inflammatory activity. However, the effects of topically applied SSA on FLG expression in inflamed skin remain unclear. This study aimed to evaluate the biological activity of SSA in restoring reduced FLG expression. The effect of SSA on FLG expression in HaCaT cells was assessed through various biological methods, including reverse transcription PCR, quantitative real-time PCR, immunoblotting, and immunofluorescence staining. TNFα and IFNγ decreased FLG mRNA, cytoplasmic FLG protein levels, and FLG gene promoter–reporter activity compared to the control groups. However, the presence of SSA restored these effects. A series of FLG promoter–reporter constructs were generated to investigate the underlying mechanism of the effect of SSA on FLG expression. Mutation of the AP1-binding site (mtAP1) in the −343/+25 FLG promoter–reporter abrogated the decrease in reporter activities caused by TNFα + IFNγ, suggesting the importance of the AP1-binding site in reducing FLG expression. The SSA treatment restored FLG expression by inhibiting the expression and nuclear localization of FRA1 and c-Jun, components of AP1, triggered by TNFα + IFNγ stimulation. The ERK1/2 mitogen-activated protein kinase signaling pathway upregulates FRA1 and c-Jun expression, thereby reducing FLG levels. The SSA treatment inhibited ERK1/2 activation caused by TNFα + IFNγ stimulation and reduced the levels of FRA1 and c-Jun proteins in the nucleus, leading to a decrease in the binding of FRA1, c-Jun, p-STAT1, and HDAC1 to the AP1-binding site in the FLG promoter. The effect of SSA was evaluated in an animal study using a BALB/c mouse model, which induces human atopic-dermatitis-like skin lesions via the topical application of dinitrochlorobenzene. Topically applied SSA significantly reduced skin thickening, immune cell infiltration, and the expression of FRA1, c-Jun, and p-ERK1/2 compared to the vehicle-treated group. These results suggest that SSA can effectively recover impaired FLG levels in inflamed skin by preventing the formation of the repressor complex consisting of FRA1, c-Jun, HDAC1, and STAT1. [ABSTRACT FROM AUTHOR]

Fibroblast Growth Factors and their receptors (FGFRs) comprise a cell signaling module that can stimulate signaling by Ras and the kinases Raf, MEK, and ERK to regulate animal development and homeostatic functions. In Caenorhabditis elegans, the sole FGFR ortholog EGL-15 acts with the GRB2 ortholog SEM-5 to promote chemoattraction and migration by the sex myoblasts (SMs) and fluid homeostasis by the hypodermis (Hyp7). Cell-specific differences in EGL-15 signaling were suggested by the phenotypes caused by egl-15(n1457), an allele that removes a region of its C-terminal domain (CTD) known to bind SEM-5. To determine how mutations altered EGL-15 activity in the SMs and Hyp7, we used the kinase reporter ERK-KTR to measure activation of the ERK ortholog MPK-1. Consequences of egl-15(n1457) were cell-specific, resulting in loss of MPK-1 activity in the SMs and elevated activity in Hyp7. Previous studies of Hyp7 showed that loss of the CLR-1 phosphatase causes a fluid homeostasis defect termed "Clear" that is suppressed by reduction of EGL-15 signaling, a phenotype termed "Suppressor of Clear" (Soc). To identify mechanisms that permit EGL-15 signaling in Hyp7, we conducted a genetic screen for Soc mutants in the clr-1; egl-15(n1457) genotype. We report the identification of SOC-3, a protein with putative SEM-5-binding motifs and PH and PTB domains similar to DOK and IRS proteins. In combination with the egl-15(n1457) mutation, loss of either soc-3 , the GAB1 ortholog soc-1 , or the SHP2 ortholog ptp-2 , reduced MPK-1 activation. We generated alleles of soc-3 to test the requirement for the SEM-5-binding motifs, finding that residue Tyr356 is required for function. We propose that EGL-15-mediated SM chemoattraction relies solely on the direct interaction between SEM-5 and the EGL-15 CTD. In Hyp7, EGL-15 signaling uses two mechanisms: the direct SEM-5 binding mechanism; and an alternative, CTD-independent mechanism involving SOC-3, SOC-1, and PTP-2. This work demonstrates that FGF signaling uses distinct, tissue-specific mechanisms in development, and identifies SOC-3 as a potential adaptor that facilitates Ras pathway activation by FGFR. [Display omitted] • The C. elegans FGFR EGL-15 uses cell-specific signaling mechanisms in development. • EGL-15 signaling requires the C-terminal domain in sex myoblast but not hypodermis. • A genetic screen was used to discover soc-3 , a gene required for EGL-15 signaling. • SOC-3 contains a GRB2 binding site and domains similar to DOK/IRS family proteins. [ABSTRACT FROM AUTHOR]

Background and purpose: The overexpression of carnitine palmitoyl transferase 1A (CPT1A) is related to the poor prognosis of breast cancer, and it can promote the utilization of fatty acids by mitochondria and maximize triphosphate (ATP) production. However, the role of CPT1A in breast cancer metastasis is still unclear. This study aimed to explore the mechanism that mitochondrial dysfunction and CPT1A/extracellular signal-regulated kinase (ERK) signaling pathway in breast cancer jointly regulate the malignant behavior of breast cancer. Methods: The lentivirus system and shRNA tools were used to overexpress or knock down CPT1A in human breast cancer cell lines MDA-MB-231 and MCF7, and the cells were divided into NC group, CPT1A group and shCPT1A group. The invasion ability of cells was detected by transwell assay, and the protein expressions of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), ERK1/2 and CPT1A were analyzed by Western blot. The mitochondrial morphology of MDA-MB-231 and MCF7 cell lines was analyzed using mitochondrial red staining, and mitochondrial respiratory capacity was analyzed by oxygen consumption rate. Results: Compared with cells expressing control vector, overexpression of CPT1A resulted in enhanced invasion abilities of MCF7 cells and MDA-MB-231 cells (P

Objective To explore the expression and significance of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway in vascular tissue of intimal hyperplasia after arteriovenous fistula (AVF) and explore the effect of suppressing MAPK/ERK pathway on autophagy and proliferation of myofibroblasts. Methods From January 2023 to March 2023, 6 patients undergoing reconstruction of AVF were selected. Vascular tissues of patients with severe intimal hyperplasia were collected in observation group while normal collateral venous tissues requiring ligation in control group. General data were collected before operation and inner diameter and intima thickness of vessels were measured by ultrasound. The vascular expression levels of phosphorylation-extracellular regulated protein kinase 1/2 (p-ERK1/2), myosin-like B cell lymphoma/leukemia-2 interacting protein 1 (Beclin-1) and proliferating cell nuclear antigen (PCNA) proteins were detected by Western blot (WB) and correlation with venous intimal thickness was examined. Myofibroblast was cultured in uremic sera and exposed to ERK1/2 inhibitor corynoxeine. Total protein and RNA of cells were extracted and the expression changes of Beclin-1 and PCNA detected by WB and real-time polymerase chain reaction (PCR). Results Insufficient blood flow appeared during dialysis. Gray scale ultrasound indicated that venous intima thickened obviously [(0.143±0.014) cm vs (0.097±0.016) cm] at the stenosis of AVF and average diameter of vascular lumen was (0.166±0.028) cm. As compared with normal collateral vascular tissues, the expressions of p-ERK1/2, Beclin-1 and PCNA proteins were markedly up-regulated in vascular tissues of severe intimal hyperplasia and the differences were statistically significant (P

Introduction: Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is related to the pathogenesis of various retinopathies including age-related macular degeneration (AMD). Oxidative stress is the major factor that induces degeneration of RPE cells associated with the etiology of AMD. Objectives: Sodium iodate (NaIO3) generates intracellular reactive oxygen species (ROS) and is widely used to establish a model of AMD due to the selective induction of retinal degeneration. This study was performed to clarify the effects of multiple NaIO3-stimulated signaling pathways on EMT in RPE cells. Methods: The EMT characteristics in NaIO3-treated human ARPE-19 cells and RPE cells of the mouse eyes were analyzed. Multiple oxidative stress-induced modulators were investigated and the effects of pre-treatment with Ca2+ chelator, extracellular signal-related kinase (ERK) inhibitor, or epidermal growth factor receptor (EGFR) inhibitor on NaIO3-induced EMT were determined. The efficacy of post-treatment with ERK inhibitor on the regulation of NaIO3-induced signaling pathways was dissected and its role in retinal thickness and morphology was evaluated by using histological cross-sections and spectral domain optical coherence tomography. Results: We found that NaIO3 induced EMT in ARPE-19 cells and in RPE cells of the mouse eyes. The intracellular ROS, Ca2+, endoplasmic reticulum (ER) stress marker, phospho-ERK, and phospho-EGFR were increased in NaIO3-stimulated cells. Our results showed that pre-treatment with Ca2+ chelator, ERK inhibitor, or EGFR inhibitor decreased NaIO3-induced EMT, interestingly, the inhibition of ERK displayed the most prominent effect. Furthermore, post-treatment with FR180204, a specific ERK inhibitor, reduced intracellular ROS and Ca2+ levels, downregulated phospho-EGFR and ER stress marker, attenuated EMT of RPE cells, and prevented structural disorder of the retina induced by NaIO3. Conclusions: ERK is a crucial regulator of multiple NaIO3-induced signaling pathways that coordinate EMT program in RPE cells. Inhibition of ERK may be a potential therapeutic strategy for the treatment of AMD.

BACKGROUND: Bushen Yiqi Huayu granule has the effect of replenishing qi and eliminating blood stasis, tonifying kidney and smoothing collages, and is often used in the treatment of osteoporosis. At present, there are few studies on the effect of Bushen Yiqi Huayu granule on the callus angiogenesis of osteoporotic fractures. OBJECTIVE: To explore the mechanism of Bushen Yiqi Huayu granule by up-regulating extracellular signal-regulated kinase/mitogen-activated protein kinases (ERK/MAPK) signaling pathway to improve callus angiogenesis after fracturing in osteoporotic rats. METHODS: (1) Bone marrow mesenchymal stem cells from osteoporotic SD rats and bone marrow monocytes from C57BL/6 mice were collected. MTT assay was used to detect different doses of Bushen Yiqi Huayu granule on bone mesenchymal stem cell toxicity. Bone mesenchymal stem cells and bone marrow monocytes were cultured in the medium supplemented with 0 and 1.5 mg/mL Bushen Yiqi Huayu granules, respectively, and osteogenic differentiation and osteoclast differentiation were carried out in vitro. (2) 144 SD rats were randomly divided into sham operation group, model group, granule group and granule+PD98059 group with 36 rats in each group. The osteoporotic model was established in the model group, granule group and granule+PD98059 group, and only part of the adipose tissue near the ovary was removed in the sham operation group. 8 weeks later, all rats received left tibial osteotomy. In the granule+PD98059 group, 5 g/kg Bushen Yiqi Huayu granule was given intragastrically, and 0.3 mg/kg PD98059 was injected into the tail vein. The granule group was given 5 g/kg Bushen Yiqi Huayu granule, and the same volume of normal saline was injected into the tail vein. The sham operation group and model group were given an equal volume of normal saline, and the caudal vein was injected with an equal volume of normal saline. Drug administration was conducted once a day for 8 weeks. Fracture healing and callus angiogenesis were observed by X-ray, micro-computed tomography and microvascular perfusion angiography. Bone mineral density and mechanical strength of the callus were measured. The tibia was observed by hematoxylin-eosin staining and Masson staining. Western blot assay and immunohistochemistry were used to examine the expression of ERK/MAPK signaling pathway-related molecules. RESULTS AND CONCLUSION: (1) Compared with the 0 mg/mL group, the alkaline phosphatase activity, mineralization level, p-ERK1/2 and p-p38 MAPK expression levels of osteocytes increased (P < 0.05), while the density and volume of osteoclasts decreased in the 1.5 mg/mL group (P < 0.05). (2) Compared with the sham operation group, the fracture healing degree of the granule group was similar at 4 and 8 weeks, but there was no significant difference in LaneSandhu score, total callus volume, mineralized callus volume, mineralized callus volume/total callus volume, trabecular thickness, vascular number, spacing, thickness, vascular volume/total volume, bone mineral density, ultimate load of callus, stiffness, p-ERK1/2, p-p38 MAPK, p-IκB-α, and p-p65 expression levels (P > 0.05). In the model group and granule+PD98059 group, fracture healing was slow, Lane-Sandhu score, total callus volume, mineralized callus volume, mineralized callus volume/total callus volume, trabecular thickness, vascular number, thickness, vascular volume/total volume, bone mineral density, ultimate load of callus, stiffness, p-ERK1/2, p-p38 MAPK, p-IκB-α, and p-p65 expression levels decreased (P < 0.05), and vascular spacing increased (P < 0.05), compared with the sham operation group. (3) It is indicated that Bushen Yiqi Huayu granule can improve fracture healing, promote callus angiogenesis and alleviate the symptoms of osteoporosis by enhancing the expression of the ERK/MAPK signaling pathway-related molecules in osteoporotic rats. [ABSTRACT FROM AUTHOR]

Background and purpose: The overexpression of carnitine palmitoyl transferase 1A (CPT1A) is related to the poor prognosis of breast cancer, and it can promote the utilization of fatty acids by mitochondria and maximize triphosphate (ATP) production. However, the role of CPT1A in breast cancer metastasis is still unclear. This study aimed to explore the mechanism that mitochondrial dysfunction and CPT1A/extracellular signal-regulated kinase (ERK) signaling pathway in breast cancer jointly regulate the malignant behavior of breast cancer. Methods: The lentivirus system and shRNA tools were used to overexpress or knock down CPT1A in human breast cancer cell lines MDA-MB-231 and MCF7, and the cells were divided into NC group, CPT1A group and shCPT1A group. The invasion ability of cells was detected by transwell assay, and the protein expressions of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), ERK1/2 and CPT1A were analyzed by Western blot. The mitochondrial morphology of MDA-MB-231 and MCF7 cell lines was analyzed using mitochondrial red staining, and mitochondrial respiratory capacity was analyzed by oxygen consumption rate. Results: Compared with cells expressing control vector, overexpression of CPT1A resulted in enhanced invasion abilities of MCF7 cells and MDA-MB-231 cells (P＜0.05), while knockdown of shCPT1A resulted in decreased invasion abilities of cells (P＜0.05). Compared with NC group, the length of mitochondrial branches in MDAMB- 231 and MCF7 cells in CPT1A group was significantly shorter (P＜0.05), and the expressions of ERK1/2 and PGC-1α increased significantly (P ＜0.05). In shCPT1A group, the length of mitochondrial branches in MDA-MB-231 and MCF7 cells increased significantly (P ＜0.05), and the expressions of ERK1/2 and PGC-1α decreased significantly (P＜0.05). In addition, compared with NC group, the cellular basis, maximum respiratory capacity and ATP production of MDA-MB-231 in CPT1A group increased significantly (P ＜0.05), while the cellular basis, maximum respiratory capacity and ATP production of MDA-MB-231 in shCPT1A group decreased significantly (P＜0.05). Conclusion: ERK1/2-PGC-1α activated by CPT1A Signaling pathway plays a key role in mitochondrial division mediated breast cancer cell metastasis. [ABSTRACT FROM AUTHOR]

Ulva prolifera is the most common specie causative to green tide, and its growth is sensitive to temperature stress. However, the mechanisms of U. prolifera response to temperature stress remain elusive. In this study, high temperature (36 °C) stimulus promoted the death of unformed cell wall protoplasts and delayed the division of formed cell wall protoplasts, while low-temperature (4 °C) stimulus did not, suggesting that the mechanisms of the response of U. prolifera to high and low-temperature stresses are different. Transcriptome results show that proliferation-related genes were differentially expressed under high and low-temperature stresses, especially the proliferating cell nuclear antigen (PCNA) and cyclins (CYCs). Subsequently, the interaction between PCNA and Cyclin A was confirmed by Co-immunoprecipitation, yeast two-hybrid, and so on. Furthermore, high- and low-temperature stresses induced the expression of PCNA and Cyclin A in varying of degrees, and activated extracellular signal-regulated kinase (ERK) signal pathway. These results suggest, PCNA, Cyclin A, and ERK signal pathway played important roles in the resistance of U. prolifera to temperature stress. Interestingly, high-temperature stress induced an increase of miR-2916 in abundance, and exhibiting reverse expression of PCNA; and PCNA was target gene of miR-2916, suggesting that miR-2916 protected U. prolifera from high-temperature stress via post-transcriptionally regulation of PCNA. This study laid a foundation for understanding the function of PCNA and Cyclin A, moreover, it has a guiding significance to explore the mechanisms of the response to temperature stress from proliferation-related genes regulatory networks in U. prolifera. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a type of autoimmune disease that results in immune disorder and excessive inflammatory response due to a reduction of self-tolerance. Invariant natural killer T (iNKT) cells can effectively alleviate clinical symptoms and hyper-inflammation in RA, but their mechanism of action is not well-defined. This study aims to investigate the mechanism of iNKT cell therapy for RA. We established a DBA/1 mouse model for RA and treated it with specific iNKT cells. A cytometric bead array was used to measure the amounts of cytokines in the serum. Flow cytometry was then employed to identify different subsets of helper T cells (Th), the frequency of conventional dendritic cells (cDC), the expression of CD80, CD86, programmed cell death ligand 1 (PD-L1), and PD-L2 on cDC surfaces, and associated pathway proteins. iNKT cell treatment reduced Th1/Th2 and Th17/ regulatory T (Treg) cell ratios while increasing interleukin-4 (IL-4) and IL-10. It enhanced the generation of immature cDCs, and it upregulated the level of PD-L2 by stimulating the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Meanwhile, it activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and inhibited the nuclear factor kappa B (NF-κB) pathway. According to our findings, iNKT cell treatment increased the expression of phosphates STAT3 in lymph node cDC, causing them to upregulate PD-L2 molecules. While activating the ERK1/2 pathway and inhibiting the NF-κB pathway, tolerogenic cDC was produced, restoring immune homeostasis and correcting excessive inflammation. These results deliver new insights into the treatment of RA by iNKT cells. [ABSTRACT FROM AUTHOR]

Activation of ERK is critical for NaIO 3 -induced multiple signaling pathways to coordinate EMT in RPE cells. [Display omitted] • NaIO 3 was demonstrated to induce EMT in human ARPE-19 cells and in RPE cells of the mouse eyes as an AMD model. • Ca2+, EGFR, and ERK signaling pathways responded to NaIO 3 -induced EMT in ARPE-19 cells. • Post-treatment with FR180204 downregulated multiple NaIO 3 -induced signaling pathways leading to EMT. • ERK was shown to play a critical role in promoting oxidative stress-induced EMT of ARPE-19 cells in contrast to MEK. • FR180204 restored the RPE integrity and retinal morphology compromised by NaIO 3 in mouse retina. Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is related to the pathogenesis of various retinopathies including age-related macular degeneration (AMD). Oxidative stress is the major factor that induces degeneration of RPE cells associated with the etiology of AMD. Sodium iodate (NaIO 3) generates intracellular reactive oxygen species (ROS) and is widely used to establish a model of AMD due to the selective induction of retinal degeneration. This study was performed to clarify the effects of multiple NaIO 3 -stimulated signaling pathways on EMT in RPE cells. The EMT characteristics in NaIO 3 -treated human ARPE-19 cells and RPE cells of the mouse eyes were analyzed. Multiple oxidative stress-induced modulators were investigated and the effects of pre-treatment with Ca2+ chelator, extracellular signal-related kinase (ERK) inhibitor, or epidermal growth factor receptor (EGFR) inhibitor on NaIO 3 -induced EMT were determined. The efficacy of post-treatment with ERK inhibitor on the regulation of NaIO 3 -induced signaling pathways was dissected and its role in retinal thickness and morphology was evaluated by using histological cross-sections and spectral domain optical coherence tomography. We found that NaIO 3 induced EMT in ARPE-19 cells and in RPE cells of the mouse eyes. The intracellular ROS, Ca2+, endoplasmic reticulum (ER) stress marker, phospho-ERK, and phospho-EGFR were increased in NaIO 3 -stimulated cells. Our results showed that pre-treatment with Ca2+ chelator, ERK inhibitor, or EGFR inhibitor decreased NaIO 3 -induced EMT, interestingly, the inhibition of ERK displayed the most prominent effect. Furthermore, post-treatment with FR180204, a specific ERK inhibitor, reduced intracellular ROS and Ca2+ levels, downregulated phospho-EGFR and ER stress marker, attenuated EMT of RPE cells, and prevented structural disorder of the retina induced by NaIO 3. ERK is a crucial regulator of multiple NaIO 3 -induced signaling pathways that coordinate EMT program in RPE cells. Inhibition of ERK may be a potential therapeutic strategy for the treatment of AMD. [ABSTRACT FROM AUTHOR]

Typical agonists of G protein-coupled receptors (GPCRs), including muscarinic acetylcholine receptors (mAChRs), activate both G-protein and β-arrestin signaling systems, and are termed balanced agonists. In contrast, biased agonists selectively activate a single pathway, thereby offering therapeutic potential for the specific activation of that pathway. The mAChR agonists carbachol and pilocarpine are known to induce phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) via G-protein-dependent and -independent pathways, respectively. We investigated the involvement of β-arrestin and its downstream mechanisms in the ERK1/2 phosphorylation induced by carbachol and pilocarpine in the human salivary ductal cell line, HSY cells. HSY cells were stimulated with pilocarpine or carbachol, with or without various inhibitors. The cell lysates were analyzed by western blotting using the antibodies p44/p42MAPK and phosphor-p44/p42MAPK. Western blot analysis revealed that carbachol elicited greater stimulation of ERK1/2 phosphorylation compared to pilocarpine. ERK1/2 phosphorylation was inhibited by atropine and gefitinib, suggesting that mAChR activation induces transactivation of epidermal growth factor receptors (EGFR). Moreover, inhibition of carbachol-mediated ERK1/2 phosphorylation was achieved by GF-109203X (a PKC inhibitor), a βARK1/GRK2 inhibitor, barbadin (a β-arrestin inhibitor), pitstop 2 (a clathrin inhibitor), and dynole 34-2 (a dynamin inhibitor). In contrast, pilocarpine-mediated ERK1/2 phosphorylation was only inhibited by barbadin (a β-arrestin inhibitor) and PP2 (a Src inhibitor). Carbachol activates both G-protein and β-arrestin pathways, whereas pilocarpine exclusively activates the β-arrestin pathway. Additionally, downstream of β-arrestin, carbachol activates clathrin-dependent internalization, while pilocarpine activates Src. [Display omitted] • Muscarinic receptors agonists carbachol and pilocarpine induce phosphorylation of ERK1/2. • Carbachol activates two pathways, the G protein- and β-arrestin-mediated pathways. • Pilocarpine preferentially activates β-arrestin-mediated pathways. • Carbachol and pilocarpine activate different pathways downstream of β-arrestin in HSY cells. [ABSTRACT FROM AUTHOR]

Abstract Heat-killed Lactiplantibacillus plantarum L-137 (HK L-137) has been suggested to enhance the intestinal barrier in obese mice, leading to improvement of metabolic abnormalities and adipose tissue inflammation, and in healthy humans with overweight, leading to improvement of systemic inflammation. However, its detailed mechanism of action has not been clarified. Therefore, this study investigated the effects of HK L-137 on the permeability of rat small intestinal epithelial IEC-6 cells, tight junction-related gene and protein expression and localization, and intracellular signaling pathways involved in barrier function. Treatment of IEC-6 cells with HK L-137 for 26 h significantly reduced the permeability to fluorescein isothiocyanate-dextran (FD-4). HK L-137 also increased gene and protein expression of zonula occludens-1 (ZO-1), an important tight junction protein, without affecting the localization. Furthermore, inhibition of the extracellular signal-regulated kinase (ERK)1/2 pathway in IEC-6 cells canceled the HK L-137-related reduction in permeability to FD-4. Phosphorylation of ERK in IEC-6 cells was induced 15 min after the addition of HK L-137. These results suggest that HK L-137 reduces intestinal permeability partly through activating the ERK pathway and increasing expression of the ZO-1 gene and protein. Enhancement of intestinal barrier function with HK L-137 might be effective in preventing and treating leaky gut, for which no specific therapeutic tool has been established.

• ERK activity plays a crucial role in regulating cortical spreading depolarization in male rodents. • CSD synergistically activates ERK1/2 and induces IL-1β production in the ipsilateral cerebral cortex of male rat. • Elevation of pERK and IL-1β production induced by CSD is predominantly TRPA1 channel-dependent. Extracellular signal-regulated kinase (ERK) are serine/threonine-selective proteins and ERK1/2 can be phosphorylated in peripheral and central brain regions after cortical spreading depolarization (CSD) and calcitonin gene-related peptide; However, it remains unclear about whether and how ERK activity modulates CSD that correlates to migraine aura. Here, we determined the role of ERK in regulating CSD and explored the underlying mechanism involving transient receptor potential ankyrin 1 (TRPA1), a stress-sensing cation channel. CSD was recorded using intrinsic optical imaging in mouse brain slices, and electrophysiology in rats. Phosphorylated ERK (pERK1/2) and interleukin-1β (IL-1β) protein levels were detected using Western blot or enzyme-linked immunosorbent assay, respectively. IL-1β mRNA level was detected using qPCR. The results showed that an ERK inhibitor, SCH77298, markedly prolonged CSD latency and reduced propagation rate in mouse brain slices. Corresponding to this, CSD induction increased levels of cytosolic pERK1/2 in ipsilateral cerebral cortices of rats, the elevation of which correlated to the level of IL-1β mRNA. Mechanistic analysis showed that pre-treatment of an anti-TRPA1 antibody reduced the cytosolic pERK2 level but not pERK1 following CSD in cerebral cortices of rats and this level of pERK2 correlated with that of cerebral cortical IL-1β protein. Furthermore, an ERK activator, AES16-2M, but not its scrambled control, reversed the prolonged CSD latency by a TRPA1 inhibitor, HC-030031, in mouse brain slices. These data revealed a crucial role of ERK activity in regulating CSD, and elevation of pERK and IL-1β production induced by CSD is predominantly TRPA1 channel-dependent, thereby contributing to migraine pathogenesis. [ABSTRACT FROM AUTHOR]

We previously demonstrated that orally supplemented Bifidobacterium breve MCC1274 (B. breve MCC1274) mitigated Alzheimer's disease (AD) pathologies in both 7-month-old AppNL-G-F mice and wild-type mice; thus, B. breve MCC1274 supplementation might potentially prevent the progression of AD. However, the possibility of using this probiotic as a treatment for AD remains unclear. Thus, we investigated the potential therapeutic effects of this probiotic on AD using 17-month-old AppNL-G-F mice with memory deficits and amyloid beta saturation in the brain. B. breve MCC1274 supplementation ameliorated memory impairment via an amyloid-cascade-independent pathway. It reduced hippocampal and cortical levels of phosphorylated extracellular signal-regulated kinase and c-Jun N-terminal kinase as well as heat shock protein 90, which might have suppressed tau hyperphosphorylation and chronic stress. Moreover, B. breve MCC1274 supplementation increased hippocampal synaptic protein levels and upregulated neuronal activity. Thus, B. breve MCC1274 supplementation may alleviate cognitive dysfunction by reducing chronic stress and tau hyperphosphorylation, thereby enhancing both synaptic density and neuronal activity in 17-month-old AppNL-G-F mice. Overall, this study suggests that B. breve MCC1274 has anti-AD effects and can be used as a potential treatment for AD. [ABSTRACT FROM AUTHOR]

Growth differentiation factor-15 (GDF-15) is proposed to be strongly associated with several cardiovascular diseases, such as heart failure and atherosclerosis. Moreover, some recent studies have reported an association between GDF-15 and platelet activation. In this study, we isolated peripheral blood platelets from healthy volunteers and evaluated the effect of GDF-15 on adenosine diphosphate (ADP)-induced platelet activation using the platelet aggregation assay. Subsequently, we detected the expression of GDF-15-related receptors on platelets, including the epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), transforming growth factor-beta receptor I (TGF-βRI), transforming growth factor-beta receptor II (TGF-βRII), glial-cell-line-derived neurotrophic factor family receptor α-like (GFRAL), and those rearranged during transfection (RET). Then, we screened for GDF-15 receptors using the GDF-15-related receptor microarray comprising these recombinant proteins. We also performed the immunoprecipitation assay to investigate the interaction between GDF-15 and the receptors on platelets. For the further exploration of signaling pathways, we investigated the effects of GDF-15 on the extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and Janus kinase 2 (JAK2) pathways. We also investigated the effects of GDF-15 on the ERK and AKT pathways and platelet aggregation in the presence or absence of RET agonists or inhibition. Our study revealed that GDF-15 can dose-independently inhibit ADP-induced human platelet aggregation and that the binding partner of GDF-15 on platelets is GFRAL. We also found that GDF-15 inhibits ADP-induced AKT and ERK activation in platelets. Meanwhile, our results revealed that the inhibitory effects of GDF-15 can be mediated by the GFRAL/RET complex. These findings reveal the novel inhibitory mechanism of ADP-induced platelet activation by GDF-15. [ABSTRACT FROM AUTHOR]

Particulate matter (PM), released into the air by a variety of natural and human activities, is a key indicator of air pollution. Although PM is known as the extensive health hazard to affect a variety of illness, few studies have specifically investigated the effects of PM10 exposure on schizophrenic development. In the present study, we aimed to investigate the impact of PM10 on MK-801, N-methyl-D-aspartate (NMDA) receptor antagonist, induced schizophrenia-like behaviors in C57BL/6 mouse. Preadolescent mice were exposed PM10 to 3.2 mg/m3 concentration for 4 h/day for 2 weeks through a compartmentalized whole-body inhalation chamber. After PM10 exposure, we conducted behavioral tests during adolescence and adulthood to investigate longitudinal development of schizophrenia. We found that PM10 exacerbated schizophrenia-like behavior, such as psychomotor agitation, social interaction deficits and cognitive deficits at adulthood in MK-801-induced schizophrenia animal model. Furthermore, the reduced expression levels of brain-derived neurotrophic factor (BDNF) and the phosphorylation of BDNF related signaling molecules, extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB), were exacerbated by PM10 exposure in the adult hippocampus of MK-801-treated mice. Thus, our present study demonstrates that exposure to PM10 in preadolescence exacerbates the cognitive impairment in animal model of schizophrenia, which are considered to be facilitated by the decreased level of BDNF through reduced ERK-CREB expression.

The transition of stem cells from quiescence to proliferation enables tissues to self-repair. The signaling mechanisms driving these stem-cell-status decisions are still unclear. Ca2+ and the extracellular signal-regulated kinase (Erk1/2) are two signaling pathways that have the potential to coordinate multiple signals to promote a specific cellular response. They both play important roles during nervous system development but their roles during spinal cord and muscle regeneration are not fully deciphered. Here we show in Xenopus laevis larvae that both Ca2+ and Erk1/2 signaling pathways are activated after tail amputation. In response to injury, we find that Erk1/2 signaling is activated in neural and muscle stem cells and is necessary for spinal cord and skeletal muscle regeneration. Finally, we show in vivo that Erk1/2 activity is necessary for an injury-induced increase in intracellular store-dependent Ca2+ dynamics in skeletal muscle-associated tissues but that in spinal cord, injury increases Ca2+ influx-dependent Ca2+ activity independent of Erk1/2 signaling. This study suggests that precise temporal and tissue-specific activation of Ca2+ and Erk1/2 pathways is essential for regulating spinal cord and muscle regeneration.

Mitochondria play a key role in the cerebral ischemia-reperfusion injury. Although the extracellular signal-regulated kinase 1/2 inhibitor PD98059 (PD) is a selective and reversible flavonoid that can protect the mitochondria in a rat model of cardiac arrest/cardiopulmonary resuscitation, its role requires further confirmation. In this study, we investigated whether PD could maintain mitochondrial homeostasis and decrease reactive oxygen species (ROS) production in neuroblastoma (SH-SY5Y) cells exposed to oxygen–glucose deprivation/reperfusion (OGD/R). PD improved the mitochondrial morphology and function, reversed the increase in ROS production and cell apoptosis, and reduced total-superoxide dismutase and Mn-superoxide dismutase activities induced by OGD/R. PD decreases ROS production and improves mitochondrial morphology and function, protecting SH-SY5Y cells against OGD/R-induced injury.

Filaggrin (FLG) is an essential structural protein expressed in differentiated keratinocytes. Insufficient FLG expression contributes to the pathogenesis of chronic inflammatory skin diseases. Saikosaponin A (SSA), a bioactive oleanane-type triterpenoid, exerts anti-inflammatory activity. However, the effects of topically applied SSA on FLG expression in inflamed skin remain unclear. This study aimed to evaluate the biological activity of SSA in restoring reduced FLG expression. The effect of SSA on FLG expression in HaCaT cells was assessed through various biological methods, including reverse transcription PCR, quantitative real-time PCR, immunoblotting, and immunofluorescence staining. TNFα and IFNγ decreased FLG mRNA, cytoplasmic FLG protein levels, and FLG gene promoter–reporter activity compared to the control groups. However, the presence of SSA restored these effects. A series of FLG promoter–reporter constructs were generated to investigate the underlying mechanism of the effect of SSA on FLG expression. Mutation of the AP1-binding site (mtAP1) in the −343/+25 FLG promoter–reporter abrogated the decrease in reporter activities caused by TNFα + IFNγ, suggesting the importance of the AP1-binding site in reducing FLG expression. The SSA treatment restored FLG expression by inhibiting the expression and nuclear localization of FRA1 and c-Jun, components of AP1, triggered by TNFα + IFNγ stimulation. The ERK1/2 mitogen-activated protein kinase signaling pathway upregulates FRA1 and c-Jun expression, thereby reducing FLG levels. The SSA treatment inhibited ERK1/2 activation caused by TNFα + IFNγ stimulation and reduced the levels of FRA1 and c-Jun proteins in the nucleus, leading to a decrease in the binding of FRA1, c-Jun, p-STAT1, and HDAC1 to the AP1-binding site in the FLG promoter. The effect of SSA was evaluated in an animal study using a BALB/c mouse model, which induces human atopic-dermatitis-like skin lesions via the topical application of dinitrochlorobenzene. Topically applied SSA significantly reduced skin thickening, immune cell infiltration, and the expression of FRA1, c-Jun, and p-ERK1/2 compared to the vehicle-treated group. These results suggest that SSA can effectively recover impaired FLG levels in inflamed skin by preventing the formation of the repressor complex consisting of FRA1, c-Jun, HDAC1, and STAT1.

Extracellular signal-regulated kinase (ERK) has been implicated in mammalian testicular and epididymal development. This study aimed to investigate ERK expression in the immature and mature testes and epididymides of bulls. We evaluated ERK expression using immunoblot analysis and immunohistochemistry. Immunoblot analysis revealed that immature bull testes and epididymides had higher phosphorylated ERK (pERK) expression than mature bull testes and epididymides. pERK immunoreactivity was higher in immature epididymides than in immature testes. pERK was localised mostly in spermatogonia, undifferentiated sustentacular (Sertoli) cells, and interstitial (Leydig) cells in immature testes, as well as in some spermatocytes and spermatids in mature testes. In immature epididymides, the body and tail had higher pERK expression than the head, whereas pERK was broadly distributed throughout the stereocilia, basal cells, and connective tissues. pERK distribution in the head of mature epididymides was similar to that in immature epididymides, whereas few connective tissue cells were expressed in the body and tail of mature epididymides. Collectively, these results suggest that ERK is expressed in the testis and epididymis of immature and mature bulls with varying intensities, and the role of ERK in male reproductive organs may include the specific function of its development.

Ketamine is an ionic glutamic acid N-methyl-d-aspartate receptor (NMDAR) antagonist commonly used in clinical anesthesia, and its rapid and lasting antidepressant effect has stimulated great interest in psychology research. However, the molecular mechanisms underlying its antidepressant action are still undetermined. Sevoflurane exposure early in life might induce developmental neurotoxicity and mood disorders. In this study, we evaluated the effect of ketamine against sevoflurane-induced depressive-like behavior and the underlying molecular mechanisms. Here, we reported that A2AR protein expression was upregulated in rats with depression induced by sevoflurane inhalation, which was reversed by ketamine. Pharmacological experiments showed that A2AR agonists could reverse the antidepressant effect of ketamine, decrease extracellular signal-regulated kinase (ERK) phosphorylation, reduce synaptic plasticity, and induce depressive-like behavior. Our results suggest that ketamine mediates ERK1/2 phosphorylation by downregulating A2AR expression and that p-ERK1/2 increases the production of synaptic-associated proteins, enhancing synaptic plasticity in the hippocampus and thereby ameliorating the depressive-like behavior induced by sevoflurane inhalation in rats. This research provides a framework for reducing anesthesia-induced developmental neurotoxicity and developing new antidepressants. [ABSTRACT FROM AUTHOR]

Placenta accreta is a high-risk condition causing obstetric crisis and hemorrhage; however, its pathogenesis remains unknown. We aimed to identify the factors contributing to trophoblast invasiveness and angiogenic potential, which in turn drive the pathogenesis of placenta accreta. We focused on the transforming growth factor (TGF)-β1-Smad pathway and investigated the intrinsic relationship between the time- and dose-dependent inhibition of the ubiquitinating enzyme UCHL5 using bAP15, a deubiquitinase inhibitor, after TGF-β1 stimulation and the invasive and angiogenic potential of two cell lines, gestational choriocarcinoma cell line JEG-3 and trophoblast cell line HTR-8/SVneo. UCHL5 inhibition negatively regulated TGF-β1-induced Smad2 activation, decreasing extravillous trophoblast invasiveness. Smad1/5/9 and extracellular signal-regulated kinase (ERK) were simultaneously activated, and vascular endothelial growth factor was secreted into the trophoblast medium. However, extravillous trophoblast culture supernatant severely impaired the vasculogenic potential of human umbilical vein endothelial cells. These results suggest that the downstream ERK pathway and Smad1/5/9 potentially regulate the TGF-β1-Smad pathway in extravillous trophoblasts, whereas Smad2 contributes to their invasiveness. The abnormal invasive and angiogenic capacities of extravillous cells, likely driven by the interaction between TGF-β1-Smad and ERK pathways, underlie the pathogenesis of placenta accreta. [ABSTRACT FROM AUTHOR]

Sphingosine 1-phosphate (S1P) is one of the lipid mediators involved in diverse physiological functions. S1P circulates in blood and lymph bound to carrier proteins. Three S1P carrier proteins have been reported, albumin, apolipoprotein M (ApoM) and apolipoprotein A4 (ApoA4). The carrier-bound S1P exerts its functions via specific S1P receptors (S1PR1-5) on target cells. Previous studies showed several differences in physiological functions between albumin-bound S1P and ApoM-bound S1P. However, molecular mechanisms underlying the carrier-dependent differences have not been clarified. In addition, ApoA4 is a recently identified S1P carrier protein, and its functional differences from albumin and ApoM have not been addressed. Here, we compared the three carrier proteins in the processes of S1P degradation, release from S1P-producing cells and receptor activation. ApoM retained S1P more stable than albumin and ApoA4 in the cell culture medium when compared in the equimolar amounts. ApoM facilitated theS1P release from endothelial cells most efficiently. Furthermore, ApoM-bound S1P showed a tendency to induce prolonged activation of Akt via S1PR1 and S1PR3. These results suggest that the carrier-dependent functional differences of S1P are partly ascribed to the differences in the S1P stability, S1P-releasing efficiency and signaling duration. [ABSTRACT FROM AUTHOR]

Background and aim: It has been reported that acupuncture at GB34 can enhance neurogenesis in the subventricular zone (SVZ) of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the signaling pathway that plays a critical role in neurogenesis needs to be established. Herein, we investigated the neurogenesis-promoting pathway mediated by acupuncture, focusing on extracellular signal-regulated kinase (ERK) signaling. Experimental procedure: Male 10-week-old C57BL/6 mice were intraperitoneally injected with 30 mg/kg MPTP once daily for 5 days. Subsequently, mice were intraperitoneally injected with 50 mg/kg bromodeoxyuridine (BrdU), and electroacupuncture (EA) was performed at GB34 and BL60 for 3 weeks. The survival of dopaminergic neurons in the nigrostriatal pathway, cell proliferation in the SVZ, and expression levels of brain-derived neurotrophic factor (BDNF) and phosphorylated ERK (pERK) were evaluated. Results and conclusion: MPTP induced dopaminergic neuronal death in the nigrostriatal pathway, and reduced the number of BrdU-positive and BrdU/doublecortin double-positive cells in the SVZ; these parameters were restored by EA. Moreover, EA prevented MPTP-induced reduction in striatal expression of BDNF and pERK. These results indicate that EA could prevent dopaminergic neuronal death in the nigrostriatal pathway and restore neurogenesis in the SVZ, which may be attributed to the activation of the BDNF-ERK pathway.

Abstract Background Hypoxia is associated with many respiratory diseases, partly due to the accumulation of edema fluid and mucus on the surface of alveolar epithelial cell (AEC), which forms oxygen delivery barriers and is responsible for the disruption of ion transport. Epithelial sodium channel (ENaC) on the apical side of AEC plays a crucial role to maintain the electrochemical gradient of Na+ and water reabsorption, thus becomes the key point for edema fluid removal under hypoxia. Here we sought to explore the effects of hypoxia on ENaC expression and the further mechanism related, which may provide a possible treatment strategy in edema related pulmonary diseases. Methods Excess volume of culture medium was added on the surface of AEC to simulate the hypoxic environment of alveoli in the state of pulmonary edema, supported by the evidence of increased hypoxia-inducible factor-1 expression. The protein/mRNA expressions of ENaC were detected, and extracellular signal-regulated kinase (ERK)/nuclear factor κB (NF-κB) inhibitor was applied to explore the detailed mechanism about the effects of hypoxia on epithelial ion transport in AEC. Meanwhile, mice were placed in chambers with normoxic or hypoxic (8%) condition for 24 h, respectively. The effects of hypoxia and NF-κB were assessed through alveolar fluid clearance and ENaC function by Ussing chamber assay. Results Hypoxia (submersion culture mode) induced the reduction of protein/mRNA expression of ENaC, whereas increased the activation of ERK/NF-κB signaling pathway in parallel experiments using human A549 and mouse alveolar type 2 cells, respectively. Moreover, the inhibition of ERK (PD98059, 10 µM) alleviated the phosphorylation of IκB and p65, implying NF-κB as a downstream pathway involved with ERK regulation. Intriguingly, the expression of α-ENaC could be reversed by either ERK or NF-κB inhibitor (QNZ, 100 nM) under hypoxia. The alleviation of pulmonary edema was evidenced by the administration of NF-κB inhibitor, and enhancement of ENaC function was supported by recording amiloride-sensitive short-circuit currents. Conclusions The expression of ENaC was downregulated under hypoxia induced by submersion culture, which may be mediated by ERK/NF-κB signaling pathway.

Objective: To explore the effects of microRNA-495 （miR-495） regulating mitogen-activated protein kinase （MAPK）/extracellular signal-regulated kinase （ERK） signaling pathway on neuronal autophagy after spinal cord injury （SCI） in rats. Methods: According to 10 rats in each group, 50 SD rats were randomly divided into sham group, SCI group, agomir-NC group, miR-495 agomir group, miR-495 agomir+Anisomycin group（ p38-MAPK specific activator Anisomycin）, the SCI model was established by hitting the T10 segment of the spinal cord, and injected with agomir-NC, miR-495 agomir, and Anisomycin into the subarachnoid space of the spinal cord of rats in the corresponding group 3 days（ 20 nmol/d） before modeling, sham group and SCI group were injected with the same amount of normal saline; qRT-PCR was used to detect the expression of miR-495 in spinal cord tissue; motor function BBB score was used to evaluate the recovery of nerve function; HE staining and acridine orange staining were used to observe the histopathology and neuronal autophagy of rat spinal cord, respectively; ELISA method was used to detect the expression of inflammatory factors in spinal cord tissue; Western blot was used to detect the expression of autophagy and MAPK/ERK pathway proteins in spinal cord tissue. Results: Compared with sham group, the expression of miR-495 and BBB score of the SCI group were significantly lower, the spinal cord histopathology, number of autophagosomes, levels of TNF-α, IL-1β, IL-6, LC3-Ⅱ/LC3-Ⅰ, Beclin-1, p-ERK1/2/ERK1/2 and pc-fos/c-fos were significantly increased （P<0.05）; compared with SCI group, the expression of miR-495 and BBB score of miR-495 agomir group was significantly increased, the spinal cord histopathology, number of autophagosomes, levels of TNF-α, IL-1β, IL-6, LC3-Ⅱ/LC3-Ⅰ, Beclin-1, p-ERK1/2/ERK1/2 and pc-fos/c-fos were significantly reduced （P<0.05）; Anisomycin reverses the inhibitory effect of miR-495 agomir on neuronal autophagy after SCI in rats. Conclusion: The overexpression of miR-495 may inhibit neuronal autophagy in rats after SCI by inhibiting the activation of MAPK/ERK signaling pathway. [ABSTRACT FROM AUTHOR]

Introduction: Damage to retinal pigment epithelium (RPE) cells caused by oxidative stress is closely related to the pathogenesis of several blinding retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and other inherited retinal degenerative conditions. However, the mechanisms of this process are poorly understood. Hence, the goal of this study was to investigate hydrogen peroxide (H2O2)-induced oxidative damage and protective role of peroxiredoxin 6 (PRDX6) protein via EGFR/ERK signaling pathway in RPE cells. Methods: Cells from a human RPE cell line (ARPE-19 cells) were treated with H2O2, and then cell viability was assessed using the methyl thiazolyl tetrazolium assay. Cell death and reactive oxygen species (ROS) were detected by flow cytometry. The levels of PRDX6, epidermal growth factor receptor (EGFR), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected by Western blot assay. PRDX6 and EGFR were also detected via immunofluorescence staining. Results: Our results show that H2O2 inhibited cell viability, induced cell death, and increased ROS levels in ARPE-19 cells. It was also found that H2O2 decreased the levels of PRDX6, EGFR, and phosphorylated ERK but increased the levels of phosphorylated P38MAPK and JNK. PRDX6 overexpression was found to attenuate H2O2-induced inhibition of cell viability and increased cell death and ROS production in ARPE-19 cells. PRDX6 overexpression also increased the expression of EGFR and alleviated the H2O2-induced decrease in EGFR and phosphorylated ERK. Moreover, inhibition of epidermal growth factor-induced EGFR and ERK signaling in oxidative stress was partially blocked by PRDX6 overexpression. Discussion: Our findings indicate that PRDX6 overexpression protects RPE cells from oxidative stress damage caused by decreasing ROS production and partially blocking the inhibition of the EGFR/ERK signaling pathway induced by oxidative stress. Therefore, PRDX6 shows promise as a therapeutic target for the prevention of RPE cell damage caused by oxidative stress associated with retinal diseases. [ABSTRACT FROM AUTHOR]

Adipose tissue-derived stem cells (ASCs) have important clinical significance as regulators of skin scar tissue regeneration. ASCs inhibit keloid formation and increase insulin-like growth factor-binding protein-7 (IGFBP-7) expression. However, whether ASCs inhibit keloid formation through IGFBP-7 remains unclear. We aimed to assess the roles of IGFBP-7 in keloid formation. We analyzed the proliferation, migration, and apoptosis of keloid fibroblasts (KFs) treated with recombinant IGFBP-7 (rIGFBP-7) or by co-culture with ASCs using CCK8 assays, transwell assays, and flow cytometry, respectively. In addition, immunohistochemical staining, quantitative polymerase chain reaction, human umbilical vein endothelial cell tube formation, and western blotting experiments were used to assess keloid formation. IGFBP-7 expression was significantly lower in keloid tissues than that in normal skin tissues. Stimulation of KFs with rIGFBP-7 at different concentrations or by co-culture with ASCs resulted in decreased KF proliferation. Additionally, KF stimulation with rIGFBP-7 resulted in increased apoptosis of KFs. IGFBP-7 also reduced angiogenesis in a concentration-dependent manner, and stimulation with different rIGFBP-7 concentrations or co-culture of KFs with ASCs inhibited the expression of transforming growth factor-β1, vascular endothelial growth factor, collagen I, interleukin (IL)-6, IL-8, B-raf proto-oncogene (BRAF), mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK) in KFs. Collectively, our findings suggested that ASC-derived IGFBP-7 prevented keloid formation by inhibiting the BRAF/MEK/ERK signaling pathway. • Keloid tissues have lower IGFBP-7 expression than normal skin tissues. • IGFBP-7 inhibits keloid fibroblast proliferation and scar angiogenesis. • IGFBP-7 increases apoptosis and reduces inflammation in keloid fibroblasts. • ASC-derived IGFBP-7 inhibits the BRAF/MEK/ERK signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Although a large percentage of chronic pain patients consume alcohol to manage their pain, there is a significant gap in knowledge regarding the mechanisms underlying the antinociceptive effects of alcohol. Methods: To determine the longitudinal analgesic effects of alcohol, we utilized the complete Freund's adjuvant (CFA) model of inflammatory pain in adult female and male Wistar rats. Both somatic and negative motivational aspects of pain were measured using the electronic von Frey (mechanical nociception) system, thermal probe test (thermal nociception), and mechanical conflict avoidance task (pain avoidance‐like behavior). Tests were conducted at baseline and 1 and 3 weeks following intraplantar CFA or saline administration. At both time points post‐CFA, animals were treated with each of three doses of alcohol (intraperitoneal; 0, 0.5, and 1.0 g/kg) over separate days in a Latin square design. Results: Alcohol produced dose‐dependent mechanical analgesia and antihyperalgesia in females but only antihyperalgesia in males. Although alcohol continued to attenuate CFA‐induced decreases in both thermal and mechanical nociceptive thresholds between 1 and 3 weeks post‐CFA, it appeared less effective at increasing thresholds 3 weeks after CFA induction. Conclusions: These data suggest that individuals may develop tolerance to alcohol's ability to alleviate both somatic and negative motivational symptoms of chronic pain over time. We also discovered sex‐specific neuroadaptations in protein kinase A‐dependent phosphorylation of GluR1 subunits and extracellular signal‐regulated kinase (ERK 1/2) phosphorylation in nociceptive brain centers of animals receiving an alcohol challenge 1 week post‐CFA. Together, these findings illustrate a sex‐specific regulation of behavioral and neurobiological indices of persistent pain by alcohol. [ABSTRACT FROM AUTHOR]

Seasonal changes in food intake and adiposity in many animal species are triggered by changes in the photoperiod. These latter changes are faithfully transduced into a biochemical signal by melatonin secreted by the pineal gland. Seasonal variations, encoded by melatonin, are integrated by third ventricular tanycytes of the mediobasal hypothalamus through the detection of the thyroid‐stimulating hormone (TSH) released from the pars tuberalis. The mediobasal hypothalamus is a critical brain region that maintains energy homeostasis by acting as an interface between the neural networks of the central nervous system and the periphery to control metabolic functions, including ingestive behavior, energy homeostasis, and reproduction. Among the cells involved in the regulation of energy balance and the blood–hypothalamus barrier (BHB) plasticity are tanycytes. Increasing evidence suggests that anterior pituitary hormones, specifically TSH, traditionally considered to have unitary functions in targeting single endocrine sites, display actions on multiple somatic tissues and central neurons. Notably, modulation of tanycytic TSH receptors seems critical for BHB plasticity in relation to energy homeostasis, but this needs to be proven. [ABSTRACT FROM AUTHOR]

Background: N-linoleyltyrosine (NITyr), one of the anandamide analogs, exerts activity via the endocannabinoid receptors (CB1 and CB2), which showed antitumor effects in various tumors. Therefore, we speculated that NITyr might show anti-non-small cell lung cancer (NSCLC) effects via the CB1 or CB2 receptor. The purpose of the investigation was to reveal the anti-tumor ability of NITyr on A549 cells and its mechanisms. Methods: The viability of A549 cells was measured by MTT assay, and the cell cycle and apoptosis were both examined by flow cytometry; in addition, cell migration was tested by wound healing assay. Apoptosis-related markers were measured by immunofluorescence. The downstream signaling pathways (PI3K, ERK, and JNK) of CB1 or CB2 were examined through Western blotting. The expressions of CB1 and CB2 were detected by immunofluorescence. Finally, the AutoDock software was used to validate the binding affinity between the targets, such as CB1 and CB2, with NITyr. Results: We found that NITyr inhibited cell viability, hindered the cell cycle, resulted in apoptosis, and inhibited migration. The CB1 inhibitor, AM251, and the CB2 inhibitor, AM630, weakened the aforementioned phenomenon. The immunofluorescence assay suggested that NITyr upregulated the expression of CB1 and CB2. Western blot analysis indicated that NITyr upregulated the expression of p-ERK, downregulated the expression of p-PI3K, and did not affect p-JNK expression. In conclusion, NITyr showed a role in inhibiting NSCLC through the activation of CB1 and CB2 receptors involved in PI3K and ERK pathways. [ABSTRACT FROM AUTHOR]

Extracellular signal-regulated kinase (ERK) has been implicated in mammalian testicular and epididymal development. This study aimed to investigate ERK expression in the immature and mature testes and epididymides of bulls. We evaluated ERK expression using immunoblot analysis and immunohistochemistry. Immunoblot analysis revealed that immature bull testes and epididymides had higher phosphorylated ERK (pERK) expression than mature bull testes and epididymides. pERK immunoreactivity was higher in immature epididymides than in immature testes. pERK was localised mostly in spermatogonia, undifferentiated sustentacular (Sertoli) cells, and interstitial (Leydig) cells in immature testes, as well as in some spermatocytes and spermatids in mature testes. In immature epididymides, the body and tail had higher pERK expression than the head, whereas pERK was broadly distributed throughout the stereocilia, basal cells, and connective tissues. pERK distribution in the head of mature epididymides was similar to that in immature epididymides, whereas few connective tissue cells were expressed in the body and tail of mature epididymides. Collectively, these results suggest that ERK is expressed in the testis and epididymis of immature and mature bulls with varying intensities, and the role of ERK in male reproductive organs may include the specific function of its development. [ABSTRACT FROM AUTHOR]

Objective: This study aims to explore the effect and mechanism of total flavonoids of patriniae radix on hippocampal neuron apoptosis in neonatal rats with hypoxic⁃ischemic encephalopathy (HIE). Methods: Seventy⁃two neonatal rats were divided into sham operation group, model group, chelerythrine group (5 mg/kg), and total flavonoids of patriniae radix low⁃(50 mg/kg)and high⁃(100 mg/kg) dose groups, total flavonoids of patriniae radix + chelerythrine group (100 mg/kg total flavonoids of patriniae radix+5 mg/kg chelerythrine), with 12 rats in each group. The HIE model was constructed by ligating the right common carotid artery and hypoxic treatment for 2.5 h. Rats in the chelerythrine group were intraperitoneally injected with chelerythrine, and the total flavonoids of patriniae radix groups were given the corresponding doses of total flavonoids of patriniae radix by gavage. Rats in the total flavonoids of patriniae radix + chelerythrine group were injected intraperitoneally with chelerythrine while giving total flavonoids of patriniae radix by gavage, once daily for 7 consecutive days. The neurological deficit score was used to detect the nerve function of rats. Hematoxylin⁃eosin (HE) staining was used to observe the pathological changes in the hippocampus of brain tissue. TdT⁃mediated dUTP nick end labeling (TUNEL)/neuronal nuclei antigen (NeuN) fluorescent double staining was used to observe the apoptosis of hippocampal neurons. Western blot was used to detect the expression of protein kinase C (PKC)/extracellular signal⁃regulated kinase (ERK) pathway and apoptosis⁃related proteins in brain tissue. Oxygen⁃glucose deprivation/reperfusion (OGD/R)was used to construct a cell model. CCK⁃8 and lactate dehydrogenase (LDH)assays were performed to analyze the neuronal effect of total flavonoids of patriniae radix on primary hippocampal neurons. Cell apoptosis was analyzed by flow cytometry, and Western blot was used to detect the expression of apoptosis⁃related proteins. Results：In the in vivo study, compared with the sham operation group, the rats in the model group showed different degrees of neurological deficits, and the neurological deficit score, the numbers of NeuN+ TUNEL+ cells, the expression of Bax and Caspase⁃3 in brain tissue increased significantly, while the expression of Bcl⁃2, the ratios of p⁃PKC/PKC and p⁃ERK1/2/ERK1/2 reduced significantly (P<0.05). The neurons in the CA1 area of the hippocampus were swollen and decreased in number. Compared with the model group, the neurological function of the total flavonoids of patriniae radix low ⁃dose and high⁃dose group improved, and the neurological deficit score, the numbers of NeuN+ TUNEL+ cells, the expression of Bax and Caspase⁃3 in brain tissue reduced significantly, while the expression of Bcl⁃2, the ratios of p⁃PKC/PKC and p⁃ERK1/2/ERK1/2 increased significantly (P<0.05). The neurons in the CA1 area of the hippocampus were increased in density. And on the basis of the intervention of total flavonoids of patriniae radix, combined with chelerythrine can significantly reduce the inhibitory effect of total flavonoids of patriniae radix on hippocampal neuronal apoptosis after HIE. In an in vitro study, total flavonoids of patriniae radix increased cell viability after OGD/R injury, reversed neuronal damage and reduced hippocampal neuronal apoptosis, while increasing hippocampal neuronal p⁃PKC/ PKC and p⁃ERK1/2 /ERK1/2 ratio (P < 0.05), verified the activation of PKC/ERK pathway by total flavonoids of patriniae radix in OGD/R⁃induced primary hippocampal neurons. Conclusion: Total flavonoids of patriniae radix can inhibit hippocampal neuron apoptosis in neonatal rats with HIE, and its mechanism may be related to the activation of PKC/ERK pathway. [ABSTRACT FROM AUTHOR]

Aims: The clinical use of brain stimulation is attractive for patients who have side effects or tolerance. However, studies on insular cortex (IC) stimulation are lacking in neuropathic pain. The present study aimed to investigate the effects of IC stimulation (ICS) on neuropathic pain and to determine how ICS modulates pain. Methods: Changes in pain behaviors were observed following ICS with various parameters in neuropathic rats. Western blotting was performed to assess molecular changes in the expression levels of phosphorylated extracellular signal‐regulated kinase (pERK), neurons, astrocytes, and microglia between experimental groups. Immunohistochemistry was performed to investigate the colocalization of pERK with different cell types. Results: The most effective pain‐relieving effect was induced at 50 Hz–120 μA in single trial of ICS and it maintained 4 days longer after the termination of repetitive ICS. The expression levels of pERK, astrocytes, and microglia were increased in neuropathic rats. However, after ICS, the expression levels of pERK were decreased, and colocalization of pERK and neurons was reduced in layers 2–3 of the IC. Conclusion: These results indicated that ICS attenuated neuropathic pain by the regulation of pERK in neurons located in layers 2–3 of the IC. This preclinical study may enhance the potential use of ICS and identify the therapeutic mechanisms of ICS in neuropathic pain. [ABSTRACT FROM AUTHOR]

Cellular prion protein (PrPC) is a widely expressed glycosylphosphatidylinositol-anchored membrane protein. Scrapie prion protein is a misfolded and aggregated form of PrPC responsible for prion-induced neurodegenerative diseases. Understanding the function of the nonpathogenic PrPC monomer is an important objective. PrPC may be shed from the cell surface to generate soluble derivatives. Herein, we studied a recombinant derivative of PrPC (soluble cellular prion protein, S-PrP) that corresponds closely in sequence to a soluble form of PrPC shed from the cell surface by proteases in the A Disintegrin And Metalloprotease (ADAM) family. S-PrP activated cell-signaling in PC12 and N2a cells. TrkA was transactivated by Src family kinases and extracellular signal-regulated kinase 1/2 was activated downstream of Trk receptors. These cell-signaling events were dependent on the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1), which functioned as a cell-signaling receptor system in lipid rafts. Membrane-anchored PrPC and neural cell adhesion molecule were not required for S-PrP-initiated cell-signaling. S-PrP promoted PC12 cell neurite outgrowth. This response required the NMDA-R, LRP1, Src family kinases, and Trk receptors. In Schwann cells, S-PrP interacted with the LRP1/NMDA-R system to activate extracellular signal-regulated kinase 1/2 and promote cell migration. The effects of S-PrP on PC12 cell neurite outgrowth and Schwann cell migration were similar to those caused by other proteins that engage the LRP1/NMDA-R system, including activated α2-macroglobulin and tissue-type plasminogen activator. Collectively, these results demonstrate that shed forms of PrPC may exhibit important biological activities in the central nervous system and the peripheral nervous system by serving as ligands for the LRP1/NMDA-R system.

Retinal ganglion cell apoptotic death is the main pathological characteristic of glaucoma, which is the leading cause of irreversible blindness. Disruption of Ca2+ homeostasis plays an important role in glaucoma. Voltage-gated Ca2+ channel blockers have been shown to improve vision in patients with glaucoma. However, whether and how voltage-gated Ca2+ channels are involved in retinal ganglion cell apoptotic death are largely unknown. In this study, we found that total Ca2+ current densities in retinal ganglion cells were reduced in a rat model of chronic ocular hypertension experimental glaucoma, as determined by whole-cell patch-clamp electrophysiological recordings. Further analysis showed that L-type Ca2+ currents were downregulated while T-type Ca2+ currents were upregulated at the later stage of glaucoma. Western blot assay and immunofluorescence experiments confirmed that expression of the CaV1.2 subunit of L-type Ca2+ channels was reduced and expression of the CaV3.3 subunit of T-type Ca2+ channels was increased in retinas of the chronic ocular hypertension model. Soluble tumor necrosis factor-α, an important inflammatory factor, inhibited the L-type Ca2+ current of isolated retinal ganglion cells from control rats and enhanced the T-type Ca2+ current. These changes were blocked by the tumor necrosis factor-α inhibitor XPro1595, indicating that both types of Ca2+ currents may be mediated by soluble tumor necrosis factor-α. The intracellular mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and nuclear factor kappa-B signaling pathway mediate the effects of tumor necrosis factor-α. TUNEL assays revealed that mibefradil, a T-type calcium channel blocker, reduced the number of apoptotic retinal ganglion cells in the rat model of chronic ocular hypertension. These results suggest that T-type Ca2+ channels are involved in disrupted Ca2+ homeostasis and apoptosis of retinal ganglion cells in glaucoma, and application of T-type Ca2+ channel blockers, especially a specific CaV3.3 blocker, may be a potential strategy for the treatment of glaucoma.

Abstract Background Cataplexy is a loss of muscle tone that can lead to postural collapse, disturbing the daily life of narcolepsy patients; it is often triggered by positive emotions such as laughter in human patients. Narcolepsy model mice also show cataplexy, and its incidence increases in response to positive emotion-inducing stimuli such as chocolate and female courtship. Although such observation indicates a positive emotion-related nature of cataplexy in narcolepsy mice, they also show cataplexy without any apparent triggering stimulus ~ (spontaneous cataplexy). Therefore, we hypothesized that some spontaneous cataplexy in narcoleptic mice might indicate the remembering of happy moments. Results To test our hypothesis, we did a conditioned place preference test on orexin/hypocretin neuron-ablated (ORX-AB) mice, one of the animal models of human narcolepsy, and counted the number of cataplexy-like behaviors. ORX-AB mice successfully remembered the chocolate-associated chamber, and the number of cataplexy-like behaviors significantly increased in the chocolate-associated chamber but not in the control chamber. In addition, ORX-AB mice remembered the aversive odor-associated chamber and avoided entering without affecting the number of cataplexy-like behaviors. Finally, similar activation of the nucleus accumbens, a positive emotion-related nucleus, was observed during both spontaneous and chocolate-induced cataplexy behaviors. Conclusions These results support our hypothesis and will promote the usefulness of a narcolepsy mice model in emotion research and serve as a basis for a better understanding of cataplexy in narcolepsy patients.

Abstract Background Tyrosine kinase (TK) plays a crucial role in the pathogenesis of idiopathic pulmonary fibrosis. Here, we aimed to investigate whether radotinib (Rb) could inhibit pulmonary fibrosis by inhibiting TK in vitro and in vivo. Methods The antifibrotic effects of Rb in transforming growth factor-β (TGF-β)1-stimulated A549 cells were determined using real-time polymerase chain reaction, western blotting, and immunocytochemistry assays. Rb inhibition of bleomycin-induced lung fibrosis in Sprague Dawley (SD) rats was determined by histopathological and​ immunohistochemical analyses. Rb-interfering metabolites were analyzed using LC-MS/MS. Results Rb concentrations of up to 1000 nM did not affect the viability of A549 cells, but Rb (30 nM) significantly reduced expression of TGF-β1 (10 ng/mL)-induced ECM factors, such as Snail, Twist, and F-actin. Rb also regulated TGF-β1-overexpressed signal cascades, such as fibronectin and α-smooth muscle actin. Furthermore, Rb attenuated the phosphorylation of Smad2 and phosphorylation of kinases, such as, extracellular signal-regulated kinase, and protein kinase B. In the inhibitory test against bleomycin (5 mg/kg)-induced lung fibrosis, the Rb (30 mg/kg/daily)-treated group showed a half-pulmonary fibrosis region compared to the positive control group. In addition, Rb significantly reduced collagen type I and fibronectin expression in the bleomycin-induced fibrotic region of SD rats. Further, the identified metabolite pantothenic acid was not altered by Rb. Conclusion Taken together, these results indicate that Rb inhibits TGF-β1-induced pulmonary fibrosis both in vitro and in vivo. These findings suggest that Rb may be an effective treatment for pulmonary fibrosis-related disorders and idiopathic pulmonary fibrosis.

Background: Oncogenic mutations in BRAF genes are found in approximately 5–10% of colorectal cancers. The majority of BRAF mutations are located within exons 11–15 of the catalytic kinase domains, with BRAF V600E accounting for more than 80% of the observed BRAF mutations. Sensitivity to BRAF- and mitogen-activated protein kinase (MEK) inhibitors varies depending on BRAF mutations and tumor cell types. Previously, we newly identified, BRAF L525R-mutation, in the activation segment of the kinase in colorectal cancer patient. Here, we characterized the function of the BRAF L525R mutation. Methods: HEK293 cells harboring a BRAF mutation (V600E or L525R) were first characterized and then treated with cetuximab, dabrafenib, and selumetinib. Cell viability was measured using WST-1 assay and the expression of proteins involved in the extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signaling pathways was evaluated using western blot analysis. Results: The MEK inhibitor selumetinib effectively inhibited cell proliferation and ERK phosphorylation in BRAF L525R cells but not in BRAF V600E cells. Further studies revealed that AKT phosphorylation was reduced by selumetinib in BRAF L525R cells but not in BRAF V600E cells or selumetinib-resistant BRAF L525R cells. Moreover, the AKT inhibitor overcame the selumetinib resistance. Conclusions: We established a model system harboring BRAF L525R using HEK293 cells. BRAF L525R constitutively activated ERK. AKT phosphorylation caused sensitivity and resistance to selumetinib. Our results suggest that a comprehensive network analysis may provide insights to identify effective therapies. [ABSTRACT FROM AUTHOR]

Aim To observe the effect of edaravone dexborneol (EDDE) on neuronal apoptosis in rats with cerebral ischemia-reperfusion, and to explore its mechanism. Methods SD rats were randomly divided into sham group, middle cerebral artery occlusion (MCAO) group, low-dose EDDE group (EDDE-L group, 3 mg/kg), and high-dose EDDE group (EDDE-H group, 6 mg/kg), chelerythrine (CHE, PKC inhibitor) group (5 mg/kg CHE), EDDE+CHE group (6 mg/kg EDDE+5 mg/kg CHE), with 15 rats in each group. Except for the sham group, the rats in the other groups were given the suture method to construct the MCAO model. The rats in each group were scored for neurological deficits; after 24 hours of reperfusion, TTC staining was used to detect the cerebral infarct volume of rats; HE staining was used to observe the pathological damage of cortical nerve cells; TUNEL staining was used to detect cortical nerve cells apoptosis; immunohistochemical method was used to detect the expression of Bcl-2 and Bax in the cortex nerve cells; Western blot was used to detect the expression of Caspase-3, cleaved Caspase-3 and protein kinase C (PKC) / extracellular signal-regulated kinase (ERK) pathway related proteins (PKC, p-PKC, ERK1/2 and p-ERK1/2) in brain tissue. Results Compared with sham group, the neurological deficit score, the percentage of cerebral infarction volume and the apoptosis rate of nerve cells increased, the positive expression of Bax increased and the ratio of cleaved Caspase-3/Caspase-3 increased (all P<0. 05), the positive expression of Bcl-2 decreased, the ratio of Bcl-2/Bax, p-PKC/PKC and p-ERK1/2/ERK1/2 decreased in brain tissue (all P<0. 05), the cerebral cortex cells were sparsely arranged, swelling and vacuolar degeneration of nerve cells were obvious, the nucleus shrank and the tissue becomes necrotic in MCAO group. Compared with MCAO group, the neurological deficit score, cerebral infarction volume percentage and apoptosis rate of nerve cells decreased, the positive expression of Bax decreased, the ratio of cleaved Caspase-3/Caspase-3 decreased (all P<0. 05), the positive expression of Bcl-2 increased, the ratio of Bcl-2/Bax, p-PKC/PKC and the ratio of p-ERK1/2/ERK1/2 increased in brain tissue (P<0. 05) in the EDDE-L group and EDDE-H group, the pathological damage of the cerebral cortex was improved to varying degrees, the number of nerve cells increased, the vacuolar degeneration was reduced, the nucleus was clearer, and the EDDE-H group was better than the EDDE-L group. CHE could eliminate the neuroprotective effect of EDDE. Conclusion EDDE cloud inhibit neuronal apoptosis and reduce cerebral ischemia/reperfusion injury, and its mechanism may be related to the activation of PKC/ERK pathway. [ABSTRACT FROM AUTHOR]

Objective：To discuss the effect of ghrelin on the differentiation of the adipose mesenchymal stem cells (ADSCs) into the neurons, and to clarify its possible mechanism. Methods：The ADSCs were randomly divided into blank group, neural differentiation inducer group, and ghrelin group (given 600 μg·L-1 Ghrelin), U0126 group (given 40 ng·L-1 U0126), Ghrelin+U0126 group (given 600 μg·L-1 Ghrelin+40 ng·L-1 U0126), and Ghrelin receptor blocker D-Lys3-GHRP-6 (D-Lys3-GHRP-6) group (given 10－10 g·L-1 D-Lys3-GHRP-6). The pathomorphology of the cells in various groups was observed under inverted microscope; the positive expression rates of neurofilament protein (NF) -200 and tubulin (Tuj-1) in the cells in various groups were detected by immunofluorescence method; the expression levels of the mitogen activated protein kinase (MAPK) /extracellular signal-regulated kinase (ERK) pathway proteins, neuron specific nuclear protein (NeuN), Tuj-1, neuron specific enolase (NSE), NF, growth hormone secretagogne receptor (GHSR), fetal liver kinase 1 (Flk1), and CD29 proteins in the cells in various groups were detected by Western blotting method. Results：On the 10th day after cell culture, the ADSCs in blank group showed the fusions of long spindle and spiral shapes; the body of the cells in neural differentiation inducer group contracted, and the long spindle like cell body was transformed into the round-like shape, the protrusion length extended, and the number of neuron-like cells was increased; in Ghrelin group, the number of body protrusions of the cells was increased and the number of round-like like cells was increased; there was a small amount of cell body contraction and circular transformation in U0126 group and D-Lys3-GHRP-6 group. The immunofluorescence assay results showed that compared with blank group, the positive expression rates of NF-200 and Tuj-1 in the cells in neural differentiation inducer group were increased (P<0. 05); compared with neural differentiation inducer group, the positive expression rates of NF-200 and Tuj-1 in the cells in Ghrelin group were increased (P<0. 05), while the positive expression rates of NF-200 and Tuj-1 in the cells in U0126 group and D-Lys3-GHRP-6 group were decreased (P<0. 05); compared with Ghrelin group, the positive expression rates of NF-200 and Tuj-1 in the cells in U0126 group, D-Lys3-GHRP-6 group, and Ghrelin+U0126 group were decreased (P< 0. 05). The Western blotting results showed that compared with blank group, the expression levels of NSE, NeuN, Tuj-1, NF, and GHSR proteins, and the ratios of phosphorylated MAPK (p-MAPK) / MAPK and phosphorylated ERK (p-ERK) /ERK in the cells in neural differentiation inducer group were increased (P<0. 05), while the expression levels of Flk1 and CD29 proteins were decreased (P< 0. 05); compared with neural differentiation inducer group, the expression levels of NSE, NeuN, Tuj-1, NF, and GHSR proteins, and the ratios of p-MAPK/MAPK and p-ERK/ERK in the cells in Ghrelin group were increased (P<0. 05), while the expression levels of Flk1 and CD29 proteins were decreased (P< 0. 05); the expression levels of NSE, NeuN, Tuj-1, NF, and GHSR proteins, and the ratios of p-MAPK/ MAPK and p-ERK/ERK in the cells in U0126 and D-Lys3-GHRP-6 groups were decreased (P<0. 05), and the expression levels of Flk1 and CD29 proteins were increased (P<0. 05); compared with Ghrelin group, the expression levels of NSE, NeuN, Tuj-1, NF, and GHSR proteins, and the ratios of p-MAPK/MAPK and p-ERK/ERK in the cells in U0126 group, D-Lys3-GHRP-6 group, and Ghrelin+U0126 group were decreased (P<0. 05), while the expression levels of Flk1 and CD29 proteins were increased (P<0. 05). Conclusion：Ghrelin can induce the differention of the ASCs into the neurons, and it mechanism is related to activating the MAPK/ERK pathway. [ABSTRACT FROM AUTHOR]

As a biased agonist, peptide angiotensin II (Ang II) type 1 (AT1) receptor ligand antagonizes Ang II-stimulated G protein signaling but stimulates several kinase pathways. Here, we developed a non-peptide AT1 receptor compound as a biased ligand. We synthesized three non-peptide AT1 receptor ligands (R239470, R781253, and R794847) as candidates of biased ligands. Extracellular signal-regulated kinase (ERK) 1/2 activation and inositol phosphate (IP) production were measured using a cell system that overexpressed AT1 receptors (wild-type, L112A, Q257A, Y292A, and N295A receptors). We also examined the modes of receptor–ligand binding using a competition binding study. The Kd values of R239470, R781253, and R794847 for the AT1 wild-type receptor were 0.8, 21, and 48 nM, respectively, as assessed in a competition binding study. Those of R239470, R781253, and R794847 for the L112A receptor were 37, 23, and 31 nM, respectively. R781253 and R794847 decreased and increased IP production, respectively, whereas R239470 did not change IP production. R781253 and R794847, but not R239470, activated ERK1/2. In conclusion, R239470, R781253, and R794847 act as a neutral antagonist, an inverse agonist, and an agonist with regard to IP production, respectively. On the other hand, R781253 and R794847, but not R239470, are agonists toward ERK1/2 activation. Thus, we developed a non-peptide AT1 receptor compound as a biased ligand. [ABSTRACT FROM AUTHOR]

Background: Hypoxia is associated with many respiratory diseases, partly due to the accumulation of edema fluid and mucus on the surface of alveolar epithelial cell (AEC), which forms oxygen delivery barriers and is responsible for the disruption of ion transport. Epithelial sodium channel (ENaC) on the apical side of AEC plays a crucial role to maintain the electrochemical gradient of Na+ and water reabsorption, thus becomes the key point for edema fluid removal under hypoxia. Here we sought to explore the effects of hypoxia on ENaC expression and the further mechanism related, which may provide a possible treatment strategy in edema related pulmonary diseases. Methods: Excess volume of culture medium was added on the surface of AEC to simulate the hypoxic environment of alveoli in the state of pulmonary edema, supported by the evidence of increased hypoxia-inducible factor-1 expression. The protein/mRNA expressions of ENaC were detected, and extracellular signal-regulated kinase (ERK)/nuclear factor κB (NF-κB) inhibitor was applied to explore the detailed mechanism about the effects of hypoxia on epithelial ion transport in AEC. Meanwhile, mice were placed in chambers with normoxic or hypoxic (8%) condition for 24 h, respectively. The effects of hypoxia and NF-κB were assessed through alveolar fluid clearance and ENaC function by Ussing chamber assay. Results: Hypoxia (submersion culture mode) induced the reduction of protein/mRNA expression of ENaC, whereas increased the activation of ERK/NF-κB signaling pathway in parallel experiments using human A549 and mouse alveolar type 2 cells, respectively. Moreover, the inhibition of ERK (PD98059, 10 µM) alleviated the phosphorylation of IκB and p65, implying NF-κB as a downstream pathway involved with ERK regulation. Intriguingly, the expression of α-ENaC could be reversed by either ERK or NF-κB inhibitor (QNZ, 100 nM) under hypoxia. The alleviation of pulmonary edema was evidenced by the administration of NF-κB inhibitor, and enhancement of ENaC function was supported by recording amiloride-sensitive short-circuit currents. Conclusions: The expression of ENaC was downregulated under hypoxia induced by submersion culture, which may be mediated by ERK/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Exposure to surgery with anesthesia early in life may lead to abnormal behavior, learning, and memory in humans. Pre-clinical studies have suggested a critical role of glial cell-derived neurotrophic factor (GDNF) in these effects. We hypothesize that the inhibition of extracellular signal-regulated kinase (ERK)-cAMP response element-binding protein (CREB) pathway contributes to GDNF decrease and the dysfunction of learning and memory. To address this hypothesis, 7-day-old Sprague–Dawley male and female rats were subjected to right carotid artery exposure (surgery) under sevoflurane anesthesia. Their learning and memory were tested by the Barnes maze, and novel object recognition tests started 23 days after the surgery. Blood and brain were harvested at various times after surgery for biochemical analyses. Rats with surgery and anesthesia performed poorly in the Barnes maze and novel object recognition tests compared with control rats. Rats with surgery had a decreased GDNF concentration in the brain and urine. The concentrations of urine GDNF were negatively correlated with the performance of rats in a delayed memory phase of the Barnes maze test. Surgery increased proinflammatory cytokines in the blood and brain. Intracerebroventricular injection of GDNF attenuated the increased inflammatory response in surgery rats. Surgery inhibited ERK and CREB. Inhibiting ERK reduced GDNF and induced poor performance in the Barnes maze and novel object recognition tests of rats without surgery. Surgery also increased brain-derived natriuretic peptide (BNP) in the brain. Intracerebroventricular injection of BNP inhibited ERK and CREB, reduced GDNF, and impaired learning and memory. Surgery, ERK inhibition, and BNP reduced the expression of synaptic proteins. Our results suggest that surgery increases BNP that inhibits ERK-CREB signaling to reduce GDNF, which leads to an unbalanced inflammatory response and a reduced synaptic protein expression for the development of postoperative cognitive dysfunction. Key messages: Surgery increases BNP and decreases ERK/CREB signaling to reduce GDNF. The increase in BNP and decrease in ERK/CREB signaling contribute to postoperative cognitive dysfunction. GDNF reduction contributes to neuroinflammatory response after surgery. Urine GDNF concentrations are negatively corrected with poor spatial memory performance. [ABSTRACT FROM AUTHOR]

The main treatment strategy for ischemic diseases caused by conditions such as poor blood vessel formation or abnormal blood vessels involves repairing vascular damage and encouraging angiogenesis. One of the mitogen-activated protein kinase (MAPK) signaling pathways, the extracellular signal-regulated kinase (ERK) pathway, is followed by a tertiary enzymatic cascade of MAPKs that promotes angiogenesis, cell growth, and proliferation through a phosphorylation response. The mechanism by which ERK alleviates the ischemic state is not fully understood. Significant evidence suggests that the ERK signaling pathway plays a critical role in the occurrence and development of ischemic diseases. This review briefly describes the mechanisms underlying ERK-mediated angiogenesis in the treatment of ischemic diseases. Studies have shown that many drugs treat ischemic diseases by regulating the ERK signaling pathway to promote angiogenesis. The prospect of regulating the ERK signaling pathway in ischemic disorders is promising, and the development of drugs that specifically act on the ERK pathway may be a key target for promoting angiogenesis in the treatment of ischemic diseases.