Decreased Protein Expression Research Articles

Doxazosin Attenuates Development of Testosterone Propionate-induced Prostate Growth by regulating TGF-β/Smad Signaling Pathway, Prostate-specific Antigen Expression and Reversing Epithelial-mesenchymal Transition in Mice and Stroma Cells.

Finasteride and doxazosin are used for the treatment of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS). Epithelial-mesenchymal transition (EMT) plays an important role in BPH, little is known about the growth inhibition and anti-fibrosis effects of doxazosin on the regulation of EMT and morphology in the prostate. The present study examined the effects of doxazosin on testosterone propionate (TP)-induced prostate growth in vivo and in vitro and its impact on the EMT and TGF-β/Smad signaling pathway. Doxazosin (5 or 10 mg/kg) and finasteride (10 mg/kg) were administered orally for 28 days in TP-induced mice. The prostate index (prostate/body weight ratio), morphological characteristics and the protein expression of the prostate were examined. We further examined the effects of doxazosin and finasteride on the EMT and TGF-β/Smad signaling pathway in mice and in human prostate stroma cell (WPMY-1). The prostate wet weight, prostate index decreased after treatment. Doxazosin (5 or 10 mg/kg), finasteride (10 mg/kg) or a combination (doxazosin + finasteride) were shown to reverse the pathological and morphological characteristics of the prostate. Doxazosin and finasteride inhibited TP-induced prostate growth, EMT, and the TGF-β/Smad signaling pathway by downregulating the expression of TGF-β1, TGFBR2, p-Smad2/3, N-cadherin, vimentin, fibronectin and α-SMA, whereas expression of E-cadherin was increased after treatment with either doxazosin or finasteride. Doxazosin (1-50 μM) inhibited normal human prostate stroma cell growth (WPMY-1) after 48 h with or without testosterone treatment. Doxazosin also regulated the EMT and proteins related to the TGF-β/Smad signaling pathway in WPMY-1 cells after 24 h. Additionally, doxazosin decreased protein expression of the prostate specific antigen both in vivo and in vitro. This study demonstrated that doxazosin inhibits prostate growth by regulating the EMT and TGF-β/Smad signaling pathways in the prostate This finding suggests that doxazosin has potential as a new signaling pathway for the treatment of BPH.

Prostatic androgen receptor signaling shows an age-related and lobe-specific alteration in mice

Benign prostatic hyperplasia (BPH) is an age-related disease that affects millions of aging males globally. While the pathogenesis of BPH remains incompletely understood, emerging evidence suggests a pivotal role for the androgen receptor (AR) in mediating prostate growth and function. Understanding age-related AR signaling alteration may inform novel BPH treatments. Here, we analyzed the prostatic protein expressions of AR, NKX3.1, and Ki-67 in young (2 months) and aged (24 months) mice. We also examined the potential mechanism of AR protein expression. Compared to young mice, decreased AR and NKX3.1 protein expression was observed in the anterior prostate (AP) and ventral prostate (VP) of aged mice, indicating reduced AR signaling in these prostate lobes. Additionally, we observed decreased protein expression of proliferation maker Ki-67 in aged AP, VP, and dorsal-lateral prostate (DLP), with no difference in apoptosis as compared to young counterparts. We conclude that prostatic androgen receptor signaling shows an age-related and lobe-specific alteration in mice.

Necroptosis contributes to deoxynivalenol-induced liver injury and inflammation in weaned piglets

BackgroundThe aim of this study was to investigate the role of necroptosis in deoxynivalenol (DON)-induced liver injury and inflammation in weaned piglets.MethodsIn Exp. 1, 12 weaned piglets were divided into 2 groups including pigs fed basal diet and pigs fed diet contaminated with 4 mg/kg DON for 21 d. In Exp. 2, 12 weaned piglets were divided into 2 groups including control piglets and piglets given a gavage of 2 mg/kg body weight (BW) DON. In Exp. 3, 24 weaned piglets were used in a 2 × 2 factorial design and the main factors including necrostatin-1 (Nec-1) (DMSO or 0.5 mg/kg BW Nec-1) and DON challenge (saline or 2 mg/kg BW DON gavage). On 21 d in Exp. 1, or at 6 h post DON gavage in Exp. 2 and 3, pigs were killed for blood samples and liver tissues. Liver histology, blood biochemical indicators, and liver inflammation and necroptosis signals were tested.ResultsDietary or oral gavage with DON caused liver morphological damage in piglets. Dietary DON led to hepatocyte damage indicated by increased aspartate transaminase (AST) activity and AST/alanine aminotransferase (ALT) ratio, and DON gavage also caused hepatocyte damage and cholestasis indicated by increased AST and alkaline phosphatase (AKP) activities. Dietary DON caused liver necroptosis indicated by increased protein abundance of total receptor interacting protein kinase 3 (t-RIP3) and total mixed lineage kinase domain-like protein (t-MLKL). Moreover, DON gavage increased mRNA expression of interleukin (IL)-6 and IL-1β in liver. DON gavage also induced liver necroptosis demonstrated by increased protein abundance of t-RIP3, phosphorylated-RIP3 (p-RIP3), t-MLKL and p-MLKL. However, pretreatment with Nec-1, a specific inhibitor of necroptosis, inhibited liver necroptosis indicated by decreased protein expression of t-RIP3, p-RIP3, t-MLKL and p-MLKL. Nec-1 pretreatment reduced liver morphological damage after DON gavage. Pretreatment with Nec-1 also attenuated liver damage induced by DON indicated by decreased activities of AST and AKP. Furthermore, Nec-1 pretreatment inhibited liver mRNA expression of IL-6 and IL-1β after DON challenge.ConclusionsOur data demonstrate for the first time that necroptosis contributes to DON-induced liver injury and inflammation in piglets.

Jinhua Hecha – A fermented green tea improves sleep via regulating glutamatergic synapses and gut microbiota

Quality sleep is crucial for preserving the internal equilibrium of the body. Sleep problems have become a predominant global health concern in recent years. Jinhua Hecha (JHHC) is a fermented green tea characterized by high amino acid and low tea polyphenols. In animal experiments, the potential beneficial effects of JHHC on sleep quality were evaluated. 50 mice were categorized into five groups: the normal control (NC) group, the FFZR positive control group, the JHHCG (2.3 g/kg) group, the JHHCZ (1.6 g/kg) group, and the JHHCD (0.7 g/kg) group. The sleep experiments revealed that mice from the JHHC group exhibited a notably extended duration of sleep following the intraperitoneal injection of pentobarbital sodium, with a shortened sleep latency after injection of barbital sodium. These results suggest that JHHC is beneficial for sleep. Mechanistically, transcriptome KEGG enrichment analysis revealed that differentially expressed genes were significantly enriched in the glutamatergic synapse signaling pathway. Immunohistochemistry and multiple immunofluorescence techniques showed decreased protein expressions of GLS, VGLUT2, GluA2, GluA4, GluK1, and mGluR1 in the colon of the JHHC group. Furthermore, an increase in beneficial bacteria such as Lactobacillus, Ligilactobacillus, and Akkermansia, as well as a decrease in Alistipes, were observed in the JHHC group. These results indicate that JHHC can improve sleep, with potential mechanisms involving gut microbiota maintenance and glutamate level reduction.

Structural and Functional Implications of Polyarginine Addition to Green Fluorescent Protein Expression in Saccharomyces cerevisiae INVSc1

Green Fluorescent Protein (EGFP) is widely used as a reporter gene, aiding in protein recovery and transduction studies. In this study, EGFP was tagged with eleven arginine residues (PolyR) and six histidine residues (His-tag) for purification. The aim was to enhance the synthesis of EGFP-PolyR in Saccharomyces cerevisiae and evaluate the effects of polyarginine modification on protein stability and expression levels. The expression of EGFP and EGFP-PolyR in S. cerevisiae was assessed through fluorescence measurements and protein levels. Structural analyses were conducted using in silico tools to investigate changes in beta strands and helices, which were validated through Western blots. Results showed that EGFP-PolyR maintained similar fluorescence levels to EGFP, but with notable structural changes. EGFP-PolyR's final beta strand terminates at Ala228, compared to Gly229 in EGFP, affecting the beta sheet's stability. Structural modifications also included altered helix lengths, with a longer helix 10 and shorter helix 9 in EGFP-PolyR. These alterations, along with shifts in helix-helix interactions, contribute to destabilization. Additionally, EGFP-PolyR exhibited unique gamma coils absent in EGFP, further differentiating its structure. The structural changes led to decreased protein expression and solubility, as indicated by Western blot analysis, with EGFP-PolyR showing significantly lower expression levels. The findings suggest that EGFP-PolyR is prone to aggregation and misfolding, characteristics often associated with aggregation-prone proteins.In conclusion, the polyarginine modification significantly impacts the structural integrity, stability, and solubility of EGFP. While fluorescence is retained, these changes hinder protein detectability and purification, highlighting the importance of considering structural alterations when modifying reporter proteins for experimental use.

Generating snoRNA-guided Programmable 2'- O -methylation.

Small nucleolar RNAs (snoRNAs) are critical in guiding post-transcriptional modifications like 2'- O -methylation (Nm), which play crucial roles in downstream processes such as splicing and translation. This study tests a novel method for Nm validation, addressing a significant gap in modern Nm research, and offers insight into the intricacies of snoRNA-guided Nm. While mapping of Nm modifications has seen significant improvement within the past decade, no major techniques have been able to validate these potential sites. Additionally, many mapping techniques lack consensus among proposed Nm sites, especially on mRNAs. Without a proper validation technique, Nm research lags compared to its peer post-transcriptional modifications. The RNase H-based Nm-VAQ assay used here quantifies 2'- O -methylation at single nucleotide resolution across various RNA species including rRNA, snRNA, and mRNA. Its optimization for mRNA allows for an unprecedented way to study the effects of Nm modifications in low abundance transcripts. Utilizing this, the study also explores the potential of creating synthetic snoRNAs to guide Nm modifications. Exogenous snoRNAs are shown to rescue Nm in genetic knockout models and can be mutated to guide Nm at any location along the target RNA transcript. Preliminary work indicates that synthetic snoRNAs demonstrate the ability to modify luciferase, impacting translation efficiency. Targeting an exon increases mRNA abundance but decreases protein expression, consistent with previous findings on Pxdn mRNA. These findings set the scene for novel understanding of the relationship between snoRNA abundance, 2'- O -methylation efficiency, and Nm's impact on gene expression.

Abstract B042: The impact of opioids on the pancreatic tumor immune microenvironment

Abstract Pancreatic cancer is a highly lethal malignancy with a 5-year survival rate of 12.8%. Due to the high prevalence of cancer-related pain, 75% of pancreatic cancer patients are prescribed opioids. Opioids, which signal through the G protein-coupled receptor (GPCR) mu-opioid receptor (MOR), have peripheral effects outside of their analgesic function. Importantly, several studies have correlated opioids with worsened survival of patients receiving immunotherapy. However, the mechanism behind this is unknown. Immunotherapy is largely ineffective in pancreatic cancer, due in part to the tumor microenvironment (TME) characterized by dense, desmoplastic stroma, with cancer-associated fibroblasts (CAFs) comprising up to 90% of the tumor volume. CAFs play a large role in immunosuppression and therapy resistance, such as through promoting infiltration of immunosuppressive cells, resulting in reduced presence of antitumor immune cells and impaired immunotherapy efficacy. While the role of CAFs in promoting immunosuppression in the tumor microenvironment is well characterized, the effect of opioids on this phenomenon is unknown. We now provide evidence that MOR directly regulates CAF function. Specifically, knockdown of MOR in CAFs reduces gene expression of alpha-smooth muscle actin (aSMA) and leukemia inhibitory factor. MOR knockdown in CAFs also decreases protein expression of aSMA and type 1a collagen. We hypothesize that opioids promote MOR-dependent CAF activation and secretion of pro-inflammatory cytokines, resulting in a more immunosuppressive TME, and thus impaired immunotherapy response. The long-term goal of this work is to determine the role of opioids on CAFs in the pancreatic TME, as well as the role of MOR in promoting immunosuppression and the resulting value of MOR as a target to improve immunotherapy efficacy. This research is clinically significant because it could uncover a novel mechanism driving immunosuppression caused by a medication commonly prescribed to almost all pancreatic cancer patients. Citation Format: Kathryn Maraszek, Michael Feigin. The impact of opioids on the pancreatic tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B042.

Effects of Shugan Tiaoshen acupuncture on anxiety-like behavior and PKC/ERK/CREB pathway in the bed nucleus of the stria terminalis in rats with post-traumatic stress disorder

To observe the effect of Shugan Tiaoshen acupuncture (acupuncture for soothing the liver and regulating the spirit) on the protein kinase C/extracellular signal-regulated kinase/cAMP response element-binding protein (PKC/ERK/CREB) signaling pathway in the bed nucleus of the stria terminalis (BNST) of rats with post-traumatic stress disorder (PTSD), and to explore the mechanism of acupuncture on alleviating anxiety and fear in PTSD. Fifty SPF-grade male SD rats were randomly divided into a blank group (10 rats) and a PTSD model group (40 rats). The PTSD model was induced by using a combination of closed electric shock and forced exhaustive swimming. Thirty successfully modeled rats were randomly assigned to a model group, a medication group, and an acupuncture group, with 10 rats in each group. The rats in the medication group were treated with paroxetine hydrochloride solution by gavage, once daily for 12 consecutive days. The rats in the acupuncture group were treated with acupuncture at "Baihui" (GV 20) and bilateral "Neiguan" (PC 6), "Shenmen" (HT 7), "Taichong" (LR 3). "Baihui" (GV 20) was needled daily, while the other acupoints were alternately needled on the left side on odd days and the right side on even days, once daily for 12 consecutive days. Anxiety and fear behaviors changes were assessed by using the open field test and elevated plus maze test. Histological changes in the BNST were observed by using HE staining and Nissl staining. The expression of PKC, phosphorylated PKC (p-PKC), ERK1/2, phosphorylated ERK1/2 (p-ERK1/2), and p-CREB proteins in the BNST were detected by using Western blot. Compared with the blank group, the model group showed decreased time and total distance spent in the center of the open field and on the open arms of the elevated plus maze (P<0.05); the BNST tissues in the model group exhibited a reduced number of neurons, disorganized cell arrangement, cell shrinkage, nuclear condensation, abnormal neuronal structure, uneven Nissl staining, and reduced Nissl bodies. The model group showed increased protein expression of p-PKC and p-PKC/PKC ratio (P<0.05) and decreased protein expression of p-ERK1/2, p-CREB, and p-ERK1/2/ERK1/2 ratio (P<0.05). Compared with the model group, the medication group and the acupuncture group showed increased time and total distance spent in the center of the open field and on the open arms of the elevated plus maze (P<0.05); the BNST tissues showed increased number of neurons, more organized cell arrangement, improved neuronal structure, and increased Nissl bodies; the medication group and the acupuncture group also showed decreased p-PKC protein expression and p-PKC/PKC ratio (P<0.05) and increased p-ERK1/2, p-CREB protein expression, and p-ERK1/2/ERK1/2 ratio (P<0.05). Shugan Tiaoshen acupuncture could alleviate anxiety and fear behaviors in PTSD rats, and improve neuronal damage in the BNST. The mechanism may be related to the regulation of the PKC/ERK/CREB signaling pathway in the BNST.

Increased susceptibility to diet-induced obesity in female mice impairs ovarian steroidogenesis: The role of elevated leptin signalling on nodal activity inhibition in theca cells

ObjectivesSusceptibility to obesity in humans is driven by the intricate interplay of genetic, environmental and behavioural factors. Moreover, the mechanisms linking maternal obesity to infertility remain largely understudied. In this study, we investigated how variable susceptibility to obesity in mice affects ovarian steroidogenesis, with a particular focus on the leptin-mediated dysregulation of Nodal signalling pathway in theca cells (TC). MethodsC56BL/6J (B6) and 129S1/SvlmJ (129) mice, models of maternal obesity (MO), were fed a chow diet (CD) and a high fat diet (HFD) for 16 weeks. To investigate the contrasting effects of leptin on ovarian steroidogenesis, B6 mice pharmacologically treated with leptin for 16 days on CD were used to model hyperleptinemia, while homozygous ob/ob (−/−) mice with genetic leptin deficiency, also on a CD, were used to examine the effects of obesity in the absence of leptin. Following the characterisation of the mouse phenotype, gonadal fat (GON), whole ovaries (WO), ovarian TC and granulosa cell (GC) fractions were collected for mRNA transcription and protein expression analysis. Finally, in vitro treated ovarian explants obtained from B6 mice were used to further elucidate the effects of Nodal on steroidogenesis. ResultsThe significant gain in body weight (BW) and fat mass (FM) in HFD-fed B6 mice (p < 0.05), was associated with increased mRNA transcription of the adipose tissue expansion genes Polymerase I and transcript release factor (Cavin), Secreted frizzled-related protein 5 (Sfrp5) and Mesoderm specific transcript (Mest) in GON (p < 0.05). Furthermore, the HFD-fed B6 mice presented also impaired glucose metabolism and insulin sensitivity (p < 0.05). In contrast, the HFD-fed 129 mice exhibited no changes in BW and FM, maintaining glucose and insulin metabolism. At the ovarian level, decreased protein expression of Steroidogenic Acute Regulatory Protein (StAR) in WO obtained from HFD-fed B6 mice (p = 0.05), was followed by reduced transcription of key steroidogenic genes like Star and Cytochrome P450 17a1 (Cyp17a) in TC (p < 0.05). Furthermore, the transcription of Nodal and its receptors was downregulated (p < 0.05), whereas mRNA levels of Suppressor of cytokine signalling 3 (Socs3) and SMAD family member 7 (Smad7) were upregulated in TC obtained from HFD-fed B6 mice (p < 0.05). No changes were seen in the genes regulating steroidogenesis, Nodal signalling, or Socs3 and Smad7 activity in the ovaries of HFD-fed 129 mice. Importantly, the pharmacological treatment of lean mice with leptin, upregulated the ovarian transcription of Socs3 and Smad7, while downregulating Nodal and its receptors (p < 0.05). Finally, in vitro pharmacological inhibition of Nodal signalling pathway in ovarian explants isolated from CD-fed B6 mice decreased the transcription of Star and Cyp17a in TC (p < 0.05), whereas Nodal treatment of explants obtained from HFD-fed B6 mice restored the transcription of both genes (p < 0.05). ConclusionsIncreased susceptibility to obesity in MO is associated with systemic hyperleptinemia and hypoestrogenism due to compromised ovarian steroidogenesis, largely driven by the inhibitory effects of leptin-Smad7 pathway on Nodal signalling activity in the TC compartment of ovarian follicles.

Western diet promotes the progression of metabolic dysfunction-associated steatotic liver disease in association with ferroptosis in male mice.

Non-alcoholic fatty liver disease (NAFLD), now referred to as metabolic dysfunction-associated steatotic liver disease (MASLD), is a silent killer that often progresses to metabolic dysfunction-associated steatohepatitis (MASH). To date, there are no pharmacological treatments for MASLD. While obesity is a major cause of the development and progression of MASLD, the underlying mechanisms remain unclear. We hypothesize that ferroptosis, a recently discovered nonapoptotic iron-dependent form of cell death, is activated during the progression of MASLD and may be a potential target for treating MASLD. Using a murine model of Western diet-induced obesity, C57BL/6J male mice were exposed to a long-term (36 weeks) Western diet. Controls were maintained with a standard chow diet. Western diet-induced obesity was confirmed by increased body mass index (BMI). Histopathological analysis demonstrated the progression of MASLD to MASH in the obese group, which was accompanied by significant hepatic iron deposition, oxidative damage, and lipid peroxidation. Hepatic ferroptosis was further confirmed by decreased protein expression of glutathione peroxidase 4 (GPX4) and increased acyl-CoA synthetase long-chain family member 4 (ACSL4), markers of ferroptosis. These findings suggest that ferroptosis is a potential mechanism involved in the pathogenesis of MASLD in male mice.

The effect of biocide chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT) mixture on C2C12 muscle cell damage attributed to mitochondrial reactive oxygen species overproduction and autophagy activation

ABSTRACT The mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMIT/MIT) is a biocide widely used as a preservative in various commercial products. This biocide has also been used as an active ingredient in humidifier disinfectants in South Korea, resulting in serious health effects among users. Recent evidence suggests that the underlying mechanism of CMIT/MIT-initiated toxicity might be associated with defects in mitochondrial functions. The aim of this study was to utilize the C2C12 skeletal muscle model to investigate the effects of CMIT/MIT on mitochondrial function and relevant molecular pathways associated with skeletal muscle dysfunction. Data demonstrated that exposure to CMIT/MIT during myogenic differentiation induced significant mitochondrial excess production of reactive oxygen species (ROS) and a decrease in intracellular ATP levels. Notably, CMIT/MIT significantly inhibited mitochondrial oxidative phosphorylation (Oxphos) and reduced mitochondrial mass at a lower concentration than the biocide amount, which diminished the viability of myotubes. CMIT/MIT induced activation of autophagy flux and decreased protein expression levels of myosin heavy chain (MHC). Taken together, CMIT/MIT exposure produced damage in C2C12 myotubes by impairing mitochondrial bioenergetics and activating autophagy. Our findings contribute to an increased understanding of the underlying mechanisms associated with CMIT/MIT-induced adverse skeletal muscle health effects.

Construction of a tumor mutational burden-derived LncRNA prognostic computational framework associated with therapy sensitivity in skin cutaneous melanoma

BackgroundSkin cutaneous melanoma (SKCM) poses a significant public health challenge due to its aggressive nature and limited treatment options. To address this, the study introduces the Tumor Mutational Burden-Derived Immune lncRNA Prognostic Index (TILPI) as a potential prognostic tool for SKCM.MethodsTILPI was developed using a combination of gene set variation analysis, differential expression analysis, and COX regression analysis. Additionally, functional experiments were conducted to validate the findings, focusing on the effects of STARD4-AS1 knockdown on SKCM tumor cell behavior. These experiments encompassed assessments of tumor cell proliferation, gene and protein expression, migration, invasion, and in vivo tumor growth.ResultsThe results demonstrated that knockdown of STARD4-AS1 led to a significant reduction in tumor cell proliferation and impaired migration and invasion abilities. Moreover, it resulted in the downregulation of ADCY4, PRKACA, and SOX10 gene expression, as well as decreased protein expression of ADCY4, PRKACA, and SOX10. In vivo experiments further confirmed the efficacy of STARD4-AS1 knockdown in reducing tumor growth.ConclusionsThis study elucidates the mechanistic role of STARD4-AS1 and its downstream targets in SKCM progression, highlighting the importance of the ADCY4/PRKACA/SOX10 pathway. The integration of computational analysis with experimental validation enhances the understanding of TILPI and its clinical implications. Overall, the findings underscore the potential of novel computational frameworks like TILPI in predicting and managing SKCM, particularly through targeting the ADCY4/PRKACA/SOX10 pathway.

Naringin alleviates fluoride-induced neurological impairment: A focus on the regulation of energy metabolism mediated by mitochondrial permeability transition pore

The neurological impairment induced by fluoride is associated with mitochondrial dysfunction. Normal mitochondrial permeability transition pore (mPTP) opening plays a pivotal role in mitochondrial function. However, it remains unclear whether p53-dependent mPTP-related mitochondrial apoptosis is associated with fluoride-induced neurological impairment, and the alleviation of naringin on those. In vivo, NaF-treated rats had impaired learning and memory abilities, damaged hippocampal structure, and higher respiratory exchange rates (RER). In vitro, the increased apoptosis rates, excessive opening of mPTP, and decreased mitochondrial membrane potential (MMP) were observed in PC12 cells treated with NaF. The protein expressions of p53, CytoC, and cleaved caspase 3 were significantly increased in hippocampi of rats treated with 50 mg/L and 100 mg/L NaF and in 40 mg/L and 80 mg/L NaF-treated PC12 cells, while the protein expression of CypD remains stable. And the changes of p53 and CypD were also confirmed by the immunofluorescence staining in vivo. After inhibiting the expression of p53 with pifithrin-α and p53-siRNA, the decreased apoptosis rates and mPTP opening, increased MMP, and decreased protein expressions of p53, CytoC, and cleaved caspase 3 were observed in NaF-treated PC12 cells. Rats, treated with NaF and naringin, had alleviated impaired neurological function, and had lower RER than rats treated with NaF alone. And compared with those in the NaF group, the decreased apoptosis rates and mPTP opening, and increased MMP were also found in PC12 cells treated with NaF and naringin. Furthermore, hippocampi of rats and PC12 cells treated with NaF and naringin had decreased protein expressions of p53, CytoC, and cleaved caspase 3. Our results indicate that fluoride activates the p53-dependent mPTP-related mitochondrial apoptosis, which then affects energy metabolism, resulting in neurological impairment. Additionally, naringin can alleviate this damage, and further studies on the potential health benefits of naringin are needed.

Integration of metabolomics and transcriptomics to reveal metabolic characteristics and key targets associated with lncRNA Vof-16 in H19-7 cells

Cognitive disorders represent one of the most common chronic complications of diabetes. Our previous study has demonstrated that long non-coding RNA (lncRNA) Vof-16 is upregulated in the hippocampal tissue of streptozotocin (STZ)-induced diabetic rats. Despite this finding, the specific roles and underlying mechanisms of lncRNA Vof-16 in diabetes-related cognitive dysfunction remain largely unexplored. To elucidate the mechanism involved, lncRNA Vof-16 was overexpressed in rat hippocampal cell line H19-7 through lentivirus transfection. We integrated metabolomics and transcriptomics approaches to identify potential targets and metabolic pathways influenced by lncRNA Vof-16. Key proteins and pathways were subsequently validated using western blotting and immunofluorescence staining. Transcriptomics indicated that lncRNA Vof-16 overexpression may modulate autophagic activity in H19-7 cells. Metabolomic profiling revealed that the primary differential metabolic pathways included trehalose degradation, tryptophan metabolism, vitamin B6 metabolism, glycolysis, pterine biosynthesis, and the pentose phosphate pathway. Ingenuity Pathway Analysis (IPA) of gene-metabolite networks demonstrated that the high lncRNA Vof-16 expression group exhibited a significantly higher association with autophagy compared to the low lncRNA Vof-16 expression group. Western blot results confirmed that lncRNA Vof-16 overexpression led to decreased protein expression levels of ATG3 and ATG12. Specifically, lncRNA Vof-16 reduces autophagy in hippocampal neurons by targeting the elevated levels of phospho-p70S6K, a downstream effector of mTORC1, potentially contributing to the pathogenesis of diabetic cognitive impairment. The construction of gene-metabolite networks may offer promising new strategies for addressing the growing issue of diabetic cognitive impairment.

SIRT2 deacetylates and decreases the expression of FOXM1 in colon cancer.

New FOXM1-specific inhibitors with the potential to be used for therapeutic purposes are under extensive research. We hypothesized that deacetylation of FOXM1 would decrease protein expression, thus providing novel therapeutic management of colon cancers. Immunostaining was used to determine FOXM1 and SIRT2 expressions in human colon cancer tissue microarrays (n = 90) from Stage I to Stage IV. SIRT2-FOXM1 interaction was evaluated in colon cancer cells using immunoprecipitation. Deacetylation of FOXM1 via SIRT2 was determined using in vitro deacetylation assays. FOXM1 could be hyper-acetylated when p300 and pCAF histone acetyltransferases were administered alongside deacetylase inhibitors. We detected that SIRT2 and FOXM1 physically interacted, and SIRT2 deacetylated FOXM1 in vitro. SIRT2 overexpression led to a significant decrease while knockdown of SIRT2 increased the FOXM1 expression in HCT116 human colon carcinoma cells. In the analysis of 90 human colorectal cancer samples, high SIRT2 expression was observed in about 49% of colorectal cancer, intermediate in 29%, and low or no staining in 22%. Strong SIRT2 expression was found to be negatively associated with the FOXM1 staining in our clinical cohort. This study reveals a molecular interaction and association between SIRT2 and FOXM1 expression in colon cancer cell lines and human colon cancer samples, and suggests that targeting SIRT2 activity using small molecule modulators may be a promising therapeutic approach for colorectal cancer.

The role of nicotinamide riboside in the preservation of retinal ganglion cells using an in vitro glutamate-induced excitotoxicity model

Delaying or preventing the loss of retinal ganglion cells (RGCs) in glaucoma is needed for vision preservation. Glutamate-mediated neurotoxicity arises from the excessive stimulation of N-methyl-D-aspartate membrane receptors by glutamate. This overstimulation, occurring specifically in RGCs, triggers a progressive deterioration of the optic nerve that ultimately leads to the vision loss in glaucoma. Our previous investigation demonstrated that nicotinamide riboside (NR) effectively preserved RGCs in multiple mouse models of glaucoma. To investigate the precise role of NR concerning RGCs which remains uncertain, a glutamate-induced excitotoxicity RGCs damage model was established using R28 cells in this study. Results showed that NR treatment could not only prevent the decrease in cell viability but also effectively inhibit the apoptosis of R28 cells induced by glutamate, as proven by flow cytometry and expression of key pro-apoptotic proteins. Additionally, it significantly attenuated oxidative stress induced by glutamate, as evaluated by the production of inflammatory factors, reactive oxygen species (ROS) and mitochondrial ROS (mtROS). Furthermore, NR elevated the intracellular nicotinamide adenine dinucleotide (NAD+) levels in R28 cells. Lastly, we used RNA-seq to reveal the underlying mechanism of NR protection. Combining the results of RNA-seq and Western blot, we found that NR also restored the decreased protein expression of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator (PGC1α) induced by glutamate. These findings strongly indicated that NR exhibits a protective effect against R28 cell apoptosis in a glutamate-induced excitotoxicity RGCs damage model. This protective effect is likely mediated through the activation of the SIRT1/PGC1α pathway, achieved by increasing intracellular NAD + levels.

Interference of FZD2 suppresses proliferation, vasculogenic mimicry and stemness in glioma cells via blocking the Notch/NF‑κB signaling pathway.

Frizzled family protein 2 (FZD2) is widely associated with tumor development and metastasis. The present study aimed to gain an insight into the role and regulatory mechanism of FZD2 in glioma. The expression level of FZD2 in normal astrocyte and glioma cells was determined by reverse transcription-quantitative PCR and western blotting, and cell transfection was conducted for FZD2 expression knockdown. Malignant behaviors including cell proliferation, migration and invasion, vasculogenic mimicry (VM) and cell stemness were determined using Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine (EdU) staining, colony formation, wound healing, Transwell, 3D culturing and sphere formation assays. The expression levels of proteins related to stemness, epithelial-mesenchymal transition (EMT) and Notch/NF-κB signaling were measured by western blotting. Then, the Notch agonist, Jagged-1 (JAG), was adopted for rescue experiments. The results demonstrated that FZD2 was highly expressed in glioma cells. Interference of FZD2 expression suppressed the proliferation of glioma cells, as evidenced by the reduced cell viability and the number of EdU+ cells and colonies. Meanwhile, the reduced sphere formation ability and decreased protein expression of Nanog, Sox2 and Oct4 following FZD2 knockdown confirmed that FZD2 repressed cell stemness in glioma. Additionally, FZD2 knockdown suppressed the migration, invasion, EMT and VM formation capabilities of glioma cells, and also blocked the Notch/NF-κB signaling pathway. Furthermore, activation of Notch by JAG treatment partially reversed the aforementioned FZD2 knockdown-mediated changes in glioma cell malignant behaviors. In conclusion, FZD2 may contribute to glioma progression through activating the Notch/NF-κB signaling pathway, providing a plausible therapeutic target for the treatment of glioma.

Methotrexate-Loaded solid lipid nanoparticles enhance the viability of cutaneous flaps: potential for surgical wound healing

Skin flaps are employed to cover cutaneous denuded surfaces, but ensuing flap necrosis often occurs. Previously, rats with myocardial infarction treated with lipid-core nanoparticles (LDE) loaded with methotrexate (MTX) improved myocardial irrigation and reduced necrosis. Here, the aim was to investigate the efficacy of LDE-MTX to preserve the viability of cutaneous flaps and its implications for surgical wound healing. Twenty-eight male rats were divided into 4 groups: (1) LDE, injected intraperitoneally with LDE only; (2) MTX (1 mg/Kg commercial MTX): (3) LDE-MTX (1 mg/Kg MTX associated with LDE), and controls without treatment. LDE, MTX or LDE-MTX were repeated after 2 days. Then, flap surgery (9x3cm) was performed on the dorsal region. Injections were continued every other day until day 7 when animals were euthanized. LDE-MTX treatment improved the total viable area of the flaps with a fourfold increase in blood flow and reduced inflammatory cell number (p < 0.001), accompanied by decreased protein expression of pro-inflammatory factors. SOD-1 was higher in LDE-MTX-treated rats (p < 0.05). In conclusion, LDE-MTX treatment achieved total viability of cutaneous flaps, with increased irrigation and diminished local inflammation. LDE-MTX may offer efficient and cost-effective prevention of cutaneous flaps and treatment for wounds from surgical procedures to be tested in future clinical studies.

USP15-Mediated Deubiquitination of FKBP 5 and Activation of the αIIbβ3 Signaling Pathway Regulate Thrombosis in Mice.

Platelets have the hemostatic function, and their aberrant activation is associated with occlusive thrombus formation. Plasma exosomes are rich in platelets containing ubiquitin-specific peptidase 15 (USP15). Herein, we aim to explore the effect of USP15 on thrombosis, as well as expounding whether USP15 acts as an upstream target of FK506 binding protein 5 (FKBP5) to regulate occlusive thrombus formation. Washed human platelets were treated with thrombin for measurement of USP15 and FKBP5 expressions. USP15 loss/gain-of-function variant in HEK293 cells was performed by cell transfection, and the interaction between USP15 and FKBP5 was examined using immunoprecipitation and ubiquitination assays. Mice with USP15-knockout platelets (Plt USP15-/-) were modeled, and subjected to calculation of bleeding time, artery thrombosis imaging and clot retraction assay. FKBP5 expression and the inhibitor of nuclear factor kappa B kinase subunit epsilon (IKBKE)/phosphatidylinositol 3-kinase (PI3K)/Rap1 pathway in wild-type and Plt USP15-/- mice-derived platelets were detected using Western blot. The activation of αIIbβ3 in washed platelets was analyzed using flow cytometry. USP15 and FKBP5 expressions were upregulated in platelets after thrombin treatment. Following transfection of USP15 knockdown and USP15 overexpression plasmids into HEK293 cells, FKBP5 protein expression was downregulated by USP15 knockdown while being upregulated by USP15 overexpression. USP15 bound to FKBP5 and protected FKBP5 against ubiquitination. Knockdown of platelet USP15 prolonged bleeding time, inhibited arterial thrombosis and delayed clot retraction in mice. Knockdown of platelet USP15 also decreased protein expressions of FKBP5, IKBKE and Rap1, p-PI3K/PI3K ratio, and activation of αIIbβ3 in mice. USP15 knockdown in platelets affects thrombosis in mice by promoting the instability of FKBP5 to repress the activation of IKBKE/PI3K/Rap1 pathway-mediated αIIbβ3.

Extracorporeal Shock Wave and Melatonin Alleviate Joint Capsule Fibrosis after Knee Trauma in Rats by Regulating Autophagy.

Joint contracture is a common clinical problem affecting joint function. Capsule fibrosis plays a pivotal role in the progression of Joint contracture. Previous studies have reported that autophagy plays a regulatory role in visceral fibrosis. This study aimed to investigate whether extracorporeal shock wave therapy (ESWT) and melatonin alleviate joint capsule fibrosis in rats with extended knee joint contracture by regulating autophagy. A rat knee joint extension contracture model was made. Then, the rats were treated with ESWT, melatonin, ESWT + melatonin, or ESWT + melatonin + mTOR agonist for 4 weeks. The range of motion (ROM) of the knee joints was measured. Joint capsules were collected and observed for pathological changes by H&E and Masson staining. LC3B protein expression was evaluated by immunofluorescence staining. TGF-β1, MMP-1, Col-Ⅰ, Col-Ⅲ, LC3, ATG7, Beclin1, p-AMPK, p-mTOR and p-ULK1 protein expressions were measured by Western blot assay. The intervention groups had significantly improved ROM of knee joint (P < 0.05), significantly improved pathological changes on HE and Masson staining, significantly decreased protein expressions of TGF-β1, MMP-1, Col-Ⅰ, Col-Ⅲ and pmTOR (P < 0.05), and significantly increased protein expressions of LC3B, LC3II/LC3I ratio, ATG7, Beclin1, p-AMPK, and p-ULK1 (P < 0.05). Among these groups, the effects demonstrated by the ESWT + melatonin group were the best. With the mTOR agonist supplement, the therapeutic effects of extracorporeal shock waves and melatonin were significantly reduced. ESWT plus melatonin alleviated knee joint capsule fibrosis in rats by regulating autophagy.