Protein Signaling Pathway Research Articles

Tanshinone II A Facilitates Chemosensitivity of Osteosarcoma Cells to Cisplatin via Activation of p38 MAPK Pathway.

To examine the mechanism of action of tanshinone II A (Tan II A) in promoting chemosensitization of osteosarcoma cells to cisplatin (DDP). The effects of different concentrations of Tan II A (0-80 µ mol/L) and DDP (0-2 µ mol/L) on the proliferation of osteosarcoma cell lines (U2R, U2OS, 143B, and HOS) at different times were examined using the cell counting kit-8 and colony formation assays. Migration and invasion of U2R and U2OS cells were detected after 24 h treatment with 30 µ mol/L Tan II A, 0.5 µ mol/L DDP alone, and a combination of 10 µ mol/L Tan II A and 0.25 µ mol/L DDP using the transwell assay. After 48 h of treatment of U2R and U2OS cells with predetermined concentrations of each group of drugs, the cell cycle was analyzed using a cell cycle detection kit and flow cytometry. After 48 h treatment, apoptosis of U2R and U2OS cells was detected using annexin V-FITC apoptosis detection kit and flow cytometry. U2R cells were inoculated into the unilateral axilla of nude mice and then the mice were randomly divided into 4 groups of 6 nude mice each. The 4 groups were treated with equal volume of Tan II A (15 mg/kg), DDP (3 mg/kg), Tan II A (7.5 mg/kg) + DDP (1.5 mg/kg), and normal saline, respectively. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell-related gene and signaling pathway expression were detected by RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis. p38 MAPK signaling pathway proteins and apoptotic protein expressions were detected by Western blot. In vitro studies have shown that Tan II A, DDP and the combination of Tan II A and DDP inhibit the proliferation, migration and invasion of osteosarcoma cells. The inhibitory effect was more pronounced in the Tan II A and DDP combined treatment group (P<0.05 or P<0.01). Osteosarcoma cells underwent significantly cell-cycle arrest and cell apoptosis by Tan II A-DDP combination treatment (P<0.05 or P<0.01). In vivo studies demonstrated that the Tan II A-DD combination treatment group significantly inhibited tumor growth compared to the Tan II A and DDP single drug group (P<0.01). Additionally, we found that the combination of Tan II A and DDP treatment enhanced the p38 MAPK signaling pathway. Western blot assays showed higher p-p38, cleaved caspase-3, and Bax and lower caspase-3, and Bcl-2 expressions with the combination of Tan II A and DDP treatment compared to the single drug treatment (P<0.01). Tan II A synergizes with DDP by activating the p38/MAPK pathway to upregulate cleaved caspase-3 and Bax pro-apoptotic gene expressions, and downregulate caspase-3 and Bcl-2 inhibitory apoptotic gene expressions, thereby enhancing the chemosensitivity of osteosarcoma cells to DDP.

MicroRNAs in Congenital Diaphragmatic Hernia: Insights into Prenatal and Perinatal Biomarkers and Altered Molecular Pathways: microRNAs in Congenital Diaphragmatic Hernia for Pathway Analysis and Prognostic Biomarkers

Congenital diaphragmatic hernia (CDH) is characterized by a diaphragmatic defect, leading to herniation of abdominal organs into the chest, lung compression, and impaired lung development, often resulting in pulmonary hypertension and lung hypoplasia. Prenatal imaging techniques like ultrasound and MRI provide anatomical predictors of outcomes, but their limitations necessitate novel biomarkers for better prognostic accuracy. This study aims to identify unique circulating maternal, fetal, and neonatal microRNAs (miRNAs) that can distinguish CDH pregnancies from healthy controls and assess their potential as markers of disease severity. We conducted a prospective study involving third-trimester maternal blood, amniotic fluid, cord blood, and neonatal blood samples from pregnancies complicated by CDH and healthy controls. miRNA expression was analyzed using RNA-sequencing, and random forest analysis identified miRNAs distinguishing CDH survivors from non-survivors. Pathway enrichment analyses were performed to explore the biological relevance of differentially expressed miRNAs. Significant miRNA expression differences were observed between CDH and control samples across all sample types. In infant blood, 148 miRNAs were up-regulated, and 36 were down-regulated in CDH cases. Pathway analysis revealed that dysregulated miRNAs in CDH targeted pathways related to protein binding, transcription regulation, and signaling pathways implicated in pulmonary hypertension and lung hypoplasia. Random forest analysis identified miRNAs in maternal blood (miR-7850-5p_L-1R+2, miR-942-3p, and miR-197-3p) that distinguished CDH survivors from non-survivors, with an ROC area under the curve of 1.0. Circulating miRNAs in maternal blood offer promising biomarkers for predicting CDH outcomes. miRNAs from infant blood provide mechanistic insights and potential targets for therapeutic intervention in critical pathways of pulmonary hypertension and lung hypoplasia. Further studies with larger cohorts are needed to validate these findings and explore the clinical application of miRNA biomarkers in CDH management.

Heterogeneity of Endothelial Cells Impacts the Functionality of Human Pancreatic In Vitro Models.

Endothelial cells (ECs) play a crucial role in maintaining tissue homeostasis and functionality. Depending on their tissue of origin, ECs can be highly heterogeneous regarding their morphology, gene and protein expression, functionality, and signaling pathways. Understanding the interaction between organ-specific ECs and their surrounding tissue is therefore critical when investigating tissue homeostasis, disease development, and progression. In vitro models often lack organ-specific ECs, potentially limiting the translatability and validity of the obtained results. The goal of this study was to assess the differences between commonly used EC sources in tissue engineering applications, including human umbilical vein ECs (HUVECs), human dermal microvascular ECs (hdmvECs), and human foreskin microvascular ECs (hfmvECs), and organ-specific human pancreatic microvascular ECs (hpmvECs), and test their impact on functionality within an in vitro pancreas test system used for diabetes research. Utilizing high-resolution Raman microspectroscopy and Raman imaging in combination with established protein and gene expression analyses and exposure to defined physical signals within microfluidic cultures, we identified that ECs exhibit significant differences in their biochemical composition, relevant protein expression, angiogenic potential, and response to the application of mechanical shear stress. Proof-of-concept results showed that the coculture of isolated human islets of Langerhans with hpmvECs significantly increased the functionality when compared with control islets and islets cocultured with HUVECs. Our study demonstrates that the choice of EC type significantly impacts the experimental results, which needs to be considered when implementing ECs into in vitro models.

Mutations in the Bone Morphogenetic Protein signaling pathway sensitize zebrafish and humans to ethanol-induced jaw malformations.

In this study, we apply a unique combination of zebrafish-based approaches and human genetic and facial dysmorphology analyses to resolve the cellular mechanisms driven by the ethanol-sensitive Bmp pathway.

PRRSV non-structural protein 5 inhibits antiviral innate immunity by degrading multiple proteins of RLR signaling pathway through FAM134B-mediated ER-phagy.

Viruses employ various evasion strategies to establish prolonged infection, with evasion of innate immunity being particularly crucial. Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant pathogen in swine industry, characterized by reproductive failures in sows and respiratory distress in pigs of all ages, leading to substantial economic losses globally. In this study, we found that the non-structural protein 5 (Nsp5) of PRRSV antagonizes innate immune responses via inhibiting the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs), which is achieved by degrading multiple proteins of RIG-I-like receptor (RLR) signaling pathway (RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7). Furthermore, we showed that PRRSV Nsp5 is located in endoplasmic reticulum (ER), where it promotes accumulation of RLR signaling pathway proteins. Further data demonstrated that Nsp5 activates reticulophagy (ER-phagy), which is responsible for the degradation of RLR signaling pathway proteins and IFN-I production. Mechanistically, Nsp5 interacts with one of the ER-phagy receptor family with sequence similarity 134 member B (FAM134B), promoting the oligomerization of FAM134B. These findings elucidate a novel mechanism by which PRRSV utilizes FAM134B-mediated ER-phagy to elude host antiviral immunity.IMPORTANCEInnate immunity is the first line of host defense against viral infections. Therefore, viruses developed numerous mechanisms to evade the host innate immune responses for their own benefit. PRRSV, one of the most important endemic swine viruses, poses a significant threat to the swine industry worldwide. Here, we demonstrate for the first time that PRRSV utilizes its non-structural protein Nsp5 to degrade multiple proteins of RLR signaling pathways, which play important roles in IFN-I production. Moreover, FAM134B-mediated ER-phagy was further proved to be responsible for the protein's degradation. Our study highlights the critical role of ER-phagy in immune evasion of PRRSV to favor replication and provides new insights into the prevention and control of PRRSV.

High expression of CNOT6L contributes to the negative development of type 2 diabetes.

Type 2 diabetes (T2D) is a chronic metabolic disorder characterized by reduced responsiveness of body cells to insulin, leading to elevated blood sugar levels. CNOT6L is involved in glucose metabolism, insulin secretion regulation, pancreatic beta-cell proliferation, and apoptosis. These functions may be closely related to the pathogenesis of T2D. However, the exact molecular mechanisms linking CNOT6L to T2D remain unclear. Therefore, this study aims to elucidate the role of CNOT6L in T2D. The T2D datasets GSE163980 and GSE26168 profiles were downloaded from the Gene Expression Omnibusdatabase generated by GPL20115 and GPL6883.The R package limma was used to screen differentially expressed genes (DEGs). A weighted gene co-expression network analysis was performed. Construction and analysis of the protein-protein interaction (PPI) network, functional enrichment analysis, gene set enrichment analysis, and comparative toxicogenomics database (CTD) analysis were performed. Target Scan was used to screen miRNAs that regulate central DEGs. The results were verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), and blood glucose measurements in mice. A total of 1951 DEGs were identified. GO and KEGG enrichment analysis revealed that differentially expressed genes were mainly enriched in the insulin signaling pathway, ECM-receptor interaction, and PPAR signaling pathway. Metascape analysis indicated enrichment primarily in the cAMP signaling pathway and enzyme-linked receptor protein signaling pathway. WGCNA analysis yielded 50 intersecting genes. PPI network construction and algorithm identification identified two core genes (CNOT6L and GRIN2B), among which CNOT6L gene was associated with multiple miRNAs. CTD analysis revealed associations of core genes with type 2 diabetes, diabetic complications, dyslipidemia, hyperglycemia, and inflammation. WB and RT-qPCR results showed that in different pathways, CNOT6L protein and mRNA levels were upregulated in type 2 diabetes. CNOT6L is highly expressed in type 2 diabetes mellitus, and can cause diabetes complications, inflammation and other physiological processes by regulating miRNA, PPAR and other related signaling pathways, with poor prognosis. CNOT6L can be used as a potential therapeutic target for type 2 diabetes.

Mechanism of the cardioprotective effect of empagliflozin on diabetic nephropathy mice based on the basis of proteomics

Diabetic nephropathy affects a significant proportion of individuals with diabetes, and its progression often leads to cardiovascular disease and infections before the need for renal replacement therapy arises. Empagliflozin has been shown to have various protective effects in cardiovascular disease studies, such as improving diabetic myocardial structure and function, and reducing myocardial oxidative stress. However, the impact of empagliflozin on cardiac protein expression and signaling pathways has not been comprehensively analyzed. To address this gap, we conducted proteome analysis to identify specific protein markers in cardiac tissue from the diabetes model group, including Myh7, Wdr37, Eif3k, Acot1, Acot2, Cat, and Scp2, in cardiac tissue from the diabetes model group. In our drug model, empagliflozin primarily modulates the fat-related metabolic signaling pathway within the heart. Empagliflozin downregulated the protein expression levels of ACOX1, ACADVL and CPT1A in the model group. Overall, our findings demonstrate that empagliflozin provides cardiac protection by targeting metabolic signaling pathways, particularly those related to fat metabolism. Moreover, the identification of cardiac biomarkers in a mouse model of diabetic nephropathy lays the foundation for further exploration of disease biomarkers in cardiac tissue.

NSUN5 promotes tumorigenic phenotypes through the WNT signaling pathway and immunosuppression of CD8+ T cells in gastric cancer

NSUN5, a key member of the M5C methylation family, plays a significant role in fundamental biological processes like cell proliferation and differentiation. However, its specific function and mechanisms in gastric cancer remain insufficiently understood. Initially, we examined NSUN5's differential expression in gastric cancer versus normal tissues, along with survival trends, associated signaling pathways, and immune infiltration using the TCGA database. Subsequently, we conducted immunohistochemistry experiments to assess NSUN5 expression in gastric cancer tissues. Gain-and loss-of-function experiments were carried out to investigate NSUN5's impact on the proliferation, stemness, and migratory capabilities of gastric cancer cells, as well as the expression of vital proteins in pertinent signaling pathways. Our findings demonstrate that NSUN5 is not only overexpressed in gastric cancer tissues, but also positively associated with tumor stage and inversely linked with patient prognosis. NSUN5 promotes the in vitro proliferation, stemness, and migration of gastric cancer cells, and the in vivo growth of these cells, chiefly through the activation of the WNT/β-catenin signaling pathway. Additionally, NSUN5 appears to diminish the infiltration of CD8+ T cells in gastric cancer, contributing to immune evasion. In conclusion, NSUN5 functions as a proto-oncogene in the progression of gastric cancer and may serve as a potential therapeutic target.

Active state structures of a bistable visual opsin bound to G proteins

Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light perception across the animal kingdom. Opsins covalently bind to the chromophore 11-cis retinal, which isomerizes to the all-trans isomer upon photon absorption, causing conformational changes that result in receptor activation. Monostable opsins, responsible for vision in vertebrates, release the chromophore after activation and must bind another retinal molecule to remain functional. In contrast, bistable opsins, responsible for non-visual light perception in vertebrates and for vision in invertebrates, absorb a second photon in the active state to return the chromophore and protein to the inactive state. Structures of bistable opsins in the activated state have proven elusive, limiting our understanding of how they function as bidirectional photoswitches. Here we present active state structures of a bistable opsin, jumping spider rhodopsin isoform-1 (JSR1), in complex with its downstream signaling partners, the Gi and Gq heterotrimers. These structures elucidate key differences in the activation mechanisms between monostable and bistable opsins, offering essential insights for the rational engineering of bistable opsins into diverse optogenetic tools to control G protein signaling pathways.

Bidirectional effects of morphine on pancreatic cancer progression via the p38/JNK pathway

Cancer patients commonly use morphine to alleviate advanced pain. Studies have shown that morphine may influence and intervene in the malignancy of various cancers, but its role and effects on pancreatic cancer are less studied. This study aims to examine how morphine affects pancreatic cancer and its possible mechanisms. In vitro experiments were conducted using the CCK-8 experiment, colony formation experiment, EdU test, wound healing experiment, and transwell migration and invasion experiment. Tumor xenograft tests were employed to investigate the in vivo impact of morphine on pancreatic cancer. The Western blot (WB) assay was used to detect possible changes in key proteins of the related signaling pathway. Our experimental results showed that low concentrations of morphine (25 µM) promoted the progression of pancreatic cancer, while high concentrations of morphine (100 µM) inhibited its progression. Further, we demonstrated that morphine may interfere with the progression of pancreatic cancer by acting on the p38/JNK signaling pathway. Morphine may affect pancreatic cancer progression through the p38/JNK pathway in a bidirectional manner at different concentrations.

Intratumoural microorganism can affect the progression of hepatocellular carcinoma.

Several recent studies have confirmed that intratumoural microorganisms can affect the occurrence and development of hepatocellular carcinoma (HCC); however, their role in tumor progression remains unclear. Hence, there is a need for further research on the role of intratumoural microorganisms in HCC. To investigate the changes in intratumoural microorganisms in HCC and the effect of Propionibacterium on HCC progression. HCC and normal liver tissue specimens were subjected to fluorescence in situ hybridization (FISH). After performing 16S rRNA sequencing on HCC and peritumoral tissues to analyze the differences between the two groups. Propionibacterium was cocultured with HCC cells in vitro. Changes in cell proliferation and migration capacity were evaluated. The expression of NF-κB pathway related proteins in tumor cells was compared. The orthotopic liver implantation model and the subcutaneous xenograft model were constructed. liver tissues and subcutaneous tumors were collected 2 weeks later. FISH demonstrated the presence of microorganisms in HCC and normal liver tissues. 16S rRNA sequencing revealed an abundance of Lysobacter, Lachnospiraceae, Pseudomonas, and Lactobacillus in HCC tissues. The distribution and abundance of Propionibacterium showed differences between HCC and peritumoral tissues (P < 0.05). In vitro studies demonstrated that Propionibacterium and its metabolite propionic acid (PA) inhibited the proliferation and migration of HCC cells (P < 0.05). The expression of the proteins in NF-κB signaling pathway also decreased in HCC cells (P < 0.05). Microorganisms in HCC and normal liver tissues displayed significant disparities. The PA-producing bacterium Propionibacterium in HCC exerts an effect on the NF-κB pathway, thereby affecting the biological behavior of HCC.

Dose-Response Effect of Various Concentrations of Cl-Containing Water-Soluble Derivatives of C60 Fullerenes on a Selective Regulation of Gene Expression in Human Embryonic Lung Fibroblasts (HELF).

The new synthesized water-soluble derivatives of C60 fullerenes are of a great interest to researchers since they can potentially be promising materials for drug delivery, bioimaging, biosonding, and tissue engineering. Surface functionalization of fullerene derivatives changes their chemical and physical characteristics, increasing their solubility and suitability for different biological systems applications, however, any changes in functionalized fullerenes can modulate their cytotoxicity and antioxidant properties. The toxic or protective effect of fullerene derivatives on cells is realized through the activation or inhibition of genes and proteins of key signaling pathways in cells responsible for regulation of cellular reactive oxygen species (ROS) level, proliferation, and apoptosis. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay was used to assess cells viability. Flow cytometry analyses was applied to measure proteins levels in human embryonic lung fibroblasts (HELF) cells. HELF is a standard, stable and well described human cell line that can be passaged many times. Quantitation of ROS was assessed using H2DCFH-DA. Fluorescence images were obtained using microscopy. Expression of BCL2, CCND1, CDKN2A, BRCA1, BAX, NFKB1, NOX4, NRF2, TBP (reference gene) was analyzed using real-time Polymerase chain reaction (PCR). We found that high and low concentrations of fullerene C60 derivatives with the five residues of potassium salt of 6-(3-phenylpropanamido)hexanoic (F1) or 6-(2-(thiophen-2-yl)acetamido)hexanoic (F2) acid and a chlorine atom attached directly to the cage cause diametrically opposite activation of genes and proteins of key signaling pathways regulating the level of oxidative stress and apoptosis in HELF. High concentrations of F1 and F2 have a genotoxic effect, causing NADPH oxidase 4 (NOX4) expression activation in 24-72 hours (2-4 fold increase), ROS synthesis induction (increase by 30-40%), DNA damage and breaks (2-2.5 fold 8-oxodG level increases), and activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (by 40-80%) against the background of reduced NF-E2-related factor 2 (NRF2) expression (by 20-45%). Low concentrations of F1 and F2 produced a cytoprotective effect: in 24-72 hours they reduce the oxidative DNA damage (by 20-40%), decrease the number of double-strand DNA breaks (by 20-30%), increase the level of anti-apoptotic proteins and enhance the antioxidant response activating the NRF2 expression (NRF2 gene expression increases 1.5-2.3 fold, phosphorylated form of the NRF2 protein increases 2-3 fold). Obtained results show that in low doses studied fullrens may serve as perspective DNA protectors against the damaging genotoxic factors.

Regulation of the NF-κB/NLRP3 signalling pathway by Shenghui Yizhi decoction reduces neuroinflammation in mice with Alzheimer’s disease

Background Shenghui Yizhi Decoction (SHYZD) has exhibited the capacity to enhance cognitive function and learning abilities in individuals diagnosed with Alzheimer’s disease (AD) while ameliorating pre-existing neuroinflammation. Nevertheless, the precise mechanism underlying its therapeutic effects on AD remains to be elucidated. Methods Twenty-four male SAMP8 mice were randomly divided into three groups, and eight male SAMR1 mice were used as a blank control, to examine their learning and spatial memory abilities. The expression of amyloid β1-42 (Aβ1-42) was detected by immunohistochemical staining of hippocampal tissue. ELISA was used to detect the interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) expressions. Real time PCR was used to detect NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cysteine protease-1 (Caspase-1), and IL-1β mRNA expression. Western blot was used to detect nuclear factor kappa-B (NF-κB), inhibitor of NF-κB α (IκBα), IκB kinase α (IKKα), NLRP3, Caspase-1, and IL-1β protein expression. Results In this study, SAMP8 mice, employed as an AD model, displayed markedly diminished abilities in terms of spatial localization, navigation, and spatial exploration when compared to the blank control group. Additionally, there was a substantial upregulation of Aβ1-42 expression in the hippocampus of these mice, along with a significant increase in the levels of inflammation-associated factors, including IL-1β, IL-6, TNF-α, NLRP3, Caspase-1, as well as the NF-κB pathway-related proteins, namely, NF-κB, IκBα, and IKKα. Moreover, after treatment with positive drugs (donepezil hydrochloride) and SHYZD, the learning abilities of the mice exhibited significant improvements. Furthermore, the hallmark AD protein Aβ1-42, inflammatory factors, and NF-κB/NLRP3 signalling pathway proteins were significantly reduced. These findings collectively suggest that SHYZD exerts a therapeutic effect on AD. Conclusion In summary, the specific molecular mechanisms through which SHYZD alleviates AD and the potential role for SHYZD in the NF-κB/NLRP3 signalling pathway are identified in this study.

A Strong Candidate Gene for Nonsyndromic Intellectual Disability Phenotype: SGSM3.

SGSM proteins are small modulator proteins interacting with proteins in the RAS signaling pathway. Studies with mouse and human tissues indicated that SGSM genes were highly expressed in the brain and could be expressed at different levels at different stages of development in fetal and adult brain tissue. It was first reported by Birnbaum et al. that the SGSM3 gene might be associated with a Mendelian inherited disease in families of Ashkenazi Jews with clinical manifestations of intellectual disability (ID). In this study, a novel homozygous stop-gain (NM_015705.6: c.1576C>T: p.(Arg526Ter)) variation was detected in the SGSM3 gene in two siblings with short stature and ID findings. The report of two cases with bi-allelic LOF variants in the SGSM3 gene from different populations with similar clinical manifestations strengthens the potential of this gene as a candidate gene for the nonsyndromic ID phenotype. Functional studies are required to investigate the signaling pathways affected by SGSM3 gene variations to produce the ID phenotype and their effect on the functioning of neurons.

Zhachong Shisanwei pill drug-containing serum protects H2O2-Induced PC12 cells injury by suppressing apoptosis, oxidative stress via regulating the MAPK signaling pathway.

Zhachong Shisanwei Pill (ZSP) is a classical Mongolian formula that combines 13 types of Chinese medicinal materials and has been used for treating ischemic stroke (IS) for centuries. However, the underlying molecular mechanisms have yet to be fully elucidated. The aim of this study is to explore potential mechanism of ZSP on nerve cells in cerebral ischemic injury. To simulate the pathological process of oxidative stress following IS, an injury model using PC12 cells was induced with hydrogen peroxide (H2O2). Afterward, PC12 cells were treated with ZSP medicated serum at low, medium, and high doses. Various assays were conducted to assess cell viability and oxidative stress indicators, including lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP). Cell apoptosis was evaluated through morphological assessment and flow cytometry. Additionally, the expression levels of apoptosis-related proteins (Bcl-2, Bax, Caspase-9, Caspase-3, PARP) and signaling pathway proteins (JNK, phosphorylated JNK, ERK, phosphorylated ERK, p38, and phosphorylated p38) were measured using automated Western blotting. Our findings indicate that ZSP medicated serum preconditioning improves the condition of PC12 cells injured by H2O2. Specifically, it increased cell survival rates and reduced LDH release. Additionally, ZSP treatment decreased ROS levels and MDA content, while enhancing the activity of SOD and CAT in the injured PC12 cells. ZSP also reversed the depolarization of mitochondrial membrane potential and protected cells from apoptosis by modulating the expression of apoptosis-related proteins, including Bcl-2, Bax, Caspase-9, Caspase-3, and PARP. Furthermore, the overactivation of the MAPK signaling pathway due to H2O2-induced injury was inhibited, as evidenced by the downregulation of phosphorylated JNK, ERK, and p38 levels. Mongolian medicine ZSP demonstrates protective effects against H2O2-induced oxidative stress and apoptosis in PC12 cells. The underlying mechanism may involve the inhibition of the MAPK signaling pathway, enhancement of antioxidant enzyme activity, reduction of intracellular peroxidation levels, and suppression of intrinsic apoptosis pathways.

Global A-to-I RNA editing during myogenic differentiation of goat MuSCs.

RNA editing, especially A-to-I editing sites, is a common RNA modification critical for stem cell differentiation, muscle development, and disease occurrence. Unveiling comprehensive RNA A-to-I editing events associated with myogenesis of the skeletal muscle satellite cells (MuSCs) is essential for extending our knowledge of the mechanism underpinning muscle development. A total of 9,632 RNA editing sites (RESs) were screened in the myoblasts (GM), myocytes (DM1), and myotubes (DM5) samples. Among these sites, 4,559 A-to-I edits were classified and further analyzed. There were 3,266 A-to-I sites in the protein-coding region, out of which 113 missense sites recoded protein. Notably, five A-to-I sites in the 3' UTR of four genes (TRAF6, NALF1, SLC38A1, ENSCHIG00000019092) altered their targeted miRNAs. Furthermore, a total of 370 A-to-I sites with different editing levels were detected, including FBN1, MYH10, GSK3B, CSNK1D, and PRKACB genes. These genes were predominantly enriched in the cytoskeleton in muscle cells, the hippo signaling pathway, and the tight junction. Furthermore, we identified 14 hub genes (TUFM, GSK3B, JAK2, RPSA, YARS1, CDH2, PRKACB, RUNX1, NOTCH2, CDC23, VCP, FBN1, RARS1, MEF2C) that potentially related to muscle development. Additionally, 123 stage-specific A-to-I editing sites were identified, with 43 sites in GM, 25 in DM1, and 55 in DM5 samples. These stage-specific edited genes significantly enriched essential biological pathways, including the cell cycle, oocyte meiosis, motor proteins, and hedgehog signaling pathway. We systematically identified the RNA editing events in proliferating and differentiating goat MuSCs, which was crucial for expanding our understanding of the regulatory mechanisms of muscle development.

8672 Disrupted Glucocorticoid Receptor Mediated Signalling Causes a Primary Cilia Defect in the Fetal Mouse Renal Tubule

Abstract Disclosure: J. Lao: None. K.L. Short: None. J. Ng: None. D.L. Cottle: None. T.J. Cole: None. Primary cilia are microtubule-based organelles that protrude from cell membranes to mediate diverse developmental signalling pathways and senses extracellular stimuli to maintain tissue homeostasis. We show that glucocorticoid (GC) signalling via the glucocorticoid receptor (GR) is a regulator of normal primary cilia formation in kidney renal tubules. RNA sequencing of whole E18.5 fetal kidney from mice with global deletion of the GR (GR-null) compared to littermate wild-type controls identified significantly reduced mRNA levels of key ciliogenesis-related genes. Further analysis of whole fetal kidney RNA by real-time-qPCR confirmed reduced mRNA levels for the cilia-associated proteins Ccp110 (fold -2.18), Cep97 (fold -1.78), Cep290 (fold -2.91), Kif3a (fold -1.82) and Rpgr (fold -1.90). Confocal microscopy revealed abnormal, stunted primary cilia in renal proximal tubules, collecting ducts and podocytes in GR-null or in renal tubule conditional GR-deleted mice, created using the HoxB7 Cre-driver allele. Primary cilia length was significantly decreased in kidney proximal tubule cells in GR-null mice (5.01 ± 0.11μm) compared with wildtype controls (6.20 ± 0.15μm). Furthermore, scanning electron microscopy of E18.5 GR-null kidney further confirmed aberrant primary cilia morphology in the proximal renal tubules and the absence of an apical brush border. Activation of GR signalling with the synthetic GC dexamethasone in cultured mouse IMCD3 kidney tubule cells significantly increased primary cilia length (2.89 ± 0.04μm) compared to vehicle controls (2.46 ± 0.28μm), an effect blocked by the GR antagonist RU486 (vehicle + RU486: 2.06 ± 0.02μm, dexamethasone + RU486: 2.00 ± 0.02μm). Aurora kinase A (AURKA) is a known regulator of primary cilia formation via the AKT cell signalling pathway and distinct proteins at different stages of the mitotic cell cycle. AURKA protein levels were downregulated in GR-null kidney and in IMCD3 cells which suggest GR regulates AURKA to control the assembly and disassembly of primary cilia. Together, these results demonstrate that GC signalling via the GR is required for normal primary ciliogenesis in the developing renal tubule and suggests that synthetic GR agonists may provide a novel therapeutic option for human ciliopathies such as those observed in forms of polycystic kidney disease. Presentation: 6/2/2024

In Vitro and In Silico Evaluation of Caffeic and Ferulic Acids Involvement in the Translocation of Glucose Transporter 4

Background: Insulin is a key hormone in our systems. Upon binding of insulin to its receptors in fat and muscle tissues, tens of proteins in the insulin signaling pathway are involved in the process of GLUT4 vesicle recruitment to the Plasma Membrane (PM) and the absorption of serum glucose. Deficits in the aforementioned pathway lead to insulin resistance and eventually to Type II Diabetes Mellitus. Objective: We appreciate the contribution of phytochemicals in the treatment of diabetes. Yet, in vitro and in silico studies are needed to validate the safety and efficacy of the phytochemicals, plus their action mechanisms. Methods: Herein, we tested two phytochemicals, caffeic acid and ferulic acid in vitro and in silico. We shed light on the insulin signaling proteins as plausible therapeutic targets using in silico studies, via AutoDock and SwissADME. Results: Results obtained in vitro indicate that Caffeic Acid (CA) increased GLUT4 translocation at 125μM by 31% in the absence of insulin, and 24.5% in presence of insulin, when compared to the control. Ferulic Acid (FA) was less potent as an enhancer of GLUT4 translocation. Best docking results were found for the binding of the phytochemicals CA and FA to PDK1, AKT, IRS1 and PTEN proteins of the insulin signaling, with comparable results. Conclusion: These findings indicate that CA and FA possess a limited anti-diabetic potency by increasing GLUT4 trafficking to the PM in skeletal muscles. These results suggest that these compounds are candidates for further investigation in pre-clinical and clinical stages of drug discovery.

The Multifaceted Roles of Hippo-YAP in Cardiovascular Diseases.

The Hippo-yes-associated protein (YAP) signaling pathway plays a crucial role in cell proliferation, differentiation, and death. It is known to have impact on the progression and development of cardiovascular diseases (CVDs) as well as in the regeneration of cardiomyocytes (CMs). However, further research is needed to understand the molecular mechanisms by which the Hippo-YAP pathway affects the pathological processes of CVDs in order to evaluate its potential clinical applications. In this review, we have summarized the recent findings on the role of the Hippo-YAP pathway in CVDs such as myocardial infarction, heart failure, and cardiomyopathy, as well as its in CM development. This review calls attention to the potential roles of the Hippo-YAP pathway as a relevant target for the future treatment of CVDs.

Flurbiprofen axetil is involved in basal-like breast cancer metastasis via suppressing the MEK/ERK signaling pathway.

Flurbiprofen axetil is commonly utilized in clinical practice as one of the nonsteroidal anti-inflammatory drugs (NSAIDs) and is included in multimodal analgesia regimens postbreast cancer surgery. Numerous NSAIDs have been studied for their potential to both promote and inhibit cancer. Given the variability in their effects on tumors, further investigation into the specific role of flurbiprofen axetil is warranted. Therefore, the primary objective of this study was to assess the impact of flurbiprofen axetil on basal-like breast cancer (BLBC) metastasis and elucidate the underlying molecular mechanisms involved. The BLBC metastasis mouse model was established by caudal vein injection of tumor cells. The lung metastasis of breast cancer in mice and the effect of flurbiprofen axetil were assessed by in vivo bioluminescence imaging, hematoxylin and eosin staining and immunohistochemistry. In vitro, the results of flurbiprofen axetil on the proliferation, migration, and invasion of MDA-MB-231 human breast cancer cells and BT-549 human breast cancer cells were assessed by colony formation assay and transwell assay. The effects of flurbiprofen axetil on several tumor metastasis-related signaling pathway proteins were examined by western blot, and the reversal extent of the flurbiprofen axetil effect by Ro 67-7476 (ERK phosphorylation agonist) was detected by transwell assay. The results showed that flurbiprofen axetil significantly inhibited BLBC lung metastasis in mice. Flurbiprofen axetil similarly inhibited breast cancer cell migration and invasion in vitro but did not affect their proliferation. Mechanistic investigations have revealed that flurbiprofen axetil exerts a noteworthy inhibitory influence on the MEK/ERK pathway while exhibiting no significant alteration in the expression of other pathway proteins intricately associated with epithelial-mesenchymal transition. In conclusion, the inhibitory effect of flurbiprofen axetil on BLBC metastasis is characterized by its selectivity in targeting the MEK/ERK signaling pathway rather than exerting a broad impact on the global signaling pathway.