Beclin-1-Derived Peptide MP1 Attenuates Renal Fibrosis by Inhibiting the Wnt/ß-Catenin Pathway.

Renal fibrosis is distinguished by the abnormal deposition of extracellular matrix and progressive loss of nephron function, with a lack of effective treatment options in clinical practice. In this study, we discovered that the Beclin-1-derived peptide MP1 significantly inhibits the abnormal expression of fibrosis and epithelial-mesenchymal transition-related markers, including α-smooth muscle actin, fibronectin, collagen I, matrix metallopeptidase 2, Snail1, and vimentin both in vitro and in vivo. H&E staining was employed to evaluate renal function, while serum creatinine (Scr) and blood urea nitrogen (BUN) were used as main indices to assess pathologic changes in the obstructed kidney. The results demonstrated that daily treatment with MP1 during the 14-day experiment significantly alleviated renal dysfunction and changes in Scr and BUN in mice with unilateral ureteral obstruction. Mechanistic research revealed that MP1 was found to have a significant inhibitory effect on the expression of crucial components involved in both the Wnt/ß-catenin and transforming growth factor (TGF)-ß/Smad pathways, including ß-catenin, C-Myc, cyclin D1, TGF-ß1, and p-Smad/Smad. However, MP1 exhibited no significant impact on either the LC3II/LC3I ratio or P62 levels. These findings indicate that MP1 improves renal physiologic function and mitigates the fibrosis progression by inhibiting the Wnt/ß-catenin pathway. Our study suggests that MP1 represents a promising and novel candidate drug precursor for the treatment of renal fibrosis. SIGNIFICANCE STATEMENT: This study indicated that the Beclin-1-derived peptide MP1 effectively mitigated renal fibrosis induced by unilateral ureteral obstruction through inhibiting the Wnt/ß-catenin pathway and transforming growth factor-ß/Smad pathway, thereby improving renal physiological function. Importantly, unlike other Beclin-1-derived peptides, MP1 exhibited no significant impact on autophagy in normal cells. MP1 represents a promising and novel candidate drug precursor for the treatment of renal fibrosis focusing on Beclin-1 derivatives and Wnt/ß-catenin pathway.

Inhibitory effects of bioactive compounds on UVB-induced photodamage in human keratinocytes: modulation of MMP1 and Wnt signaling pathways.

UVB radiation significantly threatens skin health, contributing to wrinkle formation and an elevated risk of skin cancer. This study aimed to explore bioactive compounds with potential UVB-protective properties. Using in silico analysis, we chose compounds to reduce binding energy with matrix metalloproteinase-1 (MMP1). Piperitoside, procyanidin C1, and mulberrofuran E emerged as promising candidates through this computational screening process. We investigated the UVB-protective efficacy of the selected compounds and underlying mechanisms in human immortalized keratinocytes (HaCaT). We also investigated the molecular pathways implicated in their action, focusing on the transforming growth factor (TGF)-ß and wingless-related integration site (Wnt)/ß-catenin signaling pathways. In UVB-exposed HaCaT cells (100 mJ/cm2 for 30 min), piperitoside, procyanidin C1, and mulberrofuran E significantly reduced reactive oxygen species (ROS) and lipid peroxides, coupled with an augmentation of collagen expression. These compounds suppressed MMP1, tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase (iNOS) expression, while they concurrently enhanced collagen-1 (COL1A1), ß-catenin (CTNNB1), and superoxide dismutase type-1 (SOD1) expression. Furthermore, Wnt/ß-catenin inhibitors, when administered subsequently, partially counteracted the reduction in MMP1 expression and alleviated inflammatory and oxidative stress markers induced by the bioactive compounds. In conclusion, piperitoside, procyanidin C1, and mulberrofuran E protected against UVB-induced damage in HaCaT cells by inhibiting MMP1 expression and elevating ß-catenin expression. Consequently, these bioactive compounds emerge as promising preventive agents for UVB-induced skin damage, promoting skin health.

Molecular mechanism of triptolide in myocardial fibrosis through the Wnt/ß-catenin signaling pathway.

Objective. Myocardial fibrosis (MF) is a common manifestation of end-stage cardiovascular diseases. Triptolide (TP) provides protection against cardiovascular diseases. This study was to explore the functional mechanism of TP in MF rats via the Wnt/ß-catenin pathway. Methods. The MF rat model was established via subcutaneous injection of isoproterenol (ISO) and treated with low/medium/high doses of TP (L-TP/M-TP/H-TP) or Wnt agonist BML-284. Cardiac function was examined by echocardiography. Pathological changes of myocardial tissues were observed by HE and Masson staining. Col-I/Col-III/Vimentin/α-SMA levels were detected by immunohistochemistry, RT-qPCR, and Western blot. Collagen volume fraction content was measured. Expression levels of the Wnt/ß-catenin pathway-related proteins (ß-catenin/c-myc/Cyclin D1) were detected by Western blot. Rat cardiac fibroblasts were utilized for in vitro validation experiments. Results. MF rats had enlarged left ventricle, decreased systolic and diastolic function and cardiac dysfunction, elevated collagen fiber distribution, collagen volume fraction and hydroxyproline content. Levels of Col-I/Col-III/Vimentin/α-SMA, and protein levels of ß-catenin/c-myc/Cyclin D1 were increased in MF rats. The Wnt/ß-catenin pathway was activated in the myocardial tissues of MF rats. TP treatment alleviated impairments of cardiac function and myocardial tissuepathological injury, decreased collagen fibers, collagen volume fraction, Col-I, Col-III, α-SMA and Vimentin levels, HYP content, inhibited Wnt/ß-catenin pathway, with H-TP showing the most significant effects. Wnt agonist BML-284 antagonized the inhibitive effect of TP on MF. TP inhibited the Wnt/ß-catenin pathway to repress the proliferation and differentiation of mouse cardiac fibroblasts in vitro. Conclusions. TP was found to ameliorate ISO-induced MF in rats by inhibiting the Wnt/ß-catenin pathway.

PCAT-1' s role in wound healing impairment: Mitochondrial dysfunction and bone marrow stem cell differentiation inhibition via PKM2/ß-catenin pathway and its impact on implant osseo-integration.

This study focused on unravelling the role of PCAT-1 in wound-healing process, particularly its impact on regenerative and osteogenic abilities of mesenchymal stem cells (MSCs). We delved into how PCAT-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) and interacts with pivotal molecular pathways, especially ß-catenin and PKM2, using human bone marrow-derived MSCs. MSCs were cultured under specific conditions and PCAT-1 expression was modified through transfection. We thoroughly assessed several critical parameters: MSC proliferation, mitochondrial functionality, ATP production and expression of wound healing and osteogenic differentiation markers. Further, we evaluated alkaline phosphatase (ALP) activity and mineral deposition, essential for bone healing. Our findings revealed that overexpressing PCAT-1 significantly reduced MSC proliferation, hampered mitochondrial performance and lowered ATP levels, suggesting the clear inhibitory effect of PCAT-1 on these vital wound-healing processes. Additionally, PCAT-1 overexpression notably decreased ALP activity and calcium accumulation in MSCs, crucial for effective bone regeneration. This overexpression also led to the reduction in osteogenic marker expression, indicating suppression of osteogenic differentiation, essential in wound-healing scenarios. Moreover, our study uncovered a direct interaction between PCAT-1 and the PKM2/ß-catenin pathway, where PCAT-1 overexpression intensified PKM2 activity while inhibiting ß-catenin, thereby adversely affecting osteogenesis. This research thus highlights PCAT-1's significant role in impairing wound healing, offering insights into the molecular mechanisms that may guide future therapeutic strategies for enhancing wound repair and bone regeneration.

Kinesin family member 23 knockdown inhibits cell proliferation and epithelial-mesenchymal transition in esophageal carcinoma by inactivating the Wnt/ß-catenin pathway.

Kinesin family member 23 (KIF23) serves as a tumor-promoting gene with prognostic values in various tumors. However, the role of KIF23 in esophageal carcinoma (ESCA) progression is largely unknown. The overlapping differentially expressed genes (DEGs) in GSE12452, GSE17351, and GSE20347 datasets were identified via GEO2R tool and Venn diagram software. KIF23 expression was analyzed using GSE12452, GSE17351, and GSE20347 datasets, GEPIA database, and qRT-PCR. Cell proliferation was assessed by CCK-8 and EdU incorporation assays. Gene set enrichment analysis (GSEA) analysis was performed to investigate the pathways associated with the regulatory mechanisms of KIF23 in ESCA. The expression of E-cadherin, vimentin, N-cadherin, and matrix metalloproteinase-9 (MMP-9) and alternation of Wnt/ß-catenin pathway were detected by western blot analysis. We identified two overlapping upregulated DEGs, among which KIF23 was selected for subsequent experiments. KIF23 was overexpressed in ESCA samples and cells, and knockdown of KIF23 retarded cell proliferation in ESCA cells. Besides, KIF23 knockdown suppressed epithelial-mesenchymal transition (EMT) process in ESCA cells, as evidenced by the increase of E-cadherin expression and the reduction of vimentin, N-cadherin, and MMP-9 expression. GSEA analysis suggested that Wnt signaling pathway was the significant pathway related to KIF23. Moreover, we demonstrated that KIF23 silencing inhibited the Wnt/ß-catenin pathway in ESCA cells. Activation of Wnt/ß-catenin pathway by SKL2001 reversed the effects of KIF23 silencing on cell proliferation and EMT in ESCA cells. In conclusion, KIF23 knockdown inhibited the proliferation and EMT in ESCA cells through blockage of Wnt/ß-catenin pathway.

PFT-α protects the blood-brain barrier through the Wnt/ß-catenin pathway after acute ischemic stroke.

The dysfunction of blood-brain barrier (BBB) plays a pivotal role in brain injury and subsequent neurological deficits of ischemic stroke. The current study aimed to examine the potential correlation between p53 inhibition and the neuroprotective effect of on the BBB. Rat middle cerebral artery occlusion and reperfusion model (MCAO/R) and oxygen-glucose deprivation/re-oxygenation model (OGD/R) were employed to simulate cerebral ischemia-reperfusion (CI/R) injury occurrence in vivo and in vitro. mNSS and TTC staining were applied to evaluate neurological deficits and brain infarct volumes. Evans blue (EB) staining was carried out to examine the permeability of BBB. RT-qPCR and Western blot to examine the mRNA and protein levels. Cell viabilities were detected by CCK-8. Flow cytometry and ELISA assay were employed to examine apoptosis and neuroinflammation levels. TEER value and sodium fluorescein were carried out to explore the permeability of HBMEC cells. PFT-α inhibited P53 and promoted the expression of ß-catenin and cyclin D1, which were reversed by DKK1. PFT-α inhibited neurological deficits, brain infarct volume, neuroinflammation, apoptosis, and BBB integrity than the MCAO/R rats; however, this inhibition was reversed by DKK1. PFT-α promoted OGD/R-induced cell viability in NSCs, and suppressed inflammation and apoptosis, but DKK1 weakened the effect of PFT-α. PFT-α increased OGD/R-induced TEER values in cerebrovascular endothelial cells, inhibited sodium fluorescein permeability, and increased the mRNA levels of tight junction protein, but they were all attenuated by DKK1. PFT-α protects the BBB after acute ischemic stroke via the Wnt/ß-catenin pathway, which in turn improves neurological function.

ACVRL1 drives resistance to multitarget tyrosine kinase inhibitors in colorectal cancer by promoting USP15-mediated GPX2 stabilization.

BACKGROUND: Multitarget tyrosine kinase inhibitors (mTKIs) such as Regorafenib and Sorafenib have already been approved for the treatment of many solid tumours. However, the efficacy of mTKIs in colorectal cancer (CRC) is limited; the underlined mechanism remains largely elusive. Our study was aimed to find out the resistance mechanism of mTKIs in CRC. METHODS: RNA sequencing was used to identify the expression of Activin A receptor-like type 1 (ACVRL1) under the treatment of mTKIs. Gain/loss-of-function experiments were performed to assess the biological function of ACVRL1 in resistance to mTKIs. The underlying mechanisms of ACVRL1-mediated mTKI resistance were investigated by using liquid chromatography-mass spectrometry assays (LC-MS), co-immunoprecipitation assays (Co-IP), chromatin immunoprecipitation assays, ubiquitination assays, dual luciferase reporter assays, etc. RESULTS: RNA sequencing identified the activation of ACVRL1 under the treatment of mTKIs in CRC cells. ACVRL1 knockdown and overexpression significantly affects the sensitivity of CRC cells to mTKIs both in vitro and vivo. Mechanistically, we found the ß-catenin/TCF-1-KCNQ1OT1/miR-7-5p axis mediated the activation of ACVRL1. Furthermore, LC-MS assays indicated the interaction between ACVRL1 and glutathione peroxidase 2(GPX2) protein. IP assay defined ACVRL1 truncation (282-503aa) could be responsible for interacting with GPX2, and rescue experiments with ACVRL1 truncations confirmed the importance of this interaction in driving mTKI resistance. Co-IP assays confirmed that ACVRL1 associates with ubiquitin-specific peptidase 15(USP15) which directly deubiquinates GPX2 at the K187(K, lysine) site, leading to the accumulation of GPX2 protein. Rescue experiments performed with the lysine mutants in GPX2 CRISPR knockout cell model confirmed the importance of GPX2 K187 mutant. As a result, the increased ROS clearance and decreased cell apoptosis eventually lead to mTKI resistance in CRC. CONCLUSIONS: Our results demonstrate that the Wnt/ß-catenin/KCNQ1OT1/miR-7-5p/ACVRL1/GPX2 biological axis plays a vital role in CRC, targeting which may be an effective approach for overcoming mTKI resistance.

Chlorogenic Acid Inhibits Epithelial-Mesenchymal Transition and Invasion of Breast Cancer by Down-Regulating LRP6.

Epithelial-mesenchymal transition (EMT) is a crucial biologic process for breast cancer metastasis, and inhibition of EMT could be an effective approach to suppress metastatic potential of mammary cancer. High expression of low-density lipoprotein receptor-related protein 6 (LRP6) is usually observed in breast carcinoma and predicts poor prognosis. In the present study, we investigated whether chlorogenic acid (CA) can inhibit the EMT of breast cancer cells and underlying molecular mechanism. We found that CA treatment transformed MCF-7 cell morphology from spindle shape (mesenchymal phenotype) to spherical shape (epithelial phenotype). CA clearly increased epithelial biomarkers' expression (E-cadherin and ZO-1) but decreased mesenchymal proteins' expression (ZEB1, N-cadherin, vimentin, snail, and slug). In addition, CA attenuated MMP-2 and MMP-9 activities and inhibited cell migration and invasion. CA downregulated the expression of LRP6 in MCF-7 cells. Knockdown LRP6 with siRNA repressed cell mobility and invasion, wheras overexpression of LRP6 promoted EMT and antagonized the EMT inhibitory effect of CA on MCF-7 cells. Furthermore, CA directly interacted with Wnt/ß-catenin signaling coreceptor LRP6 and reduced LRP6, p-LRP6, and ß-catenin expression levels in MCF-7 cells. In vivo study revealed that CA notably reduced tumor volume and tumor weight. CA decreased the expression of LRP6, N-cadherin, ZEB1, vimentin, MMP2, MMP9, and increased the expression of E-cadherin and ZO-1. In conclusion, CA inhibited EMT and invasion of breast cancer by targeting LRP6. SIGNIFICANCE STATEMENT: CA, the familiar polyphenol compound in traditional Chinese medicine, repressed EMT and weakened cellular mobility and invasion in MCF-7 cells. The mechanism studies demonstrated that CA could inhibit EMT and invasion of MCF-7 cells via targeting LRP6. Additionally, CA restrained tumor growth and xenograft tumor EMT in vivo. The EMT inhibitory property of CA warrants further studies of CA as a drug candidate for the therapy of metastatic breast carcinoma.

NRF1 knockdown alleviates lipopolysaccharide-induced pulmonary inflammatory injury by upregulating DKK3 and inhibiting the GSK-3ß/ß-catenin pathway.

Excessive inflammatory injury is the main cause of the incidence of severe neonatal pneumonia (NP) and associated deaths. Although dickkopf-3 (DKK3) exhibits anti-inflammatory activity in numerous pathological processes, its role in NP is still unknown. In this study, human embryonic lung WI-38 and MRC-5 cells were treated with lipopolysaccharide (LPS) to induce inflammatory injury of NP in vitro. The expression of DKK3 was downregulated in LPS-stimulated WI-38 and MRC-5 cells. DKK3 overexpression decreased LPS-induced inhibition of cell viability, and reduced LPS-induced apoptosis of WI-38 and MRC-5 cells. DKK3 overexpression also reduced LPS-induced production of pro-inflammatory factors such as ROS, IL-6, MCP-1, and TNF-α. Nuclear respiratory factors 1 (NRF1) knockdown was found to upregulate DKK3 and inactivate the GSK-3ß/ß-catenin pathway in LPS-injured WI-38 and MRC-5 cells. NRF1 knockdown also suppressed LPS-induced inhibition on cell viability, repressed LPS-induced apoptosis, and inhibited the accumulation of ROS, IL-6, MCP-1, and TNF-α in LPS-injured WI-38 and MRC-5 cells. DKK3 knockdown or re-activation of the GSK-3ß/ß-catenin pathway reversed the inhibitory effects of NRF1 knockdown on LPS-induced inflammatory injury. In conclusion, NRF1 knockdown can alleviate LPS-triggered inflammatory injury by regulating DKK3 and the GSK-3ß/ß-catenin pathway.

Short- and long-term polystyrene nano- and microplastic exposure promotes oxidative stress and divergently affects skin cell architecture and Wnt/beta-catenin signaling.

Nano- and microplastic particles (NMP) are strong environmental contaminants affecting marine ecosystems and human health. The negligible use of biodegradable plastics and the lack of knowledge about plastic uptake, accumulation, and functional consequences led us to investigate the short- and long-term effects in freshly isolated skin cells from mice. Using fluorescent NMP of several sizes (200 nm to 6 µm), efficient cellular uptake was observed, causing, however, only minor acute toxicity as metabolic activity and apoptosis data suggested, albeit changes in intracellular reactive species and thiol levels were observed. The internalized NMP induced an altered expression of various targets of the nuclear factor-2-related transcription factor 2 pathway and were accompanied by changed antioxidant and oxidative stress signaling responses, as suggested by altered heme oxygenase 1 and glutathione peroxide 2 levels. A highly increased beta-catenin expression under acute but not chronic NMP exposure was concomitant with a strong translocation from membrane to the nucleus and subsequent transcription activation of Wnt signaling target genes after both single-dose and chronic long-term NMP exposure. Moreover, fibroblast-to-myofibroblast transdifferentiation accompanied by an increase of α smooth muscle actin and collagen expression was observed. Together with several NMP-induced changes in junctional and adherence protein expression, our study for the first time elucidates the acute and chronic effects of NMP of different sizes in primary skin cells' signaling and functional biology, contributing to a better understanding of nano- and microplastic to health risks in higher vertebrates.

Strontium-modified titanium substrate promotes osteogenic differentiation of MSCs and implant osseointegration via upregulating CDH2.

OBJECTIVES: Our previous studies showed that strontium (Sr)-modified sand-blasted, large grit, acid etched titanium surface (Sr-SLA) is beneficial for osseointegration; however, the supporting mechanisms have not been explored in detail. MATERIALS AND METHODS: Whole-transcriptome RNA sequencing of peri-implant bone tissue was performed, and CDH2 was selected as a key mediator of Sr-SLA-mediated osseointegration. To test this hypothesis, a lentivirus-mediated vector targeting the silencing of the CDH2 gene was used in mesenchymal stem cells (MSCs) prior to seeding on Ti substrates. The effects of CDH2 interference on MSCs vitality, differentiation, and ß-catenin signaling activity were evaluated. In vivo, a recombinant adeno-associated virus 9 vector carrying an artificial siRNA that target CDH2 (AAV9-CDH2i) was intravenously injected in mice, followed by tibial surgery with implant placement. Osseointegration were monitored using micro-CT analysis. RESULTS: CDH2 expression in MSCs on Sr-SLA was higher than the control group, which was in parallel with the enhanced cell migration, adhesion, and upregulation of early osteogenic markers. Knocking down CDH2 in MSCs resulted in decreased cell viability and osteogenic differentiation, and the elevated biocompatibility and osteoinductive effect of Sr-SLA were greatly diminished. Surprisingly, Sr-SLA-induced upregulation of CDH2 was not followed by restriction of ß-catenin signaling because Sr-SLA also promoted the expression and nuclear translocation of ß-catenin. Systemic administration of AAV9-CDH2i effectively knocked down CDH2 expression in bone marrow cells, and in turn, inhibited bone formation induced by Sr-SLA. CONCLUSIONS: The results indicated that CDH2 is required for Sr-SLA-mediated bone regeneration, which reveals a new mechanism to explain the osteoinductive effect of Sr-SLA. Thus, biomaterial modifications targeting CDH2 may help improve early osseointegration and bone healing.

HIV gp120/Tat protein-induced epithelial-mesenchymal transition promotes the progression of cervical lesions.

BACKGROUND: Human immunodeficiency virus (HIV) infection is associated with an elevated incidence of cervical cancer, and accelerated disease progression, but the underlying mechanisms are not well understood. This study aimed to investigate the relationship between HIV infection and epithelial-mesenchymal transition (EMT) in cervical cancer. METHODS: Tissue samples from HIV-positive and negative patients with cervical intraepithelial neoplasia (CIN) and cervical cancer were analyzed for EMT-related proteins. Human cervical cancer SiHa cells were treated with HIV Tat and gp120 proteins to test their effects on EMT, migration, and invasion. RESULTS: HIV-positive patients had lower E-cadherin and cytokeratin, and higher N-cadherin and vimentin levels than HIV-negative patients. HIV Tat and gp120 proteins induced EMT, migration, and invasion in SiHa cells. Transcriptome sequencing analysis revealed that, compared to the control group, the protein-treated group showed upregulation of 22 genes and downregulation of 77 genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed the involvement of the Wnt signaling pathway in EMT. Further analysis of gene expression related to this pathway revealed upregulation of DVL1, TCF7, KRT17, and VMAC, while GSK3ß, SFRP2, and CDH1 were downregulated. Immunofluorescence assay demonstrated that HIVgp120 and Tat proteins treatment induced elevated ß-catenin expression with nuclear accumulation in SiHa cells. CONCLUSIONS: The treatment of SiHa cells with HIV Tat and gp120 proteins induces EMT and activates the Wnt/ß-catenin pathway, suggesting that the Wnt/ß-catenin pathway may play a crucial role in promoting EMT progression in cervical lesion tissues of HIV-infected patients.

Effect of DNA aptamer through blocking of negative regulation of Wnt/ß-catenin signaling in human hair follicle dermal papilla cells.

BACKGROUND: When Wnt binds to the N-terminal of Frizzled, a conformational change occurs in the C-terminal of Frizzled, which binds to Dishevelled1 (Dvl1), a Wnt signaling component protein. When Dvl1 binds to the C-terminal of Frizzled, the concentration of ß-catenin increases and it enters the nucleus to transmit cell proliferation signals. CXXC-type zinc finger protein 5 (CXXC5) binds to the Frizzled binding site of Dvl1 and interferes with Dvl1-Frizzled binding. Therefore, blocking CXXC5-Dvl1 binding may induce Wnt signal transduction. MATERIALS AND METHODS: We used WD-aptamer, a DNA aptamer that specifically binds to Dvl1 and interferes with CXXC5-Dvl1 interaction. We confirmed the penetration of WD-aptamer into human hair follicle dermal papilla cells (HFDPCs) and measured ß-catenin expression following treatment with WD-aptamer in HFDPCs, wherein Wnt signaling was activated by Wnt3a. In addition, MTT assay was performed to investigate the effect of WD-aptamer on cell proliferation. RESULTS: WD-aptamer penetrated the cell, affected Wnt signaling, and increased ß-catenin expression, which plays an important role in signaling. Additionally, WD-aptamer induced HFDPC proliferation. CONCLUSION: CXXC5-associated negative feedback of Wnt/ß-catenin signaling can be regulated by interfering with CXXC5-Dvl1 interaction.

miR-181b-1-3p affects the proliferation and differentiation of chondrocytes in TD broilers through the WIF1/Wnt/ß-catenin pathway.

Thiram is a plant fungicide, its excessive use has exceeded the required environmental standards. It causes tibial dyschondroplasia (TD) in broilers which is a common metabolic disease that affects the growth plate of tibia bone. It has been studied that many microRNAs (miRNAs) are involved in the differentiation of chondrocytes however, their specific roles and mechanisms have not been fully investigated. The selected features of tibial chondrocytes of broilers were studied in this experiment which included the expression of miR-181b-1-3p and the genes related to WIF1/Wnt/ß-catenin pathway in chondrocytes through qRT-PCR, western blot and immunofluorescence. The correlation between miR-181b-1-3p and WIF1 was determined by dual luciferase reporter gene assay whereas, the role of miR-181b-1-3p and WIF1/Wnt/ß-catenin in chondrocyte differentiation was determined by mimics and inhibitor transfection experiments. Results revealed that thiram exposure resulted in decreased expression of miR-181b-1-3p and increased expression of WIF1 in chondrocytes. A negative correlation was also observed between miR-181b-1-3p and WIF1. After overexpression of miR-181b-1-3p, the expression of ACAN, ß-catenin and Col2a1 increased but the expression of GSK-3ß decreased. It was observed that inhibition of WIF1 increased the expression of ALP, ß-catenin, Col2a1 and ACAN but decreased the expression of GSK-3ß. It is concluded that miR-181b-1-3p can reverse the inhibitory effect of thiram on cartilage proliferation and differentiation by inhibiting WIF1 expression and activating Wnt/ß-catenin signaling pathway. This study provides a new molecular target for the early diagnosis and possible treatment of TD in broilers.

Further insights into the impact of rebamipide on gentamicin-induced nephrotoxicity in rats: modulation of SIRT1 and ß-catenin/cyclin D1 pathways.

Gentamicin (GM) is an effective antibiotic administered to treat acute Gram-negative infections. Nevertheless, its clinical application is limited due to nephrotoxicity. Therefore, our research aimed to investigate the potential renoprotective impact of rebamipide (RBM), a gastroprotective drug, on GM-induced kidney damage in rats, as well as putative nephroprotective pathways. RBM was orally administered (100 mg/kg/d for 14 d) commencing 7 d before the administration of GM (100 mg/kg/d, intraperitoneally). Nephrotoxicity was elucidated, and the silent information regulator 1 (SIRT1) and ß-catenin/cyclin D1 pathways were assessed. GM induced a significant elevation in the serum levels of creatinine, blood urea nitrogen (BUN), and kidney injury molecule-1 (KIM-1), as well as the relative kidney index. In addition, GM increased lipid peroxidation and lowered total antioxidant capacity (TAC) level and superoxide dismutase (SOD) activity. GM administration also demonstrated a significant amplification in tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), nuclear factor-κappa B p65 (NF-κB p65), p38 mitogen-activated protein kinase (p38 MAPK), and caspase-3 kidney levels, as well as B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 ratio. Notably, RBM treatment amended all these changes induced by GM. Furthermore, the potential role of SIRT1 and ß-catenin-dependent signaling pathways in GM-induced renal injury was assessed. Our findings showed that GM-treated rats demonstrated a substantial decrease in SIRT1, nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) along with an increase in ß-catenin, forkhead box O-3a (FOXO-3a), and cyclin D1 protein expressions. RMB treatment markedly attenuated the deterioration caused by GM on these pathways. Additionally, RBM alleviated the GM-induced deleterious kidney tissue histopathology. In conclusion, our findings have verified that RBM can halt GM-induced renal injury by partly modulating SIRT1 and ß-catenin pathways.

Dendrobium officinale Polysaccharide (DOP) Promotes Hair Regrowth in Testosterone-Induced Bald Mice.

BACKGROUND: Androgenetic alopecia can affect up to 70% of males and 40% of females; however, certain therapeutic medications offer partial and transitory improvement but with major side effects. Dendrobium officinale polysaccharide (DOP) has been reported to improve androgen-related hair loss in mice, but the molecular mechanism remains unclear. OBJECTIVES: To explore the effects of DOP on androgenetic alopecia. METHODS: In this study, testosterone was subcutaneously administered to shave dorsa skin of mice to establish androgenetic alopecia; the effects of DOP in androgenetic alopecia were explored by DOP administration. RESULTS: Testosterone treatment extended the time of skin growing dark and hair growing, decreased the mean numbers of follicles in skin tissues, decreased ß-catenin and cyclin D1 levels, and elevated testosterone, DHT (dihydrotestosterone), and 5α-reductase levels. In contrast, DOP administration shortened skin growing dark and hair growing times, promoted follicle cell proliferation, increased follicle numbers, increased ß-catenin and cyclin D1 levels, and decreased testosterone, DHT, and 5α-reductase levels. CONCLUSION: DOP application significantly improved testosterone-induced hair follicle miniaturization and hair loss, possibly through affecting the Wnt signaling and hair follicle stem cell functions. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Chlorpyrifos-induced dysregulation of synaptic plasticity in rat hippocampal neurons.

Chlorpyrifos (CPF) is a widely used organophosphorus pesticide. Increasing evidence has shown that exposure to CPF in early life might induce neurodevelopmental disorders, but the pathogenesis remains uncertain. Synaptic plasticity plays a crucial role in neurodevelopment. This study aimed to investigate the effect of CPF on synaptic plasticity in hippocampal neurons and establish the cellular mechanism underlying these effects. Using CPF-exposed rat and primary hippocampal neurons model, we analyzed the impact of CPF on the synaptic morphology, the expression level of a presynaptic protein, a postsynaptic protein and ionotropic glutamate receptors (iGluRs), as well as the effects on the Wnt/ß-catenin pathway. We found that the synapses were shortened, the spines were decreased, and the expression of synaptophysin (Syp), postsynaptic density-95 (PSD-95), GluN1, GluA1 and Wnt7a, as well as active ß-catenin in primary hippocampal neurons was decreased. Our study suggests that CPF exposure induced dysregulation of synaptic plasticity in rat hippocampal neurons, which might provide novel information regarding the mechanism of CPF-induced neurodevelopmental disorders.

Panobinostat enhances NK cell cytotoxicity in soft tissue sarcoma.

Sarcoma is a rare and heterogeneous class of mesenchymal malignancies with poor prognosis. Panobinostat (LBH589) as one of histone deacetylase (HDAC) inhibitors has demonstrated anti-tumor activity in patients with sarcoma, but its mechanisms remains unclear. Here, we found that LBH589 alone inhibited the proliferation and colony formation of soft tissue sarcoma (STS) cell lines. Transcriptome analysis showed that treatment with LBH589 augmented the NK cell-mediated cytotoxicity. Quantitative real-time PCR and flow cytometric analysis (FACS) further confirmed that LBH589 increased the expression of NKG2D ligands MICA/MICB. Mechanistically, LBH589 activated the Wnt/ß-catenin pathway by upregulating the histone acetylation in ß-catenin promoter. In vitro co-culture experiments and in vivo animal experiments showed that LBH589 increased the cytotoxicity of natural killer (NK) cells while Wnt/ß-catenin inhibitor decreased the effects. Our findings suggest that LBH589 facilitates the anti-tumor effect of NK cells, highlights LBH589 an effective assistance drug in NK cell-based immunotherapies.

Cytochrome P450 2E1-dependent hepatic ethanol metabolism induces fatty acid-binding protein 4 and steatosis.

BACKGROUND: Hepatic steatosis is an early pathology of alcohol-associated liver disease (ALD). Fatty acid-binding protein-4 (FABP4, a FABP not normally produced in the liver) is secreted by hepatocytes in ALD and stimulates hepatoma proliferation and migration. This study sought to investigate the mechanism[s] by which hepatic ethanol metabolism regulates FABP4 and steatosis. METHODS: Human hepatoma cells (HepG2/HuH7) and cells stably transfected to express cytochrome P450 2E1 (CYP2E1), were exposed to ethanol in the absence or presence of chlormethiazole (a CYP2E1-inhibitor; CMZ) and/or EX-527 (a sirtuin-1 [SIRT1] inhibitor). The culture medium was analyzed for ethanol metabolism and FABP4 protein abundance. Cells were analyzed for FABP4 mRNA expression, SIRT1 protein abundance, and neutral lipid accumulation. In parallel, cells were analyzed for forkhead box O1 [FOXO1], ß-catenin, peroxisome proliferator-activated receptor-α [PPARα], and lipin-1α protein abundance in the absence or presence of ethanol and pharmacological inhibitors of the respective target proteins. RESULTS: CYP2E1-dependent ethanol metabolism inhibited the amount of SIRT1 protein detected, concomitant with increased FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation, effects abolished by CMZ. Analysis of pathways associated with lipid oxidation revealed increased FOXO1 nuclear localization and decreased ß-catenin, PPARα, and lipin-1α protein levels in CYP2E1-expressing cells in the presence of ethanol. Pharmacological inhibition of SIRT1 mimicked the effects of ethanol, while inhibition of FOXO1 abrogated the effect of ethanol on FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation in CYP2E1-expressing cells. Pharmacological inhibition of ß-catenin, PPARα, or lipin-1α failed to alter the effects of ethanol on FABP4 or neutral lipid accumulation. CONCLUSION: CYP2E1-dependent ethanol metabolism inhibits SIRT1-FOXO1 signaling, which leads to increased FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation. These data suggest that FABP4 released from steatotic hepatocytes could play a role in promoting tumor cell expansion in the setting of ALD and represents a potential target for therapeutic intervention.

Metformin activates Wnt/ß-catenin for the treatment of diabetic osteoporosis.

BACKGROUND: With the deepening of social aging, the incidence rate of osteoporosis and diabetes continues to rise. More and more clinical studies show that diabetes is highly correlated with osteoporosis. Diabetes osteoporosis is considered as a metabolic bone disease of diabetes patients. This study aims to explore the role and mechanism of metformin (Met) in diabetic osteoporosis. METHODS: Mouse MC3T3-E1 cells were treated with Met (0.5 mM) and exposed to high glucose (HG, 35 mM). The cells were cultured in an osteogenic medium for osteogenic differentiation, and the cell proliferation ability was determined using Cell Counting Kit-8; Alkaline phosphatase (ALP) activity detection and alizarin red staining were utilized to evaluate the effect of Met on MC3T3-E1 osteogenic differentiation. Western blot was used to detect the expressions of osteogenesis-related proteins (Runx2 and OCN) as well as Wnt/ß-catenin signaling pathway-related proteins in MC3T3-E1 cells. RESULTS: HG inhibited proliferation and calcification of MC3T3-E1 cells, down-regulated ALP activity, and the expression of Runx2 and OCN in MC3T3-E1 cells. Meanwhile, the activity of the Wnt/ß-catenin signaling pathway was inhibited. Met treatment was found to significantly stimulate the proliferation and calcification of MC3T3-E1 cells under HG conditions, as well as increase the ALP activity and the protein expression level of Runx2 and OCN in the cells. As a result, osteogenic differentiation was promoted and osteoporosis was alleviated. Apart from this, Met also increased the protein expression level of Wnt1, ß-catenin, and C-myc to activate the Wnt/ß-catenin signaling pathway. CONCLUSION: Met can stimulate the proliferation and osteogenic differentiation of MC3T3-E1 cells under HG conditions. Met may also treat diabetic osteoporosis through Wnt/ß-catenin activation.