Decreased mRNA Levels Research Articles

Loss of the Endothelial Glycocalyx Component EMCN Leads to Glomerular Impairment.

EMCN (endomucin), an endothelial-specific glycocalyx component, was found to be highly expressed by the endothelium of the renal glomerulus. We reported an anti-inflammatory role of EMCN and its involvement in the regulation of VEGF (vascular endothelial growth factor) activity through modulating VEGFR2 (VEGF receptor 2) endocytosis. The goal of this study is to investigate the phenotypic and functional effects of EMCN deficiency using the first global EMCN knockout mouse model. Global EMCN knockout mice were generated by crossing EMCN-floxed mice with ROSA26-Cre mice. Flow cytometry was used to analyze infiltrating myeloid cells in the kidneys. The ultrastructure of the glomerular filtration barrier was examined by transmission electron microscopy, whereas urinary albumin, creatinine, and total protein levels were analyzed from freshly collected urine samples. Expression and localization of EMCN, EGFP, CD45, CD31, CD34, podocin, and albumin were examined by immunohistochemistry. Mice were weighed regularly, and their systemic blood pressure was measured using a noninvasive tail-cuff system. Glomerular endothelial cells and podocytes were isolated by fluorescence-activated cell sorting for RNA sequencing. Transcriptional profiles were analyzed to identify differentially expressed genes in both endothelium and podocytes, followed by gene ontology analysis. Protein levels of EMCN, albumin, and podocin were quantified by Western blot. The EMCN-/- mice exhibited increased infiltration of CD45+ cells, with an increased proportion of Ly6GhighLy6Chigh myeloid cells and higher VCAM-1 (vascular cell adhesion molecule 1) expression. EMCN-/- mice displayed albuminuria with increased albumin in the Bowman's space compared with the EMCN+/+ littermates. Glomeruli in EMCN-/- mice revealed fused and effaced podocyte foot processes and disorganized endothelial fenestrations. We found no significant difference in blood pressure between EMCN knockout mice and their wild-type littermates. RNA sequencing of glomerular endothelial cells revealed downregulation of cell-cell adhesion and MAPK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated kinase) pathways, along with glycocalyx and extracellular matrix remodeling. In podocytes, we observed reduced VEGF signaling and alterations in cytoskeletal organization. Notably, there was a significant decrease in both mRNA and protein levels of podocin, a key component of the slit diaphragm. Our study demonstrates a critical role of the endothelial marker EMCN in supporting normal glomerular filtration barrier structure and function by maintaining glomerular endothelial tight junction and homeostasis and podocyte function through endothelial-podocyte crosstalk.

Preservation of Murine Whole Eyes With Supplemented UW Cold Storage Solution: Anatomical Considerations.

Retinal ganglion cell (RGC) apoptosis and axon regeneration are the principal obstacles challenging the development of successful whole eye transplantation (WET). The purpose of this study was to create a neuroprotective cocktail that targets early events in the RGC intrinsic apoptotic program to stabilize RGCs in a potential donor eye. University of Wisconsin (UW) solution was augmented with supplements known to protect RGCs. Supplements targeted tyrosine kinase signaling, histone deacetylase activity, K+ ion efflux, macroglial stasis, and provided energy support. Modified UW (mUW) solutions with individual supplements were injected into the vitreous of enucleated mouse eyes, which were then stored in cold UW solution for 24 hours. Histopathology, immunostaining of individual retinal cell types, and analysis of cell-specific messenger RNAs (mRNAs) were used to identify supplements that were combined to create optimal mUW solution. UW and mUW solutions reduced ocular edema and focal ischemia in globes stored in cold storage. Two major issues were noted after cold storage, including retinal detachment and reduction in glial fibrillary acidic protein staining in astrocytes. A combination of supplements resolved both these issues and performed better than the individual supplements alone. Cold storage resulted in a reduction in cell-specific mRNAs, even though it preserved the corresponding protein products. Eyes treated with optimal mUW solution exhibited preservation of retinal and cellular architecture, but did display a decrease in mRNA levels, suggesting that cold storage induced cellular stasis. Application of optimal mUW solution lowers an important barrier to the development of a successful whole eye transplantation procedure.

CSIG-01. DECREASING EXPRESSION OF TRAF PROTEINS WITH INCREASING MALIGNANCY WITH LOWEST EXPRESSION IN IDH-WILDTYP GLIOBLASTOMA

Abstract The tumor necrosis factor receptor-associated factors (TRAF) are cytoplasmatic adapter proteins that can interact directly with the intracellular domains of cell surface receptors, such as the TNF receptor superfamily. TRAFs are thus involved in cellular processes like proliferation or apoptosis. Seven members are known; all expressed in the cytosol of various cell and tissue types. Increased levels of TRAF4 in GBM tissue are related to cell proliferation and migration. About 25 % of grade I meningioma show mutations in TRAF7, which are associated with a lower risk of malignant transformation. Therefore expression patterns of all seven TRAF members are evaluated in different glioma grades. Following tumor samples were obtained during neurosurgery and shock frozen in liquid nitrogen: peritumoral tissue, astrocytoma and oligodendroglioma (G2, G3) and glioblastoma (primary and recurrent). Transcription rate of TRAF1-7 was measured via qPCR. Correlation of TRAF expression level and patient overall survival was evaluated using TCGA datasets of the TCGA-LGG” and “TCGA-GBM” projects. With the exception of TRAF5, a decreased mRNA level with increasing malignancy could be found for all TRAFs (TRAF1 p=n.s.; TRAF2 p=0.0026; TRAF3 p<0.0001; TRAF4 p=0.0021; TRAF6 p<0.0001; TRAF7 p<0.0001). IDHwt glioblastoma showed significant lower expression level compared to IDHmut glioblstoma in all TRAFs except for TRAF1 (TRAF2 p<0.0001; TRAF3 p=0.0346; TRAF4 p<0.0001; TRAF6 p=0.0447; TRAF7 p=0.0081). TRAF5 again showed a reverse effect (p=0.0065). Correlation of TRAF expression level in low grade glioma and patient survival revealed longer overall survival with low TRAF1,2,4 and TRAF5 expression (p<0.0001, p= 0.0178, p=0.0307, p<0.0001). No difference in survival rate in high grade glioma could be found.

Perinatal bisphenol A exposure has an age- and dose-dependent association with thyroid allostasis adaptive response, as well as anxiogenic-depressive-like and asocial behaviors in juvenile and adult male rats

Thyroid hormones are essential for brain development, and a shortage throughout the fetal and postnatal periods can result in mood disorders. Perinatal exposure to bisphenol A (BPA) affects thyroid activity and dependent processes indirectly during pregnancy or early postnatal life. This is particularly important because it may cause changes in tissue ontogeny, increasing the risk of developing disorders later in life. The study aimed to investigate the consequences of thyroid hormone deficiency on anxiety, social, and depressive behaviors, as well as disruption in thyroid peroxidase (TPO) gene expression, which influences the NF-κB/Nrf-2/HO-1/iNOS signaling pathway, leading to oxidative stress, inflammation, and DNA fragmentation in perinatal BPA exposure (PND18), and whether these effects can be observed in juvenile (PND60) and adult (PND95) male offspring rats. BPA increased anxiety-like behavior while decreasing sucrose preference and sociability on a choice task between novel conspecific male rats and enhanced immobility on the forced swim test. Perinatal exposure to BPA causes thyroid insult by overproducing ROS, increasing iNOS, and NF-κB levels—these effects, in turn, down-regulate Nrf-2/HO-1 signaling, resulting in DNA fragmentation within thyroid tissues. Furthermore, perinatal BPA exposure for 60 and 95 days resulted in a significant fold decrease in TPO mRNA levels in the thyroid tissues, with an insignificant fold rise in TPO expression levels in BPA 50–60. In conclusion, the present study found that perinatal BPA exposure induced thyroid allostasis-adaptive response by inhibiting the NF-κB/Nrf-2/HO-1/iNOS signaling pathway and altering the transcriptional expression of TPO, where TSH reinforced a possible association with TPO activity, disrupting thyroid hormone synthesis in juvenile rats and gradual deterioration reaching the adult stage.

Milk fat globule membrane attenuates acute Salmonella typhimurium infection induced mouse model of colitis and ameliorates intestinal immunity

Research investigating the effects of milk fat globule membranes (MFGM) on Salmonella typhimurium-induced colitis, intestinal barrier integrity, and gut-associated immune responses remains scarce. This study aimed to investigate the potential of dietary MFGM to alleviate S. typhimurium-induced colitis in mice. Male C57BL/6J mice were administered S. typhimurium to induce colitis and were then subjected to dietary interventions with either bovine MFGM (BMFGM) or goat MFGM (GMFGM). Both BMFGM and GMFGM mitigated S. typhimurium-induced colitis, as evidenced by increased body weight, decreased mRNA levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β, and IL-6) in the colon, reduced intestinal permeability, and increased mRNA expression of tight junction-associated genes (Claudin-1 and ZO-1). Additionally, there was a reduction in the number of S. typhimurium in the mesenteric lymph nodes and an improvement in CD4 T cell activation status. Furthermore, liver and spleen injury was reduced, and serum levels of pro-inflammatory cytokines (IFN-γ, IL-1β, IL-4, IL-2, IL-6, and TNF-α) were decreased by both type of MFGM. Overall, BMFGM and GMFGM effectively alleviated S. typhimurium-induced colitis and associated hepatic and splenic injuries.

Impaired Development of Collagen Antibody-Induced Arthritis in Rab44-Deficient Mice.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by immune cell-mediated joint inflammation and subsequent osteoclast-dependent bone destruction. Collagen antibody-induced arthritis (CAIA) is a useful mouse model for examining the inflammatory mechanisms in human RA. Previously, we identified the novel gene Rab44, which is a member of the large Rab GTPase family and is highly expressed in immune-related cells and osteoclasts. In this study, we induced CAIA in Rab44-knockout (KO) mice to investigate the effects of Rab44 on inflammation, cell filtration, and bone destruction. Compared with wild-type (WT) mice, Rab44-KO mice showed reduced inflammation in arthritis under CAIA-inducing conditions. Rab44-KO CAIA mice exhibited reduced cell filtration in the radiocarpal joints. Consistent with these findings, Rab44-KO CAIA mice showed decreased mRNA levels of arthritis-related marker genes including genes for inflammation, cartilage turnover, bone formation, and bone absorption markers. Rab44-KO CAIA mice exhibited predominant infiltration of M2-type macrophages at inflammatory sites and reduced bone loss compared to WT CAIA mice. These results indicate that Rab44 deficiency reduces the progression of inflammation in CAIA in mice.

An initial investigation of transcutaneous delivery of plasmid DNA encoding interleukin-10 for the treatment of psoriatic skin conditions

Psoriasis is a chronic immune-mediated skin disorder characterized by intense local inflammation, epidermal hyperplasia, and leukocyte infiltration. Current treatment approaches for psoriasis aim to alleviate symptoms and prevent disease progression, including systemically administered drugs with whole body side effects. Despite some advances in psoriasis treatment, success has been quite limited. To begin to address this challenge, we undertook an initial investigation of whether transcutaneous delivery of an endogenous anti-inflammatory cytokine could provide an effective, local treatment of psoriatic-like skin conditions. To do this, we utilized a previously documented rodent model of psoriasis, induced via a single topical application of Imiquimod (IMQ) to the shaved back of rats. The therapeutic approach used for this initial investigation was delivery of plasmid DNA encoding rat interleukin-10 (pDNA-rIL10), a non-viral gene therapy approach previously shown to be effective in suppressing neuroinflammatory disorders after localized delivery either intracerebrally or intrathecally. Translation of this CNS therapeutic for use in psoriatic-like skin disorders required reformulation to enable transcutaneous delivery. Toward that end, pDNA-rIL10 was topically applied in Lipoderm HMW, a base explicitly designed to deliver higher molecular weight compounds into skin. Here we show that a single topical application of pDNA-rIL10 in Lipoderm HMW was effective in decreasing mRNA levels of pro-inflammatory cytokines as well as reducing the recruitment of T-cells to IMQ-treated skin. Furthermore, this transcutaneous IL-10 gene therapy decreased signs of skin inflammation, reflected by reduced erythema. Moreover, the results provide an initial indication that IL10 may stimulate hair regrowth in psoriatic-like skin.

Vitamin E improves oxidative stress, apoptosis, and steroidogenesis impairment in glyphosate-induced mice

Background Glyphosate (Gly) is a nonselective pesticide with high potential to toxic effects on the reproductive system. Recent studies suggest that Vitamin E can indeed have a positive impact on the reproductive system, while Gly, a nonselective pesticide, has been linked to significant risks of toxicity on reproductive health. It’s crucial to be mindful of the potential impacts of such substances on the reproductive system. Methods Seventy female mice were categorized into seven groups.: (1) control, (2) olive oil (as Vit E solvent), (3) Vit E, (4) L-Gly, (5) H-Gly, (6) L-Gly + Vit E, and (7) H-Gly + Vit E. Various factors related to oxidative stress, antioxidant activity, steroid hormones, and mRNA expression levels of genes involved in apoptosis and steroidogenesis were assessed. Results After treatment with both doses of Gly, the malondialdehyde level significantly increased in comparison to the control group. Conversely, the activity of catalase and the levels of glutathione, estrogen, and progesterone decreased. Treatment of mice with both doses of Gly resulted in increased mRNA levels of Bax, caspase-3, and caspase-9 genes, as well as a decreased mRNA level of the Bcl-2 gene and factors involved in steroidogenesis (StAR and 3-β-HSD) comparison to the control group. Conclusion These results offer that vitamin E could help improve some of the adverse effects of Gly in the ovaries of female mice.

Activation of nuclear receptor pregnane-X-receptor protects against abdominal aortic aneurysm by inhibiting oxidative stress

Abdominal aortic aneurysm (AAA) is a life-threatening condition, but effective medications to prevent its progression and rupture are currently lacking. The nuclear receptor pregnane-X-receptor (PXR) plays a crucial role in vascular homeostasis. However, the role of PXR in AAA development remains unknown. We first detected the PXR expression in human and murine AAA tissues by RT-qPCR and Western blot. To investigate the potential role of PXR in the development of AAA, we used adeno-associated virus-mediated overexpression of PXR and pharmacological activation of PXR by ginkgolide A (GA) in mouse AAA models induced by both angiotensin II (AngII) and calcium phosphate [Ca3(PO4)2]. The underlying mechanism was further explored using RNA-sequencing and molecular biological analyses. We found a significant decrease in both mRNA and protein levels of PXR in both human and murine aortic smooth muscle cells from AAA tissues, accompanied with phenotypic switching of vascular smooth muscle cell and increased oxidative stress. PXR overexpression in abdominal aortas and GA treatment successfully suppressed AAA formation in both mouse AAA models. RNA-sequencing data revealed that PXR activation inhibited gamma-aminobutyric acid type A receptor subunit alpha3 (GABRA3) expression. Additional mechanistic studies identified that PXR suppressed AAA through mitigating GABRA3-induced reactive oxygen species (ROS) generation and subsequent phosphorylation of c-Jun N-terminal kinase (JNK). Interestingly, p-JNK was found to induce ubiquitin-proteasome degradation of PXR. In summary, our data unveiled, for the first time, the protective role of PXR against AAA pathogenesis by inhibiting oxidative stress. These findings suggested PXR as a promising therapeutic target for AAA.

Effects of PPARG on the proliferation, apoptosis, and estrogen secretion in goat granulosa cells

As a member of peroxisome proliferator-activated receptor (PPAR) family, PPARG has been reported to be involved in glucolipid metabolism in various species. However, the function of PPARG in estradiol (E2) synthesis, apoptosis, and proliferation in goat ovarian granulosa cells (GCs) is unclear. In this study, we found that goat PPARG was expressed in GCs of all grades of follicles, and localized in the cytoplasm and nucleus of GCs. Transfection of small interfering RNA-PPARG2 (si-PPARG2) decreased E2 synthesis and the abundances of HSD3B and CYP19A1 mRNA and protein. It also promoted cell apoptosis with significant increases in the ratio of BAX/BCL-2 and Caspase3 mRNA and protein. Meanwhile, cell cycle was inhibited by si-PPARG2 transfection, accompanied by decreased mRNA levels of CDK4, CKD6, MYC, CCND1, CCND2, PCNA, and CCNB, increased mRNA level of P53, and decreased protein levels of CDK4, MYC, and CCND1. Furthermore, PPARG interference affected the mRNA and protein abundances of CREB as well as the phosphorylation of CREB but not PKA. In conclusion, our data suggest that PPARG plays an important role in regulating E2 synthesis, cell apoptosis, and proliferation of goat GCs, including the CREB expression and phosphorylation. These results provide evidences for the in-depth study of PPARG in the regulation of goat GCs function.

MiR-24 regulates obstructive pulmonary disease in rats via S100A8

Purpose Chronic obstructive pulmonary disease (COPD) is a persistent inflammatory disorder characterized by minor airway inflammation and emphysema involving various cell types and cytokines. MicroRNAs (miRNAs) have emerged as critical regulators in the pathogenesis of lung diseases. This study investigates the impact of microRNA-24 (miR-24) on airway inflammatory responses in a rat model of COPD. Materials and methods The model was established by combining cigarette smoke exposure and lipopolysaccharide stimulation, and rat lung tissues were transfected with adeno-associated viruses overexpressing miR-24. Pathological changes in the lung were assessed using hematoxylin and eosin staining. Levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-6, and interleukin-8, were measured using enzyme-linked immunosorbent assay. Expression of miR-24 and S100A8 was detected through quantitative reverse transcription PCR, while protein levels of S100A8, Toll-like receptor 4 (TLR4), and myeloid differentiation primary response 88 (MyD88) were assessed using western blotting. Bioinformatics analysis and dual-luciferase reporter assay were performed to determine the relationship between S100A8 and miR-24. Results The results demonstrated the downregulation of miR-24 in rats with COPD, and its overexpression resulted in a significant decrease in S1008 mRNA levels. Additionally, the protein level of S100A8 was significantly increased in the lung tissues of COPD rats. The upregulation of miR-24, however, not only inhibited the protein expression of S100A8, TLR4, and MyD88 in lung tissues but also reduced the release of pro-inflammatory cytokines in the plasma and bronchoalveolar lavage fluid, thereby attenuating inflammatory responses and pathological injuries in the lung. Conclusions Our data suggest that miR-24 attenuates airway inflammatory responses in COPD by inhibiting the TLR4/MyD88 pathway via targeting S100A8.

Zinc pyrithione ameliorates colitis in mice by interacting on intestinal epithelial TRPA1 and TRPV4 channels

AimsAlthough zinc pyrithione (ZPT) has been studied as topical antimicrobial and cosmetic consumer products, little is known about its pharmacological actions in gastrointestinal (GI) health and inflammation. Our aims were to investigate the effects of ZPT on transient receptor potential (TRP) channels and Ca2+ signaling in intestinal epithelial cells (IECs) and its therapeutic potential for colitis. Main methodsDigital Ca2+ imaging and patch-clamp electrophysiology were performed on human colonic epithelial cells (HCoEpiC) and rat small intestinal epithelial cells (IEC-6). The transcription levels of proinflammatory cytokines such as IL-1β were detected by RTq-PCR. Dextran sulfate sodium (DSS) was used to induce colitis in mice. Key findingsZPT dose-dependently induced Ca2+ signaling and membrane currents in IECs, which were attenuated by selective blockers of transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 4 (TRPV4) channels, respectively. Interestingly, Ca2+ entry via TRPA1 channels inhibited the activity of TRPV4 channels in HCoEpiC, but not vice versa. ZPT significantly promoted migration of IECs by activating TRPA1 and TRPV4 channels. ZPT reversed lipopolysaccharides (LPS)-induced changes in mRNA expression of TRPA1 and TRPV4. Moreover, ZPT decreased mRNA levels of pro-inflammatory factors promoted by LPS in HCoEpiC, which were restored by selective TRPA1 blocker. In whole animal studies in vivo, ZPT significantly ameliorated DSS-induced body weight loss, colon shortening and increases in stool score, serum calprotectin and lactic acid (LD) in mouse model of colitis. SignificanceZPT exerts anti-colitic action likely by anti-inflammation and pro-mucosal healing through TRP channels in IECs. The present study not only expands pharmacology spectrum of ZPT in GI tract, but also repurposes it to a potential drug for colitis therapy.

Effects of treadmill exercise on endoplasmic reticulum protein folding and endoplasmic reticulum-associated protein degradation pathways in APP/PS1 mice

A hallmark of Alzheimer's disease (AD) is the disruption of protein homeostasis (proteostasis), manifested by the misfolding and aggregation of proteins. Molecular chaperones and the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway in the ER are essential for correct protein folding and degradation of misfolded proteins respectively, thus contributing to the maintenance of proteostasis. The present study aimed to investigate whether the beneficial effects of exercise in an AD mice model is associated with changes in ER protein folding and ERAD. APP/PS1 transgenic and wild-type mice were subjected to treadmill exercise for three months. The levels of molecular chaperones, specifically protein disulfide isomerases (PDIs) and heat shock proteins (HSPs), as well as ERAD-associated molecules were analyzed in the hippocampus. The result revealed a decrease in mRNA levels of PDIA2, PDIA3, PDIA4, PDIA5, PDIA6, HSPA1B, HSPA8, HSP90B1, DNAJB2, CRYAB, and CNX, an increase in mRNA levels of HSPA5 and HSPH1, an increase in protein levels of HERPUD1, and a decrease in protein levels of VCP in APP/PS1 mice. However, following a 3-month treadmill exercise regimen, an increase in mRNA levels of PDIA2, PDIA4, PDIA6, HSPA1A, HSPA8, HSP90AB1, and DNAJB2, as well as an increase in protein levels of VCP and DERL2, and a decrease in protein levels of HERPUD1 were noted. Overall, our findings indicate that disruptions in hippocampal ER protein folding and ERAD pathways may be implicated in AD, with exercise serving as a regulator of these pathways.

Betaine Induces Apoptosis and Inhibits Invasion in OSCC Cell Lines.

Betaine, known as trimethylglycine, is a non-toxic natural substance reported to affect cancer cell responses. This study delves into the impact of betaine on the survival, proliferation, and invasion of oral squamous cell carcinoma (OSCC) cells in vitro. Human OSCC cells (HSC-4 and HSC-7) were subjected to varying concentrations of betaine, and their viability and proliferation were assessed through colourimetric MTT and colony-forming unit assays. Cell cycle progression and cell apoptosis were also investigated using flow cytometry, while cell migration and invasion were examined using a transwell migration assay, and the mRNA expression was evaluated by a quantitative polymerase chain reaction. Finally, proteomic analysis was conducted through liquid chromatography-tandem mass spectrometry on the extracted protein component of the cells. Results indicate that betaine effectively suppressed OSCC proliferation and colony formation. It triggered early apoptosis without disrupting cell cycle progression, reduced cell migration, and inhibited invasion. Betaine exposure led to significantly decreased mRNA levels of MMP1, MMP2, and MMP9 while downregulating FN1, a gene linked to epithelial-to-mesenchymal transition. Proteomic analysis revealed 9240 differentially expressed up/downregulated proteins in cells treated with betaine. The significantly upregulated proteins were associated with ATP-binding cassette (ABC) transporters, while the down-regulated proteins were associated with G protein-coupled receptors (GPCR) ligand binding. In conclusion, betaine exhibits potent anti-cancer properties by attenuating OSCC cell proliferation and mitigating invasion. Exploring this natural product as an adjunct for managing oral squamous cell carcinoma shows promise, although further investigations are needed to fully elucidate its functionality.

Tristetraprolin affects invasion-associated genes expression and cell motility in triple-negative breast cancer model.

Tristetraprolin (TTP) is an RNA-binding protein that negatively regulates its target mRNAs and has been shown to inhibit tumor progression and invasion. Tumor invasion requires precise regulation of cytoskeletal components, and dysregulation of cytoskeleton-associated genes can significantly alter cell motility and invasive capability. Several genes, including SH3PXD2A, SH3PXD2B, CTTN, WIPF1, and WASL, are crucial components of the cytoskeleton reorganization machinery and are essential for adequate cell motility. These genes are also involved in invasion processes, with SH3PXD2A, SH3PXD2B, WIPF1, and CTTN being key components of invadopodia-specialized structures that facilitate invasion. However, the regulation of these genes is not well understood. This study demonstrates that ectopic expression of TTP in MDA-MB-231 cells leads to decreased mRNA levels of CTTN and SH3PXD2A, as well as defects in cell motility and actin filament organization. Additionally, doxorubicin significantly increases TTP expression and reduces the mRNA levels of cytoskeleton-associated genes, enhancing our understanding of how doxorubicin may affect the transcriptional profile of cells. However, doxorubicin affects target mRNAs differently than TTP ectopic expression, suggesting it may not be the primary mechanism of doxorubicin in breast cancer (BC) treatment. High TTP expression is considered as a positive prognostic marker in multiple cancers, including BC. Given that doxorubicin is a commonly used drug for treating triple-negative BC, using TTP as a prognostic marker in this cohort of patients might be limited since it might be challenging to understand if high TTP expression occurred due to the favorable physiological state of the patient or as a consequence of treatment.

Radiofrequency field inhibits RANKL-induced osteoclast differentiation in RAW264.7 cells via modulating the NF-κB signaling pathway

ABSTRACT In this study, we investigated the inhibitory effects of radiofrequency exposure on RANKL-induced osteoclast differentiation in RAW264.7 cells, along with the underlying mechanisms. RAW264.7 cells were subjected to radiofrequency exposure at three distinct power densities: 50 µW/cm2, 150 µW/cm2, and 450 µW/cm2. The results showed that, among the three dosage levels, exposure to 150 µW/cm2 of radiofrequency radiation significantly reduced the proliferation capacity of RAW264.7 cells. RF exposure at three power densities resulted in significant increases in the level of osteoclast apoptosis and notable decreases in osteoclast differentiation. Notably, the most pronounced effects on apoptosis, differentiation in RAW 264.7 cells were observed at the 150 µW/cm2 power density. These effects were accompanied by concurrent decreases in mRNA and protein levels of osteoclast-specific genes, including RANK, NFATc1, and TRACP. Furthermore, radiofrequency exposure at power density of 150 µW/cm2 induced a significant decrease in cytoplasmic NF-κB protein levels while increasing its nuclear fraction, thereby counteracting the effects of RANKL-induced NF-κB activation. These data suggest that radiofrequency exerts inhibitory properties on RANKL-induced NF-κB transcriptional activity, subsequently indirectly suppressing the expression of downstream NF-κB target genes, such as NFATc1 and TRACP. In conclusion, our study demonstrates that radiofrequency radiation effectively inhibits osteoclast differentiation by modulating the NF-κB signaling pathway. These findings have important implications for potential therapeutic interventions in osteoporosis.

Simultaneous Silencing of Gut Nucleases and a Vital Target Gene by Adult dsRNA Feeding Enhances RNAi Efficiency and Mortality in Ceratitis capitata.

Ceratitis capitata, known as the Mediterranean fruit fly (Medfly), is a major dipteran pest significantly impacting fruit and vegetable farming. Currently, its control heavily relies mainly on chemical insecticides, which pose health risks and have effects on pollinators. A more sustainable and species-specific alternative strategy may be based on double-stranded RNA (dsRNA) delivery through feeding to disrupt essential functions in pest insects, which is poorly reported in dipteran species. Previous reports in Orthoptera and Coleoptera species suggested that dsRNA degradation by specific nucleases in the intestinal lumen is among the major obstacles to feeding-mediated RNAi in insects. In our study, we experimented with three-day adult feeding using a combination of dsRNA molecules that target the expression of the ATPase vital gene and two intestinal dsRNA nucleases. These dsRNA molecules were recently tested separately in two Tephritidae species, showing limited effectiveness. In contrast, by simultaneously feeding dsRNA against the CcVha68-1, CcdsRNase1, and CcdsRNase2 genes, we observed 79% mortality over seven days, which was associated with a decrease in mRNA levels of the three targeted genes. As expected, we also observed a reduction in dsRNA degradation following RNAi against nucleases. This research illustrates the potential of utilizing molecules as pesticides to achieve mortality rates in Medfly adults by targeting crucial genes and intestinal nucleases. Furthermore, it underscores the importance of exploring RNAi-based approaches for pest management.

7,8‑dihydroxyflavone reduces lipid peroxidation, proinflammatory cytokines, and mediators in chemically induced‑phenylketonuria model.

Phenylketonuria (PKU) stems from a rare genetic metabolic imbalance attributed to an insufficiency in the enzyme phenylalanine hydroxylase. Within the context of PKU, brain‑derived neurotrophic factor (BDNF) plays a pivotal role in brain function. 7,8‑dihydroxyflavone (7,8‑DHF) operates as a tropomyosin receptor kinase B (TrkB) agonist, mimicking the effects of BDNF. This study aimed to examine the effects of administering 7,8‑DHF in chemically‑induced rat models specifically induced to simulate PKU chemically. The rats were subcutaneously injected with phenylalanine and p‑chlorophenylalanine, a phenylalanine hydroxylase inhibitor, along with 7,8‑DHF. The injections began on the 2nd day after birth and continued until the 10th day. Levels of interleukin‑1β (IL‑1β), interleukin‑6 (IL‑6), interleukin‑33 (IL‑33), BDNF, malondialdehyde (MDA), monoamine oxidase (MAO), and superoxide dismutase (SOD) in the brain tissues were quantified using the enzyme‑linked immunosorbent assay (ELISA). Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was performed to assess the gene expressions of inducible nitric oxide synthase (iNOS), nuclear factor kappa beta (NF‑κB), caspase‑3, nuclear factor erythroid 2‑related factor 2 (Nrf2), heme oxygenase‑1 (HO‑1), and BDNF. The results showed a decrease in mRNA levels of iNOS, IL‑1β, IL‑6, and lipid peroxidation in the group that received 7,8‑DHF. These results indicate that administering 7,8‑DHF has the potential to reduce brain damage in PKU by lowering proinflammatory cytokine levels and lipid peroxidation in PKU models. Thus, 7,8‑DHF, as a small molecule, might offer a promising adjunct therapeutic approach for PKU.

Human urine-derived stem cells: potential therapy for psoriasis-like dermatitis in mice

Over the past decade, significant advancements in stem cell research led by mesenchymal stem cells (MSCs) have facilitated their practical application in clinical settings, including inflammatory skin diseases. Urine-derived stem cells (USCs) are obtained from healthy human urine in a noninvasive approach with properties similar to mesenchymal stem cells (MSCs). However, the therapeutic potential of USCs for inflammatory skin diseases has not yet been fully explored. Herein, we report the therapeutic effects of USCs-derived culture supernatants on mice with psoriasis-like dermatitis using our originally established human USCs model. We examined the isolation of USCs from human urine using a simple centrifugation process. Cell markers related to MSCs-like cell were positive for CD29, CD44, CD73, CD90, and negative for HLA-DR, CD34, and CD45 by FACS analysis. Differentiation assays revealed that the cells possessed the capability to differentiate into adipocytes, chondrocytes, and osteocytes. USCs-conditioned medium (CM) treatment significantly suppressed the severity of dermatitis in imiquimod (IMQ)-treated psoriasis mice model. Histopathological examination revealed that USCs-CM treatment attenuated epidermal thickness and the numbers of infiltrating inflammatory cells, including neutrophils, T-cells, and macrophages in dermatitis-affected areas in IMQ-treated psoriasis mice. Furthermore, USCs-CM treatment decreased mRNA levels of IL-17A, IL-17F, and IL-23p19 was reduced in dermatitis area. In summary, our findings revealed new potential strategies for utilizing USCs and USCs-CM as therapeutic agents for inflammatory skin diseases, including psoriasis.

Abstract MP02: Inhibition of RORγt Ameliorates Angiotensin II-Induced Blood-Brain Barrier Disruption and Microglia Activation by Reducing Interleukin-17A Production in Rats

Inflammation-induced blood-brain barrier (BBB) disruption and microglial activation in the autonomic and cardiovascular brain centers contribute largely to sympathetic excitation and hypertension. We recently reported that interleukin (IL)-17A, a major mediator of tissue inflammation, promotes BBB disruption, neuroinflammation, and sympathetic activation in angiotensin (ANG) II-induced hypertension and myocardial infarction-induced heart failure. The nuclear receptor retinoid-related orphan receptor γt (RORγt) has been identified as a master transcription factor regulating the differentiation of T helper 17 cells, the primary producer of IL-17A. The present study sought to determine whether inhibition of RORγt to suppress IL-17A production can ameliorate BBB disruption and attenuate microglia activation in ANG II-induced hypertension model. SD rats were treated with a 2-week subcutaneous (SC) infusion of ANG II (150 ng/kg/min) via mini-pumps, combined with daily SC injections of RORγt inhibitor digoxin or vehicle (VEH). Compared to the VEH control, IL-17A levels in plasma and cerebrospinal fluid were significantly (*P<0.05) elevated in ANG II+VEH rats and were reduced in ANG II + digoxin rats. In ANG II+VEH rats, BBB permeability, measured by fluorescein isothiocyanate-dextran (FITC) 10 kDa, was elevated (6.82±0.61%* vs. 2.36±0.28% FITC10 extravasation), along with increased mRNA levels of caveolin-1 (a transcytosis marker, 2.26±0.39* vs. 1.07±0.17) and decreased mRNA levels of mfsd2a (a BBB-specific lipid transporter and inhibitor of transcytosis, 0.55±0.04* vs. 1.06±0.09) in the hypothalamic paraventricular nucleus (PVN). However, BBB permeability and mRNA levels of caveolin-1 and mfsd2a in the PVN were reversed in ANG II + digoxin rats. Immunostaining revealed that both caveolin-1 and mfsd2a were primarily located in the endothelial BBB within the PVN. Microglia activation, assessed by the number of branches (26±2* vs. 37±4) and total branch length (184±15* vs. 250±23), as well as the mRNA levels of tumor necrosis factor-α (2.78±0.45* vs. 1.04±0.12) and IL-1β (2.57±0.53* vs. 1.03±0.14) in the PVN were augmented in ANG II+VEH rats but were attenuated (40-58%*) in ANG II + digoxin rats. Additionally, ANG II-induced hypertension and sympathetic tone were attenuated by digoxin. These data suggest that inhibiting RORγt diminishes BBB disruption, microglial activation and sympathetic excitation induced by ANG II, likely via reducing IL-17A production.