Terminal Labeling Research Articles

Hypomethylation of IL6ST promotes development of endometriosis by activating JAK2/STAT3 signaling pathway.

Endometriosis is a chronic inflammatory disorder characterized by presence of endometrial tissue outside the uterine cavity. Immunohistochemical analysis (IHC) revealed markedly elevated expression of IL6ST in endometrial tissue of patients with ovarian endometriosis. Level of methylation of IL6ST is diminished in patients with endometriosis, whereas level of mRNA expression is markedly elevated by RT-PCR. Cell Counting Kit-8, Transwell, Terminal deoxynucleotidyl transferase dUTP nick end labeling assays substantiated endometrial stromal cells stably transfected with 3*FLAG-IL6ST plasmid exhibited enhanced viability, augmented invasive capacity, and notable reduction in apoptosis rates. Furthermore, IL6ST facilitated progression of endometriosis by activating mitogen-activated protein kinase 9/Signal Transducer and Activator of Transcription 3 signaling pathway. Western blot analysis revealed significantly elevated protein levels of p-JAK2/JAK2, p-STAT3/STAT3, HIF-1α, and VEGF in IL6ST overexpression group. Conversely, JAK2/STAT3 inhibitor WP1066 had markedly reduced p-JAK2 and p-STAT3 protein levels in IL6ST overexpression group. Inhibiting JAK2/STAT3 signaling pathway had mitigating effect on proliferative and invasive enhancement of endometrial stromal cells, as well as inhibition of apoptosis induced by IL6ST. These findings offer novel potential targets and strategies for the treatment of endometriosis.

Apoptosis in kidney tissue of senior and geriatric cats with chronic kidney disease

Apoptosis, an important pathological event associated with kidney disease progression, is expected to be a therapeutic target in chronic kidney disease (CKD). However, its role in naturally occurring CKD in aged cats remains unclear. Therefore, here, we investigated kidney tissues from aged cats (≥10 years) with or without azotemic CKD to evaluate apoptotic events using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The positive TUNEL signals of the renal cells were quantified and statistically analyzed for correlation with the severity of plasma creatinine (pCre) concentration, renal lesions (glomerulosclerosis, interstitial cell infiltration, peritubular capillaries, and interstitial fibrosis), and oxidative damage of the kidney tissue. Oxidative damage was evaluated using immunohistochemistry for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxynonenal (4-HNE). In the TUNEL assay, regardless of azotemia, positive nuclear signals were observed in the tubular epithelial and intraluminal cells, interstitial infiltrating cells, and glomerular cells. Quantitative TUNEL scores showed no significant differences between the azotemic and non-azotemic groups in any compartment of the kidney tissues. In the azotemic group, TUNEL scores did not correlate with pCre or renal lesion severity. However, the scores showed a significant positive correlation with the scores of 8-OHdG and 4-HNE. These findings suggest that apoptosis associated with oxidative damage in renal tissue is an initial pathological event that leads to CKD, rather than a change following CKD progression, in aged cats. Inhibiting apoptosis by antioxidant treatment may be a key strategy to prevent the development of CKD.

Anti-inflammatory effect of sea buckthorn in an HCl-induced cystitis rat model.

Although the mechanism underlying interstitial cystitis/bladder pain syndrome (IC/BPS) remains unclear, oxidative stress is suggested to be implicated in IC/BPS development. Sea buckthorn (SB; Hippophae rhamnoides L.) contains several compounds with antioxidant properties. In addition, intravesical application of hydrochloric acid (HCl) in rats induces histological changes similar to those observed in humans with IC. Therefore, the aim of this study was to evaluate the anti-inflammatory effects of SB in an HCl-induced rat cystitis model. Twenty 8-week-old female Sprague-Dawley rats were instilled with HCl in their bladders to create an IC/BPS model. The model rats were divided into three groups and orally administrated distilled water (control, n=4), concentrated SB (n=8), or pentosan polysulfate (PPS, n=8) daily. Pathologic inflammation grade (H&E staining), number of mast cells per square millimeter (toluidine blue staining), fibrotic changes (Masson's trichrome staining), and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling staining) of bladder tissue samples were compared among the groups. Compared to the control group, the SB and PPS groups showed reduced edema (5.25±0.96 vs. 2.25±0.46 vs. 2.50±0.54, p=0.004, p=0.005, respectively), number of mast cells (12.5±3.6 vs. 6.8±1.9 vs. 6.6±1.8, p=0.010, p=0.002, respectively), ratio of fibrotic submucosal tissue (63.9%±7.0% vs. 43.6%±9.9% vs. 40.5%±5.2%, p<0.001, p<0.001, respectively), and ratio of apoptotic nucleus (40.7%±11.7% vs. 7.7%±6.5% vs. 5.1%±4.9%, p<0.001, p<0.001, respectively). SB exhibited anti-inflammatory effects comparable to those of PPS in the HCl-induced chemical cystitis model.

Pretreatment with tetramethylpyrazine alleviated the impairment of learning and memory induced by sevoflurane exposure in neonatal rats

Sevoflurane impairs learning and memory of the developing brain. However, strategies to mitigate these detrimental effects have been scarce. Herein, we investigated whether tetramethylpyrazine could alleviate the impairment of learning and memory and its underlying mechanisim in sevoflurane-exposed neonatal rats. Postnatal 7-day Sprague-Dawley (SD) rats or primary hippocampal neurons were pretreated with tetramethylpyrazine and then exposed to sevoflurane. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and lactate dehydrogenase (LDH) assays were used to detect neuronal injury. Learning and memory function were evaluated by novel object recognition and Morris water maze tests. Long-term potentiation (LTP) was recorded to evaluate synaptic plasticity electrophysiologically in the hippocampal slices. Golgi-Cox staining or PSD95 immunochemistry was used to detect the morphology of dendritic spines. Western blotting was employed to assess the expressions of cleaved Caspase-3, PSD95, NMDAR1, NMDAR2A and NMDAR2B in the hippocampus or cultured neurons. It was found that neonatal exposure of sevoflurane impaired learning and memory, increased neuronal apoptosis, altered the morphology of dendritic spine, upregulated the expressions of NMDAR2A and PSD95, and induced LTP deficits. Pretreatment with tetramethylpyrazine not only alleviated impairment of learning and memory, but also improved sevoflurane-induced changes in neuronal damage, dendritic spine morphology, NMDAR2A and PSD95 expressions, as well as LTP. These findings indicated that pretreatment with tetramethylpyrazine alleviated the impairment of learning and memory induced by sevoflurane through improvement of hippocampal synaptic plasticity in neonatal rats.

Increased Expression of MST1 in Patients With Epilepsy and in a Rat Model of Epilepsy.

Mammalian sterile20-like kinase 1 (MST1), a serine/threonine kinase frequently expressed, has emerged as pivotal modulator of multiple physiological and pathological conditions such as cellular growth, programmed cell death, oxidative stress, neurodegeneration, inflammation, and synaptic plasticity in the central nervous system. Various neurological diseases are associated with the activation of MST1. Epilepsy is a severe neurological disorder characterized by abrupt abnormal electrical activity in the brain and recurring spontaneous seizures. The most common pathological discoveries in patients and animal models with epilepsy are neuronal death, inflammation, neurodegeneration, neurogenesis, and axonal regrowth. The purpose of this study was to assess the levels of MST1 in serum and cerebrospinal fluid (CSF) specimens obtained from individuals diagnosed with epilepsy. In addition, it aimed to explore the expression pattern of MST1 in brain tissues of epileptic rats. We used enzyme-linked immunosorbent assay to measure the levels of CSF and serum MST1 in 10 epilepsy patients and 9 control patients. After creation of epilepsy models with healthy male Sprague-Dawley rats using lithium and pilocarpine, the expression of MST1 in the temporal cortex and hippocampus was evaluated at different time points (6h, 24h, 3days, 7days, 14days, and 30days after seizures) using immunofluorescence, immunohistochemistry, and Western blotting. In patients with epilepsy, the levels of CSF-MST1 were elevated (593.90±16.28 vs. 560.40±19.42pg/mL, p<0.05) compared to the control group. Accordingly, the serum-MST1 levels were 583.40±19.70pg/mL in the epilepsy group and 555.70±20.14pg/mL in the control group, demonstrating a statistically significant distinction (p<0.05). Levels of MST1 in CSF and serum could be of diagnostic help. Neuronal apoptosis in temporal cortex and hippocampus of epileptic rats was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling staining. MST1 was expressed in the neuronal membrane and cytoplasm of the temporal cortex and hippocampus. The expression of MST1 increased after seizures, showing a relatively high level within 30days and reaching its highest point on the seventh day after status epilepticus. The findings of this study indicate that the increased expression of MST1 protein in patients with epilepsy and epileptic rats might play a role in the development of epilepsy.

Human umbilical cord mesenchymal stem cells improve the ovarian function through oxidative stress-mediated PERK/eIF-2α/ATF4/CHOP signaling in premature ovarian insufficiency mice.

Premature ovarian insufficiency (POI) is a refractory disease that severely affects female fertility. The PERK/eIF-2α/ATF4/CHOP pathway is one of the classical pathways involved in the unfolded protein response to endoplasmic reticulum stress by regulating protein synthesis and promoting apoptosis. This study aimed to investigate the functional role and mechanism of human umbilical cord mesenchymal stem cells (hUCMSCs) in the POI animal model through the PERK/eIF-2α/ATF4/CHOP pathway. Forty-five sexually mature female C57 mice were divided into a blank control group, POI model group, and hUCMSCs intervention group. To establish the POI model, mice received intraperitoneal injections of cyclophosphamide (CTX) (70mg/kg) daily for 14 consecutive days, while the control group received saline only. In the hUCMSC intervention group, mice were given hUCMSCs on days 14 and 28, based on CTX modeling in the POI model group. The hUCMSCs were isolated, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) fluorescent dye, and tail vein-injected, and the distribution of the DiR signal was monitored in the mice using a fluorescence imaging detection method. The ovarian tissues were hematoxylin and eosin stained to observe the ovarian structure, and the number of primordial follicles were counted. An enzyme-linked immunosorbent assay was used to detect the serum levels of estradiol, anti-mullerian hormone, and follicle-stimulating hormone. Terminal deoxynucleotidyl transferase dUTP nick end labeling was used to detect the apoptosis of granulosa cells (GCs). The reactive oxygen species (ROS) content of ovarian tissue was detected by flow cytometry assay. The RNA expression of PERK, eIF-2α, ATF4, and CHOP was determined by quantitative real-time polymerase chain reaction, and protein levels of the targets were determined by western blot and immunohistochemistry. We identified hUCMSCs using surface antigenic markers (CD90, CD44, CD105, and CD73), and osteoblasts and chondroplast differentiation assays. Our studies demonstrated that hUCMSC intervention significantly restored ovarian function by improving the irregular estrous cycle, increasing the number of follicles, decreasing ROS, and inhibiting GC apoptosis in POI mice. Moreover, hUCMSCs suppressed CTX-induced PERK/eIF-2a/ATF4/CHOP pathway activation. HUCMSCs can migrate to the damaged ovaries of POI mice, and improve the ovarian function of POI mice by inhibiting oxidative stress, down-regulating the expression of the PERK/eIF-2α/ATF4/CHOP pathway, and reducing the apoptosis of GCs.

CLEC7A Knockdown Alleviates Ischemic Stroke by Inhibiting Pyroptosis and Microglia Activation.

Ischemic stroke (IS) is the leading cause of mortality worldwide. Herein, we aimed to identify novel biomarkers and explore the role of C-type lectin domain family 7 member A (CLEC7A) in IS. Differentially expressed genes (DEGs) were screened using the GSE106680, GSE97537, and GSE61616 datasets, and hub genes were identified through construction of protein-protein interaction networks. An IS model was established by middle cerebral artery occlusion and reperfusion (MCAO/R). Neural function was assessed using triphenyl tetrazolium chloride, hematoxylin-eosin, and terminal deoxynucleotidyl transferase-mediated nick-end labeling. A cell counting kit was used to detect cell viability following oxygen-glucose deprivation/reperfusion (OGD/R). Inflammatory factors were detected using enzyme-linked immunosorbent assay. The mRNA and protein expression levels were detected using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Fc fragment of Immunoglobulin G (IgG) receptor IIIa (FCGR3A), Fc fragment of Immunoglobulin E (IgE) receptor Ig (FCER1G), Complement component 5a receptor 1 (C5AR1), CLEC7A, Plasminogen activator, urokinase (PLAU), and C-C motif chemokine ligand 6 (CCL6) were identified as important hub genes, from which CLEC7A was selected as the primary subject of this study. The activation of microglia and pyroptosis were observed in MCAO/R model with increased levels of interleukin (IL)-1β, IL-18, tumor necrosis factor-α, and lactate dehydrogenase. CLEC7A knockdown was found to promote cell viability in BV2 cells and inhibiting pyroptosis in HT22 cells. CLEC7A knockdown in microglia also decreased infarct volume and neurological deficit scores, and alleviated injury and neuronal apoptosis in IS rats. CLEC7A knockdown inhibited pyroptosis and microglial activation in the MCAO/R model. A pyroptosis activator reversed the effect of CLEC7A knockdown on the viability of OGD/R-treated HT22 cells. CLEC7A is a promising biomarker of IS. CLEC7A knockdown alleviates IS by inhibiting pyroptosis and microglial activation.

Aggravated liver steatosis in a modern dietary mouse model via long-term treatment of SiO2 nanoparticles in drinking water

SiO2 nanoparticles (SNPs), which are abundant in water and are used for various applications, for example, as food additives and anticaking agents, are of growing concern because of rising exposure to human health. Research has reported low potential side effects in animal models treated with SNPs; however, a few in vivo studies have shown cause for concern. Presently, high-fat foods have changed our lives and increased the incidence rates of fatty liver, obesity, and overweight, and high-fat foods issue is prevalent in our modern society. To understand the rising SNPs exposure in life and modern dietary habits combined effect, we design experiments to study this research. Institute of Cancer Research mice fed a normal or high-fat diet were treated with different concentrations of SNPs for long-term effects. Blood and liver tissue were collected and prepared for blood biochemical assays, histology analysis, silicon and triglycerides (TGs) accumulation, immunohistochemistry, fibrosis staining, and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining to analyze the influence of the combination of SNPs and a high-fat diet. This research found that the presence of SNPs in drinking water with the consumption of a high-fat diet was associated with the accumulation of SNPs and TGs in liver tissue, elevated aspartate aminotransferase and alanine aminotransferase levels in serum, activation of fibrosis and inflammation, increased oxidative stress through 4-hydroxynonenal, and the development of liver steatosis. The results showed that the long-term effect of SNPs in drinking water might induce liver steatosis, particularly under modern dietary habits such as a high-fat diet. This study investigated the interactions between environmental nanoparticles, such as the long-term risk of exposure to SNPs, and dietary factors, suggesting a significant risk to liver health, especially in human health.

Deciphering the synergistic role of tyrosyl-tRNA synthetase 1 and yes-associated protein 1: Catalysts of malignant progression in hepatocellular carcinoma

Objective: Hepatocellular carcinoma (HCC) represents a severe and aggressive malignancy with a poor prognosis, characterized by high incidences of illness and death, making it a critical issue for global health. Tyrosyl-tRNA synthetase 1 (YARS1) is known to be upregulated across various cancers and is considerably linked to tumorigenesis. However, the detailed functions and molecular mechanisms of YARS1 in HCC remain unclear. This research explores the expression of YARS1 in HCC and its role in promoting tumor progression through the yes-associated protein 1 (YAP1) pathway. Material and Methods: The potential role and diagnostic significance of YARS1 and YAP1 in HCC were analyzed using relevant datasets. Subsequently, we constructed HCC cell lines with stable knockdown or overexpression of YARS1. In vitro, we used Cell Counting Kit-8 and colony formation assays to examine cell proliferation, terminal deoxynucleotidyl transferase dUTP nick end labeling assays to detect apoptosis, and Transwell migration and invasion assays to assess cell metastasis. Western blotting was employed to analyze the molecular mechanisms. Finally, we developed a lung metastasis model for HCC to assess the impact of YARS1 and YAP1 on tumor spread in a living organism, as well as their interrelationship. Results: The findings revealed a notable increase in YARS1 expression in HCC tumors, associated with a worse prognosis. In vitro, YARS1 overexpression significantly increased HCC cell proliferation and metastasis when reducing apoptosis (P &lt; 0.001). In addition, YARS1 overexpression accelerated HCC growth in vivo. Further experiments demonstrated that silencing YAP1 effectively reversed the effects of YARS1 on HCC cell invasion (P &lt; 0.01), apoptosis inhibition (P &lt; 0.01), and metastasis (P &lt; 0.001). Conclusion: In summary, this research reveals that YARS1 enhances the malignant progression of HCC through the activation of the YAP1 signaling pathway. Elevated levels of YARS1 in HCC are strongly linked to poor prognosis, indicating that YARS1 might serve as a new therapeutic target for HCC. Future studies should investigate additional mechanisms of YARS1 in HCC and create targeted therapies to improve outcomes for HCC patients.

Novel Dynamic Organ Storage System Enhances Liver Graft Function in a Porcine Donation After Circulatory Death Model.

Donation after circulatory death (DCD) livers face increased risks of critical complications when preserved with static cold storage (SCS). Although machine perfusion (MP) may mitigate these risks, its cost and logistical complexity limit widespread application. We developed the Dynamic Organ Storage System (DOSS), which delivers oxygenated perfusate at 10°C with minimal electrical power requirement and allows real-time effluent sampling in a portable cooler. In a porcine DCD model, livers were preserved using DOSS or SCS for 10 hours and evaluated with 4 hours of normothermic MP, with n = 5 per group. After 4 hours of normothermic MP, the DOSS group demonstrated significantly lower perfusate lactate (p = 0.023), increased perfusate fibrinogen (p = 0.005), higher oxygen consumption (p = 0.018), greater bile production (p = 0.013), higher bile bicarbonate levels (p = 0.035) and bile/perfusate sodium ratio (p = 0.002), and lower hepatic arterial resistance after phenylephrine administration (p = 0.018). Histological analysis showed lower apoptotic markers in DOSS-preserved livers, with fewer cleaved caspase-3 (p = 0.039) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL; p = 0.009) positive cells. These findings suggest that DOSS can enhance DCD allograft function during transport, offering potential clinical benefits and contributing to the expansion of the donor pool.

Wanted: Dead or Alive Cells with Propidium Iodide Staining in Liver Tissue.

This study demonstrates the effectiveness of propidium iodide as a reliable marker for detecting dead or dying cells in frozen liver tissue sections. By comparing propidium iodide staining with the widely used Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, both methods showed consistent results in disease models such as alcohol-induced fibrosis and Western diet-induced fatty liver. Additionally, propidium iodide was successfully co-stained with other fluorescent markers, like phalloidin (for actin filaments) and antibodies targeting collagen, enabling detailed spatial analysis of dying cells within tissue. This multiplex approach allows for a deeper understanding of tissue organization and cell death localization, particularly in complex conditions like liver fibrosis. Moreover, our results suggest that propidium iodide staining can be applied beyond current models, offering a more accessible and cost-effective alternative to traditional methods, like TUNEL. Furthermore, its integration with other markers enables simultaneous analysis of immune responses and tissue damage, making it a powerful tool for future studies on liver disease and other inflammatory conditions. This technique has the potential to advance research into disease mechanisms and improve the evaluation of novel therapeutic strategies targeting tissue regeneration and inflammation control.

Effect of 2,5-hexanedione on rat ovarian granulosa cell apoptosis involves endoplasmic reticulum stress-dependent m-TOR signaling pathway

ABSTRACT Occupational exposure to N-hexane/2,5-hexanedione (2,5-HD) was found to adversely affect reproductive functions in females. However, there are few studies regarding the mechanisms underlying reproductive system damage initiated by 2,5-HD. Several studies demonstrated that 2,5-HD exerts hormonal dysfunctions in females by promoting apoptosis using rat ovarian granulosa cells (GCs) as a model. The endoplasmic reticulum (ER) plays a key role in cellular processes such as protein folding and modification, Ca2+ storage, and lipid synthesis, which are known to involve the activation of stress (ERS)-dependent m-TOR signaling pathway. Thus, the aim of this study was to examine the effects of 2,5-HD on ER and the associated activation of stress (ERS)-dependent m-TOR signaling pathway resulting in consequent apoptosis of ovarian GCs. Data demonstrated that after intraperitoneal treatment with 100, 200, or 400 mg/kg 2,5-HD for 6 consecutive weeks, 5 times per week, a decrease in body weight, ovarian weight, and relative ovary weight was found. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed that 2,5-HD promoted apoptosis of ovarian GCs, which involved enhanced relative protein expression levels of m-TOR/p-mTOR. Our findings demonstrated that 2,5-HD (1) elevated expression levels of pro-apoptosis-related genes Bax and Caspase 3, (2) decreased expression levels of the anti-apoptosis gene Bcl-2, and (3) activated the protein expression of glucose-regulatory protein 78 (GRP78), inositol-requiring enzyme-1 (IRE1), and c-Jun terminal kinase (JNK) associated with increased apoptosis. Evidence indicates that chronic exposure to 2,5-HD induced apoptosis of ovarian GCs, and the possible mechanism underlying this effect involves the ERS-dependent m-TOR signaling pathway.

Vaspin inhibits ferroptosis: A new hope for treating myocardial ischemia–reperfusion injury

Objective: Myocardial ischemia–reperfusion injury (MIRI) is a critical pathological basis for cardiovascular diseases. In recent years, the effect of ferroptosis on MIRI has attracted extensive attention. Vaspin, an adipose tissue-derived serine protease inhibitor, has multiple biological functions, including anti-inflammatory and antioxidant effects. This study aims to investigate the molecular mechanism by which vaspin alleviates MIRI by regulating hypoxia-inducible factor-1α (HIF-1α) and ferroptosis signaling pathways. Material and Methods: A mouse model of myocardial ischemia/reperfusion (I/R) and a hypoxia/reoxygenation (H/R) model was used to evaluate the protective effects of vaspin on MIRI. The mechanism by which ferroptosis is modulated by the vaspin/HIF-1α signaling pathway was investigated by constructing a vaspin overexpression adenoviral vector. Myocardial infarct size and histological changes were assessed using triphenyltetrazolium chloride and hematoxylin–eosin staining. Ferroptosis-related proteins were detected by Western blot assay, and apoptosis and reactive oxygen species levels were analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. Iron content in myocardial tissue and cells was measured by enzyme-linked immunosorbent assay. Results: Myocardial I/R increased myocardial infarct size and serum lactate dehydrogenase (LDH) levels compared with the control group, indicating severe myocardial injury. Western blot results showed that MIRI reduced endogenous vaspin and HIF-1α levels and inhibited glutathione peroxidase 4. In vivo and in vitro vaspin overexpression treatment reduced infarct size, decreased LDH levels, inhibited ferroptosis pathway activity, and alleviated oxidative stress levels in myocardial tissues. In the H/R model, vaspin overexpression upregulated HIF-1α, inhibited ferroptosis markers, and reduced apoptosis and iron deposition. However, inhibiting HIF-1α reversed the cardioprotective and anti-ferroptotic effects of vaspin. Conclusion: Vaspin inhibits ferroptosis and upregulates the HIF-1α signaling pathway to mitigate myocardial I/R injury. The vaspin/HIF-1α pathway could be a potential target for MIRI prevention and treatment and offers fresh perspectives on ischemic heart disease management. Vaspin could be a novel cardioprotective agent that plays a significant role in the prevention and treatment of cardiovascular diseases.

Butorphanol Alleviates Hypoxia/Reoxygenation-Induced Myocardial Injury by Activating Wnt/β-Catenin Signal Pathway.

Ischemic heart disease remains a global health problem with high morbidity and mortality. Butorphanol, as a novel opioid, has been discovered with its cardioprotective properties. The purpose of this study was to explore that butorphanol can alleviate hypoxia/reoxygenation (H/R)-induced myocardial injury by activating the Wnt/β-catenin signal pathway. In this study, Cell Counting Kit-8 (CCK-8), lactate dehydrogenase (LDH) kit, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and Western blotting experiments were performed to observe butorphanol cardioprotective function and expression of Wnt signaling pathway proteins. For the main result, anti-apoptotic effect and higher expression of Wnt signaling pathway proteins were observed with the increasing concentrations of butorphanol. Compared with the H/R group, higher cellular viability, lower LDH release, and smaller apoptotic cell population were found in the butorphanol group. In addition, expression levels of apoptosis-related protein Bax and Cleaved Caspase-3 decreased, and increased expression of Bcl-2 were observed. Conversely, the protective effects of butorphanol were attenuated in the XAV939 group. In summary, butorphanol attenuates hypoxia/reoxygenation-induced myocardial injury by activating the Wnt/β-catenin signal pathway. Our work provides a theoretical basis for butorphanol's myocardial protective function.

Wild-Type p53 Regulates Apoptosis of Human Breast Cancer Cells.

The tumor suppressor wild-type p53 is known for its role in inducing apoptosis in tumor cells. This study investigated the relationship between wild-type p53 and protein phosphatase 1 (PP1) and caspase in promoting apoptosis of breast cancer cells. Human breast cancer cell lines MCF-7 and MDA-MB-231 obtained from the American Type Culture Collection were used in this study. Small interference RNAs (Si-RNA) and plasmids were used to regulate wild-type p53 expression in these two tumor cell lines through liposome-mediated transfection. GSK-2830371 (PP1 inhibitor) and zVAD (Caspase inhibitor) were employed to further verify the PP1 activating function of wild-type p53 in Caspase-dependent MCF-7 and MDA-MB-231 apoptosis. PP1 activity was quantitatively detected by phosphorus colorimetric assay. Co-immunoprecipitation (Co-IP), flow cytometry assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Western blot, the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR), and immunofluorescence staining were used to analyze cell apoptosis degree and marker protein expression. The expression level of PP1 in the breast cancer cells was successfully regulated by cell transfection. The phosphatase activity was increased, and obvious apoptotic cytological characteristics were observed in p53-overexpressed breast cancer cells. p53 knockdown/overexpression increased/decreased the level of B cell lymphoma 2 (Bcl-2), and decreased/increased levels of Caspase-3, cleaved Caspase-3, cleaved Caspase-8, Cytochrome C (Cyt-C), Truncated BID (tBid), Bcl-2-associated X (Bax), and cell apoptosis (p < 0.01). The promotion of proteins and apoptosis induced by p53 overexpression was reversed by GSK-2830371 or zVAD. Wild-type p53 might promote Caspase-dependent apoptosis of human breast cancer cells through PP1 activation.

MiR-490-3p promotes cell apoptosis and cell-cycle arrest in osteosarcoma via the modulation of CDCA8/ATF3 by targeting NUSAP1.

Micro RNA-490-3p (miR-490-3p) is associated with a variety of malignancies. However, the role of miR-490-3p in osteosarcoma and its underlying mechanism are not yet fully understood. This study aimed to explore the role and the mechanism of miR-490-3p in osteosarcoma. MiR-490-3p and nucleolar and spindle-associated protein 1 (NUSAP1) expression in osteosarcoma was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8 (CCK-8), wound-healing, and transwell assays were used to detect cell proliferation, migration and invasion. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and western blot were used to evaluate the cell apoptosis. Flow cytometry was used to assess the cell cycle. In addition, luciferase reporter assay was used to confirm the binding of miR-490-3p and NUSAP1. Western blot and RT-qPCR was used to examine cell division cycle associated 8 (CDCA8) and activating transcription factor 3 (ATF3) expression. MiR-490-3p expression was significantly decreased in the osteosarcoma cells. Following the transfection with miR-490-3p mimic, it was found that 143B cell proliferation, migration, and invasion were inhibited, while the cell apoptotic levels and cell-cycle arrest were promoted, accompanied with decreased B cell lymphoma protein-2 (Bcl-2) protein expression, and increased protein expressions of Bcl-2-associated X (Bax), cleaved caspase-3, and cleaved caspase-9. In addition, miR-490-3p was found to bind to NUSAP1, and negatively regulate NUSAP1 expression. NUSAP1 upregulation reversed the inhibitory effects of miR-490-3p overexpression on cell proliferation, migration, and invasion, and the promoting effects on cell apoptosis and cell-cycle arrest in osteosarcoma. Moreover, miR-490-3p was identified to mediate CDCA8/ATF3 by targeting NUSAP1. MiR-490-3p upregulation inhibited cell proliferation and metastasis but promoted the cell apoptosis and cell-cycle arrest in osteosarcoma via the regulation of CDCA8/ATF3 by targeting NUSAP1. Thus, miR-490-3p might be a potential therapeutic target for the treatment of osteosarcoma.

Effect of Aconitum diphtheria on the Proliferation, Apoptosis, and Inflammatory Response of Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Rheumatoid arthritis (RA), a highly disabling autoimmune disease, is characterized by joint damage and synovial hyperplasia. This paper was designed to explore the effect and mechanisms of Aconitum diphtheria on the proliferation and apoptosis of RA fibroblast-like synoviocytes (RA-FLS). First, RA-FLS was treated with different doses of A. diphtheria extracts. Then, an inverted biological microscope was adopted to observe the RA-FLS morphology. Subsequently, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining, Cell Counting Kit-8 and western blot were utilized to analyze the apoptosis, proliferation, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway-related proteins in RA-FLS, respectively. The experimental results disclosed that, after treatment of RA-FLS with A. diphtheria extracts, the cells became round and the intercellular space was increased. Besides, the RA-FLS proliferation was effectively inhibited by A. diphtheria extracts, while the apoptosis was promoted. Additionally, A. diphtheria extracts could effectively downregulate the expression levels of p-NF-κB (p50), NF-κB (p50), p-NF-κB (p65), and NF-κB (p65). Collectively, A. diphtheria can effectively inhibit RA-FLS proliferation and promote apoptosis in a dose-dependent manner. Similarly, A. diphtheria is able to downregulate p-NF-κB and NF-κB protein expression, but there is no dose-response relationship.

Cnaphalocrocis medinalis granulovirus regulates apoptosis by targeting AIF1 and ASPP1 through tca-miR-3885-5p and tca-miR-3897-3p to promote infection

Cnaphalocrocis medinalis granulovirus (CnmeGV) is a potential biocontrol agent for C. medinalis which is a major rice pest. However, its insecticidal efficacy is slow due to cell apoptosis. This study investigated the role of miRNAs in CnmeGV-mediated apoptosis. Small RNA sequencing and qRT-PCR identified miRNAs tca-miR-3885-5p and tca-miR-3897-3p, which initially increased and then decreased post-infection, but remained higher than controls. This trend was opposite to the changes in midgut apoptosis levels detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and DNA ladder assays. Compared to the group treated with CnmeGV alone, agomirs increased the CnmeGV-induced larval mortality, reduced midgut apoptosis, whereas antagomirs had the opposite effects. We found that the upregulation of CnmeGV replication induced by agomirs initially increased and then decreased, while the apoptosis inducer PAC-1 compensated for the weakening trend of CnmeGV replication upregulation induced by agomirs in the later stages of infection. Results indicated the virus hijacks these miRNAs to inhibit early apoptosis, later requiring apoptosis for systemic infection from the midgut. Agomirs treatment and dual-luciferase assays showed these miRNAs functioned via apoptosis-inducing factor 1 (AIF1) and apoptosis-stimulating protein of p53 1 (ASPP1) mRNA expression. This study highlights the role of these miRNAs in infection and provides insights for developing viral insecticide enhancers.

A New Perspective for Improving COPD: Ginsenoside Rg3 Links SIRT1 to Inhibit Mitochondrial Autophagy.

Chronic obstructive pulmonary disease (COPD) is a prevalent yet manageable respiratory condition. However, treatments presently used normally have side effects and cannot cure COPD, making it urgent to explore effective medications. The ginsenoside Rg3 (Rg3) has been shown to have anti-inflammatory and anti-tumor properties and can improve COPD. The primary objectives of this investigation were to explore the impact of Rg3 on COPD and delve into the associated mechanisms. In vitro models exposed human bronchial epithelial cells (BEAS-2B) to cigarette smoke extract (CSE), and in vivo models induced COPD in mice through chronic inhalation of cigarette smoke (CS). Sirtuin 1 (SIRT1) expression was regulated via cell transfection or mice infection with recombinant lentiviruses. SIRT1 mRNA levels were quantified using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and SIRT protein levels were assessed by western blot or enzyme-linked immunosorbent assays (ELISA). Mitophagy was evaluated by light chain 3 (LC3) II/I and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) levels, and apoptosis was determined using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Lung function was measured with the Buxco system, and inflammation was assessed via interleukin 6 (IL-6) and keratinocyte-derived cytokine (KC) levels in bronchial alveolar lavage fluid. Lung morphological impairments were determined through Hematoxylin and Eosin (H&E) staining and mean linear intercept (MLI) measurement. In BEAS-2B cells, CSE treatment caused a decrease in SIRT1 expression (p < 0.01) and an increase in LC3 II/I (p < 0.01) and PINK1 (p < 0.01), which were all reversed by Rg3 (p < 0.01), with 20 μM Rg3 performing the best and being used subsequently. CSE increased apoptosis of BEAS-2B cells (p < 0.01), which was reversed by Rg3 (p < 0.01). Upregulated SIRT1 further decreased levels of LC3 II/I (p < 0.001), PINK1 (p < 0.001), and cell apoptosis (p < 0.001) for CSE- and Rg3-treated cells, whereas downregulated SIRT1 reversely increased levels of LC3 II/I (p < 0.001), PINK1 (p < 0.001), and cell apoptosis (p < 0.001). The establishment of COPD caused a decrease in SIRT1 mRNA (p < 0.001), SIRT1 protein (p < 0.001), and lung functions (p < 0.001) whereas IL-6 (p < 0.001), KC (p < 0.001), lung impairment, and MLI (p < 0.001) were increased; all of these effects were reversed by Rg3 (p < 0.001). Moreover, the Rg3-induced reversion was furthered by SIRT1 upregulation (p < 0.001) and was disrupted by SIRT1 downregulation (p < 0.001). Rg3, through activation of SIRT1, suppresses mitophagy and apoptosis, ameliorates COPD, and improves lung functions.

Effect of daphnetin, the coumarin derivative isolated from Daphne genus, on Helicobacter pylori adhesion to gastric epithelial cells.

Adherence of Helicobacter pylori to the surface of the gastric mucosa is the initial and crucial step for its survival and colonization in the harsh conditions of the stomach. We had previously demonstrated that daphnetin has anti-adhesion effect. This study aims to explore the mechanisms of daphnetin to reduce H. pylori adhesion to gastric epithelial cells (GES-1). Fluorescence microscopy and urease assay were used to observe and validate the anti-adhesion effect of daphnetin. Terminal deoxynucleotidyl transferase dUTP nick end labeling, comet assay and agarose gel-based assay were conducted to evaluate the level of DNA damage. Quantitative real-time polymerase chain reaction, western blotting, electrophoretic mobility shifts assay and enzyme-linked immunosorbent assay were performed to investigate the mechanisms of the anti-adhesion effect of daphnetin. Our results showed that daphnetin decreased H. pylori adhesion to GES-1 in time- and dose-dependent manners. The mechanisms by which daphnetin inhibits H. pylori adhesion involved the inducing of DNA double-strand breaks, the up-regulating of recA transcription leading to RecA binding at 1018-1597 site in the babA promoter, the decreasing of babA/babB transcription ratio, the decreasing of BabA expression and its interaction with Lewis b antigen. Our results suggested that daphnetin significantly inhibits H. pylori adhesion to GES-1 through the RecA-BabA pathway. To our knowledge, this is the first report on the mechanisms of daphnetin affecting H. pylori adhesion to GES-1.