Sensory information, including auditory feedback, is used by talkers to maintain fluent speech articulation. Current models of speech motor control posit that speakers continually adjust their motor commands based on discrepancies between the sensory predictions made by a forward model and the sensory consequences of their speech movements. Here, in two within-subject design experiments, we used a real-time formant manipulation system to explore how reliant speech articulation is on the accuracy or predictability of auditory feedback information. This involved introducing random formant perturbations during vowel production that varied systematically in their spatial location in formant space (Experiment 1) and temporal consistency (Experiment 2). Our results indicate that, on average, speakers’ responses to auditory feedback manipulations varied based on the relevance and degree of the error that was introduced in the various feedback conditions. In Experiment 1, speakers’ average production was not reliably influenced by random perturbations that were introduced every utterance to the first (F1) and second (F2) formants in various locations of formant space that had an overall average of 0 Hz. However, when perturbations were applied that had a mean of +100 Hz in F1 and −125 Hz in F2, speakers demonstrated reliable compensatory responses that reflected the average magnitude of the applied perturbations. In Experiment 2, speakers did not significantly compensate for perturbations of varying magnitudes that were held constant for one and three trials at a time. Speakers’ average productions did, however, significantly deviate from a control condition when perturbations were held constant for six trials. Within the context of these conditions, our findings provide evidence that the control of speech movements is, at least in part, dependent upon the reliability and stability of the sensory information that it receives over time. Painted on the window of a café in the Norrmalm district of Stockholm is information to help customers find their way in. Within an arrow pointing to the left is the text, “Entrance 8,47 M”. What makes this signage funny is its precision. Knowing the door’s location to the hundredth of a meter when you are steps away from entering is excessive and it makes passersby smile when they see it. People have an intuitive feel for what information they need and how precise it should be.Current models of the control of actions include sensory information that is used to coordinate the movements accurately or is needed to maintain the stability of the motor system  or Condition, χ2(6) = 492.82, p < 0.001. The Best Fit Model was a significantly better fit to the data than another alternative model that did not include the interaction between Condition and Phase, χ2(4) = 288.24, p < 0.001.In the LMM analysis of speakers’ normalized F1 speech production values, the Best-Fit Model produced a significantly better fit to the data than a null model that only included the random effects, χM = −14.02, SE = 2.95) and Return  phases than they were during the Baseline phase , all ps < 0.001. The main effect of Condition was not significant. However, there was a significant interaction between Condition and Phase. Adjusting for multiple comparisons, pairwise tests showed that there were significant mean differences between the F1 values produced by speakers during the Perturbation phase of the F1/F2 Coupled Random Perturbation Condition , and the Perturbation phases of the F1/F2 Random Perturbation Condition  and F1-Only Random Perturbation Condition , both ps < 0.001. In the F1-Only Random Perturbation Condition, speakers’ F1 values were significantly more negative during the Return phase  than during the Baseline phase , p < 0.001. In the F1/F2 Coupled Random Perturbation Condition, speakers’ F1 values were also significantly more negative during the Return phase  than during the Baseline phase , p < 0.001. Thus, on average, speakers did not reliably compensate for random F1 perturbations that had a relative overall average of 0 Hz. However, when random F1 perturbations had an average that deviated from zero, speakers demonstrated significant compensatory behavior. In two conditions, speakers’ average F1 production also remained significantly negative as compared to the Baseline phase following the restoration of their natural auditory feedback during the Return phase. A full list of pairwise comparisons and their significance values are provided in the Results from the Best-Fit Model revealed a significant Phase effect. Pairwise comparisons using the Bonferroni correction revealed that speakers’ normalized F1 values were significantly more negative during the Perturbation (2(8) = 373.2, p < 0.001. An alternative model that did not have the Condition effect failed to converge. The Best-Fit Model significantly outperformed alternative models that did not have the Phase effect , or the interaction between Condition and Phase , both ps < 0.001.The Best-Fit Model predicting speakers’ normalized F2 production was a significantly better fit to the data than a null model that only had the random effects, χM = 38.26, SE = 6.99) than during the Perturbation phases of the F1/F2 Random Perturbation Condition  and the F1-Only Random Perturbation Condition , both ps < 0.001. In the F1-Only Random Perturbation Condition, there were significant mean differences between speakers’ F2 values produced during the Baseline phase  and Return phase , and between the Perturbation phase  and the Return phase, both ps < 0.001. In the F1/F2 Coupled Perturbation Condition, speakers’ average F2 production also significantly differed in the Baseline phase  as compared to the Return phase , and in the Perturbation phase  as compared to the Return phase, p < 0.001. Hence, as in the F1 model, speakers’ compensatory behavior in F2 was most pronounced during the F1/F2 Coupled Random Perturbation Condition, where average relative perturbation magnitudes deviated from zero. A full list of pairwise comparisons and their significance values are provided in the Results from the Best-Fit Model in F2 revealed that the main effects of Condition and Phase were not significant. However, there was a significant interaction between Condition and Phase. Pairwise comparisons using the Bonferroni correction revealed that, on average, speakers’ F2 production was significantly more positive during the Perturbation phase of the F1/F2 Coupled Random Perturbation Condition  = 3.14, p = 0.056, ηp = 0.156. However, there was a significant Phase effect, F = 5.20, p = 0.025, ηp = 0.234. Follow-up comparisons revealed that, on average, speakers were significantly less variable in F1 during the Baseline phase  than they were during the Perturbation  and Return  phases, both ps < 0.029. The difference in within-speaker F1 variability in the Perturbation and Return phases was not significant, p = 0.552. The interaction between Condition and Phase was also not significant, F = 12.52, ηp = 0.038, p = 0.609. Average within-subject variability in Experiment 1 is shown in Two repeated-measures ANOVAs  were carried out to examine the influence of Condition  and Phase  on average within-subject speech production variability . In the F1 model, the Phase effect violated the sphericity assumption, Mauchly’s Test of Sphericity, F = 0.819, p = 0.450, ηp = 0.046] or Phase, F = 52.92, p = 0.323, ηp = 0.064. The interaction between Condition and Phase was also not significant, F = 0.608, p = 0.585, ηp = 0.035.There were no significant effects in the F2 model. Within-speaker standard deviation in F2 did not significantly differ by Condition  or Phase, χ2(12) = 429.23, p < 0.001. The Best-Fit Model was also a significantly better fit to the data than an alternative model that did not have the interaction term, χ2(8) = 127.85, p < 0.001.The Best-Fit Model used to predict speakers’ normalized F1 production values in Experiment 2 produced the best fit to the data and included a maximal random-effects structure with random intercepts for speakers. Including random slopes for condition and phase led to model convergence errors. The Best Fit-Model also included the fixed effects of Condition, Phase, and their interaction term. The Best-Fit Model significantly outperformed a null model that only included the maximal random-effects structure, χ2(14) = 499.25, M = −9.18, SE = 2.43) than during the Return phase  and Baseline phase , all ps < 0.001. The main effect of Condition was not significant. However, there was a significant interaction between Condition and Phase, which was mainly qualified by significant differences between phases of the Step Condition and phases of all other conditions. Notably, speakers’ normalized F1 values were significantly more negative during the Perturbation phase of the Step Condition  than they were during the Perturbation phases of the Control Condition , Condition One , Condition Three  and Condition Six , all ps < 0.001. Speakers’ mean F1 values were also significantly more negative during the Perturbation phase of Condition Six than they were during the Perturbation phase of the Control Condition, p = 0.039. Pairwise differences between the Perturbation phases of all other conditions were not significant. Speakers’ mean F1 values produced during the Return phase of the Step Condition  were also significantly more negative than those produced during the Return phase of all other conditions, all ps < 0.001. A full list of pairwise comparisons is provided in the Results from the Best-Fit Model indicated that there was a significant Phase effect. Pairwise comparisons using the Bonferroni correction indicated that speakers’ normalized F1 values were significantly more negative during the Perturbation phase  = 222.54, p < 0.001] or Phase, χ2(12) = 520.20, p < 0.001. The Best-Fit Model significantly outperformed an alternative model that did not include the interaction between Condition and Phase, χ2(8) = 156.21, p < 0.001.The Best-Fit Model used to predict speakers’ normalized F2 productions included a maximal random effects structure with random intercepts for speakers. It also included fixed effects of Condition, Phase, and their interaction term. The Best-Fit Model significantly outperformed a null model that only included the maximal random-effects structure, χ2(14) = 584.43, M = 42.47, SE = 3.90) than they were during the Perturbation phases of the Control Condition , Condition One , Condition Three , and Condition Six , all ps < 0.001. Speakers’ mean F2 values were significantly more negative during the Perturbation phase of Condition One than they were during the Perturbation phases of the Control Condition and Condition Six, both ps < 0.001. Speakers’ mean F2 values were significantly more positive during the Perturbation phase of Condition Six than they were during the Perturbation phase of Condition Three, p = 0.004. As in the F1 model, there were also a number of significant mean differences between formant values produced during the Return phases of different conditions. A full list of pairwise comparisons is provided in the In the Best Fit Model for F2, the main effects of Condition and Phase were not significant. However, there was a significant interaction between these effects. As in the F1 model, the interaction was mainly explained by significant differences between phases of the Step Condition and phases of all other conditions. Importantly, speakers’ average F2 values were significantly more positive during the Perturbation phase of the Step Condition  = 0.631, p = 0.642, ηp = 0.032. However, there was a significant main effect of Phase, F = 4.85, p = 0.013, ηp = 0.203. Pairwise comparisons showed that speakers’ F1 productions were significantly more variable during the Perturbation phase (MSD = 31.64) than they were during the Baseline phase (MSD = 28.47), p = 0.018. There were no statistically significant differences in within-speaker variability between the Baseline and Return (MSD = 29.75) phases (p = 0.172), nor between the Perturbation and Return phases, p = 0.052. The interaction between Condition and Phase was only marginally significant, F = 2.00, p = 0.050, ηp = 0.095. Using the Bonferroni correction to adjust for multiple comparisons, it was determined that none of the interaction comparisons were significant, all ps > 0.059. Notably, there was no significant difference in within-subject variability between the Baseline (MSD = 30.42), Perturbation (MSD = 29.16), and Return (MSD = 28.65) phases of the Control Condition, all ps > 0.05.Two repeated-measures ANOVAs  were conducted to examine whether within-speaker speech production variability  differed by Condition  and Phase . One outlier in F1 that was more than three standard deviations from the mean was Winsorized and replaced with the next highest value in the dataset. The F1 model revealed that speakers’ mean speech production variability did not significantly differ by Condition, ps < 0.05. The Greenhouse-Geisser correction was thus used in making decisions about significance. As in the F1 model, the main effect of Condition was not significant, F = 0.499, p = 0.664, ηp = 0.026. However, there was a significant main effect of Phase, F = 5.31, p = 0.016, ηp = 0.218. Follow-up comparisons revealed that speakers were significantly more variable in F2 during the Perturbation phase (MSD = 51.44) than they were during the Baseline phase (SDM = 45.12), p = 0.012. Within-speaker production variability did not significantly differ between the Baseline and Return (MSD = 48.59) phases (p = 0.106), nor between the Perturbation and Return phases, p = 0.054. The interaction between Condition and Phase was not significant, F = 2.25, p = 0.060, ηp = 0.106. A visual depiction of the Phase effect in F1 and F2 is shown in In the F2 model, the within-subjects effect of Condition and the interaction between Condition and Phase violated the sphericity assumption, Mauchly’s Test of Sphericity, The spectra shown in One possible explanation for compensation being significantly more pronounced in the Perturbation phase of the Step Condition and Condition Six than in Condition One and Condition Three is that the feedback error was held constant for a greater number of trials in these two conditions and thus, the error correction system was responding to more stable and predictable conditions.3. Correlations could not be computed for the Step or Control Conditions due to the F1/F2 perturbation values being held constant throughout the entire Perturbation phases. Correlations were computed at four lags: zero (simultaneous), one, three, and five trials. Our reasoning was that a comparison between simultaneous and time-lagged correlations would provide insights into whether the error correction system was operating instantaneously, or whether it was integrating information over time.We computed a series of bivariate correlations between F1/F2 perturbation values that were applied in the Perturbation phase of Condition One, Condition Three, and Condition Six and average normalized F1/F2 production values across all subjects from these three conditionsA visual depiction of the average results from the bivariate correlations in F1 and F2 are shown in As can be seen in each condition, the average simultaneous correlation values are much lower  than the average lag correlation values. This is particularly the case in Condition Three and Condition Six, where the feedback perturbations were applied in a more consistent and stable manner during the Perturbation phase.As in Experiment 1, only the introduction of perturbations that consistently deviated from baseline in direction and magnitude produced significant shifts across the Perturbation phase. The step change compensations resembled those observed in other studies that introduced such perturbations e.g., . The difFor both F1 and F2, variability increased in the Perturbation phases of all conditions, and this was particularly true for F2. This finding contrasts with other studies that have not shown an increase in variability in perturbation phases e.g., . NotablyThe experiments presented here are part of a broad literature in speech, limb, and eye movements that examine a subtype of motor learning called adaptation. Adaptive responses are designed to maintain the accuracy and stability of movements that are already learned when environmental conditions change, or when sensory perception is noisy. Adaptation is thus usually studied in paradigms that focus on reducing error following some form of perturbation. While compensatory response to auditory feedback perturbations is well documented, here we examined the speech compensation when the sensory feedback returned unpredictable errors.Across a number of different interpretations of randomness in feedback, our results indicate that the use of auditory feedback in speech motor control is governed by the relevance of the feedback. Talkers acted, on average, like random feedback was irrelevant and average performance did not change from no perturbation conditions. The exceptions to this summary were the three conditions that showed consistent error signals . In each of these conditions, the error introduced to the feedback was relatively consistent over a span of utterances. This finding is consistent with other indicators that the auditory feedback system eschews correcting the error when the deviation is too large  or if thAn outstanding issue is whether there is some flexibility in the use of auditory feedback in speech control. The increase in variability in the Perturbation phases of the current experiments may be indicative of a destabilizing effect of the random perturbations. While our repeated measures designs and the repetitive nature of our protocols are possible explanations as well, within-subject variability did not significantly differ in F1 or F2 in the Control Condition in Experiment 2. However, the heightened variability in the Return phase of Experiment 1 is consistent with this possibility. While we are using the relative variability as a measure of the system’s organization of auditory feedback processing, there are other possible contributions to changes in variability. Auditory feedback processing as studied in the laboratory setting has many of the characteristics of phenomena that have driven concerns about the Reliability Paradox (see recent symposium at the Psychonomics Society 2021 meeting). There are a number of phenomena which are robust at a group average level but are not always apparent at the individual subject level see . Test–reAnother contributing factor in formant-feedback processing is error in measurement , particuOverall, the present results are consistent with a control system that takes into account the statistics of the sensory environment. Two of the conditions point to this conclusion. In Experiment 1, the F1/F2 Coupled Random Perturbation Condition had a mean perturbation value that differed from the baseline value across the 30 trials. This restricted or biased random error signal generated a compensatory response reflecting the average. In Experiment 2, keeping the perturbation constant for six trials also produced differential response from the pattern of responses for shorter perturbations. Our lag correlation analysis in Experiment 2 is also indicative of a control system that is not instantaneously responsive to introduced error. Rather, it appears to be sensitive to the consistency and reliability of the error, integrating information and initiating compensatory behavior over a longer time span. In the context that we are testing, more specific studies focused on the predictability shown in these conditions and how the nervous system computes the consistency are warranted .https://osf.io/n4pgf.The raw data supporting the conclusions of this article are publicly available on OSF here: The studies involving human participants were reviewed and approved by the Health Sciences Research Ethics Board at Western University (Experiment 1) and the General Research Ethics Board at Queen’s University (Experiment 2). All participants provided their written informed consent prior to participating in these studies.DRN collected the data for Experiment 2, performed data analyses for both experiments, and assisted with the writing and editing of this manuscript. TM collected the data for Experiment 1. DWP performed a portion of the data analysis, implemented the experimental system, and helped with manuscript editing. KGM contributed to all aspects of this manuscript. All authors contributed to the article and approved the submitted version.


Ubiquitin-specific protease 39(USP39) plays an important role in modulating pre-mRNA splicing and ubiquitin-proteasome dependent proteolysis as a member of conserved deubiquitylation family. Accumulating evidences prove that USP39 participates in the development of hepatocellular carcinoma (HCC). However, little is known about the mechanism especially deubiquitinating target of USP39 in regulating hepatocellular carcinoma (HCC) growth. Here, we prove that USP39 promotes HCC cell proliferation and migration by directly deubiquitin β-catenin, a key molecular of Wnt/β-catenin signaling pathway whose abnormal expression or activation results in several tumors, following its co-localization with USP39. In this process, the expression of E3 ligase TRIM26, which is proved to restrain HCC in our previous research, shows a decreasing trend. We further demonstrate that TRIM26 pre-mRNA splicing and maturation is inhibited by USP39, accompanied by its reduction of ubiquitinating β-catenin, facilitating HCC progression indirectly. In summary, our data reveal a novel mechanism in the progress of HCC that USP39 promotes the proliferation and migration of HCC through increasing β-catenin level via both direct deubiquitination and reducing TRIM26 pre-mRNA maturation and splicing, which may provide a new idea and target for clinical treatment of HCC. As the fifth most common cancer in the world, hepatocellular carcinoma (HCC) endangers human health seriously and causes the second highest mortality rate in the world –4. In AsEmerging studies have shown that Wnt/β-catenin signaling pathway plays an important role in EMT and tumor progression. β-catenin activation is observed in 20–35% of hepatocellular carcinoma (HCC) cases, which also indicates that β-catenin accumulation is closely associated with HCC progression and poor prognosis –8. AlthoThe ubiquitin-specific peptidase 39 (USP39) belongs to the family of deubiquitinating enzymes (DUB), containing the amino-terminal zinc-finger ubiquitin-binding domain (ZNF-UBP) and the carboxy-terminal ubiquitin hydrolase domain (USP) . USP39 iIt was once thought that USP39 has no DUB activity due to the lack of conserved active sites  in the deubiquitinating domain . HoweverTRIM26 belongs to the TRIM family of E3 ligases, participating in diverse cellular processes such as cell proliferation, DNA repair, and innate immunity –26. As aIn this work, we investigate the functional role of USP39 in the development of HCC in vitro, and reveal that USP39 has dual functions in regulating β-catenin in HCCs. We first verify USP39 directly interact with β-catenin protein and further find that USP39 can stabilize β-catenin protein by deubiquitylation. In detail, on one hand, USP39 decreases β-catenin degradation through its deubiquitination function to promote HCC progression. On the other, USP39 promotes the proliferation and invasion of HCC cells by inhibiting splicing of TRIM26, thus affecting the ubiquitin of β-catenin induced by TRIM26. Collectively, our data suggest that USP39 has deubiquitinating and RNA splicing activity, promoting HCC progression by directly or indirectly regulating β-catenin. These findings provide a novel mechanism for understanding the progression of HCC, and may provide inspiration on further finding promising therapeutic targets against human HCC.2. All cell lines were authenticated  and were verified to be mycoplasma negative.Human hepatoma carcinoma cell lines SK-hep-1, PLC/PRF/5, and HepG2 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences, China. SK-hep-1 was cultured in MEM  supplemented with 10% fetal bovine serum . PLC/PRF/5 and HepG2 were cultured in DMEM (Hyclone) supplemented with 10% fetal bovine serum . All cells were maintained at 37 °C in a humidified chamber with 5% COPlasmids to knock down USP39 or TRIM26 were purchased from Genechem . The sequences of the shRNA for human TRIM26 mRNA are as follows: shRNA#1 GATGGATATGACGACTGGGAA and shRNA#2 GCTGCTGAGAGACTTGGAATA. Target sequences for human USP39 mRNA are as followed: shRNA#1 TTCCAGACAACTATGAGAT and shRNA#2 TTTGGAAGAGGCGAGATAA. The β-catenin knockdown plasmid was constructed on the plko.1 plasmid. Target sequences for human β-catenin mRNA as followed: shRNA#1 GGTTAATAAGGCTGCAGTTAT and shRNA#2 GCTTATGGCAACCAAGAAAGC. Plasmids were transferred into HEK 293T cells by PEI to generate lentivirus. After 48 h, the supernatant was collected and added onto cells in culture supplemented with 8 μg/ml polybrene.For ectopic expression of TRIM26 and USP39, Containing the full-length trim26 fragment of pcmv-N-Myc plasmid (Myc-TRIM26) was constructed and sequenced. Then these plasmids were transferred into HCC cells via liposomes .Nuclear and cytoplasmic fractions from different cell populations were isolated using Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer’s instructions . Nuclear and cytoplasmic proteins were analyzed by western blotting. Tubulin and lamin B were used for normalization.Cells were lysed with RIPA lysis and Extraction buffer containing a protease inhibitor cocktail , Protein lysates(20 μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes . Membrane was blocked in 5% skim milk and incubated overnight with specific primary antibodies at 4 °C followed by incubation with secondary antibodies for 1 h at room temperature. Proteins on membranes were visualized using Electrochemiluminescence  and signals were detected with the ChemiDocTM MP Imaging System . The primary antibodies used in Western blotting are as follows: anti-USP39  , TRIM26  , non-phosphor (active) b-catenin   and β-actin  .c-myc (1:1000) (proteintech), cyclin D1(1:1000) (proteintech).Cells were cultured in six-well plates, and fixed with 4% paraformaldehyde for 10 min at room temperature. Gently wash the sample three times with PBS, then permeabilized with 0.2% Triton X-100  for 10 min on ice. Samples were blocked with 5% bovine serum albumin for 1 h, and incubated with primary antibody at 4 °C overnight. Primary antibody was detected with fluorescence-conjugated rabbit/mouse secondary antibody  for 1 h in dark conditions. Cell nuclei were stained with DAPI  for 10 min. Images were obtained under fluorescence microscope .Total RNA was isolated from cells using TRIzol reagent. . Isolated total RNA was quantified and transcribed into cDNA using a reverse transcription kit . Quantitative RT-PCR was used to measure mRNA expression levels of the tested genes. GAPH was used as a control, and the relative fold change of the target gene was calculated by the 2−ΔΔCq method. Primer sequence information for specific genes is listed in Supplementary Materials Table .HCC cell viability was detected using a cell proliferation assay kit . Cells were seeded into 96-well plates with or without ICG-001at a density of 5000 cells/well. At indicated time points, 20 μL of MTT reagent  was added to each well and then incubated for 4 h at 37 °C. The cell proliferation curves were determined by measuring optical density (OD) at a wavelength of 570 nm.HCC cells were resuspended in serum-free medium and seeded at a density of 50,000 per well in the upper chamber of a 24-well plate. Medium containing 20% serum was added to the lower chamber and ICG-001 reagents were added to both of upper and lower chamber. After the cells were incubated for 24 h, the remaining cells on the upper surface of the chamber were removed by cotton swabs. Cells that migrated to the lower surface of the chamber were fixed with 4% paraformaldehyde and stained with crystal violet. The chamber was observed and photographed under a microscope, and each experiment was repeated three times.Cells were seeded in 6-well plates at a density of 500 cells/well with ICG-001(yeasen). Cells were cultured in cell chambers for 2 weeks then cells were fixed with 4% paraformaldehyde and stained with crystal violet . Colony were counted and normalized to the control. Each experiment was repeated three times.Cells were seeded in 6-well plates and scratch was made by a 20 μl pipette tip after cells were grown to 80% confluence. Cells were then incubated in medium containing 2% FBS and ICG-100. Gap size was measured 24 h later. Cell migration was normalized to the control and each experiment was repeated three times.Cells were lysed by RIP lysate containing RNase (Roche) out and Protease inhibitor cocktail (Roche), and 5% cell lysate was used as input. The remaining cell lysate was incubated with protein A/protein G agarose beads overnight at 4 °C. The agarose beads were washed three times, and total RNA was extracted according to the method for cell total RNA extraction. Total RNA was reversed into cDNA, and mRNA levels were quantified by qRT-PCR.Immunoprecipitation experiments were performed according to previously established methods . Cells wE. coli BL21 and purified. Cell lysate was obtained by expressing HA-β-catenin in HEK 293T cells, and the cell lysate and purified GST-tagged protein were incubated at 4 °C for 4 h. Add SDS-loading buffer to the sample and boil for 5 min, then detect protein ubiquitination by western blotting.The GST-tagged plasmid was transformed into Cells were transfected with Flag/HA-Ubiquitin and indicated plasmids for in vivo ubiquitination assay, which was performed as previously described . In shorParaffin sections (4 μm thick) were first incubated in an oven at 60 °C for 2 h, then deparaffinized with xylene and rehydrated through gradient ethanol immersion. Boiled for antigen retrieval with 0.01 mol/L citrate buffer solution (pH 6.0) for 30 min. Endogenous peroxidase blocking reagent  was added for 10 min to block non-specific antibody binding. Sections were also blocked in 1% fetal calf serum for 10 min, and then incubated with the primary antibody in humidified chamber overnight at 4 °C, and treated with a streptavidin-peroxidase-conjugated second antibody  for 30 min at room temperature. The immune complexes were visualized with a Vectastatin DAB kit (Vector Laboratories). Staining intensity scoring was performed in a blinded manner by two uninvolved scientists.n = 6), shTRIM26 group (n = 6) and shTRIM26+shb-catenin group (n = 6). SK-hep-1 cells (1 × 106) stably transduced with shTRIM26 and shTRIM26+shβ-catenin were injected subcutaneously into the flanks of 6–8-week-old male BALB/c nude mice, respectively. Tumor length (a) and width (b) were measured with a vernier caliper every 3–4 days, and then tumor volume was calculated using the formula: 1/2ab2. The mice were sacrificed on the 30th day after injection, and tumors were isolated. Tumor tissues were stored at −80 °C and fixed with 4% paraformaldehyde for immunohistochemistry (IHC).The nude mouse xenograft model: 6–8-week-old male nude mice (18–20 g) were purchased from the National Model Animal Center of Nanjing University and maintained in a specific pathogen-free environment with free access to food and water and a 12-h light/dark cycle. The mice were randomly divided into a control group (n = 4), shTRIM26 group (n = 4), control+ICG-001 group (n = 4), shTRIM26+ICG-001 group (n = 4). 1 × 106 Sk-hep-1 cells were injected into nude mice of each group through the caudal vein, and the mice were sacrificed 40 days later to observe liver metastasis. No blinding was done for animal studies.For extrahepatic metastasis model: 6–8-week-old male nude mice (18–20 g) were purchased from the National Model Animal Center of Nanjing University and maintained in a specific pathogen-free environment with free access to food and water and a 12-h light/dark cycle. The mice were randomly divided into a control group . A P value of <0.05 was considered statistically significant. Asterisks  stand for P < 0.05, P < 0.01, P < 0.001 and P < 0.0001, respectively.Each experiment was repeated independently at least three times. Continuous variables were presented as mean and standard deviation (SD). Unpaired two-tailed student’s To elucidate whether USP39 regulate β-catenin expression, cell proliferation, cell cycle, and tumor growth in HCC cells, knockdown and overexpression of USP39, β-catenin inhibitor ICG-001 were performed in HCC cell lines (SK-hep-1 and PLC/PRF/5). The efficiency of knockdown and complementation of USP39 was confirmed by quantitative real‐time PCR (qRT‐PCR) Fig.  and westUSP39 has the function of deubiquitination and regulating RNA splicing, and data above has proved USP39 increasing HCC progression through Wnt/β-catenin pathway. However, it is still unclear which role USP39 plays in the process. To determine how USP39 regulates β-catenin, qRT-PCR analyses of β-catenin mRNA level was performed in hepatoma cells with knockdown or overexpression of USP39. We found depletion Fig.  or overeOn account of the fact that β-catenin protein level was decreased with no change on mRNA level, and USP39 is a deubiquitinating enzyme, we hypothesized whether USP39 de-ubiquitinate and stabilize β-catenin. To test this, we first proved that a combination between USP39 and β-catenin exists by immunoprecipitation in PLC/RCF/5 cells (Supplementary Fig. To further confirm whether the reduction of β-catenin is mediated through the ubiquitin-proteasome pathway, cells were treated with proteasome inhibitor MG132. As expected, downregulation of β-catenin was dramatically reversed in USP39 knockdown SK-hep-1 cells treated with MG132 Fig. . FurtherConsidering β-catenin was proved to be a substrate of de-ubiquitinase USP39, and our previous research showed that TRIM26 balances the regulation of USP39 to its substrate, thus we assumed TRIM26 plays the same role during USP39 stabilizing β-catenin. To prove this assumption, we generated USP39 knockdown, TRIM26 knockdown and double-knockdown cell lines separately. Results showed that knockdown of USP39 led to an obvious decrease of β-catenin in HCC cells, whereas USP39 and TRIM26 double-knockdown significantly replenished β-catenin levels compared with USP39 deficit only Fig. . FurtherAccording to our previous findings, TRIM26 reduced the proliferation and migration of HCC cells. Here, to clarify whether TRIM26 regulates HCC progression via β-catenin, we generated TRIM26 and β-catenin knockdown HCC cell lines, and the knockdown efficiency was confirmed by qRT-PCR and WB (Supplementary Fig. To examine the effect of ICG-001 on CRC metastasis, we executed extrahepatic metastasis model in nude mice. Results showed that mouse injected with TRIM26 knock down SK-hep-1 cells showed more severe liver metastasis compared with the control group. The ICG-001 treatment significantly relieved metastatic outgrowth to the liver in both TRIM26 knock down and control group Fig. . To asseOur previous study indicated that de-ubiquitinase USP39 and E3 ligase TRIM26 function in an antagonistic but not a competitive pattern, and play key roles in controlling ZEB1 stability to determine the HCC progression . HoweverIn this study we note that knockdown of USP39 induced an increase in TRIM26 protein level Fig. . To explUSP39 has been reported to be involved in pre-mRNA splicing and maturation , 34. BasWe first examined the effect of USP39 on the levels of TRIM26 mRNA by using qRT-PCR, discovering that knockdown of USP39 increased transcript level of TRIM26. Fig. . Using tSince TRIM26 possesses ubiquitin ligase activity , 36, we To examine whether TRIM26 affects the stability of β-catenin, CHX was performed to inhibit the synthesis of new proteins. Results showed that knockdown of TRIM26 prolonged the half-life of β-catenin Fig. . ConsistCurrent studies on cancer have shown that ubiquitination-deubiquitination signaling pathway plays a key role in the regulation of cell homeostasis, especially in the occurrence and development of tumors, and its dysregulation will lead to tumor deterioration . In the Evidence showed that dysregulation of Wnt signaling contributes to the progression of hepatocellular carcinoma . Liu et As an important regulator of RNA splicing, USP39 participated in mRNA splicing in the process of cell proliferation . In addiAlthough our previous study found that knockdown of USP39 could increase the protein level of TRIM26, no further exploration was performed to the mechanism of USP39 regulating TRIM26. In this study, our RNA sequencing data of USP39 overexpression in HCT116 cells revealed a significant difference in alternative splicing events in exon 4 of TRIM26 compared to controls. We confirmed that USP39 deletion increased the alternate splicing transcript of TRIM26 without significant changes in its constitutive splicing. More importantly, RNA immunoprecipitation showed that USP39 is bound to TRIM26 mRNA. We demonstrated for the first time that USP39 regulates TRIM26 pre-mRNA splicing and maturation, and the decrease in TRIM26 protein level caused by USP39, was at least in part by inhibiting the splicing of its pre-mRNA.Considering the relationship between USP39 and TRIM26 in our previous study, we also wondered whether USP39 and TRIM26 could balance the ubiquitination level of β-catenin. Double knockdown of TRIM26 and USP39 eliminated the reduction of β-catenin caused by USP39 silencing. According to our previous data, knockdown of TRIM26 promoted HCC proliferation, colony formation, migration, and invasion. We double knockdown TRIM26 and β-catenin or ICG-001 treated in HCC cells, detecting that β-catenin knockdown and ICG-001 knockdown reduced the proliferation, colony formation, migration and invasion of HCC compared to TRIM26 knockdown only. Based on the above studies, we demonstrated that TRIM26 was partially involved in the regulation of β-catenin by USP39 and involved in the progression of HCC.Because of the E3 ubiquitin ligase activity of TRIM26, we considered TRIM26 has the function of promoting β-catenin ubiquitination and degradation. Immunofluorescence assay showed co-localization of TRIM26 and β-catenin, and immunoprecipitation assay confirmed that TRIM26 is directly bound to β-catenin. Finally, by immunoprecipitation and GST pull-down assay, we demonstrated that TRIM26 ubiquitinates β-catenin in HCC cells.Taken together, our experimental data support the mechanism by which USP39 regulates HCC cell proliferation and migration through the TRIM26/β-catenin axis. The elucidation of this mechanism will provide new clues for the development of hepatocellular carcinoma drugs.supplementary figure 1supplementary figure 2supplementary figure 3supplementary figure 4supplementary figure 5supplementary figure legendsSupplementary Table 1Original Data Fileaj-checklist


The aim of the current study is to investigate the impact of introducing micro- and nanoparticle MgO as a filler into epoxy resin on the radiation shielding abilities of the prepared samples. To this end, we performed a gamma-radiation spectroscopy experiment with the help of an HPGe detector and Am-241, Cs-137, and Co-60 sources. We evaluated the particle size effect (PSE) and detected the maximum PSE value with the addition of 50 wt% MgO particles, indicating that nanoparticle MgO was more successful in shielding against incoming radiation than microparticle MgO. We compared the half-value layer (HVL) for the samples with 10 wt%, 20 wt%, and 30 wt % micro-MgO and nano-MgO and found that the HVL values were lower for the nanoparticle samples than for the microparticles samples, confirming that smaller particle sizes enhanced the shielding ability of the samples against radiation. The MFP results showed that epoxy matrices containing micro-MgO, for all investigated energies, resulted in higher MFP values that those containing nano-MgO. With the continued use of radiation technology, it is important to contain radiation and prevent it from coming into contact with humans and the environment to the greatest extent possible. Long-term exposure to ionizing radiation can lead to serious health hazards, which is why radiation shields are often used to prevent any harmful effects ,2,3,4. REpoxy resins are a good candidate for radiation shields owing to their low cost, high strength, low toxicity, resistance to corrosion, excellent mechanical properties, and low shrinkage. ,10,11 Th2O3 epoxy and Fe2O3 epoxy samples against high-energy gamma rays. These additives were chosen because of their relatively high atomic numbers, resulting in an improvement in the attenuation abilities of the composites with increasing filler content. Li, R. et al. [3O4, which was then added to epoxy resin and tested. Sahin, N. et al. [2O3, SiO2, and Al2O3 in the stone substantially enhanced the shielding abilities of the resulting composites. Additionally, many studies have centered around the effects of introducing tungsten oxide, by itself and with other fillers, into epoxy resin and investigating the resulting effects on the properties of the composite. In 2015, Chang et al. examined the effects of the amalgamation of tungsten elements on an epoxy matrix to identify the radiation shielding and mechanical features of epoxy resin. The radiation shielding properties of tungsten/epoxy composites increase with the increment of incorporated tungsten filler. An increase in the quantity of tungsten elements in the epoxy matrix amplifies the radiation protection ability of the tungsten/epoxy matrix [3 on epoxy composites was studied by Azman et al. in 2013, within the energy range of 10 KeV–100 KeV. The inclusion of nano-sized WO3 in epoxy composites resulted in an increased reduction capability compared to the inclusion of micro-sized WO3 in the energy range of 10 KeV–25 KeV. The size of WO3 in the epoxy matrix was associated with only negligible differences with respect to the shielding ability at higher energies in the range of 20 KeV–40 KeV. The inclusion of WO3 in the epoxy matrix enhanced the mechanical properties of the composite up to a certain concentration, with a negative effect on the mechanical properties associated with further addition of WO3 beyond that specific level [2O3/epoxy, and Fe2O3/epoxy at energies of 0.662, 1.173, and 1.333 MeV. The gamma reduction ability of epoxy resins has been reported to increase with increased concentrations of the studied filler [2O3- and -WO3-reinforced composites in an attempt to combine previous research on Bi2O3 epoxy composites with WO3 epoxy composites. After experimentally testing the composites, they found that the shields with high amounts of Bi2O3 and WO3 were highly effective against low-energy photons, which are typically found in medicinal applications. The nano-WO3 composites also had an advantage over their microparticle counterparts; however, this advantage was mostly present at lower energies. Azman, N. et al. [2 microparticles treated with a coupling agent into an epoxy matrix. The WB2 epoxy samples were found to be better at absorbing gamma radiation than the WB samples.Many approaches have been taken to further understand the properties of modified epoxy composites against ionizing radiation. Chang, L. et al.  studied . et al.  blended . et al.  also use. et al.  used a Yy matrix . The effic level . In 2021d filler . Karabul. et al.  introducIn recent years, nanoparticle fillers have been increasingly tested, as opposed to previously used microparticle fillers, due to their high concentration of particles per gram. A smaller particle size leads to a more uniform distribution in the resin and an increased surface area to interact with, which often correlates with an improved shielding ability. This enhancement is especially noticeable at lower energies, where nanoparticle fillers tend to have a considerable advantage over their microparticle counterparts ,24. The The traditional casting method was used to form new samples to study the attenuation coefficients and their efficiency against photons. These samples consisted of a mixture of epoxy and magnesium oxide (MgO). A transparent epoxy resin and its hardener were selected, and micro and nano-sized MgO particles were used as filler. The raw materials were purchased from local stores. The purity of the micro-MgO particles was 97.8, with an average particle size of 60 ± 4 μm. MgO nanoparticles were purchased from Nano Tech company with a purity of 99.8 %, and according to TEM imaging, the average particle size was 20 ± 5 nm. The characteristics of micro- and nano-MgO were studied in . The epoSamples were prepared according to the percentages mentioned in t) was placed, and the count rate (N) was calculated; then, the sample was removed under the same conditions and arrangements, and the free count rate (N0) was calculated. The LAC was calculated experimentally using the following formula [To experimentally measure the linear attenuation factor, HPGe detector and three radioactive point sources were used to obtain four energies covering low, medium, and high regions ,27. The  formula ,30.(1)LLAC results and calculated according to protocols described in previous works [The experimental results of epoxy samples reinforced with MgO microparticles were compared with the XCOM results . The masus works ,34,35,36LAC) values, as well as those simulated by XCOM software for E-mMgO10, E-mMgO20, E-mMgO30, E-mMgO40, and E-mMgO50 epoxy samples. The aim of this step was to validate the experimental setup. The value of R2 for all studied epoxy matrix samples was between 0.9971–0.9998, which is very close to 1, validating the precision of the experimental setup utilized in this study.E-mMgO10, E-mMgO20, E-mMgO30, E-mMgO40, and E-mMgO50 epoxy matrix samples were prepared with 10, 20, 30, 40, and 50 wt% micro-MgO as filler material, respectively, to evaluate the effect on shielding properties against radiation. In this study, the experimental LAC values and the theoretical XCOM values for the micro-MgO epoxy samples were evaluated and compared. In order to elucidate the impact of the particle size of the filler material on the epoxy matrix with respect to radiation attenuation, the PSE factor was determined according to Equation (2), utilizing the same amount of nano- and microparticles:E-mMgO10, E-nMgO10, E-mMgO20, E-nMgO20, E-mMgO30, and E-nMgO30 epoxy matrix samples were prepared by adding 10 wt%, 20 wt%, and 30 wt% of micro-MgO and nano-MgO, respectively, to the epoxy matrix. At any incident photon energy level, a PSE value of more than one indicates that the inclusion of nanoparticles provides better shielding efficiency than the inclusion of the same amount of microparticles. E-mMgO10, E-nMgO10, E-mMgO20, E-nMgO20, E-mMgO30, and E-nMgO30 epoxy matrix samples containing 10 wt%, 20 wt%, and 30 % micro-MgO and nano-MgO as filler were prepared to judge their shielding properties against radiation. We evaluated the impact of micro-MgO and nano-MgO on the epoxy matrix HVL; results are presented in LAC) values with those simulated by XCOM software to validate the experimental setup. Without changing the quantity of filler MgO filler particles, we observed PSE values > 1 at photon energies of 0.060 MeV, 0.662 MeV, 1.173 MeV, and 1.332 MeV, indicating that epoxy resins with nano-MgO filler had better defensive capabilities against radiation than epoxy resins with micro-MgO as filler. The nano-MgO epoxy samples had lower HVL values than the micro-MgO epoxy samples, indicating that a smaller particle size of the filler enhances the radiation defensive quality of the resulting epoxy composite. Moreover, epoxy matrices containing lower concentrations of MgO filler had lower MFP values, with lower MFP values associated with epoxy matrix samples containing nano-MgO filler as opposed to those containing micro-MgO. Therefore, an increase in the concentration of MgO filler enhances the defensive capacity of the epoxy matrix against incident radiation, and nano-sized additives enhance the shielding ability the matrix more than micro-sizes additives.In this study, we tested six samples consisting of a mixture of epoxy and magnesium oxide (MgO) to assess the radiation-shielding ability of the samples against incident photons. We also evaluated the effect of size variation of MgO particles included in the epoxy resin. We combined concentrations of 10, 20, 30, 40, and 50 wt% micro-MgO with epoxy resin, as well as concentrations of 10 wt%, 20 wt%, and 30 wt% nano-MgO with epoxy resin. We compared experimentally obtained linear attenuation coefficient (


Pulmonary fibrosis, a chronic lung disease caused by progressive deterioration of lung tissue, is generated by several factors including genetic and environmental ones. In response to long-term exposure to environmental stimuli, aberrant tissue repair and epithelial cell-to- mesenchymal cell transition (EMT) trigger the subsequent progression of pulmonary fibrotic diseases. The Aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by ligands providing lung dysfunction when activated by environmental toxins, such as polycyclic aromatic hydrocarbons. Our previous study demonstrated that AhR mediates α-SMA expression by directly binding to the α-SMA (fibroblast differentiation marker) promoter, suggesting the role of AhR in mediating fibrogenic progression. Here we follow the hypothesis that macrophage infiltrated microenvironments may trigger inflammation and subsequent fibrosis. We studied the expression of cytokines in RAW 264.7 cells by AhR activation through an ELISA assay. To investigate molecular events, migration, western blotting and zymography assays were carried out. We found that AhR agonists such as TCDD, IP and FICZ, promote the migration and induce inflammatory mediators such as TNF-α and G-CSF, MIP-1α, MIP-1β and MIP-2. These cytokines arbitrate EMT marker expression such as E-cadherin, fibronectin, and vimentin in pulmonary epithelial cells. Expression of proteins of MMPs in mouse macrophages was determined by zymography, showing the caseinolytic activity of MMP-1 and the gelatinolytic action of MMP-2 and MMP-9. Taken together, the present study showed that AhR activated macrophages create an inflammatory microenvironment which favours the fibrotic progression of pulmonary epithelial cells. Such production of inflammatory factors was accomplished by affecting the Wnt/β-catenin signalling pathway, thereby creating a microenvironment which enhances the epithelial-mesenchymal transition, leading to fibrosis of the lung. Pulmonary fibrosis, a chronic lung disorder , is indiThe activation of myofibroblasts and their differentiation is one of the crucial steps in the progression of fibrosis . InflammMacrophages are found in most tissues of the body and play important roles in both acute and chronic pulmonary pathologies such as cytotoxicity and fibrosis . They bep-dioxins such as the compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)  and 37 °C overnight incubation to permit the substrate’s digestion of the proteinases. Following this, the gel was stained using 0.5% Coomassie brilliant blue R-250 (40% methanol and 10% acetic acid) for 30 min. A destaining was performed with 40% methanol and 10% acetic acid. The appearance of bands of clear lysis against the dark background indicated proteolytic activity. The bands were quantified using Image J software.The gelatinolytic and caseinolytic activities of macrophage cultures were determined by performing zymography following a similar method as previously described . 1 × 1055 cells/well were seeded in a culture medium at a confluence of 90% and treated with AhR agonists and antagonists as specified above and using a PBS washing process. Afterwards, a RIPA cell lysis buffer was used for cell lysis. A cytoplasmic and nuclear extraction kit was used for cytosolic and nuclear protein extraction . The concentration of protein was determined using a BCA protein assay kit . Using 10–12 percent SDS-PAGE, equivalent amounts of protein were separated. After this, the material was transferred to a nitrocellulose membrane (Amersham Biosciences). TBST-Tris buffered saline-tween20 , comprising 5% non-fat milk, was used to block the membrane. BSA/TBST (1%) was used as a diluent for primary antibody incubation. It was then incubated in 5% non-fat milk HRP-conjugated secondary antibodies. The image was developed through chemoluminescence [In 60 mm plates, MLE-12 cells amounting 5 × 10nescence  and was p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.The values are based on the results of at least three separate studies in each condition ± SEM. One-way analysis of variance (one way ANOVA) was used to analyze the data. Differences were deemed as significant when: * The cell signals and the inflammatory response are generally mediated by a number of cytokines . PreviouThe observation that TCDD induced the expression of cytokines and chemokines upon activation of AhR let us speculate whether cytokines would subsequently be induced by other AhR ligands such as IP and FICZ. To prove this, the effects of AhR agonists were evaluated using ELISA assays, which quantified the produced cytokines individually. The effects of AhR agonists on the secretion of cytokines G-CSF, TNF-α, MIP-1α, MIP-1, MIP-2 in RAW 264.7 cells were analyzed by ELISA assay. As illustrated in In the case of the macrophage inflammatory proteins, all three AhR agonists were found to be significantly increased, but a comparison among them provided varied results. As shown in −8 M), IP (10−7 M), and FICZ (10−7 M). MLE-12 cells were seeded for 10–12 h and were treated with RAW 264.7 conditioned medium for 24 h . A general marker for EMT differentiation is cell migration . To detefor 24 h . As showMMP activity is central to ECM remodeling injury . The firAn additional clear band of proteolytic activity at 43 kDa was identified as MMP-1 in a casein-gel zymography b. HoweveEpithelial-to-mesenchymal transition (EMT) is defined as the process where cells lack their cell-to-cell adhesion and polarity and turn into cells with mesenchymal properties . They thβ-catenin is an important factor for cell-to cell adhesion, where it operates as a potent linkage amid E-cadherin and the cytoplasmic actin cytoskeleton . It is tPolycyclic aromatic hydrocarbon (PAH) exposure is known to promote lung cancer development via AhR signalling . The funOur data showed that cytokine production by macrophages through AhR-activation, in part, induces pulmonary epithelial cell migration and differentiation. The present study pursued how AhR agonist treatment affected the expression of molecules involved in ECM metabolism during lung fibrosis. Consistent with the literature ,16, our In the present study, all three AhR agonists, TCDD, IP and FICZ, induced the release of the cytokines TNF-α, MIP-1α, MIP-1β, and MIP-2. IP was found to markedly enhance the level of cytokines when compared to other agonists. Our hypothesis is supported by these results. Accumulating evidence revealed that macrophages enhance the migration and cytokine release and therefore, lead to pulmonary fibrosis. Recent studies indicated that macrophages boost the advancement of pulmonary fibrosis ,38. SincA multitude of cytokines and growth hormones, such as IL-1 and TNF-α, affect MMP transcription . A recenEMT is required for embryogenesis, chronic inflammation, and tumor progression, as well as wound healing. Abnormal EMT can lead to fibrosis. The cells changed during this phase, affecting their physiological functions, migration and adhesion in concert with basal and neighbouring cells . The enh+2 route, which is where protein kinase C (PKC) is initiated. The activation of the canonical pathway, namely β-catenin/TCF-dependent pathway is heavily reliant on β-catenin [Cadherins are receptors for transmembrane adhesion that facilitate homophilic associations by establishing adherent bindings in between either one or more domains of the immunoglobulin in their extracellular area. They also link to actin microfilaments in an indirect manner in the cytoplasm through α and β-catenin. They encourage the creation of adherens junctions and the formation of permanent cell-cell interactions . The cha-catenin ,52. The -catenin . Results-catenin . However-catenin . Accordi-catenin .A few studies also suggest that unusual Wnt-β-catenin signalling pathway activation happened to be critical for regulating the pulmonary fibrogenesis process and to cause fibroblasts and mesenchymal stem cells of lungs to transform into myofibroblasts initiating lung fibrosis by increasing collagen fiber secretion and ECM deposition. It was discovered that in bronchiolar lesions, β-catenin nuclearizes abnormally increased EMT and promoted pulmonary fibrogenesis in injured lungs ,48,54,55Our study discovered that treatment with AhR agonists stimulated macrophage cells and induced cytokine production and ECM degradation. We confirmed that pro-inflammatory cytokines released by the macrophage cell initiate the EMT process and macrophages were important for the progression of pulmonary fibrosis relying on AhR agonists. The present study used a cytokine array to discover cytokines that were differently expressed after AhR agonist treatment in macrophage cells to investigate mechanisms of EMT and pulmonary fibrotic progression. Remarkable upregulations of TNF-α, G-CSF, MIP-1α, MIP-1β and MIP-2  were demThe present study signifies that β-catenin in Wnt/β-catenin signalling is responsible for the pathogenesis of pulmonary fibrosis and fibrotic disease progression. Collectively, our study explored EMT progression induced by pro-inflammatory cytokines released by macrophages upon AhR activation and the role of Wnt/β-catenin in pulmonary fibrosis pathogenesis. Whether there is a cross-talk between MMP signalling and the machineries of the Wnt-β-catenin pathway along the AhR axis remains to be solved.The activation of the Aryl hydrocarbon receptor induces pro-inflammatory cytokines, which along with potential AhR agonists are responsible for creating a microenvironment that promotes the migration of macrophages and mediates the expression of EMT biomarkers. In particular, we found that macrophages promote EMT through activating Wnt/β-catenin signalling. In order to identify the mechanisms or the pathways involved in the activation of Wnt/β-catenin signalling, further research should develop strategies for their identification. Given the plethora of AhR ligands and its cell type-specific regulatory functions, the role of AhR in the generation of fibrosis could be through different mechanisms. Reporter gene assays could be used to look for other possible pathways that might be involved.


Bronchopulmonary dysplasia is a serious and lifelong pulmonary disease in premature neonates that influences around one-quarter of premature newborns. The wingless-related integration site /β-catenin signaling pathway, which is abnormally activated in the lungs with pulmonary fibrosis, affects cell differentiation and lung development.Newborn rats were subjected to hyperoxia exposure. Histopathological changes to the lungs were evaluated through immunohistochemistry, and the activation of disheveled and Wnt /β-catenin signaling pathway components was assessed by Western blotting and real-time PCR. The abilities of proliferation, apoptosis and migration were detected by Cell Counting Kit-8, flow cytometry and scratch wound assay, respectively.P < 0.05; 14th day: P < 0.01) and malondialdehyde significantly increased  after hyperoxia exposure. Protein and mRNA expression levels of β-catenin, Dvl-1, CTNNBL1 and cyclin D1 were significantly upregulated by hyperoxia exposure on 7th day (P < 0.01) and 14th day (P < 0.01). In hyperoxic conditions, Dvl-l downregulation and Dvl-l downregulation + MSAB treatment significantly increased the proliferation rates, decreased the apoptosis rates and improved the ability of cell migration. In hyperoxic conditions, Dvl-l downregulation could decrease the mRNA expression levels of GSK3β, β-catenin, CTNNBL1 and cyclin D1 and decrease the protein relative expression levels of GSK3β, p-GSK3β, β-catenin, CTNNBL1 and cyclin D1.Contrasting with normoxic lungs, hyperoxia-exposed lungs demonstrated larger alveoli, fewer alveoli and thicker alveolar septa. Superoxide dismutase activity was significantly decreased (7th day: We confirmed the positive role of Dvl-1 and the Wnt/β-catenin signaling pathway in promoting BPD in hyperoxia conditions and provided a promising therapeutic target.The online version contains supplementary material available at 10.1186/s12860-023-00465-6. Bronchopulmonary dysplasia (BPD) is a serious and lifelong pulmonary disease in premature neonates that impacts a quarter of premature newborns –3 . Pret2+ signaling pathway [Current research shows that the Wnt/β-catenin signaling pathway can regulate the normal development of multiple organs in the body. It is important that the discovery of its molecular mechanism in the lung, using targeted therapy to treat BPD without causing damage to other organs. Dishevelled-1 (Dvl-1) had been reported to mediate three signaling pathways, including the canonical Wnt/β-catenin signaling pathway, the non-canonical Wnt/β-catenin signaling pathway and Wnt/Ca pathway . In the  pathway . Recent  pathway , 13. In  pathway . The Wnt pathway , 16.In this study, we explore the role of Dvl-1 and the Wnt/β-catenin signaling pathway in the occurrence and development of BPD. It is demonstrated that the downregulation of Dvl-l relieves hyperoxia-induced AECII apoptosis by inhibiting the Wnt/β-catenin signalling pathway. Our study provides a new perspective and theoretical basis for the clinical treatment of hyperoxia-induced lung injury through Dvl-1. It is helpful to supple treatment of hyperoxia lung injury except supportive therapy currently.In the present study, rats exposed to 85% O2 had inhibited alveolar development, which was correlated with the pulmonary morphological changes of BPD Fig. . A minimP < 0.01) and were also significantly higher on the 14th day than those on the 7th day(P < 0.01)  and MDA was significantly increased  after hyperoxia exposure as shown in Table In the hyperoxia group, IHC scores of β-catenin, Dvl-1, and cyclin D1 were significantly higher than those of the control group on the 7th day (P < 0.01), but there was no significant difference after hyperoxia exposure on the 3rd day. The protein expression levels of β-catenin, CTNNBL1 and cyclin D1 were significantly upregulated after hyperoxia exposure on the 3rd day(P < 0.01), the 7th day(P < 0.01) and the 14th day(P < 0.01) .As shown in Fig. P < 0. 01) and on the 14th day (P < 0. 01).The results of the real-time PCR analysis indicated that the mRNA expression levels of β-catenin, Dvl-1, CTNNBL1 and cyclin D1 were significantly upregulated by hyperoxia exposure on the 7th day (P < 0.01) but there was no significant difference in Dvl-l downregulation between the hyperoxia condition group and the control group (P > 0.05)  Fig. .Fig. 3DvP < 0.01). After 24 h of hyperoxia exposure, Dvl-l downregulation and MSAB treatment improved ACEII cell migration (P < 0.05), which indicated that the combined treatment of Dvl-l downregulation and MSAB relieved hyperoxia-induced inhibition of ACEII cell migration  which suggested that hyperoxia could induce ACEII cells apoptosis. However, a reverse effect occurred in the si-Dvl-l, si-Dvl-l + hyperoxia and si-Dvl-l + hyperoxia + MSAB groups, in which the cell apoptosis rates were significantly lower than they were in the hyperoxia group (P < 0.01). Although the apoptosis rate of the si-Dvl-l + hyperoxia group was lower than that of the si-Dvl-l + hyperoxia + MSAB group, there was no significant difference(P > 0.05). Our results indicated that Dvl-l downregulation inhibited hyperoxia-induced apoptosis  and the early apoptosis rate (Q3). After transient transfection and subsequent culture for 24 h, the apoptosis rate of the hyperoxia group was significantly higher than that of the control group (ceP > 0.0. Our resP < 0.01). Compared with the hyperoxia group, the mRNA relative expression levels of GSK3β, β-catenin, CTNNBL1, and cyclin D1 in the si-Dvl-l + hyperoxia and si-Dvl-l + hyperoxia + MSAB groups were significantly deceased . We also assessed the effect of Dvl-l downregulation on the above protein expressions in hyperoxia conditions. Dvl-l downregulation could decrease the protein expression levels of GSK3β, p-GSK3β, β-catenin, CTNNBL1 and cyclin D1 (P < 0.01). Interestingly, MSAB, a Wnt/β-catenin signaling pathway inhibitor, enhanced the inhibition effect of Dvl-l downregulation on the protein expression levels of GSK3β, p-GSK3β, β-catenin, CTNNBL1 and cyclin D1. These results of real-time PCR and Western blotting analysis collaboratively suggested that Dvl-l downregulation could inhibit hyperoxia-induced Wnt/β-catenin signaling pathway activation in ACEII cells -conjugated secondary antibody and RIPA lysis buffer ; BCA protein and, StarSignal chemiluminescent assay kits ; StarScript II First-strand cDNA Synthesis Mix with gDNA Remover, 2 × RealStar Green Fast Mixture (with ROXII), and TRIGene ; polyvinylidene difluoride (PVDF) membranes ; GSK3β, p-GSK3β, cyclin D1, Dvl-1, and β-catenin primary antibodies ; GAPDH primary antibody ; PCR primers ; MDA and SOD assay kits ; immunohistochemistry kit , rat AECII cells ; Roswell Park Memorial Institute (RPMI) 1640, trypsin, penicillin, and streptomycin ; fetal bovine serum ; lipofectamine 3000 ; AnnexinV-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit and Cell Counting Kit-8 ; and MSAB .2 from the beginning of birth) and the control group . The number of rats used in each group is shown in Table All animal experimental procedures were approved by the Ethics Committee of Animalsof the Affiliated Wuxi Children’s Hospital of Nanjing Medical University. Sprague Dawley (SD) rats were obtained from the Nanjing Agriculture University. A total of 36 newborn SD rats were randomly selected into two groups, hyperoxia group , and the supernatant was collected for assays. MDA and SOD assays were performed using assay kits, according to their manufacturers’ instructions.Total protein was separated from lung tissues with radio immunoprecipitation assay (RIPA), and quantified with a bicinchoninic acid assay (BCA) protein assay kit. After dilution with loading buffer, the separated proteins were boiled for 5 min. Twenty micrograms of protein samples were separated using 12.5% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes. The membranes were probed overnight at 4 °C with primary antibodies against Dvl-1 (1:1000 dilution), β-catenin (1:1000 dilution), GSK3β (1:1000 dilution), p-GSK3β (1:1000 dilution), and CTNNBL1 (1:1000 dilution) and cyclin D1 (1:1000 dilution) and GAPDH (1:1000 dilution) proteins and subsequently hybridized with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The protein bands were detected using an ECL advanced system (Millipore) and quantified using Photoshop software.2O2 to block endogenous peroxidase activity. The treated sections were placed into an EDTA-Tris buffer solution and microwaved for 20 min, blocked with serum, and incubated overnight at 4°Cwith Dvl-1 primary antibody (1:200 dilution), β-catenin primary antibody (1:200 dilution) and cyclin D1 primary antibody (1:200 dilution). After sequential incubation with a biotin-labeled secondary antibody and streptavidin-peroxidase, sections were developed using 3,3′-diaminobenzidine (DAB) and dehydrated. IHC score was determined semi-quantitatively by multiplication of the positive fraction with the grayscale value according to the following system: a) positive fraction was categorized as 0 (no staining), 1+ (≤10%), 2+ (>10% and <50%), and 3+ (≥50%); b) intensity was graded as 0 (no staining), 1 (low staining), 2 (medium staining), and 3 (strong staining).The lungs were fixed with 4% formalin for 24 h at room temperature. The fixed lungs were sequentially dehydrated, vitrified, embedded in paraffin, fixed, cut into 5 μM thick sections. The sections were dewaxed with dimethylbenzene, hydrated with gradient alcohol according to the manufacturer’s instructions and treated with 3% HTotal RNA was extracted using TRIGene reagent according to the manufacturer’s instructions. The purified mRNA was reverse transcribed into cDNA using PrimeScript™ RT reagent kit with gDNA Eraser and real-time PCR was performed using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus). Data were standardized to the endogenous expression of GAPDH. The sequences of the primers are listed in Table 2 atmosphere. The cell culture medium included 10%(v/v) FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. AECII cells were divided into the following five group: AECII cells group (control group), AECII cells hyperoxia exposure group (hyperoxia group), AECII cells Dvl-l downregulation treatment group (si-Dvl-l group), AECII cells Dvl-l downregulation + hyperoxia exposure group (si-Dvl-l + hyperoxia group), and AECII cells Dvl-l downregulation + hyperoxia exposure + 5 μM MSAB treatment group (si-Dvl-l + hyperoxia + MSAB group). When reaching 70% confluency, the transfected AECII cells were treated with lipo3000 and Dvl-l siRNA oligonucleotides and then cultured in serum-free RPMI-1640 medium for 6 h. The cell culture plate was then placed into a sterile modular incubator chamber  and exposed to hyperoxia (85% O2 and 5% CO2) for 24 h.Rat AECII cells were cultured in RPMI-1640 medium at 37 °C in a 5% CO5 cells/mL), after respectively different treatments, were cultured for 24 h, and then prepared as cell suspensions using culture medium. The cell suspension (100 μL) was inoculated into 96-well plates and cultured for 24 h. The solution (10 μL) was added to each well and incubated for 2 h at 37 °C. The absorbance of each well was measured by microplate reader  at a wavelength of 450 nm. The data analysis was reported in a previous study [The proliferation rates of AECII cells in the five groups were detected using CCK-8 assay. AECII cells . A P value < 0.05 was considered statistically significant.The experimental data were expressed as mean ± standard deviation values. Statistical analysis was conducted by SPSS 21.0 software . The unpaired Student’s t-test was used to compare differences between the two groups. A Additional file 1.Additional file 2.


Type-II diabetes mellitus (T2DM) is a major risk factor for cognitive impairment. Protecting the brain environment against inflammation, and neurodegeneration, as well as preservation of the BBB veracity through modulating the crosstalk between insulin/AKT/GSK-3β and Wnt/β-catenin signaling, might introduce novel therapeutic targets.This study aimed at exploring the possible neuroprotective potential of vitamin D3 (VitD) and/or rosuvastatin (RSV) in T2DM-induced cognitive deficits.T2DM was induced by a high-fat sucrose diet and a single streptozotocin (STZ) dose. Diabetic rats were allocated into a diabetic control and three groups treated with RSV , VitD , or their combination.Administration of VitD and/or RSV mitigated T2DM-induced metabolic abnormalities and restored the balance between the anti-inflammatory, IL 27 and the proinflammatory, IL 23 levels in the hippocampus. In addition, they markedly activated both the canonical and noncanonical Wnt/β-catenin cassettes with stimulation of their downstream molecular targets. VitD and/or RSV upregulated insulin and α7 nicotinic acetylcholine (α7nACh) receptors gene expression, as well as blood-brain barrier integrity markers including Annexin A1, claudin 3, and VE-cadherin. Also, they obliterated hippocampal ApoE-4 content, Tau hyperphosphorylation, and Aβ deposition. These biochemical changes were reflected as improved behavioral performance in Morris water maze and novel object recognition tests and restored hippocampal histological profile.The current findings have accentuated the neuroprotective potential of VitD and RSV and provide new incentives to expand their use in T2DM-induced cognitive and memory decline. This study also suggests a superior benefit of combining both treatments over either drug alone. Type-II diabetes mellitus (T2DM) is a major risk factor for cognitive impairment –3. InsulAt the molecular level, impaired insulin signaling may promote amyloid-β (Aβ) deposition and Tau hyperphosphorylation via brain insulin resistance, which disturbs insulin signaling at the blood-brain barrier (BBB) level , 12 throThe canonical pathway is activated when the Wnt-5a ligand binds to its receptor thus phosphorylating β-catenin at serine (S) 675. As a consequence, β-catenin accumulates in the cytosol and subsequently translocates to the nucleus where it promotes Wnt target genes expression . ConversEmerging evidence also suggests that blood-brain barrier (BBB) integrity is crucial in the pathology of neurodegeneration and cognitive impairment. BBB disruption resulting from multiple neuroinflammatory events that interrupt tight junctions is a marked feature of cognitive defects . Thus, pRosuvastatin (RSV) is an HMG-CoA reductase inhibitor used in the management of dyslipidemia . Lowerin3 (VitD), a well-known secosteroid hormone, exerts both genomic and non-genomic actions; these actions cooperate by crosstalk between several signaling pathways. It has been increasingly implicated in the pathophysiology and the progression of many neurological diseases [Vitamin Ddiseases  includindiseases  and ischdiseases . Currentdiseases  and thatdiseases , 32. In diseases , 33, 34.To this end, the present study aims at investigating the possible benefits of VitD and/or RSV in rats in T2DM-induced cognitive and memory loss. Additionally, this work addresses the potential modulatory role of the crosstalk between insulin and Wnt/β-Catenin cassettes, and their downstream targets in the observed beneficial outcomes.ad libitum. They were exposed for 12 h light/dark cycle and controlled temperature (25±5֯C). The study protocol was approved by the Research Ethics Committee of the Faculty of Pharmacy, Cairo University, Cairo, Egypt (PT-2310) and the Faculty of Pharmacy  along the lines of the Guide for the Care and Use of Laboratory Animals  [Adult male Sprague Dawley rats (150–180 g) were purchased from the breeding colony of the National Institute of Research . Rats were kept under standardized laboratory conditions with food and water R, 2001) .Streptozotocin (STZ) and RSV were purchased from Sigma-Aldrich Co., St. Louis, MO, USA; VitD was obtained from Medical Union Pharmaceuticals Co., Cairo, Egypt; cholesterol and long-acting human insulin (Monotard) were obtained from Middle East Co., Cairo, Egypt, and Eli Lilly Co., USA, respectively. Sucrose and lard were obtained from commercial sources and were of the highest analytical grade.Cai et al [th week, a single sub-diabetogenic dose of STZ  dissolved in 0.09 M citrate buffer solution (pH 4.8), was given after an overnight fast. Animals were then maintained on a 5% glucose solution for 24 h. A normal-control  group was kept on a conventional pellet diet and water ad libitum was run concomitantly. The T2DM model was considered successful when the random blood sugar level was above 200 mg/dl at the beginning of the 7th week [Forty rats (approximately 5–6 weeks in age) were fed a high-fat sucrose diet (HFSD) for 11 weeks, according to the method of ai et al , with sl7th week .After establishing the model (week 7), HFSD-fed animals were randomly allocated into four groups (10 rats/ each); T2DM, T2DM + VitD, T2DM + RSV, and T2DM + VitD + RSV. Then, the rats were treated daily for 5 weeks (weeks 7–11) with the drugs along with HFSD. The dose of VitD was 500 IU/kg/day; PO , while tth week, all animals were subjected to the novel object recognition and Morris water maze tests to assess learning ability, and cognitive and memory impairment.At the beginning of the 11NORT is used to assess long-term memory and cognition . It consMWMT assesses spatial learning . It is a®, Egypt). Low-density lipoprotein cholesterol (LDL-C) was calculated according to the Friedewald equation: TC−(HDL cholesterol+1/5 TGs) [®, USA) and , respectively. Homeostasis model assessment for insulin resistance (HOMA-IR) was estimated according to the following equation:After the last dose of the drugs, animals were fasted for 12 h, anesthetized with thiopental  and blood samples were collected from the heart following chest opening. Serum was separated by centrifugation at 3000 rpm for the estimation of glucose, total cholesterol (TC), triglycerides (TGs), and high-density lipoprotein cholesterol (HDL-C) using colorimetric assay kits (SPECTRUM1/5 TGs) . Free fa/ml/405.Following the collection of blood samples, brains (n = 4) were dissected and preserved in 10% formalin in saline for histopathological and immunohistochemical studies. Hippocampi from the remaining rats (n = 6) were excised and stored at -80°C. The left hippocampus was homogenized in ice-cold saline to prepare 10% homogenate to be assayed using the ELISA technique. While the right hippocampi were divided into two subsets. One subset (3 rats) was homogenized in a radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitors and was divided into aliquots for Western blotting analysis and the other one (3 rats) was submerged overnight in RNA lysis solution for the qRT-PCR assay.The Bradford assay was used for the estimation of the protein content of the homogenized samples .®, USA); with catalog numbers: MBS704680, CSB-E08465r, MBS263133, MBS451608 and MBS2703236, respectively. All procedures were performed according to the manufacturers’ instructions. The results are presented as ng/mg protein for ApoE-4, VE-cadherin and pg/mg protein for Claudin-3, IL-23, IL-27.Interleukin-23 (IL-23), interleukin-27 (IL-27), apolipoprotein E type-4 allele (ApoE-4), claudin-3 and vascular endothelial cadherin (VE-cadherin) contents were determined using ELISA kits , pGSK-3β , p-AKT , RhoA , Rac1 , pS675 β–catenin  and pS37 β–catenin  Horseradish peroxidase-conjugated goat anti-rat immunoglobulin  was used as the secondary antibody, which is a Horseradish peroxidase-conjugate (cat#: NBP1-75304). Immunoreactivity was detected by CCD camera-based imager and band intensities of the target proteins were normalized against the control sample (β-actin) (cat#: MA1115) using Chemi Doc MP Imager. Results are expressed as arbitrary units against β-actin.Following protein quantification of hippocampal tissue , protein extracts were separated by SDS gel electrophoresis and then transferred to nitrocellulose membrane. The blots were probed with antibodies  specific for Wnt-5a , Total RNA was extracted from hippocampal sections using SV total RNA isolation system  and the purity of RNA was verified at 260 nm by spectrophotometer. The extracted RNA was conversely transcribed into cDNA using RT-PCR kit  according to the manufacturer’s guidelines. Gene expression levels were assessed by SYBR Green-based Real Time Quantitative PCR method. Hippocampi were fixed in 10% phosphate-buffered formalin for 72 h. Tissue specimens were embedded in paraffin wax and sectioned at 5 μm thickness and stained with hematoxylin and eosin. Stained sections were blindly examined under a light electric microscope  and photographed with a CCD camera-based imager. Coronally cut sections (4 μm) were also prepared for immunohistochemical staining of Amyloid-β (Aβ) using polyclonal Aβ antibody 4702 (1:1500) and monoclonal Aβ antibodies 6E10  and 4G8 . Diaminobenzidine was used for staining plaque-associated immunoreactivity. The severity of the injury was semi-quantitatively scored as 0 (no staining), 1+ (<10 plaques), 2+ (>10 scattered plaques), 3+ (most of the hippocampus stained), or 4+ .p < 0.05 for all statistical tests.Data are expressed as means ± SD. For parametric analysis, multiple comparisons were performed using a one-way analysis of variance (ANOVA) test followed by Tukey’s Multiple Comparison Test. For non-parametric data, Kruskal–Wallis followed by Dunn’s multiple comparisons tests was used. GraphPad Prism software package, version 7  was used to carry out all statistical tests. The level of significance was fixed at (A) and 67% in the percentage of time spent exploring the new object (B), indicating long-term memory deterioration. Treatment with either VitD or RSV improved the discrimination index and shortened the time spent exploring the familiar object compared to the T2DM group. The combination group significantly restored the abovementioned parameters to near normal values.As revealed in (C). Additionally, in the probe test, T2DM rats spent 54% less time in the target quadrant (D) searching for the missing platform. Treatment with VitD, RSV, and their combination significantly decreased the mean escape latency, and the time spent in the target quadrant compared to T2DM, indicating improved spatial learning and memory tasks.In the MWMT, the mean escape latency (MEL) was increased by 2.5 folds compared to the NC group (B) compared to the NC group (A). Hirano bodies which are a main feature of neurodegeneration were also detected. Additionally, immunostaining of the hippocampal area (F-J) revealed a huge deposition of Aβ  compared to normal rats (F). The VitD-treated group displayed fewer Aβ plaques (H) with a moderate Aβ expression , while in the RSV-treated group, there were only a few numbers of plaques (I) with faint staining of Aβ . Interestingly, the combined treatment (E) showed the normal structure of glial cells and pyramidal cells with no expression of Aβ (J). The observed Aβ staining score is portrayed in panel K.As shown in (A), a twofold rise in serum glucose level (B), and a threefold increase in free fatty acids (C) as compared to the NC group. Conversely, administration of either VitD or RSV resulted in a significant reduction in glucose and FFAs levels, compared to T2DM. However, serum insulin level was significantly reduced by RSV treatment only. In the combination treatment, a more pronounced attenuation of the abovementioned parameters was reached as compared to either drug alone. HOMA-IR values in diabetic rats were drastically elevated to 9.7 times the NC group (D), while combined VitD and RSV therapy changed it to 3.4 times.As cleared in (A), threefold elevation in LDL-C (B), and a twofold increase in TC (C) accompanied by a marked 53.5% reduction in HDL-C (D) in comparison to the NC group. Notably, treatment with VitD significantly decreased TGs, LDL-C, and TC together with a profound boost in HDL-C levels. In parallel, administration of RSV markedly reduced TGs, LDL-C, and TC levels, but failed to raise the level of HDL-C to any significant extent. Again, combined treatment with VitD and RSV resulted in a more favorable effect on the previous parameters.(A), a fall of nearly 63% of Wnt5a (B), 68% of pS9 GSK-3β (C) and a 73% of pS675 β-catenin (D). This was paralleled by a six-fold upregulation of pS37 β-catenin (E) as compared to the NC group. Treatment with either VitD or RSV significantly decreased ApoE-4 content and upregulated Wnt5a, pS9GSK-3β, and pS675 β-catenin together with a downregulation in pS37 β-catenin as compared to T2DM group. Notably, the combined therapy showed more prominent improvement over either drug alone.As expressed in Diabetic rats presented an obvious 52.3% and 60.5% reduction in RhoA  and Rac1T2DM rats manifested a 45.7%, 73%, and 69.8% decline in the hippocampal claudin 3 , VE-cadh(A), and a 63.3% reduction in the anti-inflammatory cytokine, IL-27 (B) as compared to the NC group. Treatment with VitD, RSV, or their combination showed significant anti-inflammatory effects through reducing IL-23 and elevating IL-27 levels, compared to the T2DM group.(A), accompanied by a 79% and 69% decline in the gene expression of insulin (B) and α7nACh (C) receptors as compared to the NC group. Treatment with either VitD or RSV alone lowered the level of p-tau and elevated the expression of both receptors, compared to T2DM. Again, the combined treatment was superior to either drug alone.As shown in Because of the epidemiological evidence for an increased risk of dementia and mild cognitive impairment in patients with diabetes, VitD and RSV were given to diabetic rats either alone or combined to investigate their protective potential in T2DM-induced memory deficits. This effect was partly attributed to (1) halting of T2DM-associated metabolic dysfunction, (2) modulation of the crosstalk between hippocampal insulin and noncanonical Wnt/β-catenin cassette, (3) stimulation of the canonical Wnt/β-catenin signaling pathway, (4) mitigation of neuroinflammation and preservation of BBB integrity, (5) improvement of memory and cognitive abilities, and 6) restoration of the hippocampal histological architecture.Peripheral insulin resistance is accompanied by central manifestations like defective insulin signaling , neuroinThe primary finding was that treatment of diabetic animals with VitD or RSV markedly improved T2DM-induced metabolic abnormalities in line with other reports , 26, 28.p-AKT and p-GSK-3β with reduced Tau hyperphosphorylation and Aβ deposition as shown in Figs Notably, the combined treatment with VitD and RSV provoked greater outcomes on the disrupted metabolic profile than either one alone. Interestingly, modulation of these metabolic abnormalities was reflected centrally and could be related to the ability of VitD and/ or RSV to improve defective insulin signaling by increasing the gene expression of hippocampal insulin receptors and protein expression of Findings revealed that the protective effect of VitD in asthma , colon cpS675 β-catenin, as well as reduced ApoE-4 hippocampal levels. Hence, the enhanced Wnt/β-catenin signaling with subsequent stimulation of its nuclear targets could pin down a key mechanism by which VitD or RSV may improve T2DM provoked hippocampal injury and associated cognitive and memory impairment.As for the canonical Wnt/β-catenin cassette, it was activated following the administration of VitD, RSV, or their combination. Inhibition of canonical Wnt/β-catenin pathway leads to enhanced phosphorylation of β-catenin by GSK-3β that mediated its ubiquitination and proteasomal degradation as observed herein . HoweverAmong activated Wnt/ β-catenin transcriptional targets are genes encoding for tight junction proteins Annexin A1  and clauChen et al. [Another important downstream target for Wnt /β-catenin signaling is α7 nicotinic acetylcholine receptor (α7nAChR)  whose don et al. . Remarkan et al. –93.The findings of the current work showed that VitD or RSV increased the anti-inflammatory, IL 27 and decreased the proinflammatory, IL 23 cytokines’ levels. This was further augmented by the co-administration of both drugs. Regulating the expression of these pivotal cytokines is one of the Wnt/β-catenin downstream signaling  roles inTaken altogether, the current study accentuated the neuroprotective potential of VitD and/or RSV in ameliorating T2DM-induced hippocampal insult and accompanied behavioral alterations. These protective effects include modulation of the intersection between insulin/AKT/GSK-3β and canonical/non-canonical Wnt/β-catenin trajectories, as well as mitigation of neuroinflammation with subsequent improvement in memory and cognitive defects, as well as restoration of the hippocampal histological profile. The present work provides novel incentives for the use of RSV and/or VitD to slow down T2DM-induced neuronal injury. Further studies are warranted to determine their benefits in clinical practice.It is important to remember that even though insulin resistance is the core pathology of diabetes, there are several metabolic consequences that should also be taken into consideration. In addition, effects of the drugs used on the signaling pathways were studied in the whole hippocampal region; further studies may be needed to determine which sub-regions are responsible for the observed outcomes. Furthermore, apart from the studied pathways, more cascades need to be assessed to elucidate other mechanisms by which the examined agents can act.S1 File(PDF)Click here for additional data file.


The prognosis of pancreatic cancer is poor, with a 5-year survival rate of less than 10%. Studies have shown that chemokines in the tumour microenvironment are often altered, which is associated with immune infiltration and the prognosis and survival of pancreatic cancer patients.Multiomics and bioinformatics tools were used to clarify CXC chemokine expression and its role in the pancreatic ductal adenocarcinoma (PDAC) immune microenvironment.Most CXC chemokines were upregulated in pancreatic cancer and correlated with patient prognosis. CXC chemokines can activate cancer-related signalling pathways and affect immune infiltration. Furthermore, most CXC chemokines were significantly correlated with the abundance of macrophages, neutrophils and dendritic cells. CXCL5 was selected as a hub gene, and a variety of immune checkpoints, including PD-1/PD-L1 and CTLA-4, were identified.Our study provides novel insights into CXC chemokine expression and its role in the PDAC immune microenvironment. These results can provide more data about prognostic biomarkers and therapeutic targets of PDAC. Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignant tumour and accounts for more than 85% of pancreatic cancers. In 2020, cancer statistics data showed that there were 60,430 new cases and 48,220 deaths related to pancreatic cancer . Due to The CXC family of chemokines (CXCL1 to CXCL17) are small (8 to 10 kDa) secreted proteins that are crucial for inflammation and antitumour immunity. These small proteins are secreted not only by tumour cells but also by leukocytes, fibroblasts, endothelial cells and epithelial cells, which can induce the directional migration of neutrophils and lymphocytes and regulate tumour-associated angiogenesis and cancer cell metastasis , 5. Li YTherefore, in this study, we analysed the mRNA expression of the CXC chemokine family and its correlation with hallmarks of PDAC. We further clarified the correlation between CXC chemokines and immune cell infiltration in the TME of pancreatic cancer, which will help to better understand the CXC chemokine family and improve treatment designs and the accuracy of prognosis for patients with PDAC.P value < 0.05, fold-change > 2, and a gene rank of top 10%. Then, in Gene Expression Profiling Interactive Analysis (GEPIA), we used a |Log2FC| cut-off of 1 and a P value cut-off of 0.05 to detect CXC chemokine expression levels. We further analysed the relative expression levels of CXC chemokines and their expression levels in different pathological stages and T stages.We detected the mRNA expression of CXC chemokines in PDAC using three databases . First, P value.In GEPIA, we evaluated the prognostic significance of CXC chemokines in PDAC using a Kaplan–Meier curve . In the With cBioPortal, we analysed CXC chemokine genetic alterations in 295 pancreatic adenocarcinoma samples  dataset, and Nature Communications). In the TCGA, we explored the potential coexpression of differentially expressed CXC chemokines. In STRING and GeneMANIA, the potential interactions of these CXC chemokines were detected.In GSCALite, we detected cancer-related pathways and CXC chemokine drug sensitivity in PDAC samples . In the P < 0.05. Then, we also submitted CXC chemokines and the correlated genes to Metascape to perform pathway and process enrichment with a minimum overlap of 3, P < 0.01 and a minimum enrichment of 1.5.Enrichment analysis of CXC chemokines in PDAC was conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) 6.8, GEPIA, Metascape and GeneMANIA. First, GEPIA was used to extract the top five genes correlated with each CXC chemokine. Next, these correlated genes and CXC chemokines were used to explore the function of CXC chemokines in PDAC and to perform enrichment analysis (including gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein–protein interaction (PPI) network analysis). The analysis was conducted with the R programs “clusterProfiler” and “ggplot2” with Immune infiltration analysis of CXC chemokines in PDAC was performed using the Tumor IMmune Estimation Resource (TIMER) and the TCGA. We evaluated the correlation between CXC chemokines and immune cells, immune score and stromal score using Spearman analysis. The analysis between CXC chemokines and the immune score and stromal score was used to estimate the algorithm.In view of the expression and prognostic implications of CXC chemokines in pancreatic cancer, we selected CXCL5 as the hub gene for the correlation analysis. The correlations between CXCL5 and 66 common immune checkpoints, including 23 immunoinhibitors and 43 immunostimulators, in the TCGA database were evaluated using Spearman analysis.We first explored the expression of 16 CXC chemokines in PDAC using ONCOMINE and GEPIA. ONCOMINE data showed that in addition to CXCL1, CXCL4, CXCL11 and CXCL12, the mRNA levels of the other 12 CXC chemokines were significantly increased in pancreatic cancer  had worse disease-free survival than those with low expression , CXCL9 , CXCL10 , and CXCL17  had worse overall survival  of PDAC patients using GEPIA. Based on data from GEPIA, we found that PDAC patients with high expression of CXCL17 , DNA damage response, cell cycle, and apoptosis pathways). The results showed that most CXC chemokines are related to the activation of apoptosis and the EMT pathway and the inhibition of the DNA damage response pathway and the hormone AR pathway Fig. E. The drDue to the vital function of CXC chemokines in PDAC, we performed GO function, KEGG pathway and PPI network enrichment analyses using DAVID 6.8, GEPIA, Metascape and GeneMANIA. First, GEPIA was used to obtain the top five correlated genes for each CXC chemokine Table , and theAccording to the TIMER results, most CXC chemokines show a significant correlation with the abundance of macrophages, neutrophils and dendritic cells Fig. . With thThere were 5 immune checkpoints with correlation coefficients greater than 0.4: MICB, TNFSF13B, HAVCR2, LGALS9, and CD86; other immune checkpoints also had a certain correlation with CXCL5  combined with pembrolizumab can prolong the time on trial treatment compared with their last standard treatment and activate the Th1 immune response in patients with metastatic colorectal and pancreatic cancer . A CXCR4In addition, many studies have revealed that certain CXC chemokines play an important role in many cancers, along with other diseases. For example, CXCL2 is related to bone erosion in rheumatoid arthritis . CXCL14 Overall, our study provides novel insights into CXC chemokine expression and its role in the PDAC immune microenvironment. These results may provide more data about prognostic biomarkers and therapeutic targets for PDAC.