TdT-mediated dUTP Nick-end Labeling Assay Research Articles

FOXD2-AS1 promotes malignant cell behavior in oral squamous cell carcinoma via the miR-378 g/CRABP2 axis

BackgroundOral squamous cell cancer (OSCC) is a prevalent malignancy in oral cavity, accounting for nearly 90% of oral malignancies. It ranks sixth among the most common types of cancer worldwide and is responsible for approximately 145,000 deaths each year. It is widely accepted that noncoding RNAs participate cancer development in competitive regulatory interaction, knowing as competing endogenous RNA (ceRNA) network, whereby long non-coding RNA (lncRNA) function as decoys of microRNAs to regulate gene expression. LncRNA FOXD2-AS1 was reported to exert an oncogenic role in OSCC. Nevertheless, the ceRNA network mediated by FOXD2-AS1 was not investigated yet. This study aimed to explore the effect of FOXD2-AS1 on OSCC cell process and the underlying ceRNA mechanism.MethodsFOXD2-AS1 expression in OSCC cells were determined via reverse transcription and quantitative polymerase chain reaction. Short hairpin RNA targeting FOXD2-AS1 was transfected into OSCC cells to silence FOXD2-AS1 expression. Then, loss-of-function experiments (n = 3 each assay) were performed to measure cell proliferation, apoptosis, migration, and invasion using colony formation, TdT-mediated dUTP Nick-End Labeling, wound healing and Transwell assays, respectively. RNA binding relation was verified by RNA immunoprecipitation and luciferase reporter assays. Rescue experiments were designed to validate whether FOXD2-AS1 affects cell behavior via the gene cellular retinoic acid binding protein 2 (CRABP2). Statistics were processed by GraphPad Prism 6.0 Software and SPSS software.ResultsFOXD2-AS1 was significantly upregulated in Cal27 and SCC9 cells (6.8 and 6.4 folds). In response to FOXD2-AS1 knockout, OSCC cell proliferation, migration and invasion were suppressed (approximately 50% decrease) while OSCC cell apoptosis was enhanced (more than two-fold increase). FOXD2-AS1 interacted with miR-378 g to alter CRABP2 expression. CRABP2 upregulation partly rescued (*p < 0.05, **p < 0.01, ***p < 0.001) the inhibitory impact of FOXD2-AS1 depletion on malignant characteristics of OSCC cells.ConclusionFOXD2-AS1 enhances OSCC malignant cell behaviors by interacting with miR-378 g to regulate CRABP2 expression.

N-acetylcysteine Attenuates Cigarette Smoke-induced Alveolar Epithelial Cell Apoptosis through Reactive Oxygen Species Depletion and Glutathione Replenish In vivo and In vitro.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. N-acetylcysteine (NAC) is well known for its antioxidant properties, along with potential protective effects on COPD. However, the molecular mechanism of NAC against the apoptosis of alveolar epithelial cells (AECs) in COPD remains unclear. This study aimed to explore the anti-apoptosis effect of NAC in COPD mice and alveolar epithelial cells. In the present study, the mouse model of COPD was established by cigarette smoke (CS), and mouse alveolar epithelial (MLE-12) cells were treated with cigarette smoke extract (CSE). TdT-mediated dUTP nick-end labeling (TUNEL) assay, reverse transcription polymerase chain reaction (RT-PCR), and western blot were performed to evaluate the effects of NAC on apoptosis, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction. Meanwhile, LButhionine- sulfoximine (BSO), a glutathione (GSH) inhibitor, was used to uncover the mechanism of COPD treatment by NAC. We found that NAC pretreatment could attenuate the protein levels of apoptosis, ER stress, and mitochondrial dysfunction-related genes caused by CS in vivo. Meanwhile, CSE could decrease MLE-12 cell viability, which was prevented by apoptosis inhibitor ZVAD-FMK but not necroptosis inhibitor necrostatin-1. Pretreatment of MLE-12 cells with NAC increased cellular GSH levels, inhibited cellular and mitochondrial reactive oxygen species (ROS) accumulation, and decreased protein level of apoptosis, ER stress, and mitochondrial dysfunctionrelated genes. Moreover, experiment results showed that BSO could completely reverse the beneficial effects of NAC. Our study confirmed that NAC can attenuate CS-induced AEC apoptosis via alleviating ROS-mediated ER stress and mitochondrial dysfunction pathway, and the mechanism was found to be related to replenishing the cellular GSH content.

Ethanolic extract of Arctium lappa leaves alleviates cerebral ischemia reperfusion-induced inflammatory injury via HDAC9-mediated NF-κB pathway

BackgroundIschemic stroke (IS) is a major cause of mortality and disability worldwide. Inflammatory response is crucial in the pathogenesis of tissue injury in cerebral infarction. Arctium lappa leaves are traditionally used to treat IS. PurposesTo investigate the neuroprotective effects and molecular mechanisms of the ethanolic extract of A. lappa leaves (ALLEE) on cerebral ischemia-reperfusion (CIR). MethodsMiddle cerebral artery obstruction reperfusion (MCAO/R) rats and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model were used to evaluate ALLEE pharmacodynamics. Various methods, including neurological function, 2,3,5-triphenyltetrazolium chloride, hematoxylin and eosin, and Nissl, enzyme-linked immunosorbent, and TdT-mediated dUTP nick-end labeling assays, were used to analyze the neuroprotective effects of ALLEE in vitro and in vivo. The major chemical components and potential target genes of ALLEE were screened using network pharmacology. Molecular docking, western blotting, and immunofluorescence analyses were performed to confirm the effectiveness of the targets in related pathways. ResultsALLEE exerted potent effects on the MCAO/R model by decreasing the neurological scores, infarct volumes, and pathological features (p < 0.01). Furthermore, network pharmacology results revealed that the treatment of IS with ALLEE involved the regulation of various inflammatory pathways, such as the tumor necrosis factor (TNF) and chemokine signaling pathways. ALLEE also played key roles in targeting key molecules, including nuclear factor (NF)-κBIA, NF-κB1, interleukin (IL)-6, TNF-α and IL1β, and regulating the histone deacetylase (HDAC)-9-mediated signaling pathway. In vivo and in vitro analyses revealed that ALLEE significantly regulated the NF-κB pathway, promoted the phosphorylation activation of NF-κB P65, IκB and IKK (p < 0.01 or p < 0.05), and decreased the expression levels of the inflammatory factors, IL-1β, IL-6 and TNF-α (p < 0.01). Moreover, ALLEE significantly decreased the expression of HDAC9 (p < 0.01) that is associated with inflammatory responses. However, HDAC9 overexpression partially reversed the neuroprotective effects of ALLEE and its suppressive effects on inflammation and phosphorylation of NF-κB (p < 0.01). ConclusionsIn conclusion, our results revealed that ALLEE ameliorates MCAO/R-induced experimental CIR by modulating inflammatory responses via the inhibition of HDAC9-mediated NF-κB pathway.

Bioinformatic Identification of Signaling Pathways and Hub Genes in Vascular Dementia.

Vascular dementia (VaD) is a prevalent neurodegenerative disease characterized by cognitive impairment due to cerebrovascular factors, affecting a significant portion of the aging population and highlighting the critical need to understand specific targets and mechanisms for effective prevention and treatment strategies. We aimed to identify pathways and crucial genes involved in the progression of VaD through bioinformatics analysis and subsequently validate these findings. We conducted differential expression analysis, Weighted Gene Co-expression Network Analysis (WGCNA), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) analysis. We utilized pheochromocytoma 12 (PC12) cells to create an in vitro oxygen-glucose deprivation (OGD) model. We investigated the impact of overexpression and interference of adrenoceptor alpha 1D (ADRA1D) on OGD PC12 cells using TdT-mediated dUTP nick-end labeling (TUNEL), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot (WB), and Fluo-3-pentaacetoxymethyl ester (Fluo-3 AM) analysis. We found 187 differentially expressed genes (DEGs) in the red module that were strongly associated with VaD and were primarily enriched in vasoconstriction, G protein-coupled amine receptor activity, and neuroactive ligand-receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and cell adhesion. Among these pathways, we identified ADRA1D as a gene shared by vasoconstriction, G protein-coupled amine receptor activity, and neuroactive ligand-receptor interaction. The TUNEL assay revealed a significant decrease in PC12 cell apoptosis with ADRA1D overexpression (p < 0.01) and a significant increase in apoptosis upon silencing ADRA1D (p < 0.01). RT-qPCR and WB analysis revealed elevated ADRA1D expression (p < 0.001) and decreased phospholipase C beta (PLCβ) and inositol 1,4,5-trisphosphate receptor (IP3R) expression (p < 0.05) with ADRA1D overexpression. Moreover, the Fluo-3 AM assessment indicated significantly lower intracellular Ca2+ levels with ADRA1D overexpression (p < 0.001). Conversely, interference with ADRA1D yielded opposite results. Our study provides a new perspective on the pathogenic mechanisms of VaD and potential avenues for therapeutic intervention. The results highlight the role of ADRA1D in modulating cellular responses to OGD and VaD, suggesting its potential as a target for VaD treatment.

Validation of hyperthermia as an enhancer of chemotherapeutic efficacy: insights from a bladder cancer organoid model

Objective This study aimed to evaluate the combined efficacy of hyperthermia and chemotherapy using a bladder cancer organoid model and to explore hyperthermia-related molecular pathways. Method Tumor organoids were generated by embedding RT4 bladder cancer cells into Matrigel. The resulting organoids were treated with pirarubicin or gemcitabine at 37 °C or 42 °C. Proliferation was determined by Ki67 immunofluorescence staining, and apoptosis was assessed using a TdT-mediated dUTP nick end labeling (TUNEL) assay. RNA sequencing was used to identify the differentially expressed genes. Results Bladder cancer organoids were successfully established and exhibited robust proliferative abilities. Treatment with gemcitabine or pirarubicin under hyperthermic conditions caused pronounced structural damage to the organoids and increased cell death compared to that in the normothermically treated group. Furthermore, Ki67 labeling and TUNEL assays showed that the hyperthermia chemotherapy group showed a significantly reduced proliferation rate and high level of apoptosis. Finally, RNA sequencing revealed the IFN-γ signaling pathway to be associated with hyperthermia. Conclusion Overall, hyperthermia combined with chemotherapy exerted better therapeutic effects than those of normothermic chemotherapy in grade 1-2 non-muscle-invasive bladder cancer, potentially through activation of the IFN-γ-JAK-STAT pathway.

The promotion of sestrin2/AMPK signaling by HIF-1α overexpression enhances the damage caused by acute myocardial infarction

ObjectiveAcute myocardial infarction (AMI), is a serious form of coronary heart disease. The present study sought to investigate the impact of HIF-1α on AMI, along with its fundamental mechanism.MethodsSprague-Dawley (SD) rats were used to conduct an AMI model. 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining was used examine the region of myocardial infract area at various time intervals. Protein expression levels were detected using western blotting. The rats were randomly divided into sham, model, negative control (NC), HIF-1α overexpression (HIF-1α-OE), and HIF-1α-OE+ si-sestrin2 groups. We examined the impact of HIF-1α overexpression on AMI rats using Haematoxylin-Eosin (H&E) staining, TTC staining, enzyme-linked immunosorbent assay (ELISA), TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, and immunohistochemistry (IHC) staining.ResultsAccording to the TTC findings, the region affected by myocardial infarction reached its peak at day 14. Based on the results from the western blot analysis, the levels of HIF-1α and sestrin2 were found the minimum on day 28. Subsequently, we discovered that the overexpression of HIF-1α rescued the cardiac function parameters, improved the morphology of myocardial tissue, and mitigated inflammation. Furthermore, the overexpression of HIF-1α led to a reduction in the levels of MDA and an increase in the levels of SOD. Moreover, the overexpression of HIF-1α resulted in a decrease in cellular apoptosis. This result was confirmed by the expression levels of Bcl-2 and Bax. Nevertheless, the defensive impact of elevated HIF-1α expression was somewhat counteracted by the suppression of sestrin2. In terms of mechanism, the overexpression of HIF-1α enhanced the levels of sestrin2 and the protein adenosine monophosphate activated kinase (AMPK).ConclusionOur research suggests that the overexpression of HIF-1α may rescue the damage to myocardial tissue, and this effect is associated with the sestrin2/AMPK signaling pathway. Our study provides a novel comprehension of the protective effects of HIF-1α overexpression on AMI.

High Uric Acid Promotes Atherosclerotic Plaque Instability by Apoptosis Targeted Autophagy.

Acute rupture or erosion of unstable atherosclerotic plaques is a major cause of adverse consequences of atherosclerotic cardiovascular disease, often leading to myocardial infarction or stroke. High uric acid (HUA) is associated with the increasing risk of cardiovascular events and death. However, the mechanism by which HUA promotes atherosclerosis and whether HUA affects plaque stability are still unclear. We constructed an atherosclerotic Apoe-/- mouse model with HUA. The progression of atherosclerosis and plaques was determined by Oil Red O staining, hematoxylin and eosin (H&E) staining, and Masson staining. TdT-mediated dUTP nick-end labeling assay and immunohistochemistry were used to observe the changes of apoptosis and autophagy in plaques, respectively. Then, we validated the in vivo results with RAW 264.7 cell line. HUA promoted atherosclerosis and exacerbated plaque vulnerability, including significantly increased macrophage infiltration, lipid accumulation, enlarged necrotic cores, and decreased collagen fibers. HUA increased cell apoptosis and inhibited autophagy in plaques. In vitro results showed that HUA decreased cell viability and increased cell apoptosis in foam cells macrophages treated with oxidized low-density lipoprotein. An activator of autophagy, rapamycin, can partially reverse the increasing apoptosis. HUA promoted atherosclerosis and exacerbated plaque vulnerability, and HUA facilitates foam cell apoptosis by inhibiting autophagy.

AQP3-mediated activation of the AMPK/SIRT1 signaling pathway curtails gallstone formation in mice by inhibiting inflammatory injury of gallbladder mucosal epithelial cells

BackgroundInflammatory injury of gallbladder mucosal epithelial cells affects the development of cholelithiasis, and aquaporin 3 (AQP3) is an important regulator of inflammatory response. This study reports a mechanistic insight into AQP3 regulating gallstone formation in cholelithiasis based on high-throughput sequencing.MethodsA mouse model of cholelithiasis was induced using a high-fat diet, and the gallbladder tissues were harvested for high-throughput sequencing to obtain differentially expressed genes. Primary mouse gallbladder mucosal epithelial cells were isolated and induced with Lipopolysaccharides (LPS) to mimic an in vitro inflammatory injury environment. Cell biological phenotypes were detected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, flow cytometry, Cell Counting Kit-8 (CCK-8) assay, and Trypan blue staining. In addition, enzyme linked immunosorbent assay (ELISA) determined the production of inflammatory factors in mouse gallbladder mucosa.ResultsWhole-transcriptome sequencing data analysis identified 489 up-regulated and 1007 down-regulated mRNAs. Bioinformatics analysis revealed that AQP3 was significantly down-regulated in mice with cholelithiasis. AQP3 might also confer an important role in LPS-induced gallbladder mucosal injury. Overexpression of AQP3 activated the AMPK (adenosine monophosphate-activated protein kinase) / SIRT1 (sirtuin-1) signaling pathway to reduce LPS-induced inflammatory injury of the gallbladder mucosa epithelium, thereby ameliorating gallbladder damage and repressing gallstone formation in mice.ConclusionData from our study highlight the inhibitory role of AQP3 in gallbladder damage and gallstone formation in mice by reducing inflammatory injury of gallbladder mucosal epithelial cells, which is achieved through activation of the AMPK/SIRT1 signaling pathway.

Simvastatin inhibits proliferation and promotes apoptosis of oral squamous cell carcinoma through KLF2 signal

ObjectivesThis study aimed to explore the role and specific mechanism of the cholesterol-lowering drug simvastatin in inhibiting oral squamous cell carcinoma (OSCC). MethodsThe proliferation, apoptosis, and migration levels of OSCC cells were detected by CCK8, quantitative real-time polymerase chain reaction, Western blot, colony formation, TdT-mediated dUTP Nick-End Labeling assay, and wound healing assay. The inhibitory effect of simvastatin in vivo was detected by a mouse xenograft tumor model. Immunohistochemistry and immunofluorescence staining were used to assess the KLF2 and β-catenin expressions in cells and tissues. ResultsKLF2 expression in OSCC cells and tissues was downregulated. The addition of KLF2 inducer, GGTI298, inhibited the proliferation and migration of OSCC cells. Simvastatin played a role in inhibiting the proliferation and promoting the apoptosis of OSCC cells. Moreover, it inhibited β-catenin expression and promoted KLF2 expression in OSCC cells. KLF2 siRNA reversed the effect of simvastatin on the proliferation and apoptosis of OSCC cells. ConclusionsKLF2, as a tumor suppressor gene, may be an important marker for diagnosing and treating OSCC. Simvastatin inhibits the progression of OSCC by regulating the KLF2 signal.

Intravenous Delivery of miR-21 Inhibitor in Mice With Head and Neck Squamous Cell Carcinoma.

Synthetic miRNA inhibitors have recently attracted considerable interest as potential therapeutic agents for head and neck squamous cell carcinoma. However, due to the lack of evidence, no attempts have been made to deliver these inhibitors intravenously for squamous cell carcinoma. This study investigated whether intravenous administration of a miR-21 inhibitor with lipid nanoparticles could suppress HNSCC in xenograft mice. Head and neck squamous cell carcinoma xenograft mice were intravenously injected with Invivofectamine 3.0® containing either a miR-21 inhibitor or a control inhibitor, using a modified protocol for nucleic acid encapsulation. Quantitative PCR was used to measure the expression level of intratumoral miR-21. And TdT-mediated dUTP nick-end labeling (TUNEL) immunohistochemistry was used to assess cell death. Intravenous injection of miR-21 inhibitor significantly inhibited head and neck squamous cell carcinoma growth and miR-21 expression in tumor tissue compared to the control inhibitor. TUNEL assay showed significant apoptosis of tumor cells after intravenous administration of miR-21 inhibitor. Intravenous delivery of a miR-21 inhibitor with lipid nanoparticles is a promising approach for miRNA-targeted therapy of head and neck squamous cell carcinoma.

Shenqisherong pill ameliorates neuronal apoptosis by inhibiting the JNK/caspase-3 signaling pathway in a rat model of cervical cord compression

Ethnopharmacological relevanceThe Shenqisherong (SQSR) pill is an empirical prescription of traditional Chinese medicine (TCM), which originated from the National Chinese Medical Science Master, Shi Qi. It has been widely used in the treatment of cervical spondylotic myelopathy (CSM) and promote the recovery of spinal cord function, but underlying molecular mechanism remains unclear. Aim of the studyThe objective of this study was to confirm the neuroprotective effects of the SQSR pill. Materials and methodsA rat model of chronic compression at double-level cervical cord was used in vivo. The protective role of SQSR pill on CSM rats was measured by Basso, Beattie, and Bresnahan (BBB) locomotor scale, inclined plane test, forelimb grip strength assessment, hindlimb pain threshold assessment, and gait analysis. The levels of reactive oxygen species (ROS) were examined by Dihydroethidium (DHE) staining and 2′,7′-Dichlorofluorescein (DCF) assay, and apoptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL) assay. The expression of apoptosis proteins was evaluated by immunofluorescence staining and Western blot. ResultsSQSR pill could facilitate locomotor function recovery in rats with chronic cervical cord compression, reduce local ROS in the spinal cord and downregulate the c-Jun-N-terminal kinase (JNK)/caspase-3 signaling pathway. In addition, the SQSR pill could protect primary rat cortical neurons from glutamate-treated toxicity in vitro by reducing the ROS and downregulating the phosphorylation of JNK and its downstream factors related to neuronal apoptosis meditated by the caspase cascade. Then, the neuroprotective effect was counteracted by a JNK activator. ConclusionsTogether, SQSR pill could ameliorate neuronal apoptosis by restraining ROS accumulation and inhibiting the JNK/caspase-3 signaling pathway, indicating that SQSR pill could be a candidate drug for CSM.

Carbon Monoxide Releasing Molecule-3 Alleviates Oxidative Stress and Apoptosis in Selenite-Induced Cataract in Rats via Activating Nrf2/HO-1 Pathway

Purpose This study investigated the protective effect of carbon monoxide releasing molecule-3 (CORM-3), the classical donor of carbon monoxide, on selenite-induced cataract in rats and explore its possible mechanism. Methods Sprague-Dawley rat pups treated with sodium selenite (Na2SeO3) were chosen as the cataract model. Fifty rat pups were randomly divided into 5 groups: Control group, Na2SeO3 (3.46 mg/kg) group, low-dose CORM-3 (8 mg/kg/d) + Na2SeO3 group, high-dose CORM-3 (16 mg/kg/d) + Na2SeO3 group, and inactivated CORM-3 (iCORM-3) (8 mg/kg/d) + Na2SeO3 group. The protective effect of CORM-3 was tested by lens opacity scores, hematoxylin and eosin staining, TdT-mediated dUTP nick-end labeling assay, and enzyme-linked immunosorbent assay. Besides, quantitative real-time PCR and western blotting were used for mechanism validation. Results Na2SeO3 induced nuclear cataract rapidly and stably, and the achievement ratio of Na2SeO3 group was 100%. CORM-3 alleviated lens opacity of selenite-induced cataract and attenuated the morphological changes of the rat lens. The levels of antioxidant enzymes GSH and SOD in rat lens were also increased by CORM-3 treatment. CORM-3 significantly reduced the ratio of apoptotic lens epithelial cells, besides, CORM-3 decreased the expression of Cleaved Caspase-3 and Bax induced by selenite and increased the expression of Bcl-2 in rat lens inhibited by selenite. Moreover, Nrf-2 and HO-1 were upregulated and Keap1 was downregulated after CORM-3 treatment. While iCORM-3 did not exert the same effect as CORM-3. Conclusions Exogenous CO released from CORM-3 alleviates oxidative stress and apoptosis in selenite-induced rat cataract via activating Nrf2/HO-1 pathway. CORM-3 may serve as a promising preventive and therapeutic strategy for cataract.

MiR-185-5p / ATG101 axis alleviated intestinal barrier damage in intestinal ischemia reperfusion through autophagy

ObjectiveIntestinal ischemia-reperfusion (II/R) is a common pathological injury in clinic, and the systemic inflammatory response it causes will lead to multiple organ damage and functional failure. miR-185-5p has been reported to be a regulator of inflammatory response and autophagy, but whether it participates in the regulation of autophagy in II/R is still unclear. Therefore, we aimed to explore the mechanism of miR-185-5p regulating intestinal barrier injury in (II/R). MethodsCaco-2 cells was induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to establish II/R model. The superior mesenteric artery of C57BL/6 mice was clamped for 45 min and then subjected to reperfusion for 4 h for the establishment of II/R mice model. miR-185-5p mimic, miR-185-5p inhibitor, pcDNA-autophagy-related 101 (ATG101) were respectively transfected into Caco-2 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess miR-185-5p expression. Western blot detected the level of ATG101 and tight junction-associated proteins ZO1, Occludin, E-cadherin, β-catenin, as well as autophagy markers ATG5, ATG12, LC3Ⅰ/Ⅱ, Beclin1 and SQSTM1. Transepithelial electrical resistance (TEER) values was detected by a resistance meter. FITC-Dextran was performed to measure cell permeability. 5-ethynyl-2’-deoxyuridine (EDU) staining measured cell proliferation. Transmission electron microscope was conducted to observe autophagosomes. Hematoxylin & eosin (H&E) staining observed the damage of mice intestinal. Immunohistochemistry (IHC) measured the percentage of ki67 positive cells. TdT-mediated dUTP nick-end labeling (TUNEL) assay assessed cell apoptosis in intestinal tissues of II/R. Dual-luciferase assay verified the targeting relationship between miR-185-5p and ATG101.Results miR-185-5p was overexpressed in OGD/R-induced Caco-2 cells and intestinal tissues of II/R mice. Knocking down miR-185-5p markedly promoted autophagy and TEER values, reduced cell permeability, and alleviated intestinal barrier damage. ATG101 was a target of miR-185-5p, and overexpression of ATG101 promoted autophagy and dampened OGD/R-induced intestinal barrier damage. Overexpression of miR-185-5p reversed the effect of overexpressed ATG101 on OGD/R-induced Caco-2 cells. ConclusionKnockdown of miR-185-5p enhanced autophagy and alleviated II/R intestinal barrier damage by targeting ATG101.

Rhynchophylline alleviates endothelial injury in spontaneously hypertensive rats by regulating PPARγ mediated iron death

Purpose: To determine the effects of rhynchophylline (Rhy) on the progression of cardiovascular diseases in rats.&#x0D; Methods: Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR, 7 week old, weighing 180 – 200 g) were used in this study. The rats were divided into WKY, SHR, SHR+ Rhy (30 mg/kg), and SHR+ Rhy (50 mg/kg). WKY rats were used as the normal control group which had free access to distilled water. The rats were then orally administered Rhy (30 and 50 mg/kg daily by gavage). For inhibition of PPARr, T0070907 was given at a dose of 2 mg/kg for 24 h. The blood pressure of control, spontaneously hypertensive rats (SHR), and SHR rhynchophylline (Rhy) rats were measured. While PPARr levels in the hypertensive rats were assessed by Western blot. Cell apoptosis in response to SHR and Rhy were evaluated by TdT-mediated dUTP nick-end labeling (TUNEL) and Western blot assays, whereas oxidative stress and inflammatory response in SHR rats treated with Rhy were determined by enzyme linked immunosorbent assay (ELISA) and Immunoblot assays, respectively.&#x0D; Results: Rhy decreased hypertension and up-regulated PPARγ in SHR rats (p &lt; 0.01). In addition, Rhy improved SHR endothelial cell apoptosis by inducing PPARγ, and also produced reduction in proinflammatory factor secretion by inducing PPARγ. Furthermore, Rhy mitigated oxidative stress and iron death by inducing PPARγ (p &lt; 0.001).&#x0D; Conclusion: Rhynchophylline provides a promising method in alleviating endothelial injury in SHR rats by modulating PPARγ-mediated inflammation and oxidative stres. We therefore thought Rhy alleviated endothelial injury and could serve as a promising drug.

Mouse nerve growth factor suppresses neuronal apoptosis in valproic acid-induced autism spectrum disorder rats by regulating the phosphoinositide-3-kinase/serine/threonine kinase signaling pathway.

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by deficits in social communication and restrictive behaviors. Mouse nerve growth factor (mNGF), a neurotrophic factor, is critical for neuronal growth and survival, and the mNGF treatment is considered a promising therapy for neurodegeneration. In light of this, we aimed to evaluate the effect of mNGF on neurological function in ASD. An ASD rat model was established by intraperitoneal injection of valproic acid (VPA). Social behavior, learning, and memory of the rats were measured. TdT-mediated dUTP Nick-end labeling and Nissl assays were performed to detect neuronal apoptosis and survival in the hippocampus and prefrontal cortex. Apoptosis-related proteins and oxidative stress markers were detected. mNGF improved locomotor activity, exploratory behavior, social interaction, and spatial learning and memory in VPA-induced ASD rats. In the hippocampus and prefrontal cortex, mNGF suppressed neuronal apoptosis, increased the number of neurons, superoxide dismutase, and glutathione levels, and decreased reactive oxygen species, nitric oxide, TNF-α, and IL-1β levels compared with the VPA group. In addition, mNGF increased the levels of Bcl-2, p-phosphoinositide-3-kinase (PI3K), and p-serine/threonine kinase (Akt), and decreased the levels of Bax and cleaved caspase-3, while the PI3K inhibitor LY294002 reversed these effects. These data suggest that mNGF suppressed neuronal apoptosis and ameliorated the abnormal behaviors in VPA-induced ASD rats, in part, by activating the PI3K/Akt signaling pathway.

Rice tapetum differentiation is sensitive to downregulation of OsUCH3, a ubiquitin C-terminal hydrolase.

The tapetum is a special tissue within the stamen that supplies nutrients to support microspore differentiation. Many genes have been identified to function in tapetum differentiation (Astrand et al., 2021). Ubiquitin C-terminal hydrolase (UCH) is a type of cysteine protease involved in protein deubiquitination process (Mevissen and Komander, 2017) and plays roles in gonadal transformation and spermatogenesis in animals (Luo et al., 2009; Kwon et al,. 2004; Wang et al., 2006). OsUCH3 (Os02g43760) was highly expressed in early rice stamen cells (Chen et al., 2015), the OsUCH3 expression was first observed in all cells of anther primordium and then gradually concentrated into the germ cells and tapetum cells, and OsUCH3 protein exhibits stronger ubiquitin C-terminal hydrolase enzymatic activity (Wang et al., 2018). These data suggested that OsUCH3 is likely associated with stamen development. To explore whether OsUCH3 plays roles in rice stamen development, we constructed transgenic lines to down-regulate OsUCH3 expression through RNAi vector as described previously with primers (ccACTAGTATGGAGGATGCTCATTCC and TcGGATCCCACAACTTTCGAAAGAGC) (Liu et al., 2005) (Figure 1a–g) and the OsUCH3-targeting CRISPR-Cas9 mutant lines (Figure 1h). CRISPR-Cas9 osuch3 mutant lines exhibited the same male-sterile phenotype as OsUCH3R. In observation of the overall morphology, there was no significant difference at vegetable development (Figure 1a). However, low seed setting rate was observed in the RNAi lines (Figure 1b) and CRISPR-Cas9 osuch3 mutant (Figure 1h). Alexander staining showed that OsUCH3R lines had abnormal pollen (Figure 1d) with the latter being indicated by the significant reduction in pollen grain number (Figure 1e, g). The RT-qPCR results revealed that OsUCH3 expression levels were significantly reduced in OsUCH3R transgenic rice (Figure 1f). To examine whether OsUCH3 affected stamen development, we pollinated OsUCH3R pistils with wild-type pollen grains, which rescued the sterility of OsUCH3R. The result suggested that the low seed setting rate resulted mainly from down-expression of OsUCH3 (Figure 1i). Morphological analysis revealed abnormal tapetum differentiation and locule collapse were observed in the OsUCH3R lines from stage 5 (S5) through 12 (S12) (Figure 1j). This observation suggested that the downregulation of OsUCH3 caused a defect in the tapetum differentiation and stamen development. In contrast to the degeneration of the tapetum that was observed in the WT from stages 7 (S7) to 9 (S9) (Figure 1k–m), the tapetum in the OsUCH3R lines was not properly degenerated during these stages (Figure 1n–p) as revealed by transmission electron microscopy (TEM) analysis. In addition, TEM observations revealed that the secondary cell walls of the endothecium cells were thinner in the OsUCH3R lines than in the wild type (Figure 1p, red arrowhead). We found green fluorescence in wild-type tapetal cells from stages 7 to 9 (Figure 1q-t) using the terminal deoxynucleotidyl transferase-mediated TdT-mediated dUTP Nick-End Labeling (TUNEL) assay (Chen et al., 2018). No such TUNEL signals were detected in OsUCH3R tapetal cells during the same developmental period (Figure 1u–x). These results suggested that DNA degradation during tapetum differentiation after stage 7 was abnormal in OsUCH3R. Numerous genes were known being involved in tapetum differentiation (Chen et al., 2018). GAMYB4 was up-regulated while the expression of YY1, OsCP1 and UDT1 exhibited no change in the OsUCH3R lines (Figure 1y). OsUCH3 is lower expressed in shoot tips and leaf primordia. While no differential expression of SAM genes OsWOX3, the expression of OSH1 and OsWUS was significantly decreased in the OsUCH3R lines (Figure 1y). These changes demonstrated the link between abnormal tapetum differentiation in the OsUCH3R lines with the changes in the expression levels of known tapetum genes. The phenotypes we found in OsUCH3R lines and CRISPR mutants demonstrated that tapetal differentiation is sensitive to the change of expression level of OsUCH3 and protein deubiquitination is an importance factor for proper tapetum differentiation. This finding provided a new opportunity not only in deciphering the regulatory network of tapetum differentiation at protein level, but in manipulating male sterility for agricultural applications. Sequence data from this article can be found in the GenBank/EMBL data library. This work was supported by grants from the Chinese Transgenic Project (grant no. CARS-01-06, 2016ZX08010001 and 2016ZX08009-003) and the National Natural Science Foundation of China (grant no. 31630006). The authors declare no competing interests. D.H.W., Z.H.X. and S.N.B. designed the research and wrote the manuscript. D.H.W. performed most of the experiments. N.L. and S.D.Y. provided an assistance for mutant genotyping and plant material fixation. Z.S.C and Y.N.L provided technical assistance for transgenic plant analyse. Z.H.L helped the semithin sectioning.

Eriodictyol regulated ferroptosis, mitochondrial dysfunction, and cell viability via Nrf2/HO-1/NQO1 signaling pathway in ovarian cancer cells.

This study aimed to investigate the antitumor effect and the underlying molecular mechanism of eriodictyol on ovarian cancer cells.CaoV3 and A2780 were exposed to eriodictyol at different concentrations of 0-800 μM. Cell apoptosis and viability were determined by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay and Cell Counting Kit-8 (CCK-8) assay, respectively. Mitochondrial membrane potentialwas evaluated by flow cytometers with a JC-1 detection kit. Fe2+ content was evaluated using an iron assay kit. The section of tumor tissues was observed using hematoxylin-eosin (H&E) staining and nuclear factor erythroid 2-related factor 2 (Nrf2) expression was detected by immunohistochemistry (IHC) staining.Eriodictyol suppressed cell viability and induced cell apoptosis of CaoV3 and A2780 cells. Half maximal inhibitory concentration (IC50 ) value of CaoV3 at 24and 48 h was (229.74 ± 5.13) μM and (38.44 ± 4.68) μM, and IC50 value of A2780 at 24and 48 h was (248.32 ± 2.54) μM and (64.28 ± 3.19) μM. Fe2+ content and reactive oxygen species production were increased and protein levels of SLC7A11 and GPX4 were decreased by eriodictyol. Besides, eriodictyol reduced the ratio of JC-1 fluorescence ratio, glutathione and malondialdehyde contents but elevated Cytochrome C level. Nrf2 phosphorylation were obviously downregulated by eriodictyol. Finally, eriodictyol suppressed tumor growth, aggravated mitochondrial dysfunction and downregulated Nrf2 expression in tumor tissue in mice.Eriodictyol regulated ferroptosis, mitochondrial dysfunction and cell viability via Nrf2/HO-1/NQO1 signaling pathway in ovarian cancer.

Inhibition of miR-182-5p Targets FGF9 to Alleviate Osteoarthritis.

The pathogenesis of osteoarthritis (OA) is complex and there is no specific drug for treatment. The aim of this study was to identify the molecular targets of OA therapy, focusing on the expression and biological functions of miR-182-5p and its target genes in OA. miR-182-5p and fibroblast growth factor 9 (FGF9) were overexpressed or knocked down in IL-1β-induced chondrocytes. An OA knee model was performed by surgically destroying the medial meniscus. The gene expression of miR-182-5p and FGF9 was calculated. The protein FGF9 was tested by western blotting. Cell counting kit-8 (CCK8), plate cloning assay, and flow cytometry were conducted to evaluate cell proliferation and apoptosis. The expression of inflammatory factors, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and interleukin (IL)-8, was evaluated using enzyme-linked immunosorbent assay (ELISA). Dual-luciferase reporter assays validated the targeting relationship between miR-182-5p and FGF9. Hematoxylin-eosin (HE) and safranin O-fast Green (S-O) staining were utilized to access cartilage damage. Ki67 expression in cartilage was detected using immunohistochemistry (IHC). TdT-mediated dUTP nick-end labeling (TUNEL) assays were used to calculate the apoptosis rate of cartilage. The expression of miR-182-5p was upregulated, and FGF9 was downregulated in the IL-1β-induced chondrocytes. OA chondrocytes proliferation ability in the miR-182-5p mimics group was decreased, and the apoptosis rate and inflammatory factor were increased. Transfection with miR-182-5p inhibitor increased the proliferative ability and decreased the apoptosis rate in the IL-1β-induced chondrocytes. Transfection with miR-182-5p inhibitor reversed IL-1β-induced inflammatory factor release in chondrocytes. Targeted binding sites existed between miR-182-5p and FGF9. After overexpression of FGF9, the miR-182-5p effect on OA chondrocytes was reversed. The hyaline cartilage thickness and proteoglycan content decreased in OA rats, and this was reversed by miR-182-5p inhibitor treatment. miR-182-5p expression levels were increased in OA chondrocytes and regulated chondrocyte proliferation, apoptosis, and inflammation by targeting FGF9. miR-182-5p is a potential gene for OA treatment.

Enriched Environment Attenuates Ferroptosis after Cerebral Ischemia/Reperfusion Injury via the HIF-1α-ACSL4 Pathway.

Enriched environment (EE) has been proven to be an effective intervention strategy which can improve neurofunctional recovery following cerebral ischemia/reperfusion (I/R) injury. However, it still needs further investigation for the underlying mechanisms. Recently, it has been shown that ferroptosis played an essential role in the pathophysiological development of ischemic stroke (IS). This study is aimed at investigating whether EE plays a neuroprotective role by attenuating ferroptosis after cerebral I/R injury. We used middle cerebral artery occlusion/reperfusion (MCAO/R) to build a model of cerebral I/R injury. To evaluate the effect of EE on neurological recovery, we used the modified neurological severity score (mNSS) and the Morris water maze (MWM). We used the western blot to detect the protein levels of glutathione peroxidase 4 (GPX4), hypoxia-inducible factor-1α (HIF-1α), and acyl-CoA synthetase long-chain family member 4 (ACSL4). We used the quantitative real-time PCR (qRT-PCR) to measure the mRNA levels of ACSL4 and inflammatory cytokines including tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and interleukin 1 beta (IL-1β). The occurrence of ferroptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL) assay, diaminobenzidine- (DAB-) enhanced Perls' staining, iron level assays, and malondialdehyde (MDA) level assays. The results verified that EE enhanced functional recovery and attenuated ferroptosis and neuroinflammation after cerebral I/R injury. EE increased the expression of HIF-1α while inhibited the expression of ACSL4. Our research indicated that EE improved functional recovery after cerebral I/R injury through attenuating ferroptosis, and this might be related to its regulation of the neuroinflammation and HIF-1α-ACSL4 pathway.

Interactive effects of temperature and salinity on the apoptosis, antioxidant enzymes, and MAPK signaling pathway of juvenile pufferfish (Takifugu fasciatus)

Takifugu fasciatus is an anadromous fish widely distributed in East Asia and often experiences fluctuations in temperature and salinity. This study investigated the effects of the interactions between temperature and salinity on apoptosis, antioxidant enzymes, and mitogen-activated protein kinase (MAPK) signaling pathways in the liver of T. fasciatus. The temperature (25, 21, 17, and 13 ℃) and salinity (0, 10, and 20 ppt salinity) were conducted with three replicates for each treatment. Quantitative reverse transcription-PCR (qRT-PCR), western blotting, and TdT-mediated dUTP Nick-End Labeling (Tunel) assays were used to detect mRNA expression, protein levels, and apoptosis levels. The expressions of caspase 3, 7, 9, and the apoptosis index increased in T. fasciatus as the temperature decreased, while the expression of B-cell lymphoma-2 (BCL-2) decreased. The correlation results in this study showed that after the exposure to 10 ppt salinity at 13 and 17 ℃, the glutathione peroxidase (GSH-PX) activity and malondialdehyde (MDA) concentration, caspase3, 7, and 9 expressions, and apoptosis index were decreased as compared with those in the control. In addition, there were no deaths and no significant change in apoptosis in the group at 21 ℃, and there were a small number of deaths in the 20 ppt, 25 ℃ groups. Correspondingly, MDA concentration and total superoxide dismutase (T-SOD) activity, caspase7 and 9 expressions, and apoptosis index in the liver were significantly higher in the 20 ppt, 25 ℃ groups than in the 0 ppt, 25 ℃, and 10 ppt, 25 ℃ groups. In addition, salinity had no significant influence on the expression of p-p38 MAPK and its phosphorylation ratio (p-p38/p38) at low temperatures but significantly affected its downstream transcription factors (ATF2, ElK-1, MEF2, and P53). Our study provides a reference for healthy cultures of T. fasciatus.