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BACKGROUND: Psoriasis, a chronic inflammatory skin disorder, is frequently linked with metabolic, cardiovascular, and psychological comorbidities. Recent research has highlighted the correlation between psoriasis and major depressive disorder (MDD); however, the underlying mechanism remains unclear. METHODS: Commonly differentially expressed genes (DEGs) in psoriasis and MDD were identified and visualized using data from the GEO database. Subsequently, functional enrichment analysis was conducted using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Genemania. The hub gene was selected through LASSO and Random Forest algorithms, validated in clinical tissues using Student's t-test and Receiver Operating Characteristic curve. To investigate the hub gene's function in disease phenotype, we established imiquimod (IMQ)-induced psoriasiform dermatitis and chronic unpredictable mild stress (CUMS) mouse models. Lentiviral shRNA interference was topically applied in mice, and downstream pathways were validated at the mRNA and protein levels. RESULTS: A total of 395 overlapping DEGs were identified from GSE121212 and GSE54568 datasets, and twenty core genes were extracted. Functional enrichment analysis revealed that the core genes were significantly associated with the Wnt signaling pathway, neurodegeneration, and energy metabolism. CD19 was identified as the hub gene through algorithms, and external validation showed remarkable AUC values of 0.69 and 0.74, respectively. The level of CD19 increased significantly in IMQ-treated and CUMS-treated mice. Suppression of CD19 significantly alleviated the phenotypes of IMQ-induced psoriasiform dermatitis and CUMS-induced depressive-like behaviors by regulating the PPARγ/ß-catenin/Wnt3a pathway. CONCLUSION: CD19 may serve as a common biomarker or therapeutic target of psoriasis and MDD via PPARγ/ß-catenin/Wnt3a pathway.

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BACKGROUND: Wnt proteins are crucial for embryonic development, stem cell growth, and tissue regeneration. Wnt signaling pathway is activated when Wnt proteins bind to cell membrane receptors. METHODS AND RESULTS: We employed a luciferase reporter assay in HEK293STF cells to measure Wnt protein-induced signaling. We observed that Wnt3a uniquely promotes the Wnt/ß-catenin pathway through positive cooperativity. Additionally, MFH-ND, a molecular mimic of Wnt ligands, markedly increased Wnt3a-induced signaling in a dose-responsive manner. This suggests that various Wnt ligands can synergistically enhance Wnt pathway activation. CONCLUSIONS: The study suggests the likelihood of various Wnt ligands coexisting in a single signalosome on the cell membrane, providing new insights into the complexities of Wnt signaling mechanisms.

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BACKGROUND: The colorectal adenoma undergoes neoplastic progression via the normal epithelium-adenoma-adenocarcinoma sequence as reported in the Vogelgram. The hazard of developing a tumor is deeply associated with the number and size of adenomas and their subtype. Adenomatous polyps are histologically categorized as follows: approximately 80-90% are tubular, 5-15% are villous, and 5-10% are tubular/villous. Given the higher risk of a malignant transformation observed in tubular/villous adenomas, patients diagnosed with adenomatous polyposis are at an improved risk of developing CRC. The Wnt/ß-catenin pathway plays a key role in the onset of colorectal adenoma; in particular, intestinal cells first acquire loss-of-function mutations in the APC gene that induce the formation of adenomas. METHODS: Wnt/ß-catenin pathway APC, Wnt3a, Wnt5a, LEF1, and BCL9 genes and protein expression analyses were conducted by qRT-PCR and western blot in 68 colonic samples (polyps and adjacent mucosa) from 41 patients, of which 17 were affected by FAP. Ten normal colonic mucosal samples were collected from 10 healthy donors. RESULTS: In this study, both the APC gene and protein were less expressed in the colon tumor compared to the adjacent colonic mucosa. Conversely, the activated ß-catenin was more expressed in polyps than in the adjacent mucosa. All results confirmed the literature data on carcinomas. A statistically significant correlation between Wnt3a and BCL9 both in polyps and in the adjacent mucosa underlines that the canonical Wnt pathway is activated in early colon carcinogenesis and that the adjacent mucosa is already altered. CONCLUSION: This is the first study analyzing the difference in expression of the Wnt/ß-catenin pathway in human colorectal adenomas. Understanding the progression from adenomas to colorectal carcinomas is essential for the development of new therapeutic strategies and improving clinical outcomes with the use of APC and ß-catenin as biomarkers.

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BACKGROUND & AIMS: Humans with WNT2B deficiency have severe intestinal disease, including significant inflammatory injury, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis. METHODS: We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the baseline histology and health of the small intestine and colon, and the impact of inflammatory challenge using dextran sodium sulfate (DSS). We also evaluated human intestinal tissue. RESULTS: Mice with WNT2B deficiency had normal baseline histology but enhanced susceptibility to DSS colitis because of an increased early injury response. Although intestinal stem cells markers were decreased, epithelial proliferation was similar to control subjects. Wnt2b KO mice showed an enhanced inflammatory signature after DSS treatment. Wnt2b KO colon and human WNT2B-deficient organoids had increased levels of CXCR4 and IL6, and biopsy tissue from humans showed increased neutrophils. CONCLUSIONS: WNT2B is important for regulation of inflammation in the intestine. Absence of WNT2B leads to increased expression of inflammatory cytokines and increased susceptibility to gastrointestinal inflammation, particularly in the colon.

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BACKGROUND: The current treatment of osteogenesis imperfecta (OI) is imperfect. Our study thus delves into the potential of using Dickkopf-1 antisense (DKK1-AS) to treat OI. METHODS: We analysed serum DKK1 levels and their correlation with lumbar spine and hip T-scores in OI patients. Comparative analyses were conducted involving bone marrow stromal cells (BMSCs) and bone tissues from wild-type mice, untreated OI mice, and OI mice treated with DKK1-ASor DKK1-sense (DKK1-S). RESULTS: Significant inverse correlations were noted between serum DKK1 levels and lumbar spine (correlation coefficient = - 0.679, p = 0.043) as well as hip T-scores (correlation coefficient = - 0.689, p = 0.042) in OI patients. DKK1-AS improved bone mineral density (p = 0.002), trabecular bone volume/total volume fraction (p < 0.001), trabecular separation (p = 0.010), trabecular thickness (p = 0.001), trabecular number (p < 0.001), and cortical thickness (p < 0.001) in OI mice. DKK1-AS enhanced the transcription of collagen 1α1, osteocalcin, runx2, and osterix in BMSC from OI mice (all p < 0.001), resulting in a higher von Kossa-stained matrix area (p < 0.001) in ex vivo osteogenesis assays. DKK1-AS also reduced osteoclast numbers (p < 0.001), increased ß-catenin and T-cell factor 4 immunostaining reactivity (both p < 0.001), enhanced mineral apposition rate and bone formation rate per bone surface (both p < 0.001), and decreased osteoclast area (p < 0.001) in OI mice. DKK1-AS upregulated osteoprotegerin and downregulated nuclear factor-kappa B ligand transcription (both p < 0.001). Bone tissues from OI mice treated with DKK1-AS exhibited significantly higher breaking force compared to untreated OI mice (p < 0.001). CONCLUSIONS: Our study elucidates that DKK1-AS has the capability to enhance bone mechanical properties, restore the transcription of osteogenic genes, promote osteogenesis, and inhibit osteoclastogenesis in OI mice.

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Infectious hematopoietic necrosis virus (IHNV) is a serious pathogen that causes great economic loss to the salmon and trout industry. Previous studies showed that IHNV alters the expression patterns of splenic microRNAs (miRNAs) in rainbow trout. Among the differentially expressed miRNAs, miRNA146a-3p was upregulated by IHNV. However, it is unclear how IHNV utilizes miRNA146a-3p to escape the immune response or promote viral replication. The present study suggested that one multiplicity of infection (MOI) of IHNV induced the most significant miR-146a-3p expression at 1 day post infection (dpi). The upregulation of miR-146a-3p by IHNV was due to viral N, P, M, and G proteins and relied on the interferon (IFN) signaling pathway. Further investigation revealed that Wingless-type MMTV integration site family 3a (WNT3a) and G1/S-specific cyclin-D1-like (CCND1) are the target genes of miRNA-146a-3p. The regulation of IHNV infection by miRNA-146a-3p is dependent on WNT3a and CCND1. MiRNA-146a-3p was required for the downregulation of WNT3a and CCND1 by IHNV. Moreover, we also found that WNT3a and CCND1 are novel proteins that induce the type-I IFN response in RTG-2 cells, and both of them could inhibit the replication of IHNV. Therefore, IHNV-induced upregulation of miRNA-146a-3p promotes early viral replication by suppressing the type-I IFN response by targeting WNT3a and CCND1. This work not only reveals the molecular mechanism of miRNA-146a-3p during IHNV infection but also provides new antiviral targets for IHNV.

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AICP is a crucial process that maintaining tissue homeostasis and regeneration. In the past, cell death was perceived merely as a means to discard cells without functional consequences. However, during regeneration, effector caspases orchestrate apoptosis, releasing signals that activate stem cells, thereby compensating for tissue loss across various animal models. Despite significant progress, the activation of Wnt3a by caspase-3 remains a focal point of research gaps in AICP mechanisms, spanning from lower to higher regenerative animals. This inquiry into the molecular intricacies of caspase-3-induced Wnt3a activation contributes to a deeper understanding of the links between regeneration and cancer mechanisms. Our report provides current updates on AICP pathways, delineating research gaps and highlighting the potential for future investigations aimed at enhancing our comprehension of this intricate process.

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Regeneration is a multifaceted biological phenomenon that necessitates the intricate orchestration of apoptosis, stem cells, and immune responses, culminating in the regulation of apoptosis-induced compensatory proliferation (AICP). The AICP context of research is observed in many animal models like in Hydra, Xenopus, newt, Drosophila, and mouse but so far not reported in earthworm. The earthworm Perionyx excavatus is used in the present study to understand the relationship between AICP-related protein expression and regeneration success in different conditions (normal regeneration and abnormal multiple bud formation). Initially, the worms are amputated into five equal portions and it is revealed that regeneration in P. excavatus is clitellum independent and it gives more preference for anterior regeneration (regrowth of head portion) than for posterior regeneration (regrowth of tail portion). The posterior segments of the worm possess enormous regeneration ability but this is lacking in anterior segments. Alkaline phosphate, a stem cell marker, shows strong signals throughout all the posterior segments but it decreases in the initial 1st to 15th anterior segments which lack the regeneration ability. While regenerating normally, it was suggested that the worm follow AICP principles. This is because there was increased expression of apoptosis signals throughout the regeneration process along with constant expression of stem cell proliferation response together with cellular proliferation. In amputated posterior segments maintained in vitro, the apoptosis signals were extensively detected on the 1st day. However, on the 4th and 6th days, caspase-3 and H2AX expression are significantly suppressed, which may eventually alter the Wnt3a and histone H3 patterns that impair the AICP and result in multiple bud formation. Our results suggest that AICP-related protein expression pattern is crucial for initiating proper regeneration.

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We previously reported the neurotoxic effects of arsenic in the hippocampus. Here, we explored the involvement of Wnt pathway, which contributes to neuronal functions. Administering environmentally relevant arsenic concentrations to postnatal day-60 (PND60) mice demonstrated a dose-dependent increase in hippocampal Wnt3a and its components, Frizzled, phospho-LRP6, Dishevelled and Axin1 at PND90 and PND120. However, p-GSK3-ß(Ser9) and ß-catenin levels although elevated at PND90, decreased at PND120. Additionally, treatment with Wnt-inhibitor, rDkk1, reduced p-GSK3-ß(Ser9) and ß-catenin at PND90, but failed to affect their levels at PND120, indicating a time-dependent link with Wnt. To explore other underlying factors, we assessed epidermal growth factor receptor (EGFR) pathway, which interacts with GSK3-ß and appears relevant to neuronal functions. We primarily found that arsenic reduced hippocampal phosphorylated-EGFR and its ligand, Heparin-binding EGF-like growth factor (HB-EGF), at both PND90 and PND120. Moreover, treatment with HB-EGF rescued p-GSK3-ß(Ser9) and ß-catenin levels at PND120, suggesting their HB-EGF/EGFR-dependent regulation at this time point. Additionally, rDkk1, LiCl (GSK3-ß-activity inhibitor), or ß-catenin protein treatments induced a time-dependent recovery in HB-EGF, indicating potential inter-dependent mechanism between hippocampal Wnt/ß-catenin and HB-EGF/EGFR following arsenic exposure. Fluorescence immunolabeling then validated these findings in hippocampal neurons. Further exploration of hippocampal neuronal survival and apoptosis demonstrated that treatment with rDkk1, LiCl, ß-catenin and HB-EGF improved Nissl staining and NeuN levels, and reduced cleaved-caspase-3 levels in arsenic-treated mice. Supportively, we detected improved Y-Maze and Passive Avoidance performances for learning-memory functions in these mice. Overall, our study provides novel insights into Wnt/ß-catenin and HB-EGF/EGFR pathway interaction in arsenic-induced hippocampal neurotoxicity.

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BACKGROUND: Chemobrain is widespread in breast cancer patients receiving chemotherapy. However, the exact mechanism, especially the associated signalling pathway, is not currently clear. This study was to evaluate the behavioural changes in breast cancer mice after chemotherapy and to further explore the role of Wnt3a/glycogen synthase kinase (GSK3ß)/ß-catenin signalling in chemobrain. METHODS: MMTV-PyMT(+) breast cancer mice were injected intraperitoneally with doxorubicin (4 mg/kg) once a week for three weeks to establish a chemobrain model. The Morris water maze (MWM) and novel object recognition (NOR) tests were performed to assess the learning and memory ability. Electron microscopy was used to observe the structural changes in the hippocampal CA1 region. The brain tissue of breast cancer mice after chemotherapy was taken out for mRNA-seq detection. Then, the expression levels and phosphorylation of key proteins in the Wnt3a/GSK3 ß/ß-catenin signalling pathway were evaluated through Western blotting (WB) and immunofluorescence. RESULTS: Doxorubicin-induced spatial and short-term memory impairment was observed in breast cancer mice, and obvious neuronal damage could be seen in the hippocampal CA1 region. Immunofluorescence staining for GSK3ß was increased. Wnt signalling pathway is highly enriched from mRNA-seq analysis, with GSK3ß genes at important nodes. The relative protein levels of p-PI3K, p-AKT, p-GSK3 ß, Wnt3a and TCF-1 were decreased significantly, while the p-ß-catenin level was increased. After injection of the GSK3ß inhibitor sb216763 (1 ng/0.5 µl/side), hippocampal neuronal injury was alleviated to some extent, and the changes in the expression of proteins upstream and downstream of this signalling pathway were reversed. CONCLUSION: Wnt3a/GSK3 ß/ß-catenin signalling is likely involved in doxorubicin-induced memory impairment. This result provides basic evidence for the further study of chemobrain in breast cancer.

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BACKGROUND: Worldwide cleft lip with or without a cleft palate (CL/P) is the most common craniofacial birth defect. Apart from changes in facial appearance, additionally affected individuals often suffer from various associated comorbidities requiring complex multidisciplinary treatment with overall high expenses. Understanding the complete pathogenetic mechanisms of CL/P might aid in developing new preventative strategies and therapeutic approaches, help with genetic counselling, and improve quality of life. Many genes have been associated with the development of orofacial clefts; however, the majority require further research. Based on the role of PAX7, PAX9, SHH, SOX3, WNT3A, and WNT9B in orofacial development, the intention was to use chromogenic in situ hybridization to detect the six genes in postnatal CLP-affected palatine tissue and compare their distribution within the tissue samples. RESULTS: Statistically significant differences in the distribution of PAX7, PAX9, WNT3A, and WNT9B were observed. In total, 19 pairs of moderate to very strong positive correlations were noted. CONCLUSIONS: Changes in the cleft-affected palatine epithelium primarily seem to be associated with the PAX7 gene; however, PAX9, WNT3A, WNT9B, and SOX3 role seems to be more limited. Whilst connective tissue changes seem to depend on PAX7 only, SHH seems to participate individually and indistinctly. Numerous positive correlations reflect the complicating interactions of the pathways and their components in the orofacial cleft morphopathogenesis.

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OBJECTIVE: The present study aimed at evaluating the potential contribution of Phosphatase and Tensin Homolog (PTEN) and its gene polymorphism (PTEN rs701848 T/C) in relation to Wingless/integrase-1 (Wnt) signaling in childhood epilepsy and the impact of antiepileptic medications on their serum levels. METHODS: This study included 100 children with epilepsy (50 pharmacoresistant and 50 pharmacoresponsive) and 50 matched controls. All subjects had their genotypes for the PTEN rs701848T/C polymorphism assessed using TaqManTM assays and real-time PCR. By using the sandwich ELISA technique, the blood concentrations of PTEN and Wnt3a were measured. RESULTS: Serum Wnt3a levels in epileptic patients were significantly higher than in the control group, p < 0.001. Children with epilepsy who received oxcarbazepine had considerably lower serum Wnt3a levels than those who didn't, p < 0.001.With an AUC of 0.71, the cutoff value for diagnosing epilepsy as serum Wnt3a > 6.2 ng/mL has a sensitivity of 55% and a specificity of 80%. When compared to controls, epileptic children had considerably more (TT) genotype and less (TC and CC) genotypes, p < 0.05 for all. Epileptic children had significantly higher (T) allele frequency than controls, p = 0.006 with OR (95%CI) = 1.962(1.206-3.192). Pharmacoresistant epileptic children had significantly higher (TT) genotype compared to pharmacoresponsive type (p = 0.020). CONCLUSION: We originally found a strong association between PTEN rs701848 T/C and childhood epilepsy, in particular pharmacoresistant type. Serum Wnt3a levels increased in epilepsy, but were not significantly different between different alleles of PTEN. In pharmaco-responsive children Wnt3a levels differed significantly between the different PTEN genotypes. Antiepileptics may affect Wnt3a levels.

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Low molecular weight collagen peptide (LMWCP) is a collagen hydrolysate derived from fish. We investigated the effects of LMWCP on hair growth using human dermal papilla cells (hDPCs), human hair follicles (hHFs), patch assay, and telogenic C57BL/6 mice, while also examining the underlying mechanisms of its action. LMWCP promoted proliferation and mitochondrial potential, and the secretion of hair growth-related factors, such as EGF, HB-EGF, FGF-4, and FGF-6 in hDPCs. Patch assay showed that LMWCP increased the neogeneration of new HFs in a dose-dependent manner. This result correlated with an increase in the expression of dermal papilla (DP) signature genes such as, ALPL, SHH, FGF7, and BMP-2. LMWCP upregulated phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) and ß-catenin, and nuclear translocation of ß-catenin, and it increased the expression of Wnt3a, LEF1, VEGF, ALP, and ß-catenin. LMWCP promoted the growth of hHFs and increased the expression of ß-catenin and VEGF. Oral administration of LMWCP to mice significantly stimulated hair growth. The expression of Wnt3a, ß-catenin, PCNA, Cyclin D1, and VEGF was also elevated in the back skin of the mice. Furthermore, LMWCP increased the expression of cytokeratin and Keratin Type I and II. Collectively, these findings demonstrate that LMWCP has the potential to increase hair growth via activating the Wnt/ß-catenin signaling pathway.

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BACKGROUND: Regeneration is a highly complex process that requires the coordination of numerous molecular events, and identifying the key ruler that governs is important to investigate. While it has been shown that TCTP is a multi-functional protein that regulates cell proliferation, differentiation, apoptosis, anti-apoptosis, stem cell maintenance, and immune responses, but only a few studies associated to regeneration have been reported. To investigate the multi-functional role of TCTP in regeneration, the earthworm Perionyx excavatus was chosen. METHODS: Through pharmacological suppression of TCTP, amputation, histology, molecular docking, and western blotting, the multi-function role of TCTP involved in regeneration is revealed. RESULTS: Amputational studies show that P. excavatus is a clitellum-independent regenerating earthworm resulting in two functional worms upon amputation. Arresting cell cycle at the G1/S boundary using 2 mM Thymidine confirms that P. excavatus execute both epimorphosis and morphallaxis regeneration mode. The pharmacological suppression of TCTP using buclizine results in regeneration suppression. Following the combinatorial injection of 2 mM Thymidine and buclizine, the earthworm regeneration is completely blocked, which suggests a critical functional role of TCTP in morphallaxis. The pharmacological inhibition of TCTP also suppresses the key proteins involved in regeneration: Wnt3a (stem cell marker), PCNA (cell proliferation) and YAP1 (Hippo signalling) but augments the expression of cellular stress protein p53. CONCLUSION: The collective results indicate that TCTP synchronously is involved in the process of stem cell activation, cell proliferation, morphallaxis, and organ development in the regeneration event.

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ObjectiveTo observe the effect of Shengxiantang （SXT） on cell senescence mediated by wingless/integrated （Wnt）3a/β-catenin pathway in rats with idiopathic pulmonary fibrosis （IPF） and reveal the possible mechanism in improving lung function of IPF rats. MethodA total of 32 SPF level SD rats were randomly divided into sham group， model group， pirfenidone group， and SXT group. The IPF rat model was established by intratracheal instillation of bleomycin （0.005 g·kg-1）. The following day after surgery， rats in the SXT group were given the aqueous solution of SXT granules （0.78 g·kg-1）， and the pirfenidone group was given pirfenidone suspension （0.05 g·kg-1）. The other groups were given deionized water （10 mL·kg-1） for 28 consecutive days. Lung tissue was collected after the lung function was measured. The pathological changes of the lung tissue were observed by hematoxylin-eosin （HE） and Masson staining， and then the Szapiel score and Ashcroft score were performed. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect telomere length. Western blot was applied to detect the expressions of epithelial-mesenchymal transformation （EMT） markers ［α-smooth muscle actin （α-SMA） and E-cadherin］， telomere reverse transcriptase （TRET）， aging-related proteins （p53 and p21）， senescence-associated secretory phenotype ［interleukin-6 （IL-6） and matrix metalloproteinase-1 （MMP-1）］， and key proteins of Wnt signaling pathway ［Wnt3a， glycogen synthase kinase-3β （GSK-3β）， β-catenin， Cyclin D1， and c-Myc］. ResultCompared with those in the Sham group， peak expiratory flow （PEF） and minute ventilation volume （MV） in the model group were significantly decreased （P<0.01）， and the frequency of respiratory （f） was significantly increased （P<0.01）. The Szapiel score， Ashcroft score， and protein expression of α-SMA， p53， p21， IL-6， MMP-1， Wnt3a， GSK3β， β-catenin， Cyclin D1， and c-Myc were increased （P<0.01）. The expressions of E-cadherin and TERT， as well as telomere length were significantly decreased （P<0.01）. Compared with those in the model group， PEF and MV in the SXT group were significantly increased （P<0.01）， while f was significantly decreased （P<0.01）. The Szapiel score， Ashcroft score， and protein expression of α-SMA， p53， p21， IL-6， MMP-1， Wnt3a， GSK3β， β-catenin， Cyclin D1， and c-Myc were significantly decreased （P<0.05， P<0.01）. Nevertheless， the expression of E-cadherin and TERT， as well as telomere length were significantly increased （P<0.01）. ConclusionSXT presents a significant protective effect on lung function in IPF rats， and the prescription may act on the Wnt3a/β-catenin signaling pathway to regulate cell senescence induced by TERT to inhibit EMT.

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The specific molecular mechanism of neuroprotective effects of wnt-3a on spinal cord injury (SCI) has not been elucidated. In our study, we evaluated the recovery of motor function after SCI by BBB, observed neuronal apoptosis by western blot and TUNEL, observed the changes of neuronal inflammation by western blot and immunofluorescence staining, and observed the changes of motoneurons and spinal cord area in the anterior horn of the spinal cord via Nissl and HE staining. We found that wnt-3a could significantly promote the recovery of motor function, reduce the loss of motor neurons in the anterior horn of the spinal cord, promote the recovery of injured spinal cord tissue, inhibit neuronal apoptosis and inflammatory response, and ultimately promote neuronal function after SCI. However, when XAV939 inhibits the wnt/ß-catenin signaling pathway, the neuroprotective effects of wnt-3a are also significantly inhibited. The above results together indicated that wnt-3a exerts its neuroprotective effect on after SCI via activating the wnt/ß-catenin signaling pathway.

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PURPOSE: The role of Wnt signaling in oncogenesis and drug resistance is well known. Receptor-interacting protein kinase (RIPK4) contributing to the increased activity of many signaling pathways, including Wnt/ß-catenin, may be an important target for designing new drugs for metastatic melanoma, but its role in melanoma is not fully understood. METHODS: We tested the effect of genetic manipulation of RIPK4 (CRISPR/Cas9) on xenograft growth. In addition, immunohistochemistry was used to detect active ß-catenin, Ki67 and necrosis in xenografts. Wnt signaling pathway activity was examined using Western blot and Top-Flash. The effect of RIPK4 knockout on melanoma cells in vitro stimulated Wnt3A on wound overgrowth, migration and invasion ability was then evaluated. RESULTS: Our study showed that CRISPR/Cas9-mediated RIPK4 knockout (KO) significantly reduced tumor growth in a mouse model of melanoma, particularly of WM266.4 cells. RIPK4 KO tumors exhibited lower percentages of Ki67+ cells as well as reduced necrotic area and decreased levels of active ß-catenin. In addition, we observed that RIPK4 knockout impaired Wnt3A-induced activation of LRP6 and ß-catenin, as manifested by a decrease in the transcriptional activity of ß-catenin in Top-Flash in both tested melanoma cell lines, A375 and WM266.4. Prolonged incubation (48 h) with Wnt3A showed reduced level of MMP9, C-myc, and increased SOX10, proteins whose transcription is also dependent on ß-catenin activity. Moreover, RIPK4 knockout led to the inhibition of scratch overgrowth, migration and invasion of these cells compared to their controls. CONCLUSION: RIPK4 knockdown inhibits melanoma tumor growth and Wnt3A stimulated migration and invasion indicating that RIPK4 might be a potential target for melanoma therapy.

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OBJECTIVES: This study aimed to investigate the functions of 19 types of Wnt ligands during the process of osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs), with particular attention to WNT3A and WNT4. MATERIALS AND METHODS: The expression levels of 19 types of Wnt ligands were examined using real-time quantitative polymerase chain reaction (real-time qPCR) during hPDLSCs osteogenic differentiation at 7, 10, and 14 days. Knockdown of WNT3A and WNT4 expression was achieved using adenovirus vectors, and conditioned medium derived from WNT3A and WNT4 overexpression plasmids was employed to investigate their roles in hPDLSCs osteogenesis. Osteogenic-specific genes were analyzed using real-time qPCR. Alkaline phosphatase (ALP) and alizarin red S activities and staining were employed to assess hPDLSCs' osteogenic differentiation ability. RESULTS: During hPDLSCs osteogenic differentiation, the expression of 19 types of Wnt ligands varied, with WNT3A and WNT4 showing significant upregulation. Inhibiting WNT3A and WNT4 expression hindered hPDLSCs' osteogenic capacity. Conditioned medium of WNT3A promoted early osteogenic differentiation, while WNT4 facilitated late osteogenesis slightly. CONCLUSION: Wnt ligands, particularly WNT3A and WNT4, play an important role in hPDLSCs' osteogenic differentiation, highlighting their potential as promoters of osteogenesis. CLINICAL RELEVANCE: Given the challenging nature of alveolar bone regeneration, therapeutic strategies that target WNT3A and WNT4 signaling pathways offer promising opportunities. Additionally, innovative gene therapy approaches aimed at regulating of WNT3A and WNT4 expression hold potential for improving alveolar bone regeneration outcomes.

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MicroRNAs and the WNT signaling cascade regulate the pathogenetic mechanisms of atherosclerotic coronary artery disease (CAD) development. OBJECTIVE: To evaluate the expression of microRNAs (miR-21a, miR-145, and miR-221) and the role of the WNT signaling cascade (WNT1, WNT3a, WNT4, and WNT5a) in obstructive CAD and ischemia with no obstructive coronary arteries (INOCA). METHOD: The cross-sectional observational study comprised 94 subjects. The expression of miR-21a, miR-145, miR-221 (RT-PCR) and the protein levels of WNT1, WNT3a, WNT4, WNT5a, LRP6, and SIRT1 (ELISA) were estimated in the plasma of 20 patients with INOCA (66.5 [62.8; 71.2] years; 25% men), 44 patients with obstructive CAD (64.0 [56.5; 71,0] years; 63.6% men), and 30 healthy volunteers without risk factors for cardiovascular diseases (CVD). RESULTS: Higher levels of WNT1 (0.189 [0.184; 0.193] ng/mL vs. 0.15 [0.15-0.16] ng/mL, p < 0.001) and WNT3a (0.227 [0.181; 0.252] vs. 0.115 [0.07; 0.16] p < 0.001) were found in plasma samples from patients with obstructive CAD, whereas the INOCA group was characterized by higher concentrations of WNT4 (0.345 [0.278; 0.492] ng/mL vs. 0.203 [0.112; 0.378] ng/mL, p = 0.025) and WNT5a (0.17 [0.16; 0.17] ng/mL vs. 0.01 [0.007; 0.018] ng/mL, p < 0.001). MiR-221 expression level was higher in all CAD groups compared to the control group (p < 0.001), whereas miR-21a was more highly expressed in the control group than in the obstructive (p = 0.012) and INOCA (p = 0.003) groups. Correlation analysis revealed associations of miR-21a expression with WNT1 (r = -0.32; p = 0.028) and SIRT1 (r = 0.399; p = 0.005) protein levels in all CAD groups. A positive correlation between miR-145 expression and the WNT4 protein level was observed in patients with obstructive CAD (r = 0.436; p = 0.016). Based on multivariate regression analysis, a mathematical model was constructed that predicts the type of coronary lesion. WNT3a and LRP6 were the independent predictors of INOCA (p < 0.001 and p = 0.002, respectively). CONCLUSIONS: Activation of the canonical cascade of WNT-ß-catenin prevailed in patients with obstructive CAD, whereas in the INOCA and control groups, the activity of the non-canonical pathway was higher. It can be assumed that miR-21a has a negative effect on the formation of atherosclerotic CAD. Alternatively, miR-145 could be involved in the development of coronary artery obstruction, presumably through the regulation of the WNT4 protein. A mathematical model with WNT3a and LRP6 as predictors allows for the prediction of the type of coronary artery lesion.

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Renal cell carcinoma, shorted as RCC is a well-known urological cancer with high level of morbidity and mortality. Although the regulatory role of the spindle microtubule assembly factor (ASPM) in tumor progression has been established, its relationship to the development of RCC remains unclear. To determine the significance of this gene in RCC, we examined its expression in RCC patients in the TCGA database and compared ASPM level between clinical samples of normal tissues and RCC tissues collected at our center. The prognostic relevance of ASPM was assessed by generating Kaplan-Meier survival curves and log-rank functions. Following alteration of ASPM expression using sh-ASPM or oe-ASPM transfection, RCC cell characteristics were evaluated through CCK-8, Transwell, and colony formation assays. Western blot analysis was conducted to measure levels of genes affected by ASPM, and rescue experiments were performed to explore the involvement of Wnt3a signaling in ASPM-mediated malignancy in RCC. Our findings indicate that ASPM is upregulated in RCC samples, and its levels are associated with the long-term survival of RCC patients. ASPM promotes the migration, proliferation, and invasiveness of RCC cells, and the Wnt3a pathway may be implicated in this process. In conclusion, these results indicate that ASPM contributes to the cancer progression of RCC by targeting the Wnt3a signaling pathway.