Melanoma is the most aggressive skin malignancy with increasing incidence worldwide. Pannexin1 (PANX1), a member of the pannexin family of channel-forming glycoproteins, regulates cellular processes in melanoma cells including proliferation, migration, and invasion/metastasis. However, the mechanisms responsible for coordinating and regulating PANX1 function remain unclear. Here, we demonstrated a direct interaction between the C-terminal region of PANX1 and the N-terminal portion of β-catenin, a key transcription factor in the Wnt pathway. At the protein level, β-catenin was significantly decreased when PANX1 was either knocked down or inhibited by two PANX1 blockers, Probenecid and Spironolactone. Immunofluorescence imaging showed a disrupted pattern of β-catenin localization at the cell membrane in PANX1-deficient cells, and transcription of several Wnt target genes, including MITF, was suppressed. In addition, a mitochondrial stress test revealed that the metabolism of PANX1-deficient cells was impaired, indicating a role for PANX1 in the regulation of the melanoma cell metabolic profile. Taken together, our data show that PANX1 directly interacts with β-catenin to modulate growth and metabolism in melanoma cells. These findings provide mechanistic insight into PANX1-mediated melanoma progression and may be applicable to other contexts where PANX1 and β-catenin interact as a potential new component of the Wnt signaling pathway. Blocking or reducing PANX1 in melanoma cells decreases the levels of β-catenin and suppresses β-catenin transcriptional activity. Moreover, depletion of PANX1 attenuated the mitochondrial respiratory activity of melanoma cells. Our findings underline the molecular mechanisms through which PANX1 regulates melanoma tumorigenesis and suggests that PANX1 may be a new interactor in the Wnt signaling pathway and can potentially be a target for the treatment of malignant melanoma.Pannexin 1 is a member of a glycoprotein family PANX 1, 2, and 3) that oligomerizes to establish large pore channels between the intracellular and extracellular space for cell communication , and 3 t. PANX1 mA, left panel). To investigate whether this correlation is restricted to melanoma or applies to other types of cancers, we analyzed TCGA data of 1108 biopsies taken from patients with breast cancer. We observed that, similar to melanoma, mRNA expression of PANX1 significantly correlates with that of β-catenin . Of note, both PANX1 and β-catenin have been implicated in survival of metastatic breast cancer cells and poor patient outcome . We observed that PANX1 is expressed in all cell lines tested but most abundant in 131/4-5B1 cells at both the protein and mRNA levels (B and C). A375-P cells with PANX1 knockdown were used to validate the specificity of the antibody. We chose 131/4-5B1 cells to conduct immunoprecipitation studies, incubating protein lysates with specific antibodies against the carboxyl terminal region of PANX1. Endogenous PANX1 co-immunoprecipitated (co-IP) with β-catenin in 131/4-5B1 human melanoma cells . In contrast, no β-catenin precipitated with rabbit IgG, validating the specificity of the interaction . Reciprocal analysis revealed that endogenous PANX1 specifically co-IP with endogenous β-catenin from 131/4-5B1 human melanoma cells .To determine whether there is an interaction between PANX1 and β-catenin in melanoma cells, we first analyzed a panel of melanoma biopsies from the Cancer Genome Atlas (TCGA) and found a modest, yet significant, correlation between PANX1 and β-catenin mRNA (CTNNB1) in 471 patients with malignant melanoma . Calmodulin co-IP with PANX1 in a Ca2+-dependent manner . The interaction between calmodulin and PANX1 in melanoma cells represents an additional layer of complexity in the modulation of Ca2+ signaling through PANX1.PANX1 has been shown to act as a Caeticulum , 7, provpoptosis . The molic cells . Calmoduic cells , 25, 26.Fig. 1E, A. CalmodD, whether PANX1 and β-catenin bind directly or through a protein complex. To address this question, we performed an in vitro analysis using a transcription and translation (TNT) system and purified recombinant proteins. Selected regions of PANX1  were expressed using the TNT and labeled with [35S]methionine. The PANX1 constructs were incubated with Maltose-Binding Protein (MBP)-tagged β-catenin. After washing, complexes were resolved by SDS-PAGE, and gels were dried and processed by autoradiography. Analogous to findings with co-IPs (D), full-length PANX1 binds directly to purified β-catenin in vitro . Analysis of two fragments of PANX1 revealed minimal binding of the middle segment (M) of PANX1 (amino acids 128–203) to β-catenin . In contrast, amino acids 288 to 426, which constitute the C-terminal segment (C) of PANX1, exhibit robust binding to β-catenin . Binding specificity was confirmed by the absence of bands from samples incubated with MBP alone (B and C). The expression levels of TNT products were comparable among samples . We investigated whether the C-terminal region of PANX1 (aa 288–426) is homologous to any other proteins. The results using NCBI blast analysis revealed that the 138 aa in the C-terminus region of PANX1, where β-catenin binds, is specific to PANX1 among different species , middle and C-terminal  and the C-terminal (amino acids 666–781) regions of β-catenin. His-tagged purified fragments of β-catenin were incubated with lysates of HEK293 cells expressing myc-PANX1-C. Complexes were isolated using Talon Metal Affinity Resin. PANX1 binds exclusively to the N-terminal region of β-catenin (amino acids 1–137) (D and E). No binding is detected to the R1 or C constructs of β-catenin or to Talon beads alone. Collectively, these data demonstrate that the C-terminal region of PANX1 binds to the N-terminal portion of β-catenin.In addition, we conducted s 1–137) , D and EPANX1 gene. Western blotting confirmed the loss of PANX1 expression (A). Deletion of the PANX1 gene from A375-P and A375-MA2 cells caused a substantial reduction in the abundance of β-catenin and its downstream effector Microphthalmia-Associated Transcription Factor . Since the growth rate of PANX1 knockout cells in culture was severely reduced and cells were difficult to maintain for experimental procedures, we knocked down PANX1 in A375-P cells using shRNA. The levels of PANX1 mRNA in cells transfected with control shRNA were comparable with those in nontransfected A375-P cells (B). In contrast, PANX1 mRNA in cells that were transfected with either of two different shRNA constructs against PANX1 (PANX1shRNA-B and D) was 3.2 ± 0.04-fold lower than control cells (B). Similarly, the amount of PANX1 protein was decreased by 4.2 ± 0.02-fold compared with control samples . Analogous to our observations with PANX1 KO cells, the abundance of β-catenin was significantly reduced in PANX1 knockdown melanoma cells . To evaluate whether loss of PANX1 affects the β-catenin mRNA, we performed quantitative RT-qPCR. A375-P cells transfected with shRNA against β-catenin were used as control (E). Levels of β-catenin mRNA among nontransfected, control shRNA and PANX1 shRNA transfected A375-P cells were comparable (E). These data indicate that PANX1 likely contributes to the posttranscriptional modification and/or stability of β-catenin protein in melanoma cells and does not have a significant impact on β-catenin mRNA levels.To investigate the effect of PANX1 on β-catenin function in melanoma cells, we generated PANX1 knockout (PANX1 KO) A375-P and A375-MA2 cells using the CRISPR/Cas9 system. A double nicking strategy was employed to reduce the likelihood of off-target effects. In this method, paired guide RNAs with the ability to cleave single strands were used, which resulted in specific double-strand breaks within the pression A. Deletior MITF, A. Since -P cells B. In conol cells B. Simila control E. Levelsmparable E. These F). In contrast, in PANX1-deficient cells, PANX1 is decreased and less abundant at the cell surface and areas of cell–cell contact. In addition, the pattern of β-catenin staining at cell borders is also irregular and disrupted when PANX1 is reduced in melanoma cells (F).Next, we validated our findings by immunofluorescence analysis in PANX1-deficient melanoma cells. In cells transfected with control shRNA, β-catenin is abundant at the cell membranes where it partially colocalizes with membrane-bound PANX1 F. In conma cells F.Mitf-M, a specific isotype in melanocytes, contains β-catenin-binding sites (A and C). MITF protein was significantly reduced in PANX1-deficient melanoma cells (C and G). Similarly, qPCR analysis revealed a significant reduction in the mRNA of MITF when PANX1 is knocked down in A375-P cells (G). MITF expression is modulated through β-catenin and another key effector in the Wnt pathway called lymphoid enhancer-binding factor 1 (LEF1) in melanoma cells . In addition, mRNA levels of Axin2, which is a common β-catenin target gene, were substantially reduced upon PANX1knockdown but did not reach statistical significance (G).MITF is a melanocyte lineage-specific transcription factor that is linked to plasticity of melanoma cells and has key roles in proliferation, migration, and invasiveness of melanoma cells , 28. Theng sites . To asseng sites , A and Cma cells , C and G-P cells G. MITF ema cells , 31, 32.ll lines , 34, 35,ll lines , 37, 38.ll lines G. In addificance G.H). These results demonstrate that expression of several key effectors of the Wnt signaling pathway, including β-catenin, is reduced in PANX1-deficent melanoma cells.Wnt/β-catenin signaling is a major regulator of melanoma proliferation , 39, 40.Fig. 3A) and mRNA levels (B) after β-catenin shRNA KD, as expected. Interestingly, the levels of both PANX1 protein and mRNA were substantially reduced in β-catenin-deficient melanoma cells (A and C). The 1.9-fold decrease in PANX1 mRNA when β-catenin was knocked down was the same as knocking down PANX1 using PANX1 shRNA (C). Knocking down β-catenin in melanoma cells also reduced MITF mRNA by 2.8-fold (D). The role of Wnt/β-catenin/MITF pathway in melanoma cell proliferation is well established (E). The effect of β-catenin reduction on PANX1 subcellular localization was investigated using immunofluorescence analysis (F). Our findings suggest that in β-catenin knocked down A375-P cells, PANX1 is reduced, particularly in its localization at the cell surface and areas of cell–cell contact, and shows a diffuse intracellular distribution (F).The effect of shRNA knockdown (KD) of β-catenin on the expression of PANX1 in melanoma cells was evaluated in A375-P cells. Western blotting and qPCR confirmed reduced β-catenin protein A and mRNA levels B after βma cells , A and CX1 shRNA C. Knocki2.8-fold D. The roablished , 30. In ablished E. The efanalysis F. Our firibution F.Figure H). To assess the effect of long-term exposure to PANX1 blockers, we incubated A375-MA2 cells with PBN for 72 h. Quantification revealed that PBN decreases protein levels of both PANX1 (by 20%) and β-catenin (by 30%) (A and B). Spironolactone (SPL) was recently shown to specifically block PANX1 (C and D). SPL caused about 37% ± 11 reduction in PANX1 and 47% ± 8 reduction in β-catenin levels (C and D). qPCR showed that neither PBN nor SPL significantly altered PANX1 mRNA , which suggests that long-term exposure to blockers likely affects PANX1 at the protein level.We previously reported that long-term exposure to PANX1 channel blockers, namely Carbenoxolone (CBX) and Probenecid (PBN), reduces the growth rate of melanoma cells evident after 3 days in culture , a phenoFig. 3(by 30%) , A and Bck PANX1 . Incubatck PANX1 , C and Dn levels , C and DE, right panel), suggesting that PBN likely suppresses β-catenin not only through reduction of PANX1 but also through inhibition of β-catenin mRNA transcription. Immunofluorescence analysis of A375-P cells demonstrated that both CBX and PBN reduce PANX1 levels and localization at the cell membrane  and altered the cell membrane localization of β-catenin  in a manner similar to either PANX1 or β-catenin knockdown cells . Interestingly, 72 h exposure to CBX and PBN also alters calmodulin subcellular localization in melanoma cells causing more accumulation of calmodulin around nuclear compartments (B). Given the association of PANX1 and calmodulin , it is likely that depletion of PANX1 alters the subcellular localization of its binding partners, including calmodulin. Overall, our findings suggest that long-term exposure to PANX1 blockers reduces PANX1 and β-catenin proteins and changes their subcellular localization in melanoma cells.Treatment with PBN but not SPL reduced the β-catenin mRNA levels . PANX1-deficient cells exhibited significantly lower basal respiration than cells transfected with control shRNA . Furthermore, following the injection of carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), an electron transport chain uncoupling agent, maximal respiration  and spare respiratory capacity  were also decreased in PANX1 knockdown cells. These results suggest that PANX1-deficient cells exhibit suppressed mitochondrial metabolism, which may explain reduced proliferation and the previously reported reduction in the migration of these cells . In-depth analysis of possible intracellular interactions between PANX1 and β-catenin will provide better understanding of PANX1-mediated signaling. An important finding in our study is a direct interaction between PANX1 and β-catenin. Binding to β-catenin is mediated through the C-terminal region of PANX1, which has been shown to interact with actin and actin-interacting protein Arp3   is an alternative way for increasing its activity, which can happen even without signaling inputs from Wnt ligands . Dissociation of β-catenin from the cell border is likely to increase the exposure of cytoplasmic β-catenin to degradation, which is an important regulatory mechanism of Wnt/β-catenin in cancer .PANX1 mRNA expression z-scores (RNA Seq V2 RSEM) and CTNNB1 mRNA expression z-scores (RNA Seq V2 RSEM) were generated using data in 2. Trypsin  was used to dissociate cells from culture dishes.Human melanoma cells lines, A375P (ATCC CRL-3224), A375-MA2 (ATCC CRL-3223), A2058 (ATCC CRL-11147) were obtained from ATCC while cell line 131/4-5B1 was a gift from Dr Kerbel  and cultProtein lysates were extracted with: 1% Triton X-100, 150 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, or a RIPA buffer , 0.5% sodium deoxycholate). Each buffer contained 1 mM sodium fluoride, 1 mM sodium orthovanadate, and half of a tablet of complete-mini EDTA-free protease inhibitor (Roche). Protein was quantified by bicinchoninic acid (BCA) assay (Thermo Scientific). Protein lysates (40 μg) were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane using an iBlotTM System (Invitrogen). Membranes were blocked with 3% bovine serum albumin (BSA) with 0.05% Tween-20 in 1X phosphate buffer saline (PBS), and incubated with anti-human PANX1 antibody  , anti-huCarbenoxolone disodium salt  and water-soluble Probenecid  were dissolved in Hanks’s Balanced Salt Solution  to develop stock concentrations of each compound. Spironolactone was purchased from Selleckchem (#52-01-7).et al., (addgene.org) containing guide RNA sequences for human PANX1 in a 6-well plate. PANX1 gRNAs were designed with http://tools.genome-engineering.org (sequences GTTCTCGGATTTCTTGCTGA and CTCCGTGGCCAGTTGAGCGA). Twenty-four hours post transfection, cells were selected with 1 μg/ml Puromycin for 72 h. Following selection, cells were screened for PANX1 levels by western blot. Plasmids were a gift from Feng Zhang (Addgene plasmid #48140 and #62987).PANX1 knockout cells were generated using CRISPR/Cas9 D10A following Ran et al.,  protocolA375-P cells were transfected with two constructs (PANX1 shRNA-B and PANX1 shRNA-D) from Origene PANX1 human 29-mer shRNA kit in pRS vector (#TR302694) (sequence: 5’-CGCAATGCTACTCCTGACAAACCTTGGCATGTCAAGAGCATGCCAAGGTTTGTCAGGAGTAGCATTGTT-3’) plus a pLKO.1 sh β-catenin.1248 (CCGGAGGTGCTATCTGTCTGCTCTACTCGAGTAGAGCAGACAGATAGCACCTTTTTT) (#19761) GFP shRNA cassette (5’GCCCGCAAGCTGACCCTGAAGTTCATTCAAGAGATGAACTTCAGGGTCAGCTTGCTTTTT-3’) from Addgene (#30323) as a control. Control shRNA transfected cells were evaluated for PANX1 and β-catenin expression compared with nontransfected cells. Single cell colony of PANX shRNA-expressing cells from two constructs (B and D) were selected with puromycin and examined for PANX1 knockdown (KD). Stable knockdown samples showed 80 to 90% reduction in PANX1 expression. Cells were maintained under puromycin selection pressure and periodically examined for effective PANX1 knockdown by western blot. Similarly, single cell colony selected β-catenin shRNA transfected cells were examined for 80 to 90% reduction in β-catenin expression. Cells were maintained under puromycin selection pressure and periodically examined for effective β-catenin knockdown by western blotting. Experiments were conducted after verifying at least a 70 to 85% knockdown of PANX1 and β-catenin protein levels by western blotting.35S]Methionine-labeled TNT products were synthesized using the TNT quick coupled transcription/translation system (Promega) essentially as described previously (NT Quick Master mix and 20 μCi of [35S]methionine (PerkinElmer Life Sciences) for 90 min at 30 °C. TNT products were confirmed by SDS-PAGE and autoradiography before being used in pull-down assays. To identify the region of β-catenin that binds to PANX1, TNT products of PANX1 were incubated with maltose-binding protein (MBP)-β-catenin  for 3 h at 4 °C. Complexes were washed five times with wash buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 1% Triton X-100 and separated by SDS-PAGE. The gels were dried, and autoradiography was performed. To identify the region of PANX1 that binds to β-catenin, portions of PANX1 were expressed with TNT. Radiolabeled products were incubated with MBP-β-catenin and processed as described above.[eviously . BrieflypcDNA-PANX1(HCVp) was purchased from Addgen (Plasmid#87698). pcDNA3-myc-PANX1-C was used to make PCR product of PANX1-C with pcDNA-PANX1 as template and 5’CGGGATCCGCTCCCGTGGTTGTCTACACGCTG-3’ as positive primer, 5’-CCGGAATTCTCTAGATCAGCAAGAAGAATCCAGAAGTCTC-3’ as negative primer. The PCR product was cut with BamH I and Xba I and inserted into pcDNA3-myc at BamH I and Xba I site.E. Coli and purified with Talon Metal Affinity Resin according to the manufacturer’s protocol.pPET28a-TEV-hβ-Catenin-N (amino acids 1–137) (Plasmid#17203), pPET28a-TEV-hβ-Catenin-R1  (Plasmid#17200) and pPET28a-hβ-Catenin-C (amino acids 666–781) (Plasmid#17204) were purchased from Addgene. His-tagged proteins were expressed in BL21 strain of For binding assays, pcDNA3-myc-PANX1-C was transfected into HEK293 cells. Cells were harvested 72 h post transfection using Buffer A . Cells were lysed using sonication and spun down. The supernatants were precleared with Talon beads for 1 h at 4 °C. Equal amounts of protein lysate were incubated with His-tagged β-catenin-N, -R1 or -C attached to Talon beads, or empty Talon beads alone (negative control). Samples were rotated at 4 °C for 3 h and washed 5x with Buffer A. Then, samples were resuspended in 30 μl SDS-Sample buffer. Proteins were separated by 15% SDS-PAGE. The gel was cut below 25 kDa. The upper part was stained with Coomassie blue. The lower part was transferred to PVDF membranes and probed with anti-myc antibody (Millipore anti-Myc-Tag Cat# 06-549).g for 10 min at 4 °C. Supernatants were precleared with protein A-Sepharose beads for 1 h. Equal amounts of protein lysate were incubated with protein A-Sepharose beads and anti-PANX1 polyclonal antibodies or anti-β-catenin monoclonal antibody for overnight at 4 °C. Rabbit IgG and mouse IgG were used as controls for polyclonal and monoclonal antibody immunoprecipitations, respectively. Samples were washed five times with Buffer A, resolved by SDS-PAGE, and Quick Western detection kit (LiCore #926-69100) was used to detect the protein processed by western blotting.131/4-5B1 or A375-P cells were plated in 10-cm dishes to reach 80% confluence. The following day, the cells were washed with ice-cold PBS and lysed with 500 μl of Buffer A  with either 1 mM CaCl2 (+) or EGTA(−) supplemented with complete protease and phosphatase inhibitors (Roche). Lysates were subjected to two rounds of sonication for 10 s each, and insoluble material was precipitated by centrifugation at 13,000Cells were cultured at 1000/well onto a 96-well plate. Cell Proliferation Reagent WST-1 (Sigma Aldrich) was used to assess the growth rate of cells according to manufacturer’s instructions. Measurements at 450 nm and at a 690 nm were taken on an Epoch microplate spectrometer (Biotek).To measure mRNA, cells were cultured for 24 h. Then total RNA was isolated from the cells using an RNA isolation kit (Qiagen). In total, 250 ng of RNA was reverse transcribed to cDNA using a high-capacity cDNA reverse transcriptase kit (Applied Biosystems#4374966) according to the manufacturer’s instructions. RT-qPCR was performed using SYBR Green PCR Master Mix (BioRad#1725274) and 200 nM forward and reverse primers. The primers used human PANX1: 5’- AACCGTGCAATTAAGGCTG -3’ (forward); and 5’- GGCTTTCAGATACCTCCCAC -3’ (reverse); β-catenin: 5’- AAAATGGCAGTGCGTTTAG -3’ (forward); and 5’- TTTGAAGGCAGTCTGTCGTA -3’ (reverse); MITF: 5’- CCTTCTCTTTGCCAGTCCATCT -3’ (forward); and 5’- GGACATGCAAGCTCAGGACT -3’ (reverse); and Axin2: 5’-GAGTGGACTTGTGCCGACTTCA-3’ (forward); 5’- GGTGGCTGGTGCAAAGACATAG-3’ (reverse); and Lef1: 5’- CCTGGTCCCCACACAACTG-3’ (forward); and 5’- GGCTCCTGCTCCTTTCTCTG-3’ (reverse); RT-qPCR enzyme activation was initiated for 10 min at 95 °C and then amplified for 40 cycles of a two-step PCR (15 s at 95 °C and 1 min at 60 °C). All samples were assayed at least in duplicate, and YWHAZ was used as reference control. The results were analyzed using the ΔΔCT method.Cells were grown on glass coverslips and were fixed 72 h posttransfection using ice-cold 8:2 methanol:acetone for 15 min at 4 °C and blocked with 2% BSA-PBS. Coverslips were incubated with anti-human PANX1 antibody  and anti-mouse β-catenin (1:450 BD Biosciences# 610153). Nuclei was stained with Hoechst 33342 (1:1000), Alexa Fluor 647 anti-mouse and 555 goat anti-rabbit IgG  were used as secondaries, and mounted using Airvol  prior to imaging. Immunofluorescence images were obtained using a Zeiss LSM 800 Confocal Microscope with a Plan-Apochromat 63x/1.40 Oil DIC objective (Carl Zeiss).2 prior to running the assay. A mitochondrial stress test kit  was used to determine basal respiration, maximal respiration, and spare respiratory capacity following the manufacturer’s instructions. OCR was normalized to total protein/well using a DC protein assay  or a bicinchoninic acid (BCA) assay (Thermo Scientific), and Agilent Seahorse Wave Desktop and XF Cell Mito Stress Test Report Generator software were used for data processing.Control shRNA and PANX-1 shRNA malignant melanoma cells were seeded at a density of 25,000 to 30,000 cells/well in an XFe24 cell culture microplate  pretreated with 0.01% Poly-L-Lysine (Millipore #A-005-C) 1 to 2 days prior to running the assay. On the day of the assay, cell culture medium was replaced with Seahorse XF DMEM medium, pH 7.4  supplemented with 10 and 25 mM glucose , 4 mM glutamine , and 1 mM pyruvate . Cells were incubated in XF DMEM for one h at 37 °C, 0% COAll data are representative of at least three independent experiments conducted with three technical replicates unless otherwise mentioned in the figure legends. Statistical analyses were performed using GraphPad Prism software (version 8.0). Error bars indicate mean ± standard error mean. The details of statistical analysis are provided in figure legends.spenuela@uwo.ca).All the data described in the study is contained within the article. Raw data available upon request (Dr Silvia Penuela, Western University, This article contains The authors declare no conflict of interest of any kind with the contents of this article.


Gastrocnemius and Tibialis anterior muscles unraveled that the level of Grm2 expression is higher in Tibialis MNs compared to Gastrocnemius MNs suggesting a specific mGluR gene expression profile in these two MN pools. Finally, we assessed the functional impact of mGluR modulation on electrically induced bouts of fictive locomotion in the isolated spinal cord preparation of P1-P3 mice, and in vivo during spontaneous episodes of swimming activity in both P1-P3 and P8-P12 mouse pups. We observed that the mGluR agonists induced distinct and specific effects on the motor burst amplitudes and period of the locomotor rhythms tested and that their actions are function of the developmental stage of the animals. Altogether our data show that the metabotropic glutamatergic system exerts a complex neuromodulation in the developing spinal lumbar motor networks and provide new insights into the expression and modulation of ADSP in MNs.The present study explores the impact of metabotropic glutamate receptor (mGluR) activation on activity-dependent synaptic plasticity (ADSP) and the intrinsic membrane properties of lumbar motoneurons (MNs) using a combination of biochemical, pharmacological, electrophysiological and behavioral techniques. Using spinal cord slices from C57BL/6JRJ mice at two developmental stages, 1-3 and 8-12 postnatal days , we found that ADSP expressed at glutamatergic synapses between axons conveyed in the ventrolateral funiculus (VLF) and MNs, involved mGluR activation. Using specific agonists of the three groups of mGluRs, we observed that mGluR stimulation causes subtype-specific and developmentally regulated modulation of the ADSP and synaptic transmission at VLF-MN synapses as well as the intrinsic membrane properties of MNs. RT-qPCR analysis revealed a downregulation of mGluR gene expression with age in the ventral part of the lumbar spinal cord. Interestingly, the selective harvest by laser microdissection of MNs innervating the  MoreoveExperiments were performed on 326 new-born C57BL/6JRJ mice, aged postnatal day 1 (P1) to P3 and P8 to P12, without consideration of sex. All procedures were carried out in strict accordance with the guidelines of the European Committee Council Directive and approved by the local ethic committee of the University of Bordeaux and the French Agriculture and Forestry Ministry for handling animals . All efforts were made to minimize animal suffering and reduce the number of animals used.2 2.50; MgSO4 1.3; NaH2PO4 0.58; NaHCO3 25; glucose 10; equilibrated with 95% O2, 5% CO2 adjusted to pH 7.4 at room temperature (23°C). Recordings were performed after a recovery period of at least one hour.1-3 postnatal days mouse pups were anesthetized with 4% isoflurane until reflexes were lost and eviscerated after decapitation. Spinal cords were isolated via laminectomy, sectioned at the thoracic level 1 (T1) and placed ventral side up in the recording chamber. Dissection and recording procedures were performed under continuous superfusion with artificial cerebrospinal fluid (aCSF) containing the following (in mM): NaCl 130; KCl 3; CaCl2 0,5, MgCl2 7, NaH2PO4 1,15, NaHCO3 26, glucose 11 and sucrose 205, bubbled with 95% O2, 5% CO2 and sectioned into transverse slices (350 μm) with a vibroslicer . Slices were allowed to recover in oxygenated aCSF (see above) for at least 1 h at 30°C before being transferred to the recording chamber.Lumbar spinal cords from P1-P3 and P8-P12 mice were dissected free in an ice-cold sucrose-based saline solution containing the following (in mM): KCl 2, CaClin vitro spinal cord preparation, motor output was recorded extracellularly using a custom-made differential AC amplifier from the right and left lumbar 2 , rL5 and lL5 ventral roots using glass suction electrodes filled with aCSF. Bouts of fictive locomotion were evoked by electrical stimulation  of the VLF using a concentric bipolar electrode (Phymep) positioned between the T9 and T11 levels . Voltage- or current- clamp recordings were obtained using a Multiclamp 700B amplifier . Data acquisition and analysis were performed using Axograph software. All intracellular recordings were made in high cation aCSF containing 7.5 mM CaCl2 and 8 mM MgSO4 in order to decrease polysynaptic transmission. Throughout recordings, GABAergic and glycinergic inputs were blocked with gabazine and strychnine (1 μM each), respectively.Whole-cell patch clamp recordings from putative MNs located throughout the lumbar spinal cord enlargement, identified by their relatively large soma (superior to 20 μm) and location in the lamina IX, were made under visual control with a Multiclamp 700B (Axon Instruments), using glass microelectrodes (3–6 MΩ) filled with the following (in mM): K Gluconate 120, KCl 20, MgClf-I) was calculated and fitted with linear functions to compute the slope of the linear part (first three points) of the function. MNs were held at −60 mV by bias current application ranging from −500 pA to + 150 pA in current clamp mode.Input membrane resistance was determined from voltage-current curves obtained from MNs held at −60 mV. After-hyperpolarization (AHP) parameters were computed from single action potential (AP) elicited by injecting a brief depolarizing current pulse . MN firing behavior was studied using series of depolarizing current pulse injections with increasing amplitude. Then, the instantaneous frequency of firing , a group I/II metabotropic glutamate receptor (mGluR) antagonist MCPG ; a group I mGluR agonist DHPG ; a group II mGluR agonist LY354740 [-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid; 0.5 μM] and a group III mGluR agonist L-AP4 . The concentration of the different mGluR agonists used in the present study was determined by performing dose-response curves to reach similar inhibitory effects on the glutamatergic synaptic transmission . The glutamate transporter inhibitor TBOA  was purchased from Tocris , and the glycine receptor antagonist strychnine (1 μM) from Sigma-Aldrich .All drugs were prepared as stock solutions, aliquoted and frozen until use. The following pharmacological agents from Abcam  were used: gabazine . Animals were left to recover from anesthesia until spontaneous movement production and then placed into a tank filled with warm (ca. 35°C) water. Spontaneous swimming activity was recorded during approximately 10 s. Electromyographic signals were amplified (x 5000) using custom-made amplifiers, and filtered at 2 kHz. Data acquisition and analysis were performed using Axograph software. After three sessions of control swimming activity spaced 5 min apart, mice were randomly assigned to the following four treatment groups: vehicle (aCSF), DHPG (20 mg/kg), LY354740 (0.5 mg/kg), or L-AP4 (10 mg/kg). These concentrations were determined by an extrapolation from the concentrations used for intracellular recordings by a simple conversion based on mouse pup weight. The DHPG concentration has to be increased due to an absence of effects in vivo, while the LY354741 concentration was reduced due to a complete blockage of the swimming activity in pups (data not shown). Solutions were injected subcutaneously using an Hamilton syringe  at a volume of 0.3 μL for P3 and 1 μL for P8-P10 animals. Swimming activity was recorded 5, 10, 20 and 30 min after the injection. Between each session, mouse pups were placed on a heating pad.Animals were anesthetized with isoflurane and implanted with bipolar electrodes  directly pinned into the RNA NucleoSpin®RNA Plus extraction kit . RNA quality and quantity were analyzed using Agilent RNA 2200 Screen Tape system  and DS-11 spectrometer  respectively.The ventral lumbar part of the spinal cord or individual L2 and L5 ventral segments were isolated from isoflurane-anesthetized mouse pups and immediately frozen at −80°C until use. During all procedures, care was taken to avoid RNA degradation. Total RNA was extracted from samples using the Gastrocnemius or Tibialis Anterior muscles, MNs were backfilled with cholera toxin ß-subunit (CTB) conjugated with Alexa Fluor 594 or Alexa Fluor 488 . Both muscles of both hindlimbs were injected in anesthetized P0 mice. After 12 h of migration, the lumbar spinal cord was dissected out from anesthetized animals, cryosectioned into longitudinal sections (20 μm) and collected on polyethylene-naphthalate membrane RNase-free microscope slides. Immediately after dehydration in successive baths of ethanol at increasing concentrations, labeled MN somas were dissected at 63x magnification using a PALM laser microdissection and capture system . Gastrocnemius or Tibialis MNs were collected in separate tubes with adhesive caps for no longer than 30 min per slide to limit RNA degradation. The collected material was then treated with lysis buffer and stored at −80°C until RNA extraction. In the case of laser capture microdissection (LCM) samples, the ReliaPrep™ RNA Cell Miniprep System from Promega  was used for RNA extraction. The RNA integrity and quantity were checked by the Bioanalyser 2100  and the Nanodrop 1000 , respectively.To perform RT-qPCR from identified MNs innervating the GoScript™ Reverse Transcription Kit (Promega) by standard protocols. Samples were distributed in duplicate using the Eppendorf epMotion 5073 automated pipetting. PCR was performed with the GoTaq Master Mix from Promega and the CFX384 real-time PCR detection system . Cycling parameters for the qPCR reaction included a 3 min hot start followed by 40 cycles of denaturation at 90°C for 10 s, annealing at 60°C for 30 s, and elongation at 72°C for 30 s. Sdha (succinate dehydrogenase) and actine ß (Actß) were used as internal controls. Two technical replicates were run for each RT-qPCR experiments and relative expression levels were calculated using the 2–ΔΔCt method after normalization to the Sdha housekeeping gene. The primers sequences used  was carried out to test for significant effects between the different drugs for unpaired observations. Two-way or RM two-way ANOVAs followed by Sidak’s multiple comparison tests were performed to evaluate drug and age effects. Chi-square tests were used to compare motoneuron proportions displaying STD, LTD or no change in the different conditions tested. In the text and figures, all data are expressed as means ± SEM. Statistical significance level was set at n = 18 in the presence of MCPG, p = 0.5, Wilcoxon matched-pairs test) in P1-P3 mice. At excitatory glutamatergic synapses, mGluRs, in contrast to NMDA and AMPA receptors that are enriched in the postsynaptic density, are localized perisynaptically and have been shown to be activated when high concentrations of glutamate are reached in the synaptic cleft  applied to the VLF in slices triggered excitatory postsynaptic currents (VLF-EPSCs) in lumbar MNs held at −60 mV . As previc cleft . We theric cleft . Howeveric cleft . These rIt has been previously shown that the activation of extrasynaptic mGluRs by high synaptic concentrations of glutamate that spill over the synaptic terminals is a major determinant of synaptic plasticity expressed in supraspinal structures . We thusn = 14, MCPG −42.4 ± 4%, n = 12 and STD: control −59.3 ± 4%, n = 17 and MCPG −34.6 ± 8%, n = 3 . Altogether these data indicate that mGluRs are activated during VLF-HFS and contribute to the expression of ADSP at VLF-MN synapses.To amplify the glutamate release induced by HFS and to further activate potential perisynaptic mGluRs at VLF-MN synapses, VLF-HFS was applied in the presence of TBOA. As shown in %, n = 3 . In cont%, n = 3 : controlTo assess the separate roles of the three different groups of mGluRs in the modulation of ADSP at VLF-MN synapses, we tested the impact of exposure to specific mGluR agonists, DHPG, LY354740, and L-AP4 for mGluRI, mGlurII, and mGluRIII, respectively (see section “DISCUSSION”). The agonists were applied to the slices throughout recording periods. 10 min after the onset of each agonist’s superfusion, a 10 min period of stable low frequency (0.03 Hz) VLF stimulation was applied prior to VLF-HFS application. In P1-P3 mice, as in the control condition , in the p = 0.45). Moreover, the magnitudes of depression of VLF-EPSC amplitudes attained during LTD or STD were not significantly different in control condition and in the presence of each of the three mGluR agonists  relationship in lumbar MNs by applying depolarizing current pulses injected in series . During this protocol, two different types of responses were observed when spike threshold was reached: 22 out of 68 (32%) P1-P3 MNs and 29 out of 51 (43%) P8-P12 MNs exhibited only a solitary spike at the beginning of the depolarizing pulse (f-I relationships in the two age ranges examined (f-I slope). In P8-P10 MNs, however, DHPG and L-AP4 . As illustrated in f-I relationships of MNs, which have been shown to be closely correlated with AHP amplitudes (We then examined whether the after-spike hyperpolarization (AHP), a major target of neuromodulators in MNs, was modified in the presence of the mGluR agonists. In control conditions, the AHP amplitude values were found not to be significantly different between P1-P3 and P8-P12 MNs  was found to be significantly higher than in P1-P3 MNs , although at least four subtypes, mGluR5, mGluR2, mGluR3 and mGluR7, were significantly down-regulated during the second postnatal week  which have been shown to contain predominantly flexor and extensor MNs, respectively  and 783 Gastrocnemius MNs (from 5 P1 mice) were collected and purified . With this experimental approach, all the genes tested were detectable in both MN subtypes with the exception of Grm4 and RT-qPCR analysis revealed that the level of Grm2 expression was significantly lower in Gastrocnemius MNs compared to Tibialis MNs . This finding therefore indicated that extensor and flexor MNs exhibit slightly, albeit significantly, different mGluR expression profiles.In a next step, we quantified and compared mGluR gene expression levels in laser-microdissected MNs that had been retrogradely labeled from the ankle extensor muscle r muscle . It shoupression . The mRNAs the ADSP expressed at VLF-MN synapses is dependent on the flexor or extensor identity of MNs, we sought whether the effects of the mGluR agonists differ between MNs exhibiting different ADSP profiles at VLF-MN synapses after VLF-HFS. We found that the mGluR-mediated modulations of VLF-EPSC amplitude values, EPSC2/EPSC1 ratio, input membrane resistance and AHP amplitude were similar whatever the ADSP expressed by MNs and at the two developmental stages tested (data not shown).in vitro spinal cord preparation of new-born rodents, bouts of fictive locomotion can be induced by 50 Hz stimulation of the VLF and recorded from lumbar ventral roots (n = 14 preparations) and L2 burst amplitudes .In the al roots . We thenal roots . In contal roots , numbersplitudes  across iplitudes . As indith stimulation in about 50% of the preparations tested . The subcutaneous injection of aCSF had no effect on the parameters of monitored swimming activity, either of P3 .SB conceived the study. CQ and SB designed and performed the experiments and analyzed the data. CQ, J-RC, and SB wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Our re-evaluation of the fossil and lithic material from Shukbah Cave confounds such assumptions due to the identification of a Neanderthal molar tooth alongside Nubian Levallois cores and points at the site2. Hallinan and colleagues3 question this finding, instead supporting the use of Nubian Levallois technology as a fossile directeur to track expansions of Homo sapiens. We tackle these critiques, highlighting the problematic foundations in the assertion that Nubian Levallois technology is a unique, discrete entity, resulting in its misuse to support simplistic culture-historical narratives.An exclusive connection between 6, but both here and elsewhere it is evident that major sedimentary phases were resolved. The provenance of the Neanderthal molar and faunal records from the site both support a late Middle Palaeolithic timeframe, with no wider evidence from the site currently supporting substantive, earlier occupations. Garrod6 directly cautioned the rare presence of artefacts from distinctly later timeframes and younger technological systems within the Layer D assemblage may be attributed to the incised upper contact of Layer D, and is consistent with the 11 younger intrusive elements identified by Hallinan and colleagues3.Garrod’s excavation notes and publications on Shukbah indicate that finer stratigraphic diversity was recognised within the breccia deposits6, from a chaîne opératoire analysis by Callander7 that included the Rockefeller collection, and our attribute-based analysis of 707 artefacts2. Callander illustrates the uneven distribution of artefacts from Shukbah D between multiple major collections, with a notably higher proportion of retouched pieces and lower proportion of Levallois elements present in the small Rockefeller collection than elsewhere. We therefore suggest that the examination of a small and particularly biased sample by Hallinan and colleagues gives misleading insight into the character of the Shukbah D assemblage, as evident when contrasted with studies of much larger sample sizes by Garrod6, Callander7, and ourselves2. While we cannot preclude the possibility of earlier occupation of the site, the incidence rate of artefacts Hallinan and colleagues3 consider characteristic of such episodes (listed as LP [n = 3] or early MP [n = 7]) is extremely low (0.8%) amongst the artefacts recovered by Garrod (n = 1235), and may have been substantially lower had artefact retention practices been more comprehensive. Defining the presence of whole cultural phases based on single or rare artefacts, whilst overlooking the overwhelming majority of an assemblage, is questionable. On these terms, then, it would appear that any mixing of the Shukbah D assemblage has been minimal. The combined presence of a dominant focus on Levallois point production alongside centripetal and bidirectional flake production in the Shukbah D assemblage is directly comparable to other, dated, late Middle Palaeolithic sites, such as Kebara10, which were occupied by Neanderthals and contain comparable faunal records to Shukbah6. Assessments made by multiple researchers clearly demonstrate the classic late Middle Palaeolithic character of the Shukbah Layer D assemblage with a predominant focus on Levallois point production.A late Middle Palaeolithic attribution of the assemblage is consistent with the typological character of the Layer D assemblage as reported by Garrod6. Hallinan and colleagues3 present partial quotes from Garrod’s description to suggest that the Neanderthal molar derived from the contact between Layers D and B. Garrod unequivocally attributed the Neanderthal molar to the intact breccia deposits that are ascribed to Layer D: “A few human fragments were found in D, viz., a small portion of a temporal bone and a molar tooth, both from the base of the hummock on the edge of the pit”6. The tooth was overlain by ca. 2 m of Layer D breccias on the right side of the hummock with no direct contact with Layer B, contra Hallinan and colleagues3. Distinct stratigraphic contexts are described by Garrod for other human fossils potentially associated with abraded deposits6, which have no bearing on the secure attribution of the Neanderthal molar tooth to the Layer D breccias that yielded the late Middle Palaeolithic stone tool assemblage.The provenance of the molar tooth is clearly described by Garrod11, the parameters of which we used to identify the presence of Nubian Levallois technology in the Shukbah D assemblage. Hallinan and colleagues3 critique our predominant presentation of artefacts in plan (78%). Our approach is consistent with contemporary reporting of Nubian technology from Arabia by Usik and colleagues11, in the Levant by Goder-Goldberger and colleagues13, and in South Africa by Hallinan and Shaw14, where 65–95% of cores are only illustrated in plan. The broadest range of definitions of Nubian Levallois technology share focus on description of flaking surface scar patterns1, which are best observed in plan.We documented nine cores in plan, as well as multiple views of two cores in SI Fig. 2 that visibly meet the four criteria set out by3 assert both the distinct form of Nubian Levallois reduction and a consensus in the criteria for its definition, following Usik and colleagues11. This overlooks considerable debate in the literature1. Usik and colleagues11 identify discrete modes of preparation of the flaking surface for Type 1, 1/2, and 2 Nubian Levallois cores, that Crassard and Hilbert15 suggest unnecessarily formalises plasticity, given that cores examined today are static elements of a past dynamic system. Goder-Goldberger and colleagues12 highlight that a definition that maintains the distinctness of Nubian technology is critical to its use as an “archaeological missing link”12, yet Usik and colleagues11 indicate that as distal flaking surfaces become flatter Nubian cores can be seen as “grading into bidirectional cores or recurrent cores”, Hallinan & Shaw14 identify cores with ‘Nubian affinity’ but not enough to confidently identify them as Nubian, and Rose and colleagues16 identify that “ there is overlap between Nubian Type 2 core preparation and some preferential point-producing Levallois reduction systems in the Levantine Mousterian”. Given this broad recognition that Nubian Levallois technology can grade into other Levallois approaches, some appraisal must be made of how and where to divide this spectrum of variability, as well as why.Hallinan and colleagues11, nor do they demonstrate them to be discrete from other Levallois methods. For instance, it is not established that there is a discrete distribution of distal flaking surface angle of Nubian and other Levallois cores that can be simply divided at 120 degrees, and in practice this quantitative threshold is exceeded elsewhere by Hallinan and co-authors14 in identification of Nubian cores. Nevertheless, to answer the Hallinan and colleagues3 critique the distal ridge angles on ten cores from Shukbah D stored at UCL have been measured. The angles range from 83° to 120°, with a mean of 102.4°,meaning that they are consistent with the particular descriptions set out by Usik and colleagues11 and favoured by Hallinan and colleagues3. Likewise, Hallinan and Shaw14 identify the difficulty in applying the shape categories, arguing instead for the need for quantitative analyses, which again should be established through comparative study if argued to be distinct features of Nubian Levallois technology. This remarkable absence of comparative studies prohibits acceptance that Nubian Levallois technology is inherently discrete from other Levallois methods.No comparative basis to identify the categories chosen to define Nubian Levallois technology are set out by Usik and colleagues3 contest whether Nubian Levallois end products can be identified, although they are commonly identified in the recent literature. For instance, Rose and colleagues indicate that Nubian Levallois reduction “results in the signature Nubian Levallois point”16, Goder-Goldberger and colleagues indicate “Other recognizable artifacts associated with the Nubian reduction sequence are the specific core shaping blades and the end-products”12 and that "the Nubian flaking system is unique and includes identifiable cores, preparation blanks and end products"13, whilst Hallinan and Shaw identify Nubian products from "various reduction strategies including Type 2, 1/2 and, to a lesser extent, Type 1 Nubian methods" from an appraisal of dorsal scar patterns14. Indeed, the need for quantitative study between Nubian and other Levallois points was highlighted as a "major limitation in the study of Nubian technology"14. Our identification of Nubian Levallois products is comparable to that set out by Hallinan and Shaw14 and provides a testable hypotheses for further analyses: that the use of Nubian Levallois reduction schemes has no significant impact upon the key metric attributes we studied in comparison to other Levallois points.Hallinan and colleagues17 for the presence of Nubian Levallois technology. Contra Hallinan and colleagues3, who argue we overlook the potential role of raw material procurement on driving variability, we suggested that the immediate availability of larger clasts at the southern Arabian sites of TH123b and TH383 best explains the distinct variability observed in comparison with other sites, highlighting the larger size of these cores and the role of differential reduction intensity.We undertook extensive quantitative comparative studies of Late Pleistocene Middle Palaeolithic and Middle Stone Age toolkits across the Levant, Arabia and eastern Africa, a considerable research endeavour evident from the scarcity of similar analyses at comparable scope and scale. Indeed direct reappraisal of existing assemblages such as Aduma, rather than reliance on existing reports, is critical to evaluate diversity of Levallois reduction approaches; in this instance, we rejected previous assertions13), perhaps more so than Garrod’s excavation practices. These factors may have limited scope for comparative analyses to date but should similarly limit the use of this technology as a fossil directeur to track human expansions. Increasing sample sizes within and between sites will undoubtedly enhance precision of comparative studies, yet our results provide the means to test key hypotheses—whether Nubian Levallois is discrete from other Levallois technologies. Hallinan and colleagues3 argue that incorrect identification of Nubian technology best explains why considerable overlap exists between our Nubian and Other Levallois categories for Shukbah and other sites. However, this overlooks comparable results from other metric analyses15 as well as more extensive analyses involving refitting studies18, which support our results and demonstrate Nubian and ‘classic’ Levallois can be variants of a single technological approach.Nubian Levallois technology is best known from undated surface sites, typically occurring in low frequencies compared to other reduction methods. A narrow focus on Nubian Levallois technology has led to biased sampling practices that can result in the destruction of assemblage integrity (e.g.11 determined the criteria they set out is opaque, prohibiting clear differentiation from other Levallois methods. Hallinan and colleagues3 identify a core within the Shukbah D assemblage that meets key criteria set out by Usik and colleagues11. However as further ‘in-depth technological analyses’ are deemed necessary to resolve whether or not the core results from ‘a true Nubian reduction scheme’, it is apparent that these preferred criteria alone do not provide sufficient analytical utility to identify Nubian reduction schemes. This problem is exacerbated by Hallinan and colleagues3 asserting a single definition for the identification of Nubian Levallois cores, overlooking plurality in the literature, whilst simultaneously highlighting such plurality in previous studies as prohibiting the identification of Nubian Levallois point blanks. If, as Hallinan and colleagues3 assert, “a unified definition of Nubian technology is fundamental to a better understanding of its significance”, we consider it essential that such definitions are derived from comparative studies. In the absence of clear comparative studies that demonstrate the unique, discrete qualities of Nubian Levallois technology, we support suggestions that it forms part of a spectrum of variability amongst Levallois reduction approaches often associated with semi-arid landscapes18, which is consistent with its independent appearance in areas including southern Africa19. This provides the simplest explanation for its appearance at Shukbah, as well as the results of our comparative study. In contrast, the use of Nubian Levallois technology, and particularly its appearance in undated surface sites and the absence of robust association between artefacts and human fossils, to track the expansions of a discrete biological population, Homo sapiens, remains problematic. As a result, the use of Nubian Levallois technology by other populations that employ Levallois reduction methods and focus on the production of points is, perhaps, unsurprising.Amongst the plurality of definitions of Nubian Levallois technology in the literature, the basis upon which Usik and colleagues


The flexural strength of the epoxy nanocomposite was very high even at a low silica content because of the strong interactions between the PDMS-treated fillers and the epoxy matrix. These strong interfacial interactions originate from the attractive forces between the polymer and the filler. Therefore, the polymer nanocomposite containing the PDMS-treated fumed silica is shown to be sufficiently commercially promising.The objectives of this study are to improve the thermal and mechanical properties of epoxy/fumed silica composite with different surface treatments of fumed silica. The addition of silica nanoparticles improved the thermal stability of the composite and slowed down the pyrolysis process. The crosslinking density and T In engineering structures, components such as factory piping are exposed to high temperatures; hence, these materials should exhibit high durability, chemical resistance, and thermal stability. As an important requirement for industries, design of high-strength materials with heat resistance has been prioritized . PolymerThe binders used in most polymer composites are composed of a thermosetting resin and epoxy. Epoxy resins are widely used in various industrial applications, such as paints, coatings, composite materials, 3D printing, and adhesives, because of their excellent mechanical properties, heat resistance, insulation, and adhesive strength. Moreover, it is possible to control their curing rate by adding various curing agents. However, low conductivities and high coefficients of thermal expansion are the major disadvantages of epoxy resins. Further, this material has a weak external-impact withstanding capability because of its high crosslinking density due to the formation of a 3D network structure. Therefore, when an epoxy composite is used in combination with a material having a different coefficient of thermal expansion, such as a metal, peeling or cracking may occur because of their different thermal deformations. In recent times, many studies have been conducted to improve the fluidity and enhance the impact reinforcement effects of epoxy resins by introducing fumed silica particles ,20,21,22g) and crosslinking density were determined by dynamic mechanical analysis (DMA). Moreover, a bending test was performed using a universal testing machine to study the mechanical properties of the epoxy nanocomposite.In this study, fumed silica with different surface properties was physically dispersed in an epoxy resin, and then the viscosity of the resulting mixture was measured to confirm the dispersibility of fumed silica in epoxy resin. The curing behavior of the mixture was analyzed by differential scanning calorimetry (DSC). The thermal stability of the cured epoxy nanocomposite was confirmed through thermogravimetric analysis (TGA), and its glass transition temperature  were used as the curing agents. The molecular structures of the epoxy and polyether diamines are shown in A bisphenol A-type epoxy resin  containing diglycidyl ether of bisphenol A (DGEBA) with butyl glycidyl ether (BGE) was used. Butyl glycidyl ether (BGE) and polyether diamines  and PDMS-treated fumed silica  were provided by Evonik Industries .Fumed silica , followed by stirring at 23 °C for 3 min and ultrasonication . After cooling, a curing agent was added and the defoaming process was performed for 1 min. Next, epoxy/fumed silica mixture with the curing agents was initially cured at 80 °C in a Teflon sheet for 3 h, and the solvent was evaporated, followed by post-curing at 150 °C for 2 h .−1 using a BERTEX80 FTIR spectrometer  to identify the functional groups present in fumed silica. After the sample was dried at 160 °C for 24 h, a KBr pellet was prepared for the FTIR characterization. The number of scans was 32, and the resolution was 4 cm−1. Field-emission scanning electron microscopy  was employed to analyze the structure of fumed silica (Fourier transform infrared (FTIR) spectroscopy was conducted between 4000 and 400 cmd silica . The mor3 s−1, and the results were analyzed to obtain the rheological properties of the fumed silica–epoxy resin mixture.The difference in the dispersibilities of the fumed silica that underwent different surface treatments was determined by measuring the viscosity of the untreated and the treated fumed silica and epoxy resin mixture at room temperature using a rheometer  equipped with parallel plates, each with a diameter of 25 mm. Steady shear measurement was then performed at a shear rate range of 0.01 to 10To examine the thermal stability of the composites, TGA of the epoxy/fumed silica composites was performed using a TGA 4000 system . The TGA measurements were conducted at a heating rate of 10 °C/min, over a temperature range of 30–800 °C, under a constant nitrogen flow atmosphere. Specimens were measured only once for each content. The curing behavior of the epoxy/fumed silica and epoxy/PDMS-treated fumed silica was examined by DSC  at a heating rate of 10 °C/min (from 30 to 300 °C) under a nitrogen flow of 20 mL/min. Three specimens were measured.The temperature dependences of the dynamic storage modulus (G’) and tan δ values of the epoxy/fumed silica composites were evaluated by DMA  in the film-tension mode over a temperature range of 35–200 °C, at a heating rate of 5 °C/min, strain rate of 0.1%, and frequency of 1 Hz. The dimensions of the specimens were 60 mm × 12 mm × 3 mm. The flexural properties of the composites were determined using a three-point bend test. The epoxy/fumed silica composites were measured using a 10 kN universal testing machine  at a crosshead speed of 0.1 mm/s. The flexural specimens had dimensions of 60 mm× 12 mm × 3 mm. The tests were performed at a temperature of 25 ± 1 °C and humidity of 50 ± 2% RH, in accordance with the ASTM D790 standard methods.In this study, a DGEBA-type epoxy resin diluted in BGE was used as the epoxy resin, and polyether diamines were used as the crosslinkers. −1, respectively. The spectrum also showed absorption peaks at 3435 and 1631 cm−1, which could be assigned to the stretching and bending vibrations of the OH group on the silica surface. In the FTIR spectrum of the PDMS-treated fumed silica, additional peaks at 2960 (assigned to C–H stretching in CH3), 1262 (CH3 symmetric bending in Si–CH3), and 804 cm−1 (-CH3 rocking in Si–CH3) were observed, which confirmed the existence of different functional groups in the PDMS-treated fumed silica [As shown in d silica ,25,26,272O3 composite material decreased as the shear rate increased [After sufficiently dispersing the fumed silica with different surface functional groups in the epoxy resin (YD-115) through mixing and ultrasonication, the steady shear viscosities of the mixtures with different silica contents were measured c,d. As sncreased ,28. Compp, decreased with increase in the epoxy/fumed silica composite filler content. The Tp decreased from 132.5 to 129 °C as the PDMS-treated fumed silica content was increased from 1 to 5 wt.%, respectively, as shown in The curing reaction of the epoxy composites was investigated by DSC. The morphological characteristics of the epoxy/fumed silica composites with 3 wt.% fumed silica were analyzed by TEM. f is the char yield (%) of epoxy/fumed silica composites, and W0 is the char yield (%) of pure epoxy. The untreated fumed silica rapidly formed char upon heating to high temperatures. Owing to the good dispersibility of PDMS-treated fumed silica, only a small amount of char is formed in the beginning; however, there is a steep increase in the char formation when the silica content is increased to 5 wt.%.To determine the thermal stability, TGA of the epoxy/fumed silica composite was performed. The TGA curves of the pure epoxy and epoxy/fumed silica composites showed similar thermal degradation tendencies as evident from 5 was defined as the temperature at which the mass percentage of the epoxy/fumed silica composite decreased by approximately 5% , and Tf is the final experimental temperature (800 °C). For the analysis, the TGA plot was divided into three regions represented by S1, S2 and S3, as shown in The integral procedural decomposition temperature (IPDT), proposed by Doyle, is related to the volatile fraction of polymers and can be utilized to estimate the overall intrinsic thermal stability of polymers during decomposition. In this study, the IPDT was calculated from the TGA curve using the following equations ,37,38,39Jin, F.L. et al. and Chiang, C.L. et al. showed that nano inorganic particle improve the thermal stability of pure epoxy. Liu, Y.L. et al. reported epoxy-silica hybrid composites have an increase in IPDT with increasing silica content. It means that silica particles contribute to the level-up of IPDT while delaying the thermal decomposition rate of the organic part of the organic-inorganic hybrid composite ,36,39. Tg of the epoxy composite containing silica nanoparticles is higher than that of the pure epoxy resin by approximately 5 °C [g of the epoxy/fumed silica composites increased because of the PDMS treatment-induced interfacial interactions between the fumed silica particles and the epoxy matrix.The effect of silica nanoparticles with different surface functional groups on the viscoelastic properties of the crosslinked polymers was analyzed using DMA . The stoely 5 °C ,42,43,44Flexural tests were performed to confirm the mechanical properties of the epoxy composite material. The interaction between the nanofiller particles and the matrix was confirmed by applying a continuous force to the structure. 5, char content, and IPDT of the composites were analyzed. The addition of silica nanoparticles improved the thermal stability of the composite and slowed down the pyrolysis process. The crosslinking density and Tg of the epoxy/fumed silica composites increased because of the interfacial interaction between the PDMS-treated fumed silica particles and the epoxy matrix. The flexural strength of the epoxy nanocomposite was very high even at a low silica content, because of the strong interactions between the PDMS-treated fillers and the epoxy matrix. These strong interfacial interactions originate from the attractive forces between the polymer and the filler. Therefore, surface-treated fumed silica is a promising filler material for industrial applications.The dispersibility of fumed silica particles in a polymer matrix was confirmed by measuring the viscosity of a mixture of fumed silica and epoxy resin with different surface properties. Depending on the surface treatment of fumed silica, shear thinning behavior and Newtonian behavior was observed in the composite. In particular, the initial viscosity of the PDMS-treated silica mixture increased with increase in the silica content. In the curing reaction of the mixture, the maximum temperature at the exothermic peak decreased as the content of the fumed silica filler was increased. These results show that the fumed silica filler has a catalytic effect on the epoxy curing reaction. Additionally, the Td


Epoxy resins, due to their high stiffness, ease of processing, good heat, and chemical resistance obtained from cross-linked structures, have found applications in electronics, adhesives coatings, industrial tooling, and aeronautic and automotive industries. These resins are inherently brittle, which has limited their further application. The emphasis of this study is to improve the properties of the epoxy resin with a low-concentration (up to 0.4% by weight) addition of Multi-Walled Carbon Nanotubes (MWCNTs). Mechanical characterization of the modified composites was conducted to study the effect of MWCNTs infusion in the epoxy resin. Nanocomposites samples showed significantly higher tensile strength and fracture toughness compared to pure epoxy samples. The morphological studies of the modified composites were studied using Scanning Electron Microscopy (SEM). Nanoscience paves new avenues in the field of the scientific community as well as industry. In the case of civil engineering, a substantial interest can be observed in nano-sized materials, such as nano-silica powder, nanofibers, and nanotubes intended for structural applications . NanofibGantayat et al.  develope2/g .The material used in the present investigation is Epoxy resin and MWCNTs. The epoxy resin used to carry out this work is Bisphenol-A (L-12) and associated hardener L-aliphatic amine (K-6) . MWCNTs were manufactured by the Chemical Vapor Deposition method , which rPure epoxy nanocomposites loaded with MWCNTs in proportions of 0.1–0.4 wt.% in the incremental steps of 0.1 to matrix weight were synthesized. The steps followed during the preparation of these nanocomposites are shown in 2 is applied for the curing specimen to obtain a flat plate. The mixture is then cooled for 2 h. Finally, MWCNTs reinforced epoxy is poured into the mold and cured at room temperature [First, MWCNTs, 250 gm of epoxy resin, 25 gm of curing agent, along with 0.5 mL of degassed solvent are mixed to form a mixture. The mixture is sonicated using a bath for 120 min to minimize the agglomeration of MWCNTs and to ensure uniform dispersal of NPs in the matrix. As the epoxy resin passed between three rollers, it was subjected to a shear force that untangled the NPs and dispensed the dispersed NPs into the epoxy resin. The curing agent is added to the mixture and stirred for 5 min at a speed of 350 rpm by a mechanical agitator. The mixture is then placed in a vacuum chamber coupled in an oven for 20 min and then exposed to atmospheric pressure for 10 min. This process is employed to reduce the viscosity of the epoxy resin that relieves any trapped bubbles in the agitated mixture, thereby enhancing the fluidity. A pressure of 150 kN/mperature . NanocomTensile testing of the PE and nanocomposites were carried out as per ASTM D 3039 standard using a dog bone-shaped specimen shown in The composite beams were assessed for strength-deflection criteria. The results were weighed against the values of the pristine specimens. The outcomes are presented in The load curves are shown in 2-sp3. For 0.3 wt.% of MWCNTs infusion in the resin matrix, tensile strength attained was 63.0 N/mm2 against neat epoxy of 24.8 N/mm2, an upsurge of 154%. The dosage of MWCNTs beyond 0.3 wt.% up to 0.4 wt.% tensile strength decreased, and this could be due to the sword in sheath mechanism of failure  means the stress was primarily concentrated on the outside walls of the MWCNTs when compared to the inner walls before rupture [The reasons could be due to the high aspect ratios of MWCNTs which form the significant factor in ductility enhancement. This can also be attributed to the flexibility of the MWCNTs and the rehybridization capacity of the carbon atoms in the degree of sp rupture .The experiment fits the best curve for polynomial regression when the degree of freedom was taken to 5, as shown in The tensile stress vs. strain plots is depicted in The tensile strength of all samples increased with the addition of the MWCNTs, and the changes in strength were almost similar . From thThe experiment fits the best curve for linear regression, as shown in Flexural properties of PE and nanocomposites were determined using the three-point bending method as per ASTM D 790 standard. The specimen used for conducting the test are as shown in The tests were conducted on Tinius Olsen UTM (10 kN capacity) using a crosshead speed of 1 mm/min with a width of 30 mm. The flexural modulus and strength were determined from these tests. Five specimens from each variation were tested, and the results were averaged. A support gap to depth ratio of 16:1 is maintained. The loading is made till the rupture of the external surface of the sample or till a strain reaches a maximum value of 5%, whichever is earlier. A strain extent of 0.01 mm/min is maintained to apply a gradual load on the trial sample. The machine was calibrated so that the fault in the capacity measurement does not vary more than ±1% of the max load that is to be quantified.The load-deflection plots obtained from the three-point bending tests of PE and MWCNTs loaded epoxy composite are shown in Morphological studies, such as SEM and EDX, are conducted to study the effective interfacial adhesion between the holding matrix and the strength fillers . ScanninThe rupture ace of epoxy composites altered by MWCNTs was characterized by SEM to study the dispersion of the MWCNTs in the holding matrix. EDX analyses were conducted on the selected samples to verify the metallic constituents of CNTs that influence mechanical properties under study. The metallic traces present in the fillers are shown in From the above experiment, we can conclude that the highly flexible behavior of CNTs has made them an ideal reinforcing phase. In the case of a tensile test, comparing the results of plain polymer with composites reinforced with epoxy/MWCNTs, that is, in the case of specimen A3 (0.3 wt.%), it is observed that an increase in tensile strength by 61% as compared to the unfilled epoxy resin. This could be due to the aspect ratio of MWCNTs, flexibility, and rehybridization capacity of the planar graphene sheet that makes up MWCNTs. Composites with a higher percentage of CNTs showed declining trends of tensile strength, which can be attributed to the non-homogeneous dispersal of MWCNTs. In the case of flexural strength, comparing the results of plain polymer with composites reinforced with epoxy/MWCNTs, that is, in the case of specimen A4, it is observed that an increase in flexural strength by 60% as compared to the PE specimen is observed. This is due to the hindrance of the crack growth at the micro-level by bridging the crack at the nano-level itself. The reinforcement beyond A3 (0.3 wt.%), that is, A4, has shown deteriorating trends in the strength features. This may be because of nanotube waviness with the increase in filler content. Composites with a higher % of CNTs showed declining trends of tensile and flexural strength. SEM analysis shows the dispersion of MWCNTs in the polymer matrix, and EDX shows the presence of other elements in the sample, which will affect the strength of the composites.


Dynamic mechanical analysis (DMA) is one of the most common methods employed to study a material’s viscoelastic properties. The effect of thermal aging on plain epoxy and a fique fabric-reinforced epoxy composite was investigated by comparing the mass loss, morphologies, and DMA properties of aged and unaged samples. In fact, thermal aging presents a big challenge for the high-temperature applications of natural fiber composites. In this work, both plain epoxy and fique fabric-reinforced epoxy composite were found to have different molecular mobility. This leads to distinct transition regions, with different changes in intensity caused by external loadings from time-aging. Three exponentially modified Gauss distribution functions (EMGs) were applied to loss factor curves of fique fabric-reinforced epoxy composite and plain epoxy, which allowed identifying three possible mobility ranges. From these results it was proposed that the thermal degradation behavior of natural fibers, especially fique fiber and their composites, might be assessed, based on their structural characteristics and mechanical properties. One of the most common methods used to measure a material’s viscoelastic properties is the dynamic mechanical analysis (DMA). Based on DMA it is possible to have information about the temperature- and time-dependent behavior ,2, as weThe importance of the accelerated aging studies has been recognized as a research method, which serves the purpose of determining the usability conditions and period of a product . NaturalIt has been discussed that reinforcement of natural fibers in the thermoplastics and thermoset matrices offers several benefits such as having low cost, being ecofriendly renewable, and causing less damage to processing equipment compared to synthetic reinforcements . This diThe use of NFRPC is expected to grow in the future for specific applications, for example, use of light-weight natural fiber composites in ballistic armor ,32,33. IFurcraea andina [Among the lesser known natural fibers that have been investigated as reinforcement of polymer composites stands the fique fiber extracted from the South American plant a andina ,35,36. Ia andina ,22,37.A recent investigation disclosed the superior ballistic performance of fique fabric as personal armor in comparison to worldwide-used synthetic aramid fabric ,39,40. AHowever, for successful application of these composites as ballistic armor their change under aging at a limit degradation temperature must be evaluated. Hence, the present work investigated the accelerated thermal aging of 40 vol% fique fabric-reinforced epoxy composite and the matrix itself, here named as plain epoxy (PE).2, and volume density, 1.53 g/cm3, were measured according to the corresponding standards in a previous article [The fique fabric,  article . The mat3, in a Buehler Isomet cutoff machine  using a diamond blade. Methods of accelerated thermal aging, as further described, were applied to these specimens.The 40 vol% fique fabric–epoxy matrix composite was fabricated by placing the previously dried fabric piece as ply layers inside a steel mold, and DGEBA-TETA resin was also poured in. Laminated plates of 150 × 120 × 3.2 mm were made by the compression molding process and cured for 24 h at room temperature, inside the mold, under a 5-ton load. For DMA tests, these plates were cut into specimens, with dimensions of 50 × 13 × 3.2 mmThe fique fabric–epoxy composite and plain epoxy samples were subjected to an accelerated aging test performed in air chambers  at 170 °C for progressively increasing time lengths . The aging temperature of 170 °C was selected as the limit temperature before degradation in both the epoxy ,42 and t0 h (no aging): control sample;Aging temperature: 170 °C (T); andAging time: 72, 120, 240 h.The mass and dimensions, particularly the thickness, of the fique fabric–epoxy composite samples were measured before and after aging using a 0.001-g precision Gehaka scale and a 0.01-mm Mitutoyo digital caliper, respectively.The microstructural properties, damage mechanisms, and degradation were monitored using scanning electron microscopy (SEM) images of the plain epoxy and fique fabric composite, in aged and unaged states. A model Quanta FEG250 FEI microscope Thermofisher Scientific, Hillsboro, OR, USA, operating with secondary electrons at 28 kV was used. Samples were gold-sputtered for electron conduction. The study focused on the accelerated aging of the surface layer of the composite and the plain epoxy, which means no “in core” analysis was intended.TGA curves of epoxy and fique fabric composites were carried out in a Shimadzu equipment, Tokyo, Japan. Samples were crushed and put in a platinum crucible to be analyzed under nitrogen atmosphere in a temperature range from 20 °C to 700 °C at a heating rate of 10 °C/min.DMA was used to characterize the temperature-dependent viscoelastic properties of the materials described in In the present investigation, the effects of thermal aging on the morphology and thermo-viscoelastic-mechanical properties were evaluated at 170 °C for different exposure times.It is well known that epoxy properties are strongly dependent on the usage conditions, such as time and temperature. When exposed to temperatures of about 100–200 °C, polymers suffer from softening, creep, distortion, and deterioration of mechanical properties . Figure The aging of polymers in an oxidative environment, such as the one employed in this study, leads to a heterogeneous degradation, which is governed by the diffusion of oxygen through the polymer . PreviouSEM images of fique fabric-reinforced epoxy composite unaged and thermally aged are shown in Thermal oxidation of the fique fabric–epoxy composite introduced damage at the fiber–matrix interfaces, which caused the formation of microcracks and degraded the structural property. It was possible to note that all exposure times promoted the fiber–matrix debonding and fiber fibrillation due to the degradation of less stable components of the fique fibers as well as shrinkage of the epoxy. The induced thermal stress may be relieved by crack formation in the matrix and fiber failure . Both maAll samples of the fique fabric, plain epoxy, and fique fabric composites revealed modification in the macrostructural level by the color change. A brownish layer formation at the sample surface was also reported in the literature ,49,53,54The dynamic mechanical relaxation behavior of fique fabric-reinforced epoxy composites and plain epoxy annealed at 170 °C up to 240 h and unaged was investigated. The main DMA parameters for both aged and unaged 40 vol% fique fabric composite and plain epoxy are shown in One should notice that the E’ value of the FC-T72 was the highest among the conditions due to an increase of the chain mobility during the glassy/rubbery transition . By contFurthermore, the results indicated a more pronounced effect of exposure time on aged composite samples in comparison to plain epoxy ones. It might have been caused by the fiber–matrix interface weakening on the composites. Nevertheless, for both fique fabric composites and plain epoxy, an increase of Tg was observed for the samples aged up to 120 h. The results revealed an increase of up to 45 °C on Tg, the same as found by Inamdar et al. .g. This provided the thermal energy to activate monomers to further crosslink and then the storage modulus achieved a plateau associated with final crosslinking reactions [Another point worth noting in eactions .It is well known that another noticeable effect of the 3D molecular arrangement of the polymer matrix is the main peak in the curve of loss modulus (E”). The loss factor (tan δ) is the E”/E’ ratio, which represents the mechanical damping or molecular internal friction of a viscoelastic configuration. The effect of time aging on the tan δ for the fique fabric-reinforced epoxy composite and plain epoxy is shown in i); (2) the half peak width at half height of the Gaussian part of corresponding EMG (wi); (3) the temperature at peak maximum of Gaussian part of the EMG (Tc1); and (4) the relaxation parameter in the exponential part of the EMG (Toi) [2 coefficient. The number of fit parameters obtained was equal to i, wi, Tci, Toi). DMA investigations revealed distinct changes in the shape of the loss factor (tan δ) curve. One can note that the tan δ curves, in MG (Toi) ,8,9. FigMG (Toi) ,8,9. Aft1, Tc2, and Tc3) increased with aging time. The same process was considered for fique fabric composite, and the EMGs’ fit parameters are presented in i) was caused by (1) hindrance in mobility, (2) increase of stiffness, and (3) increase of crosslinking. In addition, the peak temperature (Tci) of the Gauss part can be interpreted as Tg of the relaxation free transition, i.e., transition without the exponential part; one has a pure Gauss distribution. By contrast, when the exponential relaxation (Toi) was more pronounced, the higher was the residual internal friction.The sum of peak areas is considered the total area of the loss factor curve, and for almost all conditions for both plain epoxy and fique fabric composites these values decreased. This means that restrictions on chains’ mobility increased. Furthermore, a small peak area  phenomenon. The DLO facilitates the formation of a heterogeneous oxidized layer such that the regions near the air-exposed surfaces are heavily oxidized compared to the interior regions, which show reduced or minimal oxidation . In summFrom these results it is proposed that the thermal degradation behavior of natural fibers, especially fique fiber, and their composites might be assessed, based on their structural characteristics and mechanical properties.Hemicellulose loss and oxidation are believed to be the main mechanisms for the microstructure changes that occur during thermal aging of fique fabric-reinforced epoxy composites. All samples of fique fabric, plain epoxy, and fique fabric composites revealed physical aspect changes at the macrostructural level, exhibiting a formation of a brownish layer at the sample surface.Mass loss rapidly increased at the initial stage of thermal aging, mainly due to the evaporation of moisture and the volatilization of residual low-molecular-weight substances. The samples thermally exposed for 120 h showed the higher mass loss, ∼14%, and high shrinkage, ~15%, which might be associated with the outgassing of resin oxidation by-products.In DMA analyses, we observed significant property changes after aging. The most notable changes occurred in the storage modulus (E’) curves. For samples aged for 72 h, a significant decrease in E’ modulus was observed for both the plain epoxy and the fique fabric composite. In addition, an increase of Tg was observed for the samples aged up to 120 h. By contrast, the fique fabric composite aged with 240-h aging time exhibited a lower E’ modulus, which indicated the degradation at the fiber–matrix interface, also verified in the SEM analyses. Additionally, it is worth mentioning the presence of a broad peak around 98 °C for the unaged plain epoxy, which was attributed to the post-curing reaction given by the incomplete crosslinking.The loss factor results were modeled with three exponentially modified Gauss functions in order to get a molecular interpretation of the involved phenomena, which can be analyzed by the areas of the second and third transition peaks or the corresponding temperature values of the assigned Gauss peaks. The fique fabric composites presented smaller peak areas compared to the plain epoxy, which might be explained by the higher hindrance in mobility, an increase of stiffness, and an increase of crosslinking.


Diabetic nephropathy (DN) is the leading cause of the end-stage renal disease (ESRD). The proliferation and apoptosis of mesangial cells induced by the activated Wnt/β-catenin pathway is crucial in DN. Trigonelline (TRL) is an alkaloid that has been shown to decrease proteinuria and protect the renal function in DN. However, the effect of TRL on the Wnt/β-catenin pathway of mesangial cells is unclear.As a cellular DN model, human mesangial cells (HMCs) were treated with high-glucose (HG). β-Catenin plasmid and control knockdown plasmids were transfected into HG-treated HMCs as β-catenin pcDNA and β-catenin siRNA groups, respectively. Cell viability was measured by MTT assay. Flow cytometry was used to detect the cell cycle. Cell apoptosis was evaluated by flow cytometry and terminal dUTP transferase nick end labeling (TUNEL) assay. mRNA expression of Wnt1, Wnt3a, Wnt4, Wnt5a, β-catenin, TCF4, Cyclin D1, and CDK4 were detected by qRT-PCR. Protein expression of Wnt4, Wnt5a, nucleus-β-catenin, TCF4, Cyclin D1, and CDK4 were detected by western blotting.TRL significantly inhibited HG-induced HMCs viability over three-time points measured . In addition, TRL suppressed the levels of fibronectin (FN) and collagen IV (Col IV) in HG-stimulated HMCs. Furthermore, TRL efficiently inhibited the activation of the Wnt/β-catenin signaling pathway in HG-stimulated HMCs. Taken together, these data indicated that TRL inhibited HG-induced HMCs proliferation and ECM expression via the modulation of the Wnt signaling pathway.TRL reduces HG-induced cell injury by regulating the Wnt/β-catenin signaling pathway. Diabetic nephropathy (DN) has become the third-largest threat to human health after cancer . There iThe Wnt/β-catenin signaling pathway comprises Wnt ligand–protein outside the cell, receptors on the cell membrane, signaling part in the cytoplasm, and transcription regulation part in the nucleus . The maiTrigonella foenum-graecum L. (fenugreek), a traditional Chinese herb, and its components are beneficial in preventing and treating diabetes and central nervous system disease. Trigonelline (TRL) is an active component in the extract of fenugreek seeds. Trigonelline has hypoglycemic, hypolipidemic, neuroprotective, and it has been shown to alleviate kidney damage in STZ-diabetic rats [tic rats , 19. Whed-glucose  and placed in a 37 °C, 5% CO2 incubator. The cells were sub cultured and every two days and divided into seven groups. Group NG was treated with normal glucose , group HG was treated with high glucose , group A was treated with a medium dose of TRL , group B was transfected with β-catenin siRNA and then treated with TRL (100 μM), group C was transfected with β-catenin pcDNA, group D was transfected with β-catenin pcDNA then treated with TRL (100 μM), and group E was treated with the Wnt/β-catenin signaling pathway inhibitor ICG-001 , group A, B, C, D, E groups were incubated in high glucose containing media.The American Type Culture Collection provided HMCs. The cells were cultured in 5.5 mm 3/well) were plated in 96-well plates, serum-starved overnight and grouped by NG and HG. Then divided into individual TRL concentration administration groups  for different time durations . 24.5 mM mannitol (MA) was added to the NG medium as an osmotic control. After 24 h, 48 h and 72 h, 100 µL MTT  was added to each well. After 4 h, 150 μL DMSO (Sigma-Aldrich) was added to each well. The absorbance of each well was measured using a microplate reader  with a reference wavelength of 490 nm.Exponentially growing cells  for 20 min. Incubation with rabbit monoclonal antibodies against β-catenin  at 4 °C overnight. Then, the bound antibody was detected with horseradish peroxidase (HRP)-anti-rabbit IgG and diaminobenzidine (DAB) and then counterstained with hematoxylin. The percentage of positively stained areas in the glomeruli was analyzed by computer image.HMC was cultured in groups on a six-well plate. After the cells reached approximately 80% confluence, 2.5 µg of plasmids expressing CTNNB1  were transfected into the HMCs to overexpress β-catenin using Lipofectamine 2000 reagent  following the manufacturer’s protocol. For β-catenin knockdown, 75 pmol of small interfering RNA (siRNA) against CTNNB1  was transfected into the HMCs using Lipofectamine 2000 reagent. The siRNA sequences are as follows: si-CTNNB1 (Human) sense 5′-GGGUUCAGAUGAUAUAAAUTT-3′ and antisense 5′-AUUAUAUCAUCUGAACCCAG-3′. The siRNA/liposome and pcDNA/liposome complexes were added to the serum-free medium and the complexes were formed at room temperature for 20 min. After 4 h of incubation, the medium containing HG (30 mM) was added and the pregnancy continued. After 48 h of incubation, cells were harvested to detect β-catenin expression and related targets.−ΔΔCt method. Each sample was tested in triplicate. Primer sequences are shown in Table TRIzol Reagent . PCR was executed on the 7500 FAST Real-Time PCR System for 45 cycles . The thermocycling conditions were as follows: initial melting at 94 °C for 30 s followed by 45 cycles of 94 °C for 5 s, 51 °C for 15 s, and 72 °C The 10% sec. The GAPDH mRNA was amplified simultaneously and used as a loading control. The relative amounts of mRNA were determined by the 2Total Protein Extraction Kit . For separation, an equal amount of protein was loaded onto a 12% sodium dodecyl sulfate–polyacrylamide gel. The protein was then transferred to a polyvinylidene fluoride membrane  and blocked with 5% skim milk for 1 h. Incubate the membrane and primary antibodies overnight at 4 °C. The next day, HRP-conjugated anti-rabbit IgG secondary antibody  was incubated for 1 h. Images were captured using the Tanon 4200 Automatic Chemiluminescence Image Analysis System  and analyzed using Image-Pro Plus 6.0.The HMC was inoculated to a 6-well plate with a density of 1 × 104. After 48 h, PBS was washed twice and collected, fixed in 70% cold ethanol at 4 °C overnight. Next, the fixed cells were resuspended at room temperature with 50 g/mL Propidium iodide (PI).  binding buffer in 30 min. Then flow cytometry for detection.  ModFit LT V3.3.11  was used to determine the proportion of G0/G1, S and G2/M cells.g for 5 min; gently aspirate the supernatant and mix well with 1 mL PBS. Annexin V—FITC and PI (5 μL each) were then added to tubes, mixed gently, and incubated for 15 min. Then, 300 mL of binding buffer was added and apoptosis was detected within 1 h.Annexin v-FITC/PI kit detected cell apoptosis.  Cells were washed with pre-cooled PBS and collected, then fixed at 4 °C overnight in 70% ethanol. After 72 h of incubation, cells were collected and centrifuged at 4 °C and 100×The TUNEL apoptosis detection kit was used . DNA strand breakage was evaluated by fluorescence labeling of the terminal dUTP notch (TUNEL). DAPI solution  used nuclear staining. TUNEL positive cells were counted in the defined area and their density was calculated.Data are presented as mean ± SEM with n denoting the sample size in each group. Differences between groups were analyzed by variance analysis or rank sum test. One-way ANOVA tests were used to compare three or more independent groups. SPSS software was used to test the normality of the data and statistical analysis . We considered a p-value < 0.05 as statistically significant.To examine the effect of TRL on HG-induced HMCs, HMCs were cultured and the MTT assay was used to detect cell proliferation for different periods . Cells cultured under HG conditions for 24 h increased faster than the untreated cells P < 0.01), and the difference was still significant after 48 h (P < 0.01). However, the proliferation rate of HG-treated cells decreased after 72 h. Treatment with mannitol did not alter cell growth at any time tested, suggesting that the HG-induced HMC proliferation was not due to high osmotic pressure. Compared to the proliferation rate in the HG group, that in the cells treated with TRL at a concentration of 25 to 800 μM decreased significantly (P < 0.05), and the effect was concentration-dependent  for these experiments to choose the optimum concentration. As shown in Fig. The above results demonstrate the beneficial effects of TRL against HG-induced over-proliferation of HMCs. To shed light on how TRL mediates these effects, we use the Wnt/β-catenin pathway inhibitor ICG-001 and study the expression of nucleus-β-catenin, Wnt5a, and TCF4. We found that TRL had the same impact on these signals as ICG-001. These results indicate that TRL might influence the survival of HMCs by inhibiting the Wnt pathway. Next, we chose to transfect HMCs with plasmids expressing β-catenin transiently. We found that the signals of the Wnt/β-catenin pathway increased significantly but decreased when treated with TRL at the same time. These results revealed that HG might lead to an increase in β-catenin.Additionally, TRL reduced the upregulation of the mRNA and protein levels of β-catenin. Compared with that observed for the HG group, the expression of each signal factor was significantly down-regulated in the 100 μM of TRL and the β-catenin siRNA group , and the expression level was also significantly reduced compared to that of group A treated with TRL alone (Fig. Flow cytometry analysis was performed further to examine the role of TRL in HMC cell-cycle progression. As shown in Fig. As shown in Fig. Interestingly, group C had more DNA fragmentation than the HG group Fig. B. This dDN is a glomerular sclerosis disease caused by the abnormal metabolism of diabetes mellitus. High glucose quickly affects mesangial cells, which in turn cause cellular stress and damage . The proThe development of DN is associated with the abnormal proliferation of mesangial cells, and HG conditions could induce the proliferation of mesangial cells. In this experiment, the effect of TRL on the expansion of HMCs was studied by MTT assays. We found that the absorbance increased significantly in the HG group at 48 h, suggesting that hyperglycemia stimulated excessive proliferation of HMCs within the experimental dose range. TRL could inhibit the HMCs hyperproliferation induced by HG in a concentration-dependent manner. After the HG treated cells were cultured for 72 h, cell proliferation slowed and cell viability significantly decreased. However, TRL could enhance HMC activity at this time. Since HG conditions had the most significant effect on the addition of HMCs at 48 h, this time point was selected to study further the effect of TRL on the proliferation of HMCs under HG and understand its mechanism.There are three phases of the cell cycle: G0/G1 phase, DNA synthesis-related S phase, and G2/M phase , 28. CycThe Wnt/β-catenin signaling pathway plays an important role in the pathogenesis of DN. Furthermore, numerous studies demonstrated that specific blockade of the Wnt/β-catenin pathway prevented DN progression, including abolishing cell proliferation and ECM expression in glomerular HMCs , 30. TheWnt/β-catenin signaling is required to protect glomerular mesangial cells from high glucose-mediated apoptosis –33. β-CaIn short, results demonstrated that HG condition and β-catenin overexpression could activate the Wnt/β-catenin signaling pathway. At the same time, TRL inhibited Wnt/β-catenin signaling by regulating the expression of β-catenin, TCF4, and other related factors. TRL could down-regulate the expression of Cyclin D1 and CDK4, stopping the cell cycle at the G1 phase, inhibiting abnormal proliferation is induced by HG and over-expression of β-catenin. TRL inhibited apoptosis by down-regulating β-catenin signaling. This provides a novel insight into understanding the molecular and signaling pathways through which TRL suppresses HG-induced cell over-proliferation and apoptosis. Thus, treatment with TRL and the modulation of the Wnt/β-catenin pathway represent novel therapeutic strategies for the treatment of DN in the clinical setting.