Extracellular Signal-regulated Protein Kinase Research Articles

Follistatin drives neuropathic pain in mice through IGF1R signaling in nociceptive neurons.

Neuropathic pain is a debilitating chronic condition that lacks effective treatment. The role of cytokine- and chemokine-mediated neuroinflammation in its pathogenesis has been well documented. Follistatin (FST) is a secreted protein known to antagonize the biological activity of cytokines in the transforming growth factor-β (TGF-β) superfamily. The involvement of FST in neuropathic pain and the underlying mechanism remain largely unknown. Here, we report that FST was up-regulated in A-fiber sensory neurons after spinal nerve ligation (SNL) in mice. Inhibition or deletion of FST alleviated neuropathic pain and reduced the nociceptive neuron hyperexcitability induced by SNL. Conversely, intrathecal or intraplantar injection of recombinant FST, or overexpression of FST in the dorsal root ganglion (DRG) neurons, induced pain hypersensitivity. Furthermore, exogenous FST increased neuronal excitability in nociceptive neurons. The biolayer interferometry (BLI) assay and coimmunoprecipitation (co-IP) demonstrated direct binding of FST to the insulin-like growth factor-1 receptor (IGF1R), and IGF1R inhibition reduced FST-induced activation of extracellular signal-regulated kinase (ERK) and protein kinase B (AKT), as well as neuronal hyperexcitability. Further co-IP analysis revealed that the N-terminal domain of FST exhibits the highest affinity for IGF1R, and blocking this interaction with a peptide derived from FST attenuated Nav1.7-mediated neuronal hyperexcitability and neuropathic pain after SNL. In addition, FST enhanced neuronal excitability in human DRG neurons through IGF1R. Collectively, our findings suggest that FST, released from A-fiber neurons, enhances Nav1.7-mediated hyperexcitability of nociceptive neurons by binding to IGF1R, making it a potential target for neuropathic pain treatment.

Chrysene contribution to bronchial asthma: Activation of TRPA1 disrupts bronchial epithelial barrier via ERK pathway

BackgroundElevated polycyclic aromatic hydrocarbon (PAH) levels are associated with exacerbation of asthma. Chrysene is one of the most prevalent unsubstituted PAHs in the environment. Transient receptor potential ankyrin 1 (TRPA1) can be used as a chemoreceptor to detect inhaled stimuli and plays an important role in the occurrence and deterioration of asthma. Whether exposure to a high concentration of chrysene in the environment can activate TRPA1 and contribute to the development of asthma, potentially through the dysfunction of the bronchial epithelial barrier, remains unclear. MethodsA cell-based assay was performed to verify the downregulation of the expression of E-cadherin and tight junction (TJ) proteins by chrysene in bronchial epithelial cells to explore the role of chrysene-mediated TRPA1 activation in the regulation of TJ protein expression through the extracellular signal-regulated protein kinase (ERK) pathway. Animal tests were conducted to determine whether chrysene could enhance airway hyperresponsiveness (AHR) induced by house dust mites (HDMs) and disrupt barrier function, thereby contributing to asthma. ResultsThe cell-based assay revealed that chrysene could disrupt the function of the bronchial epithelial barrier and decrease the expression levels of E-cadherin, zonula occludens-1 (ZO-1), occludin, and claudin-5 through the ERK pathway. Chrysene induced airway epithelial barrier dysfunction primarily through TRPA1 instead of transient receptor potential vanilloid 1. TRPA1 knockdown was able to attenuate chrysene-induced downregulation of TJ protein expression and downregulate ERK activation (p-ERK). Compared with exposure to HDM alone, coexposure to chrysene and HDM resulted in an increased incidence of AHR, disruption of barrier function, and eosinophilic inflammatory responses in a mouse model of asthma. Coexposure to chrysene and HDM increased TRPA1 expression. The animal test verified that the TRPA1 inhibitor HC030031 could suppress chrysene and HDM-induced asthma in mice. ConclusionsOur findings showed that chrysene contributed to the breakdown of the function of the bronchial epithelial barrier through the TRPA1–ERK axis and therefore acted as an adjuvant to contribute to asthma.

Hyperthermia-mediated cell death via deregulation of extracellular signal-regulated kinase and c-Jun NH2-terminal kinase signaling

Hyperthermia is a promising anticancer treatment that induces heat stress, thereby stimulating various signal transduction pathways to maintain cellular homeostasis. Mitogen-activated protein kinases (MAPKs) associate various extracellular stimuli with cytoplasmic and nuclear mediators through a three-tiered cascade of kinases, including MAPKs, MAP2Ks, and MAP3Ks. In mammals, three major groups of MAPKs have been characterized: extracellular signal-regulated protein kinases (ERK1/2), p38 MAPKs (α, β, γ, and δ), and c-Jun NH2-terminal kinases (JNK1/2/3). Each group of MAPKs is activated by heat and exhibits distinct biological functions. Recent studies have indicated that in hyperthermia, MAPK signaling pathways regulate cell survival and death in unique ways. This review offers a concise overview of the MAPK signaling pathway, specifically ERK and JNK, focusing on their relevance in cancer, interplay with heat shock proteins or phosphatases, and current understanding of the MAPK signaling pathway in hyperthermia.

Punicalagin as a novel selective aryl hydrocarbon receptor (AhR) modulator upregulates AhR expression through the PDK1/p90RSK/AP-1 pathway to promote the anti-inflammatory response and bactericidal activity of macrophages

Aryl hydrocarbon receptor (AhR) plays an important role in inflammation and immunity as a new therapeutic target for infectious disease and sepsis. Punicalagin (PUN) is a Chinese herbal monomer extract of pomegranate peel that has beneficial anti-inflammatory, antioxidant and anti-infective effects. However, whether PUN is a ligand of AhR, its effect on AhR expression, and its signaling pathway remain poorly understood. In this study, we found that PUN was a unique polyphenolic compound that upregulated AhR expression at the transcriptional level, and regulated the AhR nongenomic pathway. AhR expression in lipopolysaccharide-induced macrophages was upregulated by PUN in vitro and in vivo in a time- and dose-dependent manner. Using specific inhibitors and siRNA, induction of AhR by PUN depended on sequential phosphorylation of 90-kDa ribosomal S6 kinase (p90RSK), which was activated by the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphoinositide-dependent protein kinase (PDK)1 pathways. PUN promoted p90RSK-mediated activator protein-1 (AP-1) activation. AhR knockout or inhibitors reversed suppression of interleukin (IL)-6 and IL-1β expression by PUN. PUN decreased Listeria load and increased macrophage survival via AhR upregulation. In conclusion, we identified PUN as a novel selective AhR modulator involved in AhR expression via the MEK/ERK and PDK1 pathways targeting p90RSK/AP-1 in inflammatory macrophages, which inhibited macrophage inflammation and promoted bactericidal activity.

Enhanced activation of signaling pathway by recombinant human adiponectin from genome-edited chickens

Adiponectin (ADPN) exerts various cellular and metabolic functions by activating signaling pathways, including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways, the protein kinase B (Akt) pathway, and the p38 mitogen-activated protein kinase (MAPK) pathway. However, generating functional recombinant human adiponectin (hADPN) in bacterial or mammalian cells is challenging. Although ADPN agonist peptides have been developed, problems like stability, solubility, and affinity for receptors remain. Recently, a genome-edited chicken bioreactor system was established, ensuring efficient ADPN production with optimal post-transcriptional modifications. We assessed the ability of egg white (EW)-derived hADPN, commercial hADPN, various ADPN agonist peptides, and globular ADPN on activation of the ERK1/2, Akt, and p38 MAPK pathways. EW-derived hADPN, abundant in hexamers and high molecular weight multimers, significantly phosphorylated ERK1/2 in serum-starved HEK293 cells after 15 min of treatment. Comparative analysis revealed that EW-derived hADPN and commercial hADPN induced greater phosphorylation of ERK1/2, Akt, and p38 MAPK than ADPN agonist peptides and globular ADPN, with EW-derived hADPN showing the highest activation. In summary, the finding that EW-derived hADPN strongly activates the ERK1/2, Akt, p38 MAPK signaling pathways highlights that an ADPN production system based on genome-edited chickens is an advantageous alternative to existing methods.

Role of hedgehog signaling in the pathogenesis and therapy of heterotopic ossification.

Heterotopic ossification (HO) is a pathological process that generates ectopic bone in soft tissues. Hedgehog signaling (Hh signaling) is a signaling pathway that plays an important role in embryonic development and involves three ligands: sonic hedgehog (Shh), Indian hedgehog (Ihh) and desert hedgehog (Dhh). Hh signaling also has an important role in skeletal development. This paper discusses the effects of Hh signaling on the process of HO formation and describes several signaling molecules that are involved in Hh-mediated processes: parathyroid Hormone-Related Protein (PTHrP) and Fkbp10 mediate the expression of Hh during chondrogenesic differentiation. Extracellular signal-regulated kinase (ERK), GNAs and Yes-Associated Protein (YAP) interact with Hh signaling to play a role in osteogenic differentiation. Runt-Related Transcription Factor 2 (Runx2), Mohawk gene (Mkx) and bone morphogenetic protein (BMP) mediate Hh signaling during both chondrogenic and osteogenic differentiation. This paper also discusses possible therapeutic options for HO, lists several Hh inhibitors and explores whether they could serve as emerging targets for the treatment of HO.

Potential Skin Health Benefits of Abalone By-Products Suggested by Their Effects on MAPKS and PI3K/AKT/NF-kB Signaling Pathways in HDF and HaCaT Cells.

Abalone, a marine edible gastropod with nutritional value, is a popular seafood delicacy worldwide, especially in Asia; however, viscera by-products are generally discarded during processing. Therefore, we investigated the skin health benefits of abalone viscera ultrasonic extract (AVU) in human dermal fibroblasts (HDFs) and human keratinocyte (HaCaT) cells. AVU showed valuable protein contents, indicating that it is a worthy and safe material for industrial application. AVU increased collagen synthesis production and messenger RNA (mRNA) expression of Collagen Type I Alpha 1, 2, and 3 chains through the transforming growth factor beta/suppressor of mother against the decapentaplegic pathway in HDF cells. AVU also increased hyaluronic acid production, upregulated Hyaluronan Synthases 1, 2, and 3, filaggrin and aquaporin3 mRNA levels, and downregulated hyaluronidase mRNA levels in HaCaT cells. Furthermore, mechanistic studies showed that AVU increased the phosphorylation of extracellular signal-regulated kinase, p38, and cyclic AMP response-binding protein activation. AVU activated the transcription factors, phosphoinositide 3-kinase, protein kinase B, and nuclear factor kappa B cell p65 and downregulated the degranulation of inhibitory kappa B in HaCaT cells. Studies of hyaluronic acid production in AVU by inhibiting EKR, p38 and NF-κB have shown that p38 MAPK and NF-κB signaling are pivotal mechanisms, particularly in the AVU. These results demonstrated that AVU produced from by-products may improve skin health and may thus be used as a functional food and cosmetics ingredient.

Dietary plants for oral cancer prevention and therapy: A review of preclinical and clinical studies.

Oral cancer is a disease with high mortality and rising incidence worldwide. Although fragmentary literature on the anti-oral cancer effects of plant products has been published, a comprehensive analysis is lacking. In this work, a critical and comprehensive evaluation of oral cancer preventative or therapeutic effects of dietary plants was conducted. An exhaustive analysis of available data supports that numerous dietary plants exert anticancer effects, including suppression of cell proliferation, viability, autophagy, angiogenesis, invasion, and metastasis while promoting cell cycle arrest and apoptosis. Plant extracts and products target several cellular mechanisms, such as the reversal of epithelial-to-mesenchymal transition and the promotion of oxidative stress and mitochondrial membrane dysfunction by modulation of various signaling pathways. These agents were also found to regulate cellular growth signaling pathways by action on extracellular signal-regulated kinase and mitogen-activated protein kinase, inflammation via modulation of cyclooxygenase (COX)-1, COX-2, and nuclear factor-κB p65, and metastasis through influence of cadherins and matrix metalloproteinases. In vivo studies support these findings and demonstrate a decrease in tumor burden, incidence, and hyperplastic and dysplastic changes. Clinical studies also showed decreased oral cancer risk. However, high-quality studies should be conducted to establish the clinical efficacy of these plants. Overall, our study supports the use of dietary plants, especially garlic, green tea, longan, peppermint, purple carrot, saffron, tomato, and turmeric, for oral cancer prevention and intervention. However, further research is required before clinical application of this strategy.

Impact of compression forces on different mesenchymal stem cell types regarding orthodontic indication.

The potential of stem cells, for example upper periodontal ligament stem cells from the maxilla (u-PDLSC) and from the mandible (l-PDLSC), adipose-derived mesenchymal stem cells (AD-MSC), and bone marrow-derived mesenchymal stem cells (BM-MSC), with respect to periodontal remodeling and orthodontic treatment is of great importance. In this work, we focus on the comprehensive adaptability of different stem cell types to mechanical forces with the aim to better understanding cell behavior and to refine a new mechanistic approach to investigate periodontal remodeling. We comprehensively analyze stem cells and observe distinct morphological and proliferation changes under compression in dependence on stem cell type. The cell signaling of extracellular signal-regulated kinase (ERK) and protein kinase B, also called AKT, and their respective phosphorylation shows diverse responses to compression. Additionally, vascular endothelial growth factor and hepatocyte growth factor secretion were reduced under mechanical stress in all cell types, with cell-specific variations. Osteoprotegerin secretion was reduced under compression, particularly in u-PDLSC. At least, diverse soluble receptors of NF-kB-ligand secretion patterns among cell types under pressure were observed, providing crucial insights into bone metabolism. These findings offer a deeper understanding of the behavior of mesenchymal stem cells under mechanical stimuli, highlighting their roles in bone remodeling, wound healing, and tissue regeneration in orthodontic and regenerative medicine contexts. Our results underscore the potential of u-PDLSC, l-PDLSC, and AD-MSC in periodontal regeneration, with AD-MSC showing notable resilience under compression, indicating its promising role for further investigation for orthodontic research. While these findings are encouraging, further research is essential to fully comprehend the mechanism of stem cell-based periodontal therapies.

Inhibiting FGFR by toadflax reverses erlotinib resistance in nonsmall cell lung cancer

This study aims to demonstrate the effect of toadflax (bufalin) on erlotinib resistance in nonsmall cell lung cancer (NSCLC) by inhibiting the fibroblast growth factor receptor (FGFR). The microfluidic mobility transferase and caliper mobility-shift assays were employed to detect the FGFR inhibition by bufalin and the binding reversibility. Further, the inhibitory effects of bufalin were determined in HCC827 and HCC827/ER cells in vitro, investigating relative FGFR overexpression by quantitative reverse transcriptase-PCR (RT-qPCR) and FGFR downstream proteins, that is, FGFR substrate 2 (FRS2), extracellular signal-regulated kinase (ERK), and S6 by western blot analysis. Finally, HCC827/ER-inoculated xenograft tumors were constructed to observe the effects of bufalin and bufalin + erlotinib intervention on tumor growth. Bufalin inhibited FGFR by reversibly binding to FGFR1. In addition, the western blot analysis indicated a significant reduction in the expression levels of FGFR, FRS2, ERK, and S6 proteins in HCC827 and HCC827/ER cells, increasing the expression levels of apoptotic caspase-3 and poly-(ADP-ribose) polymerase proteins. Bufalin + erlotinib combination significantly inhibited the apoptosis of HCC827/ER cells and subsequent tumor growth in vivo. In addition, FGFR overexpression significantly reversed the sensitivity of bufalin to HCC827/ER cells, promoting the value-addition of HCC827/ER cells. Further, bufalin + erlotinib significantly reduced the growth of erlotinib-resistant HCC827/ER tumors, induced apoptosis, and inhibited the expression of FGFR and p-ERK proteins. These findings indicated that bufalin could reverse the erlotinib resistance in NSCLC by inhibiting the FGFR expression.

Abstract Mo124: New Rho-kinase Inhibitor Reduces Diastolic Dysfunction induced by Estrogen Depletion in Spontaneously Hypertensive Rats

Heart failure with preserved ejection fraction (HFpEF) observed in women with estrogen depletion is a consequence of the reduction of cardioprotection and endothelial dysfunction. Rho-kinase (ROCK) signaling pathway plays a role in cardiac dysfunction consequent to female aging. This work investigated the effects of a new synthetic ROCK inhibitor (iROCK) after oral administration in spontaneously hypertensive rats (SHR) submitted to oophorectomy (OVX). Experimental protocols were approved Animal Care and Use Committee at Universidade Federal do Rio de Janeiro. Under ketamine (80 mg/kg, i.p.) and xylazine (15 mg/kg, i.p.) anesthesia, female SHR submitted to OVX had cardiac function assessed after 8 weeks and compared to SHR-Sham. Eight weeks after OVX, SHR were randomly divided in groups, which were orally treated with either vehicle (DMSO) or iROCK (10 mg/kg) during 2 weeks. Novel iROCK displays potent inhibitory activity for ROCK1 and ROCK2 isoforms, with IC50 of 1.3 μM and 1.7 μM, respectively. At the end of treatment, hemodynamic parameters were evaluated using echocardiography and LV catheterization. Cardiac tissues collected for determining the expression of mitogen-activated protein kinases p38MAPK and extracellular signal-regulated kinase (ERK-1/2) using Western blot technique. After 8 weeks of estrogen depletion, SHR displayed HFpEF because ejection fraction (EF) was 85.2 ± 0.9%, while a reduction in EF from 83.0 ± 2.7 to 64.9 ± 0.9% was only detected after 12 weeks of OVX. Altered filling pressure confirmed the diastolic dysfunction after OVX, because the ratio of rapid mitral flow (E) to rapid wave of mitral annulus tissue Doppler (e') increased from 12.1 ± 1.3 in SHR-Sham to 19.3 ± 0.5. LV end diastolic pressure (LVEDP) increased from 13.4 ± 1.3 to 21.1 ± 4.9 in SHR after 8 weeks post-OVX. Oral administration of iROCK (10 mg/kg) reduced both E/e’ to 16.2 ± 0.5 and LVEDP to 6.4 ± 0.7 mmHg. OVX in SHR increased cardiac p38 MAPK and ERK-1/2 contents by 2.3 and 1.3 fold from SHR-Sham values. In contrast, iROCK reduced p38 overexpression but not altered ERK-1/2 content. Since pro-inflammatory cytokines and oxidative stress activate p38, the recovery of its expression after treatment, indicates that iROCK could reduce the inflammatory component of HF. In conclusion, the new iROCK recovered LV diastolic function induced by estrogen depletion in SHR, through the reduction of myocardial inflammation.

Therapeutic targeting of siRNA/anti-cancer drug delivery system for non-melanoma skin cancer. Part I: Development and gene silencing of JAK1siRNA/5-FU loaded liposome nanocomplexes

Non-melanoma skin cancer (NMSC) is one of the most prevalent cancers, leading to significant mortality rates due to limited treatment options and a lack of effective therapeutics. Janus kinase (JAK1), a non-receptor tyrosine kinase family member, is involved in various cellular processes, including differentiation, cell proliferation and survival, playing a crucial role in cancer progression. This study aims to provide a more effective treatment for NMSC by concurrently silencing the JAK1 gene and administering 5-Fluorouracil (5-FU) using liposome nanocomplexes as delivery vehicles. Utilizing RNA interference (RNAi) technology, liposome nanocomplexes modified with polyethylene imine (PEI) were conjugated with siRNA molecule targeting JAK1 and loaded with 5-FU. The prepared formulations (NL-PEI) were characterized in terms of their physicochemical properties, morphology, encapsulation efficiency, in vitro drug release, and stability. Cell cytotoxicity, cell uptake and knockdown efficiency were evaluated in human-derived non-melanoma epidermoid carcinoma cells (A-431). High contrast transmission electron microscopy (CTEM) images and dynamic light scattering (DLS) measurements revealed that the nanocomplexes formed spherical morphology with uniform sizes ranging from 80-120 nm. The cationic NL-PEI nanocomplexes successfully internalized within the cytoplasm of A-431, delivering siRNA for specific sequence binding and JAK1 gene silencing. The encapsulation of 5-FU in the nanocomplexes was achieved at 0.2 drug/lipid ratio. Post-treatment with NL-PEI for 24, 48 and 72 h showed cell viability above 80 % at concentrations up to 8.5 × 101 µg/mL. Notably, 5-FU delivery via nanoliposome formulations significantly reduced cell viability at 5-FU concentration of 5 µM and above (p < 0.05) after 24 h of incubation. The NL-PEI nanocomplexes effectively silenced the JAK1 gene in vitro, reducing its expression by 50 %. Correspondingly, JAK1 protein level decreased after transfection with JAK1 siRNA-conjugated liposome nanocomplexes, leading to a 37 % reduction in pERK (phosphor extracellular signal-regulated kinase) protein expression. These findings suggest that the combined delivery of JAK1 siRNA and 5-FU via liposomal formulations offers a promising and novel treatment strategy for targeting genes and other identified targets in NMSC therapy.

Lipopolysaccharide Triggers Luminal Acidification to Promote Defense Against Bacterial Infection in Vaginal Epithelium

The vaginal epithelium plays pivotal roles in host defense against pathogen invasion, contributing to the maintenance of an acidic microenvironment within the vaginal lumen through the activity of acid-base transport proteins. However, the precise defense mechanisms employed by the vaginal epithelium following bacterial infection remain incompletely understood. This study demonstrated that the bacterial lipopolysaccharide (LPS) potentiated net proton efflux by up-regulating the expression of Na+-H+ exchanger 1 (NHE1), without affecting other acid-base transport proteins in vaginal epithelial cells. Pharmacological inhibition or genetic knockdown of Toll-like receptor-4 (TLR4) and the extracellular-signal-regulated protein kinase (ERK) signaling pathway effectively counteracted the up-regulation of NHE1 and the enhanced proton efflux triggered by LPS in vaginal epithelial cells. In vivo studies revealed that LPS administration led to luminal acidification through the up-regulation of NHE1 expression in the rat vagina. Moreover, inhibition of NHE exhibited impaired defense against acute bacterial infection in the rat vagina. These findings collectively indicated the active involvement of vaginal epithelial cells in facilitating luminal acidification during acute bacterial infection, offering potential insights into the treatment of bacterial vaginosis.

Anti-Inflammatory Effect of Medicinal Fungus Antrodia cinnamomea Cultivated on Pinus morrisonicola Hayata

Research background. The fungus Antrodia cinnamomea, which grows on Cinnamomum kanehirae tree, has many medicinal uses. However, its cultivation using the traditional method of growing on the C. kanehirae tree is costly and time-consuming. A possible alternative method of cultivating A. cinnamomea is to use Pinus morrisonicola Hayata tree, as it contains α-terpineol, which stimulates the synthesis of triterpenoids. Experimental approach. To compare the cultivation of A. cinnamomea on P. morrisonicola and C. kanehirae, the contents of triterpenoids and antcin were determined using high-performance liquid chromatography. Anti-inflammatory effects of the extracts of each product were investigated in lipopolysaccharide (LPS)-stimulated BV-2 cells. Their mechanisms on mitogen-activated protein kinase (MAPK) signalling pathways (p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)) were determined using Western blot analysis. Results and conclusions. The results showed that the cultivation times of A. cinnamomea on P. morrisonicola and traditional C. kanehirae discs were drastically different, lasting 6 and 18 months, respectively. The concentration of triterpenoids in the corresponding fruiting bodies was (70.0±3.0) and (20.0±4.0) mg/mL, respectively. More antcins were produced in the P. morrisonicola culture. Similar anti-inflammatory effect was obtained by both cultures, which is confirmed by the reduced production of IL-1β, IL-6, COX-2 and nitrogen monoxide. Their mechanisms were confirmed by the suppression of MAPK signalling pathways. Novelty and scientific contribution. Cultivation on P. morrisonicola is an innovative and more cost-effective method for growing A. cinnamomea. The same anti-inflammatory effect is achieved in a shorter production time.

Non-integrin laminin receptor (LamR) plays a role in axonal outgrowth from chicken DRG via modulating the Akt and Erk signaling.

37/67kDa laminin receptor (LamR)/ribosomal protein SA exhibits dual function as both a ribosomal protein and cell surface receptor for laminin. LamR influences critical cellular processes such as invasion, adhesion, and migration when acting as a receptor. Despite the acknowledged importance of LamR/67LR in various cellular processes, its contribution to the peripheral nervous system development is obscure. Thus, this study investigated the biological activity of LamR in peripheral axonal outgrowth in the presence of laminin-1 or Ile-Lys-Val-Ala-Val (IKVAV) peptide, whose important role in dorsal root ganglia (DRG) axonal outgrowth we recently showed. Unexpectedly, we did not observe LamR on the surface of DRG cells or in a conditioned medium, suggesting its intracellular action in the negative regulation of DRG axonal outgrowth. Using C-terminus LamR-targeting IgG, we demonstrated the role of LamR in that process, which is independent of the presence of Schwann cell precursors (SCPs) and is mediated by extracellular signal-regulated kinase (Erk) and Protein kinase B (Akt1/2/3) signaling pathways. Additionally, we show that the action of LamR towards laminin-1-dependent axonal outgrowth is unmasked only when the activity of integrin β1 is perturbed. We believe that modulation of LamR activity provides the basis for its use for inhibiting axon growth as a potential therapeutic agent for regulating abnormal or excessive neurite growth during neurodevelopmental diseases or pathological nerve regeneration.

Apple Pomace Extract Induces Cell Proliferation and Increases Type I Collagen and Hyaluronan Production in Human Skin Fibroblasts In Vitro.

Apple pomace is the residue left after apples are squeezed. The majority of pomace produced worldwide is produced by the apple manufacturing industry, however, most of the pomace produced by the industry is discarded. Apple pomace contains functional ingredients, such as polyphenols and triterpenoids, and exerts several beneficial effects on human health; however, studies on its cosmetic effects on the skin are lacking. Therefore, herein, we investigated the effects of apple pomace extract (APE) on human skin fibroblasts (HSFs) in vitro. When HSFs were cultured with the extract for 72h, the number of HSFs increased at concentrations of 10 and 20µg/mL. Transcriptome analysis and reverse transcription-quantitative PCR results revealed that the extract upregulated the expression of hyaluronan synthase (HAS) 1, HAS2, and HAS3 and downregulated the expression of HYAL1, a gene encoding the hyaluronan-degrading enzyme, in HSFs. Additionally, enzyme-linked immunosorbent assay revealed increased amounts of factors related to skin extracellular matrix, such as type I collagen and hyaluronic acid, secreted in the culture supernatant. The western blotting results suggested that the extract induced extracellular signal-regulated kinase and protein kinase B phosphorylation in HSFs. Additionally, several GO_Terms related to mitosis were detected in the Gene Ontology analysis. This is the first study to show that APE induces the proliferation of HSFs and production of factors related to skin anti-aging.

Anti-myocardial ischemic injury effects and mechanisms of cryptotanshinone in regulating macrophage polarisation through Dectin-1 signalling pathway

The aim of this study was to investigate the potential mechanism by which cryptotanshinone(CTS) may exert its anti-myo-cardial ischemic effect through the regulation of macrophage polarization via the dendritic cell-associated C-type lectin 1(Dectin-1) signaling pathway. Male C57BL/6 mice, aged six weeks, were utilized to establish myocardial ischemia models and were subsequently divided into five groups: sham, model, CTS low-dose(21 mg·kg~(-1)·d~(-1)), CTS high-dose(84 mg·kg~(-1)·d~(-1)), and dapagliflozin(0.14 mg·kg~(-1)·d~(-1)). The cardiac function, serum enzyme levels, Dectin-1 expression, macrophage polarization, and neutrophil infiltration in the myocardial infarction area were assessed in each group. An in vitro model of M1-type macrophages was constructed using lipopolysaccharide/interfe-ron-γ(LPS/IFN-γ) stimulated RAW264.7 cells to investigate the impact of CTS on macrophage polarization and to examine alterations in key proteins within the Dectin-1 signaling pathway. In the CTS group, compared to the model group mice, there was a significant improvement in the cardiac function and myocardial injury, along with a notable increase in the ratio of M2/M1-type macrophages in the myocardial infarcted area and a decrease in neutrophil infiltration. Additionally, Dectin-1 exhibited low expression. The results of in vitro experiments demonstrated that CTS can decrease the expression of M1-type marker genes and increase the expression of M2-type marker genes. Besides, it can decrease the levels of Dectin-1 and the phosphorylation of its associated proteins, including spleen tyrosine kinase(Syk), protein kinase B(Akt), nuclear factor-kappaB p65(NF-κB p65), and extracellular signal-regulated protein kinases(ERK1/2). Additionally, CTS was found to enhance the phosphorylation of signal transducer and activator of transcription-6(STAT6). The above results suggest that CTS exerts its anti-myocardial ischemic injury effect by regulating macrophage polarization through the Dectin-1 signaling pathway.

MAPK signaling pathway participates in the regulation of intestinal phosphorus and calcium absorption in broiler chickens via 1,25-dihydroxyvitamin D3

Four experiments were performed to investigate the role of the mitogen-activated protein kinase (MAPK) signaling pathway in intestinal absorption of phosphorus (P) and calcium (Ca) in broiler chickens. Experiment 1 assessed how dietary levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) influence the gene expression of intestinal P and Ca transporters in broilers. Experiment 2 evaluated the effects of 1,25(OH)2D3 administered via intraperitoneal injection on the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Experiments 3 and 4 investigated the effect of ERK and p38MAPK inhibitors on the expression of intestinal P and Ca transporters. The findings demonstrated that broilers (1-21 days old) fed a 1,25(OH)2D3-deficient diet (0.625 µg/kg) exhibited reduced body weight, tibia P and Ca levels, and mRNA levels of P transporters (NaPi-IIb, PiT-1, and PiT-2), Ca transporters (NCX1, PMCA1b, and CaBP-D28k), vitamin D receptors (VDR), ERK, and p38MAPK in the duodenum (Experiment 1) (P < 0.05). By comparison, the growth, bone quality, and mRNA levels of genes (except for duodenal NaPi-IIb) in broilers were similar to those in broilers fed the control diet when dietary 1,25(OH)2D3 was adequate (5 µg/kg) (Experiment 1) (P > 0.05). After intraperitoneal injection of 1,25(OH)2D3, the mRNA level of jejunal NaPi-IIb and the protein level of p-p38MAPK/t-p38MAPK in broilers (9-14 days old) decreased (P < 0.05), whereas the mRNA level of CaBP-D28k and the protein level of p-ERK/t-ERK increased (Experiment 2) (P < 0.05). The mRNA and protein expression of jejunal NaPi-IIb and the protein expression of CaBP-D28k in broilers (9-17 days old) treated with the ERK inhibitor PD98059 were greater than those in the control group (Experiment 3) (P < 0.05). Similarly, compared with control broilers, broilers (9-17 days old) treated with the p38MAPK inhibitor SB203580 showed elevated mRNA expression of jejunal NaPi-IIb and CaBP-D28k (Experiment 4) (P < 0.05). These results suggest that adequate supplementation with 1,25(OH)2D3 (5 µg/kg) can restore broiler growth and bone quality by upregulating the transcription of genes involved in intestinal P and Ca absorption. Additionally, the ERK and p38MAPK signaling pathways are implicated in the modulatory effect of 1,25(OH)2D3 on the absorption of P and Ca in broilers.

Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitor Improves the Vascular Function of Arteriovenous Fistula in Rats with Hyperglycemia.

Few evidence-based medications to improve the primary patency of arteriovenous fistulas in patients with diabetes who require hemodialysis are available. We investigated whether proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) could improve arteriovenous fistula function through pleiotropic effects in a rat model of hyperglycemia. Ex vivo effects of PCSK9i on the aorta of Sprague-Dawley (SD) rats were investigated using an organ bath system. For in vivo experiments, an abdominal aortocaval (AC) fistula was generated in SD rats (200-250 g) after inducing hyperglycemia through streptozotocin administration (80 mg/kg, intraperitoneal). Alirocumab (50 mg/kg/week, subcutaneous) was administered on the day of fistula surgery and day 7. Echocardiography, blood flow through the aorta-limb, vasomotor reactivity, and serum biochemistry were examined on D14. Furthermore, enzyme-linked immunosorbent assay and immunoblotting were performed. PCSK9i induced aorta relaxation ex vivo through a potassium channel-associated mechanism. PCSK9i significantly improved blood flow and preserved endothelial function without changes in cardiac function and serum lipid levels in rats with hyperglycemia. The levels of lectin-like oxidized low-density lipoprotein receptor-1, superoxide dismutase, cyclooxygenase-2, caspase-1, and interleukin-1β were significantly reduced in the treatment group. PCSK9i decreased the ratio of phosphorylated to total p38 mitogen-activated protein kinase and extracellular signal-regulated kinase in the aorta of rats with hyperglycemia. Short-term treatment with PCSK9i preserved endothelial function, induced vascular dilatation, and increased blood flow in the AC fistula of rats with hyperglycemia. The pleiotropic mechanisms were associated with the suppression of oxidative stress and tissue inflammation during hyperglycemia.

SIRT6 Inhibits the Proliferation and Collagen Synthesis of Keloid Fibroblasts through MAPK/ERK Pathway.

Keloid, a fibroproliferative disorder, significantly impacts patients' quality of life, yet effective therapies remain elusive. This study explored the role of silent information regulator 6 (SIRT6) in modulating the proliferation, invasion, and collagen synthesis of keloid fibroblasts. Keloid and normal skin specimens were collected, and fibroblasts were isolated from the keloid tissue. SIRT6 recombinant adenovirus (Ad) was constructed to infect keloid fibroblasts to overexpress SIRT6. This study entails three groups: Control group, adenovirus-Negative Control (Ad-NC) group, and Ad-SIRT6 group. SIRT6 protein and mRNA levels were measured via Western blotting and Quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Cell viability was determined using 5-ethynyl-2'-deoxyuridine (EdU) assay. Flow cytometry was exploited to measure cell apoptosis. To investigate cell migration, wound healing assay and Transwell assay were employed. Western blotting was also utilized to study the expression levels of apoptotic proteins, collagen deposition-related proteins, and Mitogen-Activated Protein Kinases (MAPK)/extracellular regulated protein kinases (ERK) pathway-related proteins. Compared to the control and Ad-NC groups, the Ad-SIRT6 group exhibited significantly elevated SIRT6 level; diminished cell proliferation, migration and invasion; reduced protein levels of α-smooth muscle actin (α-SMA), collagen I, collagen III, phospho SMAD Family Member 3 (p-Smad3), transforming growth factor-β 1 (TGF-β1), and MAPK/ERK pathway proteins (phospho extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), phospho MAP kinase-ERK kinase (p-MEK) and phospho-c-Raf (p-c-Raf)). Treatment with epidermal growth factor (EGF), an MAPK/ERK pathway agonists, reversed the inhibitory effect of SIRT6 on cell activity and inhibited apoptosis in keloid fibroblasts. SIRT6 overexpression in keloid fibroblasts attenuates proliferation, invasion, and collagen synthesis, while fostering apoptosis, likely through the suppression of MAPK/ERK pathway activity. This suggests a potential therapeutic target for keloid treatment.