Background: Medulloblastoma is the most common pediatric malignant tumor in central nervous system. Although its prognosis has been improved enormously by the combination treatments with surgery, radiotherapy, and chemotherapy, it still could progress via invasion and distant dissemination. We aimed to investigate molecular mechanisms of medulloblastoma invasion in the current work.Methods: The gene expression profile of medulloblastoma were analyzed based on the data deposited in Gene Expression Omnibus (GEO) and filtered according to brain specific proteins in the Uniprot. Delta-catenin was identified and further analyzed about its expression and roles in the prognosis of medulloblastoma patient. The function of delta-catenin on cell invasion and migration were investigated by transwell and wound healing assay. Whether delta-catenin participates in the epithelial-mesenchymal transition (EMT) regulated invasion was also studied.Results: Delta-catenin expression was highly upregulated in tumor tissues compared to normal tissues from medulloblastoma patients in five independent, nonoverlapping cohorts. Furthermore, delta-catenin expression level was upregulated in WNT subgroup, and significantly correlated with better prognosis, and associated with metastasis through GEO database analysis. Functional assays indicated that delta-catenin inhibited medulloblastoma cell invasion and migration through regulating the key factors of EMT pathway, such as E-cadherin and vimentin.Conclusion: Delta-catenin might be a positive predictor for prognosis of medulloblastoma patients, through attenuating medulloblastoma cell invasion by inhibiting EMT pathway. The latescribed . Metastaescribed . AlthougDelta-catenin, a catenin encoded by CTNND2, is a known neuroprotein that interacts with presenilin-1 . Actuallin vitro experiments. Delta-catenin expression was associated with improved survival. Delta-catenin alleviated medulloblastoma invasion in vitro by targeting EMT pathway.Although the role of delta-catenin has been examined in glioma , there a2 fold change (FC) > 1 and p < 0.05 were considered to be included for subsequent analysis. Then the DEGs were considered as delta-catenin related compared with the brain specific protein list in the Uniprot.The Differentially Expressed Genes (DEGs) were analyzed using the GEO database  by “limma” R package. After Benjamini–Hochberg (BH) multiple test adjustment, DEGs with absolute logWe further compared delta-catenin expression use GEO datasets between normal and medulloblastoma patient tissues. Then we used the clinical information from GEO datasets and the medulloblastoma patient cohort (74) from Sun Yat-sen University Cancer Center   to analyhttps://thebiogrid.org/) is a biomedical interaction repository. The database can be used to retrieve publications on protein and genetic interactions, chemical interactions and post-translational modifications of important model organism species. Gene set enrichment analysis (GSEA) was performed using the GSEA software and its results were visualized using “Cluster Profiler” and “ggplot2” in R.The bioinformatics analysis was conducted mainly in R v. 38.0. The “combat” function of the “sav” R package was used for batch-effect correction. The “Coxph” function, and the “survival” R package, were used to estimate the relationship between delta-catenin expression and survival. We used R package “survivalROC” to plot the receiver operating characteristic (ROC) curve of overall survival and calculate the area under the curve (AUC). 0.5 generally indicates some predictive ability. The higher the AUC is, the more accurate the prediction result is. BioGRID7 , in accordance with the Helsinki Declaration.The medulloblastoma cell lines Daoy and ONS-76, and a tool cell line HEK293T were obtained from the Cell Bank of State Key Laboratory of Oncology in South China. The cells were cultured in DMEM with 10% fetal bovine serum  in a humidified incubator at 37°C with 5% COn = 74) from medulloblastoma patients who underwent surgery at Sun Yat-sen University Cancer Center from 2011 to 2020 were used for immunohistochemical staining (IHC). Medulloblastoma diagnosis was based on the World Health Organization Classification of Central Nervous System Tumors (2016). Delta-catenin expression were detected by immunohistochemistry following standard protocol as per our previous paper , and blocked with goat serum. Tissue samples were incubated overnight at 4°C with the antibody against delta-catenin . HRP-labeled goat anti-rabbit/mouse antibody was then added to the slides for 1 h at 26°C. Finally, the tissues were stained with diaminobenzidine (DAB) and counterstained with hematoxylin. The slides were visualized and photographed using an automatic slide scanner (KF-PRO-020) at ×40 magnification. Quantitative image analyses were conducted using the HALO software  using the multiplex IHC modules (Paraffin-embedded tissue samples (us paper . Briefly modules . The hisTo establish stable knockdown and overexpression (OE) lines of Daoy and ONS-76, the plasmids pSlenti-U6-shRNA (CTNND2)-CMV-EGFP-F2A-Puro-WPRE  and pEZ-Lv105-hCTNND2-Puro  were used. The plasmid was co-transfected with lentivirus packaging plasmids  into HEK293T with transfection reagent Lipofectamine 3000 . The supernatant of HEK293T was collected twice, at 48 and 72 h after transfection. Cells (Daoy and ONS-76) were infected with the filtered supernatant containing virus. Puromycin (2 μg/ml) was added for 48 h after the infection and selected for 7 days. The target sequence for delta-catenin was 5′-GCAACAACACTGCAAGCAA-3′ and 5′-GCTAAAGGCGAACACACTT-3′.−ΔΔCt method was used for quantitative analysis. The primer sequences for delta-catenin were: 5′-AGGTCCCCGTCCATTGATAG-3′ and 5′-ACTGGTGCTGCAACATCTGAA-3′. The sequences for β-actin were: 5′- CTCCATCCTGGCCTCGCTGT-3′ and 5′- GCTGTCACCTTCACCGTTCC-3′.Total RNA from the cell lines was extracted following the protocol of RNA-Quick Purification Kit . The reverse transcription were performed with HisScript III All-in-one RT SuperMix . Then real-time PCR was performed using ChamQ SYBR qPCR Master Mix , and triplicate samples were run on a Bio-Rad CFX96 qPCR system according to the manufacturer’s protocol. The Ct values for delta-catenin was normalized to β-actin, and the 2Cells were collected and washed twice in PBS, lysed in RIPA buffer  with PMSF on ice for 30 min, then centrifuged at 12,000 × g for 15 min at 4°C. Protein concentration was determined with the BCA kit . And the lysates were then subjected to SDS-PAGE (10% gel). The separated protein bands were transferred onto PVDF membrane . After been blocking in milk (5% in TBST) for 1 h at 26°C, the membrane was probed with primary antibodies, such as for delta-catenin , E-cadherin , and vimentin , beta-tubulin  and GAPDH  at 4°C for 12–16 h. After been washed in TBST for three times (15 min each time), the membranes were incubated with corresponding HRP-conjugated secondary antibodies  for 1 h at 26°C. The bands were visualized using Ncm-ECL Ultra , captured using an ChemiDoc Touch Imaging System , and quantified using ImageJ (v1.8.0).Daoy (shNC and sh delta-catenin) cells were seeded in confocal dishs 24 h in advance. Then the dishs were fixed with 4% formaldehyde for 15 min at 26°C, rinsed three times in PBS (5 min each time), and blocked in blocking buffer  at 26°C for 60 min. The cells were then incubated overnight at 4°C in diluted primary E-cadherin antibody . Then the slides were rinsed three times in PBS for 5 min each and incubated with fluorochrome-conjugated secondary antibody  for 1 h at 26°C in dark. The slides were rinsed three times again in 1× PBS for 5 min each, and DAPI was added to counter-stain the nucleus. Images were obtained using a Zeiss LSM 880 with fast Airyscan.5 cells/ml), 200 µl was inoculated into the upper chamber and 700 µl of complete medium was simultaneously added to the lower chamber. When placing the chamber, avoid air bubbles attached to the bottom of the chamber. After culturing for 15 h in incubator, the upper chamber was fixed in 4% paraformaldehyde for 30 min, then stained with 0.5% crystal violet for 7 min. The images were captured by the microscope (Nikon ECLIPSE Ni) when the chambers were dried and then counted the number of cells by ImageJ software (v1.8.0). For the invasion assay, 0.2% matrigel  was added into upper chamber before cells were seeded. For delta-catenin-overexpression (OE) cells, more cells (1 × 106 cells/ml) were suspended and culturing time extended to 24 h.Cells were suspended in serum-free DMEM (2.5 × 105 cells/well) and then the plates were placed in the incubator for pre-culture  overnight. Scratching on the cell surface with tip vertically next day and washing three times with PBS, then adding serum-free medium and placing in the incubator. Capturing images at the same point for each group in 0, 12, 24, and 48 h. The healing area between scratches were measured by ImageJ software (v1.8.0).Cells were inoculated in 6-well plate  and GraphPad Prism version 8.0 . Survival analyses were performed using the Kaplan-Meier survival curves. All the data are shown as mean and standard deviation (mean ± SD). Several thousand differentially expressed genes were pulled out in the following datasets, 2123 in GSE74195, 3182 in GSE66354 and 5960 in GSE86574 . The ovep < 0.0001). Delta-catenin expression in medulloblastoma cell was comparable with that in glioblastoma and low-grade glioma cells  is an interactive web application for gene expression analysis based on 9736 tumors and 8587 normal samples from the TCGA and the GTEx databases using the output of a standard processing pipeline for RNA sequencing data . Using oma cells .Using data from two independent and nonoverlapping medulloblastoma patient cohorts, we examined the association between delta-catenin expression level and medulloblastoma prognosis. We divided the Cavalli cohort dataset (GSE85217)  into twoIn our study, delta-catenin expression was significantly higher in WNT group of medulloblastoma than in non-WNT groups   based onin vitro.To evaluate the role of delta-catenin expression in medulloblastoma dissemination, we established stable knockdown and OE cells of Daoy and ONS-76. RT-qPCR and western blotting were used to confirm infection efficiency . TransweVia immunofluorescence, we found that knockdown of delta-catenin broke down the continuous arrangement of E-cadherin in the adjacent areas of cell to discrete and disorganized form  in delta-catenin-knockdown medulloblastoma cells and control cells. Delta-catenin knockdown significantly reduced the expression of E-cadherin and increased that of vimentin . Moreovezed form . These din vitro, and EMT pathway may be the underlying mechanism.We aimed to elucidate the molecular mechanisms of medulloblastoma invasion, to improve the therapeutic response and prolong survival in medulloblastoma patients. Our bioinformatics analysis revealed that delta-catenin expression was positively associated with survival of medulloblastoma patients. It was enriched in WNT subgroup, which had the lowest metastasis rate among the four subgroups. Delta-catenin inhibited medulloblastoma cell invasion These findings are consistent with various studies showing that delta-catenin inhibits tumors. For example, CTNND2 loss-of-function mutation was found to be common in glioblastoma. Based on analysis of TCGA data, reduced CTNND2 expression was associated with poor prognosis of glioblastoma, especially for the mesenchymal type . Delta-cHowever, delta-catenin was also reported to serve as an oncoprotein . It coulvia various pathways (Prior to now, there has been a poor understanding of the context-dependent roles of delta-catenin in cancer. Two explanations for this have been proposed. First, it may be primarily due to variation in CTNND2. In prostate cancer, wild-type and mutant delta-catenin both exhibited pro-oncogenic and tumor-suppressive functions; that study found a protein truncation, caused by a nonsense mutation of delta-catenin, mainly occurring in the cytoplasm, promoted carcinoma progression pathways . CTNND2 pathways . This maThe second possible reason might be related to epigenetic modification. Post-translational phosphorylation of delta-catenin, which alters its function substantially, has been increasingly described. Phosphorylation of delta-catenin plays an important role in determining delta-catenin’s role in neuronal development and oncogenesis . And a cMedulloblastoma, which is differ from the other pediatric brain tumor such as ependymoma and brainstem glioma , is the This study has some certain limitations. The cell line in our study are all belong to SHH subgroup . Given tIn conclusion, we found that high expression of delta-catenin, which is enriched in WNT subgroup, is a favorable prognostic factor in medulloblastoma. The EMT pathway may participate in this reduced invasion and metastasis.


Graphene oxide (GO) is a candidate for nanofillers to improve the mechanical and thermal stability of nanocomposites. In order to determine the molecular interaction to improve the mechanical properties of GO–epoxy resin composites, we investigated the relationship between GO oxidation properties and the tensile strength of the epoxy resin. With respect to GO preparation, graphite was oxidised by the Brodie or Hummers method, and the oxidised GO was reduced or chloride substituted. The X-ray photoelectron spectroscopy (XPS) spectral patterns indicate that a shorter Brodie oxidation method GO (B-GO) is associated with a higher proportion of hydroxyl groups. The oxidised GO materials, with the exception of the sample produced by the 54 h Brodie oxidation method, improved the tensile strength of the composites while the epoxy resin with reduced or chlorinated GO did not increase the tensile strength of the film. Based on XPS and elemental analyses, the improvement in the tensile strength is due to the presence of O atom based functional groups, such as hydroxyl groups, on the GO surface. The interaction between the epoxy resin and O atom based functional groups on the GO contributes to improving the tensile strength of the composites. In order to determine the molecular interaction to improve the mechanical properties of graphene oxide (GO)–epoxy resin composites, we investigated the relationship between GO oxidation properties and the tensile strength of the epoxy resin. Therefore, relatively hydrophobic nanocarbon based fillers including carbon nanotubes, fullerene, graphene and graphene oxide (GO) are added to epoxy resins.2 Specifically, GO is prepared by the oxidation of graphite powder by nitric acid or sulfonic acid, and the synthesis method is suitable for scaling-up as opposed to the oxidation of carbon nanotubes and other carbon nanomaterials. Furthermore, GO includes several chemical functionalisation processes to their surface and edge.5–21 Therefore, mechanical and chemical properties of the polymer composites are tuned based on the amount and contents of GO functional groups.Epoxy resin is a thermosetting polymer that is used as a matrix that is hardened by a crosslinking reaction between the epoxy groups of the monomer and amine groups of the hardener. It is currently used for coating materials in electrical circuits and devices due to its high electrical insulation properties and is used as a bonding agent to prevent the occurrence of cracking in the concrete used for buildings.5–19 When GO was grafted with epoxy chains, the improvement in tensile strength were also observed due to changes in the curing behaviour.20,21 However, in order to extend this technology for large scale application, the scalability of GO functionalisation processes acts as an obstacle for amine functionalisation and GO grafting methods. We suggest that molecular scale interaction between GO and epoxy resin contributes to designing GO-based composites for large scale production. The use of oxidised or reduced GO as a nanofiller allows the discussion of mechanical properties of GO-based composites based on the molecular interaction between GO and epoxy function groups.In order to enhance the crosslink network between the amine functionalised nanofiller and epoxy monomer for GO–epoxy composites, the aliphatic and aromatic amine groups were functionalised to the GO edge, and the tensile strength and glass transition temperature of the composites that contained the functionalised GO improved.In the present study, we discuss the relationship between degree of GO oxidation and tensile strength of the GO–epoxy resin composites with the aim of obtaining a critical interaction between GO filler and epoxy resin to improve the mechanical strength of GO–epoxy resin composites. In order to investigate the influence of GO functional groups on the tensile strengths of the composites, GO was prepared by employing the Brodie method or Hummers method and the dispersion of GO samples with different degrees of oxidation including reduction treatment and chloride substitution, preparation of GO thermosetting epoxy resin composite films, and tensile tests of the films.4, hydrazine monohydrate and thionyl chloride were purchased from Wako Chemical Co. Ltd. . 94% Fuming nitric acid, hydrogen peroxide and N,N-dimethylformamide (DMF) were obtained from Nakarai tesque Co. Ltd. .Graphite powder, potassium chlorate, sulfuric acid, KMnO22 The black suspension was diluted with 250 mL of deionised water and centrifuged at 8000 rpm for 20 min. Following the removal of supernatants, deionised water was added and centrifuged until the suspension obtained a pH of 7. The suspension was filtrated and dried . We obtained 0.5 h oxidised GO (0.5hB-GO), 3 h oxidised GO (3hB-GO) and 54 h oxidised GO (54hB-GO) with different degrees of oxidation.Specifically, 0.5 g of graphite powder was mixed with 10 mL of white fuming nitric acid and 4 g of potassium chlorate and oxidised for 0.5 h, 3 h or 54 h.4 and oxidised for 2 h at 35 °C.23 In order to quench the oxidation of graphite, distilled water (15 mL) and hydrogen peroxide (1.5 g) were added to the suspension and reacted for 30 min. The suspension was centrifuged at 8000 rpm for 20 min. Following the removal of supernatants, deionised water was added and centrifuged until the pH of the GO suspension corresponded to 7. The resultant powder was filtrated and dried up at 100 °C for 18 h. When the ratios of graphite/oxidant were 1, 1/3 or 1/5, the respective final products were defined as 1H-GO, 3H-GO or 5H-GO, respectively.In this method, 0.5 g of graphite powder was mixed with 15 mL of sulfuric acid and 0.5 g , 1.5 g or 2.5 g of KMnO2 atmosphere , and the resultant material is thermally reduced GO (T-rGO).In this method, 0.5hB-GO was heated in a tube furnace under a N24Specifically, 0.5hB-GO powder and filtration paper soaked with 2 mL of hydrazine monohydrate were sealed in a glass flask by using a paraffin film. We obtained hydrazine reduced GO (Hyd-rGO) by exposing GO to hydrazine vapour at 90 °C for 6 h.2 atmosphere . The refluxed mixture was cooled to room temperature, filtered at 60 °C, and dried at room temperature. The final product is termed as chloride substituted GO (0.5hCl-GO or 54hCl-GO) corresponding to the oxidation time of B-GO. Information about the GO sample is presented along with the acronyms in In this method, 2 mL of thionyl chloride was added to the 0.5hB-GO or 54hB-GO suspension. Additionally, DMF that was used as a catalyst was added in a dropwise manner to the suspension and sonicated for 30 min. The mixture was refluxed under Nλ = 1.54184 Å).25Particle size of GO samples was analysed by using an optical microscope  and the average length was approximately 20 (μm) for the optical microscopically observed samples. We did not observe any difference between the sizes of GO in different oxidation processes using an optical microscope, as depicted in Fig. S1A and B in ESI.In 0.5hB-GO, 3hB-GO, 54hB-GO, 1H-GO, 3H-GO, 5H-GO, T-rGO, Hyd-rGO, 0.5hCl-GO and 54hCl-GO of the GO samples, the GO functional groups and the percentages were analysed. The X-ray photoelectron spectroscopy (XPS) spectra were recorded by using XPS  and curve-fitted by a gauss function by using IGOR Pro . Hydroxyl group contents were calculated by comparing the peak areas of 286 eV and 282 eV in the XPS spectra.The elemental analysis of GO samples was performed at the Okayama University Advanced Science Research Centre, Department of Instrumental Analysis. We estimated the percentages of O atoms based on carbon, hydrogen and nitrogen  atomic component ratios and compared the percentage of O atoms in the GOs. The contents of the hydroxyl group of the samples were calculated based on the calculations of the O atom percentage and hydroxyl group ratio from the analyses of XPS spectra ([hydroxyl group ratio by XPS]) as shown in Specifically, 500 μl of DMF or GO suspension was added to 3 g of epoxy resin monomer , mixed for 1.5 min by using a deaerator . Additionally, 2 g of epoxy resin hardener  was also added to the GO–epoxy monomer suspension, mixed by using deaerator and degassed for 20 min by using a vacuum pump. In order to prepare cured epoxy sheet, the resin was added on a silicone sheet and degassed for 10 min. The degassed resin was sandwiched by using two silicone sheets, and a weight of 500 g was placed on the silicone sheet containing the resin for 10 min. The resin was incubated in a heater at 80 °C for 5 h, and it was completely cured at 30 °C for 18 h. Transmission images of a 0.05 wt% GO composite using an optical microscope depict very little aggregate of GO inside the film, as depicted in Fig. S1C and D of ESI.θ = 15° were observed without a peak at 2θ = 26° from graphite, suggesting graphite oxidation. Peaks corresponding to 2θ = 10° were also observed in XRD spectra of 3H-GO and 5H-GO, while peaks corresponding to 2θ = 26° and 15° were observed in the spectrum of 1H-GO, thereby suggesting the occurrence of stronger oxidation to increase the distance between graphene sheets with unoxidised graphite species.The O atom percentages for 0.5hB-GO and 54hB-GO were 31.09% and 36.48%, and the O atom percentages for hydroxyl group (wt%) of 0.5hB-GO and 54hB-GO were 21.3% and 17.1%, respectively. The results suggest that oxidised GO contains relatively higher contents of hydroxyl groups in the early stage of Brodie method, and the percentage of the epoxy group increases with increases in oxidation duration. The O atom percentage of 1H-GO was 25.1%, and this is lower than those of other oxidised GO samples.A decrease in the H atom contents of 0.5hB-GO and 1H-GO when compared to those of other oxidised samples was due to the withdrawal of a few functional groups at the surface by vacuum drying for elemental analysis. When all the O atoms of the GO with an oxidation duration exceeding 2 h correspond to the hydroxyl group, the ratio of the hydroxyl group to the carboxyl group was calculated to be 3 : 1 from the peak area of XPS spectra. The O atom percentage of Hyd-rGO was 19.94%, and the H atom content was less than 0.1%, thereby suggesting the elimination of hydroxyl group in Hyd-rGO from 0.5hB-GO.9,20 According to previous studies, as the dispersibility of the GO-based filler in a resin improved, the tensile strength of the composite increased.26 Because we could not observe any significant difference in the sizes of GO-based fillers based on the oxidising conditions, the concentration dependence that is depicted in the filler could be related with the filler dispersibility of the resin.When the GO concentration in cured epoxy resin exceeded 0.3 wt%, the tensile strengths of the GO–epoxy resin composites were same as those of neat epoxy resin or below those of neat epoxy resin. Suitable GO concentration for the tensile strength improvement corresponded to 0.015–0.06 wt%. The optimised concentration for maximum strength of the test piece was similar to those obtained in extant studies (0.1%).t-test: P = 0.03) because it is assumed that P < 0.05/5 = 0.01 corresponds to a significant difference with respect to the comparison of the five groups (neat versus other samples) as shown in R2 = 0.48). It is reported that enhanced interaction/adhesion was observed in the GO composites owing to the oxygen functional groups and the winkled and rough surface of the filler by comparing the tensile properties of graphene and GO composites, which are consistent with this week correlation, as depicted in 27 The increasing surface area of GO also contributed to the improvement in the tensile strengths of the composites because of the increasing van der Walls interaction.28,29 Furthermore, the encapsulation of graphene or rGO with surfactants can improve the tensile strength of GO–epoxy composites owing to the dispersibility and non-covalent interaction between the filler and resin.30,31 Additionally, the GO-based filler sizes that are prepared by milling and/or functionalisation processes influenced the strengths and toughness of the GO–epoxy composites.26,32,33 In this study, we consider that the size effects would not considerably impact the tensile strengths of GO-based composites based on GO sizes and dispersibility in the epoxy resin because we used the same graphite powders as the initial material for the GO preparation. The enhancement in the tensile strength of our composites could be attributed to both covalent and non-covalent interactions of the oxygen-based functional groups.−1 as indicated by the density functional theory calculation.34 The energy was considerably similar to the bonding energy between epoxy resin and oxidised aluminium surface, and this suggests that the interaction between carbon fibre and epoxy resin resembles those of oxidised aluminium/epoxy resin by hydrogen bonds. The tensile strength improvement in GO–epoxy resin composites is due to hydrogen bonds between hydroxyl group at GO surface and polar groups (hydroxyl and amine groups) in epoxy resin. Furthermore, the bonding energy of hydrogen bonds between carboxyl group of filler and hydroxyl group of epoxy resin increased to 19.1 kcal mol−1 and exceeded that of the energy containing hydroxyl group.34 Therefore, we hypothesise that H-GO with hydroxyl group and carboxyl group are preferable with respect to improving the tensile strength of epoxy resin.We also discuss the manner in which the hydroxyl group improves the tensile strength of GO–epoxy resin composites due to hydrogen bonds between GO and epoxy resin of amino group of the curing agent or the hydroxyl group of the monomer. Bonding energy between hydroxyl group of epoxy resin and carbon fibre surface was 13.8 kcal molIn this study, we compared the tensile strengths of GO–epoxy films containing oxidised GO and GO after chemical modification. The results indicated that the tensile strengths of GO–epoxy resin composites containing T-rGO, Hyd-rGO, 0.5hCl-GO and 54hCl-GO were similar to those of epoxy resin without GO. The tensile strength improvement by GO addition to epoxy resin was due to hydroxyl groups at the GO surface. The findings suggest that oxidised GO containing hydroxyl group is appropriate for improved GO–epoxy resin composites. Additionally, H-GO contains both hydroxyl group and carboxyl group, and these polar functional groups are expected to contribute to tensile strength improvement.There are no conflicts to declare.RA-008-C8RA00252E-s001


CXC chemokines are small molecules and secretory peptides, which are significantly associated with cancer progression. Aberrant expression of CXC chemokines is always observed in cancer patients. An increasing number of studies have reported that CXC chemokines play essential roles in tumor angiogenesis, tumor-promoting inflammation, and metastasis, which favor cancer promotion. The expression level of CXC chemokines is closely associated with the clinicopathological characteristics and outcomes of cancer. The specific roles of CXC chemokines and corresponding signaling pathways in cancer progression are summarized in this review. Furthermore, we have discussed the potential application of CXC chemokines for cancer targeted therapy and personalized treatment.CXC chemokines are small chemotactic and secreted cytokines. Studies have shown that CXC chemokines are dysregulated in multiple types of cancer and are closely correlated with tumor progression. The CXC chemokine family has a dual function in tumor development, either tumor-promoting or tumor-suppressive depending on the context of cellular signaling. Recent evidence highlights the pro-tumorigenic properties of CXC chemokines in most human cancers. CXC chemokines were found to play pivotal roles in promoting angiogenesis, stimulating inflammatory responses, and facilitating tumor metastases. Enhanced expression of CXC chemokines is always signatured with inferior survival and prognosis. The levels of CXC chemokines in cancer patients are in dynamic change according to the tumor contexts . Thus, CXC chemokines have great potential to be used as diagnostic and prognostic biomarkers and therapeutic targets. Currently, the molecular mechanisms underlying the effect of CXC chemokines on tumor inflammation and metastasis remain unclear and application of antagonists and neutralizing antibodies of CXC chemokines signaling for cancer therapy is still not fully established. This article will review the roles of CXC chemokines in promoting tumorigenesis and progression and address the future research directions of CXC chemokines for cancer treatment. According to global cancer statistics, there were 19.3 million new cancer cases and almost 10 million cancer deaths in 2020 around the world . A growiCXC chemokines are small secretory proteins with four highly conserved cysteine amino acid residues and the first two cysteines are separated by one non-conserved amino acid residue . Based oThe CXC chemokines  are significantly upregulated in most cancers and positively associated with cancer metastasis and chemo-resistance. Conversely, downregulation of these CXC chemokines greatly suppresses the motility of cancer cells . AltogetCXC chemokine receptors, CXCRs, are G protein-coupled and seven-transmembrane receptors for CXC chemokines ,26. The Previous studies have shown that the expression of CXCR is not only observed in tumor cells, but also in granulocytes, monocytes, mast cells, some natural killer cells , endotheThe CXCR signaling contexts are determined by CXCLs and/or tumor types. CXCRs are involved in variable signaling pathways which regulate diverse biological activities in tumor cells. CXCR2 is a major receptor for ELR+ CXCLs, including CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 , CXCL7, CXCL/CXCR signaling axes play critical roles in cancer development and persistency. Upon binding of CXCLs to CXCRs, CXCR signaling is activated that elicits a series of tumor-associated effects, either pro-tumorigenic or tumor-suppressive. Therefore, CXCR antagonists and neutralizing antibodies towards tumor-promoting chemokines or, alternatively, CXCR agonists of tumor-suppressing chemokine receptors and exogeneous recombinant protein of tumor-suppressing chemokine are able to be used for cancer treatment. There have been many studies on determination of the efficacy of CXCR antagonists and neutralizing antibodies towards CXCLs or CXCRs in various types of cancer. For example, the small-molecule antagonists, SCH-527123 and SCH-479833, that target CXCR2/CXCR1 were found to inhibit colon cancer and melanoma by decreasing neovascularization and increasing apoptosis of tumor cells ,73. The Angiogenesis is regarded as an essential process to maintain the progression of pathological conditions, including cancer and chronic inflammatory diseases . In tumoThe ELR+ members of the CXC chemokine family promote tumor angiogenesis ,22,51,54CXC chemokines are upregulated in tumorigenesis. In melanoma tumor, the level of CXCL1, CXCL2, and CXCL3 is significantly elevated, accompanied by proangiogenic activity . Most ELR- members of CXC chemokines have an anti-angiogenic function, such as CXCL9 and CXCL10 . HoweverTumor-associated inflammation is a key feature of cancers. Many studies have demonstrated that inflammation, especially chronic inflammation, promotes tumor development . Most CXWhen the tumor microenvironment is similar to chronic inflammation, the tumor tissues are surrounded with an increased number of neutrophils and deteriorate in some cases . Tumor-aIt is well known that CXC chemokines are potent drivers of leukocytes for tumor-promoting inflammation. Therefore, CXC chemokines are important regulators in the communication between tumor and leukocytes and cancer-associated inflammation. For example, neutrophil-derived IL-8 and tumor-derived CXCL1 are involved in the interaction of tumor and the LPS-stimulated neutrophils, resulting in tumor cell extravasation and distant metastasis . However, the inflammatory microenvironment of different tumors is regulated by different chemokines. In addition, different chemokines chemoattract different inflammatory cells. Therefore, CXC chemokines may be used as a therapeutical target for disrupting tumor-promoting inflammatory milieu.Metastasis is one of the major impediments for cancer treatment and is a major factor for cancer patient death. Tumor metastases begin with basement membrane degradation by matrix metalloproteinases (MMPs), such as MMP2 and MMP9, followed by tumor extravasation and dissemination, and finally colonization in new lesions. It has been reported that CXC chemokines actively participate in distant colonization of cancer cells in tumor metastasis. However, the exact mechanism by which CXC chemokines regulate tumor metastasis remains to be determined. Tumor-associated inflammation and neutrophil infiltration are critical for the establishment of tumor metastasis. It has been shown that CXC chemokines play crucial roles in neutrophil activation and recruitment. When tumor cells are co-cultured with neutrophils, the expression of many pro-metastatic genes, including chemokine receptors (CXCR4 and CXCR7), MMPs (MMP12 and MMP13), and growth factors (IL-6 and TGF-β), is significantly elevated . It was It is well known that angiogenesis is essential for tumor metastasis by providing avenues for cancer cell migration. CXCL8 is a potent mediator for tumor angiogenesis. Studies have confirmed that CXCL8 enhances vessel density and facilitates distant metastasis in melanoma. In vivo studies, it was found that CXCL8 administration increased metastasis in a melanoma mouse model . It has Epithelial–mesenchymal transition (EMT) is an important process for tumor metastasis. It was observed that CXCL1 was significantly increased in the metastatic lesions which results in an elevation of mesenchymal markers expression including β-catenin, vimentin, and N-cadherin and a decrease in epithelial marker E-cadherin expression ,34. SimiExtracellular matrix degradation is a direct cause for tumor dissemination and invasion. Extracellular matrix degradation is usually catalyzed by several proteases, such as MMPs and serine protease plasmin . Thus, MAdditionally, cell adhesion molecules also actively participate in the distant metastasis of tumor cells. Elevated expression of vascular adhesion molecule-1 (VCAM-1) was proven to potentiate CXCL13-mediated metastasis . MeanwhiFurthermore, the premetastatic niche (PMN) is an important tissue microenvironment for tumor metastasis. It has been found that CXCL1 is active in PMN formation. CXCL1 attracts hematopoietic stem/progenitor cells (HSPCs), facilitates the differentiation from HSPCs to myeloid-derived suppressor cells (MDSCs), promotes PMN formation , and enaIn addition, the chemokine–cholesterol signaling axis also plays roles in tumor metastasis. Studies have shown that the TNBC cells-derived chemokines CXCL1/CXCL2/CXCL8 stimulate cholesterol synthesis, facilitate angiogenesis, and enhance TNBC cells lung metastases .In conclusion, CXC chemokines regulate cytokine secretion and gene expression and produce and maintain a pro-metastatic microenvironment.At present, many cancers still lack reliable biomarkers for diagnosis and prognosis, which becomes a major hurdle for superior cancer treatment. Therefore, identification of new cancer biomarkers is an urgent task for cancer therapy. CXC chemokines are secretory proteins primarily in the blood, which makes CXC chemokines detectable with simple, fast, and no-invasive technical approaches. As a significant amount of CXC chemokines presents in the blood, serum samples have been widely used to detect CXC chemokine levels by ELISA . CXC cheFurthermore, dysregulated expression of CXC chemokines has been observed to be associated with the clinicopathological features and clinical outcomes of cancer patients. Overexpression of tumor-promoting CXC chemokines in cancer patients is accompanied by aggressive phenotypes  and inferior prognosis. More recent work has suggested that CXCL5 is a biomarker for poor prognosis in NSCLC patients ,86. In pIn addition, the level of CXC chemokines in tumor tissues may be used as predictive indexes for cancer prognosis. A study investigating the relationship between the levels of several CXC chemokines in tumor tissues and gastric cancer relapse has shown that the levels of CXCL1, CXCL2, CXCL5, CXCL8, CXCL11, and CXCL13 in gastric cancer patients without recurrence after treatment were significantly lower than those before treatment, and there were no changes in most of the CXC chemokines in patients with recurrence . MoreoveRecently, the therapeutic value of CXC chemokines for multiple cancers has been explored. The expression level of some CXC chemokines would change in the process of drug administration. It was observed that activation of CXCR4 by CXCL12 enforced tumor resistance to therapies by decreasing apoptotic signaling . CXCL1, CXC chemokines are differentially expressed in different types of cancer . It has Taken together, manipulating CXC chemokine levels is an attractive approach for cancer treatment. Expression profiles of CXC chemokines or circulating levels of chemokines in the blood are useful tools for cancer diagnosis and prognosis. As CXC chemokines have dual effects in carcinogenesis, such as the downregulation of the tumor-promoting CXC chemokines with RNA interference techniques or chemical inhibitors or neutralizing antibodies, and the upregulation of the tumor-suppressing CXC chemokines by exogenous expression or the addition of recombinant proteins, they may provide effective approaches for cancer therapy.CXC chemokines play important roles in tumor initiation and progression. CXC chemokines, especially ELR+ CXCLs, promote tumor neovascularization. Interaction of CXC chemokines with tumor tissues produces a tumor metastatic microenvironment. Cancer patients with tumors constitutively expressing CXC chemokines usually have a poor prognosis. Although it has been observed that CXC chemokines have significant effects on tumor proliferation, angiogenesis, migration, invasion, and metastasis, the regulatory mechanisms underlying the effects of CXC chemokine on cancer onset and development remain incompletely determined.Regulation of angiogenesis is one of the major effect of CXC chemokines on tumor progression . CXC cheCXC chemokines are pro-inflammatory mediators and are chemotactic for leukocytes . These cells are critical for inflammatory and immune responses . CXC cheIn addition to the roles of CXC chemokines in facilitating angiogenesis and modulating inflammatory and immune responses, they also regulate tumor metastasis. Many studies have shown that CXC chemokines attract neutrophils to tumor tissues, promote neovascularization, enhance the expression of pro-metastatic genes  , and theIt has been proposed that inducing angiogenesis, producing a tumor-promoting inflammatory response, and activating metastasis are hallmarks of cancer progression ,146. CXC


Over a century ago, it was found that a rapid burst of oxygen is needed and produced by the sea urchin oocyte to activate fertilization and block polyspermy. Since then, scientific research has taken strides to establish that Reactive Oxygen Species (ROS), besides being toxic effectors of cellular damage and death, also act as molecular messengers in important developmental signaling cascades, thereby modulating them. Wnt signaling pathway is one such developmental pathway, which has significant effects on growth, proliferation, and differentiation of cells at the earliest embryonic stages of an organism, apart from being significant role-players in the instances of cellular transformation and cancer when this tightly-regulated system encounters aberrations. In this review, we discuss more about the Wnt and ROS signaling pathways, how they function, what roles they play overall in animals, and mostly about how these two major signaling systems cross paths and interplay in mediating major cellular signals and executing the predestined changes during the perinatal condition, in a systematic manner. In multicellular organisms, this mediating system or cell signaling system as we know it, is much more elaborate and extensive as compared to the unicellular organisms, as the multicellular organisms are themselves able to function efficiently as a whole, owing to the intricate communication mediated by the innumerable molecules inter-playing and reprising their roles to achieve even the most basic day to day functions like respiration, growth, proliferation, differentiation, inflammation, wound healing, and death. For higher organisms with organ system levels of organization, this signaling system branches and broadens into highly efficient networks with positive and negative feedback loops, keeping themselves tightly regulated within a species-specific framework  and/or cascades of protein phosphorylation then relay the downstream signaling further. Finally, these intracellular signaling pathways evoke the expected response by going on to activate one or multiple transcription factors that would regulate specific sets of genetic expression, which are essential for an array of cellular functions , that go on to react, being highly reactive in nature, to form ozone and other peroxides. They also react to and support the formation of chemicals promoting formation of other hyper-reactive oxidants like various nitroaromatics, quinones, and other pollutants like bipyrimidiulium herbicides that are related to paraquat. ROS also participate in chain reactions of chemical formations that are again subsequently metabolized to radicals, like the phenols, aminophenols, polyhalogenated alkanes or other chemicals, that in their lifetime do release copper and iron, that could support and promote the formation of hydroxyl radicals in future. Rapid radiolysis of water molecules upon exposure to ionizing radiation like X-rays or ᵧ-rays, or even UV irradiation of Hactivity .2˙−. Mitochondrial sources add on important sources of ROS as additives. Antioxidant enzyme systems work in the body at all times, each and every second in the body, to avoid the generation of oxidative stress, which happens when the delicate balance of pro-oxidant and anti-oxidant system in the body is affected, by exogenous or endogenous means , the ETC transmembrane super-oxide generating system, contribute to the generation of ROS. The cytosolic portions of these enzymes transfer an electron from the NADPHs to an FAD co-factor, the electron is thereby passed-on to the haem group, subsequently being added to molecular oxygen, generating superoxide anion Ous means .2O2), the hydroxyl radical (.OH), the superoxide anion (O2−), and peroxynitrites (ONOO−), which are all are chemically active, in many cases exogenous, prooxidant molecules that are generated by various physical and chemical redox processes. ROS are involved in a variety of physiological and pathological processes in the cell by generating various methods of cellular survival and glycolysis . It has been established that cancer cells continue to thrive under some levels of oxidative stress, and compared with non-cancerous tissues, ROS levels are elevated in breast, colon, pancreatic, prostate, and other cancers , the category of highly active radicals including hydrogen peroxide , and act in host defense mechanisms by producing copious amounts of ROS. Fertilization event in sea urchins also triggers extracellular H2O2 production that catalyzes extracellular protein cross-linking to form a protective fertilization envelope around the oocyte , and then, research found the Wnt family of genes to be highly conserved in the animal kingdom, starting from nematodes to mammals (2+ (Wnt/Ca2+ pathway)  .via Wntless, a specialized transmembrane protein, and act as ligands for Frizzled (Fz), a G-protein coupled 7-pass transmembrane receptor. All of them bind to a cysteine-rich conserved region of Fz thus activating it, and in the canonical mode, evoke a cellular response by inhibiting the β-catenin degradation complex, that is composed of four proteins–Axin, Adenomatous Polyposis Coli (APC), GSK3β, and casein kinase 1 (CK1), thereby stabilizing β-catenin in the cytosol. The accumulated β-catenin then migrates to the nucleus to interact with T-cell factor/Lymphocyte enhancer factor (TCF/LEF) family of transcription factors, thereby activating the transcription of target genes  (The Wnt glycoprotein homologues are secreted out of the cells et genes . A majoret genes . Before et genes . The speet genes . The Ca2e (ROCK) . During e (ROCK) , includie (ROCK) .via covalent modification of redox-sensitive target proteins, at some of their specific cysteine residues. Enzymatic activities of these proteins can be reversibly modified by the oxidation of these reactive and very specific cysteine residues. Multiple emerging evidence now suggests that ROS might regulate an array of physiological parameters ranging from growth factor stimulation responses to inflammatory response generation, and very obviously, deregulations in these ROS signaling pathways may contribute to a multitude of human diseases , and other ROS as well as nitrogen oxides (Nox) might induce p21Ras via the JAKs and Src kinases with the involvement of phosphatidylinositol 3′-kinase , and some other amino acids like histidine, tyrosine, and tryptophan, ROS play the role of a significant family with varied molecular diversity, that participates in almost every signaling pathway known to scientists . What isvia this protein Dvl. Nucleoredoxin (NRX), an ubiquitously expressed protein of the Trx antioxidant family, was found to directly control Dvl activity in its reduced form, by forming a complex with Dvl, thereby, keeping it inactive in the cytoplasm . The breakthrough came when Funato et al., in 2006 discovered that ROS might be involved in the Wnt signaling mediation, and that too, ytoplasm . This inytoplasm . In thes ligands .via the activation of Vav-2, the Rac1 guanine nucleotide exchange factor  mediates the binding of profilin to actin, by forming a complex with rac1. This binding event of profilin to actin leads to cytoskeletal restructuring, and gastrulation. Thus, indirectly, ROS mediates gastrulation  pathway .2O2 elimination, and neutralization of other pro-oxidants and ROS  have been identified in mammals having six isoforms (known as of now), that are directly involved in H and ROS . Prx pro and ROS .Thus, it can be firmly suggested that ROS levels are important determinants for the functioning of Wnt pathway, and hence, must be tightly regulated to effect development correctly .ROS has been shown to mediate cellular responses towards intra-as well as extracellular conditions, such as presence or absence of cytokines and growth factors, state of nutrient deprivation, as well as hypo- and hyperoxia, all of which in turn, regulate cell proliferation, growth, differentiation, and apoptosis . The fol2 conditions. Hence, it is implicated that the early embryo must be very sensitive to ROS or ROS-like exogenous factors, which might cause fatally culminating oxidative stress. High O2 concentrations in vitro embryo culture conditions is heavily detrimental to the embryological growth, and this deterioration can be reversed by treatment with free radical scavengers  have pro-survival effects, whereas ROS derived from mitochondria (occurring at high levels) have anti-survival effects and promote apoptosis . Early eavengers . The los embryos . The eve embryos .Unfit cells require to be eliminated for precise patterning of the embryo during early development. A major way to get rid of these unfit cells is by the induction of ROS and further ROS-mediated apoptosis. Akieda et al., in 2019 reported that, when Wnt/β-catenin is abnormally activated or inhibited in a mosaic manner in normal embryos, it led to activation of ROS production and further oxidation of DNA . Their sThe study demonstrated an interesting ‘noise-cancelling system’ model for signaling gradients that is mediated by cell-competition. During AP patterning in zebrafish embryos, extent of Wnt/β-catenin activity influences cadherin and membrane β-catenin protein level gradient formation along the AP axis. Now, when cells displaying unfit Wnt/β-catenin activity appear spontaneously in the developing embryo, it produces ample noise in the gradient of Wnt/β-catenin as well as alterations in cadherin and membrane β-catenin protein levels; this leads to a significant imbalance between the unfit and neighbouring cells based on cadherin levels. Unfit cells then activate the TGF-β-Smad signaling pathway that produces ROS, and subsequently undergo a ROS-mediated apoptosis. In this very specific ‘identify, pick and eliminate’ process, the embryonic tissues successfully eliminate noise generated in the Wnt/β-catenin gradient in order to its effect proper formation, besides a robust patterning of the embryo. This morphogen noise-cancelling system mediated by apoptosis is thought to be evolutionarily conserved, as it has been found that Wnt/β-catenin as well as other basic morphogen signaling pathways utilize similar systems. The inhibition of ROS signaling actually reduces the magnitude of physiologically appearing apoptotic cells, albeit partially, which corroborate the view that these apoptotic cell populations comprise ROS activated cells that are Wnt/β-catenin defective, besides other cells that may be potentially defective in some other morphogen-signaling systems .2 concentration of 40%, until the gas balance is normalized in the blood. Li et al., in 2013 tried to understand the extent and manner in which ROS can cause oxygen toxicity to the lungs, and so they treated cells with O2 at varying concentrations at room temperature. They found that the lower O2 fraction (40%) had fared better in terms of survival rates and apoptosis than their higher O2 fraction (95%) counterparts. These good survival rates in the lower O2 fraction subsequently decreased with the increase in their O2 treatment dosage, or with increased duration of low O2 that they received. They went further to examine the relationship between Wnt signaling pathway and ROS in lung cells as it is known that the Wnt/β-catenin pathway plays critical roles in lung development in its early stages. Numerous factors in the pathway have been found to participate and play specific roles in the control and regulation of lung development, such as proliferation and differentiation of lung cells, formation of steric configuration of the lung, as well as specific development of the lung’s distal end. For example, Wnt5α gene is implicated to play a role in the development of lung vessels as well as the embryonic pulmonary parenchyma , as they provide the best possible environments for cellular culture and experimentation for the relevant studies. But clinical studies were also conducted to observe the effects of ROS in adult patients, for preliminary acquisition of information. Patients with acute oxygen deficiency have to undergo oxygen therapy, usually at a stable Orenchyma . While s2 concentration might stimulate the Wnt pathway in their early stages, thus causing premature expression of Wnt5α as well as β-catenin, both of whose levels subsided with prolonged exposure to very high concentrations of O2. Therefore, it was seen that Wnt pathway might function early in the development of hyperoxia-induced lung injury, which is contrary to the general understanding that Wnts help in cell growth and proliferation, and are activated under lower concentrations of oxidants like H2O2 and inactivated at higher concentrations, as has been seen in the previous sections. The authors attributed the low survival of high O2 fraction to oxidative damage and lung cell death. Hence, the effects of ROS mediated Wnt signaling may be diversified according to sources and cellular types, as well as the magnitude of oxidative stimulation.Thus the study suggested that in alveolar epithelial cells (AEC IIs) a higher O2+ ion concentration are downstream effects of non-canonical Wnt signaling pathway, their study showed that the same Ca2+ concentration gradient acts upstream of the ROS-mediated canonical Wnt/β-catenin pathway during neuronal differentiation , once the GFs are removed, cytoplasmic Ca2+ enters mitochondria via mitochondrial calcium uniporters (MCU), thereby increasing ROS as by-products of altered ETC activity in the mitochondria. This elevated ROS then interacts with Nrx, thus, releasing the Nrx-bound Dvl, so as to stabilise β-catenin in the cytoplasm, thereby kicking off the Wnt pathway-mediated transcription of regulatory genes for growth and differentiation. This observation was cross-checked by inhibition of ITPR1 mediated Ca2+ efflux as well as MCU mediated Ca2+ influx. Besides, the experiment was repeated with the additional treatment of NAC, a highly potent ROS-quencher. In all of the above three cases, the results were consistent. It was seen that Dvl2 was no longer robustly activated, expressed by the lower levels of nuclear β-catenin accumulation, which attenuated the expression of Wnt target genes, consequently leading to an impeded differentiation of the neuronal system. It was also seen that physiological changes in ROS metabolism altered the response magnitudes of the Wnt/β-catenin signaling pathway effectors. Thus it was concluded that ROS is involved in spatio-temporally altering cellular decisions of the neuronal progenitor cells, and effected their commitment and differentiation into neurons. In embryonic development, it was an important benchmark to be known while documenting the effects of ROS and Wnt.Rharass et al., in 2014, conducted experiments on hNPCs  which provided concrete evidence for an endogenous ROS-mediated activation of Wnt pathway, and while it was known that changes in Cantiation . In hNPCHe et al., in 2017 discovered that mothers fed with high sucrose diet prenatally, gave birth to offsprings that had higher levels of oxidative damage in the brain, in addition to higher expression of oxidative enzymes like Nox2, and lesser expression of brain-derived neurotrophic factor (BDNF) as well as genes involved in maintenance of synaptic plasticity and dendritic arbors, like Wnt2 and Wnt3a, and NR2B  in the hippocampus. Wnt signaling pathway down-regulation or disruption in the hippocampal region has been reported to cause cognitive decline along with neuronal loss, effects which are similar to oxidative stress induced neuronal aging and death. Thus, it can be suggested that there must be some link between the two signaling pathways in neuronal aging, about which, no concrete evidence has been found until date, and continues to remain an intense area of research .via a significant role play and downregulation of the Wnt pathway, indicating the importance of the pathway in neurological and overall brain development during the embryonic stage  and intellectual disability, and concluded that the mutational variants putatively causing haploinsufficiency lead to macrocephaly. The authors also proposed an opposing pathomechanism due to variants in the PH-domain of the same WDFY3, that led to microcephaly. Hence, this experiment came in as a breakthrough of how studying next-generation sequencing can actually facilitate the discovery of monogenic causes for mild to moderate neurodevelopmental delay in humans and end a long-term diagnostic blank space dilemma.In 2019, Diana De Luc and her co-workers found an important breakthrough in exome and genome sequencing experiments with array analysis, in a significant number of human individuals—in humans, the loss of function mutations of WDFY3 gene, lead to macrocephaly, ic stage . In the All of the above studies tried documenting the crosstalks of ROS with Wnt during neuronal development of the embryo, and as experiments, walked off with important results that worked as shoulders for future researchers to stand on.Like in most cells, enhanced ROS has been observed to have a toxic/inhibitory effect on hematopoietic stem cells (HSCs) by impairing their functioning. β-catenin comes into the rescue and provides protection to the cells after irradiation-induced oxidative stress . AbsenceFor the first functional HSCs to emerge, β-catenin/canonical Wnt signaling has been implicated to be an essential event. Studies conducted by Kwarteng in 2018 indicated that mostly the canonical Wnt pathway remains active in the fetal HSPCs, but both the fetal as well as adult HSPCs are potentially able to activate the non-canonical Wnt pathways. Post-transplant, β-catenin was suggested by them to metabolically regulate and maintain short-term transplantation competitiveness of the fetal HSPCs, but the switch from fetal to the adult HSCs corresponded to a downregulation of the canonical Wnt/β-catenin pathway . This su2O2 levels , besides being an integral adaptor protein for stabilizing cytoplasmic β-catenin and efficient transduction of the Wnt signal, is also a Notch signaling inhibitor. Therefore, Dvl plays a key role in making decisions pertaining to cellular fate, especially in cases where Wnt and Notch have antagonistic effects .via the TNFR1 receptor (tumor necrosis factor receptor 1)  . Studiesia TNFR1 .From various studies, it has been found that ROS-modulated signaling of Wnt and Notch regulates the development of vasculature in a variety of aspects, ranging from stem cell differentiation to vascular cell migration, like VEGF signaling, angiogenesis, and recruitment of cardiac as well as endothelial progenitor cells  Calicet.Xenopus laevis embryos that Wnt signaling pathways including both canonical as well as non-canonical, are indispensable regulators of pronephos development . Hwang and his co-workers found that, this RA-mediated differentiation also concomitantly showed a sustained increase in the levels of ROS, which by then were already known to positively influence the Wnt signaling pathway. They then hypothesized that treating F9 cells with H2O2 could activate the Wnt signaling pathway even in the absence of Wnt ligands, such that the extraembryonic endoderm is formed, and successfully tested it, to find that H2O2 treatment indeed brought about the appearance of the markers of primitive endoderm–morphological, molecular, as well as biochemical ones. They also found the canonical Wnt/β-catenin signaling to have been activated, as well as the expression of Nox4 to have been elevated. All these RA-mediated primitive endoderm differentiation markers were inhibited when the F9 cells were pretreated with antioxidants like NAC, or Nox inhibitors like DPI, prior to RA treatment  with retinoic acid (RA), which was known to initiate differentiation of the extraembryonic endoderm reatment . Thus, tNrx, as well as Trx, play important roles in embrogenesis and tissue patterning, which can be suggested from the fact that Trx1 as well as Nrx knockout mice pups either suffer lethality during implantation, or if alive with abnormalities as foetuses, they die soon around birth . Similar2O2 as well as performed Wnt6 peconditioning, and in both cases, they obtained results that mimicked that of Hwang et al., that is, similar diffentiation occurred, accompanied by ROS generation, as happens in case of RA-induced differentiation. They further found that the redox state of Nrx, as expected, dictates the formation of extraembryonic endoderm. Nrx bound to Dvl2 in the undifferentiated F9 cells, and H2O2 treatment rescued this bondage, in line with other studies. When Sandieson and his co-workers depleted Nrx levels, similar morphological changes were observed as in the case of RA-induced differentiation, and when they increased the activity of Protein Kinase A, the primitive endoderm cells were further induced to parietal endoderm cells. In line with other studies, this depletion of Nrx also led to an increased transcription of TCF/LEF targeted genes, which indicated the canonical Wnt signaling to be in action  such that senescence in these culture cells could be delayed , which they obviously concluded, to be coming from elevation of endogenous ROS from mitochondria, thereby shifting these Wnt3A-treated cells into oxidative stress. Concomitantly, they observed decrease in proliferation, along with enhanced levels of senescence markers . They re-checked their experimental findings by repeating the same experiments with the addition of NAC (N-acetyl Cysteine), a molecule which effectively normalised the aberrant parameters. Wnt mediated mitochondrial biogenesis was still underway in these NAC-treated cells, confirming the established idea that Wnt directly activated mitochondrial biogenetic process rather than as a secondary response to elevated ROS levels. But in contrast to that, elevated ROS levels were essentially required to induce Wnt-mediated senescence, thereby suggesting an upstream role of Wnt in induction of replicative senescence in these MEFs, by increment of mitochondrial biogenesis, which elevates the cellular ROS levels, and its consequential ROS-mediated cellular damage (It is a well-established fact that ROS levels, when elevated chronically, lead to accumulated effects of oxidative-stress mediated cell damage, further leading to senescence and cell death, or transformation to malignancy . But in  delayed . They obr damage  (Figure r damage .A great deal of research has gone into studying the canonical Wnt/β-catenin signaling pathway in the deepest details, and most, if not all of them have suggested that it plays a key role to induce, manage, execute, and regulate the various events that are required for normal development of the embryo, as well as cellular maintenance in adult life. Because of its main engagement in the growth, proliferation, development and differentiation processes of the cells and their progenitors, the Wnt signaling pathway has also been implicated to be one of the root causes of several human diseases, including neonatal and congenital conditions, and cancer. Stem cell research for therapy has made its foray into the minutest details of this pathway in the past few years, given its important role in maintaining all kinds of adult, embryonic, adult, as well as cancer stem cells. Hence it becomes an important task to find and learn about the regulatory mechanisms of the Wnt pathway under normal conditions, and how aberrations in these cascades can be mitigated, such that any deadly disease state can be prevented from onset or progression.ROS on the other hand have been for long, deemed as toxic molecules, leading to various diseases across all the organs of the body, leading to an overall outcome of cell damage and death in most, if not all cases. But with the broadening of research, they have increasingly come to be recognized as essential regulators of a plethora of biological functions and modulating various states of pathophysiology. They have been found to act at various nodes of signaling cascades, activating or inhibiting them, to induce significant toxic or pro-survival effects. It has been convincingly established that the oxidants could be regulated at their production source, and could be very specific in what their effects are. That the earliest organisms like bacteria, and plants heavily rely on significant aspects of oxidant signaling mechanisms and pathways, suggests that oxidant signaling mechanisms are in all probabilities, ancient, and might have been evolutionarily conserved.We have discussed here a significant role of ROS–in mediating the Wnt/β-catenin signaling cascade and its modulation. We have henceforth concluded that clear and correct functioning and cross-talk of these two signaling pathways of ROS and Wnt are essential for appropriately and robustly deciding the cell-fate. Whereas we have mostly focused on the term ‘signaling cascades’, we must keep this in mind that the cell is an active network of multiple signaling cascades working in tandem, to bring about any particular effect. And these networks are active every moment that we are living, to facilitate our effective and healthy life. Hence, more research on the ROS modulation of these cell-fate deciding signaling pathways like Wnt, as well as Notch, Hedgehog, etc., along with the cross-talks going on in these networks, would provide important clues for strategizing clinical practices, therapies, and drug discovery, for targeting the diseases that might affect foetuses and neonates during the perinatal period, as well as for later diseases like aging and amyloid induced neurodegeneration, cardiovascular diseases, liver as well as kidney ailments, and cancer.


CUX1 is a transcription factor that plays an important role in the growth and development of hair follicles. The aim of this study was to investigate the effect of CUX1 on the proliferation of Hu sheep dermal papilla cells (DPCs) to reveal the role of CUX1 in hair follicle growth and development. First, the coding sequence (CDS) of CUX1 was amplified by PCR, and then CUX1 was overexpressed and knocked down in DPCs. A Cell Counting Kit-8 (CCK8), 5-ethynyl-2-deoxyuridine (EdU), and cell cycle assays were used to detect the changes in the proliferation and cell cycle of DPCs. Finally, the effects of overexpression and knockdown of CUX1 in DPCs on the expression of WNT10, MMP7, C-JUN, and other key genes in the Wnt/β-catenin signaling pathway were detected by RT-qPCR. The results showed that the 2034-bp CDS of CUX1 was successfully amplified. Overexpression of CUX1 enhanced the proliferative state of DPCs, significantly increased the number of S-phase cells, and decreased the number of G0/G1-phase cells (p < 0.05). CUX1 knockdown had the opposite effects. It was found that the expression of MMP7, CCND1 (both p < 0.05), PPARD, and FOSL1 (both p < 0.01) increased significantly after overexpression of CUX1 in DPCs, while the expression of CTNNB1 (p < 0.05), C-JUN, PPARD, CCND1, and FOSL1  decreased significantly. In conclusion, CUX1 promotes proliferation of DPCs and affects the expression of key genes of the Wnt/β-catenin signaling pathway. The present study provides a theoretical basis to elucidate the mechanism underlying hair follicle development and lambskin curl pattern formation in Hu sheep.CUT-like homeobox 1 protein (CUX1), also called CUX, CUTL1, and CDP, is a member of the DNA-binding protein homology family. Studies have shown that  There are great differences in the quality of the lambskin of Hu sheep. According to the width of the pattern, it can be classified into four types: small waves (pattern width 0.5–1.25 cm), medium waves (pattern width 1.25–2 cm), large waves (pattern width 2 cm or more), and straight hair (no pattern). Wavy lambskin with small waves is the most desired type, and the straight hair pattern is the least desired [The hair follicle is an important accessory organ of the skin and includes both dermal and epidermal parts. The dermis contains the outer root sheath and the inner root sheath, while the epidermis includes the hair papilla and the dermal sheath. DPCs are located in the dermis of the hair follicle and are a special type of interstitial cells that exist at the base of the hair follicle, where they are surrounded by hair matrix cells, and play an important regulatory role in the formation, growth, and circulation of hair . RompolaCUX1 in mice leads to many abnormal manifestations, such as growth retardation, whisker curling, altered hair follicle morphology, and male sterility [CUX1 affects the growth and development of DPCs, previous research has found that miR-143 is a differentially expressed miRNA that may affect hair follicle development in Hu sheep [CUX1 and KRT71, thereby inhibiting the proliferation of DPCs. Therefore, we hypothesized that CUX1 may also directly affect the proliferation of DPCs and the curvature of hair growth.The homology frame CUT-like homeobox 1 protein (CUX1), also called CUX, CUTL1, and CDP, is a member of the homology domain family of DNA-binding proteins. Previous studies have shown that inactivation of terility ,7. AlthoHu sheep . It has Wnt10b regulates the growth cycle of hair follicles and is mainly expressed during the anagen phase of hair follicles and is not expressed during the regression and resting phases. During hair follicle growth, high expression of Wnt10b has been detected in the bulge of the hair follicle, while Wnt10b protein expression has been detected near the hair papilla cells. When Wnt10b is highly expressed, it inhibits the activation of downstream β-catenin in the Wnt/β-catenin signaling pathway [Wnt10b inhibits the activation of the Wnt/β-catenin signaling pathway and allows hair follicles in the resting phase to enter the anagen phase.Numerous studies have shown that many molecular signaling pathways are involved in the regulation of hair follicle growth and development, such as the Wnt/β-catenin signaling pathway, the BMP signaling pathway, the TGF-β signaling pathway, and the Notch signaling pathway ,11,12,13 pathway ,16. TherCUX1 in DPCs and whether CUX1 can regulate the Wnt/β-catenin pathway, to provide a theoretical basis to elucidate the mechanisms underlying lambskin pattern formation in lambs.In this study, we aimed to investigate the role of 2), as well as heart, liver, spleen, lung, kidney, and muscle tissue samples, were taken in triplicate, snap-frozen in liquid nitrogen, and stored at −80 °C until use.Healthy and disease-free 3-day-old lambs (Jiangsu Xuzhou Su Sheep Industry Co.) were selected for this experiment. Three pairs of full sibling individuals were divided into small waves groups and straight wool groups. Scapular skin tissue samples  and stored at −80 °C. The first strand of cDNA was synthesized by FastKing gDNA Dispelling RT SuperMix , and stored at −80 °C.Our experimental protocol was approved by the Animal Ethics Committee of Yangzhou University (NXFC2020-NF-1).2. DPCs in the growth stage were inoculated uniformly in 6-well, 12-well, and 96-well plates, and when the cells reached 50–80% confluency, they were transfected with jetPRIME reagent following the manufacturer’s instructions. RNA and protein were extracted and stored at −80 °C until use.DPCs were cultured in DMEM/F12 containing 5% fetal bovine serum and 1% penicillin–streptomycin at 37 °C with 5% COCUX1 coding sequence (CDS) were designed using Oligo 6 software based on the CUX1 sequence downloaded from NCBI. The full-length CDS was amplified using lamb skin tissue cDNA as a template. The fragment was cloned into the PEX-1 overexpression vector, and successful cloning was confirmed by double digestion with QuickCut™ XhoI  and QuickCut™ NotI . The overexpression vector were sent to the Tsingke Biotechnology Co., Ltd.  for verification.Primers to amplify the full-length CUX1 primer sequences are as follows:The PEX-1-CUX1-F: CCCaagcttATGGCGGCCAATGTGGGATCPEX-1-CUX1-R: CCGctcgagACACTGCCACAGGTCGCCGCUX1 was designed and synthesized by Shanghai Jima Pharmaceutical Co., Ltd. . The sequences are shown in The siRNA-CUX1 overexpression vector (PEX-1-CUX1), interfering RNA (siRNA-232), and negative controls (PEX-1 and siRNA-NC). At 36 h after transfection, they were washed twice with PBS, 10 μL CCK-8 reagent  was added to each well and the culture was continued at 37 °C for 2 h, then the OD450 values were measured at 0, 24, 48, and 72 h using a microplate reader . The CCK-8 results were analyzed by GraphPad Prism 8. In addition, transfected cells were processed with the Cell-Light TM EdU Apollo 567 In Vitro Kit  and photographed with an inverted fluorescence microscope . The EDU pictures were analyzed by ImagePro Plus 6.0.First, hair papilla cells were transfected with the Hair papilla cells were transfected, and after 48 h, cell suspensions were collected according to the Cell Cycle and Apoptosis Kit . Changes in DNA content were detected by flow cytometry  and the results were analyzed by ModFit.CUX1, CDK2, PCNA, CYCLIN D1, WNT10, MMP7, C-JUN, PPARD, CCND1, FOSL1, CTNNB1, and GAPDH were designed by NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 30 January 2021)) ) .TM , the reaction System: 2 × TB Green Premix Ex Taq II 12.5 μL, F 1 μL, R 1 μL, cDNA 2 μL, RNase-Free ddH2O 8.5 μL.According to the TB GreenTM Premix EX TaqReaction procedure: 95 ℃ 30 s, 95 ℃ 5 s, 60 ℃ 30 s, 40 cycles.S were transfected for 48 h, proteins were extracted with RIPA . Protein concentrations were determined with a BCA Protein Concentration Kit . After adjusting the protein concentrations with PBS, proteins were separated by SDS-PAGE in 10% gels that were prepared with the PAGE Gel Rapid Preparation Kit . Then the proteins were transferred to PVDF membranes, which were blocked in 5% skimmed milk powder solution at room temperature for 1 h. Next, the membranes were incubated with primary antibody   , washed,, China) . Protein-∆∆CT method. The independent sample t-test was used to perform variance and the one-way ANOVA was used to perform variance. The value of p < 0.05 represented a significant difference; p < 0.01 represented an extremely significant difference.SPSS 17.0 and Excel were used to analyze the experiment data. Graphs were plotted with GraphPad Prism 8. Data are represented as the mean ± standard error of the mean(SEM). The RT-PCR results used the 2CUX1 expression levels in heart, liver, spleen, lung, kidney, dorsal muscle, and skin samples were determined by RT-qPCR. CUX1 expression was relatively high in the skin, suggesting that CUX1 may be involved in the regulation of hair follicle development , respectively. CUX1 mRNA expression (p < 0.01) (p < 0.05)  A and CUX < 0.05) B,C were CUX1 knockdown fragments. CUX1 mRNA levels were determined by RT-qPCR. The siRNA-232 group showed the strongest reduction in CUX1 mRNA expression (p < 0.05)  B,C. BaseCUX1 overexpression on the proliferation of DPCs. When CUX1 was overexpressed, the mRNA expression levels of the cell proliferation genes CDK2, PCNA, and CYCLIN D1 were increased, and the highest proliferation rate was reached at 72 h (p < 0.01)   C. The ch reduced D,G. Afte < 0.05) E,F. ThesCUX1 knockdown plasmid. After CUX1 knockdown, the mRNA expression levels of CDK2, PCNA, and CYCLIN D1 were significantly decreased (p < 0.05) (p < 0.05). This difference was highly significant at 72 h after transfection   A, and th < 0.05) B. The CCsfection C. The chelevated D,G. The  < 0.05) E,F. ThesCUX1 on the Wnt/β-catenin signaling pathway, the expression of key genes in the Wnt/β-catenin signaling pathway  after CUX1 overexpression and knockdown was determined by RT-PCR. CUX1 overexpression significantly increased the mRNA expression levels of MMP7, CCND1 (both p < 0.05), PPARD, and FOSL1 (both p < 0.01)  and mRNA expression levels of C-JUN, PPARD, CCND1, and FOSL1 were significantly decreased (p < 0.01)  A. After  < 0.01) B. The mRHu sheep are a unique white lamb breed in China, known for producing lamb skins with wavy patterns. In recent years, due to the emphasis on meat quality and reproductive performance, as well as the introduction of large quantities of foreign bloodlines in the production area, the wavy pattern characteristic of lamb skins has gradually disappeared and the quality of lamb skins has gradually deteriorated. The curvature of wool is a key factor in determining the quality of Hu sheep lamb skins, yet the molecular regulatory mechanisms determining wool curvature are not clear. Therefore, it is important to study the molecular mechanisms regulating wool bending for the conservation of lamb skin germplasm resources of Hu sheep.DPCs are the regulatory center of hair follicle growth and development, and the number and type of DPCs are decisive for follicle growth, wool type, and wool curvature. It has been suggested that the main cause of hair follicle curvature is the autonomous functioning of multiple papilla centers formed in the dermal papillae, which likely contributes to asymmetric hair growth curvature. It has also been suggested that curly hairs originate from a hair bulb surrounded by a large number of proliferating cells, which cause curved hair growth due to the uneven distribution of their proliferation space. Since DPCs have great potential to proliferate and differentiate and are present in the center of the hair bulb, we investigated the causes of curly growth in wool from the perspective of hair papilla cell proliferation.CUX1 belongs to a family of homologous structural domain transcription factors that are widely expressed in a variety of tissues and have important roles in organ development, cell proliferation, and differentiation [CUX1 can be directly regulated as a target gene by miRNAs that are involved in the regulation of cell proliferation and differentiation [CUX1 expression and participate in the developmental process of macrophage differentiation [CUX1 [CUX1 to regulate cardiomyocyte proliferation and differentiation and cardiac development [CUX1 is a cell-cycle regulator that plays an important role in the development of several tissues. In addition, it has been shown that CUX1 can affect the regulation of the hair growth cycle in mice, while loss of its exons leads to hair curvature [CUX1 in mice, Luong and Ellis mutated different C-terminal positions of CUX1. Mutant mice exhibited retarded growth and had abnormal hair development. This indicates that CUX1 plays an important role in hair follicle growth and development [CUX1 in DPCs, we analyzed whether CUX1 can regulate the proliferation of DPCs by overexpressing and knocking down CUX1. Overexpression of CUX1 promoted mRNA and protein expression of related proliferative genes, while the opposite observations were made after CUX1 knockdown. The proliferation status and cell-cycle progression of DPCs were detected by CCK8, cell-cycle, and EdU assays, and it was found that overexpression of CUX1 promoted the proliferation of DPCs, while knockdown of CUX1 inhibited the proliferation of DPCs. These results suggest that CUX1 can promote the proliferation of DPCs.ntiation ,18. Studntiation ,20,21. Fntiation ; miR-122on [CUX1 ; and miRelopment . Meanwhiurvature ,26. To eelopment . To veriCUX1 plays its important regulatory role by affecting the Wnt/β-catenin signaling pathway. CUX1 can regulate β-catenin expression and activate the Wnt/β-catenin signaling pathway in gliomas, and, when CUX1 is knocked down, the expression of β-catenin at the mRNA and protein levels is suppressed. In addition, a TOP/FOP assay showed that downregulation of CUX1 significantly inhibited the activity of the Wnt/β-catenin signaling pathway [CUX1 can regulate Wnt/β-catenin to exert its biological function in DPCs, we overexpressed and knocked down CUX1 and then detected the mRNA expression levels of WNT10, MMP7, C-JUN, PPARD, CCND1, FOSL1, and CTNNB1. CUX1 upregulated key Wnt/β-catenin genes, thus regulating the proliferation of DPCs.The Wnt/β-catenin signaling pathway plays a critical role in hair growth and development. It was found that  pathway . To inveCUX1 can affect the expression of the cell proliferation genes CDK2, PCNA, and CYCLIN D1 to some extent, promote the proliferation of DPCs and regulate the expression of key genes in the Wnt/β-catenin signaling pathway. The present study provides a basis for the elucidation of the molecular mechanisms underlying lambskin pattern formation.In conclusion, we showed that


To test this hypothesis, we used adenine-induced CKD rats and angiotensin II (AngII)-induced HK-2 and NRK-49F cells. Treatment with SKI inhibited renal function decline, hypertension and renal fibrosis. Mechanistically, SKI abrogated the increased protein expression of multiple RAS elements, including angiotensin-converting enzyme and angiotensin II type 1 receptor, as well as Wnt1, β-catenin and downstream target genes, including Snail1, Twist, matrix metalloproteinase-7, plasminogen activator inhibitor-1 and fibroblast-specific protein 1, in adenine-induced rats, which was verified in AngII-induced HK-2 and NRK-49F cells. Similarly, our results further indicated that treatment with rhein isolated from SKI attenuated renal function decline and epithelial-to-mesenchymal transition and repressed RAS activation and the hyperactive Wnt/β-catenin signalling pathway in both adenine-induced rats and AngII-induced HK-2 and NRK-49F cells. This study first revealed that SKI repressed epithelial-to-mesenchymal transition by synchronously targeting multiple RAS elements by blocking the hyperactive Wnt/β-catenin signalling pathway.Chronic kidney disease (CKD) is a major worldwide public health problem. The increase in the number of patients with CKD and end-stage kidney disease requesting renal dialysis or transplantation will progress to epidemic proportions in the next several decades. Although blocking the renin-angiotensin system (RAS) has been used as a first-line standard therapy in patients with hypertension and CKD, patients still progress towards end-stage kidney disease, which might be closely associated with compensatory renin expression subsequent to RAS blockade through a homeostatic mechanism. The Wnt/β-catenin signalling pathway is the master upstream regulator that controls multiple intrarenal RAS genes. As Wnt/β-catenin regulates multiple RAS genes, we inferred that this pathway might also be implicated in blood pressure control. Therefore, discovering new medications to synchronously target multiple RAS genes is necessary and essential for the effective treatment of patients with CKD. We hypothesized that Shenkang injection (SKI), which is widely used to treat CKD patients, might ameliorate CKD by inhibiting the activation of multiple RAS genes  The worldwide increase in the number of chronic kidney disease (CKD) patients could be reflected in the rising number of end-stage renal disease patients who undergo renal replacement treatment, such as dialysis and transplantation . CKD aff1R), after renal damage , safflower (Carthamus tinctorius L.), Astragalus , and red sage . SKI quality was determined by fingerprint analysis and high-performance liquid chromatography (HPLC) as described previously  have been demonstrated to improve CKD and protect against renal fibrosis by inhibiting RAS activation and/or the Wnt/β-catenin signalling pathway . Shenkaneviously . The maieviously . SKI caneviously . With iteviously . The finrtension . We hypo1R , Wnt1 , active β-catenin , β-catenin , Snail1 , Twist , MMP-7 , PAI-1  and FSP1  were purchased from Santa Cruz Biotechnology , Proteintech Company , Abcam Company  and BD Transduction Laboratories . Glyceraldehyde-3-phosphate dehydrogenase  and α-tubulin (11224-1-AP) were purchased from Proteintech Company .SKI was obtained from Shijishenkang Pharmaceutical Company Ltd. . Adenine (purity ≥99.0%) was purchased from Sigma‒Aldrich Company Ltd. . Chrysophanol, emodin and rhein in SKI were isolated and identified and were purchased from Chengdu Pufei De Biotech Co., Ltd. . The purities of chrysophanol, emodin and rhein were 99.12%, 98.83%, and 99.25%, respectively. Primary antibodies against collagen I , α-SMA, , fibronectin , E-cadherin , ACE , ATn = 8/group): control, CKD, low-dose SKI-treated CKD, medium-dose SKI-treated CKD, high-dose SKI-treated CKD, chrysophanol-treated CKD, emodin-treated CKD and rhein-treated CKD. Except for the control group, the other groups of CKD rats were orally administered adenine (200 mg/kg/d) for 3 weeks. The treatment groups were administered SKI , chrysophanol (30 mg/kg/d), emodin (100 mg/kg/d) or rhein (150 mg/kg/d) for 3 weeks. After 3 weeks, individual rats were placed in metabolic cages to collect 24-h urine. The rats were anaesthetized with 10% urethane, and then blood and kidneys were collected for subsequent analysis. All animal care and experimental procedures were approved by the Ethics Committee for Animal Experiments of Northwest University.Six- to eight-week-old male Sprague‒Dawley rats (body weight 180–210 g) were purchased from the Central Animal Breeding House of Xi’an Jiaotong University . The rats underwent an adaptation for 1 week, during which they were fed commercial feed. The rats were administered adenine by gavage as previously described . The ratSystolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by rat tail plethysmography . The levels of serum creatinine were determined by using an Olympus AU6402 automatic analyser.3, 110 mg/L sodium pyruvate, 15 mM HEPES and 1,000 mg/L glucose supplemented with 10% foetal bovine serum  at 37°C with 5% CO2.Human kidney proximal tubular epithelial cells (HK-2 cells), normal rat kidney proximal tubular epithelial cells (NRK-52E cells) and normal rat kidney interstitial fibroblast cells (NRK-49F cells) were purchased from the China Centre for Type Culture Collection. The cells were cultured in DMEM-F12 containing 4 mM L-glutamine, 1.2 g/L NaHCO4 cells/well. After 24 h, HK-2, NRK-52E and NRK-49F cells were treated with SKI  for 24 h. In addition, HK-2 cells were treated with rhein  for 24 h. Subsequently, CCK-8 was added to each well and incubated at 37°C for 3 h. The absorbance was measured at 450 nm on a microplate reader . There were six replicates for each experimental condition.A cell counting kit-8  assay was used to evaluate the viability of HK-2, NRK-52E and NRK-49F cells. The cells were resuspended and seeded in 96-well plates at a density of 1×10Different concentrations of SKI  or rhein (10 μM) were used to treat HK-2 cells stimulated by AngII (1.0 μM). In addition, SKI (1.0 mg/ml) or rhein (10 μM) was used to treat NRK-49F cells stimulated by AngII (1.0 μM). The angiotensin II type 1 receptor blocker losartan  was used as a standard positive control.The effects of different concentrations of SKI on the morphological changes of HK-2 cells treated with AngII (1.0 μM) were visualized with a Leica Microsystems CMS GmbH  using ToupView 3.7 software.1R, Wnt1, β-catenin and FSP1, in paraffin sections of kidney tissues was performed as previously described . A two-tailed unpaired Student’s in vivo experiments. Oral administration of exogenous adenine increased SBP and DBP in rats, which was accompanied by activation of the RAS, including the upregulation of intrarenal ACE and AT1R protein expression in adenine-induced rats  and examined cell viability. As shown in in vitro.High blood pressure led to RAS activation in adenine-induced rats. Therefore, we used AngII stimulation to investigate the effect of SKI on HK-2 cells. As shown in Our results further showed that treatment with SKI significantly inhibited the protein expression of collagen I and E-cadherin in AngII-induced HK-2 cells . Additioin vivo experiment demonstrated that SKI inhibited intrarenal RAS activation in adenine-induced rats. We further investigated the effect of SKI on the RAS in AngII-induced HK-2 cells. The upregulation of AT1R protein expression was observed in AngII-induced HK-2 cells, while SKI significantly abrogated the increase in AT1R protein expression, which was consistent with the inhibitory effect of losartan  mice  mice . Klotho UO) mice . Collect1R in rats with salt-sensitive hypertension induced by a high-salt and high-fat diet, but this formula did not affect the activities of plasma renin, ACE or AngII  Wolf (Polyporaceae), a well-known edible mushroom, is typically used in functional foods, nutraceuticals, dietary supplements and medications. P. cocos exerted various pharmacological effects, such as anti-inflammatory, antioxidant, diuretic and antifibrotic effects. We have isolated and identified a number of novel tetracyclic triterpenoids, such as poricoic acid ZC, poricoic acid ZD, poricoic acid ZE, poricoic acid ZG, poricoic acid ZH, poricoic acid ZI, poricoic acid ZM and poricoic acid ZP, from P. cocos (1R, in TGF-β1- and AngII-induced HK-2 cells and/or UUO mice. Treatment with these compounds could also inhibit the protein expression of Wnt1 and β-catenin, as well as downstream target genes, including Snail1, Twist, MMP-7, PAI-1, and FSP1 (A. rhizome, a diuretic TCM, exerts renoprotective effects (A. rhizome and could ameliorate renal injury by repressing multiple RAS elements by inhibiting the hyperactive Wnt/β-catenin signalling pathway (in vitro experiment indicated that pachymic acid B from P. cocos, alisol B 23-acetate from A. rhizome and ergone from P. umbellatus could inhibit the protein expression of Snail1, Twist, MMP-7, PAI-1, and FSP1 (via the Wnt/β-catenin signalling pathway. In addition, several publications have demonstrated the inhibitory effects of isolated compounds on the hyperactive Wnt/β-catenin signalling pathway in diabetes-induced renal injury. For example, rhein is an anthraquinone that is extracted from rhubarb, which is one of the major components of SKI. Treatment with rhein inhibited the upregulation of Wnt1 protein expression and the phosphorylation of β-catenin and GSK3β in db/db mice with DN (In vitro experiments from the same research group further revealed that treatment with rhein could inhibit the upregulation of Wnt3a, β-catenin and phosphorylated GSK3β protein expression in high glucose-induced podocytes (A. mongholicus, which is another herb in SKI. Treatment with astragaloside IV inhibited the protein expression of Wnt1 and β-catenin in DN rats induced by a high-fat diet and streptozotocin (Tripterygium wilfordii significantly inhibited the hyperglycaemia-induced expression of Wnt1 and β-catenin at both the mRNA and protein levels in diabetic rats induced by streptozotocin (Coprinus comatus could attenuate the protein expression of Wnt1 and β-catenin in DN rats induced by a high-fat diet and streptozotocin (Sinomenium acutum, inhibited the expression of profibrotic proteins, including α-SMA and fibronectin, in TGF-β1-treated HEK293 human embryonic kidney cells and ameliorated tubulointerstitial fibrosis by inhibiting the protein expression of α-SMA and fibronectin in UUO mice, which was associated with the inhibition of β-catenin protein expression in TGF-β1-treated HEK293 cells (In contrast to TCM formulas, our previous studies have indicated that a number of isolated compounds from diuretic TCMs, such as  pathway . P. cocoP. cocos . Furtherand FSP1 . In addiand FSP1 . Similar effects . 25-O-Me pathway . In addiand FSP1 . In addiand FSP1 . Increasand FSP1 . Our preand FSP1 . Taken t with DN . In vitrodocytes . Astragaozotocin . Similarozotocin . Moreoveozotocin . Sinomen93 cells . CollectIn summary, our current study was the first to demonstrate that SKI could lower elevated blood pressure, improve impaired renal function and ameliorate renal fibrosis. Mechanistically, SKI could lower hypertension and abolish RAS activation by inhibiting the hyperactive Wnt/β-catenin signalling pathway. Three anthraquinones blocked multiple RAS elements by inhibiting the hyperactive Wnt/β-catenin signalling pathway. Inhibiting multiple RAS genes by blocking the activated Wnt/β-catenin signalling pathway might be considered a therapeutic strategy by which TCMs can be used to protect against renal fibrosis.