Activation of CX3CR1 in microglia plays an important role in the development of neuropathic pain. Here, we investigated whether neuropathic pain could be attenuated in spinal nerve ligation (SNL)-induced rats by reducing microglial activation through the use of poly (PLGA)-encapsulated CX3CR1 small-interfering RNA (siRNA) nanoparticles. After confirming the efficacy and specificity of CX3CR1 siRNA, as evidenced by its anti-inflammatory effects in lipopolysaccharide-stimulated BV2 cells in vitro, PLGA-encapsulated CX3CR1 siRNA nanoparticles were synthesized by sonication using the conventional double emulsion (W/O/W) method and administered intrathecally into SNL rats. CX3CR1 siRNA-treated rats exhibited significant reductions in the activation of microglia in the spinal dorsal horn and a downregulation of proinflammatory mediators, as well as a significant attenuation of mechanical allodynia. These data indicate that the PLGA-encapsulated CX3CR1 siRNA nanoparticles effectively reduce neuropathic pain in SNL-induced rats by reducing microglial activity and the expression of proinflammatory mediators. Therefore, we believe that PLGA-encapsulated CX3CR1 siRNA nanoparticles represent a valuable new treatment option for neuropathic pain. Neuropathic pain is caused by various lesions or diseases of the somatosensory system . The assIn general, when a nerve is damaged, neurotransmitters such as glutamate, ATP, and substance P are released form presynaptic terminals of the primary afferent neurons in the dorsal root ganglia . ReleaseThe correlation between neuropathic pain and microglia is well established, with strong evidence of both morphological and pathological mechanisms of disease . In the CX3CL1 is a neurotransmitter with a unique structure belonging to the chemokine family, with signaling mediated via ligation to its cognate receptor, CX3CR1. Unlike other chemokines, CX3CL1 is primarily expressed in the kidney, heart, lung, and brain, with no expression seen in leukocytes ,13. ManyRecently, gene therapies based on small interfering RNAs (siRNA) that silence disease-related genes have attracted considerable attention due to their potential to treat genetic disorders and other intractable conditions . siRNA hPoly (PLGA) is a Food and Drug Administration (FDA)-approved method for delivering siRNA to specific targets . PLGA haIn this study, we examined the use of PLGA-encapsulated CX3CR1 siRNA nanoparticles for the treatment of neuropathic pain in an SNL model. To test our hypothesis, we investigated whether PLGA-encapsulated CX3CR1 siRNA nanoparticles can reduce pain behavior in SNL-induced neuropathic pain rats.Before generating PLGA-encapsulated CX3CR1 siRNA nanoparticles, we confirmed the efficacy and specificity of CX3CR1 siRNA in microglial cells. First, to evaluate whether CX3CR1 siRNA can effectively reduce the CX3CR1 protein level in microglial cells, the experimental group was treated with BV2 cells with CX3CR1 siRNA for two days, while the control group used scrambled (sc) siRNA. CX3CR1 protein levels were then detected by Western blot, and the CX3CR1/ACTB ratio was quantified. The expression of CX3CR1 protein was reduced by approximately 50% in CX3CR1 siRNA-treated rats compared to the control group A,B.Lipopolysaccharide (LPS) activates various cell types, including microglial cells, resulting in the transcription of a wide range of proinflammatory mediators . Based oSeveral factors were considered when selecting a gene delivery system to effectively deliver CX3CR1 siRNA to microglia in the spinal cord, including efficacy, cost, safety, and convenience. In this study, we used PLGA, a substance that has already have been proven biodegradable and biocompatible in humans by the US Food and Drug Administration (FDA) . PLGA naTo evaluate pain behavior caused by neuropathic pain, we used SNL, a well-established model for evaluating neuropathic pain in rats . Before After SNL surgery, microglia are activated in the ipsilateral dorsal horn of the spinal cord. Activation of microglia on the contralateral side was not observed. To assess the activation of microglia, tissue sections were immuno-stained using an anti-Iba 1 antibody. In the sham group, microglia were not activated in the dorsal horn on either side of the spinal cord; however, in the SNL group, the microglia on the ipsilateral side was dramatically activated in the dorsal horn as well as in the ventral horn compared to the contralateral side at 3 days post-operation C,D.To investigate whether PLGA-encapsulated CX3CR1 siRNA nanoparticles can alleviate pain behavior caused by neuropathic pain, sc siRNA or PLGA-encapsulated CX3CR1 siRNA nanoparticles were injected into the intervertebral space of SNL-induced rats on the fourth day, when the pain was stabilized after surgery, using a Hamilton syringe A. The meTo confirm whether PLGA-encapsulated CX3CR1 siRNA nanoparticles reduced the expression of CX3CR1 in the spinal cord, L5 spinal dorsal horn tissues were collected at 6 days post-intrathecal injection that had the best pain relief effect. CX3CR1 siRNA treatment significantly reduced the expression of CX3CR1 protein, as determined by Western blot using anti-CX3CR1 antibodies C,D. TogeIncreased expression of CX3CR1 in microglia plays an important role in neuropathic pain. Thus, we hypothesized that inhibiting the expression of CX3CR1 using PLGA-encapsulated CX3CR1 siRNA nanoparticles could reduce microglia activation and alleviate neuropathic pain. As expected, microglial activation was significantly reduced, in terms of both the absolute number and cell density, in the ipsilateral dorsal horn on day 3 post-injection in the CX3CR1 siRNA group compared to the sc siRNA group A,B.Next, we examined changes in neuropathic pain-related inflammatory genes’ expression. Spinal dorsal horn tissues on the ipsilateral side were collected separately on day 3 post-injection, and the mRNA levels of proinflammatory genes including TNF-α, IL-1β, IL-6, and COX-2 were measured by qRT-PCR. mRNA levels of these proinflammatory genes were significantly decreased in the group injected with PLGA-encapsulated CX3CR1 siRNA nanoparticles compared to the group injected with sc siRNA-encapsulated PLGA nanoparticles E.Together, these data indicate that PLGA-encapsulated CX3CR1 siRNA nanoparticles inhibit the expression of CX3CR1, thereby inhibiting microglial activation and the expression of proinflammatory genes and alleviating neuropathic pain in SNL-induced rats. Neuropathic pain is a chronic condition that can be difficult to treat. Various methods have been developed to treat neuropathic pain, including drug therapies, such as analgesics, antiepileptic drugs, tricyclic antidepressants (TCA), serotonin-norepinephrine reuptake inhibitors (SNRIs), and opioids, as well as mechanical interventions such as nerve blockage, spinal cord stimulation, and intrathecal pumps; however, the effectiveness of these treatments remains unclear . The devActivated microglia in the spinal cord play an important role in the development of neuropathic pain. Activated microglia have been shown to induce and sustain neuropathic pain by stimulating secretion of various proinflammatory factors, such as TNF-α, IL-1β, and IL-6. Therefore, reduction of microglial activation represents a promising therapeutic target for the reduction of neuropathic pain. Drugs that reduce the activity of microglia include minocycline and pentoxifylline. While effective, these drugs have the potential to generate cytotoxicity when used at high doses  and haveCX3CR1 is upregulated in spinal microglia upon nerve injury and is involved in the development and maintenance of neuropathic pain . In addiIn this study, nanoparticles were used to effectively deliver CX3CR1 siRNA. Nanoparticles are a suitable system for most drug administration routes. Various natural and synthetic polymers have been studied for several years to improve the biocompatibility and biodegradability of nanoparticles. Among them, PLGA used in this study is the most commonly used polymer because of its biocompatibility and resorption through natural pathways. As such, research on nanoparticles’ manufacturing continues. As a result, nanoparticles are attractive mediators for gene silencing and are an effective means of treating neuropathic pain due to their various advantages, including efficient cell uptake, sustained intracellular drug release, favorable safety profiles, low cost, and ease of manufacture. However, the disadvantage of PLGA nanoparticles is that siRNA encapsulation efficiency and delivery power are lower than that of recombinant viruses. Therefore, further research will be needed.Sprague-Dawley rats  were purchased form Daehan Bio Link  and placed in the study habitat 1 week before the experiment to allow for adaptation to the environment. Rats were housed three per cage in a controlled environment with a 12 h light/dark cycle and free access to food and water. This study was approved by the Animal Care and Use Committee at the Chungnam National University (CNUH-019-A0040) and followed the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain . The num2 humidified incubator. Scrambled siRNA or CX3CR1 siRNA was introduced into BV2 cells using the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific).BV2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). All cultures were kept at 37 °C in a 5% COSpinal cords collected for the purpose of immunostaining were cryopreserved at –80 °C after fixing in 4% PFA and cryoprotected in 30% sucrose for 2–3 days. Thereafter, spinal cords were sectioned coronally at 35 µm using a freezing microtome, and sections were stored in PBS. Nuclei were counterstained with Hoechst 33342 and visualized with a confocal microscope. The immune densities in the graphs were quantified using Image J software.Total RNA from BV2 cells was isolated using TRIzol reagent according to the manufacturer’s protocol . RNA concentrations were quantified using a Nanodrop spectrometer (Thermo scientific). cDNA was prepared from total RNA using a commercially available kit . Quantitative polymerase chain reaction (qPCR) was performed using the AriaMax Real-time PCR system (Agilent Technologies) with the TOPreal qPCR 2X premix (SYBR Green with low ROX). The primers used for rat TNF-α, IL-1β, and COX2 were as follows: TNF-α 5′-AGATGTGGAACTGGCAGAGG-3′, and antisense 5′-CCCATTTGGGAACTTCTCCT-3′; IL-1β, 5′-CAGCAGCATCTCGACAAGAG-3′, and antisense 5′-CATCATCCCACGAGTCACAG-3′ COX2, 5′-CAGTATCAGAACCGCATTGCC-3′, and antisense 5′-GAGCAAGTCCGTGTTCAAGGA-3′.g for 10 min at 4 °C, washed once with deionized RNAase-free water, resuspended in water, and freeze-dried. The physical characteristics were then analyzed with the Zetasizer Nano ZS  and electron microscopy.PLGA nanoparticles from the Nanoglia Company  were prepared as reported previously, with minor modifications . BrieflyNeuropathic pain in rats was achieved through the widely used L5 nerve root spinal nerve ligation (SNL) method ,26. RatsThe pain behavior test was evaluated using manual von Frey filaments , as described previously ,32,33. RIntrathecal injection was performed as follows . Rats wet-test and one-way analysis of variance (ANOVA), followed by an appropriate multiple comparison test. p-values ≤ 0.05 were considered statistically significant. *p-values < 0.05 and the data were analyzed by the Shapiro–Wilk test for normality. All statistical analyses were performed using GraphPad Prism 6 .The data are expressed as the mean ± standard error of the mean (SEM). Statistical significance in comparisons of multiple groups was assessed by Our data indicate that PLGA-encapsulated CX3CR1 siRNA nanoparticles effectively reduce neuropathic pain in SNL-induced rats by reducing microglial activity and expression of proinflammatory mediators. Therefore, we believe that PLGA-encapsulated CX3CR1 siRNA nanoparticles are valuable as a new treatment for neuropathic pain.


Wnt/β-catenin signaling plays vital role in the regulation of cellular proliferation, migration, stem cells cell renewal and genetic stability. This pathway is crucial during the early developmental process; however, the distinct role of Wnt/β-catenin signaling during pre-implantation period of bovine embryonic development is obscure. Here, we evaluated the critical role of Wnt/β-catenin pathway in the regulation of bovine blastocyst (BL) development and hatching. 6 bromoindurbin-3’oxime (6-Bio) was used to stimulate the Wnt signaling. Treatment with 6-Bio induced the expression of peroxisome proliferator-activated receptor-delta (PPARδ). Interestingly, the PPARδ co-localized with β-catenin and form a complex with TCF/LEF transcription factor. This complex potentiated the expression of several Wnt directed genes, which regulate early embryonic development. Inhibition of PPARδ with selective inhibitor 4-chloro-N-(2-{[5-trifluoromethyl]-2-pyridyl]sulfonyl}ethyl)benzamide (Gsk3787) severely perturbed the BL formation and hatching. The addition of Wnt agonist successfully rescued the BL formation and hatching ability. Importantly, the activation of PPARδ expression by Wnt stimulation enhanced cell proliferation and fatty acid oxidation (FAO) metabolism to improve BL development and hatching. In conclusion, our study provides the evidence that Wnt induced PPARδ expression co-localizes with β-catenin and is a likely candidate of canonical Wnt pathway for the regulation of bovine embryonic development. The development of oocyte into an implantation competent blastocyst (BL) undergoes a series of progressive developmental events. During this process the classical developmental signaling cascades participate to ensure the successful implantation and subsequently improve the pregnancy outcomes . A huge -Catenin ethyl)benzamide (Gsk3787)  was used to specifically inhibit the PPARδ function during the pre-implantation period of bovine embryonic development. A 5 mg powder of Gsk3787 was dissolved in DMSO to make 2.5 mM stock solution and was kept in the dark condition at −20 °C until use. Stock solution was serially diluted in phosphate buffered saline (PBS). COCs were randomly distributed into five groups (n = 50 to 55/group). Different concentrations of Gsk3787  were used in IVM and IVC medium. The study was performed in 10 to 11 reciprocal sets of independent experiments. Based on the blastocyst development and hatching rate, 15 µM dose showed maximum effect on PPARδ activity. For further analysis, 15 µM dose was used.Oocyte aspiration and in-vitro maturation procedure was performed as described previously . Brieflyg for 5 min. The pellet formed at the bottom of tube was re-suspended in 10% heparin solution prepared in in-vitro fertilization (IVF) medium  followed by incubation at 38.0 °C in a humidified atmosphere containing 5% CO2 for 15 min. Spermatozoa was diluted with IVF medium with a final concentration of 1 × 106 per mL. For each group 50 to 55 matured COCs were transferred into a 4-well dish and fertilized with 700 μL of IVF medium. These dishes were incubated in a humidified condition with 5% CO2 at 38.0 °C for 18–20 h. After fertilization, oocytes were cleared from the cumulus cells by repeated pipetting and denuded presumptive zygotes were cultured for 8 days. BLs were cultured in a SOF media supplemented with 44 μg/mL sodium pyruvate (C3H3NaO3), 14.6 μg/mL glutamine, 10 IU/mL penicillin, 0.1 mg/mL streptomycin, 3 mg/mL FBS and 310 μg/mL glutathione. At day-8, BLs were washed three times in 1 × PBS and stored at 4 °C after fixation in 4% paraformaldehyde until further analysis. For gene expression analysis, BLs were immediately snap-frozen in liquid nitrogen after washing in nuclease free water and stored at −80 °C in 1.5-mL Eppendorf tubes.For in-vitro fertilization, the bovine semen straw was thawed in water bath at 38.0 °C for 1 min and washed in 10 mL DPBS, followed by centrifugation at 750× GAPDH. For the mRNA expression analysis, experiment was performed in triplicate sets. Primers used for qRT-PCR are listed in Total RNA from BLs (n = 5 per group) was isolated using RNA isolation kit . Subsequently, cDNA synthesis and quantitative real time polymerase chain reaction (q-RT-PCR) was performed by using iScript Reverse transcriptase  and SYBR Green master mix , respectively. PCR amplification was performed with the following conditions: initial denaturation at 94 °C for 5 min followed by 40 cycles of 94 °C for 30 s, 58 °C for 30 s and 72 for 30 s. Relative mRNA expression of all genes was normalized with w/v) PFA  solution, and permeabilized with 0.5% Triton X-100. After blocking, sequentially incubated with primary antibody overnight and secondary antibody either conjugated with FITC or TRITC for 1 h at RT. Samples were washed extensively with PBS/polyvinylpyrrolidone (PVP) and the nuclei were counterstained with DAPI for 5 min at RT. Images were viewed under a confocal laser microscope . The antibodies used for immunofluorescence are listed in Immunofluorescence staining was performed as described previously . Brieflyw/v) PFA solution. BLs were washed three times with PBS and incubated with PBS/PVP containing 10 mg/mL NR solution for 3 h at room temperature in protected light condition. Stained BLs were washed 3 times with PBS/PVP solution and incubated with DAPI (1:100 (v/v) in PBS/PVP for 5min. BLs were mounted on a glass slide with an overlaid cover slip after washing twice with PBS. Thereafter, the NR lipophilic fluorescent dye (485 nm) was excited under confocal laser-scanning Olympus FluoviewFV1000 microscope. Red fluorescence (lipids) intensities were measured by using Image J  software. Normalization was performed by subtracting the background intensity from each image of the experimental groups. The lipid contents from each group is presented as mean fluorescence intensity.To analyze the accumulation of lipid droplets, a fluorescent probe  specific for intracellular lipids was used to evaluate the lipid content in the day-8 fixed BLs. NR stock solution was prepared by dissolving 1 mg/mL in DMSO, stored at room temperature under dark condition. Day-8 BLs were fixed in 4% (w/v) paraformaldehyde on the lower surface of the scrubbed membrane for 15 min at room temperature. Then stained with DAPI for 5 min. Cells that passed across the membrane were counted under a phase-contrast Olympus IX71 microscope.To quantify the effect of Wnt pathway stimulation on BL implantation potential, the spreading and invasion areas of day-8 BLs were quantified. Blastocysts were placed on an invasion chamber insert  containing polyethylene terephthalate membranes (8 µm-diameter pores) in 24-well tissue culture plates (Corning Inc. Life Sciences). The upper surface of the chamber was coated with Matrigel  and incubated for 2 h at 37 °C for drying. Three BLs per culture insert were placed in the Matrigel coated chamber containing the same medium used for embryo culture. Incubated at 37 °C for 72 h, replaced with fresh medium every 24 h at the bottom of the culture chamber. The invasion and spreading area of trophoblasts after 10 days of culture was examined under a phase contrast Olympus IX71microscope and analyzed using Image J software. Thereafter, a cotton swab was used to scrub the upper surface of the chamber insert membrane. Cells were fixed with 4% . All results for the expression level of various genes or comparison of fluorescence intensities were analyzed by using one-way analysis of variance followed by Sidak’s Multiple Comparison Test. GraphPad Prism 6.0 software package (USA) was used. Significant differences were considered at * p < 0.05; ** p < 0.01; *** p < 0.001.Statistical analysis was performed using SPSS software version 18.0 . All percentage data are presented as mean ± standard error of mean (SEM). Triplicate sets of experiment were used to obtain all the image data, and single BL image was shown as representative image from the individual group, whereas all graphical data obtained from triplicate sets of experiments are presented as the mean standard error of mean (SEM). All mean fluorescence intensities presented in the present study were quantified per BL from each group, To evaluate the stimulatory effect of specific agonist of Wnt/β-catenin pathway, we used 6-Bio during in-vitro maturation and culturing of bovine embryonic development. First, we determined the optimal dose of 6-Bio for maximal activation of Wnt signaling pathway. Exogenous stimulation of Wnt activity with 6-Bio dose improved the bovine BL formation and hatching rate . The 400The functional importance of the Wnt signaling pathway during bovine embryonic development was characterized by analyzing several pluripotency and lineage specification markers. The expression of pluripotency maker Oct4 displayed a significant up regulation in the inner cell mass in the 6-Bio treated group A,B. SimiTo further explore the role of Wnt/β-catenin pathway in the bovine embryonic development, we probed the expression of genes that are influenced by the Wnt/β-catenin pathway. The exogenous stimulation of Wnt activity strongly enhanced the expression of Cox2. Cox2 is a key enzyme in the production of prostaglandins and plays an important role in number of reproductive processes ,24. SevePPARδ expression is documented to be highly associated with Wnt signaling . CarcinoIn the early embryonic development, the cell proliferation event is accompanied by several metabolic process. The most important of these metabolic processes is the lipid metabolism . The ATPOur investigations showed that Wnt stimulated condition enhanced the lipid metabolism by up regulating PPARδ expression. To better understand the categorical role of Wnt/β-catenin signaling on lipid metabolism, we assessed the lipid droplets accumulation by Nile red staining during BL development. The immunostaining analysis displayed that the accumulated lipid content is profoundly low in 6-Bio treated group as compared to control group. PPARδ antagonist (Gsk3787) treated group exhibited a significant increase in the lipid droplets accumulation. However, treatment with Wnt agonist 6-Bio in combination with Gsk3787 reduced the lipid droplet content in the developing BL A,B. ThesWe also analyzed the effect of activated Wnt condition on BL competency for implantation. During the pre-implantation development, the BL achieves the competency to attach to the uterus. This attachment is essential for a normal and successful implantation process . To gainThe stimulation of Wnt/β-catenin activity with its agonist directly induces the expression of PPARδ in bovine embryos. This stimulation in turn influences the pre-implantation period of development and BL hatching rate by mediating the expression of direct Wnt/β-catenin targeted genes. There are many conflicting studies regarding the functional interaction of PPARδ with Wnt/β-catenin pathway. β-catenin is a transcriptional co-activator of TCF/LEF transcription factor, which triggers the expression of several genes regulated by canonical Wnt signaling pathway ,32. We wThe stimulation of Wnt activity strongly enhanced the spatial overlapping of PPARδ with β-catenin while PPARδ was randomly overlapped with β-catenin in control group as shown by immune fluorescence analysis A. This rDuring the pre-implantation development, the subsequent formation of distinctive embryonic lineages is governed by the activation of the specific signaling pathways . Wnt is Our present investigation using small-molecule agonist (6-Bio) of canonical Wnt pathway provides an evidence that activation of Wnt/β-catenin signaling is important during the pre-implantation period of in-vitro produced bovine embryos. The reciprocal stimulation with 6-Bio treatment significantly improved BL development and hatching rate A,B. 6-BiDuring early embryo development, the maintenance of cell proliferation event is regulated by the activation of several pluripotency factors and their interaction with the Wnt signaling cascade ,35,36,37Different cancerous and developmental studies reported the cross-talk between Wnt/β-catenin pathway and the nuclear hormone receptor PPARs family ,43. PPARPPARδ is critically involved in regulation of lipid metabolism and positively influence the BL hatching . InteresFinally, we propose that the stimulation of canonical Wnt activity by small molecule activator 6-Bio up regulates the downstream PPARδ expression. The PPARδ in conjunction with β-catenin makes a transcriptional co-activator complex and boost the Wnt directed genes expression during the development of bovine embryos . In summ


The use of aspirin has been associated with reduced breast cancer risk, but it is litter known if aspirin overcomes chemoresistance in triple-negative breast cancer (TNBC). Herein, we demonstrated that changes in the expression of Yes-associated protein (YAP) and β-catenin might be a promising predictive biomarker for neoadjuvant chemotherapy sensitivity in TNBC patients. Inhibition of YAP or β-catenin enhanced the cytotoxicity of the anti-microtubule agents docetaxel and vinorelbine against drug-resistant TNBC cells as well as the sensitivity of these cells to the agents in vitro and in vivo. Interestingly, aspirin not only significantly inhibited the growth of TNBC cells, but also attenuated YAP and β-catenin expression by upregulating the E3 ubiquitin ligase β-TrCP to abolished docetaxel and vinorelbine resistance. The combination of aspirin and docetaxel or vinorelbine remarkably inhibited the growth of drug-resistant TNBC cells in vitro and in vivo. Moreover, TNBC patients with high YAP and/or β-catenin expression had a higher risk of relapse or mortality than patients with low YAP and/or β-catenin expression. Collectively, our study discovered a novel role of aspirin based on its anticancer effect, and put forward some possible mechanisms of chemoresistance in TNBC. The combined use of aspirin and anti-microtubule drugs presented several promising therapeutic approaches for TNBC treatment. Despite many advances in targeted molecular therapy, the mortality rate of breast cancer remains very high. Triple-negative breast cancer (TNBC) accounts for 15–20% of all breast cancer cases and is a subtype of breast cancer characterized by the lack of oestrogen receptor (ER), progesterone receptor (PR) and ErbB-2/human epidermal growth factor receptor 2 (HER-2) expression2. Due to the lack of effective therapeutic targets, TNBC is associated with more aggressive behaviours, earlier relapse and poorer prognosis. Chemotherapy remains the mainstream treatment for TNBC patients2. Docetaxel- and vinorelbine-based regimens have proven to be greatly effective in early and advanced TNBC, respectively3. However, docetaxel or vinorelbine resistance is very common. Therefore, more effective therapeutic strategies for TNBC are urgently needed.Breast cancer had one of the highest incidences and one of the highest mortality rates of cancers that affect females in 20186. The cancer-preventing effects of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) are well established for breast cancer, and NSAIDs could impede the development of ER-positive breast cancer through the inhibition of aromatase7. The latest study found that regular aspirin use was associated with a 39% reduced risk of breast cancer, and the strength of these associations did not differ by familial risk or mutation status8. Intriguingly, in BRCA1 or BRCA2 germline mutation carriers, aspirin use was associated with a 27% or 20% reduced risk of breast cancer, respectively8. Moreover, TNBC is a subtype of breast cancer with the highest BRCA mutation rate9. The retrospective analysis found that aspirin use improved disease-free survival and reduced the risk of distant metastases in stage II and stage III TNBC patients10, but it is unclear whether aspirin could increase the sensitivity to chemotherapy drugs or reverse drug resistance in TNBC.Many epidemiological studies have found that tumour mortality decreases exponentially with an increase in aspirin intake in several cancers, including colon, breast, lung, and prostate cancers and cancers of other organs11. YAP is the major downstream effector of Hippo signalling, and members of the Hippo pathway can act as transcriptional coactivators to promote the expression of their target genes involved in proliferation and survival12. Accumulating evidence suggests oncogenic roles of YAP in cancer progression, but the role of YAP remains controversial in different subtypes of breast cancer13. The Wnt/β-catenin pathway plays a major role in embryonic growth and leads to tumorigenesis when aberrantly activated14. Many studies have indicated that this pathway is abnormally activated in the progression of several cancers, including breast cancer15. β-Catenin is stabilized and translocated to the nucleus, where it acts as a cofactor to activate the expression of target genes implicated in cell growth16. In addition, it has been reported that the Hippo/YAP and Wnt/β-catenin pathways are mutually regulated and that their crosstalk plays a vital role in many physiological and pathological processes18. However, whether YAP or β-catenin is involved in chemotherapy resistance in breast cancer remains elusive.The Hippo pathway is an evolutionarily conserved signalling pathway that plays important roles in organ size control, tissue regeneration, stem cell self-renewal and tumorigenesisIn this study, we confirm that YAP and β-catenin mediates docetaxel and vinorelbine resistance in TNBC and that aspirin could enhance the cytotoxicity of anti-microtubule drugs and reverse drug resistance. The specific mechanism is that aspirin impairs YAP and β-catenin by upregulating the E3 ubiquitin ligase β-TrCP to overcome docetaxel and vinorelbine resistance. Moreover, TNBC patients with high YAP and/or β-catenin expression had a higher risk of relapse or mortality than patients with low YAP and/or β-catenin expression. The combination of aspirin and anti-microtubule drugs might be a promising therapeutic strategy for TNBC.2 and cyclophosphamide 600 mg/m2) followed by four cycles of docetaxel (100 mg/m2). This study was approved by the Ethics Committee of the 940th Hospital of Joint Logistics Support Force of Chinese People’s Liberation Army.Thirty pairs of paraffin-embedded breast carcinoma and adjacent normal breast tissue samples, as well as 112 paraffin-embedded breast carcinoma samples, were collected and these tissues were made into tissue microarray slides . These samples were prospectively obtained from patients with breast cancer who underwent resection from January 2005 to December 2011 and were followed up for 2.1–11 years. The tissues larger than 5 cm from the tumor margin were selected and obtained as the adjacent normal tissues and these tissues were diagnosed and confirmed by pathologists as the normal tissues. Fresh tumor tissues were obtained from 40 patients with breast cancer undergoing neoadjuvant chemotherapy. All these patients received four cycles of AC (doxorubicin 60 mg/m2O2 for 5 min at room temperature. The appropriate positive and negative controls were included in each run of IHC.Immunohistochemistry (IHC) was performed on all tissue samples using biotin-streptavidin HRP detection systems. After deparaffinization with xylene and dehydration in a graded alcohol series, the tissue sections were subjected to antigen retrieval by microwaving in sodium citrate buffer for 10 min and then inhibiting endogenous peroxidase activity. After nonspecific binding was blocked, the slides were incubated with YAP (1:200) and β-catenin (1:200) antibody , Ki67  in phosphate-buffered saline (PBS) overnight at 4 °C in a humidified container. Biotinylated secondary antibodies  were then used according to the manufacturer’s recommendations. The sections were incubated with HRP-streptavidin conjugates appropriate for detecting these proteins. The brown color indicative of peroxidase activity was developed by incubation with 0.1% 3,3′-diaminobenzidine (Zhongshan Golden Bridge Biotechnology) in PBS with 0.05% HAn immunoreactivity score system based on the proportion and intensity of positively stained cells was applied. The two extensional standards taken were as follows: (1) number of positively stained cells: ≤5%, 0; 6–25%, 1; 26–50%, 2; 51–75%, 3; and >75%, 4; and (2) staining intensity: colorless, 0; pallideflavens, 1; yellow, 2; and brown, 3. Scores (1) and (2) were multiplied, and the staining grade was classified as absent (score 0), weak (score 1–4), moderate (score 5–8) or strong (score 9–12). Specimens were rescored independently by two pathologists. Moderate or strong tumor immunostaining was classified as having high expression, and absent or weak tumor immunostaining was classified as having low expression.−ΔΔCt method.The total RNA from each sample was extracted using NucleoZOL reagent  in accordance with the recommended protocol. Retrotranscription was performed with the Reverse Transcriptase M-MLV . The RT-PCR reactions were performed with a SYBR Premix Ex Taq™ kit  on a Bio-Rad CFX96 Touch system . The primers used were as follows: YAP, forward: GGTGCCACTGTTAAGGAAAGG, reverse: GTGAGGCCACAGGAGTTAGC; β-catenin, forward: CATTACAACTCTCCACAACC3, reverse: CAGATAGCACCTTCAGCAC; β-actin, forward: TGGCACCCAGCACAATGAA, reverse: CTAAGTCATAGTCCGCCTAGAAGCA. The data were analyzed using the 22 at 37 °C. Docetaxel and vinorelbine were purchased from Aosaikang and Haosen in China, respectively. Aspirin was purchased from Sigma in USA. The following antibodies were used for IHC and western blotting: YAP , β-catenin , β-TrCP, Bcl-l2, Bax , C-Myc, CyclinD1 , Ki67 , GAPDH .The human TNBC cell lines MDA-MB-231 and MDA-MB-468 were from ATCC and stored in our laboratory. The docetaxel or vinorelbine-resistant MDA-MB-231 cell lines (MDA-MB-231/DR or MDA-MB-231/VR) were treated and established with docetaxel or vinorelbine for about 6 months in our laboratory. All these cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum  in 5% CO3) were seeded in a 35 mm dishes. After 10 days, the resulting colonies were rinsed with PBS, fixed with methanol at −4 °C for 5 min, and stained with Giemsa (Sigma) for 20 min. For apoptosis assay, we used the Annexin V-FITC Apoptosis detection kit . Cells were collected and washed twice with PBS and then resuspended in 500 μl of staining solution containing fluorescein isothiocyanate (FITC)-conjugated annexin V antibody (5 μl). Subsequent to the staining, the cells were incubated with 10 μl of propidium iodide (PI) for 5 min on ice in the dark. The analyses were performed using a Navios flow cytometer (Beckman Coulter).For cell survival assay, the cells were seeded in 96-well plates for 24 h and were allowed to adhere overnight in regular growth media. After treatment with the indicated drugs or transfection for 48 h, the relative cell proliferation was measured using the Cell Counting Kit-8 . For colony formation assay, a total of cells  kit . Cells grown on coverslips were treated with different drugs for 48 h and then incubated with BrdU for 1 h and stained with an anti-BrdU antibody according to the manufacturer’s instructions. The results were analyzed using a fluorescence microscope (Olympus). For accessing cell migration ability assay, we used scratch wounding migration assay. The cells were cultured in six-well plates and reached 90% confluence; a scratch wound was created using a pipette tip. To avoid the effects of cell proliferation, the cells were treated by 5 µM mitomycin C for 2 h. The wound edges were photographed with a Nikon Eclipse TE 2000-U , and the scratch widths were analyzed using ImageJ software.The cells were lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors . The protein concentration was determined by the Bradford protein assay kit (BioRad). Proteins were resolved by SDS-PAGE and transferred to polyvinyl difluoride (PVDF) membranes (Millipore). The blots were probed with the different primary antibodies and species-matched secondary antibodies. The expression of the proteins was detected using the BioRad semidry transfer system.6 cells (in 100 μl PBS) were injected into the abdominal mammary fat pad of 4-week-old female nude mice . After the tumour volumes exceeded ~100 mm3, mice were randomized into nine groups (n = 5 per group): Group 1 (control) mice received intratumoural negative control shRNA lentiviral vector  injections and intraperitoneal phosphate-buffered saline (50 μl) injections. Group 2 (sh-YAP) mice received intratumoural sh-YAP lentiviral vector  injections. Group 3 (sh-β-catenin) mice received intratumoural sh-β-catenin lentiviral vector  injections. Group 4 (Doc) mice received intraperitoneal docetaxel injections at a dose of 8 mg/kg twice per week. Group 5 (Vin) mice received intraperitoneal vinorelbine injections at a dose of 2 mg/kg twice per week. Group 6 (sh-YAP + Doc) mice received sh-YAP lentiviral vector and docetaxel injections. Group 7 (sh-β-catenin + Doc) mice received sh-β-catenin lentiviral vector and docetaxel injections. Group 8 (sh-YAP + Vin) mice received sh-YAP lentiviral vector and vinorelbine injections. Group 9 (sh-β-catenin + Vin) mice received sh-β-catenin lentiviral vector and vinorelbine injections. Three weeks after treatment, mice were sacrificed, and tumours were resected. Additionally, a nude mouse study of drug-resistant TNBC cells was also carried out following the above methods. After the tumour volumes exceeded ~100 mm3, mice were randomized into groups (n = 5 per group): Group 1 (control) mice received intragastric and intraperitoneal phosphate-buffered saline injections. Group 2 (Doc) mice received intraperitoneal docetaxel injections at a dose of 8 mg/kg twice per week. Group 3 (Vin) mice received intraperitoneal vinorelbine injections at a dose of 2 mg/kg twice per week. Group 4 (Doc + Asp) mice received intraperitoneal docetaxel injections and intragastric aspirin at 50 mg/kg/d. Group 5 (Vin + Asp) mice received intraperitoneal vinorelbine injections and intragastric aspirin at 50 mg/kg/d. Tumour growth was monitored by caliper measurements using the following formula: V = (length × width2)/2, where L and W represent the length and width, respectively, and the survival of mice was analysed by Kaplan–Meier analysis. The animal experiments were approved by the institutional review board of West China Hospital of Sichuan University. All efforts were made to minimize the animals’ suffering and reduce the number of animals used.A total of 5 × 10t-test or Student’s t-test. All data are expressed as the mean ± standard deviation. In the clinical specimen study, the associations between YAP or β-catenin expression and categorical variables were analysed using Pearson’s chi-square test as appropriate. Correlations between YAP or β-catenin expression and the expression of other molecules were analysed by using the Spearman correlation test. Kaplan–Meier analysis was used to evaluate disease-free survival and overall survival. The effects of different variables on outcomes were assessed using a univariate Cox regression model to select proper variables for multivariate analysis and build a more reliable model. A multivariate Cox proportional hazards model was used to identify factors affecting DFS and OS. P values of less than 0.05 were considered statistically significant.Statistical analyses were performed with GraphPad Prism version 5.0  and SPSS statistical software, version 22.0 . Data from in vitro experiments were analysed statistically using a one-way analysis of variance (ANOVA), an independent samples 13. To explore the roles of YAP and β-catenin in the carcinogenesis of human breast cancer, we investigated the expression of YAP and β-catenin by IHC in 30 pairs of breast carcinoma and adjacent normal breast tissue specimens. Positive staining of YAP was found predominantly in the cytoplasm and nucleus of both normal breast cells and breast cancer cells , while β-catenin expression was higher in cancer tissues than in the paired adjacent normal tissues (P < 0.001).Oncogenic roles of β-catenin in breast cancer has been confirmed, but the role of YAP remains controversiallls Fig. . Converslls Fig. . Indeed,lls Fig. , respectlls Fig. , respectP = 0.017); in the sensitive group, YAP expression was decreased after chemotherapy (P = 0.009), but in the insensitive group, there was no significant difference between the values before and after chemotherapy. The changes in expression of β-catenin in TNBC treated with NCT were similar to the changes seen in YAP expression ; in the sensitive group, β-catenin expression was decreased after chemotherapy (P = 0.003). The data implied that changes in the expression of YAP or β-catenin might be a promising predictive biomarker for response in TNBC patients with NCT. More importantly, YAP or β-catenin might also be involved in chemotherapy resistance in TNBC treatment.It remains elusive that whether YAP or β-catenin is involved in chemotherapy resistance in breast cancer, especially in TNBC. Therefore, we examined the expression of YAP and β-catenin in TNBC before and after neoadjuvant chemotherapy (NCT). As shown in Fig. ion Fig. . Before 3) grown in nude mice. As shown in Fig. Docetaxel and vinorelbine are the main drugs for breast cancer treatment, and both drugs exert their anticancer effects by inhibiting the formation of microtubules in tumour cells. To investigate whether YAP or β-catenin affect the inhibitory effects of docetaxel or vinorelbine in TNBC cells, CCK-8 assays were performed. As shown in Fig. To assess whether YAP or β-catenin mediated docetaxel or vinorelbine resistance in TNBC cells, we designed the following assays. First, we established an MDA-MB-231 docetaxel-resistant cell line (MDA-MB-231/DR) and a vinorelbine-resistant cell line (MDA-MB-231/VR). As shown in Fig. To assess the anticancer effect of aspirin on TNBC cells, the proliferation, colony formation and migration assays were employed. The data indicated that aspirin can inhibit the growth of TNBC cells remarkably Fig. . Next, wThe above data indicated that aspirin could sensitize drug-resistant TNBC cells to docetaxel or vinorelbine in vitro, but whether these combined strategies also function in vivo was unknown. We then established drug-resistant MDA-MB-231 breast tumours grown in nude mice and divided the mice into three groups: the control group, the chemotherapy group, and the aspirin combined with chemotherapy group. As shown in Fig. 19. YAP is phosphorylated by Hippo/LATS signaling kinase, causing YAP to remain in the cytoplasm20. β-TrCP catalyses YAP ubiquitination, ultimately leading to YAP degradation20. β-TrCP also acts as a ubiquitin ligase that directly recognizes and degrades phosphorylated β-catenin21. In this part of study, the specific mechanism by which aspirin inhibits YAP or β-catenin was explored. First, we found that aspirin could decrease YAP or β-catenin expression, and as the concentration of aspirin increased, the inhibitory effects became more significant  and positive lymph node metastasis status (P = 0.036). Moreover, high β-catenin expression was correlated with positive lymph node metastasis status (P = 0.028), high histological grade (P = 0.035), and advanced TNM staging (P = 0.040). The correlation coefficients are shown in Table First, we analysed the histopathological characteristics of 112 breast cancer samples with available YAP or β-catenin protein status. The correlations between YAP or β-catenin expression and different clinical histopathological factors are presented in Table 11. The core upstream components of the Hippo pathway comprise several tumour suppressors, including Mst1/2, Sav1/WW45, Lats1/2, and Mob122. These proteins act in a kinase cascade that culminates in the phosphorylation and inactivation of YAP/TAZ22. In most types of tumours, YAP acts as an oncogene to promote the expression of its target genes involved in cell proliferation and survival23. Our laboratory has previously identified a novel positive feedback loop involving YAP and CD44, in which CD44 functions as both an upstream regulator and a downstream effector of YAP through RhoA regulation in hepatocellular carcinoma (HCC)24. We also found that the EGFR-PI3K-PDK1 pathway could activate YAP signalling, and inhibition of YAP enhanced the cytotoxicity of EGFR pathway inhibitors25. Moreover, we also observed that MEK nuclear localization, which is an alternative mechanism of KRAS-targeted-drug resistance, promotes YAP stability by sequestering β-TrCP in KRAS mutant cancer cells26. However, the roles and regulatory mechanisms of YAP remain largely unknown in some tumours, such as breast cancer.The Hippo pathway is an evolutionarily conserved signalling pathway that restricts organ size and is found in organisms ranging from Drosophila to mammals30. In contrast, another study suggested that YAP may act as an oncoprotein in breast cancer and this conclusion was drawn based on the observation that YAP expression was increased in certain breast cancers and promoted the tumor growth and radiation resistance of breast cancer32. In order to clarify these inconsistencies, we initially examined the expression of YAP in normal breast and breast cancer tissues and found that YAP expression in breast cancer tissues was higher than that in normal tissues, but there was no significant difference. Then, our data suggested that high YAP expression was correlated with smaller tumor size and positive lymph node metastasis and had not worse DFS or OS than those with low YAP expression in all breast cancer patients. Notably, subgroup analysis found that high YAP expression was correlated significantly with poor DFS or OS in TNBC patients, while there was no difference in HR+ and HER2+ subtypes. Therefore, YAP may mainly act as a tumor-promoting factor in TNBC, and could act as a promisingly prognostic marker for TNBC.Whether YAP functions as a tumor suppressor or an oncogene remains controversial in breast cancer. Some studies suggested that YAP expression was decreased in breast cancer and associated with favorable prognosis, and based on these data it has been suggested that YAP may act as a tumor suppressor in breast cancer16. β-Catenin is stabilized and translocated to the nucleus, where it acts as a cofactor to activate the expression of target genes implicated in cell cycle progression, survival, angiogenesis, and metabolism33. The interactions between the Hippo/YAP and Wnt/β-catenin pathways play a crucial role in many physical and pathological processes34. Intriguingly, the crosstalk between the two pathways is context-dependent and can result in synergism. YAP has been shown to activate the Wnt/β-catenin cascade by promoting the nuclear translocation of the tyrosine phosphatase SHP235. β-Catenin activation could promote YAP stabilization by inhibiting its proteolysis36. Moreover, β-catenin and YAP interact physically and are activated in most human hepatoblastoma tissues, and overexpression of activated forms of the two proteins in livers leads to rapid liver tumour development in mice37. Our study showed that β-catenin was overexpressed in breast cancer tissues compared with paired adjacent normal tissues and acted as an independent predictor of poor DFS and OS in breast cancer, especially for TNBC. Our results suggest that the combination of high expression of YAP and β-catenin defines a subgroup of breast cancer patients with extremely poor outcomes.Wnt/β-catenin signalling has been demonstrated to contribute to tumorigenesis and cancer stem cell plasticity38. However, drug resistance remains the key problem for treatment with docetaxel and vinorelbine. Our clinical data suggested that YAP/β-catenin might be involved in chemotherapy resistance in TNBC treatment. In the cellular experiments, we found that YAP and β-catenin counteracted the inhibitory effects of docetaxel and vinorelbine on TNBC cells. Interestingly, in a nude mouse model, tumour growth was remarkably inhibited after combined treatment with knockdown of YAP or β-catenin and chemotherapy drugs. We investigated whether YAP or β-catenin mediated docetaxel and vinorelbine resistance in TNBC cells and found that both YAP and β-catenin were key modulators of docetaxel and vinorelbine resistance. In MDA-MB-231 docetaxel- and vinorelbine-resistant cell lines, repression of YAP or β-catenin increased the sensitivity of the drug-resistant cells to docetaxel and vinorelbine. Until now, there has been little study on YAP and docetaxel resistance, and the existing studies showed that YAP expression was elevated in docetaxel-resistant prostate cancer cells and that knockdown of YAP suppressed the migration and invasion of docetaxel-resistant prostate cancer cells39. Our data were consistent with these findings, implying that YAP may be a novel regulator of docetaxel-resistance. To our knowledge, this is the first report about YAP and β-catenin participating in vinorelbine resistance and YAP or β-catenin inhibition enhancing the sensitivity of vinorelbine-resistant breast cancer cells.Docetaxel- and vinorelbine-based regimens have been confirmed to be greatly effective in breast cancer patients40. Other studies found that regular aspirin use was associated with a reduced risk in breast cancer patients, especially for high-risk populations with familial risk or a mutated status8. In addition, aspirin could also overcome tamoxifen resistance in oestrogen receptor-positive breast cancer cells41. Aspirin also induced autophagy to affect the proliferation, apoptosis and drug-resistance of tumour cells43. It is unclear whether aspirin could overcome docetaxel or vinorelbine resistance. Here, we reported for the first time that aspirin reversed docetaxel and vinorelbine resistance in vitro and in vivo in TNBC, and one possible mechanism was that aspirin reduced the expression of YAP and β-catenin by promoting β-TrCP, an E3 ubiquitin ligase. At present, it is controversy about which doses of aspirin could achieve the best antitumor effect. Different studies used different dose settings4. The aspirin concentrations (1–5 mM) used were consistent with measurements of aspirin in the serum of patients treated for chronic inflammatory diseases45. Hence, it is a very complicated process that aspirin exerts the antitumor effect in the human body and the cellular or animal studies do not fully reflect what happens in the human body. On the one hand, it is related to metabolism and distribution of aspirin in the body. On the other hand, different cancers show the different responses to aspirin. The laboratory studies just provide the possible mechanisms and directions, but it is still necessary to future study in the clinic and laboratory.In recent years, many clinical epidemiological studies have found that with an increase in aspirin intake, tumour mortality decreases exponentially in patients with colon, breast, lung, and prostate cancers and cancers of other organs19. YAP is phosphorylated by Hippo/LATS signaling kinase, causing YAP to remain in the cytoplasm20. β-TrCP catalyses YAP ubiquitination, ultimately leading to YAP degradation20. β-TrCP also acts as a ubiquitin ligase that directly recognizes and degrades phosphorylated β-catenin21. Our previous study also showed that inhibition of β-TrCP promotes YAP stability and carcinogenicity26. Interestingly, in this study we found that aspirin can upregulate β-TrCP expression to inhibit YAP and β-catenin expression and it is a possible antitumour mechanism of aspirin. Some studies found that dephosphorylation by PP2A destroys the β-TrCP binding site created by phosphorylation. As a result, β-catenin is prevented from been recruited to the β-TrCP complex for ubiquitin conjugation, leading to its stabilization and high levels of accumulation46. Interestingly, in another study, aspirin acted through PP2A inhibition to increase β-catenin phosphorylation thereby reducing Wnt/β-catenin pathway activity, and aspirin acted on the Wnt/β-catenin pathway via a proteasome-dependent mechanism induced by β-TrCP47. Our results seem to be consistent with the inhibitive effects of aspirin on β-catenin through β-TrCP. In brief, we think that on the one hand, aspirin can inhibit PP2A to make β-TrCP bind to the phosphorylation of β-catenin and promote the degradation of β-catenin; on the other hand, aspirin may upregulate β-TrCP expression directly or indirectly to promote the degradation of β-catenin and this specific mechanism need further study.β-TrCP regulates the expression of a variety of proteinsIn conclusion, this work demonstrated that both YAP and β-catenin affect the efficacy and mediate the resistance of docetaxel and vinorelbine in TNBC cells in vitro and in vivo. To our knowledge, this is the first report showing that aspirin can reverse docetaxel and vinorelbine resistance in TNBC cells in vitro and in vivo. It also suggests a possible mechanism by which aspirin impairs the expression of YAP and β-catenin by promoting β-TrCP to overcome drug resistance. Therefore, the combined use of aspirin and docetaxel or vinorelbine could be promising clinical approaches for TNBC treatment.FigureS1FigureS2FigureS3FigureS4FigureS5Table.S1Table.S2Table.S3Supplementary figure legends


However, the clinical significance and underling mechanism of δ‐catenin expression in renal cell carcinoma (RCC) remain elusive. Herein, we detected the protein expression of δ‐catenin in 28 clinical specimens of paired renal cancer tissues and normal renal tissues by Western blot analysis. δ‐Catenin expression in 58 cases of renal cell carcinoma was also examined by immunohistochemistry, and its association with clinicopathological factors was analyzed by statistical analysis. In vitro and in vivo assays were employed to further explore the biological role of δ‐catenin in RCC. The results showed that δ‐catenin was highly expressed in both clinical samples and cell lines of RCC. RCC patients with higher δ‐catenin expression had a more advanced pTNM stage and tumor stage as well as lymph nodes metastasis than those with lower expression. By regulating the nuclear translocation of β‐catenin and β‐catenin‐mediated oncogenic signals, δ‐catenin promoted proliferation and inhibited apoptosis in RCC. In vivo assay indicated δ‐catenin facilitated tumor growth in ACHN cell xenograft mouse model. Taken together, our study suggests that δ‐catenin might be considered as a novel prognostic indicator and actionable target for gene therapy in renal cell carcinoma. By regulating the nuclear translocation of β‐catenin and β‐catenin‐mediated oncogenic signals, δ‐catenin promoted proliferation and inhibited apoptosis of renal cancer cells. It is the adhesive junction‐associated protein that was originally identified to be enriched in brain tissues.β‐Catenin, a crucial member of the adherent junctions,In the present study, we detected the expression of δ‐catenin in RCC specimens by Western blot analysis and immunohistochemistry staining, and then analyzed the correlation between its expression and clinicopathological factors. To gain insights into the role of δ‐catenin in RCC, we explored whether it was implicated in proliferation and apoptosis of RCC cells. In addition, the regulation of δ‐catenin on β‐catenin and β‐catenin‐mediated oncogenic signals were also investigated. Moreover, we determined the influence of δ‐catenin on tumor growth in the xenograft mouse model. Our results illuminated that elevated δ‐catenin expression in RCC caused the activation of β‐catenin and its target genes, thereby affecting proliferation and apoptosis. This cognition of δ‐catenin may help to establish a novel targeted therapy and improve current therapies of RCC.22.1Normal human renal epithelial cells (HK‐2) and human renal cancer cell lines  were obtained from Procell Life Science & Technology  and cultured in MEM medium or RPMI‐1640 medium supplemented with 10% fetal bovine serum (BI). All the cells were maintained in a humidified incubator with 5% CO2 at 37°C.2.2g for 5 minutes at 4°C. The supernatant was a cytoplasmic extract. The pellet was resuspended with 50 μL nuclei lysis buffer  and kept in ice for 30 minutes (vortexed 15‐30 seconds at a high speed every 1‐2 minutes). Nuclear proteins were extracted by centrifugation at 12 000 × g for 10 minutes at 4°C. The supernatant was the nuclear extract. The protein concentration was assessed by Enhanced BCA Protein Assay Kit .The cells were lysed in 200 μL cytoplasmic lysis buffer A supplemented with PMSF on ice for 10‐15 minutes, and then added with 10 μL cytoplasmic lysis buffer B. The mixture was vortexed for 5 seconds and centrifuged at 12 000 × 2.3Cells were collected and lysed with RIPA lysis buffer . The protein concentrations were evaluated via Enhanced BCA Protein Assay Kit (Beyotime). Protein lysates were separated by electrophoresis on a sodium dodecyl sulfate‐polyacrylamide gel (8%‐15%), transferred onto PVDF membranes, and incubated with primary antibodies at 4°C overnight followed by goat anti‐rabbit or goat anti‐mouse IgG (Proteintech) at 37°C for 40 minutes. The protein bands were visualized by enhanced chemiluminescence (ECL). Primary antibodies used in this study were as follows: anti‐δ‐Catenin antibody , anti‐Cyclin D1 antibody , anti‐CDK6 antibody , anti‐CDK4 antibody , anti‐c‐myc antibody , anti‐Bcl2L1 antibody , anti‐cleaved‐caspase‐3 antibody , anti‐survivin antibody , anti‐β‐catenin antibody , anti‐β‐actin antibody , anti‐Histone H3 antibody .2.4A498 and ACHN cells were transfected by δ‐Catenin‐specific siRNA (5ʹ‐CCCUAGCAGUACUGACCAATT‐3ʹ and 5ʹ‐UUGGUCAGUACUGCUAGGGTT‐3ʹ) or siCtrl (5ʹ‐UUCUCCGAACGUGUCACGUTT‐3ʹ and 5ʹ ‐ACGUGACACGUUCGGAGAATT‐3ʹ) encapsulated with Lipofectamine 2000  following its transfection protocol. After 48 hours of transfection, subsequent assays were performed as described. ACHN cells were transfected by shRNA plasmids or shCtrl plasmids as the negative control with Lipofectamine 2000 for silencing δ‐catenin. After 24 hours, the cells were selected in media supplemented with 300 μg/mL G418 to obtain the stably transduced cells.2.5−∆∆Ct parameters. Sequences of real‐time PCR primers were as follows: δ‐Catenin: forward: 5ʹ‐TCTGAGAAACCTGGTGTATGG‐3ʹ, reverse: 5ʹ‐CAGTCGTCTTGCGGAGTAA‐3ʹ, β‐actin: forward: 5ʹ‐CCACTGCCGCATCCTCTT‐3ʹ, reverse: 5ʹ‐GGTCTTTACGGATGTCAACG‐3ʹ.Total RNA was isolated from RCC cells using total RNA rapid extraction kit  according to the manufacturer's protocol. The obtained RNA was reverse transcribed into cDNA by M‐MLV . The cDNA was used to perform real‐time PCR with Taq HS Perfect Mix  and SYBR Green . The data were determined by Exicycler™ 96 , calculated by 22.63 per well beforehand. After being adhered, the cells were transfected with siCtrl and δ‐catenin siRNA; and CCK‐8 reagent (Sigma) was added into the 96‐well plates with 10 µL per well and incubated for 1 hour. The optical density (OD) value was measured at 450 nm with a microplate reader (BioTek). The OD450 values at 0, 24, 48, 72, and 96 hours were tested, respectively.Cell counting kit‐8 (CCK‐8) assay was employed to detect cell viability. RCC cells were seeded into 96‐well plates with 4 × 102.7The slides of cells were fixed for 15 minutes with 4% paraformaldehyde and permeabilized with 0.1% tritonX‐100 for 30 minutes. The samples were blocked by goat serum for 15 minutes. Primary antibody (diluted to 1:200 with PBS) such us Ki67 and β‐catenin was added to the cells and incubated overnight at 4°C. After PBS washing, cells were incubated with Cy3‐labeled goat anti‐rabbit IgG  diluted in PBS (1:200) for 1 hours at room temperature. The nuclei were counterstained by DAPI. Finally, the samples were sealed by anti‐fade reagent and observed using the OLUMPUS fluorescence microscope (at 400 × magnification).2.8Flow cytometry was performed to detect the cell cycle and apoptosis of RCC cells. After transfection for 48 hours, cells were collected, treated by Cell Cycle Analysis kit (Beyotime) following the manufacturer's protocol, and detected using NovoCyte flow cytometer (ACEA Biosciencs). For the apoptosis analysis, the cells were treated with Annexin V‐FITC Apoptosis Detection Kit (Beyotime) and accessed through flow cytometer.2.9ACHN cells transfected with δ‐catenin siRNA or siCtrl were seeded in 6‐well plates. After incubation for 24 hours, the cells were transfected with 2 μg β‐catenin responsive firefly luciferase reporter plasmid TOPflash or negative control FOPflash and 0.5 μg constitutively active vector encoding Renilla luciferase using lipofectamine 2000. At 24 hours after transfection, the cells were lysed and then firefly and Renilla luciferase activities were determined using the dual luciferase reporter assay system (Promega) according to the manufacturer's instructions.2.107) with stable knockdown of δ‐catenin with shRNA plasmids or ACHN cells transfected with shCtrl plasmids were injected subcutaneously into the right flank of the nude mice. The tumor size was measured every four days, and the tumor volume was calculated according to the following: V = (width2 × length)/2. The mice were sacrificed 27 days after injection. Tumors were excised and the tumor weight was recorded.All animal experiments in this study were approved by the Institutional Animal Ethics Committee of China Medical University, and were carried according to the Guideline for the Care and Use of Laboratory Animals. Male Balb/c nude mice at the age of 6 weeks were divided into two groups randomly (6 mice per group). ACHN cells  overnight at 4°C. The slides were washed with PBS and incubated with HRP‐labeled goat anti‐rabbit antibody  for 1 hour. After washing, the slides were added with diaminobenzidine (DAB) and counterstained with hematoxylin. In the end, the sections were dehydrated by graded ethanol, sealed by neutral balsam, and visualized with microscope (at 400 × magnification).2.12P value < .05 was considered statistically significant.The data were presented as mean ± SD in the figures. Statistical analysis was performed with two‐tailed unpaired t‐test, one‐way ANOVA or two‐way ANOVA as indicated. 33.1P < .05), but not correlated with patient's gender, age, tumor size, and distant metastasis (P > .05). These results together demonstrated that the expression of δ‐catenin was enhanced in RCC tissues, which was associated with poor prognosis in RCC patients.We firstly determined the expression of δ‐catenin in human renal cancer tissues. Western blot analysis was performed to examine δ‐catenin expression from 28 cases of patients with RCC. The results showed that δ‐catenin was highly expressed in tumor tissues compared with adjacent normal tissues Figure A. Furthe3.2Next, we determined the level of δ‐catenin in normal human renal epithelial cells HK‐2) and three different RCC cell lines . As expected, δ‐catenin was highly expressed in RCC cells compared with HK‐2 cells . Tumor volume was recorded over a period of 27 days and the solid tumors were photographed and weighed after the mice were sacrificed. As shown in Figures 4It has been proven that δ‐catenin has ectopic overexpression in lung cancer and prostate cancer and promotes tumor progression,Researches conducted by Zhang et alIn this study, we found δ‐catenin promoted RCC cell proliferation by altering G1 to S phase transition in cell cycle. Furthermore, δ‐catenin restrained apoptosis of RCC cells through upregulating Bcl2L1 (a long isoform of Bcl2) that protects cells from undergoing apoptosis.Given that δ‐catenin affected subcellular β‐catenin distribution and activated the downstream effectors of β‐catenin in prostate cancer,In our study, we used RNA interference (RNAi) including transient siRNA and stable shRNA for loss‐of‐function studies to investigate the role of δ‐catenin in RCC development. One major drawback of these means is the limited utility, as evidenced by δ‐catenin‐silenced cells still could grow and preserve tumorigenesis capacity. In future research, the inducible system (such as the tetracycline‐controlled operator system) should be combined with RNAi, which could reveal the direct effects caused by the loss of δ‐catenin via comparing noninduced and induced cells, thereby accurately evaluating the crucial role of δ‐catenin in RCC cell growth and apoptosis.In summary, our study is the first to report δ‐catenin was overexpressed in tissues of RCC patients and its correlation with pTNM stage, tumor stage, and lymph nodes metastasis. In addition, knockdown of δ‐catenin can obviously decrease the proliferation capacity and induce apoptosis of RCC cells by affecting β‐catenin nuclear localization and its downstream target gene expression. Therefore, our results illustrated that δ‐catenin might be a prognostic marker and a promising therapeutic target in RCC.The authors declare that they have no competing interests.Yongsheng Song conceived and designed the study. Ju Lincheng performed the experiments and analyzed the data. Shan Liping wrote the paper. Yinbo obtained funding. All authors have read and approved the final manuscript. Click here for additional data file.


The increase of adhesion parameter b was 77.5% for A and 151.5% for B. The adhesion of both eco-adhesives was tested using the bell peel test (BPT) with the Al and CFRP adherends. When compared to REF, the average peel load for B was 17.6% (39.3%) and 58.3% (176.9%) higher for the Al and CFRP adherends, respectively. Complete adhesion failure of REF reflected the weak adhesion to both Al and CFRP, which was improved by the addition of eco-epoxy components A and B showing the presence of cohesive failure. The microhardness testing method of interface adhesion was proven to be a fast and reliable testing method, providing a qualitative indication in adhesive selection.This paper presents a new process for obtaining eco-epoxide adhesives synthesized from bio-renewable raw material (tannic acid—TA) and used for bonding lightweight materials  and carbon fiber reinforced polymer (CFRP)). Two synthesized bio-epoxy components based on TA, (A) glycidyl ether and (B) glycidyl phosphate ester of TA, were used as a replacement for the toxic epoxy component based on Bisphenol A. The effect of eco-epoxy components on the interface adhesion was measured by the determination of adhesion parameter  Nowadays, thermosetting polymers have a significant share in plastic production 20%) due to their outstanding properties . Epoxy m0% due toBisphenol A (BPA) is a petroleum based chemical that represents a building block of most epoxy materials >90%). The aromatic core of such compounds contributes to the stability, as well as high thermal and mechanical properties of epoxy materials. Bisphenol A diglycidyl ether (DGEBA) is an epoxy-functionalized component based on BPA, which is also used in the production of food-related and health products such as bottles, containers, dental materials, and products for skin care . However0%. The aEfforts have been made in adhesive technology to use bio-renewable sources in producing commercial (petroleum/based) counterparts with equal or even better performance. Abundant bio-based raw materials like lignin, tannin, and cellulose consist of numerous hydroxyl groups, increasing their reactivity, which is favorable in the production of adhesives ,19,20. IThe aim of this study is to investigate the interface adhesion of novel eco-epoxy adhesives by the addition of two types of modified tannic acid: (A) glycidyl ether and (B) glycidyl phosphate ester of TA, which are used as a bio-based replacement of the BPA-based epoxy component. The majority of structural epoxy adhesives, used in aerospace, contain a BPA component, and thus the adhesion effects were analyzed on two different substrates: aluminum (Al) and carbon fiber reinforced polymer (CFRP), which are used for lightweight structures. Methods used for characterization were the microhardness testing method, the bell peel test (BPT), and microstructural analysis of fractured surfaces. In addition, proving that the microhardness testing method of the interface adhesion is a reliable and fast testing method will enable its use as qualitative indicator in adhesive selection.N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF), phosphorus oxychloride (POCl3), glycidol, magnesium sulfate, calcium chloride—were used as received and supplied from Sigma-Aldrich Chemie Gmbh, Steinheim, Germany. The solvent used for the surface cleaning, acetone, was supplied from Sigma Aldrich, USA. The reference adhesive was selected to be a BPA-based epoxy (LG700 epoxy component and HG 700R curing agent) and was supplied from GI-NI ltd., Belgrade, Serbia .The chemicals used in the synthesis of modified tannic acid (TA)—epichlorohydrin (EPH), sodium hydroxide (NaOH), deionized water (MiliQ), chloroform, Two types of substrates were selected: aluminum alloy 2024 and CFRP composites HexPly 8552 unidirectional prepreg epoxy resin in combination with AS4 carbon fiber . CFRP laminates were manufactured in the autoclave with a curing cycle of 180 °C for 120 min at 7 bars pressure. During manufacturing, the surface of the laminates was in contact with a fluorinated ethylene propylene copolymer release film .6) and 0.5 mm for the flexible adherend [0°/90°].Aluminum and CFRP plates were cut into 5 replicates according to the standards for the bell peel test (BPT), 150 × 300 mm each . Each sa4 overnight. The toluene solution was filtrated and transferred to a flask equipped with a short distillation column, Liebig condenser, and recipient fitted well to sustain high vacuum (~1 kPa). Concomitant increase of temperature (2 °C/min) was followed by pressure decrease in order to remove all volatile residues in glycidyl ether of TA. The obtained product was a highly viscous brownish liquid.Glycidyl ether of TA was obtained via the reaction of TA and EPH at 80 °C and at 1:1.5 wt. ratio of TA to NaOH . EPH . After 10 min, glycidol was added in a dropwise manner for 2 min. Using the same time intervals (1 and 2 min), the addition was continued until the whole amount of reactants was reached. Then, the temperature was set to 85 °C with the vacuum gradually increasing, until all the chloroform was removed. The reaction was completed after 12 h and then the vacuum was increased (10 Pa) in order to remove NMP. When the reaction was finished, the product purification was performed analogously to glycidyl ether of TA. The obtained product was a highly viscous brown liquid.Glycidyl phosphate ester of TA was obtained according to a novel developed procedure for obtaining fire retardant epoxy components . At roomThe chemical structure of both types of adhesive components obtained by the modification of TA are presented in 2O3 powder  in ±45° direction b, III—ac2, H2O, N2, and O2 [, and O2 ,31. Howe, and O2 ,33,34,35, and O2 .The surface roughness parameters are analyzed as arithmetical mean height  and as maximum height profile  . ResultsThree adhesives were selected for testing the adhesion effects on both of substrates: (1) reference epoxy adhesive (REF); (2) epoxy adhesive with 15 wt.% of glycidyl ether of tannic acid (adhesive A); and (3) epoxy adhesive with 15 wt.% of glycidyl phosphate ester of TA, (adhesive B). Glass bead spacers , used for the adhesion thickness control, were mixed with the adhesives at 0.1 wt.% prior to bonding. Final thickness of samples was 210 µm ± 40 µm. The curing of the specimens was performed at room temperature for 24 h, with post-curing at 70 °C for 4 h. After curing, 25 mm wide samples were cut.TM iSTM10 spectrometer . The attenuated total reflectance (ATR) sampling technique was used, which is equipped with a DTGS detector and Golden Gate system with a diamond crystal. The OMNIC software was used to record spectra of samples in a wavelength range of 4000–500 cm−1 with a 4 cm−1 resolution. Afterwards, they were post-processed with ATR correction.FTIR spectroscopy of epoxy components used in the adhesive preparation was performed in order to confirm the obtained structure of the synthesized components and to compare the presence of the functional groups that can affect the bonding interface. The analysis was done using a NicoletThe adhesive affinity to both adherends, Al and CFRP, was assessed by measuring the wetting angle. A drop of the adhesive mixtures was placed on the aluminum adherend after the surface treatment. Five wetting angle measurements were performed using the same test conditions. The images of the samples were taken with an optical microscope , and the contact angles were determined using the image analysis software Image-Pro Plus 4.0 .P (kgf) is the applied load and d (mm) is the average length of the indentation diagonals [The microhardness of the adhesives on aluminum surfaces was characterized by the micro Vickers hardness (HV) tester Leitz, Kleinharteprufer DURIMETI, using an original quadrangular pyramid diamond indenter with an angle of 136° . In ordeiagonals .BPT was performed with the aim to study the adhesion effect of the synthesized eco-epoxy components on both adherends, Al and CFRP. The experimental procedure for BPT was based on the standard test method described in ASTM D3167 . Figure The post-mortem fractured surface of representative samples from each tested group was analyzed in order to determine the type of adhesion failure using 3D optical microscope with a wide-area 3D measurement system, type VR-5200 from Keyence, Itasca, Illinois, USA. The surface roughness parameters (Ra/Pa/Wa and Rz/Pz/Wz) and the corresponding statistical analysis were carried out by the VR-5000 Series Analyzer Software.−1 for both A and B when compared to TA. Other characteristic changes were observed as deformation vibration of epoxy C–O group at 913 cm−1 and epoxy ring C–O–C stretching vibration at 829 cm−1 [−1 and from 867 to 874 cm−1 due to the interference of phosphate and the epoxy group. This is the most significant indicator of the obtained glycidyl phosphate ester of TA. In addition, the carbonyl group vibration at ~1700 cm−1 was noticed for the TA-derived components due to the ester linkage of the TA core. The REF epoxy component showed characteristic peaks of diglycidyl ether of Bisphenol A (DGEBA): C–O deformation band centered at 915 cm−1, C–H stretching of the terminal epoxy at 3054 cm−1, broad O–H stretching vibration at ~3500 cm−1, bands of ether linkage at 1000–1100 cm−1, oxirane C–O–C stretching at 829 cm−1, oxirane C–O stretching at 913 cm−1, stretching of C–O–C ethers at 1034 cm−1, aromatic C–C stretch at 1508 cm−1, aromatic ring stretching at 1609 cm−1, aliphatic C–H, CH2, and C–H aromatic stretch at 2965–2877 cm−1, oxirane ring C–H stretching at 3054 cm−1 [FTIR analysis of the modified TA and neat TA was performed to follow the structural changes in the course of modification and to analyze the differences in the content of the functional groups that can affect the adhesive and cohesive properties of the obtained adhesives . The cha829 cm−1 . Compone054 cm−1 .Based on the presented FTIR spectra, it is clear that the obtained A and B components showed higher polarity due to the presence of hydroxyl groups which can improve the adhesion to both Al and CFRP by establishing hydrogen bonding, and Van der Waals intermolecular forces with the carbonyl group. On the other hand, REF components showed a higher amount of epoxy groups increasing the cross-linking density, and thus leading to a more brittle material.2O3 layer. A higher wetting angle of adhesive B relative to A, for both adherends, can be attributed to the increased amount of interaction by hydrogen bonding transfer of the phosphate group. Phosphorus is acting as a ligand atom transferring hydrogen bonding from a hydrogen bond donor (or antecedent atom of a hydrogen bond donor) via oxygen as atom of a ligand [Wetting theory suggests that the adhesion is a result of the molecular contact between the substrate-adhesive and the forces developed. The adhesive should have a lower surface tension than a critical surface tension of a substrate in order to establish good wetting of a solid surface . Van dera ligand . In the a ligand .b is determined as the ratio of the plastic zone radius formed under a micro-indenter and the depth of indentation, which is influenced by the applied film (adhesive), the adherend, and the established adhesion [The adhesion parameter adhesion . If a weadhesion . A modelb as a function of the replaced epoxy BPA component. An increase of the adhesion parameter b with the increase of TA content indicated an increase of adhesion with Al adherend. This phenomenon can be interpreted in the light of the establishment of a stronger hydrogen bonding on the interface with the introduction of a higher content of TA component. The hydrogen bonding transfer of the phosphate group, observed in the wetting angle measurements, affected the higher rate of the adhesion parameter b increase for adhesive B. Higher amounts of TA component in adhesives deteriorated the processability/applicability of the adhesives. Thus, the content of 15 wt.% was selected for BPT. The adhesion parameter b, for adhesive B with 15 wt.% of replacement, was 41.6% higher than for adhesive A and 153.4% higher than the REF. These results indicate the strong interface adhesion of adhesive B and its significant improvement when compared to the reference BPA-based epoxy component (REF).Fave), maximal peel strength (Fmax), and failure modes are given in Fave and Fmax showed the same tendency for both adherends, Al and CFRP, for the tested adhesives. Fave (Fmax) of adhesive A was as low as 35.3% (24.4%) and 62.5% (61.5%) on the Al and the CFRP adherends when compared to REF, respectively. Fave of adhesive B was as high as 17.6% (39.3%) and 58.3% (176.9%) on the Al and the CFRP adherends when compared to REF, respectively. Regarding the failure mode, REF showed complete adhesive failure for both Al and CFRP indicating weak adhesion. Adhesive A showed a small amount of cohesive failure . This percentage of CF was the highest for adhesive B on both Al and CFRP in comparison with A and REF adhesives.BPT was used to obtain an insight into the adhesion failure mechanisms and the peel strength of the used adhesives. The average  curves with the corresponding fractured Al surfaces after BPT for all the tested adhesives are presented in The surface roughness characterization was done on the rigid adherend, after testing, to further analyze the features of the fractured Al surface . In the b gives an indication of the interface strength, it can be compared and correlated to the amount of cohesive failure on the flexible adherend after BPT. Cohesive failure suggests that the adhesion interface forces prevailed over the cohesive forces and may be an indicator of the adhesion strength, similar to the adhesion parameter b. R2 = 0.99) fitted between the cohesive failure and the adhesion parameter b. The obtained results indicated that the fast and easy method for assessment of the adhesion quality, using the adhesion parameter b, may be used to predict the differences between the selected adhesives for the same adherend material. Nevertheless, the adhesion parameter b cannot predict the differences in peel loads, and its use might be limited only for the preselection of adhesives for specific use.Since the adhesion parameter b, bell peel test (BPT), and fractured surface analysis. This study showed that the introduction of TA-based components contributed to the increase of hydrogen bonding, which further enhanced the interface adhesion. Improvement of the interface adhesion was noticed by the increase in the adhesion parameter b, which was 77.5% higher for component A and 151.5% higher for component B. The higher contact angle of component B compared to the one of A was due to the presence of a higher extent of cohesive forces in the adhesive itself. The highest peel loads of B were influenced by both adhesive and cohesive strength. The hydrogen bonding, as a main adhesion mechanism, enhanced the adhesion strength of adhesive B. Moreover, the cohesion strength of adhesive B is improved by higher cross-linking density than adhesive A. Linear correlation between the cohesive failure and the adhesion parameter b indicated that the microhardness testing method of interface adhesion can be used as a fast and reliable testing method in the adhesive selection process. The synthesized epoxy ester phosphate derivate of TA (adhesive B) showed enhanced interfacial adhesion on both Al and CFRP, and their high potential as a replacement of the BPA component (DGEBA) was emphasized. Industrial application of obtained eco-epoxy adhesives might consider bonding of secondary/non-structural elements of lightweight structures.Two eco-epoxide components based on TA were synthesized: (A) epoxy functionalized and (B) epoxy ester phosphate derivate of TA, and used as a replacement of the BPA-based component. The effect of the synthesized bio components on the interface adhesion was tested on Al and CFRP, which are commonly used for lightweight structures, and compared to the reference BPA-based epoxide. Adhesion properties were evaluated by contact angle, adhesion parameter


Ischemic stroke has been becoming one of the leading causes resulting in mortality and adult long-term disability worldwide. Post-stroke pneumonia is a common complication in patients with ischemic stroke and always associated with 1-year mortality. Though ambroxol therapy often serves as a supplementary treatment for post-stroke pneumonia in ischemic stroke patients, its effect on functional recovery and potential mechanism after ischemic stroke remain elusive. In the present study, the results indicated that administration of 70 mg/kg and 100 mg/kg enhanced functional recovery by virtue of decreasing infarct volume. The potential mechanism, to some extent, was due to promoting NSCs differentiation into neurons and interfering NSCs differentiation into astrocytes through increasing GCase expression to activate Wnt/β-catenin signaling pathway in penumbra after ischemic stroke, which advanced basic knowledge of ambroxol in regulating NSCs differentiation and provided a feasible therapy for ischemic stroke treatment, even in other brain disorders in clinic. Previous studies have certified that administration of ambroxol could increase the brain GCase activity not only in healthy non-human primates  was dissolved in normal saline and was intraperitoneally injected for 12 days  for behavioral tests, and 7 days  for immunostaining and immunoblot after dMCAO. For mice in group sham and dMCAO + Vehicle, they only received an equal volume of normal saline as ambroxol groups at the same time point.This study was approved by The Third Military Medical University Ethics Committee  and all procedures were performed in accordance with the Chinese Animal Welfare Legislation for protection of animals used for scientific purposes. The model was established using distal middle cerebral artery occlusion (dMCAO) as previously described , then dissociated in StemPro Accutase Cell Dissociation Reagent  and incubated in the aforementioned medium. NSPCs, used for all experiments in the present study, were from passage 3 to 5.A total of 11 E14.5 C57BL/6 mice were obtained from Animal Experimental Center of Third Military Medical University and applied for cultivating primary NSCs in the present study, as previously described  model is widely used as a feasible 5/ml) were seeded in 10 μg/ml poly-L-ornithine (PLO)-pre-coated confocal culture dishes and incubated in DMEM/F12 medium supplemented with B27 and 1% GlutaMAX™ with or without Ambroxol for 10 days under humidified 5% CO2 condition at 37°C. Cardamonin  and XAV939  were dissolved in DMSO in the stock solution (10 mM), and diluted in culture medium at the final concentration. Recombinant mouse β-glucocerebrosidase  and various concentration of ambroxol were dissolved in PBS in the stock solution. All chemicals were added to the culture medium when NSCs exposed to OGD for 10 days during differentiation. For NSCs in group OGD + Vehicle, they received an equal volume of PBS and DMSO (0.1%) at the same time point as OGD groups. The culture medium was exchanged every 2–3 days.For differentiation, NSCs , and local epibiotic neural lineage cells, mice were received two intraperitoneal BrdU injections  per day for 3 consecutive days, then mice were killed 4 days after the last injection.To tell the difference of DCX5/ml) cultured in poly-L-ornithine (PLO)-pre-coated confocal culture dishes or frozen brain sections (−20°C) from each group were post-fixed using 4% paraformaldehyde (PFA) in 0.01 M phosphate buffer saline (PBS) for 1 h at room temperature after rinsed twice with PBS. Then, the samples were blocked with 10% normal goat serum supplemented with 0.5% Triton X-100  in PBS for 30 min at room temperature. Afterward, samples were immersed in rabbit polyclonal anti-GBA , mouse monoclonal anti-MAP2 , rat monoclonal anti-GFAP , mouse monoclonal anti-β-catenin , mouse monoclonal anti-BrdU , rabbit polyclonal anti-DCX , rabbit polyclonal anti-GFAP , mouse monoclonal anti-DCX , mouse monoclonal anti-Nestin  or Alexa Fluor® 647-conjugated mouse monoclonal anti-Nestin  overnight at 4°C. Then, samples were incubated with Alexa Fluor® 555 or 488-conjugated secondary antibody  at room temperature for 1 h. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole  for 10 min at room temperature. Subsequently, samples were mounted onto glass slides and images were captured by a confocal microscope  and evaluated using Zen 2011 software .For immunostaining, NSCs  twice for 10 min. Afterward, they were incubated in 3% HFor immunostaining analyzing, the samples were obtained from penumbra  using the WesternBright ECL Kits . Densitometric measurement of each membrane was performed using Image Lab™ software .Brain tissues from penumbra or cell samples were homogenized in 200 μl ice-cold RIPA  supplemented with protease inhibitor cocktail . Then, lysate was collected after centrifugation at 10,000 g at 4°C for 20 min. Afterward, the protein concentration was determined by an enhanced BCA Protein Assay Kit . Proteins (50 μg for brain tissue and 20 μg for cell sample) were separated by 10% SDS-PAGE under reducing conditions and electro-blotted to polyvinylidene difluoride  membranes. Then, the membranes were immersed in 5% (w/v) non-fat dry milk (Beyotime Institute of Biotechnology) in TBS with Tween-20 (TBST) at room temperature for 2 h. Afterward, the membranes were cut out at different parts according to a pre-stained protein molecular ladder  to allow separate detection of proteins migrating at the same distance, and were incubated in primary antibodies, rabbit polyclonal anti-GBA , mouse monoclonal anti-β-catenin , rabbit polyclonal anti-β-catenin , rabbit polyclonal anti-phospho-β-catenin , rabbit polyclonal anti-non-phospho (active)-β-catenin , rabbit monoclonal anti-Axin2 , rabbit polyclonal anti-DCX , rabbit polyclonal anti-GFAP  or mouse monoclonal anti-GAPDH  overnight at 4°C. Subsequently, the membrane was incubated in corresponding horseradish peroxidase (HRP)-conjugated secondary antibody after rinsed twice with TBST. All membranes were visualized by a ChemiDoc™ XRS−ΔΔCq method. Primer sequences used in the present study were as follows:After the NSCs were exposed to different concentration of ambroxol under OGD for 8 h, the total RNA was extracted using TaKaRa MiniBEST Universal RNA Extraction Kit  according to the manufacturer's instructions. Then, 1 μg RNA was reverse transcribed into cDNA using PrimeScript RT reagent Kit with gDNA Eraser  according to the manufacturer's instructions. Subsequently, qPCR was performed using CFX96 System  with SYBR Premix Ex TaqII (Tli RNaseH Plus)  under the following condition: 95°C for 30 s, 40 cycles at 95°C for 5 sec and 60°C for 30 s. Relative mRNA levels were normalized to GAPDH and analyzed using the 2post hoc test in case of the data with a normal distribution using a Shapiro–Wilk normality test. Moreover, data failing the normality test were represented as median and interquartile range (IQR) analyzed using the Mann–Whitney U test. A p < 0.05 was considered as significant difference.The statistical analysis was carried out using SPSS v19.0 . Comparisons between two groups were analyzed using Student's t tests or analysis of variance (ANOVA), followed by Tukey's in vivo experiment.Based on our previous investigation that 35 mg/kg and 70 mg/kg ambroxol could facilitate neuronal survival and decrease white matter fiber bundle injury by suppressing microglial activation and decreasing proinflammatory cytokine accumulation in mice with intracerebral hemorrhage (ICH), finally enhance functional recovery  and GFAP+ cells were assessed in the SVZ  activity to regulate NSCs differentiation  was applied to optimize the suitable therapeutic concentration in vitro experiments. Firstly, the mRNA expression of GCase, β-catenin and Axin2-a negative mediator of Wnt/β-catenin signaling pathway inducing β-catenin degradation  model was applied to mimic Above findings suggest that there is a positive correlation between GCase and β-catenin when NSCs were exposed to OGD and with ambroxol administration. Hence, Cardamonin, an inhibitor of Wnt/β-catenin signaling pathway, was applied to investigate the role of Wnt/β-catenin signaling pathway in NSCs differentiation with/without administration of ambroxol. The results revealed that the increased expression of DCX induced by administration of 60 μM ambroxol was obviously decreased with addition of 10 μM Cardamonin . And, thGiven that Cardamonin bears the capacity of targeting various signaling molecules, transcriptional factors, cytokines and enzymes, such as mTOR, NF-κB, Akt, STAT3, COX-2, except for Wnt/β-catenin signaling pathway, XAV-93923, a tankyrase inhibitor targeting Wnt/β-catenin signaling, was used to assure the results obtained from Cardamonin. At the same time, the recombinant mouse glucocerebrosidase (10 ng/ml) was used to investigate the correlation between NSCs differentiation and activation of Wnt/β-catenin signaling pathway. The results indicated that rGCase enhanced NSCs differentiation into neurons under OGD condition, which held the similar effect on NSCs differentiation as the ambroxol using immunostaining assays . Subsequin vivo, 60 μM in vitro, respectively. Moreover, administration of ambroxol increased GCase expression to activate canonical Wnt/β-catenin signaling pathway resulting in β-catenin accumulation, which promoted NSCs differentiation into neurons, instead of astrocytes, in penumbra after ischemic stroke. Additionally, administration of ambroxol reduced the expression of p-β-catenin and Axin2. The canonical Wnt/β-catenin pathway, one of the highly conserved developmental pathways, plays a pivotal role in various physical and pathological conditions, including regulation of neurogenesis, tissue homeostasis and neural survival in the nervous system (CNS)  and casein kinase 1 (CK1) , which decreases NF-κB transcriptional activity, resulting in microglial polarization from M1 to M2 phenotype and downregulation of inflammation .HG performed most of the experiments, with assistance from CZ, YY, WC, JZ, XF, XJ, LT, and YZ. HG, YY, and CZ analyzed the results and edited figures. WC performed dMCAO and statistical analysis. JZ, XF, and LT performed cell culture and treatments. HG, YZ, CZ, and JZ performed immunoblotting and immunostaining. HG wrote preliminary draft of the manuscript. RH and HF edited the manuscript. YC and HF designed experiments and revised the manuscript. All authors approved final version of the manuscript.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.