TdT-mediated dUTP Nick End Labeling Research Articles

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.

In Vitro Wound Healing and Anticancer Effects of Ixora coccinea in Malignant Melanoma Cell Lines.

Background Ixora coccinea is a medicinal plant with many active constituents that are responsible for wound healing and have anticancer properties. Herbal extracts increase the mechanisms related to wound healing, like blood clotting, fighting infection, and epithelialization. The effect responsible for this property may be the presence of phytoconstituents like flavonoids, polyphenols, and alkaloids. Many researchers have evaluated the wound-healing effect of I. coccinea leaf extract in aqueous methanol. This study aimed to determine the in vitro wound healing and anticancer efficacy of I. coccinea leaf ethyl acetate extract and evaluate the in silico docking of the selected phytoconstituents of I. coccinea in the 2vcj protein. Materials and methods The human dermal fibroblast cell line was used to determine the rates of cell migration and proliferation for evaluating the wound-healing effect of the I. coccinea leaf ethyl acetate fraction. 4',6-diamidino-2-phenylindole (DAPI)fluorescence labeling was used to estimate the rate of cell migration. The one-step TUNEL (TdT-mediated dUTP Nick-End Labeling) in situ apoptosis kit and the annexin V-FITC/7-AAD apoptosis kit were used to perform DNA damage assays in the malignant melanoma cell line. The ethyl acetate fraction of I. coccinea leaves was analyzed for its impact on wound healing markers, including keratin-10, keratin-14, type IV collagen, and α-SMA. Results The wound-healing nature was interesting in the ethyl acetate fraction at doses of 50 µg/mL and 100 µg/mL. Both studies involved in the DNA damage study against malignant melanoma cell lines showed the cleavage of apoptotic cancer cells, which was detected using a fluorescence microscope. When compared with the control, a dose of 100 μg/ml of ethyl acetate fraction from the leaves of I. coccinea showed fibroblast migration of cells into the wound area. The statistical values were considered significant at the level of P < 0.05. An in silico docking study on the 2vcj protein revealed that selected phytoconstituents of I. coccinea resulted in good docking scores to inhibit Hsp90. Conclusion I. coccinea ethyl acetate leaf extract can inhibit the growth of malignant melanoma cell lines and promote wound healing, as shown by the study results. It might be a viable therapeutic modality for skin cancer.

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.

Influence of trimetazidine on myocardial injury in mice with diabetic cardiomyopathy

IntroductionThe prevalence of diabetes mellitus is increasing year by year globally, and diabetic cardiomyopathy (DCM), as the most common complication of type 2 diabetes mellitus, seriously affects the prognosis of patients. Trimetazidine (TMZ), as a drug affecting myocardial energy metabolism, mainly reduces the oxidation rate of β-oxidation by inhibiting 3-ketoacyl-CoA thiolase (3-KAT), a key enzyme in β-oxidation of free fatty acid (FFA), so that the energy metabolism substrate of cardiomyocytes preferentially selects glucose rather than fatty acids, increases the content of intracellular adenosine triphosphate (ATP), enhances the contractile function of cardiomyocytes, and improves the state of cellular ischemia and hypoxia. Previous studies have shown that TMZ is closely related to the activation and induction of apoptosis of the MAPK pathway and AMPK pathway, and plays a role in the treatment of diabetic cardiomyopathy, but the specific mechanism is still unclear. ObjectiveThis study aims to investigate the impact of TMZ on myocardial damage in mice exhibiting diabetic cardiomyopathy (DCM), and to furnish a laboratory foundation for the clinical treatment of diabetic cardiomyopathy. MethodMale db/db mice (6 weeks old, n = 21) and male wild-type (wt) (6 weeks old, n = 20) mice were selected for the study. The wt mice were randomly assigned to the wt group (n = 10) and wt + TMZ group (n = 10), while the remaining db/db mice were randomly allocated to the db/db group (n = 11) and db/db + TMZ group (n = 10). Following 8 weeks of feeding, the wt + TMZ group and db/db + TMZ group received TMZ via gavage, whereas the remaining groups were administered physiological saline. Periodic measurements of blood glucose, blood lipids, and myocardial enzymes were conducted in mice, with samples obtained after the 12th week for subsequent biochemical analysis, myocardial pathology assessment, immunohistochemistry, western blot analysis, and TUNEL staining (TdT-mediated dUTP Nick-End Labeling). ResultGLU, TC, TG, LDL-C, and CK-MB levels were significantly higher in db/db mice compared to wt mice (GLU: M ± SD wt 5.94 ± 0.37, db/db 17.63 ± 0.89, p < 0.05, ES = 0.991; TC: M ± SD wt 3.01 ± 0.32, db/db 6.97 ± 0.36, p < 0.05, ES = 0.972; TG: M ± SD wt 0.58 ± 0.2, db/db 1.75 ± 0.14, p < 0.05, ES = 0.920; LDL-C: M ± SD wt 1.59 ± 0.12, db/db 3.87 ± 0.14, p < 0.05, ES = 0.989; CK-MB: M ± SD wt 0.12 ± 0.01, db/db 0.31 ± 0.04, p < 0.05, ES = 0.928). HDL-C levels were significantly lower in db/db mice (M ± SD wt 1.89 ± 0.08, db/db 0.64 ± 0.09, p < 0.05, ES = 0.963). Histopathological analysis confirmed myocardial damage in db/db mice. Treatment with TMZ reduced GLU, TC, TG, LDL-C, and CK-MB levels (p < 0.05, ES > 0.9) and increased HDL-C levels compared to untreated db/db mice. Additionally, TMZ treatment significantly decreased myocardial cell apoptosis (p < 0.05, ES = 0.980). These results demonstrate the efficacy of TMZ in reversing myocardial injury in DCM mice. ConclusionTMZ can mitigate myocardial damage in db/db mice by downregulating the expression of caspase-12, a protein associated with the endoplasmic reticulum stress (ERS) cell apoptosis pathway, consequently diminishing cell apoptosis. This underscores the protective efficacy of TMZ against myocardial damage in mice afflicted with DCM.

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.

Dihydromyricetin regulates RIPK3-CaMKII to prevent necroptosis in high glucose-stimulated cardiomyocytes

BackgroundDiabetic cardiomyopathy is one common cardiovascular complication without effective treatments. Dihydromyricetin (DHY), a natural dihydroflavonol compound extracted from Ampelopsis grossedentata, possesses versatile pharmacologically important effects. In our current research, we planned to evaluate the impact and probable DHY mechanisms in high glucose (HG)-induced cardiomyocytes. MethodsPrimary cardiomyocytes were pretreated with different concentrations of DHY (0, 20, 40, 80, 160, and 320 μM) for various time (0, 1, 2, 4, 12, and 24 h). They were then stimulated for 48 h with 5.5 mmol/L normal glucose (NG) and 33.3 mmol/L high glucose (HG). Cell viability, adenosine-triphosphate (ATP) levels, and lactate dehydrogenase (LDH) release of cardiomyocytes were detected. JC-1 staining was employed to measure the mitochondrial membrane potential. MitoSOX staining and dihydroethidium (DHE) staining were applied to evaluate the oxidative stress levels. TDT mediated dUTP nick end labeling (TUNEL) was used to measure apoptotic levels. Expressions of calcium/calmodulin-dependent protein kinase II (CaMKII), phospholamban (PLB), optic atrophy 1 (OPA1), dynamin-related protein 1 (DRP1), caspase 3, mixed kinase lineage domain like protein (MLKL), receptor interacting protein kinase 3 (RIPK3), and receptor interacting protein kinase 1 (RIPK1) were detected by immunofluorescence and/or Western blot. ResultsDHY improved cell viability, enhanced ATP level, and decreased LDH content in HG-stimulated cardiomyocytes, suggesting DHY attenuating cell injury. DHY reduced number of TUNEL positive cells, inhibited RIPK3 and cleaved-caspase 3 expression, implying DHY alleviated necroptosis in HG-stimulated cardiomyocytes. DHY diminished JC-1 monomers, DHE and MitoSOX fluorescence intensity as well as DRP1 expression but increased JC-1 aggregates intensity and OPA1 expression, indicating that DHY attenuated oxidative stress in HG-stimulated cardiomyocytes. DHY also attenuated CaMKII activity by suppressed PLB phosphorylation and inhibited CaMKII oxidation in HG-stimulated cardiomyocytes. ConclusionsHG-induced cardiomyocytes injury was alleviated wherein DHY attenuated necroptosis, repressed ROS production, and inhibited CaMKII oxidation, suggesting that DHY may serve as potential agent to prevent and treat diabetic cardiomyopathy.

Regulatory mechanism of the Glabrene against non-small cell lung cancer by suppressing FGFR3.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor with limited effective treatment options. This study aimed to investigate the regulatory mechanism of Glabrene on NSCLC through its interaction with FGFR3. HCC827 cells were implanted into nude mice and treated with Glabrene. Tumor volume was monitored at 0, 3, 6, and 9 days after medical treatment. Tissue analysis included Hematoxylin and Eosin (HE) and Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) staining, as well as immunohistochemistry for Ki67, ERK1/2, and p-ERK1/2 expression. Cell viability was determined with the CCK8 method. We utilized immunofluorescence techniques to observe apoptosis, as well as the levels of E-cadherin and Vimentin expression. Cellular proliferation was determined via plate cloning assay and cellular mobility was determined via scratch assay. Cellular invasion ability was assessed via a transwell assay. mRNA and protein levels of FGFR3, MMP1, MMP9, vimentin, E-cadherin, ERK1/2, and p-ERK1/2 were detected via qPCR and Western blot. IGF-1, VEGF, and Estradiol (E2) levels were measured through Enzyme linked immunosorbent assay (ELISA). This study verified that Glabrene was capable of suppressing tumor growth in NSCLC mice, reversing tumor tissue's pathological morphology, attenuating the capacities of cancerous cells' proliferation, migration, and invasion, and leading to apoptosis. Besides, Glabrene could reduce the FGFR3 expression in HCC827 cells. Over-expression of FGFR3 promotes the proliferation of HCC827 cells, increase both contents of IGF-1, VEGF, and E2, and expressions of MMP1, MMP9, vimentin, and p-ERK1/2, while Glabrene inhibited FGFR3. Glabrene, and inhibition of FGFR3 expression were capable of decreasing FGFR3, MMP1, MMP9, vimentin, and p-ERK1/2 expression, as well as contents of IGF-1, VEGF, and E2 in model mice and HCC827 cells, and promoting the expression of E-cadherin. Glabrene has the potential as a therapeutic agent for NSCLC by reducing cancer invasion and migration through the inhibition of ERK1/2 phosphorylation and suppression of epithelial-mesenchymal transition (EMT).

WTAP-mediated N6-methyladenosine modification promotes the inflammation, mitochondrial damage and ferroptosis of kidney tubular epithelial cells in acute kidney injury by regulating LMNB1 expression and activating NF-κB and JAK2/STAT3 pathways.

Acute kidney injury (AKI) is a serious complication of sepsis patients, but the pathogenic mechanisms underlying AKI are still largely unclear. In this view, the roles of the key component of N6-methyladenosine (m6A)-wilms tumor 1 associated protein (WTAP) in AKI progression were investigated. AKI mice model was established by using cecal ligation and puncture (CLP). AKI cell model was established by treating HK-2 cells with LPS. Cell apoptosis was analyzed by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining and flow cytometry analysis. Cell viability was analyzed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The concentrations of inflammatory factors were examined with ELISA kits. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and Fe2+ levels were detected with related kits. Gene expression was detected by western blot assay or quantitative real-time polymerase chain reaction (qRT-PCR) assay. The relation between WTAP and lamin B1 (LMNB1) was verified by Methylated RNA Immunoprecipitation (meRIP) assay, RIP assay, dual-luciferase reporter assay and Actinomycin D assay. CLP induced significant pathological changes in kidney tissues in mice and promoted inflammation, mitochondrial damage and ferroptosis. LMNB1 level was induced in HK-2 cells by LPS. LMNB1 knockdown promoted LPS-mediated HK-2 cell viability and inhibited LPS-mediated HK-2 cell apoptosis, inflammation, mitochondrial damage and ferroptosis. Then, WTAP was demonstrated to promote LMNB1 expression by m6A Methylation modification. Moreover, WTAP knockdown repressed LPS-treated HK-2 cell apoptosis, inflammation, mitochondrial damage and ferroptosis, while LMNB1 overexpression reversed the effects. Additionally, WTAP affected the pathways of NF-κB and JAK2/STAT3 by LMNB1. WTAP-mediated m6A promoted the inflammation, mitochondrial damage and ferroptosis in LPS-induced HK-2 cells by regulating LMNB1 expression and activating NF-κB and JAK2/STAT3 pathways.

Jintiange capsule ameliorates glucocorticoid-induced osteonecrosis of the femoral head in rats by regulating the activity and differentiation of BMSCs

Background and aimA surplus of glucocorticoids (GC) is a main cause of non-traumatic osteonecrosis of the femoral head (ONFH), and Jintiange (JTG), as one of the traditional Chinese medicines (TCM), also plays an instrumental role in the alleviation of bone loss simultaneously. Therefore, JTG was thought to be able to reverse GC-induced ONFH (GC-ONFH) to a certain extent. Experimental procedureIn vivo, the effect of JTG on trabeculae in the subchondral bone of the femoral head was investigated using micro-computed tomography (micro-CT), TdT-mediated dUTP nick end labeling (TUNEL) and histological staining; in vitro, proliferation, viability, apoptosis, and senescence of purified bone mesenchymal stem cells (BMSCs) were examined to demonstrate the direct impact of JTG on these cells. Meanwhile after using a series of interventions, the function of JTG on BMSC differentiation could be assessed by measuring of osteogenic and adipogenic markers at levels of protein and mRNA. ResultsOur final results demonstrated that with the involvement of Wnt/β-catenin pathway, JTG was able to significantly promote osteogenesis, restrain adipogenesis, delay senescence in BMSCs, reduce osteoclast number, weaken apoptosis, and enhance proliferation of osteocytes, all of which could mitigate the progression of subchondral osteonecrosis. ConclusionAccording to the results of experiments in vitro and vivo, JTG was deemed to relieve the early GC-ONFH using the prevention of destruction of subchondral bone, which was contributed to regulating the differentiation of BMSCs and the number of osteoclasts.

Adiponectin attenuates H2O2-induced apoptosis in chicken skeletal myoblasts through the lysosomal-mitochondrial axis.

Adiponectin has previously been investigated for exerting its protective effect against myocardial injury through anti-apoptotic and anti-oxidative actions. Therefore, the present study aimed to investigate the nature and mechanism of adiponectin inhibition of H2O2-induced apoptosis in chicken skeletal myoblasts. Skeletal muscle satellite cells were differentiated and assigned into three groups. Group C was on the blank control group, group H was stimulated with the H2O2 (500μmol/L, 4h) alone group, group A + H was pre-treated with adiponectin (10μg/mL, 24h) and stimulated with the H2O2 (500μmol/L, 4h) group. Cytotoxicity inhibited by adiponectin was evaluated by the CCK-8 assay. The degree of apoptosis and oxidative damage was investigated by the TdT-mediated dUTP nick end labeling (TUNEL) and reactive oxygen species (ROS) staining assays. Oxidative stress was assessed by evaluating lipid peroxidation, superoxide dismutase, and reduced glutathione. Acridine orange (AO) staining detected lysosomal membrane permeability. The changes in mitochondrial membrane potential (MMP) were analyzed using 5,5,6,6'-tetrachloro-1,1,3,3-tetraethylimidacarbocyanine iodide (JC-1) dye under a fluorescence microscope. The lysosomal function, mitochondrial function, and apoptosis-related mRNA and protein expression levels were quantified by real-time quantitative PCR and western blot, respectively. The results suggested that adiponectin treatment attenuated H2O2-induced cytotoxicity and oxidative stress in skeletal myoblasts. Compared with H2O2 treatment, TUNEL and ROS staining demonstrated lower apoptosis upon adiponectin treatment. AO staining confirmed the amelioration of lysosomal membrane damage, and JC-1 staining revealed an increase in mitochondrial membrane potential after adiponectin treatment. At the molecular level, adiponectin treatment inhibited the expression of the lysosomal apoptotic factors cathepsin B, chymotrypsin B, and the mitochondrial apoptotic pathway cytochrome-c (cyt-c) and caspase-8; decreased the apoptotic marker gene Bax; and increased the expression of the anti-apoptotic marker gene Bcl-2. Adiponectin treatment attenuated H2O2-induced apoptosis in skeletal myoblasts, possibly by inhibiting oxidative stress and apoptosis through the lysosomal-mitochondrial axis.

Mechanism of Qili Qiangxin Capsule for Heart Failure Based on miR133a-Endoplasmic Reticulum Stress.

To investigate the pharmacological mechanism of Qili Qiangxin Capsule (QLQX) improvement of heart failure (HF) based on miR133a-endoplasmic reticulum stress (ERS) pathway. A left coronary artery ligation-induced HF after myocardial infarction model was used in this study. Rats were randomly assigned to the sham group, the model group, the QLQX group [0.32 g/(kg·d)], and the captopril group [2.25 mg/(kg·d)], 15 rats per group, followed by 4 weeks of medication. Cardiac function such as left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), the maximal rate of increase of left ventricular pressure (+dp/dt max), and the maximal rate of decrease of left ventricular pressure (-dp/dt max) were monitored by echocardiography and hemodynamics. Hematoxylin and eosin (HE) and Masson stainings were used to visualize pathological changes in myocardial tissue. The mRNA expression of miR133a, glucose-regulated protein78 (GRP78), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), X-box binding protein1 (XBP1), C/EBP homologous protein (CHOP) and Caspase 12 were detected by RT-PCR. The protein expression of GRP78, p-IRE1/IRE1 ratio, cleaved-ATF6, XBP1-s (the spliced form of XBP1), CHOP and Caspase 12 were detected by Western blot. TdT-mediated dUTP nick-end labeling (TUNEL) staining was used to detect the rate of apoptosis. QLQX significantly improved cardiac function as evidenced by increased EF, FS, LVSP, +dp/dt max, -dp/dt max, and decreased LVEDP (P<0.05, P<0.01). HE staining showed that QLQX ameliorated cardiac pathologic damage to some extent. Masson staining indicated that QLQX significantly reduced collagen volume fraction in myocardial tissue (P<0.01). Results from RT-PCR and Western blot showed that QLQX significantly increased the expression of miR133a and inhibited the mRNA expressions of GRP78, IRE1, ATF6 and XBP1, as well as decreased the protein expressions of GRP78, cleaved-ATF6 and XBP1-s and decreased p-IRE1/IRE1 ratio (P<0.05, P<0.01). Further studies showed that QLQX significantly reduced the expression of CHOP and Caspase12, resulting in a significant reduction in apoptosis rate (P<0.05, P<0.01). The pharmacological mechanism of QLQX in improving HF is partly attributed to its regulatory effect on the miR133a-IRE1/XBP1 pathway.

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.

Astragaloside IV (ASIV) Mediates Endothelial Progenitor Cell (EPC) Exosomal LINC01963 to Inhibit Pyroptosis and Oxidative Stress in High Glucose-impaired Endothelial Cells.

Hyperglycemia is widespread in the world's population, increasing the risk of many diseases. This study aimed to explore the regulatory effect and mechanism of astragaloside IV (ASIV)-mediated endothelial progenitor cells (EPCs) exosomal LINC01963 in endothelial cells (HUVECs) impaired by high glucose. Morphologies of exosomes were observed by light microscope and electron microscope. Immunofluorescence was used to identify EPCs and detect the expressions of caspase-1. LINC01963 was detected by quantitative reverse transcription PCR. NLRP3, ASC, and caspase-3 were detected by Western Blot. Nanoparticle tracking analysis was carried out to analyze the exosome diameter. High-throughput sequencing was applied to screen target lncRNAs. The proliferation of endothelial cells was measured by cell counting kit-8 assay. The apoptosis level of HUVECs was detected by flow cytometry and TdT-mediated dUTP Nick-End labeling. The levels of IL-1β, IL-18, ROS, SOD, MDA, and LDH were measured by enzyme-linked immunosorbent assay. ASIV could promote the secretion of the EPC exosome. LINC01963 was obtained by high-throughput sequencing. It was observed that high glucose could inhibit the proliferation, reduce the level of SOD, the expression of NLRP3, ASC, and caspase-1, increase the levels of IL-1β, IL-18, ROS, MDA, and LDH, and promote apoptosis of HUVECs. Whereas LINC01963 could inhibit the apoptosis of HUVECs, the expression of NLRP3, ASC, and caspase-1 increased, and the levels of IL-1β, IL-18, ROS, MDA, and LDH decreased. EPCs exosomal LINC01963 play an inhibitory role in high glucose-induced pyroptosis and oxidative stress of HUVECs. This study provides new ideas and directions for treating hyperglycemia and researching exosomal lncRNAs.

Intervention effect of Cigu Xiaozhi prescription on ceramide lipoapoptosis in non-alcoholic fatty liver disease.

To explore the mechanism of the Chinese medicine Cigu Xiaozhi prescription (, CGXZ) in the treatment of the non-alcoholic fatty liver disease (NAFLD) by detoxification and phlegm-reducing, the effect of CGXZ prescription on ceramide-mediated lipid apoptosis in Hep G2 cells with NAFLD. The experiment was randomly divided into 6 groups: normal control group, model group, CGXZ prescription medicated serum high, medium, and low dose groups, and pioglitazone positive control group. Using 500 μmol/L free fatty acid (FFA) mixture to induce Hep G2 cells to establish NAFLD cell model, respectively, with 2%, 4%, and 6% concentration of CGXZ prescription medicated serum intervention for 24 h. The changes in organelles and lipid droplet accumulation were observed under the electron microscope. Furthermore, TdT-mediated dUTP Nick-End Labeling method was used to assay hepatocyte apoptosis; Biochemical determination of glutamic-pyruvic transaminase, glutamic oxalacetic transaminase, triglycerides, and FFA levels in Hep G2 cells; the content of ceramide was determined by high-performance thin-layer chromatography. Finally, Western Blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the protein and gene expression levels, such as inducible nitric oxide synthase (iNOS), nuclear factor κB (NF-κB), B cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax). Under the electron microscope, the cells in the model group showed moderate-to-severe steatosis, and apoptotic bodies could be seen. The model group had greater improvements in the apoptosis rate (P < 0.01), and the levels of ceramide C2 and FFA in the cytoplasm (P < 0.01) than the normal control group. The protein expressions of NF-κB, iNOS, and Bax were significantly up-regulated (P < 0.05), while the Bcl-2 had no significant change (P > 0.05). Compared with the model group, the levels of ceramide C2 and FFA (P < 0.01), the protein expressions of NF-κB, iNOS, and Bax (P < 0.05) in the CGXZ prescription treatment group and pioglitazone positive control group were significantly decreased; Only the Bcl-2 protein was significantly up-regulated in the high-dose Chinese medicine group (P < 0.05). The down-regulation of Bax mRNA expression in each Chinese medicine treatment group was significantly better than in the pioglitazone positive control group (P < 0.01). The CGXZ prescription, formulated with the method of detoxification and phlegm, can inhibit lipoapoptosis in the NAFLD cell model by down-regulating the levels of ceramide C2 and FFA, which may be achieved by regulating ceramide/iNOS/NF-κB signaling pathway.

Compound Danshen dripping pills prevent early diabetic retinopathy: roles of vascular protection and neuroprotection.

Introduction: Diabetic retinopathy (DR) represents a major cause of adult blindness, and early discovery has led to significant increase in the number of patients with DR. The drugs currently used for treatment, such as ranibizumab, mainly focus on the middle and late periods of DR, and thus do not meet the clinical need. Here, the potential mechanisms by which compound Danshen Dripping Pills (CDDP) might protect against early DR were investigated. Methods: Db/db mice were used to establish a DR model. The initial weights and HbA1c levels of the mice were monitored, and retinal pathology was assessed by hematoxylin-eosin (HE) staining. The vascular permeability of the retina and thickness of each retinal layer were measured, and electroretinogram were performed together with fundus fluorescein angiography and optical coherence tomography. The levels of inflammatory factors were examined in retinal tissue, as well as those of intercellular adhesion molecule 1 (ICAM-1), IL-6, and monocyte chemoattractant protein 1 (MCP-1) in the serum using ELISA. Immunohistochemistry was used to evaluate levels of vascular endothelial growth factor (VEGF), B-cell lymphoma 2 (Bcl-2), and Bclassociated X protein (Bax). Retinal cell injury and apoptosis were examined by TdT-mediated dUTP Nick End Labeling (TUNEL) assays. Results: The data showed that CDDP significantly improved cellular disarrangement. Imaging data indicated that CDDP could reduce vascular permeability and the amplitude of oscillatory potentials (OPs), and restore the thickness of the ganglion cell layer. Moreover, CDDP reduced the expression levels of inflammatory factors in both the retina and serum. Conclusion: These findings strongly suggest that CDDP prevents early DR through vascular and neuroprotection.

Hair Follicle Damage after 100 mGy Low-Dose Fractionated X-Ray Irradiation and the Protective Effects of TEMPOL, a Stable Nitroxide Radical, against Radiation.

The effects of long-term low-dose X-ray irradiation on the outer root sheath (ORS) cells of C3H/He mice were investigated. Mice were irradiated with a regime of 100 mGy/day, 5 days/week, for 12 weeks (Group X) and the results obtained were compared to those in a non-irradiated control (Group C). Potential protection against ORS cells damage induced by this exposure was investigated by adding the stable nitroxide radical 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) at 1 mM to the drinking water of mice (Group X + TEMPOL). The results obtained were compared with Group C and a non-irradiated group treated with TEMPOL (Group C + TEMPOL). After fractionated X-ray irradiation, skin was removed and ORS cells were examined by hematoxylin and eosin staining and electron microscopy for an abnormal nuclear morphology and nuclear condensation changes. Fractionated X-irradiated mice had an increased number of ORS cells with an abnormal nuclear morphology as well as nuclear condensation changes. Sections were also immunohistochemically examined for the presence of TdT-mediated dUTP nick-end labeling (TUNEL), 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE), vascular endothelial growth factor (VEGF), nitrotyrosine, heme oxygenase 1 (HO-1), and protein gene product 9.5 (PGP 9.5). Significant increases were observed in TUNEL, 8-OHdG, and 4-HNE levels in ORS cells from mice in Group X. Electron microscopy also showed irregular shrunken ORS cells in Group X. These changes were prevented by the presence of TEMPOL in the drinking water of the irradiated mice. TEMPOL alone had no significant effects. These results suggest that fractionated doses of radiation induced oxidative damage in ORS cells; however, TEMPOL provided protection against this damage, possibly as a result of the rapid reaction of this nitroxide radical with the reactive oxidants generated by fractionated X-ray irradiation.

N-acetylcysteine protects septic acute kidney injury by inhibiting SIRT3-mediated mitochondrial dysfunction and apoptosis.

To investigate the protective effect of N-acetylcysteine (NAC) on septic acute kidney injury (SAKI) via regulating Sirtuin3 (SIRT3)-mediated mitochondrial dysfunction and apoptosis. By constructing SIRT3 knockout mice and culturing kidney tubular epithelial cells (KTECs), we assessed the changes of renal function and detected the protein expression of adenine nucleotide translocator (ANT), cyclophilin (CypD) and voltage-dependent anion channel (VDAC) using western-blotting, and simultaneously detected toll-like receptor 4 (TLR4), inhibitor of kappa B kinase (IKKβ), inhibitor of Kappa Bα (IκBα), and p65 protein expression. We observed mitochondrial damage of KTECs using a transmission electron microscope and assessed apoptosis by TdT-mediated dUTP Nick-End Labeling and flow cytometry. SIRT3 deficiency led to the deterioration of renal function, and caused a significant increase in inducible nitric oxide synthase production, a decrease in mitochondrial volume, up-regulation of TLR4, IκBα, IKKβ, and p65 proteins, and up-regulation of ANT, CypD and VDAC proteins. However, NAC significantly improved renal function and down-regulated the expression of TLR4, IκBα, IKKβ, and p65 proteins. Furthermore, SIRT3 deficiency led to a significant increase in KTEC apoptosis, while NAC up-regulated the expression of SIRT3 and inhibited apoptosis. NAC has a significant protective effect on SAKI by inhibiting SIRT3-mediated mitochondrial dysfunction and apoptosis of KTECs.

Fucoidan alleviated autoimmune diabetes in NOD mice by regulating pancreatic autophagy through the AMPK/mTOR1/TFEB pathway.

The present study investigated the effect and its underlying mechanisms of fucoidan on Type 1 diabetes mellitus (T1DM) in non-obese diabetic (NOD) mice. Twenty 7-week-old NOD mice were used in this study, and randomly divided into two groups (10 mice in each group): the control group and the fucoidan treatment group (600 mg/kg. body weight). The weight gain, glucose tolerance, and fasting blood glucose level in NOD mice were detected to assess the development of diabetes. The intervention lasted for 5 weeks. The proportions of Th1/Th2 cells from spleen tissues were tested to determine the anti-inflammatory effect of fucoidan. Western blot was performed to investigate the expression levels of apoptotic markers and autophagic markers. Apoptotic cell staining was visualized through TdT-mediated dUTP nick-end labeling (TUNEL). The results suggested that fucoidan ameliorated T1DM, as evidenced by increased body weight and improved glycemic control of NOD mice. Fucoidan down-regulated the Th1/Th2 cells ratio and decreased Th1 type pro-inflammatory cytokines' level. Fucoidan enhanced the mitochondrial autophagy level of pancreatic cells and increased the expressions of Beclin-1 and LC3B II/LC3B I. The expression of p-AMPK was up-regulated and p-mTOR1 was inhibited, which promoted the nucleation of transcription factor EB (TFEB), leading to autophagy. Moreover, fucoidan induced apoptosis of pancreatic tissue cells. The levels of cleaved caspase-9, cleaved caspase-3, and Bax were up-regulated after fucoidan treatment. Fucoidan could maintain pancreatic homeostasis and restore immune disorder through enhancing autophagy via the AMPK/mTOR1/TFEB pathway in pancreatic cells.

2'-Fucosyllactose restores the intestinal mucosal barrier in ulcerative colitis by inhibiting STAT3 palmitoylation and phosphorylation

2'-Fucosyllactose (2'-FL), the primary constituent of human milk oligosaccharides, has been identified as a potential regulator of inflammation in inflammatory bowel disease. Despite this recognition, the specific mechanisms through which 2'-FL alleviates ulcerative colitis (UC) remain ambiguous. This study seeks to investigate the potential anti-inflammatory properties of 2'-FL concerning intestinal inflammation and uncover the associated mechanisms. C57BL/6J mice were orally administered a daily dose of 500mg/kg 2'-FL for 11 consecutive days, followed by the induction of colitis using 3% (wt/vol) dextran sulfate sodium (DSS) for the final 6 days. Subsequently, a comprehensive range of techniques, including an Acyl-biotin exchange assay, fluorescein-isothiocyanate-labeled dextran assay, histopathology, ELISA, quantitative real-time PCR, Western blot, immunofluorescence staining, immunohistochemistry staining, Alcian blue-periodic acid schiff staining, TdT-mediated dUTP nick end labeling, transmission electron microscopy, iTRAQ quantitative proteomics, bioinformatics analysis, and the generation of signal transducer and activator of transcription 3 (STAT3) knockout mice, were employed to explore the relevant molecular mechanisms. Administration of 2'-FL significantly ameliorated DSS-induced colitis in mice and enhanced the integrity of the intestinal mucosal barrier. 2'-FL downregulated the phosphorylation of STAT3 and inhibited STAT3-related signaling pathways in colon tissues, which, in turn, reduced inflammatory responses. Interestingly, knockdown of STAT3 attenuated the protective effects of 2'-FL, highlighting that 2'-FL-mediated inflammatory attenuation is dependent on STAT3 expression. Additionally, 2'-FL could influence STAT3 activation by modulating the palmitoylation and depalmitoylation of STAT3. 2'-FL promotes the recovery of the intestinal mucosal barrier and suppresses inflammation in ulcerative colitis by inhibiting the palmitoylation and phosphorylation of STAT3.

Transformation of Ginsenosides by Lactiplantibacillus plantarum MB11 Fermentation: Minor Ginsenosides Conversion and Enhancement of Anti-Colorectal Cancer Activity.

The present study aimed to increase the content of minor ginsenosides and enhance the anti-colorectal cancer activity of ginsenosides via biotransformation by Lactiplantibacillus plantarum MB11 screened from fermented foods. A subcutaneous transplantation tumor model of murine colorectal cancer CT26 cells was established in mice to study the anticarcinogenic activities and mechanism of fermented total ginsenosides (FTGs). The results showed that L. plantarum MB11 fermentation increased the content of minor ginsenosides and decreased that of major ginsenosides. FTGs reduced the tumor weight and size compared with the model group. Immunofluorescence and TdT-mediated dUTP nick end labeling (TUNEL) analysis showed that FTGs significantly increase the number of caspase-3 cells in tumor tissue and induce cell apoptosis. Mechanically, FTGs activate AMPK/mTOR autophagy pathway and regulate JAK2/STAT3 and Bax/Bcl-2/caspase-3 apoptosis pathway. Overall, fermentation with L. plantarum MB11 enhanced minor ginsenosides in total ginsenosides, and FTGs induced subcutaneous transplantation tumor autophagy and apoptosis in mice.