ERK Signaling Pathway Research Articles

Revealing the improvement of diabetes by Si Wei Jiang Huang Tang San through ERK/HIF1α signaling pathway via network pharmacology

Ethnopharmacological relevanceSi Wei Jiang Huang Tang San (SWJHTS) is a traditional Tibetan medicine prescription for the treatment of urethritis, frequent urination, and urgency, composed of four traditional Chinese medicines: Curcumae longae rhizoma, Berberidis cortex, Tribuli fructus, and Phyllanthi fructus. However, whether SWJHTS exhibits hypoglycemic efficacy and its specific mechanism remain unclear. Aim of the studyIn this study, we aimed to investigate the anti-diabetic effects of SWJHTS and elucidate the underlying mechanism. Materials and methodsHPLC-MS method was used to identify the key components of four kinds of traditional Chinese medicine (Curcumae longae rhizoma, Berberidis cortex., Tribuli fructus, and Phyllanthi fructus) which composed SWJHTS and determine their structure. Normal mice and 145 mg/kg STZ-induced type 1 diabetic mice were treated with three doses of SWJTHS by oral gavage. Body weight, 24h food and water intake, fasting blood glucose, glucose tolerance and other indicators were measured to evaluate the hypoglycemic effect of SWJHTS. OMIM, Genecards and other databases were used to collect targets of diabetes, and HPLC-MS results and TCMSP database information were used to collect drug component targets. Bioinformatics methods such as pathway enrichment analysis and molecular docking were used to predict the key targets of SWJHTS. The gene and protein expressions of HIF1α and ERK signaling pathways in HepG2 cells treated with SWJHTS were detected by RT-PCR and Western blot. ResultsA total of 181 components were identified, including curcumin, palmatine, and berberine, etc. The in vivo studies showed that SWJHTS could significantly lower fasting blood glucose levels and improve the symptoms of polydipsia, polyphagia, and polyuria in diabetic mice. Furthermore, we identified HIF1α as the potential key target of SWJHTS against diabetes utilizing network pharmacology approach and in silico molecular docking. Subsequently, we experimentally confirmed that SWJHTS could suppress the high glucose-induced upregulation of HIF1α expression, which mediated the glucose consumption in HepG2 cells. The ERK signaling pathway was further found to be activated by the SWJHTS as the upstream of HIF1α. ConclusionsSWJHTS can improve glucose metabolism by targeting the ERK/HIF1α signaling pathway; hence might be a prospective anti-diabetic drug for diabetic patients as traditional Tibetan medicine.

Mycobacterium tuberculosis Mce2D protein blocks M1 polarization in macrophages by inhibiting the ERK signaling pathway

Macrophages play a pivotal role in controlling Mycobacterium infection, and the pathogen thrives in the event of immune evasion and immunosuppression of macrophages. Mammalian cell entry proteins (Mce) are required for Mycobacterium tuberculosis (M. tb) growth and the host cell's initial phagocytosis and cytokine response. Mce2D protein is one of a family of proteins that infect M. tb; however, the function and mechanism of action remain unclear. In this study, we constructed the Mce2D knockout strain using Mycobacterium smegmatis to study the function of Mce2D in the infection of macrophages. The results indicated that compared to the knockout strain, the release of proinflammatory cytokines (TNF-α and IL-1β) reduced when WT strain infected the macrophages. Moreover, Mce2D boosted the metabolism of oxidized fatty acids, increased the energy supply of TCA, and lowered the glycolysis of glucose in macrophages after bacterial infection, all of which prevented the polarization of macrophages to M1, which was driven by the fact that Mce2D blocked ERK2 phosphorylation by interacting with ERK2 through its DEF motif. This, in turn, promoted nuclear translocation of HIF-1α, allowing signal accumulation, which increased the HIF-1α transcription levels. Finally, the mouse infection experiment showed that Mce2D caused blockage of M1 polarization of alveolar macrophages, resulting in reduced bactericidal activity and antigen presentation, weakening Th1 cell-mediated immune response and helping bacteria escape the immune system. Our results reveal that Mce2D causes immune escape by blocking M1 polarization in macrophages, providing potential targets for the rational design of therapies against M. tb infection.

Efficient improvement of the proliferation, differentiation, and anti-arthritic capacity of mesenchymal stem cells by simply culturing on the immobilized FGF2 derived peptide, 44-ERGVVSIKGV-53

IntroductionThe stem cell microenvironment has been evidenced to robustly affect its biological functions and clinical grade. Natural or synthetic growth factors, especially, are essential for modulating stem cell proliferation, metabolism, and differentiation via the interaction with specific extracellular receptors. Fibroblast growth factor-2 (FGF-2) possesses pleiotropic functions in various tissues and organs. It interacts with the FGF receptor (FGFR) and activates FGFR signaling pathways, which involve numerous biological functions, such as angiogenesis, wound healing, cell proliferation, and differentiation. ObjectivesHere, we aim to explore the molecular functions, mode of action, and therapeutic activity of yet undetermined function, FGF-2-derived peptide, FP2 (44-ERGVVSIKGV-53) in promoting the proliferation, differentiation, and therapeutic application of human Wharton’s jelly mesenchymal stem cells (hWJ-MSCs) in comparison to other test peptides, canofin1 (FP1), hexafin2 (FP3), and canofin3 (FP4) with known functions. MethodsThe immobilization of test peptides that are fused with mussel adhesive proteins (MAP) on the culture plate was carried out via EDC/NHS chemistry. Cell Proliferation assay, colony-forming unit, western blotting analysis, gene expression analysis, RNA-Seq. analysis, osteogenic, and chondrogenic differentiation capacity were applied to test the activity of the test peptides. We additionally utilized three-dimensional (3D) structural analysis and artificial intelligence (AI)-based AlphaFold2 and CABS-dock programs for receptor interaction prediction of the peptide receptor. We also verified the in vivo therapeutic capacity of FP2-cultured hWJ-MSCs using an osteoarthritis mice model. ResultsCulture of hWJ-MSC onto an FP2-immobilized culture plate showed a significant increase in cell proliferation (n = 3; *p < 0.05, **p < 0.01) and the colony-forming unit (n = 3; *p < 0.05, **p < 0.01) compared with the test peptides. FP2 showed a significantly upregulated phosphorylation of FRS2α and FGFR1 and activated the AKT and ERK signaling pathways (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Interestingly, we detected efficient FP2 receptor binding that was predicted using AI-based tools. Treatment with an AKT inhibitor significantly abrogated the FP2-mediated enhancement of cell differentiation (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). Intra-articular injection of FP2-cultured MSCs significantly mitigated arthritis symptoms in an osteoarthritis mouse model, as shown through the functional tests (n = 10; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), modulation of the expression level of the pro-inflammatory and anti-inflammatory genes, and improved osteochondral regeneration as demonstrated by tissue sections. ConclusionOur study identified the FGF-2-derived peptide FP2 as a promising candidate peptide to improve the therapeutic potential of hWJ-MSCs, especially in bone and cartilage regeneration.

Biomimetic nanoparticles loaded lutein functionalized by macrophage membrane for targeted amelioration pressure overload-induced cardiac fibrosis

Lutein is a strong antioxidant with anti-inflammatory, anti-oxidative and cardioprotective effects and could be a promising candidate for the treatment of hypertensive heart disease (HHD), but is not clinically appealing because of its low oral bioavailability and main distribution in the eyes. To address this, a biomimetic drug delivery system-MMLNPs was established by coating macrophage membranes (MMs) onto lutein-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (LNPs). This study characterized the physical properties of biomimetic nanoparticles and examined the targeting capability, therapeutic effects and mechanism, and biosecurity of administering them for cardiac fibrosis therapy in the transverse aortic constriction (TAC) model and in vitro. Transmission electron microscope mapping and dynamic light scattering analysis proved that MMLNPs were spherical nanoparticles camouflaged by a layer of cell membrane and had negative zeta potential. Confocal laser scanning microscopy and flow cytometry analysis showed that MMs on the biomimetic nanoparticles hindered the phagocytosis of macrophages and facilitated the targeting of activated endothelial cells. Ex vivo fluorescence imaging experiments demonstrated the targeting of biomimetic nanoparticles to the injured heart. EdU assay indicated that MMLNPs have the same potential to inhibit angiotensin (Ang) II-induced cardiac fibroblast proliferation as free lutein. Furthermore, echocardiography showed that MMLNPs improved cardiac function and structure, and Masson staining and western blotting showed that MMLNPs ameliorated cardiac fibrosis. We found MMLNPs inhibited the interleukin (IL)-11/ERK signaling pathway which was up-regulated in the TAC model compared to the sham-operated mouse. Biochemical testing and hematoxylin and eosin staining proved that the long-term use of MMLNPs lacked biological toxicity. Collectively, MMLNPs might be a promising nanodrug delivery approach to attenuate pressure overload (PO)-induced cardiac fibrosis.

Proteomic Profiling Identifies Distinct Regulation of Proteins in Obese Diabetic Patients Treated with Metformin.

Background: Obesity and type 2 diabetes mellitus (T2DM) are characterized by underlying low-grade chronic inflammation. Metformin has been used as the first line of therapy in T2DM as it decreases hepatic glucose production and glucose intestinal absorption, enhances insulin sensitivity and weight loss, and is known to ameliorate inflammation. The mechanisms through which metformin exerts its effect remain unclear. Proteomics has emerged as a unique approach to explore the biological changes associated with diseases, including T2DM. It provides insight into the circulating biomarkers/mediators which could be utilized for disease screening, diagnosis, and prognosis. Methods: This study evaluated the proteomic changes in obese (Ob), obese diabetics (OD), and obese diabetic patients on metformin (ODM) using a 2D DIGE MALDI-TOF mass spectrometric approach. Results: Significant changes in sixteen plasma proteins (15 up and 1 down, ANOVA, p ≤ 0.05; fold change ≥ 1.5) were observed in the ODM group when compared to the Ob and OD groups. Bioinformatic network pathway analysis revealed that the majority of these altered plasma proteins are involved in distinct pathways involving acute-phase response, inflammation, and oxidative response and were centered around HNF4A, ERK, JNK, and insulin signaling pathways. Conclusions: Our study provides important information about the possible biomarkers altered by metformin treatment in obese patients with and without T2DM. These altered plasma proteins are involved in distinct pathways involving acute-phase response, inflammation, and oxidative response and were centered around HNF4A, ERK, JNK, and insulin signaling pathways. The presented proteomic profiling approach may help in identifying potential biomarkers/mediators affected by metformin treatment in T2DM and inform the understanding of metformin's mechanisms of action.

Polyphyllins in cancer therapy: A systematic review and meta-analysis of animal studies

BackgroundPolyphyllins are secondary metabolites that inhibit the growth of various tumours; however, clinical trials on their use are lacking. Hypothesis/purposeIn this study, we aimed to evaluate the antitumour efficacy of polyphyllins in animal models. Study designSystematic review and meta-analysis. MethodsElectronic bibliographic databases including PubMed, Web of Science, China Science and Technology Journal Database, Wanfang Data, and China National Knowledge Infrastructure were searched for relevant articles. The Systematic Review Centre for Laboratory Animal Experimentation's Risk of Bias tool was used to assess methodological quality. RevMan V.5.4 (Cochrane) and Stata MP 17 software were used to perform a meta-analysis. ResultsThirty articles were analysed including 33 independent experiments and 452 animals in this paper. Overall, tumour volume (standardised mean difference [SMD]: −3.35; 95 % confidence interval [CI]: −4.27 to −2.43; p < 0.00001) and tumour weight (SMD: −3.79; 95% CI: −4.75 to −2.82; p < 0.00001) were reduced by polyphyllins, which showed a good cancer therapeutic effect; mouse weight (SMD: −0.22; 95% CI: −0.61 to −0.18; p = 0.28) was insignificantly different, which indicated that polyphyllins did not affect the growth of the mice within the test range. Moreover, the molecular mechanisms of the antitumour activity of polyphyllins were explained, including the P53, NF-kB, AMPK, and ERK signalling pathways. ConclusionPolyphyllins inhibit the growth of cancers within the experimental dose. However, due to heterogeneity of the results of the included studies, more studies are needed to support this conclusion.

Cholinergic signaling of muscarinic receptors directly involves in the neuroprotection of muscone by inducing Ca2+ antagonism and maintaining mitochondrial function

Ethnopharmacological relevanceMusk, a traditional Chinese medicine, is broadly used in inducing resuscitation and refreshing the mind, activating blood and alleviating pain. It is commonly used for the treatment of ischemic stroke, and muscone is its core medicinal component. Aim of the studyThe aim of this study was to explore whether muscone ameliorates neuronal damage through cholinergic signaling of muscarinic receptors. Materials and methodsThe effects of muscone were tested in a rat model of middle cerebral artery occlusion (MCAO) as well as injured neurons induced by oxygen-glucose deprivation (OGD) in PC12 cells. Cell counting kit 8 (CCK8) assay was used to measure the cell viability, and the production of lactate dehydrogenase (LDH) and adenosine-triphosphate (ATP) were examined by kit. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), tetramethylrhodamine ethyl ester (TMRE) and Fluo-4 acetoxymethyl ester (Fluo-4 AM) staining were used to demonstrate effect of muscone on the reactive oxygen species (ROS) level, mitochondria membrane potential (MMP) and intracellular Ca2+ measurement in cells respectively, in which all of those staining was visualized by laser confocal microscope. For in vivo experiments, rats' cerebral blood flow was measured using laser Doppler blood flowmetry to evaluate the MCAO model, and a modified neurological severity score (mNSS) was used to assess the recovery of neurological function. Calculate infarct rate was measured by 2,3,5-Triphenyl Tetrazolium Chloride (TTC) staining. Except DCFH-DA and Fluo-4 AM staining, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolylcarbocyanine iodide (JC-1) staining was used to observe intracellular Ca2+ measurement in brain cells. Protein levels in cells and tissues were detected by Western blot. ResultsPretreatment with muscone significantly improved the cell viability, lactic acid production, mitochondrial membrane potential collapse and function, Ca2+ overload, ROS generation, and cell apoptosis in OGD PC12 cells. Muscone also regulated PI3K, ERK and AKT signal pathways by activating cholinergic signaling of muscarinic receptors in PC12 cells induced with OGD. More importantly, the blocking of cholinergic signaling of muscarinic receptors by atropine significantly reduces the neuroprotective effects of muscone, including the cell viability, Ca2+ efflux, and mitochondrial repair. Furthermore, muscone was found to effectively alleviate mitochondrial dysfunction and elevated levels of ROS induced by the MCAO in the brain tissue. Notably, this beneficial effect of muscone was attenuated by atropine but not by (+)-Sparteine. ConclusionsOur study indicates that muscone exerts its neuroprotective effects by activating muscarinic receptors of cholinergic signaling, thus providing a promising therapeutic target for the treatment of OGD-induced nerve injury in stroke. The findings suggest that these treatments may hold potential benefits for stroke patients.

Suppression of TBCK enhances TRAIL-mediated apoptosis by causing the inactivation of the akt signaling pathway in human renal carcinoma Caki-1 cells.

TBC1 domain-containing kinase (TBCK) protein functions as a growth suppressor in certain cell types and as a tumor promoter in others. Although TBCK knockdown increases the responsiveness of cancer cells to anticancer drugs, the detailed mechanisms by which TBCK knockdown increases susceptibility to anticancer drugs remain unknown. This study analyzed the role of TBCK in sensitivities to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and doxorubicin in human renal cancer cells. Flow cytometry was employed to evaluate the extent of apoptosis. Western blotting, transient transfection, and lentiviral infection techniques were conducted to investigate the impact of TBCK on apoptosis-related protein expression and mitogen-activated protein kinase (MAPK). TBCK knockdown in renal cancer cells inhibits ERK and Akt signaling pathways and increases TRAIL and doxorubicin sensitivity. In TBCK-knockdown Caki-1 cells, ERK and Akt phosphorylation was suppressed compared to control cell lines, and TRAIL and doxorubicin sensitivities were increased in these cells. In addition, the phosphorylation of PDK1 was suppressed in TBCK-suppressed cells, indicating that TBCK may be involved in the PDK1 and Akt signaling pathways. The introduction of dominantly active Akt into TBCK-suppressed cells restored their sensitivity to TRAIL. In addition, TBCK downregulation enhanced TRAIL sensitivity in different renal cancer cell lines. These data suggest that TBCK could potentially have a crucial function in influencing the effects of anti-cancer drugs including TRAIL by modulating the signaling pathway involving Akt and PDK1 in human renal cancer cells.

Hypoxia improves self-renew and migration of urine-derived stem cells by upregulating autophagy and mitochondrial function through ERK signal pathway

Urine-derived stem cells (USCs) are autologous stem cells with self-renewal ability and multi-lineage differentiation potential. Our previous studies have shown that hypoxia preconditioning can improve self-renewal and migration abilities of USCs by up-regulating autophagy. The purpose of this study was to investigate the specific mechanism by which hypoxia treatment promotes the biological function of USCs. We found that hypoxia treatment upregulated the expression of phosphralated ERK protein without affecting the expression of total ERK protein. Inhibiting ERK signaling with the PD98059 inhibitor decreased cell proliferation, migration and colony formation during hypoxia treatment. Hypoxia increased ATP production, mitochondrial membrane potential and mt-DNA copy number, which were reversed by inhibiting the ERK signal. Additionally, the number of autophagosomes and autophagic lysosomes was significantly lower in PD98059 group than in the hypoxia group. PD98059 treatment inhibited the up-regulation of autophagy related proteins induced by hypoxia. Therefore, this study suggests that hypoxia improves the self-renewal and migration abilities of USCs by upregulating autophagy and mitochondrial function through ERK signaling pathway. This finding may provide a new therapeutic mechanism for hypoxia pretreated USCs as a source of stem cell transplantation.

GRK2 expression and catalytic activity are essential for vasoconstrictor/ERK-stimulated arterial smooth muscle proliferation

Prolonged vasoconstrictor signalling found in hypertension, increases arterial contraction, and alters vessel architecture by stimulating arterial smooth muscle cell (ASMC) growth, underpinning the development of re-stenosis lesions and vascular remodelling. Vasoconstrictors interact with their cognate G protein coupled receptors activating a variety of signalling pathways to promote smooth muscle proliferation. Here, angiotensin II (AngII) and endothelin 1 (ET1), but not UTP stimulates ASMC proliferation. Moreover, siRNA-mediated depletion of endogenous GRK2 expression, or GRK2 inhibitors, compound 101 or paroxetine, prevented AngII and ET1-promoted ASMC growth. Depletion of GRK2 expression or inhibition of GRK2 activity ablated the prolonged phase of AngII and ET-stimulated ERK signalling, while enhancing and prolonging UTP-stimulated ERK signalling. Increased GRK2 expression enhanced and prolonged AngII and ET1-stimulated ERK signalling, but suppressed UTP-stimulated ERK signalling. In ASMC prepared from 6-week-old WKY and SHR, AngII and ET1-stimulated proliferation rates were similar, however, in cultures prepared from 12-week-old rats AngII and ET1-stimulated growth was enhanced in SHR-derived ASMC, which was reversed following depletion of GRK2 expression. Furthermore, in ASMC cultures isolated from 6-week-old WKY and SHR rats, AngII and ET1-stimulated ERK signals were similar, while in cultures from 12-week-old rats ERK signals were both enhanced and prolonged in SHR-derived ASMC, and were reversed to those seen in age-matched WKY-derived ASMC following pre-treatment of SHR-derived ASMC with compound 101. These data indicate that the presence of GRK2 and its catalytic activity are essential to enable pro-proliferative vasoconstrictors to promote growth via recruitment and activation of the ERK signalling pathway in ASMC.

BM-MSCs display altered gene expression profiles in B-cell acute lymphoblastic leukemia niches and exert pro-proliferative effects via overexpression of IFI6

BackgroundThe tumor microenvironment (TME) is a supportive environment responsible for promoting the growth and proliferation of tumor cells. Current studies have revealed that the bone marrow mesenchymal stem cells (BM-MSCs), a type of crucial stromal cells in the TME, can promote the malignant progression of tumors. However, in the adult B-cell acute lymphoblastic leukemia (B-ALL) microenvironment, it is still uncertain what changes in BM-MSCs are induced by leukemia cells.MethodsIn this study, we mimicked the leukemia microenvironment by constructing a BM-MSC–leukemia cell co-culture system. In vitro cell experiments, in vivo mouse model experiments, lentiviral transfection and transcriptome sequencing analysis were used to investigate the possible change of BM-MSCs in the leukemia niche and the potential factors in BM-MSCs that promote the progression of leukemia.ResultsIn the leukemia niche, the leukemia cells reduced the MSCs' capacity to differentiate towards adipogenic and osteogenic subtypes, which also promoted the senescence and cell cycle arrest of the MSCs. Meanwhile, compared to the mono-cultured MSCs, the gene expression profiles of MSCs in the leukemia niche changed significantly. These differential genes were enriched for cell cycle, cell differentiation, DNA replication, as well as some tumor-promoting biofunctions including protein phosphorylation, cell migration and angiogenesis. Further, interferon alpha-inducible protein 6 (IFI6), as a gene activated by interferon, was highly expressed in leukemia niche MSCs. The leukemia cell multiplication was facilitated evidently by IFI6 both in vitro and in vivo. Mechanistically, IFI6 might promote leukemia cell proliferation by stimulating SDF-1/CXCR4 axis, which leads to the initiation of downstream ERK signaling pathway. As suggested by further RNA sequencing analysis, the high IFI6 level in MSCs somewhat influenced the gene expression profile and biological functions of leukemia cells.ConclusionsBM-MSCs in the leukemia niche have varying degrees of changes in biological characteristics and gene expression profiles. Overexpression of IFI6 in BM-MSCs could be a key factor in promoting the proliferation of B-ALL cells, and this effect might be exerted through the SDF-1/CXCR4/ERK signal stimulation. Targeting IFI6 or related signaling pathways might be an important measure to reduce the leukemia cell proliferation.

Glucosamine-loaded injectable hydrogel promotes autophagy and inhibits apoptosis after cartilage injury

The ability of cartilage to regenerate and repair is limited. N-acetyl- d-glucosamine (GlcNAc) is a nutritional supplement commonly used to activate chondrocytes. To prolong the duration of action of GlcNAc and improve its curative effect after cartilage injury, a GlcNAc thermosensitive hydrogel is prepared based on Pluronic F127 (PF127). The physicochemical properties results indicate that this hydrogel is injectable and retards the release of GlcNAc. Further, the therapeutic benefits of GlcNAc hydrogel are detected through intra-articular injection in rat specimens with cartilage injury. Behavioral experiments results indicate that the rats treated with GlcNAc hydrogel had longer step lengths, smaller foot angles and slower fall times. Compared with the sham group, the expression of Sox9 was 1.5 times and the level of collagen II was 2.4 times in the hydrogel treated group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining result confirmed that the GlcNAc hydrogel reduce apoptosis by about 50%. Our results of immunohistochemical staining, Western blotting assays and enzyme activity detection all suggested that GlcNAc hydrogel reduce the expression of cleaved-caspase3 and caspase8 (Compared to the sham group, the protein contents were reduced by about 50% in the GlcNAc hydrogel group). We also found that GlcNAc hydrogel activates autophagy through ERK signal pathway. The results of Western blotting indicated that GlcNAc hydrogel increase the levels of LC3B and Becline1 (hydrogel group & sham group, LC3B: 1.56 ± 0.07 & 1.00 ± 0.14; Becline1: 1.98 ± 0.07 & 1.00 ± 0.13). Whereas, the content of P62 reduced after GlcNAc hydrogel treatment, the relative level in sham group and hydrogel group are 1.00 ± 0.02 and 0.73 ± 0.06. Our results revealed that the number of P-ERK positive cells in the hydrogel group (57.36 ± 3.56%) was higher when compared with the sham (24.82 ± 2.72%). And, the ratio of P-ERK and ERK was higher than that in the sham group (1.48 ± 0.07 & 1.00 ± 0.08). The GlcNAc thermosensitive hydrogel is a promising and sustainable drug delivery system for intra-articular injection in the treatment of cartilage injury.

UGCG modulates heart hypertrophy through B4GalT5-mediated mitochondrial oxidative stress and the ERK signaling pathway

Mechanical pressure overload and other stimuli often contribute to heart hypertrophy, a significant factor in the induction of heart failure. The UDP-glucose ceramide glycosyltransferase (UGCG) enzyme plays a crucial role in the metabolism of sphingolipids through the production of glucosylceramide. However, its role in heart hypertrophy remains unknown. In this study, UGCG was induced in response to pressure overload in vivo and phenylephrine stimulation in vitro. Additionally, UGCG downregulation ameliorated cardiomyocyte hypertrophy, improved cardiomyocyte mitochondrial oxidative stress, and reduced the ERK signaling pathway. Conversely, UGCG overexpression in cardiomyocytes promoted heart hypertrophy development, aggravated mitochondrial oxidative stress, and stimulated ERK signaling. Furthermore, the interaction between beta-1,4-galactosyltransferase 5 (B4GalT5), which catalyses the synthesis of lactosylceramide, and UGCG was identified, which also functions as a synergistic molecule of UGCG. Notably, limiting the expression of B4GalT5 impaired the capacity of UGCG to promote myocardial hypertrophy, suggesting that B4GalT5 acts as an intermediary for UGCG. Overall, this study highlights the potential of UGCG as a modulator of heart hypertrophy, rendering it a potential target for combating heart hypertrophy.

Osteogenic induction of asiatic acid derivatives in human periodontal ligament stem cells

Asiatic acid (AA) and asiaticoside, pentacyclic triterpenoid compounds derived from Centella asiatica, are known for their biological effects in promoting type I collagen synthesis and inducing osteogenesis of stem cells. However, their applications in regenerative medicine are limited due to their low potency and poor aqueous solubility. This work aimed to evaluate the osteogenic induction activity of AA derivatives in human periodontal ligament stem cells (hPDLSCs) in vitro. Four compounds were synthesised, namely 501, 502, 503, and 506. AA was used as the control. The 502 exhibited low water solubility, while the 506 compound showed the highest. The cytotoxicity analysis demonstrated that 503 caused significant deterioration in cell viability, while other derivatives showed no harmful effect on hPDLSCs. The dimethyl aminopropyl amine derivative of AA, compound 506, demonstrated a relatively high potency in inducing osteogenic differentiation. An elevated mRNA expression of osteogenic-related genes, BMP2, WNT3A, ALP, OSX and IBSP was observed with 506. Additionally, the expression of BMP-2 protein was enhanced with increasing dose of 506, and the effect was pronounced when the Erk signalling molecule was inhibited. The 506 derivative was proposed for the promotion of osteogenic differentiation in hPDLSCs by upregulating BMP2 via the Erk signalling pathway. The 506 molecule showed promise in bone tissue regeneration.

PGF2alpha Inhibits 20alpha-HSD Expression by Suppressing CK1alpha-induced ERK and SP1 Activation in the Corpus Luteum of Pregnant Mice.

Prostaglandin F2α (PGF2α) is a luteolytic hormone that promotes parturition in mammals at the end of pregnancy by reducing progesterone secretion from the corpus luteum (CL). In rodents and primates, PGF2α rapidly converts progesterone to 20α-hydroxyprogesterone (20α-OHP) by promoting 20α-hydroxysteroid dehydrogenase (20α-HSD) expression. However, the specific mechanism of 20α-HSD regulation by PGF2α remains unclear. Casein Kinase 1α (CK1α) is a CK1 family member that regulates a variety of physiological functions, including reproductive development. Here, we investigated the effects of CK1α on pregnancy in female mice. Our experiments showed that CK1α is expressed in mouse CL, and its inhibition enhanced progesterone metabolism, decreased progesterone levels, and affected mouse embryo implantation. Further, CK1α mediated the effect of PGF2α on 20α-HSD in mouse luteal cells in vitro. Our results are the first to show that CK1α affects the 20α-HSD mRNA level by affecting the ERK signalling pathway to regulate the expression of the transcription factor SP1. These findings improve our understanding of PGF2α regulation of 20α-HSD.

Melatonin enhances the ability of M2 macrophages to secrete IL10 by inhibiting Erk5 signaling pathway

BackgroundMelatonin plays a role in repairing damaged cartilage and regulating immune cells. The anti-inflammatory effect of Melatonin involves multiple pathways and molecular activation, which directly or indirectly inhibits inflammatory reaction. M2 macrophages have the ability to anti-inflammatory response and repair damaged tissues, secrete IL10 and IL-4, and participate in tissue repair and remodeling. Erk5 is a recently discovered member of the MAPK family and one of the least studied members. It plays an important role in cell differentiation, proliferation, secretion and other functions. This experiment aims to study how Melatonin affects M2 Macrophage polarization and secretion through ERK5 signaling pathway. MethodsThe RAW 264.7 macrophages were used for cell culture. The cells were cultured according to the pre-experimental results. The effects of Melatonin on M2 macrophages were comprehensively evaluated by CCK8 activity detection, RT-PCR, ELISA, cellular immunofluorescence, and WB.SD mice were selected to evaluate the effect of Melatonin on cartilage damage in rats with knee Osteoarthritis through HE staining, immunohistochemistry and immunofluorescence. ResultsMelatonin cultivates RAW 264.7 macrophages. Without affecting the polarization ratio of M2 Macrophage polarization, Melatonin may reduce Erk5 gene expression, reduce Erk5 and p-Erk5 protein synthesis, and cooperate with BIX 02189 to enhance the secretion function of existing M2 macrophages and increase the secretion of cytokines IL10. Immunohistochemistry of rat knee Osteoarthritis model confirmed that the expression of IL10 was up-regulated and the synthesis of type II collagen was enhanced, but immunofluorescence found that the polarization of M2 Macrophage polarization in subchondral bone was not obvious. ConclusionMelatonin enhances the ability of M2 macrophages to secrete IL10 by inhibiting Erk5 signaling pathway, but has no effect on M2 Macrophage polarization.

Dysfunctional endocannabinoid CB1 receptor expression and signaling contribute to skeletal muscle cell toxicity induced by simvastatin

Statins are the most prescribed lipid-lowering agents worldwide. Their use is generally safe, although muscular toxicity occurs in about 1 in 10.000 patients. In this study, we explored the role of the endocannabinoid system (ECS) during muscle toxicity induced by simvastatin. In murine C2C12 myoblasts exposed to simvastatin, levels of the endocannabinoids AEA and 2-AG as well the expression of specific miRNAs (in particular miR-152) targeting the endocannabinoid CB1 gene were increased in a time-dependent manner. Rimonabant, a selective CB1 antagonist, exacerbated simvastatin-induced toxicity in myoblasts, while only a weak opposite effect was observed with ACEA and GAT211, selective orthosteric and allosteric agonists of CB1 receptor, respectively. In antagomiR152-transfected myoblasts, simvastatin toxicity was in part prevented together with the functional rescue of CB1. Further analyses revealed that simvastatin in C2C12 cells also suppresses PKC and ERK signaling pathways, which are instead activated downstream of CB1 receptor stimulation, thus adding more insight into the mechanism causing CB1 functional inactivation. Importantly, simvastatin induced similar alterations in skeletal muscles of C57BL/6 J mice and primary human myoblasts. In sum, we identified the dysregulated expression of the endocannabinoid CB1 receptor as well as the impairment of its downstream signaling pathways as a novel pathological mechanism involved in statin-induced myopathy.

Fucoxanthin, a marine derived carotenoid, attenuates surgery-induced cognitive impairments via activating Akt and ERK pathways in aged mice

BackgroundFucoxanthin is the most abundant marine carotenoid derived from brown seaweeds, possesses antioxidant, anti-inflammatory, and neuroprotective properties, and might be benefit for the treatment of neurological disorders. Post-operative cognitive dysfunction (POCD) is a neurological symptom with learning and memory impairments, mainly affecting the elderly after surgery. However, there is no effective treatments for this symptom. PurposesIn this study, we evaluated the neuroprotective effects of fucoxanthin against POCD in aged mice after surgery. Study design and methodsThe animal model of POCD was established in 12 – 14 month aged mice with a laparotomy. Curcumin was used as a positive control. The beneficial effects of fucoxanthin on POCD was analyzed by behavioral tests. Pro-inflammatory cytokines were measured by Enzyme-linked Immunosorbent Assay (ELISA). And the expressions of key proteins in the Akt and ERK signaling pathways were analyzed by Western blotting analysis. The morphology of microglial cells and astrocytes was explored by immunohistochemical staining. The activity of antioxidant superoxide dismutase (SOD) and catalase (CAT) were measured by anti-oxidative enzyme activity assays. ResultsFucoxanthin at 100 – 200 mg/kg significantly attenuated cognitive dysfunction, with a similar potency as curcumin, in aged mice after surgery. In addition, fucoxanthin and curcumin significantly increased the expression of pAkt, prevented the activation of microglial cells and astrocytes, and inhibited the secretion of pro-inflammatory interleukin-1β (IL - 1β) and tumor necrosis factor-α (TNF-α). Furthermore, fucoxanthin and curcumin elevated the ERK pathway and potently increased the activity of antioxidant enzymes. Most importantly, U0126, an inhibitor of the ERK pathway, and wortmannin, an inhibitor of the Akt pathway, significantly abolished the cognitive-enhancing effects, as well as the inhibition of neuroinflammation and the reduction of oxidative stress, induced by fucoxanthin in aged mice after surgery. ConclusionFucoxanthin might be developed as a functional food or drug for the treatment of POCD by inhibiting neuroinflammation and enhancing antioxidant capacity via the activation of the Akt and ERK signaling pathways.

Chemical constituents from the flowers of Inula japonica and their anti-inflammatory activity

Ethnopharmacological relevanceThe flowers of Inula japonica (Inulae Flos) can be used to treat cough and asthma and remove phlegm in traditional Chinese medicine (TCM). Aim of the studyOur research aimed to obtain active components with the inhibition of inflammation and MUC5AC production to alleviate asthma symptoms from I. japonica. Materials and methodsThese compounds were separated from the MeOH extract of Inulae Flos by column chromatography over silica gel, AB-8 macroporous resin column, MPLC, and semipreparative HPLC. Their structures were elucidated by detailed spectroscopic data analysis, ECD calculations, and chemical methods. NO production was determined to evaluate anti-inflammatory activity in RAW 264.7 cells. The expression of MUC5AC, IL-1β, and IL-4 were measured in NCI–H292 cells by qRT-PCR. The anti-asthma activity assessments in vivo were performed through H & E and PAS staining, pulmonary function analysis, and cytokines determination by qRT-PCR or ELISA. The expression levels of PI3K, p-PI3K, AKT, p-AKT, MEK, p-MKE, ERK, p-MEK, and IL-1β were analyzed through western blotting. ResultsOne undescribed 1,10-seco-eudesmanolide derivative (1), two previously unreported 1,10-seco-eudesmanolide glycosides (2 and 3), and thirty-two known compounds (4–35) were obtained from Inulae Flos. Compound 11 had the most inhibitory effect against LPS-induced NO production in RAW 264.7 murine macrophages. Meanwhile, compound 11 also attenuated the increase in MUC5AC, IL-1β, and IL-4 mRNA expression in NCI–H292 cells. The results of the animal experiment confirmed that compound 11 significantly ameliorated OVA-induced asthma in a murine model of allergic asthma demonstrated by elevated pulmonary function, reduced inflammatory cell infiltration and mucus production. In addition, compound 11 significantly inhibited the levels of OVA-specific IgE in serum, of IL-4 and IL-6 in BALF, and of MUC5AC, IL-1β , IL-4, IL-5, IL-6 and IL-13 in lung tissue. Finally, compound 11 suppressed PI3K/AKT/MEK/ERK signaling pathway in lung tissue of mice. ConclusionThis study indicated that compound 11 might be a potential therapeutic candidate ameliorating airway inflammation and mucus hypersecretion via PI3K/AKT/MEK/ERK signaling pathway in allergic asthma.

Control of gastruloid patterning and morphogenesis by the Erk and Akt signaling pathways.

Many developmental processes rely on the localized activation of receptor tyrosine kinases and their canonical downstream effectors Erk and Akt, yet the specific roles played by each of these signals is still poorly understood. Gastruloids, 3D cell culture models of mammalian gastrulation and axial elongation, enable quantitative dissection of signaling patterns and cell responses in a simplified, experimentally accessible context. We find that mouse gastruloids contain posterior-to-anterior gradients of Erk and Akt phosphorylation induced by distinct receptor tyrosine kinases, with features of the Erk pattern and expression of its downstream target Snail exhibiting hallmarks of size-invariant scaling. Both Erk and Akt signaling contribute to cell proliferation, whereas Erk activation is also sufficient to induce Snail expression and precipitate profound tissue shape changes. We further uncover that Erk signaling is sufficient to convert the entire gastruloid to one of two mesodermal fates depending on position along the anteroposterior axis. In all, these data demonstrate functional roles for two core signaling gradients in mammalian development and suggest how these modules might be harnessed to engineer user-defined tissues with predictable shapes and cell fates.