Transferase biotin-dUTP Nick End Labeling Research Articles

Silencing DDX3 Attenuates Interleukin-1β-Induced Intervertebral Disc Degeneration Through Inhibiting Pyroptosis.

Intervertebral disc degeneration (IVDD) is a common disorder associated with chronic inflammation and cell death. In this study, an IVDD rat model was created through Interleukin-1β (IL-1β) injection. The degeneration of intervertebral disc tissues was assessed using magnetic resonance imaging (MRI), followed by hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining. RNA sequencing was performed to identify differentially expressed genes (DEGs) between the IVDD model and control rats. The expression levels of DEGs (DEAD-box polypeptide 3 (DDX3), lysine-specific demethylase 5D (KDM5D), interferon-induced gene-1 (IFIT1), ribosomal protein S10 (RPS10), tenomodulin (TNMD), and pentraxin 3 (PTX3)) were measured by real-time quantitative polymerase chain reaction (RT-qPCR). The regulatory effect of DDX3 on pyroptosis in IL-1β-treated nucleus pulpous (NP) cells was assessed after transfection with siRNA of DDX3. A total of 601 DEGs were identified from the IVDD model rat, and were abundant in extracellular matrix (ECM) organization, ECM-receptor interaction, and inflammatory pathways, including the PI3K-Akt, TNF, and AMPK signaling pathways. DDX3, KDM5D, and IFIT1 levels were notably elevated, whereas RPS10, TNMD, and PTX3 levels were decreased in the IL-1β-induced IVDD rat model. Moreover, silencing DDX3 promoted cell proliferation and abolished IL-1β-induced cell apoptosis and pyroptosis. This study revealed the role of DDX3 in IVDD pyroptosis, providing potential target for IVDD management.

Anticolorectal Cancer Activity of Bilobalide in Patient-Derived Colorectal Cancer Organoids and AOM/DSS Mouse Model

Bilobalide has shown strong anti-inflammatory activity. Colorectal cancer (CRC) is closely associated with inflammation. However, no studies have reported on the use of bilobalide for treating CRC. This study aims to evaluate the effect of bilobalide on CRC prevention. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (RT-qPCR), Western blotting, and immunofluorescence showed that bilobalide significantly inhibits the M2 polarization of macrophages dependent on phorbol 12-myristate 13-acetate (PMA) and interleukin-4 (IL-4). Analysis of signaling pathways showed that the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and signal transducer and activator of transcription 3 (STAT3) was regulated. In particular, human CRC organoids were established. Western blotting, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), and analysis of cell viability and morphology further supported the hypothesis that the anti-CRC effects of bilobalide could be explained by its ability to suppress M2 macrophage polarization and promote M1 transformation. C57BL/6 mice treated with azoxymethane (AOM)/dextran sodium sulfate (DSS) were divided into three groups, i.e., control, AOM/DSS, low (2.5 mg/kg), and high (5 mg/kg). High-dose bilobalide markedly inhibited the progression of CRC, as evidenced by the increased body weight, decrease in disease activity index (DAI) death rate, and alleviation of colon length reduction and tumorigenesis. According to the in vivo results, reduced levels of inflammatory cytokines in the serum included tumor necrosis factor (TNF-α), IL-6, IL-1β, and IL-10. Bilobalide reduced oxidative stress indices, lipid peroxide (LPO), and malondialdehyde (MDA) and increased reduced glutathione (GSH). In addition, the expression of proliferating cell nuclear antigen (PCNA), Ki67, cellular Myc (c-Myc), and CD206 was downregulated in the drug-treated groups, as confirmed by the immunohistochemical staining. Collectively, these results indicated that bilobalide administration improve experimental CRC by inhibiting M2 macrophage polarization and oxidative stress. Thus, bilobalide may prevent CRC and serve as a potential therapeutic target for CRC.

Curcumin alleviates myocardial inflammation, apoptosis, and oxidative stress induced by acute pulmonary embolism by regulating microRNA-145-5P/insulin receptor substrate 1 axis.

The treatment of patients with acute pulmonary embolism (APE) is extremely challenging due to the complex clinical presentation and prognosis of APE related to the patient's hemodynamic status and insufficient arterial blood flow and right ventricular overload. Protective efficacy against cardiovascular diseases of curcumin, a common natural polyphenolic compound, which has antithrombotic properties and reduces platelet accumulation in the circulation by inhibiting thromboxane synthesis has been demonstrated. However, the direct effect of curcumin on APE has rarely been studied. Therefore, the present study aimed to investigate the therapeutic potential of curcumin in APE and associated myocardial injury to provide new insights into curcumin as a promising competitive new target for the treatment of APE. A suspension of 12 mg/kg microspheres was injected intravenously into rats. An APE rat model was built. Before modeling, intragastric 100 mg/kg curcumin was given, and/or lentiviral plasmid vector targeting microRNA-145-5p or insulin receptor substrate 1 (IRS1) was injected. Pulmonary artery pressure was measured to assess right ventricular systolic pressure (RVSP). Hematoxylin and eosin (H&E) staining was performed on liver tissues and myocardial tissues of APE rats. TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) staining and immunohistochemical (IHC) staining were conducted to measure apoptosis and CyPA-CD147 expression in the myocardium, respectively. Inflammatory indices interleukin-1beta (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were measured by ELISA in cardiac tissues. RT-qPCR and Western blot were performed to determine the expression levels of related genes. In addition, by dual luciferase reporter assay and RIP assay, the relationship between microRNA-145-5p and insulin receptor substrate 1 (IRS1) was confirmed. In results: curcumin improved APE-induced myocardial injury, reduced myocardial tissue edema, and thrombus volume. It attenuated APE-induced myocardial inflammation and apoptosis, as well as reduced lung injury and pulmonary artery pressure. Curcumin promoted microRNA-145-5p expression in APE rat myocardium. MicroRNA-145-5p overexpression protected against APE-induced myocardial injury, and microRNA-145-5p silencing abolished the beneficial effects of curcumin in APE-induced myocardial injury. IRS1 was targeted by microRNA-145-5p. IRS1 silencing attenuated APE-induced myocardial injury, and enhanced therapeutic effect of curcumin on myocardial injury in APE rats. In conclusion, curcumin alleviates myocardial inflammation, apoptosis, and oxidative stress induced by APE by regulating microRNA-145-5p/IRS1 axis.

SerpinA3 Promotes Myocardial Infarction in Rat and Cell-based Models.

This study aimed to examine the role and molecular mechanism of the nuclear factor κB (NFκB)/serine protease inhibitor A3 (SerpinA3) interaction in myocardial ischemia-reperfusion (IR) injury. First, a rat model for myocardial ischemia-reperfusion injury was established, using 2,3,5-triphenyltetrazolium chloride to measure the size of the myocardial infarction. Pathological variations in myocardial tissue were detected using hematoxylin-eosin staining. Flow cytometry and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining were used to measure cell death in the rat model. The SerpinA3 mRNA and protein expressions in the myocardium of IR-model rats were remarkably higher than those in the control group. Furthermore, the oxidative, inflammatory, and apoptotic activities of the myocardial tissue of SerpinA3-knockdown (KD) rats were significantly improved compared to those in the WT group. SerpinA3-KD also contributed to the recovery of cardiac function in IR-model rats. Additionally, silencing of SerpinA3 inhibited p65 phosphorylation in myocardial tissues and reduced H2O2-induced inflammation, oxidative stress, and apoptosis in myocardial cells. The expression of SerpinA3 increased in myocardial tissue after IR stimulation. Knockdown of SerpinA3 can deactivate NF-κB and reduce inflammation, oxidative stress, and apoptosis in vivo and in vitro, thereby lessening myocardial injury caused by IR. In conclusion, SerpinA3 promotes myocardial infarction in rat and cell-based models by activating NF-κB. However, the mechanism by which increased Serpina3 expression causes downstream NF-κB activation to mediate the proposed, pathological effects in myocardial IR injury remain untested and worthy of future investigations.

Interferon-induced transmembrane protein 3 in the hippocampus: a potential novel target for the therapeutic effects of recombinant human brain natriuretic peptide on sepsis-associated encephalopathy.

This study aims to explore whether interferon-induced transmembrane protein 3 (IFITM3) is involved in recombinant human brain natriuretic peptide (rhBNP)-mediated effects on sepsis-induced cognitive dysfunction in mice. The cellular localization and expression level of IFITM3 in the hippocampus were detected. The IFITM3 overexpression was achieved using an intracranial stereotactic system to inject an adeno-associated virus into the hippocampal CA1 region of mice. Field experiments, an elevated plus maze, and conditioned fear memory tests assessed the cognitive impairment in rhBNP-treated septic mice. Finally, in the hippocampus of septic mice, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining and Immunoblot were used to detect changes in the protein expression of cleaved Caspase-8 and cleaved Caspase-3 in apoptosis-related pathways, and toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB) p65 in inflammatory pathways. Fourteen days after cecal ligation and puncture (CLP) surgery, IFITM3 localized in the plasma membrane and cytoplasm of the astrocytes in the hippocampus of septic mice, partially attached to the perivascular and neuronal surfaces, but not expressed in the microglia. The expression of IFITM3 was increased in the astrocytes and neurons in the hippocampus of septic mice, which was selectively inhibited by the administration of rhBNP. Overexpression of IFITM3 resulted in elevated anxiety levels and long-term learning and memory dysfunction, completely abolished the therapeutic effect of rhBNP on cognitive impairment in septic mice, and induced an increase in the number of neuronal apoptosis in the hippocampal CA1 region. The expression levels of cleaved Caspase-3 and cleaved Caspase-8 proteins were significantly increased in the hippocampus, but the expression levels of TLR4 and NF-κB p65 were not increased. The activation of IFITM3 may be a potential new target for treating sepsis-associated encephalopathy (SAE), and it may be one of the key anti-apoptotic mechanisms in rhBNP exerting its therapeutic effect, providing new insight into the clinical treatment of SAE patients.

PI3K/AKT/mTOR pathway and its related molecules participate in PROK1 silence-induced anti-tumor effects on pancreatic cancer.

The PI3K/AKT/mTOR (phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin) pathway can be initiated by PROK1 (prokineticin 1), but its effect and mechanism of action in pancreatic carcinoma (PC) are not fully understood. In this study, we elucidated the roles of PROK1 and its related molecules in PC in vivo. PANC-1 cells with PROK1 knockdown were injected into BALB/c nude mice. The growth and weight of the tumor were monitored and measured, which was followed by TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling), immunohistochemical staining, and hematoxylin and eosin staining. The key proteins related to proliferation, apoptosis, and the PI3K/AKT/mTOR pathway were determined by Western blotting. We also used public databases to identify the molecules related to PROK1. The reduction of PROK1 inhibited angiopoiesis and promoted apoptosis in vivo. PCNA-1, cyclin D1, and Bcl-2 decreased considerably, while Bax and cleaved caspase-3 increased significantly after PROK1 inhibition. The PI3K/AKT/mTOR signal inhibition was also closely associated with PROK1 knockdown. The possible related molecules of PROK1, such as von Willebrand factor, were screened and considered to be involved in the aberrant activation of PI3K/AKT. In conclusion, PROK1 knockdown significantly prevented tumor growth and promoted apoptosis of human PC cells in vivo, where the PI3K/AKT/mTOR pathway was probably inhibited. Therefore, PROK1, along with its related molecules, might be important targets for PC therapy.

CXCR4 enhances the inhibitory effects of bone mesenchymal stem cells on lung cell apoptosis in a rat model of smoking-induced COPD.

Chronic obstructive pulmonary disease is the 3rd leading cause of death worldwide, and the available treatments are unsatisfactory, resulting in a major economic burden. As cellular therapy is commonly used for lung disease, we investigated a treatment with CXCR4-overexpressing BMSCs in a COPD model. We extracted and purified Bone marrow mesenchymal stem cells (BMSCs) from SD rats. COPD apoptosis model was established by cigarette smoke exposure. BMSCs (1 × 106 cells per injection)were transplanted in vivo twice a month during model establishment, and alveolar rupture in the lung was assessed. Lung cell apoptosis was assessed by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) analysis, and the concentrations of apoptotic proteins in the lungs were detected by Western blotting. We successfully isolated BMSCs and established CXCR4-overexpressing BMSCs. qRT‒PCR and Western blotting detection both reveal that CXCR4 mRNA level and protein both significantly higher expression in CXCR4-BMSCs than the pBABE-BMSCs. Continuous cigarette smoke exposure caused alveolar septal rupture: In the model group, the alveolar mean linear intercept in the first month was significantly lower than that in the third month (p < 0.05). In the third month, the alveolar mean linear intercept values of the control and CXCR4-BMSC groups were lower than those of the model group (control group p < 0.01, CXCR4-BMSC group p < 0.05), and TUNEL staining revealed that the apoptosis rates of the control and CXCR4-BMSC groups were significantly lower than those of the model group (p < 0.01). Furthermore, the levels of the apoptotic proteins cleaved caspase-8, cleaved caspase-3 and cleaved PARP-1 were higher in the model group than in the control group (p < 0.05) and significantly lower in the CXCR4-BMSC group than in the model group (p < 0.05). The transplantation of CXCR4-overexpressing BMSCs during COPD model generation significantly inhibited apoptosis via the extrinsic apoptosis pathway. CXCR4 enhances the inhibitory effects of bone mesenchymal stem cells on lung cell apoptosis in a rat model of smoking-induced COPD.

DUSP12 ameliorates myocardial ischemia-reperfusion injury through HSPB8-induced mitophagy.

This study aimed to explore the role of dual specificity phosphatase 12 (DUSP12) in regulating myocardial ischemia-reperfusion (I/R) injury and the underlying mechanism. The expression of DUSP12 in myocardial tissuesand heat-shock protein beta-8(HSPB8) and mitophagy-related proteins in myocardial tissues and H9c2 cells were detected by western blot analysis. The serum creatine kinase isoenzymes (CK-MB)and lactate dehydrogenase (LDH), levels of reactive oxygen speciesand malondialdehyde,superoxide dismutase activity in myocardial tissuesand H9c2 cells, and caspase-3 activity in H9c2 cells were analyzed by corresponding assay kits. The infarct area in therat's heart was observed by triphenyl tetrazolium chloridestaining. The apoptosis of myocardial cells in myocardial tissues and H9c2 cells was detected by terminal-deoxynucleotidyl transferase dUTP-biotin nick-end labelingassay. The interaction between DUSP12 and HSPB8 was clarified by the coimmunoprecipitation assay. The transfection efficacy of si-HSPB8#1 and si-HSPB8#2 in H9c2 cells was confirmed by real-time quantitative-polymerasechain reactionand western blot analysis. As a result, DUSP12 expression was downregulated in I/R rats, which was promoted by lentivirus-expressing DUSP12. DUSP12 overexpression reduced the serumcreatine kinase isoenzymes (CK-MB) and LDH, decreased the infarct area in the rat's heart, and suppressed the apoptosis and oxidative stress in myocardial tissues. DUSP12 overexpression also upregulated the expression of HSPB8 to promote mitophagy. The coimmunoprecipitation assay indicated that DUSP12 could be combined with HSPB8. In addition, DUSP12 overexpression could inhibit hypoxia/reoxygenation-elicited apoptosis as well as oxidative stress in H9c2 cells by upregulating HSPB8 expression to promote mitophagy, which was countervailed by HSPB8 deficiency. In conclusion, DUSP12 overexpression decreased the apoptosis and oxidative stress in myocardial I/R injury through HSPB8-induced mitophagy.

TGR5 overexpression mediated by the inhibition of transcription factor SOX9 protects against hypoxia-/reoxygenation-induced injury in hippocampal neurons by activating Nrf2/HO-1 signaling

Cerebral ischemia/reperfusion (CI/R) injury is a destructive cerebrovascular disease associated with long-term disability and high mortality rates. TGR5 has been discovered in multiple human and animal tissues and to modulate a variety of physiological processes. The current study sought to reveal the function of TGR5 in CI/R injury and uncover the latent regulatory mechanism. A hypoxia/reoxygenation (H/R) model was established in mouse hippocampal HT22 cells. The TGR5 expression in the H/R-treated HT22 cells was tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blots. After TGR5 was overexpressed, Cell Counting Kit-8 assays were used to estimate cell viability, and lactate dehydrogenase (LDH) release was assessed by a LDH assay kit. Cell apoptosis was measured by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assays. Cytochrome c release was detected by immunofluorescence assays and western blots were used to analyze the protein levels of apoptosis-related factors. The oxidative stress levels were assessed by corresponding kits. Next, SOX9 expression in the H/R-treated HT22 cells was tested by RT-qPCR and western blots. The interaction between the TGR5 promoter and SOX9 was verified by luciferase reporter and chromatin immunoprecipitation assays. Subsequently, after the H/R-treated HT22 cells had been co-transfected with TGR5 overexpression and SOX9 overexpression plasmids, TGR5 expression was tested by RT-qPCR and western blots, and the above-mentioned functional experiments were repeated. Finally, the expression of Nrf2/HO-1 signaling-related proteins was examined by western blots. TGR5 expression was significantly decreased in the H/R-exposed HT22 cells. The elevation of TGR5 enhanced the viability, hindered the apoptosis, and alleviated the oxidative stress of the HT22 cells under H/R conditions. Additionally, SOX9 had a strong affinity with TGR5 promoter, and TGR5 was transcriptionally inhibited by SOX9. Further, SOX9 overexpression restored the protective role of TGR5 upregulation in H/R-induced HT22 cell injury. Additionally, TGR5 overexpression mediated by SOX9 inhibition activated Nrf2/HO-1 signaling. TGR5 was transcriptionally inhibited by SOX9, and the overexpression of TGR5 played a protective role in CI/R injury.

Down-regulation of LCN2 attenuates retinal vascular dysfunction and caspase-1-mediated pyroptosis in diabetes mellitus.

BackgroundDiabetic retinopathy (DR) is a diabetic microangiopathy with increasing incidence, which seriously threatens the quality of life of patients. This study investigated the molecular regulation mechanism of lipocalin-2 (LCN2) in DR by targeting the function of human retinal vascular endothelial cells (HRVECs).MethodsThe expression of LCN2 in the retinal tissue of diabetic and high glucose (HG)-induced HRVECs was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis and western blotting assay. After intravitreal injection of adeno-associated virus (AAV)-NC or AAV-sh-LCN2, in vivo experiments, hematoxylin and eosin (H&E) staining, and retinal trypsin digestion experiments were performed to analyze the effect of LCN2 silencing on DR retinal tissue. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining was used to evaluate apoptosis and immunohistochemical (IHC) staining was performed to detect the expressions of caspase-1. Western blot was used to detect the expressions of pyroptosis-associated proteins. After transfection of sh-NC and sh-LCN2, the function of HRVECs cells induced by HG was evaluated by wound healing assay, Transwell assay, and tube formation assay.ResultsThe expression of LCN2 was significantly up-regulated in diabetic retinal tissue and HG-induced HRVECs. In vivo experiments showed that LCN2 silencing can significantly reduce diabetic retinal injury. Cell function experiments also revealed that LCN2 silencing inhibited cell migration, invasion, and angiogenesis. Flow cytometry and immunofluorescence staining showed that downregulation of LCN2 could inhibit caspase-1 mediated pyroptosis in HG-induced HRVECs.ConclusionsDown-regulation of LCN2 can significantly inhibit cell migration, invasion, and angiopoiesis, and pyroptosis regulated by caspase-1, thus attenuating the progression of DR.

Mangosteen Inhibits Growth and Survival of Cervical Cancer Cells.

Cervical cancer is the most common cancer of the female reproductive system. Late-stage cervical cancer treatment has been largely unsuccessful, and urgent anti-cancer therapy is needed. Mangosteen, a tropical fruit, has been studied and found to be rich in xanthones, known anti-cancer compounds. This study was designed to investigate the effect of mangosteen extract (ME) on SiHa cervical cancer cells and to explore the underlying molecular mechanisms. Clonogenic survival assay, Quick Cell Proliferation Assay, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, and caspase-3 activity kits were used to investigate the in vitro role of ME treatment in SiHa cervical cancer cell growth. We further investigated the possible molecular mechanisms using RT-PCR. Statistical analysis was done with unpaired two-tailed Student's t-test and significance at p-value <0.05; each experiment was repeated three times. Our study found that the growth and proliferation of SiHa cervical cancer cells was inhibited by ME. ME also induced apoptosis in SiHa cervical cancer cells. The anti-proliferative effect of ME on cervical cancer cells was associated with statistically significant (p<0.05) down-regulation of the pro-proliferative molecules cyclin B, cyclin D and cyclin E. The pro-apoptotic effect of ME was associated with statistically significant (p<0.05) down-regulation of the anti-apoptotic molecules flice-like inhibitory protein (FLIP) and survivin. ME impedes the growth and survival of SiHa cervical cancer cells by down-regulating cyclin B, cyclin D, cyclin E as well as FLIP and survivin. ME may be a promising strategy for targeted cancer immunotherapy development.

Toxicological mechanism of large amount of copper supplementation: Effects on endoplasmic reticulum stress and mitochondria-mediated apoptosis by Nrf2/HO-1 pathway-induced oxidative stress in the porcine myocardium

Copper (Cu) is an essential micronutrient that is required by all living organisms. However, Cu can also be a potentially toxic metal if excessive dietary supplementation occurs. The current study aimed to investigate the mechanism of Cu toxicity in the cardiomyocytes of large mammal pigs. Here, we used pigs to explore Cu toxicity in the control group (10 mg/kg Cu) and treatment groups (125 mg/kg and 250 mg/kg Cu) for a period of 80 days. Consequently, we identified that large amount intake of Cu led to in oxidative damage, and activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)-mediated antioxidant pathway, indicating an imbalanced redox status in the myocardium. Furthermore, Cu exposure activated endoplasmic reticulum (ER) stress through upregulating levels of glucose-regulated protein 78 (GRP78), c-Jun N-terminal kinase (JNK), glucose-regulated protein 94 (GRP94), X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP). Additionally, mitochondrial fission and fusion homeostasis was disrupted and the copy number of mitochondrial DNA (mtDNA) was reduced under Cu exposure. Furthermore, Cu exposure could induce apoptosis, evidenced by the increased terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling (TUNEL)-positive staining, the upregulated expression levels of Cytoplasm-cytochrome C (Cytc), Bcl-2-associated X protein (Bax), and Cleaved-caspase3, and decreased expression level of B-cell lymphoma-2 (Bcl-2) and Mitochondrial-cytc. In summary, large amount of Cu could trigger Nrf2/HO-1 pathway-mediated oxidative stress, which promotes ER stress and mitochondrial damage pathways, causing apoptosis in cardiomyocytes.

Knockdown of zinc finger protein 267 suppresses diffuse large B-cell lymphoma progression, metastasis, and cancer stem cell properties

ABSTRACT Zinc finger protein 267 (ZNF267) is a member of the Kruppel-like transcription factor family, which regulates various biological processes such as cell proliferation and differentiation. However, the biological significance of ZNF267 and its potential role in diffuse large B-cell lymphoma (DLBCL) remain to be documented. Experiments were herein conducted to study the role of ZNF267 in DLBCL. real-time quantitative reverse transcription PCR and Western blotting assays were conducted to detect the expression of ZNF267 in tissues and cells. Tissue microarray and bioinformatics analyses of public data were also done to detect the expression status and clinical significance of ZNF267. Functional cell experiments including CCK8 assay, colony formation assay, 5-ethynyl-2ʹ-deoxyuridine (EDU) assay, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay, transwell assay, and wound healing assay were conducted to study the effects of ZNF267 knockdown and overexpression on cell proliferation and mobility. Xenograft assay was also conducted to confirm the effects of ZNF267 knockdown in vivo. In the present study, we found ZNF267 was significantly upregulated in DLBCL and predicted a poor survival outcome based on the bioinformatics analysis. Functionally, the knockdown of ZNF267 resulted in less cell proliferation and mobility, whereas the overexpression led to enhanced cell proliferation and mobility. Animal experiments also confirmed that ZNF267 silence contributed to less tumor growth and less lung metastasis. Further analysis showed that ZFN267 knockdown resulted in decreased epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. Our results suggest that ZNF267 is an oncogene in DLBCL and its silence could compromise the aggression of DLBCL, which makes ZNF267 a promising therapeutic target.

The effects of agomelatine on endoplasmic reticulum stress related to mitochondrial dysfunction in hippocampus of aging rat model

BackgroundAgomelatine, a novel antidepressant, is a melatonin MT receptor agonist and serotonin 5HT2C receptor antagonist. In this study, agomelatine was used to investigate the molecular mechanisms of hippocampal aging associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and apoptosis, all of which led to short-term memory impairment. MethodHippocampal aging was induced in male Wistar rats by d-galactose (D-gal) intraperitoneal injection (100 mg/kg) for 14 weeks. During the last 4 weeks of D-gal treatment, rats were treated with agomelatine (40 mg/kg) or melatonin (10 mg/kg). At the end of the experiment, all rats were assessed for short-term memory by using the Morris water maze test. Subsequently, rats were sacrified and the hippocampus was removed from each rat for determination of reactive oxygen species (ROS), malondialdehyde (MDA), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays; and immunohistochemistry related to ER stress, mitochondrial dysfunction, and apoptosis. ResultsAgomelatine suppressed the expression of the aging-related proteins P16 and receptor for advanced glycation endproducts (RAGE), the expression of NADPH oxidase (NOX) 2 and 4, and ROS production. This treatment also shifted the morphology of astrocytes and microglia toward homeostasis. Furthermore, agomelatine decreased inositol-requiring enzyme 1 (pIRE1), protein kinase R-like endoplasmic reticulum kinase (pPERK), and chaperone binding immunoglobulin protein (BiP), leading to suppression of ER stress markers C/EBP homologous protein (CHOP) and caspase-12. Agomelatine reduced Ca2+ from the ER and stabilized the mitochondrial membrane stability, which was denoted by the BCL2 Associated X (Bax)/B-cell lymphoma 2 (Bcl2) balance. Agomelatine decreased cleaved caspase-3 production and the Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive area, and glutamate excitotoxicity was prevented via suppression of N-methyl-d-aspartate (NMDA) receptor subunit expression. Agomelatine exhibited effects that were similar to melatonin. ConclusionAgomelatine improved neurodegeneration in a rat model of hippocampal aging by attenuating ROS production, ER stress, mitochondrial dysfunction, excitotoxicity, and apoptosis.

EVALUATING THE EFFICIENCY OF PHYSIOLOGICAL ICSI (PICSI) IN PATIENTS WITH DIFFERENT LEVELS OF ABNORMAL SPERM DNA FRAGMENTATION

To evaluate the efficiency of sperm selection by PICSI on males with different levels of abnormal sperm DNA fragmentation (SDF). A retrospective cohort study included 630 couples undergoing ICSI from October 2016 to October 2019. Cases were divided according to their SDF levels into: (Mild: 20.4-25%, Moderate: 25.1-30%, and Severe: >30%) and normal SDF (<20% control) groups. The study included females ≤37 years old, with a minimum of 5 mature oocytes injected and had a fresh blastocyst transfer. All males SDF levels were assessed prior to their ICSI cycles using the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. Comparisons included pre-implantation embryo development, pregnancy, and live birth rates. The Chi square test was used for the qualitative parameters and Kruskal Walis test for non-parametric quantitative parameters (SPSS version 23). We found no significant differences between the control and high SDF groups in: type of infertility, female age, male age, count, motility, abnormal morphology, smoking status, abstinence days, No. of retrieved oocytes, and No. of mature oocytes. The outcomes of ICSI versus PICSI for the different study groups are shown in Table 1.Tabled 1Study outcomesNormal SDF n=291PICSIp-valuesMild n=147Moderate n=93Severe n=99Cleavage rate (%)75.6±18.275.3±16.677±17.978.8±14.80.6Blastocyst rate (%)64.7±19.663.2±2067.1±17.764.8±18.10.82Good quality Blastocyst rate (%)37.2±23.838±27.233±22.833.8±25.10.27Fair quality Blastocyst rate (%)17±17.115.2±18.215.2±15.618.4±17.30.5Vitrified Blastocysts3.36±3.53.37±3.52.64±2.22.67±2.70.65Implantation rate (%)44.5±43.241.5±40.738.4±44.434.4±39.20.09Clinical pregnancy rate (%)*64.461.355.760.20.53Ongoing pregnancy rate (%)*54.645.245.252.30.22Miscarriage rate (%)*14.523.515.68.20.11Live birth rate (%)*53.64343.951.20.16Data are mean (SD), otherwise stated. n refers to the number of cycles. PICSI = physiological intracytoplasmic sperm injection. SDF = Sperm DNA fragmentation. * Data are percentages only. P values <0.05 are considered significant. Open table in a new tab PICSI can perform with the same efficiency at all levels of abnormal SDF, improving their reproductive outcomes to the level of the control group. A prospective randomized trial with a control of abnormal SDF is planned as a future study to confirm these findings.

Protective effects of sunlight exposure against PRK-induced myopia in infant rhesus monkeys.

Extensive clinical evidence suggests that time spent outdoors might reduce the risk of myopia. This study aimed to determine whether increasing sunlight exposure has a protective effect on hyperopic-defocus induced myopia in a non-human primate. Twelve 2-month-old rhesus monkeys were treated monocularly with photorefractive keratectomy (PRK) (4.0 D) and divided randomly into two groups: artificial light (AL; n=6) and natural light (NL; n=6). Monkeys in the AL group were reared under artificial (indoor) lighting (08:00-20:00h). Monkeys in the NL group were exposed to natural (outdoor) lighting for 4h (09:00-11:00 and 15:00-17:00h). Ocular refraction, corneal power and axial dimensions were measured before sunlight exposure and every 10days after PRK. At day 180, retinal histology and apoptosis activity were evaluated by hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase biotin (dUTP) nick end labelling (TUNEL) assay. Mean (±SD) PRK induced anisometropia was +3.11 (0.33) D. At the end of the experiment, both eyes of the NL monkeys exhibited significantly more hyperopia and shorter vitreous chamber depths (VCD), compared with AL monkeys (p<0.05). The NL group exhibited a significantly slower rate of compensation to the induced anisometropia than the AL group (p<0.05). The retinas of both groups exhibited normal histology and levels of apoptosis. Moderate sunlight exposure exerts protective effects against the myopic shift resulting from PRK-induced defocus in monkeys. These results are consistent with current clinical findings that increased outdoor exposure protects against myopia development. Sunlight exposure should serve as an independent positive factor in human myopia control.

Anti-apoptotic efficacy of Qingnao Yizhi formula in hypoxia/reoxygenation primary cortical neurons through the promotion of synaptic plasticity by modulating the NogoA-Nogo receptor/Rho-Rho kinase signaling pathway.

To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula (，QNYZ) in cultured cerebral cortical neuronal cells (CNCs) and the regulation of the NogoA-Nogo receptor (NgR)/Rho-Rho kinase (ROCK) signaling pathway. Primary cultured CNCs were randomly divided into the following groups: normal control group (N-C), hypoxia-reoxygenation group (H/R), high-dose QNYZ group (Q-H), low-dose QNYZ group (Q-L) butylphthalide (NBP) group, and Y-27632 (a selective ROCK transduction pathway inhibiter) group. Except those in the N-C group, CNCs were placed in hypoxic conditions for 24 h and then in reoxygenation conditions for 24 h. Cell media was changed every 48 h, and various assays were performed on the 7th day. Cell viability was evaluated by measuring mitochondrial dehydrogenase activity, using a CCK-8 assay, in triplicate. Synapsin (SYN) protein concentrations were evaluated by enzyme-linked immunosorbent assay. NogoA and RhoA protein expression were evaluated through Western blotting. The gene expression of NogoA, NgR, RhoA, and ROCK was evaluated by reverse transcription-polymerase chain reaction. Cell apoptosis was measured using a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. Compared with the N-C group, the cell viability of the H/R group decreased significantly (P < 0.05). The cell viability values for the Q-H and Q-L groups increased compared with that for the H/R group, and the difference was significant for the Q-H group (P < 0.05). The NogoA and RhoA protein levels and the NogoA, NgR, RhoA, and ROCK mRNA expression levels increased in the H/R group, compared with the N-C group, and decreased significantly in the Q-H and Q-L groups (P < 0.05) and in the Y-27632 group (P < 0.05) compared with the H/R group. The SYN levels in the Q-H, Q-L, and NBP groups significantly increased compared with that in the H/R group (P < 0.05). Compared with the H/R group, the numbers of apoptotic cells in the Q-H, Q-L, and NBP groups significantly decreased (P < 0.05). The presented study demonstrated that QNYZ exerted anti-apoptotic effects on H/R-induced CNCs, possibly through the modulation of the NogoA-NgR/Rho-ROCK signaling pathway and the promotion of synaptic plasticity in H/R CNCs.

Emodin protects against intestinal and lung injury induced by acute intestinal injury by modulating SP-A and TLR4/NF-κB pathway.

Objective: Our aim was to investigate the effect of emodin on intestinal and lung injury induced by acute intestinal injury in rats and explore potential molecular mechanisms.Methods: Healthy male Sprague–Dawley (SD) rats were randomly divided into five groups (n=10, each group): normal group; saline group; acute intestinal injury model group; model + emodin group; model+NF-κB inhibitor pynolidine dithiocarbamate (PDTC) group. Histopathological changes in intestine/lung tissues were observed by Hematoxylin and Eosin (H&E) and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) staining. Serum IKBα, p-IKBα, surfactant protein-A (SP-A) and toll-like receptor 4 (TLR4) levels were examined using enzyme-linked immunosorbent assay (ELISA). RT-qPCR was performed to detect the mRNA expression levels of IKBα, SP-A and TLR4 in intestine/lung tissues. Furthermore, the protein expression levels of IKBα, p-IKBα, SP-A and TLR4 were detected by Western blot.Results: The pathological injury of intestinal/lung tissues was remarkedly ameliorated in models treated with emodin and PDTC. Furthermore, the intestinal/lung injury scores were significantly decreased after emodin or PDTC treatment. TUNEL results showed that both emodin and PDTC treatment distinctly attenuated the apoptosis of intestine/lung tissues induced by acute intestinal injury. At the mRNA level, emodin significantly increased the expression levels of SP-A and decreased the expression levels of IKBα and TLR4 in intestine/lung tissues. According to ELISA and Western blot, emodin remarkedly inhibited the expression of p-IKBα protein and elevated the expression of SP-A and TLR4 in serum and intestine/lung tissues induced by acute intestinal injury.Conclusion: Our findings suggested that emodin could protect against intestinal and lung injury induced by acute intestinal injury by modulating SP-A and TLR4/NF-κB pathway.

Laminin degradation by matrix metalloproteinase 9 promotes ketamine-induced neuronal apoptosis in the early developing rat retina.

AimsDuring early development, laminin degradation contributes to the death of neurons. This study aims to investigate the role and regulation of laminin in ketamine‐induced apoptosis.MethodsWe performed terminal deoxynucleotidyl transferase biotin‐dUTP nick end labeling (TUNEL) and immunohistochemical assays to investigate the roles of the non‐integrin laminin receptor, matrix metalloproteinase 9 (MMP9) in ketamine‐induced neuronal apoptosis. In situ zymography, Western blot, and immunofluorescence were used to explore the relationships between laminin, MMP9 activity, and Zn2+. Experiments were performed using whole‐mount retinas dissected from Sprague Dawley rats.ResultsThe TUNEL and immunohistochemical assays indicated that ketamine‐induced neuronal apoptosis in early developing rat retina. Blockade of non‐integrin laminin receptor promoted ketamine‐induced apoptosis, while non‐integrin laminin receptor activation attenuated ketamine‐induced apoptosis. Ketamine‐induced laminin degradation, possibly by enhancing the activity of MMP9. MMP9 inhibition reduced ketamine‐induced apoptosis by reducing laminin degradation. Downregulation of Zn2+ attenuated the increased MMP9 activity, laminin degradation caused by ketamine and significantly reduced ketamine‐induced neuronal apoptosis.ConclusionLaminin degradation by MMP9 promoted ketamine‐induced neuronal apoptosis in early developing rat retina. The non‐integrin laminin receptor may be a pathway involved in ketamine‐induced apoptosis. Zn2+ downregulation may play a protective role against ketamine‐induced neuronal apoptosis through inhibiting MMP9 activity.

Hearing loss through apoptosis of the spiral ganglion neurons in apolipoprotein E knockout mice fed with a western diet

Age-related hearing loss (ARHL) is a neurodegenerative disease associated with an aged population. ARHL is influenced by biological factors such as aging, sex difference, and atherosclerosis. The mechanisms of ARHL caused by atherosclerosis have not been previously determined in apolipoprotein E knockout (ApoE KO) male mice. To investigate the onset and cause of the hearing loss, ApoE KO male mice were treated with a western diet (ApoE KO-WD) for 16 weeks. The lipid profile, atherosclerotic plaques throughout the aorta, and auditory brainstem response (ABR) thresholds were measured in the ApoE KO-WD male mice. The expression of S100 calcium-binding protein B (S100B), a neuronal damage biomarker, was also observed. Reactive oxygen species (ROS) and apoptosis rates were detected in the cochlea of the ApoE KO male mice. Atherosclerotic plaques on the aorta and ABR thresholds were significantly increased in the ApoE KO-WD male mice at 24 weeks of age. ABR thresholds had a statistically significant positive correlation with the area of atherosclerotic plaques (r = 0.783, p = 0.013) in male mice at 24 weeks of age. S100B protein expression and the dihydroethidium (DHE) reaction to ROS in the cochlear spiral ganglion neurons (SGNs) were significantly increased in the ApoE KO and ApoE KO-WD male mice. Cells positive for active caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) in the SGNs were significantly increased in ApoE KO-WD male mice indicating an increased rate of cellular apoptosis. In conclusion, ROS in the SGNs were activated by increased S100B expression in ApoE KO-WD male mice, and this resulted in an increased apoptosis rate. Thus, hearing loss began at 16 weeks in ApoE KO-WD male mice. Our results suggest that the ApoE KO-WD male mice are a suitable animal model for studying ARHL associated with exacerbated atherosclerosis.