BCL2/adenovirus E1B 19 kDa Interacting Protein 3 Research Articles

Expression and significance of hypoxia-inducible factor 1α and Bcl-2/adenovirus E1B19kDa-interacting protein 3 in children with traumatic brain injury

To dynamically observe the changes in hypoxia-inducible factor 1α (HIF-1α) and Bcl-2/adenovirus E1B19kDa-interacting protein 3 (BNIP3) in children with traumatic brain injury (TBI) and evaluate their clinical value in predicting the severity and prognosis of pediatric TBI. A prospective study included 47 children with moderate to severe TBI from January 2021 to July 2023, categorized into moderate (scores 9-12) and severe (scores 3-8) subgroups based on the Glasgow Coma Scale. A control group consisted of 30 children diagnosed and treated for inguinal hernia during the same period, with no underlying diseases. The levels of HIF-1α, BNIP3, autophagy-related protein Beclin-1, and S100B were compared among groups. The predictive value of HIF-1α, BNIP3, Beclin-1, and S100B for the severity and prognosis of TBI was assessed using receiver operating characteristic (ROC) curves. Serum levels of HIF-1α, BNIP3, Beclin-1, and S100B in the TBI group were higher than those in the control group (P<0.05). Among the TBI patients, the severe subgroup had higher levels of HIF-1α, BNIP3, Beclin-1, and S100B than the moderate subgroup (P<0.05). Correlation analysis showed that the serum levels of HIF-1α, BNIP3, Beclin-1, and S100B were negatively correlated with the Glasgow Coma Scale scores (P<0.05). After 7 days of treatment, serum levels of HIF-1α, BNIP3, Beclin-1, and S100B in both non-surgical and surgical TBI patients decreased compared to before treatment (P<0.05). ROC curve analysis indicated that the areas under the curve for predicting severe TBI based on serum levels of HIF-1α, BNIP3, Beclin-1, and S100B were 0.782, 0.835, 0.872, and 0.880, respectively (P<0.05), and for predicting poor prognosis of TBI were 0.749, 0.775, 0.814, and 0.751, respectively (P<0.05). Serum levels of HIF-1α, BNIP3, and Beclin-1 are significantly elevated in children with TBI, and their measurement can aid in the clinical assessment of the severity and prognosis of pediatric TBI.

Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury

BackgroundThe mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes.MethodsA 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein–Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms.ResultsA total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway.ConclusionsThe proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

The Role of BNIP3 and Blocked Autophagy Flux in Arsenic-Induced Oxidative Stress-Induced Liver Injury in Rats.

Arsenic is a widely distributed environmental toxic substance in nature. Chronic arsenic exposure can cause permanent damage to the liver, resulting in the death of poisoned patients. However, the mechanism of liver damage caused by arsenic poisoning is yet unclear. Here, four different concentrations of sodium arsenite (NaAsO2) (0mg/L (control group), 25mg/L, 50mg/L, and 100mg/L group)were established to induce liver injury in rats. Taking this into account, the relationship and potential mechanisms of oxidative stress, Bcl-2/adenovirus E1B-19-kDa-interacting protein 3 (BNIP3), and inhibition of autophagy flux in liver injury caused by arsenic poisoning were studied. The results indicated that long-term exposure to NaAsO2 could induce oxidative stress, leading to high expression of BNIP3, thereby impaired autophagy flux, and ultimately resulting in liver damage. This research provides an important basis for future research on liver damage caused by chronic arsenic exposure and prevention and treatment with BNIP3 as the target.

Mitophagy in Carcinogenesis and Tumour Progression- A New Paradigm with Emerging Importance.

The term Mitophagy has been newly concerned in reforming the metabolic landscape inside cancerous cells in addition to the interface between malignant cells as well as other major constituents of tumour microenvironment. Several profoundly interrelated systems, comprising mitochondrial dynamics and mitophagy, function in mammalian cells as vital mitochondrial regulator processes, and their consequence in neoplastic development has recently been illuminated clinically. In specific instances of cancer cells, mitochondrial-protected metabolic paths are revamped to meet expanded bioenergetics along with biosynthetic necessities of malignant cells, in addition to deal with oxidative stress. It is an exhausting task to foresee the role that mitophagy has on malignant growth cells since it relies upon various elements like cancer variability, malignant growth phase, genetic background and harmony between cell demand and accessibility. As per condition, mitophagy may have a double role as cancer suppressor for example Atg5 (autophagy related 5) or Atg7 (autophagy related 7) or execute promoter like function for instance FUNDC1 (FUN14 domain-containing protein 1), BNIP3 (BCL2/adenovirus E1B 19-kDa-interacting protein 3), PINK1 (PTEN-instigated kinase 1) etc. Tumour suppressive function of Parkin (E3 ubiquitin ligase) is likewise distinguished in mammary gland carcinoma where obstruction of mitophagy impacts tumour progression. In pancreatic cancer cells and hepatocellular carcinoma hypermethylation of the BNIP3, promoter occurs that prevent HIF-1 (Hypoxia-Inducible Factor 1) binding besides ensuing initiation of mitophagy. Since the dual role of mitophagy has in malignant growth relying upon various circumstances and cell varieties, a range of studies have been performed on mitophagy and its role in cancer progression and development is opening up a new paradigm with immense clinical importance.

Superiority of focused ion beam-scanning electron microscope tomography of cardiomyocytes over standard 2D analyses highlighted by unmasking mitochondrial heterogeneity.

BackgroundCardioprotection by preventing or repairing mitochondrial damage is an unmet therapeutic need. To understand the role of cardiomyocyte mitochondria in physiopathology, the reliable characterization of the mitochondrial morphology and compartment is pivotal. Previous studies mostly relied on two‐dimensional (2D) routine transmission electron microscopy (TEM), thereby neglecting the real three‐dimensional (3D) mitochondrial organization. This study aimed to determine whether classical 2D TEM analysis of the cardiomyocyte ultrastructure is sufficient to comprehensively describe the mitochondrial compartment and to reflect mitochondrial number, size, dispersion, distribution, and morphology.MethodsSpatial distribution of the complex mitochondrial network and morphology, number, and size heterogeneity of cardiac mitochondria in isolated adult mouse cardiomyocytes and adult wild‐type left ventricular tissues (C57BL/6) were assessed using a comparative 3D imaging system based on focused ion beam‐scanning electron microscopy (FIB‐SEM) nanotomography. For comparison of 2D vs. 3D data sets, analytical strategies and mathematical comparative approaches were performed. To confirm the value of 3D data for mitochondrial changes, we compared the obtained values for number, coverage area, size heterogeneity, and complexity of wild‐type cardiomyocyte mitochondria with data sets from mice lacking the cytosolic and mitochondrial protein BNIP3 (BCL‐2/adenovirus E1B 19‐kDa interacting protein 3; Bnip3 −/− ) using FIB‐SEM. Mitochondrial respiration was assessed on isolated mitochondria using the Seahorse XF analyser. A cardiac biopsy was obtained from a male patient (48 years) suffering from myocarditis.ResultsThe FIB‐SEM nanotomographic analysis revealed that no linear relationship exists for mitochondrial number (r = 0.02; P = 0.9511), dispersion (r = −0.03; P = 0.9188), and shape (roundness: r = 0.15, P = 0.6397; elongation: r = −0.09, P = 0.7804) between 3D and 2D results. Cumulative frequency distribution analysis showed a diverse abundance of mitochondria with different sizes in 3D and 2D. Qualitatively, 2D data could not reflect mitochondrial distribution and dynamics existing in 3D tissue. 3D analyses enabled the discovery that BNIP3 deletion resulted in more smaller, less complex cardiomyocyte mitochondria (number: P < 0.01; heterogeneity: C.V. wild‐type 89% vs. Bnip3 −/− 68%; complexity: P < 0.001) forming large myofibril‐distorting clusters, as seen in human myocarditis with disturbed mitochondrial dynamics. Bnip3 −/− mice also show a higher respiration rate (P < 0.01).ConclusionsHere, we demonstrate the need of 3D analyses for the characterization of mitochondrial features in cardiac tissue samples. Hence, we observed that BNIP3 deletion physiologically acts as a molecular brake on mitochondrial number, suggesting a role in mitochondrial fusion/fission processes and thereby regulating the homeostasis of cardiac bioenergetics.

FOXO3a protects glioma cells against temozolomide-induced DNA double strand breaks via promotion of BNIP3-mediated mitophagy

FOXO3a (forkhead box transcription factor 3a) is involved in regulating multiple biological processes in cancer cells. BNIP3 (Bcl-2/adenovirus E1B 19-kDa-interacting protein 3) is a receptor accounting for priming damaged mitochondria for autophagic removal. In this study we investigated the role of FOXO3a in regulating the sensitivity of glioma cells to temozolomide (TMZ) and its relationship with BNIP3-mediated mitophagy. We showed that TMZ dosage-dependently inhibited the viability of human U87, U251, T98G, LN18 and rat C6 glioma cells with IC50 values of 135.75, 128.26, 142.65, 155.73 and 111.60 μM, respectively. In U87 and U251 cells, TMZ (200 μM) induced DNA double strand breaks (DSBs) and nuclear translocation of apoptosis inducing factor (AIF), which was accompanied by BNIP3-mediated mitophagy and FOXO3a accumulation in nucleus. TMZ treatment induced intracellular ROS accumulation in U87 and U251 cells via enhancing mitochondrial superoxide, which not only contributed to DNA DSBs and exacerbated mitochondrial dysfunction, but also upregulated FOXO3a expression. Knockdown of FOXO3a aggravated TMZ-induced DNA DSBs and mitochondrial damage, as well as glioma cell death. TMZ treatment not only upregulated BNIP3 and activated autophagy, but also triggered mitophagy by prompting BNIP3 translocation to mitochondria and reinforcing BNIP3 interaction with LC3BII. Inhibition of mitophagy by knocking down BNIP3 with SiRNA or blocking autophagy with 3MA or bafilomycin A1 exacerbated mitochondrial superoxide and intracellular ROS accumulation. Moreover, FOXO3a knockdown inhibited TMZ-induced BNIP3 upregulation and autophagy activation. In addition, we showed that treatment with TMZ (100 mg·kg−1·d−1, ip) for 12 days in C6 cell xenograft mice markedly inhibited tumor growth accompanied by inducing FOXO3a upregulation, oxidative stress and BNIP3-mediated mitophagy in tumor tissues. These results demonstrate that FOXO3a attenuates temozolomide-induced DNA double strand breaks in human glioma cells via promoting BNIP3-mediated mitophagy.

HIF-1α Protects Granulosa Cells From Hypoxia-Induced Apoptosis During Follicular Development by Inducing Autophagy.

Owing to the avascular structure of the ovarian follicle, proliferation of granulosa cells (GCs) and development of follicles occur under hypoxia, which is obviously different from the cell survival requirements of most mammalian cells. We hypothesized that autophagy may exert an inhibitory effect on GC apoptosis. To decipher the underlying mechanism, we constructed a rat follicular development model using pregnant mare serum gonadotropin and a cell culture experiment in hypoxic conditions (3% O2). The present results showed that the autophagy level was obviously increased and was accompanied by the concomitant elevation of hypoxia inducible factor (HIF)-1α and BNIP3 (Bcl-2/adenovirus E1B 19kDa-interacting protein 3) in GCs during follicular development. The levels of Bax (Bcl2-associated X) and Bcl-2 (B-cell lymphoma-2) were increased, while the activation of caspase-3 exhibited no obvious changes during follicular development. However, inhibition of HIF-1α attenuated the increase in Bcl-2 and promoted the increase in Bax and cleaved caspase-3. Furthermore, we observed the downregulation of BNIP3 and the decrease in autophagy after treatment with a specific HIF-1α activity inhibitor (echinomycin), indicating that HIF-1α/BNIP3 was involved in autophagy regulation in GCs in vivo. In an in vitro study, we also found that hypoxia did not obviously promote GC apoptosis, while it significantly enhanced the activation of HIF-1α/BNIP3 and the induction of autophagy. Expectedly, this effect could be reversed by 3-methyladenine (3-MA) treatment. Taken together, these findings demonstrated that hypoxia drives the activation of HIF-1α/BNIP3 signaling, which induces an increase in autophagy, protecting GC from apoptosis during follicular development.

Pramipexole attenuates neuronal injury in Parkinson's disease by targeting miR-96 to activate BNIP3-mediated mitophagy

BackgroundParkinson's disease is a common neurodegenerative problem. Pramipexole (PPX) plays protective role in Parkinson's disease. Nevertheless, the mechanism of PPX in Parkinson's disease-like neuronal injury is largely uncertain. Methods1-methyl-4-phenylpyridinium (MPP+)-stimulated neuronal cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice were used as the model of Parkinson's disease. MPP+-induced neuronal injury was assessed via cell viability, lactic dehydrogenase (LDH) release and apoptosis. microRNA-96 (miR-96) and BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) abundances were examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blotting. Mitophagy was tested by Western blotting and immunofluorescence staining. MPTP-induced neuronal injury in mice was investigated via behavioral tests and TUNEL. ResultsPPX alleviated MPP+-induced neuronal injury via increasing cell viability and decreasing LDH release and apoptosis. PPX reversed MPP+-induced miR-96 expression and inhibition of mitophagy. miR-96 overexpression or BNIP3 interference weakened the suppressive role of PPX in MPP+-induced neuronal injury. miR-96 targeted BNIP3 to inhibit PTEN-induced putative kinase 1 (PINK1)/Parkin signals-mediated mitophagy. miR-96 overexpression promoted MPP+-induced neuronal injury via decreasing BNIP3. PPX weakened MPTP-induced neuronal injury in mice via regulating miR-96/BNIP3-mediated mitophagy. ConclusionPPX mitigated neuronal injury in MPP+-induced cells and MPTP-induced mice by activating BNIP3-mediated mitophagy via directly decreasing miR-96.

Crosstalk between hypoxia-sensing ULK1/2 and YAP-driven glycolysis fuels pancreatic ductal adenocarcinoma development.

Autophagy and glycolysis are two catabolic processes that manipulate pancreatic ductal adenocarcinoma (PDAC) development in response to hypoxia sensing, yet the underlying mechanism of how they are interlinked remain elusive.Methods: The functional roles of Unc-51 like kinase 1 and 2 (ULK1/2) in pyruvate kinase M2 (PKM2) transcription and glycolysis under hypoxia were assessed by chromatin immunoprecipitation, luciferase reporter, glucose consumption and lactate production assay. Co-immunoprecipitation, cellular ubiquitination, His-pulldown, in vitro protein kinase assay, immunofluorescence, immunohistochemistry, CRISPR technology, in silico studies were adopted to determine the molecular mechanism. Correlation analyses were performed in KPC (Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/+) mice and clinical samples from PDAC patients. Therapeutic potential of ULK1/2 inhibitor and 2-deoxyglucose (2-DG) or 3-bromopyruvate (3-BP) was evaluated in cell-derived xenograft (CDX) and the patient-derived xenograft (PDX) models of nude mice.Results: ULK1/2, but not ULK3, augments hypoxic glycolysis in PDAC cells mediated by PKM2 independent of BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). Mechanistically, hypoxia stimulates ULK1 to translocate into nucleus, where it interacts with and phosphorylates yes-associated protein (YAP) at Ser227, resulting in YAP stabilization through blockade of ubiquitin-proteasome system (UPS), which in turn facilitates PKM2 transcription, glycolysis, cell proliferation in vitro as well as PDAC growth in mice. ULK1/2 is positively correlated with YAP and PKM2 in tumor tissues from KPC mice and clinical samples from PDAC patients. Pharmacological deactivation of ULK1/2 potentiates the antineoplastic efficacy of 2-DG and 3-BP in CDX and PDX models.Conclusion: Our findings underscore the Ser227 autophosphorylation-dependent nuclear YAP stabilization as a central node that couples ULK1/2-initiated autophagy to hypoxic glycolysis during PDAC development and propose that targeting ULK1/2 combined with 2-DG or 3-BP might be a feasible therapeutic strategy against PDAC.

Intracellular AIBP (Apolipoprotein A-I Binding Protein) Regulates Oxidized LDL (Low-Density Lipoprotein)-Induced Mitophagy in Macrophages.

Atherosclerotic lesions are often characterized by accumulation of OxLDL (oxidized low-density lipoprotein), which is associated with vascular inflammation and lesion vulnerability to rupture. Extracellular AIBP (apolipoprotein A-I binding protein; encoded by APOA1BP gene), when secreted, promotes cholesterol efflux and regulates lipid rafts dynamics, but its role as an intracellular protein in mammalian cells remains unknown. The aim of this work was to determine the function of intracellular AIBP in macrophages exposed to OxLDL and in atherosclerotic lesions. Approach and Results: Using a novel monoclonal antibody against human and mouse AIBP, which are highly homologous, we demonstrated robust AIBP expression in human and mouse atherosclerotic lesions. We observed significantly reduced autophagy in bone marrow-derived macrophages, isolated from Apoa1bp-/- compared with wild-type mice, which were exposed to OxLDL. In atherosclerotic lesions from Apoa1bp-/- mice subjected to Ldlr knockdown and fed a Western diet, autophagy was reduced, whereas apoptosis was increased, when compared with that in wild-type mice. AIBP expression was necessary for efficient control of reactive oxygen species and cell death and for mitochondria quality control in macrophages exposed to OxLDL. Mitochondria-localized AIBP, via its N-terminal domain, associated with E3 ubiquitin-protein ligase PARK2 (Parkin), MFN (mitofusin)1, and MFN2, but not BNIP3 (Bcl2/adenovirus E1B 19-kDa-interacting protein-3), and regulated ubiquitination of MFN1 and MFN2, key components of mitophagy. These data suggest that intracellular AIBP is a new regulator of autophagy in macrophages. Mitochondria-localized AIBP augments mitophagy and participates in mitochondria quality control, protecting macrophages against cell death in the context of atherosclerosis.

LncRNA DGCR 5/miR‑27a‑3p/BNIP3 promotes cell apoptosis in pancreatic cancer by regulating the p38 MAPK pathway.

Long non-coding RNA (lncRNA) DGCR5 has been identified as a tumor suppressor in several types of cancer. However, its biological functions in pancreatic cancer (PaCa) have not yet been fully elucidated. The present study was designed to investigate the role of lncRNA DGCR5 in the regulation of PaCa cell apoptosis. For this purpose, lncRNA DGCR5, miR-27a-3p and Bcl-2/adenovirus E1B-19kDa-interacting protein 3 (BNIP3) expression levels were examined by reverse transcription-quantitative (RT-qPCR) and western blot analysis, respectively. RNA pull-down assay was used to verify DGCR5 as a target of miR-27a-3p and dual luciferase reporter assay was used to clarify whether miR-27a-3p targets the BNIP3 3′ UTR. In addition, PaCa cell apoptosis was assessed by flow cytometry. Recombinant plasmids and cell transfection were performed to modulate the endogenous expression of related genes. Thereafter, the role of DGCR5 in PaCa was analyzed using a nude mouse model of PaCa. lncRNA DGCR5 was found to be downregulated in PaCa tissues and cells. DGCR5 functioned as a decoy of miR-27a-3p, and BNIP3 was negatively regulated by miR-27a-3p. Following the transfection of DGCR5 plasmid into PaCa cells, the expression of miR-27a-3p was downregulated, and this downregulation was reversed following transfection with miR-27a-3p mimic. In addition, DGCR5 regulated the BNIP3 and p38 MAPK pathways via miR-27a-3p and promoted PaCa cell apoptosis via the miR-27a-3p/BNIP3 pathway. The results of in vivo experiments also indicated the positive effects of DGCR5 on a nude mouse model of PaCa. On the whole, the findings of the present study indicate that lncRNA DGCR5 upregulates the BNIP3 and p38 MAPK pathways via miR-27a-3p to promote PaCa cell apoptosis, thereby attenuating PaCa development.

Par-4 regulates autophagic cell death in human cancer cells via upregulating p53 and BNIP3

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that selectively induces apoptosis in cancer cells. Although the mechanism of Par-4-mediated induction of apoptosis has been well studied, the involvement of Par-4 in other mechanisms of cell death such as autophagy is unclear. We investigated the mechanism involved in Par-4-mediated autophagic cell death in human malignant glioma. We demonstrate for the first time that the tumor suppressor lipid, ceramide (Cer), causes Par-4 induction, leading to autophagic cell death in human malignant glioma. Furthermore, we identified the tumor suppressor protein p53 and BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) as downstream targets of Par-4 during Cer-mediated autophagic cell death. RNAi-mediated down-regulation of Par-4 blocks Cer-induced p53-BNIP3 activation and autophagic cell death, while upregulation of Par-4 augmented p53-BNIP3 activation and autophagic cell death. Remarkably, in many instances, Par-4 overexpression alone was sufficient to induce cell death which is associated with features of autophagy. Interestingly, similar results were seen when glioma cells were exposed to classical autophagy inducers such as serum starvation, arsenic trioxide, and curcumin. Collectively, the novel Par-4-p53-BNIP3 axis plays a crucial role in autophagy-mediated cell death in human malignant glioma.

Clinical significance of Bcl-2/adenovirus E1B19kDa interacting protein 3 in patients with hand, foot and mouth disease

Objective To investigate the expression and clinical significance of Bcl-2/adenovirus E1B19kDa interacting protein 3 (BNIP3) in serum and cerebrospinal fluid (CSF) of patients with severe hand, foot and mouth disease (HFMD). Methods Ninety children with HFMD were classified into three groups with 30 in each group: critical group (clinical stage 3), severe group (clinical stage 2) and common group (clinical stage 1, excluding encephalitis with CSF and other examinations). Another thirty healthy children were randomly selected as the control group. The levels of BNIP3 in serum and CSF were detected before and after treatment. Moreover, serum neuro-specific enolase (NSE) and S100B protein were also measured to analyze their correlation with BNIP3. Receiver operating characteristic (ROC) curve was used to evaluate the prediction efficiency of BNIP3 for the severity of HFMD. Results The levels of serum BNIP3, S100B protein and NSE in the critical group were higher than those in the other three groups (P<0.01). CSF BNIP3 level in the critical group were significantly higher than that in the common and severe groups (P<0.01). Serum BNIP3, S100B protein and NSE were significantly higher in the severe group than in common and control groups (P<0.01). CSF BNIP3 was significantly increased in the severe group as compared with that in the common group (P<0.01). After treatment, the levels of BNIP3, S100B protein and NSE in serum and BNIP3 in CSF were decreased in both critical and severe groups (P<0.01). The levels of BNIP3 in serum and CSF were positively correlated with the level of S100B protein and NSE (P<0.01). Serum BNIP3 had the highest Youden value at the cut-off value of 3.015 μg/L, with a sensitivity of 83.33% and a specificity of 90.00%, in the prediction of severe HFMD. CSF BNIP3 had the highest Youden value at the cut-off value of 1.735 μg/L, with a sensitivity of 73.33% and a specificity of 93.33%, in the prediction of severe HFMD. Conclusions BNIP3 is involved in the pathological process of brain injury in children with severe HFMD. Detection of BNIP3 helps evaluate the severity and prognosis of HFMD. Key words: Bcl-2/adenovirus E1B 19kDa interacting protein 3; Hand, foot and mouth disease; Child

Sevoflurane postconditioning alleviates hypoxia-reoxygenation injury of cardiomyocytes by promoting mitochondrial autophagy through the HIF-1/BNIP3 signaling pathway.

BackgroundSevoflurane postconditioning (SpostC) can alleviate hypoxia-reoxygenation injury of cardiomyocytes; however, the specific mechanism remains unclear. This study aimed to investigate whether SpostC promotes mitochondrial autophagy through the hypoxia-inducible factor-1 (HIF-1)/BCL2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) signaling pathway to attenuate hypoxia-reoxygenation injury in cardiomyocytes.MethodsThe H9C2 cardiomyocyte hypoxia/reoxygenation model was established and treated with 2.4% sevoflurane at the beginning of reoxygenation. Cell damage was determined by measuring cell viability, lactate dehydrogenase activity, and apoptosis. Mitochondrial ultrastructural and autophagosomes were observed by transmission electron microscope. Western blotting was used to examine the expression of HIF-1, BNIP3, and Beclin-1 proteins. The effects of BNIP3 on promoting autophagy were determined using interfering RNA technology to silence BNIP3.ResultsHypoxia-reoxygenation injury led to accumulation of autophagosomes in cardiomyocytes, and cell viability was significantly reduced, which seriously damaged cells. Sevoflurane postconditioning could upregulate HIF-1α and BNIP3 protein expression, promote autophagosome clearance, and reduce cell damage. However, these protective effects were inhibited by 2-methoxyestradiol or sinBNIP3.ConclusionSevoflurane postconditioning can alleviate hypoxia-reoxygenation injury in cardiomyocytes, and this effect may be achieved by promoting mitochondrial autophagy through the HIF-1/BNIP3 signaling pathway.

Panax Notoginseng Saponins Attenuate Myocardial Ischemia-Reperfusion Injury Through the HIF-1α/BNIP3 Pathway of Autophagy.

Panax Notoginseng Saponins (PNS) is a formula of Chinese medicine commonly used for treating ischemia myocardial in China. However, its mechanism of action is yet unclear. This study investigated the effect and the mechanism of PNS on myocardial ischemia-reperfusion injury (MIRI) through the hypoxia-inducible factor 1α (HIF-1α)/bcl-2/adenovirus E1B19kDa-interacting protein3 (BNIP3) pathway of autophagy. We constructed a rat model of myocardial injury and compared among 4 groups (n = 10, each): the sham-operated group (Sham), the ischemia-reperfusion group (IR), the PNS low-dose group, and the PNS high-dose group were pretreated with PNS (30 and 60 mg/kg, respectively). Serum creatine kinase, malonaldehyde (MDA), lactate dehydrogenase, myocardial tissue superoxide dismutase, and reactive oxygen species were detected in rats with myocardial ischemia-reperfusion after the intervention of PNS. The rat myocardial tissue was examined using hematoxylin and eosin (H&E) staining, and the mitochondria of myocardial cells were observed using transmission electron microscopy. The expressions of microtubule-associated protein light chain 3 (LC3), HIF-1α, BNIP3, Beclin-1, and autophagy-related gene-5 (Atg5) in rat myocardial tissue were detected using Western blotting. The results showed that PNS was significantly protected against MIRI, as evidenced by the decreasing in the concentration of serum CK, MDA, lactate dehydrogenase, and myocardial tissue superoxide dismutase, reactive oxygen species, the attenuation of myocardial tissue histopathological changes and the mitochondrial damages of myocardial cells, and the increase of mitochondria autophagosome in myocardial cells. In addition, PNS significantly increased the expression of LC3 and the ratio of LC3II/LC3I in rat myocardial tissue. Moreover, PNS significantly increased the expression of HIF-1α, BNIP3, Atg5, and Beclin-1 in rat myocardial tissue. The protective effect of PNS on MIRI was mainly due to its ability to enhance the mitochondrial autophagy of myocardial tissue through the HIF-1α/BNIP3 pathway.

Protective effects of ginsenoside Rb1 on H2O2-induced oxidative injury in human endothelial cell line (EA.hy926) via miR-210.

Ginsenoside Rb1 (Rb1) possesses a cardioprotective effect via mediating microRNAs (miRs), while it is unexplored whether miR-210 is regulated by Rb1 in response to oxidative stress. Human endothelial EA.hy926 cells were stimulated with H2O2 before Rb1 treatment. After transfection, cell viability, apoptosis, migration, and invasion assays were conducted. Western blot was applied to quantify protein. BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) and miR-210 were analyzed with quantitative reverse transcription polymerase chain reaction. Dual luciferase activity assay was performed. Rb1 elevated viability, migration, and invasion of H2O2-treated cells. H2O2-induced apoptosis was moderated by Rb1. miR-210 was augmented in H2O2-treated cells after Rb1 stimulation. miR-210 inhibitor abolished the positive effects of Rb1. BNIP3 was negatively modulated by miR-210 and implicated in modulating viability, apoptosis, and migration and invasion. In addition, BNIP3 modulated phosphorylation of regulators. Rb1 repressed oxidative injury via elevating miR-210. miR-210 negatively mediated BNIP3, which participated in oxidative damage via regulating mammalian targets of rapamycin (mTOR) and nuclear factor-κB (NF-κB).

Estrogen Receptor Beta (ERβ) Mediated-CyclinD1 Degradation via Autophagy Plays an Anti-Proliferation Role in Colon Cells.

Dysfunction of autophagic degradation machinery causes tumorigenesis, including colorectal cancer (CRC). Overexpression of CyclinD1 in CRC has been reported. Recent evidence also suggests that ERβ deficiency is related to the pathogenesis of CRC. Very little is known, however, about the detailed molecular mechanisms underlying the relationship among ERβ, autophagy, and CyclinD1 in CRC. Here, results showed that ERβ played an anti-proliferation role in HCT116 through impairing cell cycle but not apoptosis. Additionally, CyclinD1 accumulation was increased in response to chloroquine (CQ) or in MEF Atg7 knockout cells. Further, ERβ could inhibit the mammalian target of rapamycin (mTOR) or activate Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) to promote autophagy in HCT116. In summary, these results indicate that ERβ-mediated CyclinD1 degradation can inhibit colon cancer cell growth via autophagy.

Cytoplasmic M‑CSF facilitates apoptosis resistance by inhibiting the HIF‑1α/BNIP3/Bax signalling pathway in MCF‑7 cells.

Macrophage colony‑stimulating factor (M‑CSF), a tumour marker, is related to tumour cell anti‑apoptosis and drug resistance. However, the role of M‑CSF in MCF‑7 cells is unknown. In the present study, the effect and mechanism of M‑CSF on hypoxia‑inducible factor‑1α (HIF‑1α)/BCL2/adenovirus E1B 19kDa‑interacting protein3 (BNIP3)/Apoptosis Regulator BAX signalling in human breast cancer MCF‑7 cells were investigated. Western blotting revealed that the expression of HIF‑1α, BNIP3, Bax, caspase‑3 and caspase‑9 was lower in MCF‑7‑M cells compared to MCF‑7 and MCF‑7‑C cells treated with adriamycin (ADM). Immunoprecipitation combined with western blotting was used to detect the interaction between Bcl‑2 and BNIP3 or Bax protein. MCF‑7‑M cells had a higher amount of Bax binding to Bcl‑2 compared to MCF‑7 cells or MCF‑7‑C cells, while the amount of BNIP3 binding to Bcl‑2 was decreased in MCF‑7‑M cells. Hoechst33342 staining and flow cytometry were utilized to evaluate the effect of M‑CSF on apoptosis in MCF‑7 cells treated with ADM. Compared to ADM‑treated MCF‑7 cells, the apoptotic rate of MCF‑7‑M cells was significantly decreased. These effects were dependent on the concentration of ADM. In conclusion, cytoplasmic M‑CSF suppressed apoptosis by inhibiting the HIF‑1α/BNIP3/Bax signalling pathway, which potentiated the dissociation of Bcl‑2 from Bcl‑2‑BNIP3 compounds and the formation of Bcl‑2‑Bax compounds.

Recurrent nonsevere hypoglycemia exacerbates imbalance of mitochondrial homeostasis leading to synapse injury and cognitive deficit in diabetes.

Recurrent nonsevere hypoglycemia (RH) can lead to cognitive dysfunction in patients with diabetes, although the involved mechanisms remain unclear. Here, we aimed to investigate the mechanism underlying RH-induced cognitive deficits with a focus on mitochondrial homeostasis. To establish a model that mimicked RH in patients with type 1 diabetes (T1DM) receiving insulin therapy, streptozotocin-induced mice with T1DM were subjected to recurrent, twice-weekly insulin injections over 4 wk. We found that RH disrupted the mitochondrial fine structure, reduced the number of mitochondria, and upregulated the expression of mitochondrial dynamics and mitophagy markers, including dynamin-related protein 1 (Drp1), Bcl-2/adenovirus E1B 19-kDa-interacting protein-3 (BNIP3), and microtubule-associated protein 1 light-chain 3 (LC3) in the hippocampus of T1DM mice. Moreover, RH and chronic hyperglycemia synergistically promoted the production of reactive oxygen species, impaired mitochondrial membrane potential, and suppressed mitochondrial energy metabolism. Under diabetic conditions, RH also altered the synaptic morphology and reduced the expression of synaptic marker proteins. Long-term recognition memory and spatial memory, assessed with the Morris water maze test, were also impaired. However, these effects were largely prevented by mitochondrial division inhibitor 1, a potent and selective Drp1 inhibitor. Thus, it appears that RH exacerbates the imbalance of mitochondrial homeostasis, leading to synapse injury and cognitive deficits in diabetes. The adjustment of mitochondrial homeostasis could serve as an effective neuroprotective approach when addressing low blood sugar conditions.

Expression Analysis of caspase-6, caspase-9 and BNIP3 in Prostate Cancer:

Altered regulation of cell death is a feature of human cancer. The aim of this study was to explore whether the expression of the proapoptotic proteins caspase-6, caspase-9, and Bcl-2/adenovirus E1B-19kDa-interacting protein3 (BNIP3) is altered in prostate cancers. We analyzed the expression of caspase-6, caspase-9, and BNIP3 in 107 prostate adenocarcinoma tissues by immunohistochemistry using a tissue microarray (TMA) method. Normal glandular cells expressed caspase-6 and BNIP3 proteins in 10 (9.3%) and 9 (8.4%) prostate tissues, respectively. By contrast, the prostate cancers expressed caspase-6 and BNIP3 in 65 (60.7%) and 69 (64.5%) cases, respectively. Prostate intraepithelial neoplasia (PIN) showed caspase-6 and BNIP3 expression in 65% and 65% of cases, respectively. We observed caspase-9 expression in 40 (37.4%) normal, 8 (40%) PIN, and 45 (42.1%) cancer tissues. None of the expression of caspase-6, caspase-9 or BNIP3 was associated with pathological characteristics such as tumor size, patient age, Gleason score, or tumor stage. Our data showed that prostate cancer and PIN cells display higher expression of the proapoptotic proteins caspase-6 and BNIP3 than normal cells. Neo-expression of these proteins from the PIN stage suggests that apoptosis deregulation might occur in the early stage of prostate carcinogenesis, and that altered expression of proapoptotic proteins may be a feature of prostate cancer.