Extracellular Signal-regulated Kinase Research Articles

Intranasal LAG3 antibody infusion induces a rapid antidepressant effect via the hippocampal ERK1/2-BDNF signaling pathway in chronically stressed mice

The decline of microglia in the dentate gyrus is a new phenomenon that may explain the pathogenesis of depression, and reversing this decline has an antidepressant effect. The development of strategies that restore the function of dentate gyrus microglia in under stressful conditions is becoming a new focus. Lymphocyte-activating gene-3 (LAG3) is an immune checkpoint expressed by immune cells including microglia. One of its functions is to suppress the expansion of immune cells. In a recent study, chronic systemic administration of a LAG3 antibody that readily penetrates the brain was reported to reverse chronic stress-induced hippocampal microglia decline and depression-like behaviors. We showed here that a single intranasal infusion of a LAG3 antibody (In-LAG3 Ab) reversed chronic unpredictable stress (CUS)-induced depression-like behaviors in a dose-dependent manner, which was accompanied by an increase in brain-derived neurotrophic factor (BDNF) in the dentate gyrus. Infusion of an anti-BDNF antibody into the dentate gyrus, construction of knock-in mice with the BDNF Val68Met allele, or treatment with the BDNF receptor antagonist K252a abolished the antidepressant effect of In-LAG3 Ab. Activation of extracellular signal-regulated kinase1/2 (ERK1/2) is required for the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and BDNF decrease in the dentate gyrus. Moreover, both inhibition and depletion of microglia prevented the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and impairment of ERK1/2-BDNF signaling in the dentate gyrus. These results suggest that In-LAG3 Ab exhibits an antidepressant effect through microglia-mediated activation of ERK1/2 and synthesis of BDNF in the dentate gyrus.

The inhibitory effect of chlorogenic acid on oxidative stress and apoptosis induced by PM2.5 in HaCaT keratinocytes.

Exposure to fine particulate matter with an aerodynamic diameter of less than 2.5 μm (PM2.5) can cause oxidative damage and apoptosis in the human skin. Chlorogenic acid (CGA) is a bioactive polyphenolic compound with antioxidant, antifungal, and antiviral properties. The objective of this study was to identify the ameliorating impact of CGA that might protect human HaCaT cells against PM2.5. CGA significantly scavenged the reactive oxygen species (ROS) generated by PM2.5, attenuated oxidative cellular/organelle damage, mitochondrial membrane depolarization, and suppressed cytochrome c release into the cytosol. The application of CGA led to a reduction in the expression levels of Bcl-2-associated X protein, caspase-9, and caspase-3, while simultaneously increasing the expression of B-cell lymphoma 2. In addition, CGA was able to reverse the decrease in cell viability caused by PM2.5 via the inhibition of extracellular signal-regulated kinase (ERK). This effect was further confirmed by the use of the mitogen-activated protein kinase kinase inhibitor, which acted upstream of ERK. In conclusion, CGA protected keratinocytes from mitochondrial damage and apoptosis via ameliorating PM2.5-induced oxidative stress and ERK activation.

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.

A behind-the-scenes role of BDNF in the survival and differentiation of spermatogonia.

Mouse spermatogenesis entails the maintenance and self-renewal of spermatogonial stem cells (SSCs), which require a complex web-like signaling network transduced by various cytokines. Although brain-derived neurotrophic factor (BDNF) is expressed in Sertoli cells in the testis, and its receptor tropomyosin receptor kinase B (TrkB) is expressed in the spermatogonial population containing SSCs, potential functions of BDNF for spermatogenesis have not been uncovered. Here, we generate BDNF conditional knockout mice and find that BDNF is dispensable for in vivo spermatogenesis and fertility. However, in vitro, we reveal that BDNF-deficient germline stem cells (GSCs) exhibit growth potential not only in the absence of glial cell line-derived neurotrophic factor (GDNF), a master regulator for GSC proliferation, but also in the absence of other factors, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin. GSCs grown without these factors are prone to differentiation, yet they maintain expression of promyelocytic leukemia zinc finger (Plzf), an undifferentiated spermatogonial marker. Inhibition of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and Src pathways all interfere with the growth of BDNF-deficient GSCs. Thus, our findings suggest a role for BDNF in maintaining the undifferentiated state of spermatogonia, particularly in situations where there is a shortage of growth factors.

Pro-differentiative, Pro-adhesive and Pro-migratory Activities of Isorhamnetin in MC3T3-E1 Osteoblasts via Activation of ERK-dependent BMP2-Smad Signaling.

Osteoporosis (OP) is an epidemic bone remodeling disorder of growing relevance with the aging population. Considering that isorhamnetin (ISO), a flavonoid derived from plant, has been newly reckoned as an active ingredient in treating OP, our paper was conducted to investigate the regulatory role and mechanism of ISO in OP. CCK-8 method detected cell activity. Alkaline phosphatase (ALP) assay kit, ALP staining and alizarin red S staining measured osteogenic differentiation. RT-qPCR and Western blot examined the expressions of osteoblast-related proteins. Wound healing and cell adhesion assays severally detected cell migration and adhesion. Also, Western blot tested the expressions of extracellular signal-regulated kinase (ERK) signaling-associated proteins. As illustrated, after MC3T3-E1 pre-osteoblasts were stimulated to differentiate to osteoblasts, ISO markedly promoted the differentiation, mineralization, migration and adhesion of MC3T3-E1 osteoblasts in a concentration-dependent manner. In addition, administration of ISO functioned as an activator of ERK-dependent BMP2-Smad signaling in MC3T3-E1 osteoblasts and pretreatment with ERK inhibitor PD98059 partially compensated the impacts of ISO on MC3T3-E1 osteoblasts differentiation, mineralization, migration as well as adhesion. To be summarized, ISO might activate ERK-dependent BMP2-Smad signaling to facilitate the differentiation, mineralization, migration and adhesion of MC3T3-E1 osteoblasts, suggesting the protective potential of ISO in OP.

A comprehensive view on the fisetin impact on colorectal cancer in animal models: Focusing on cellular and molecular mechanisms.

Flavonoids, including fisetin, have been linked to a reduced risk of colorectal cancer (CRC) and have potential therapeutic applications for the condition. Fisetin, a natural flavonoid found in various fruits and vegetables, has shown promise in managing CRC due to its diverse biological activities. It has been found to influence key cell signaling pathways related to inflammation, angiogenesis, apoptosis, and transcription factors. The results of this study demonstrate that fisetin induces colon cancer cell apoptosis through multiple mechanisms. It impacts the p53 pathway, leading to increased levels of p53 and decreased levels of murine double minute 2, contributing to apoptosis induction. Fisetin also triggers the release of important components in the apoptotic process, such as second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI and cytochrome c. Furthermore, fisetin inhibits the cyclooxygenase-2 and wingless-related integration site (Wnt)/epidermal growth factor receptor/nuclear factor kappa B signaling pathways, reducing Wnt target gene expression and hindering colony formation. It achieves this by regulating the activities of cyclin-dependent kinase 2 and cyclin-dependent kinase 4, reducing retinoblastoma protein phosphorylation, decreasing cyclin E levels, and increasing p21 levels, ultimately influencing E2 promoter binding factor 1 and cell division cycle 2 (CDC2) protein levels. Additionally, fisetin exhibits various effects on CRC cells, including inhibiting the phosphorylation of Y-box binding protein 1 and ribosomal S6 kinase, promoting the phosphorylation of extracellular signal-regulated kinase 1/2, and disrupting the repair process of DNA double-strand breaks. Moreover, fisetin serves as an adjunct therapy for the prevention and treatment of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA)-mutant CRC, resulting in a reduction in phosphatidylinositol-3 kinase (PI3K) expression, Ak strain transforming phosphorylation, mTOR activity, and downstream target proteins in CRC cells with a PIK3CA mutation. These findings highlight the multifaceted potential of fisetin in managing CRC and position it as a promising candidate for future therapy development.

Baicalin ameliorates angiotensin II-induced cardiac hypertrophy and mitogen-activated protein kinase signaling pathway activation: A target-based network pharmacology approach

Baicalin, a flavonoid glycoside from Scutellaria baicalensis Georgi., exerts anti-hypertensive effects. The present study aimed to assess the cardioprotective role of baicalin and explore its potential mechanisms. Network pharmacology analysis pointed out a total of 477 potential targets of baicalin were obtained from the PharmMapper and SwissTargetPrediction databases, while 11,280 targets were identified associating with hypertensive heart disease from GeneCards database. Based on the above 382 common targets, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed enrichment in the regulation of cardiac hypertrophy, cardiac contraction, cardiac relaxation, as well as the mitogen-activated protein kinase (MAPK) and other signaling pathways. Moreover, baicalin treatment exhibited the amelioration of increased cardiac index and pathological alterations in angiotensin II (Ang II)-infused C57BL/6 mice. Furthermore, baicalin treatment demonstrated a reduction in cell surface area and a down-regulation of hypertrophy markers (including atrial natriuretic peptide and brain natriuretic peptide) in vivo and in vitro. In addition, baicalin treatment led to a decrease in the expression of phosphorylated c-Jun N-terminal kinase (p-JNK)/JNK, phosphorylated p38 (p-p38)/p38, and phosphorylated extracellular signal-regulated kinase (p-ERK)/ERK in the cardiac tissues of Ang II-infused mice and Ang II-stimulated H9c2 cells. These findings highlight the cardioprotective effects of baicalin, as it alleviates hypertensive cardiac injury, cardiac hypertrophy, and the activation of the MAPK pathway.

Protective Effects and Mechanism of Heracleum moellendorffii Hance on Alcohol-Induced Cognitive Decline in Mice.

Chronic and continuous alcohol consumption increases the risk of cognitive decline and may lead to alcohol-related dementia. We investigated the potential of Heracleum moellendorffii Hance root extract (HME) for treating alcohol-related cognitive impairment. Behavioral tests evaluated the effects of HME on cognitive function and depression. Changes in hippocampus and liver tissues were evaluated by Western blotting and H&E staining. The group treated with HME 200 mg/kg showed a significant increase in spontaneous alternation in Y-maze and a decrease in immobility in a forced swimming test (FST) compared to the vehicle-treated group. These results suggest that HME can restore memory deficits and reverse depressive symptoms caused by chronic alcohol consumption. The HME-treated group also upregulated brain-derived neurotrophic factor (BDNF), phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated cAMP response element-binding protein (CREB) in the hippocampus. Additionally, it reduced lipid vacuolation in the liver and increased the expression of aldehyde dehydrogenase 1 (ADH1). The administration of HME improves cognitive impairment and reverses depressive symptoms due to alcohol consumption, restoring neural plasticity in the hippocampus and alcohol metabolism in the liver. These findings suggest that HME is a promising treatment for alcohol-related brain disorders. Molecular mechanisms underlying the therapeutic effects of HME and its active ingredients should be investigated further.

Hepatic BMAL1 and HIF1α regulate a time-dependent hypoxic response and prevent hepatopulmonary-like syndrome

The transcriptional response to hypoxia is temporally regulated, yet the molecular underpinnings and physiological implications are unknown. We examined the roles of hepatic Bmal1 and Hif1α in the circadian response to hypoxia in mice. We found that the majority of the transcriptional response to hypoxia is dependent on either Bmal1 or Hif1α, through shared and distinct roles that are daytime determined. We further show that hypoxia-inducible factor (HIF)1α accumulation upon hypoxia is temporally regulated and Bmal1 dependent. Unexpectedly, mice lacking both hepatic Bmal1 and Hif1α are hypoxemic and exhibit increased mortality upon hypoxic exposure in a daytime-dependent manner. These mice display mild liver dysfunction with pulmonary vasodilation likely due to extracellular signaling regulated kinase (ERK) activation, endothelial nitric oxide synthase, and nitric oxide accumulation in lungs, suggestive of hepatopulmonary syndrome. Our findings indicate that hepatic BMAL1 and HIF1α are key time-dependent regulators of the hypoxic response and can provide molecular insights into the pathophysiology of hepatopulmonary syndrome.

Ursolic Acid from Perilla frutescens (L.) Britt. Regulates Zymosan-Induced Inflammatory Signaling in Raw 264.7

Background: Inflammation is considered one of the hallmarks of inflammatory diseases. Ursolic acid (UA) may exert therapeutic effects; however, the anti-inflammatory effects of UA require further study. Objectives: This study evaluated the effects of UA from Perilla frutescens (L.) Britt. leaves on pro-inflammatory activity induced by zymosan. Methods: Ursolic acid was extracted from P.frutescens (L.) Britt. leaves using ethanol, and thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) were used for UA analysis. The effect of UA on zymosan-induced cytokine production was evaluated by enzyme-linked immunosorbent assay (ELISA). Western blotting detected phosphorylation of extracellular signal-regulated kinases (ERK) 1/2, p38, and p47phox in Raw 264.7 cells. Reactive oxygen species (ROS) were measured using a specific immunofluorescent dye. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activities were assayed using a luminometer. Results: Zymosan-induced tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12 p40 in macrophages were significantly inhibited by UA (30 μg/mL, P < 0.001). In addition, UA (30 μg/mL, P < 0.001) significantly inhibited zymosan-induced phosphorylation of ERK1/2, p38, p47phox, ROS, and NADPH oxidase in the cells. Importantly, blockade of Dectin-1 using laminarin, a pure β-glucan, markedly abrogated the UA-mediated inhibition of zymosan-induced production of inflammatory cytokines, ERK1/2, p38, ROS, and p47phox phosphorylation in macrophages. Conclusions: Collectively, these data indicate that UA regulates zymosan-induced inflammatory responses and suggest novel approaches for managing excessive inflammatory responses.

A bioactive composite scaffold enhances osteochondral repair by using thermosensitive chitosan hydrogel and endothelial lineage cell-derived chondrogenic cell

Articular cartilage regeneration is a major challenge in orthopedic medicine. Endothelial progenitor cells (EPCs) are a promising cell source for regenerative medicine applications. However, their roles and functions in cartilage regeneration are not well understood. Additionally, thermosensitive chitosan hydrogels have been widely used in tissue engineering, but further development of these hydrogels incorporating vascular lineage cells for cartilage repair is insufficient. Thus, this study aimed to characterize the ability of EPCs to undergo endothelial–mesenchymal stem cell transdifferentiation and chondrogenic differentiation and investigate the ability of chondrogenic EPC-seeded thermosensitive chitosan-graft-poly (N-isopropylacrylamide) (CEPC-CSPN) scaffolds to improve healing in a rabbit osteochondral defect (OCD) model. EPCs were isolated and endothelial-to-mesenchymal transition (EndMT) was induced by transforming growth factor-β1 (TGF-β1); these EPCs are subsequently termed transdifferentiated EPCs (tEPCs). The stem cell-like properties and chondrogenic potential of tEPCs were evaluated by a series of in vitro assays. Furthermore, the effect of CEPC-CSPN scaffolds on OCD repair was evaluated. Our in vitro results confirmed that treatment of EPC with TGF-β1 induced EndMT and the acquisition of stem cell-like properties, producing tEPCs. Upon inducing chondrogenic differentiation of tEPCs (CEPCs), the cells exhibited significantly enhanced chondrogenesis and chondrocyte surface markers after 25 days. The TGF-β1-induced differentiation of EPCs is mediated by both the TGF-β/Smad and extracellular signal-regulated kinase (Erk) pathways. The CEPC-CSPN scaffold reconstructed well-integrated translucent cartilage and repaired subchondral bone in vivo, exhibiting regenerative capacity. Collectively, our results suggest that the CEPC-CSPN scaffold induces OCD repair, representing a promising approach to articular cartilage regeneration.

Immunohistochemistry Screening of Different Tyrosine Kinase Receptors in Canine Solid Tumors-Part I: Proposal of a Receptor Panel to Predict Therapies.

The use of tyrosine kinase inhibitors (TKI) has been growing in veterinary oncology and in the past few years several TKI have been tested in dogs. However, different from human medicine, we lack strategies to select patients to be treated with each TKI. Therefore, this study aimed to screen different tumor subtypes regarding TKI target immunoexpression as a predictor strategy to personalize the canine cancer treatment. It included 18 prostatic carcinomas, 36 soft tissue sarcomas, 20 mammary gland tumors, 6 urothelial bladder carcinomas, and 7 tumors from the endocrine system. A total of 87 patients with paraffin blocks were used to perform immunohistochemistry (IHC) of human epidermal growth factor receptor 2 (HER-2), epidermal growth factor receptors 1 (EGFR1), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet derived growth factor receptor beta (PDGFR-β), c-KIT, and extracellular signal-regulated kinase 1/2 (ERK1/ERK2). The immunohistochemical screening revealed a heterogeneous protein expression among histological types with mesenchymal tumors showing the lowest expression level and carcinomas the highest expression. We have demonstrated by IHC screening that HER2, EGFR1, VEGFR-2, PDGFR-β and ERK1/ERK2 are commonly overexpressed in dogs with different carcinomas, and KIT expression is considered relatively low in the analyzed samples.

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.

ERK synchronizes embryonic cleavages in Drosophila.

Extracellular-signal-regulated kinase (ERK) signaling controls development and homeostasis and is genetically deregulated in human diseases, including neurocognitive disorders and cancers. Although the list of ERK functions is vast and steadily growing, the full spectrum of processes controlled by any specific ERK activation event remains unknown. Here, we show how ERK functions can be systematically identified using targeted perturbations and global readouts of ERK activation. Our experimental model is the Drosophila embryo, where ERK signaling at the embryonic poles has thus far only been associated with the transcriptional patterning of the future larva. Through a combination of live imaging and phosphoproteomics, we demonstrated that ERK activation at the poles is also critical for maintaining the speed and synchrony of embryonic cleavages. The presented approach to interrogating phosphorylation networks identifies a hidden function of a well-studied signaling event and sets the stage for similar studies in other organisms.

Vaccarin suppresses diabetic nephropathy through inhibiting the EGFR/ERK1/2 signaling pathway.

Diabetic nephropathy (DN) is recognized as one of the primary causes of chronic kidney disease and end-stage renal disease. Vaccarin (VAC) confers favorable effects on cardiovascular and metabolic diseases, including type 2 diabetes mellitus (T2DM). Nonetheless, the potential role and mechanism of VAC in the etiology of DN have yet to be completely elucidated. In this study, a classical mouse model of T2DM is experimentally induced via a high-fat diet (HFD)/streptozocin (STZ) regimen. Renal histological changes are assessed via H&E staining. Masson staining and immunohistochemistry (IHC) are employed to assess renal fibrosis. RT-PCR is utilized to quantify the mRNA levels of renal fibrosis, oxidative stress and inflammation markers. The levels of malondialdehyde (MDA) and reactive oxygen species (ROS), as well as the content of glutathione peroxidase (GSH-Px), are measured. The protein expressions of collagen I, TGF-β1, α-SMA, E-cadherin, Nrf2, catalase, SOD3, SOD2, SOD1, p-ERK, p-EGFR (Y845), p-EGFR (Y1173), p-NFκB P65, t-ERK, t-EGFR and t-NFκB P65 are detected by western blot analysis. Our results reveal that VAC has a beneficial effect on DN mice by improving renal function and mitigating histological damage. This is achieved through its inhibition of renal fibrosis, inflammatory cytokine overproduction, and ROS generation. Moreover, VAC treatment effectively suppresses the process of epithelial-mesenchymal transition (EMT), a crucial characteristic of renal fibrosis, in high glucose (HG)-induced HK-2 cells. Network pharmacology analysis and molecular docking identify epidermal growth factor receptor (EGFR) as a potential target for VAC. Amino acid site mutations reveal that Lys-879, Ile-918, and Ala-920 of EGFR may mediate the direct binding of VAC to EGFR. In support of these findings, VAC reduces the phosphorylation levels of both EGFR and its downstream mediator, extracellular signal-regulated kinase 1/2 (ERK1/2), in diabetic kidneys and HG-treated HK-2 cells. Notably, blocking either EGFR or ERK1/2 yields renal benefits similar to those observed with VAC treatment. Therefore, this study reveals that VAC attenuates renal damage via inactivation of the EGFR/ERK1/2 signaling axis in T2DM patients.

Integration of serum pharmacochemistry with network pharmacology to reveal the potential mechanism of Yangqing Chenfei formula for the treatment of silicosis.

To explore the mechanisms of Yangqing Chenfei formula (, YCF) in the treatment of silicosis through a comprehensive strategy consisting of serum pharmacochemistry, network pharmacology analysis, and in vitro validation. An ultrahigh-performance liquid chroma-tography-tandem mass spectrometry method was used to confirm the active components in YCF-medicated serum. Then, we obtained targets for active components and genes for silicosis from multiple databases. Furthermore, a protein-protein interaction network was constructed, and Kyoto Encyclopedia of Genes and Genomes pathway and biological process analyses were conducted to elucidate the mechanisms of YCF for the treatment of silicosis. Finally, we validated the important components and mechanisms in vitro. Altogether, 19 active components were identified from rat serum after YCF administration. We identified 724 targets for 19 components, which were mainly related to inflammation [phosphatidy linositol 3 kinase/protein kinase B, forkhead box O, hypoxia inducible factor, and T-cell receptor signaling pathway, nitric oxide biosynthetic process], fibrotic processes [vascular endothelial growth factor signaling pathway, extracellular signal regulated kinase (ERK) 1 and ERK2 cascade, smooth muscle cell proliferation], and apoptosis (negative regulation of apoptotic process). In addition, 218 genes for silicosis were identified and were mainly associated with the inflammatory response and immune process [cytokine?cytokine receptor interaction, tumor necrosis factor alpha (TNF-α), toll-like receptor, and nucleotide binding oligomerization domain-like receptor signaling pathway]. Taking an intersection of active component targets and silicosis genes, we obtained 61 common genes that were mainly related to the inflammatory response and apoptosis, such as the phosphatidylinositol-3-kinase/protein kinase B signaling pathway, mitogen activated protein kinases signaling pathway, TNF signaling pathway, toll-like receptor signaling pathway, biosynthesis of nitric oxide, and apoptotic process. In the herb-component-gene-pathway network, paeoniflorin, rutin and nobiletin targeted the most genes. In vitro, paeoniflorin, rutin and nobiletin decreased the mRNA levels of inflammatory factors [interleukin (IL)-6, TNF-α, and IL-1β], suppressed p-AKT and cleaved caspase-3, and increased B cell lymphoma (Bcl)-2 protein expression in silica-induced macrophages in a concentration-dependent manner. YCF could significantly relieve the inflammatory response of silicosis via suppression of the AKT/Bcl-2/Caspase-3 pathway.

Extracellular vesicles miR-31-5p promotes pancreatic cancer chemoresistance via regulating LATS2-Hippo pathway and promoting SPARC secretion from pancreatic stellate cells.

Pancreatic cancer remains one of the most lethal malignant diseases. Gemcitabine-based chemotherapy is still one of the first-line systemic treatments, but chemoresistance occurs in the majority of patients. Recently, accumulated evidence has demonstrated the role of the tumour microenvironment in promoting chemoresistance. In the tumour microenvironment, pancreatic stellate cells (PSCs) are among the main cellular components, and extracellular vesicles (EVs) are common mediators of cell‒cell communication. In this study, we showed that SP1-transcribed miR-31-5p not only targeted LATS2 in pancreatic cancer cells but also regulated the Hippo pathway in PSCs through EV transfer. Consequently, PSCs synthesized and secreted protein acidic and rich in cysteins (SPARC), which was preferentially expressed in stromal cells, stimulating Extracellular Signal regulated kinase (ERK) signalling in pancreatic cancer cells. Therefore, pancreatic cancer cell survival and chemoresistance were improved due to both the intrinsic Hippo pathway regulated by miR-31-5p and external SPARC-induced ERK signalling. In mouse models, miR-31-5p overexpression in pancreatic cancer cells promoted the chemoresistance of coinjected xenografts. In a tissue microarray, pancreatic cancer patients with higher miR-31-5p expression had shorter overall survival. Therefore, miR-31-5p regulates the Hippo pathway in multiple cell types within the tumour microenvironment via EVs, ultimately contributing to the chemoresistance of pancreatic cancer cells.

Exploring the mechanism of action of Vanda tessellata extract for the treatment of osteoarthritis through network pharmacology, molecular modelling and experimental assays

The present study employed a comprehensive approach of network pharmacology, molecular dynamic simulation and in-vitro assays to investigate the underlying mechanism of the anti-osteoarthritic potential of Vanda tessellata extract (VTE). Thirteen active compounds of VTE were retrieved from the literature and the IMPPAT database. All of these passed the drug likeness and oral bioavailability parameters. A total of 535 VTE targets and 2577 osteoarthritis related targets were obtained. The compound-target-disease network analysis revealed vanillin, daucosterol, gigantol and syringaldehyde as the core key components. Protein-protein interaction analysis revealed BCL2, FGF2, ICAM 1, MAPK1, MMP1, MMP2, MMP9, COX2, STAT3 and ESR1 as the hub genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed AGE-RAGE signalling pathway, HIF-1 signalling pathway and ESR signalling pathway as the major signalling pathway of VTE involved in treating osteoarthritis. Molecular docking analysis showed daucosterol and gigantol to have good binding affinity with BCL2, ESR1 and MMP9, and the results were further confirmed through molecular dynamics simulation analysis. The mechanism predicted by network pharmacology was validated in vitro on IL-1β-induced SW982 synovial cells. VTE did not show any cytotoxicity and inhibited the migration of SW982 cells. VTE inhibited the expression level of IL-6, IL-8, TNF-α, PGE-2, MMP-2 and MMP-9 in a dose-dependent manner. VTE inhibited nuclear translocation of NF- κβ and suppressed phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK) of the mitogen-activated protein kinase (MAPK) signalling pathway. The results showed that VTE exerted an anti-osteoarthritic effect by a multi-target, multi-component and multi-signalling pathway approach.

Solanum melongena L. Extract Promotes Intestinal Tight Junction Re-Assembly via SIRT-1-Dependent Mechanisms.

Tight junction disruption can lead to pathogenesis of various diseases without therapeutic strategy to recover intestinal barrier integrity. The main objective of this study is to demonstrate the effect of Solanum melongena L. extract (SMLE) on intestinal tight junction recovery and its underlying mechanism. Intestinal barrier function is attenuated by Ca2+ depletion. SMLE treatment increased TER value across T84 cell monolayers. Permeability assay reveals that Ca2+ depletion promotes 4-kDa FITC-dextran permeability, but not 70-kDa FITC-dextran. SMLE suppresses the rate of 4-kDa FITC-dextran permeability, indicating that SMLE inhibits paracellular leak pathway permeability. SMLE-mediated TER increase and leak pathway suppression are abolished by neither calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor nor AMP-activated protein kinase (AMPK) inhibitor. Furthermore, mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) inhibitors have no effects on SMLE-mediated TER increase and leak pathway suppression. Interestingly, SMLE is unable to enhance TER value and diminish leak pathway permeability in T84 cell monolayers pre-treated with sirtuin-1 (SIRT-1) inhibitor. Immunofluorescence staining reveals that SMLE enhances re-assembly of tight junction proteins, including occludin and ZO-1 to intercellular space but this effect is abolished by SIRT-1 inhibitor. These data suggest that SMLE promotes intestinal tight junction re-assembly via SIRT-1-dependent manner.