BNIP3 Expression Research Articles

Association between HIF-1α, BNIP3, and autophagy in the chorionic villi of missed abortion.

To investigate the expression of autophagy mediated by the hypoxia-inducible factor 1α (HIF-1α)/BNIP3 signaling pathway in villus tissues of missed abortion and HTR-8/SVneo cells and to elucidate the association of HIF-1α and BNIP3 in autophagy of missed abortion. Villus tissues from 30 healthy women with induced abortion and 35 patients with missed abortion were collected, and HTR-8/SVneo cells were cultured under hypoxia and transfected with HIF-1α-siRNA. Real-time polymerase chain reaction was utilized to measure the mRNA levels of HIF-1α and BNIP3; Western blotting was performed to determine the protein levels of HIF-1α, BNIP3, LC3 II/I, and Beclin 1 in villus tissues and HTR-8/SVneo cells. Cellular invasion activity was detected by transwell matrigel assay. The level of autophagy was confirmed by transmission electron microscopy of autophagosome formation. The mRNA levels of HIF-1α and BNIP3 were significantly lower in the missed abortion villi than in the induced abortion samples. The protein levels of HIF-1α, BNIP3, Beclin 1, and LC3II/I were significantly decreased in villus tissues from missed abortion, and autophagosomes were significantly decreased in villus tissues from missed abortion. Under hypoxia, the mRNA expression of HIF-1α and BNIP3 was inhibited after silencing HIF-1α by RNAi, while the protein expression of HIF-1α, BNIP3, Beclin1, and LC3II/I was significantly downregulated. The number of invading cells was significantly decreased, and autophagosomes were significantly decreased after silencing HIF-1α by RNAi in HTR-8/SVneo cells. Autophagy mediated by the HIF-1α/BNIP3 signaling pathway in villous trophoblast cells may be associated with the progression and development of missed abortion.

Melatonin ameliorates acute lung injury caused by paraquat poisoning by promoting PINK1 and BNIP3 expression

Paraquat (PQ) poisoning can result in multiple organ dysfunction syndrome, mainly manifesting as acute lung injury and acute respiratory distress syndrome. No specific cure exists for PQ poisoning. However, by scavenging mitochondrial DNA (mtDNA), the damage-associated molecular pattern during PQ poisoning, mitophagy can ameliorate the downstream inflammatory pathways activated by mtDNA. Melatonin (MEL), however, can promote the expression of PINK1 and BNIP3, which are key proteins involved in mitophagy. In this study, we first explored whether MT could reduce PQ-induced acute lung injury by affecting mitophagy in animal models, and then, we studied the specific mechanism associated with this process through in vitro experiments. We also evaluated MEL intervention in the PQ group, while inhibiting the expression of PINK1 and BNIP3, to further determine whether the protective effects of MEL are associated with its effect on mitophagy. We found that when the expression of PINK1 and BNIP3 was inhibited, MEL intervention could not reduce mtDNA leakage and the release of inflammatory factors caused by PQ exposure, suggesting that the protective effect of MEL was blocked. These results suggest that by promoting the expression of PINK1 and BNIP3 and activating mitophagy, MEL can reduce mtDNA/TLR9-mediated acute lung injury during PQ poisoning. The results of this study could provide guidance for the clinical treatment of PQ poisoning to reduce associated mortality.

Abstract 1719: VHLloss enables immune checkpoint blockade therapy by boosting interferon response

Abstract Background: The von-Hippel Lindau (VHL) as a part of E3 ubiquitin ligase is a key regulator of hypoxia induced factors (HIFs). It is also characterized as a tumor suppressor gene mutated in up to 80% clear cell renal cell carcinoma (ccRCC) cases. Immune checkpoint blockade (ICB) therapy has shown improved ccRCC treatment outcomes. However, the biological roles of the VHL gene in ICB therapy are to be explored. Methods: The association between VHL gene mutation and overall survival of patients receiving ICB treatments was determined by integrating multiple human datasets. CRISPR/Cas9 was used to generate VHL knockout (KO) for in vivo tumor growth and in vitro mechanistic studies. The anti-tumor responses of genetic VHL loss in combination with anti-PD1 treatment were evaluated in three syngeneic murine tumor models. Tumor infiltrated lymphocytes and intratumoral T-cell receptor (TCR) diversity were analyzed by flow cytometry and TCR sequencing, respectively. Mitochondrial defects resulted from VHL loss were tested by JC-1 mitochondrial membrane potential assay, immunofluorescence microscopy, and qRT-PCR. VHL loss-mediated downstream signaling were further examined by western blot and bulk RNA sequencing. Results: The substantial overall survival benefits among ICB-treated patients withVHL mutations across different cancers, including RCC, in multiple human cohorts. Genetic knockout of VHL enhanced anti-PD1 therapy in three murine tumor models, including those of non-RCC origins. Moreover, VHL loss caused significant infiltration of immune effector cells and increased diversity of intratumoral TCR repertoire. VHL deficiency resulted in HIFα accumulation, increased expression of pro-mitophagy factor BNIP3, and impaired mitochondrial membrane potential that led to the cytoplasmic leakage of mitochondrial DNA (mtDNA), triggering cGAS-STING activation and type I interferon production. HIFα overexpression showed similar results as demonstrated in VHL-KO cells. In contrast, double knockdown of VHL and HIFα reversed VHL-loss induced BNIP3 upregulation, rescued mtDNA leakage to the cytoplasm, and disrupted constitutive activation of cGAS-STING signaling. Furthermore, blocking type I interferon signaling abrogated VHL-deficiency induced tumor suppression. Conclusion: Our study uncovered a novel role for VHL loss in creating an immunogenic tumor microenvironment through the activation of mtDNA-cGAS-STING pathway and type I interferon signaling in tumor cells via VHL-HIFα-BNIP3 axis, making them susceptible to ICB therapy. This finding provides a mechanistic explanation for ccRCC responsiveness to ICB therapy despite a moderate tumor mutation burden and suggests potential therapeutic strategies targeting ccRCC and other VHL-deficient tumors based on constitutive cGAS-STING and type I interferon activation. Citation Format: Meng Jiao, Xuhui Bao, Mengjie Hu, Dong Pan, Jonathan Kim, Xinjian Liu, Fang Li, Chuan-Yuan Li. VHLloss enables immune checkpoint blockade therapy by boosting interferon response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1719.

Allantopyrone A interferes with the degradation of hypoxia-inducible factor 1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Allantopyrone A is an α-pyrone metabolite that was originally isolated from the endophytic fungus Allantophomopsis lycopodina KS-97. We previously demonstrated that allantopyrone A exhibits anti-cancer, anti-inflammatory, and neuroprotective activities. In the present study, we showed that allantopyrone A up-regulated the protein expression of hypoxia-inducible factor (HIF)-1α in human fibrosarcoma HT-1080 cells. It also up-regulated the mRNA expression of BNIP3 and ENO1, but not other HIF target genes or HIF1A. Allantopyrone A did not inhibit the prolyl hydroxylation of HIF-1α, but enhanced the ubiquitination of cellular proteins. Consistent with this result, chymotrypsin-like and trypsin-like proteasome activities were reduced, but not completely inactivated by allantopyrone A. Allantopyrone A decreased the amount of proteasome catalytic subunits. Therefore, the present results showed that allantopyrone A interfered with the degradation of HIF-1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Expression of HIF1α, BNIP3, and beclin-1 in the brain of newborn and adult yaks (Bos grunniens).

As a main consumer of energy, the brain is particularly susceptible to the effects of hypoxia. However, during long-term evolution, the brain of the plateau yak developed adaptive mechanisms enabling it to maintain normal physiological conditions. A total of 20 male yaks belonging to two age groups [newborns (1-6 days old; n = 10) and adults (3-5 years old; n = 10)] were obtained, and the brain tissue was fixed and processed by standard methods. RT-qPCR, ELISA and IHC assays were used to investigate the expression and localization of HIF1α, BNIP3 and beclin-1 in the hippocampus, cerebral cortex, thalamus, medulla oblongata and cerebellum of newborn and adult yak brains and to explore their potential neuroprotective role. We found that the expression levels of HIF1α, BNIP3 and beclin-1 at the mRNA and protein levels varied in the different regions of yak brain, with the highest expression observed in the hippocampus, followed by the cerebral cortex, thalamus, medulla oblongata and the cerebellum. Moreover, the HIF1α, BNIP3 and beclin-1 expression were significantly higher in the newborn yaks' brains than in the adult yak. The IHC results showed that HIF1α, BNIP3 and beclin-1 were mainly distributed in the neurons of the cerebral cortex, hippocampus, thalamus, medulla oblongata and cerebellum. In particular, HIF1α accumulated in the nucleus and cytoplasm. Furthermore, the immunoreactivity of BNIP3 and beclin-1 was concentrated in the cytoplasm. The results indicate that the yak hippocampus and cerebral cortex may be more resistant to hypoxia than thalamus, medulla oblongata and cerebellum, and the expression of BNIP3 and beclin-1 may be regulated by HIF1α to serve a neuroprotective role in the yak's brain to adaptation to hypoxia. Additionally, the brain of adult yaks may have a higher tolerance to hypoxia than the brain of newborn yaks.

EXT418, a novel long-acting ghrelin, mitigates Lewis lung carcinoma induced cachexia in mice.

Ghrelin is a potential therapy for cachexia due to its orexigenic properties and anabolic effects on muscle and fat. However, its clinical use is limited by the short half-life of active (acylated) ghrelin (~11min in humans). EXT418 is a novel long-acting, constitutively active ghrelin analog created by covalently linking it to a vitamin D derivative. Here, we evaluated the effects and mechanisms of action of EXT418 on Lewis lung carcinoma (LLC)-induced cachexia in mice. Male C57BL/6J mice (5- to 7-month-old) were implanted with 1×106 heat-killed (HK) or live LLC cells. When the tumour was palpable, mice were injected with vehicle (T+V) or EXT418 daily (T+418 Daily, 0.25mg/kg/day) or every other day (T+418 EOD, 0.5mg/kg/EOD) for up to 14days, whereas HK-treated mice were given vehicle (HK+V). Subsets of T+418 Daily or EOD-treated mice were pair-fed to the T+V group. Body composition and grip strength were evaluated before tumour implantation and at the end of the experiment. Molecular markers were probed in muscles upon termination. In tumour-bearing mice, administration of EXT418 daily or EOD partially prevented weight loss (T+V vs. T+418 Daily, P=0.030; and vs. T+418 EOD, P=0.020). Similar effects were observed in whole body fat and lean body mass. Grip strength in tumour-bearing mice was improved by EXT418 daily (P=0.010) or EOD (P=0.008) administration compared with vehicle-treated mice. These effects of EXT418 on weight and grip strength were partially independent of food intake. EXT418 daily administration also improved type IIA (P=0.015), IIB (P=0.037) and IIX (P=0.050) fibre cross-sectional area (CSA) in tibialis anterior (TA) and EXT418 EOD improved CSA of IIB fibres in red gastrocnemius (GAS; P=0.005). In skeletal muscles, tumour-induced increases in atrogenes Fbxo32 and Trim63 were ameliorated by EXT418 treatments (TA and GAS/plantaris, PL), which were independent of food intake. EXT418 administration decreased expression of the mitophagy marker Bnip3 (GAS/PL; P≤0.010). Similar effects of EXT418 EOD were observed in p62 (GAS/PL; P=0.039). In addition, EXT418 treatments ameliorated the tumour-induced elevation in muscle Il6 transcript levels (TA and GAS/PL), independently of food intake. Il-6 transcript levels in adipose tissue and circulating IL-10 were elevated in response to the tumour but these increases were not significant with EXT418 administration. Tumour mass was not altered by EXT418. EXT418 mitigates LLC-induced cachexia by attenuating skeletal muscle inflammation, proteolysis, and mitophagy, without affecting tumour mass and partially independent of food intake.

YTHDF2 controls hexavalent chromium-induced mitophagy through modulating Hif1α and Bnip3 decay via the m6A/mRNA pathway in spermatogonial stem cells/progenitors

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, and SSC homeostasis is essential for lifelong male fertility. Currently, environmental pollution remains one of the factors affecting human reproductive health. Chromium is a prevalent metal element, and excessive exposure to hexavalent chromium (Cr (VI)) can cause male reproductive disorders. Nevertheless, the toxic effects of Cr (VI) on SSCs and the underlying mechanisms remain incompletely understood. Here, we showed that Cr (VI) exposure triggered mitophagy in mouse SSCs/progenitors in a time-dependent manner. Concurrently, Cr (VI) treatment caused reactive oxygen species (ROS) accumulation and activated the HIF1α-mediated BNIP3 expression to trigger mitophagy. In addition, Cr (VI) exposure significantly decreased the level of m6A modification. Further, we identified that YTHDF2 regulated the stability of Bnip3 and Hif1α mRNAs in an m6A-dependent manner, which was involved in Cr (VI)-induced mitophagy. Collectively, our study not only expands the mechanisms for Cr (VI)-caused male reproductive toxicity, but also provides pharmacological targets for prevention and treatment of Cr (VI)-induced male fertility impairment.

Tetramethylpyrazine Attenuates Oxygen-glucose Deprivation-induced Neuronal Damage through Inhibition of the HIF-1α/BNIP3 Pathway: from Network Pharmacological Finding to Experimental Validation

A network pharmacological analysis combined with experimental validation was used to investigate the neuroprotective mechanism of the natural product Tetramethylpyrazine(TMP). Protecting neurons is critical for acute ischemic stroke treatment. Tetramethylpyrazine is a bioactive component extracted from Chuanxiong. The neuroprotective potential of TMP has been reported, but a systematic analysis of its mechanism has not been performed. Based on the hints of network pharmacology and bioinformatics analysis, the mechanism by which TMP alleviates oxygen-glucose deprivation-induced neuronal damage through inhibition of the HIF-1α/BNIP3 pathway was verified. In this study, we initially used network pharmacology and bioinformatics analyses to elucidate the mechanisms involved in TMP's predictive targets on a system level. The HIF-1α/BNIP3 pathway mediating the cellular response to hypoxia and apoptosis was considered worthy of focus in the bioinformatic analysis. An oxygen-glucose deprivation (OGD)-induced PC12 cell injury model was established for functional and mechanical validation. Cell viability, lactate dehydrogenase leakage, intracellular reactive oxygen species, percentage of apoptotic cells, and Caspase-3 activity were determined to assess the TMP's protective effects. Transfection with siRNA/HIF-1α or pcDNA/HIF-1α plasmids to silence or overexpress hypoxia-inducible factor 1α(HIF-1α). The role of HIF-1α in OGD-injured cells was observed first. After that, TMP's regulation of the HIF-1α/BNIP3 pathway was investigated. The pcDNA3.1/HIF-1α-positive plasmids were applied in rescue experiments. The results showed that TMP dose-dependently attenuated OGD-induced cell injury. The expression levels of HIF-1α, BNIP3, and the Bax/Bcl-2 increased significantly with increasing OGD duration. Overexpression of HIF-1α decreased cell viability, increased BNIP3 expression, and Bax/Bcl-2 ratio; siRNA-HIF-1α showed the opposite effect. TMP treatment suppressed HIF-1α, BNIP3 expression, and the Bax/Bcl-2 ratio and was reversed by HIF-1α overexpression. Our study shows that TMP protects OGD-damaged PC12 cells by inhibiting the HIF-1α/BNIP3 pathway, which provides new insights into the mechanism of TMP and its neuroprotective potential.

Expression profile of the free radicals metabolism and antioxidant genes in chronic obstructive lung disease

Chronic obstructive pulmonary disease (COPD) is a multifactorial heterogeneous chronic inflammatory disease of the respiratory system. Oxidative stress plays a central role in the pathophysiology of COPD. The goal of the study is the identification of new molecular markers of COPD based on the analysis of the expression profile of genes involved to oxidative stress response and antioxidant protection in peripheral blood mononuclear cells. Methods. To identify differential gene expression in COPD we performed expression profiling of 84 oxidative stress response and antioxidant protection genes in peripheral blood mononuclear cells samples from COPD (N=10 with frequent exacerbation emphysema and bronchitis phenotype, N=10 rare exacerbation emphysema phenotype and N=10 smoking controls). RNA was isolated from PBMCs, and gene expression was assessed using RT2 Profiler PCR Arrays « Human Oxidative Stress Plus PCR Array» (Qiagen). Results. Significant changes were revealed in the expression profile of several genes in frequent exacerbation emphysema and bronchitis phenotype COPD phenotype: decreased expression of BNIP3, GCLC, PRNP, TTN, HSP90AA1 gene; and increased expression of FTH1, PRDX3, SOD2 genes. In the group of COPD patients with rare exacerbation emphysema phenotype, an increase in the transcriptional activity of most genes that play a key role in the mechanisms of cell protection from free radical damage was found: FTH1, PRDX3, SOD2, PTGR1,GPX4, GSR, PTGS1, HSPA1A, NCF1 и NCF2, PRDX5, SIRT2, SQSTM1, SRXN1, TXNRD2, LHPP. We confirmed the assumption that the blood cell transcriptome reflects the pathogenesis and systemic nature of COPD and can be considered as a source of information for assessing the severity of the disease, monitoring the effectiveness of therapy, and searching for new non-invasive biomarkers of the disease and its various phenotypes.

The Role of Apoptotic Genes and Protein-Protein Interactions in Triple-negative Breast Cancer.

Compared to other breast cancer types, triple-negative breast cancer (TNBC) has historically had few treatment alternatives. Therefore, exploring and pinpointing potentially implicated genes could be used for treating and managing TNBC. By doing this, we will provide essential data to comprehend how the genes are involved in the apoptotic pathways of the cancer cells to identify potential therapeutic targets. Analysis of a single genetic alteration may not reveal the pathogenicity driving TNBC due to the high genomic complexity and heterogeneity of TNBC. Therefore, searching through a large variety of gene interactions enabled the identification of molecular therapeutic genes. This study used integrated bioinformatics methods such as UALCAN, TNM plotter, PANTHER, GO-KEEG and PPIs to assess the gene expression, protein-protein interaction (PPI), and transcription factor interaction of apoptosis-regulated genes. Compared to normal breast tissue, gene expressions of BNIP3, TNFRSF10B, MCL1, and CASP4 were downregulated in UALCAN. At the same time, BIK, AKT1, BAD, FADD, DIABLO, and CASP9 was down-regulated in bc-GeneExMiner v4.5 mRNA expression (BCGM) databases. Based on GO term enrichment analysis, the cellular process (GO:0009987), which has about 21 apoptosis-regulated genes, is the top category in the biological processes (BP), followed by biological regulation (GO:0065007). We identified 29 differentially regulated pathways, including the p53 pathway, angiogenesis, apoptosis signaling pathway, and the Alzheimer's disease presenilin pathway. We examined the PPIs between the genes that regulate apoptosis; CASP3 and CASP9 interact with FADD, MCL1, TNF, TNFRSRF10A, and TNFRSF10; additionally, CASP3 significantly forms PPIs with CASP9, DFFA, and TP53, and CASP9 with DIABLO. In the top 10 transcription factors, the androgen receptor (AR) interacts with five apoptosis-regulated genes (p<0.0001; q<0.01), followed by retinoic acid receptor alpha (RARA) (p<0.0001; q<0.01) and ring finger protein (RNF2) (p<0.0001; q<0.01). Overall, the gene expression profile, PPIs, and the apoptosis-TF interaction findings suggest that the 27 apoptosis-regulated genes might be used as promising targets in treating and managing TNBC. Furthermore, from a total of 27 key genes, CASP2, CASP3, DAPK1, TNF, TRAF2, and TRAF3 were significantly correlated with poor overall survival in TNBC (p-value <0.05); they could play important roles in the progression of TNBC and provide attractive therapeutic targets that may offer new candidate molecules for targeted therapy. Our findings demonstrate that CASP2, CASP3, DAPK1, TNF, TRAF2, and TRAF3 were substantially associated with the overall survival rate (OS) difference of TNBC patients out of a total of 27 specific genes used in this study, which may play crucial roles in the development of TNBC and offer promising therapeutic interventions.

ET-7 EVALUATION OF HYPOXIA-TARGETING RADIOPHARMACEUTICAL64CU-ATSM FOR PET MONITORING WITH LOCAL THERAPY IN HIGH-GRADE GLIOMA MODEL

Abstract World Health Organization (WHO)-defined central nervous system (CNS) grade 4 high-grade gliomas (HGGs) are highly aggressive brain cancers characterized by the presence of hypoxia within a rapidly-growing tumor mass. Due to invasion to the surrounding brain parenchyma, these tumors commonly recur locally, despite aggressive surgical resection, and new therapeutic approaches are required for local tumor control. Here, we show a positron emission tomography (PET) integrated local therapy (PETx), to target HGGs. This technique consists of a one-step local theranostic application, followed by PET monitoring, with a hypoxia-targeting radiopharmaceutical 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM). We examined the safety and therapeutic potential of 64Cu-ATSM PETx for HGG patient-derived xenograft (PDX) tumors, which recapitulated the parent tumor phenotype of high expression of hypoxia-inducible factor-1α and BNIP3, biomarkers of tissue hypoxia. Biodistribution, dosimetry, and toxicity studies of 64Cu-ATSM local administration determined the maximum tolerated dose (MTD) to be 3.7 MBq in mouse. PETx using the MTD dose of 64Cu-ATSM indicated high tumor penetration, distribution, and retention of 64Cu-ATSM in PDX tumors, as compared to sham-treated mice. The 64Cu-ATSM PETx promoted DNA double-strand breaks, followed by apoptosis in tumors, and extensively prolonged overall survival with tolerable systemic toxicity. These findings indicate the potential of 64Cu-ATSM PETx to induce high uptake in the hypoxic tumor microenvironment, and strong therapeutic effects in PDX models. These findings establish 64Cu-ATSM PETx as a potential novel theranostic approach to facilitate local control of WHO CNS grade 4 HGGs.

4-O-Methylascochlorin-Mediated BNIP-3 Expression Controls the Balance of Apoptosis and Autophagy in Cervical Carcinoma Cells

4-O-methylascochlorin (MAC) is a 4-fourth carbon-substituted derivative of ascochlorin, a compound extracted from a phytopathogenic fungus Ascochyta viciae. MAC induces apoptosis and autophagy in various cancer cells, but the effects of MAC on apoptosis and autophagy in cervical cancer cells, as well as how the interaction between apoptosis and autophagy mediates the cellular anticancer effects are not known. Here, we investigated that MAC induced apoptotic cell death of cervical cancer cells without regulating the cell cycle and promoted autophagy by inhibiting the phosphorylation of serine-threonine kinase B (Akt), mammalian target of rapamycin (mTOR), and 70-kDa ribosomal protein S6 kinase (p70S6K). Additional investigations suggested that Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP-3), but not Hypoxia-inducible factor 1 alpha (HIF-1α), is a key regulator of MAC-induced apoptosis and autophagy. BNIP-3 siRNA suppressed MAC-induced increases in cleaved- poly (ADP-ribose) polymerase (PARP) and LC3II expression. The pan-caspase inhibitor Z-VAD-FMK suppressed MAC-induced cell death and enhanced MAC-induced autophagy. The autophagy inhibitor chloroquine (CQ) enhanced MAC-mediated cell death by increasing BNIP-3 expression. These results indicate that MAC induces apoptosis to promote cell death and stimulates autophagy to promote cell survival by increasing BNIP-3 expression. This study also showed that co-treatment of cells with MAC and CQ further enhanced the death of cervical cancer cells.

MitoTEMPOL modulates mitophagy and histopathology of Wistar rat liver after streptozotocin injection.

This study aims to explore the effect of mitoTEMPOL on histopathology, lipid droplet, and mitophagy gene expression of Wistar rat's liver after injection of streptozotocin (STZ). Twenty male Wistar rats were divided into 4 groups: Control (n=5); 100 mg/kg BW/day mitoTEMPOL orally (n=5); 50 mg/kg BW STZ intraperitoneal injection (n=5); and mitoTEMPOL+STZ (n=5). STZ was given a single dose, while mitoTEMPOL was given for 5 weeks after 1 week of STZ injection. Histopathological appearance, lipid droplets, mitophagy, and autophagy gene expression were examined after the mitoTEMPOL treatment. We found metabolic zone shifting that might be correlated with the liver activity of fatty acid oxidation in the STZ group, a decrease of lipid droplets in mitoTEMPOL and mitoTEMPOL + STZ compared with Control and STZ groups were found in this study. We also found significant changes in PINK1, Parkin, BNIP3, Mfn1, and LC3 gene expression, but no difference in Opa1, Fis1, Drp1, and p62 gene expression, suggesting a change of mitochondrial fusion rather than mitochondrial fission correlated with mitophagy. All this concluded that mitoTEMPOL could act as a modulator of mitophagy and metabolic function of the liver, thus amplifying its crucial role in preventing mitochondrial damage in the liver in the early onset of diabetes mellitus.

Grim-19 deficiency promotes decidual macrophage autophagy in recurrent spontaneous abortion.

Dysregulation of decidual macrophages leads to the occurrence of recurrent spontaneous abortion (RSA). However, the role of macrophages in RSA occurrence remains unclear. In this study, we found that the expression of Grim-19 was decreased, and the expression of autophagy related proteins Beclin1, LC3B II/I and BNIP3 was markedly upregulated in decidual macrophages of RSA patients compared with the normal pregnancy group. Furthermore, we demonstrated that downregulation of GRIM-19 increased the expression of autophagy related proteins Beclin1, LC3B II/I, BNIP3 and the proinflammatory cytokines IL1B, IL6 and TNFa in uterine mononuclear cells of GRIM-19+/- mice. The proportion of CD45+CD11b+F4/80+LC3B+ cells in GRIM-19+/- mouse uteri was significantly higher than that in WT mouse uteri. In addition, we confirmed that inhibition of Grim-19 by siRNA enhanced the expression of autophagy related proteins in RAW264.7 cells and THP-1 cells. More importantly, downregulation of Grim-19 in RAW264.7 cells promoted the release of proinflammatory cytokines and promoted phagocytic activity, which could be reversed by autophagy blockade. For THP-1-derived macrophages, the results of RNA-seq suggested that Grim-19 mainly modulates immune and inflammatory-related pathways, leading to cytokine production, and thus contributing to inflammation. Therefore, our data reveal that Grim-19 deficiency influences macrophage function, characterized by enhanced proinflammatory cytokines and phagocytic activity, and this might be regulated by autophagy. This may represent a novel mechanism for the occurrence of RSA.

Lipid droplet turnover at the lysosome inhibits growth of hepatocellular carcinoma in a BNIP3-dependent manner.

Hepatic steatosis is a major etiological factor in hepatocellular carcinoma (HCC), but factors causing lipid accumulation leading to HCC are not understood. We identify BNIP3 (a mitochondrial cargo receptor) as an HCC suppressor that mitigates against lipid accumulation to attenuate tumor cell growth. Targeted deletion of Bnip3 decreased tumor latency and increased tumor burden in a mouse model of HCC. This was associated with increased lipid in bnip3−/− HCC at early stages of disease, while lipid did not accumulate until later in tumorigenesis in wild-type mice, as Bnip3 expression was attenuated. Low BNIP3 expression in human HCC similarly correlated with increased lipid content and worse prognosis than HCC expressing high BNIP3. BNIP3 suppressed HCC cell growth by promoting lipid droplet turnover at the lysosome in a manner dependent on BNIP3 binding LC3. We have termed this process “mitolipophagy” because it involves the coordinated autophagic degradation of lipid droplets with mitochondria.

Thiamine insufficiency induces Hypoxia Inducible Factor-1α as an upstream mediator for neurotoxicity and AD-like pathology

Insufficiencies of the micronutrient thiamine (Vitamin B1) have been associated with inducing Alzheimer's disease (AD)-like neuropathology. The hypometabolic state associated with chronic thiamine insufficiency (TI) has been demonstrated to be a contributor towards the development of amyloid plaque deposition and neurotoxicity. However, the molecular mechanism underlying TI induced AD pathology is still unresolved. Previously, we have established that TI stabilizes the metabolic stress transcriptional factor, Hypoxia Inducible Factor-1α (HIF1α). Utilizing neuronal hippocampal cells (HT22), TI-induced HIF1α activation triggered the amyloidogenic cascade through transcriptional expression and increased activity of β-secretase (BACE1). Knockdown and pharmacological inhibition of HIF1α during TI significantly reduced BACE1 and C-terminal Fragment of 99 amino acids (C99) formation. TI also increased the expression of the HIF1α regulated pro-apoptotic protein, BCL2/adenovirus E1B 19 kDa protein-interacting protein (BNIP3). Correspondingly, cell toxicity during TI conditions was significantly reduced with HIF1α and BNIP3 knockdown. The role of BNIP3 in TI-mediated toxicity was further highlighted by localization of dimeric BNIP3 into the mitochondria and nuclear accumulation of Endonuclease G. Subsequently, TI decreased mitochondrial membrane potential and enhanced chromatin fragmentation. However, cell toxicity via the HIF1α/BNIP3 cascade required TI induced oxidative stress. HIF1α, BACE1 and BNIP3 expression was induced in 3xTg-AD mice after TI and administration with the HIF1α inhibitor YC1 significantly attenuated HIF1α and target genes levels in vivo. Overall, these findings demonstrate a critical stress response during TI involving the induction of HIF1α transcriptional activity that directly promotes neurotoxicity and AD-like pathology.

Swimming exercise and nano-l-arginine supplementation improve oxidative capacity and some autophagy-related genes in the soleus muscle of aging rats

The present research aims to evaluate the effect of swimming exercise and chitosan-coated l-arginine on mitochondrial oxidation, BCL2 Interacting Protein 3 (Bnip3), NIP-like protein × (Nix), B-cell lymphoma-extra-large (Bcl-xL) and autophagy-related protein light chain 3(LC3) expression in soleus muscle of aging rats. In this experimental research, 25 male Wistar rats were assigned into five groups randomly: young, old, old + Nano l-arginine (Nano L-a), old + exercise (Ex), and old + Nano l-arginine (Nano L-a) + exercise (Ex) (n = 5 in each). They performed a swimming exercise program five days a week for six weeks. To determine the relative strength for rats before and after performing these interventions, the 1repetition maximum (1RM) test was done as a pre and post-test. The exercise program started with 20 min and after four sessions, gradually increased to 60 min and this time was maintained until the completion of the training period. l-arginine coated with chitosan nanoparticles was given to the rats in the l-arginine-supplemented group via gavage at a dosage of 500 mg/kg/day, five days a week, for six weeks. Additionally, the rats in all groups were fed a normal diet (2.87 kcal/g and 15 % energy from fat). Upon the completion of the protocol implementation, the rats were sacrificed and the soleus muscle was fixed and frozen to determine hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), gene expression analysis, levels of reactive oxygen species (ROS), and total antioxidant capacity (TAC). The results from the present research indicated that swimming exercise and Nano l-arginine improve the strength and histology of muscle tissue in old rats (p < 0.05). Aging significantly increased the expression of Nix and Bnip3 (p < 0.05) and reduced the Bcl-xL gene expression (p < 0.05). The expression of LC3 protein also increased with aging (p < 0.05). Therapeutic interventions, such as combined treatment (old + Nano L-a + Ex) for old animals, reduced the amount of this protein in soleus muscle (p < 0.05). The ROS values also showed a significant reduction only in the old + Nano L-a + Ex group compared to the old group. Moreover, TAC values show a significant decrease in the old and old + Ex groups in comparison to the young group. The use of arginine supplement, especially in nano form, along with swimming exercise seems to reduce the oxidative damage to the elderly muscle tissue, which has a positive effect on the structure and function of the soleus muscle. Since these interventions only had a significant effect on LC3 protein, further studies with more diverse measurement methods for autophagy are suggested.

Autophagy Is Essential for Neural Stem Cell Proliferation Promoted by Hypoxia.

Hypoxia as a microenvironment or niche stimulates proliferation of neural stem cells (NSCs). However, the underlying mechanisms remain elusive. Autophagy is a protective mechanism by which recycled cellular components and energy are rapidly supplied to the cell under stress. Whether autophagy mediates the proliferation of NSCs under hypoxia and how hypoxia induces autophagy remain unclear. Here, we report that hypoxia facilitates embryonic NSC proliferation through HIF-1/mTORC1 signaling pathway-mediated autophagy. Initially, we found that hypoxia greatly induced autophagy in NSCs, while inhibition of autophagy severely impeded the proliferation of NSCs in hypoxia conditions. Next, we demonstrated that the hypoxia core regulator HIF-1 was necessary and sufficient for autophagy induction in NSCs. Considering that mTORC1 is a key switch that suppresses autophagy, we subsequently analyzed the effect of HIF-1 on mTORC1 activity. Our results showed that the mTORC1 activity was negatively regulated by HIF-1. Finally, we provided evidence that HIF-1 regulated mTORC1 activity via its downstream target gene BNIP3. The increased expression of BNIP3 under hypoxia enhanced autophagy activity and proliferation of NSCs, which was mediated by repressing the activity of mTORC1. We further illustrated that BNIP3 can interact with Rheb, a canonical activator of mTORC1. Thus, we suppose that the interaction of BNIP3 with Rheb reduces the regulation of Rheb toward mTORC1 activity, which relieves the suppression of mTORC1 on autophagy, thereby promoting the rapid proliferation of NSCs. Altogether, this study identified a new HIF-1/BNIP3-Rheb/mTORC1 signaling axis, which regulates the NSC proliferation under hypoxia through induction of autophagy.

Establishment of the first cell line from the small yellow croaker (Larimichthys polyactis) and its application in unraveling the mechanism of ROS-induced apoptosis under hypoxia

Cell lines play an important role in studying biological processes in vitro. In this study, we aimed to establish and characterize the first cell line isolated from the small yellow croaker (Larimichthys polyactis), an economically important marine fish in China. The small yellow croaker fry (SYCF) cells proliferated well in Leibovitz's 15 medium containing 2% antibiotics, 0.1 M NaCl, and 10% fetal bovine serum at 27 °C. Sequence analyses of mitochondrial genes confirmed the origin of SYCF cells. Furthermore, we unraveled the mechanism of hypoxia-induced apoptosis, a major threat to aquatic animals using the SYCF cell line. The HIF-PHD pathway of the SYCF cells can be activated under hypoxic stress, suggesting that this cell line is impressible for hypoxia and can serve as a superb model in vitro to study the cell physiology in L. polyactis in a hypoxic setting. We found that hypoxia results in morphological abnormalities, excess reactive oxygen species (ROS) production, and dysfunctional redox homeostasis, indicating the occurrence of oxidative stress in the SYCF cells. In addition, decreased cell viability and severe apoptosis were detected under hypoxic conditions. Through monitoring the indicators of the intrinsic and extrinsic apoptosis pathways, including the mitochondrial membrane potential, the expression of bax, bcl2, bnip3, and fas, Bcl2, and Caspase-3 protein, and activity of Caspases-3/8/9, we found that both pathways are involved. Considering that ROS-induced oxidative stress may be the main reason of apoptosis, we investigated whether the addition of an antioxidant, N-Acetyl-L-cysteine (NAC), will ameliorate the apoptosis. Interestingly, we found that both intrinsic and extrinsic apoptosis indicators were markedly recovered by adding NAC in culture media, significant remissions were also observed in the cell viability and apoptosis rate. These results suggested that hypoxia-induced apoptosis of SYCF cells may be mediated by the overproduction of ROS, and the antioxidant may be a useful material to relieve cell damage under hypoxia in fish. In general, this study established a marine fish cell line of SYCF cells, which can be used as an in vitro research model of L. polyactis and for revealing the mechanisms of ROS-induced apoptosis under hypoxic conditions in this species. This provides important insights into the cellular physiology of marine fish under hypoxia.

BNIP3 enhances pancreatic cancer cell migration and proliferation via modulating autophagy under hypoxia

Chemotherapy and immunotherapy for pancreatic ductal adenocarcinoma (PDAC) have limited success for the intricated surrounding cancer microenvironment. Hypoxic microenvironment in PDAC causes the activation of multiple different molecules and signaling pathways compared with normoxia. We studied the roles of BNIP3 for the migration and proliferation of PDAC and Panc1 cells in vitro. In the present study, we found that BNIP3 expression was elevated and enhanced the migration and proliferation of CFPAC-1 and Panc1 cells under hypoxia. The upregulation of BNIP3 was important for the autophagic activation, while inhibition of autophagy with siRNA targeting Atg5 and Atg7 impaired the hypoxia-induced cell migration and proliferation. Additionally, blocking ERK1/2 mitogen-activated protein kinase (MAPK) signaling with PD98058 significantly down-regulated BNIP3 expression, autophagic activation, as well as the migration and proliferation of CFPAC-1 and Panc1 cells under hypoxia. Collectively, our results here uncover a hitherto unknown hypoxia-BNIP3-autophagy axis in modulating the migration and proliferation and provide a potential intriguing drug target for the therapy of PDAC.