Occurrence Of Autophagy Research Articles

NLRP3 Inflammasome Simultaneously Involved in Autophagy and Phagocytosis of THP-1 Cells to Clear Aged Erythrocytes.

Autophagy and phagocytosis are two important processes that capture and digest materials found in cellular interiors and exteriors, respectively. Aged red blood cells (RBCs) are cleared by phagocytes in vivo. We focused on determining whether autophagy occurs after phagocytes swallow sunset erythrocytes, and whether the degree of autophagy is related to scavenging ability of phagocytes to erythrocytes. In addition, the ability of NLR family pyrin domain containing protein 3 (NLRP3) inflammasome to regulate erythrocyte clearance by phagocytes and its association with autophagy-related protein 16-like protein 1 (ATG16L1) are confirmed. We constructed a stable and low-NLRP3 expression THP-1 cell line using CRISPR/Cas9 technology. The analysis of erythrocyte clearance and autophagy of THP-1 cells with low NLRP3 expression showed that autophagy changes together when THP-1 engulfs aged RBCs. The occurrence of autophagy was dominated by microtubule-associated protein 1A/1B-light chain 3- (LC3-) associated phagocytosis accompanied by canonical autophagy. A negative correlation exists between the clearance of RBCs by THP-1 cells and the degree of autophagy. Downregulating the expression of NLRP3 in THP-1 cells can simultaneously inhibit the scavenging ability of THP-1 to erythrocytes and the degree of autophagy. In addition, the autophagy inhibitor bafilomycin A1 (BafA1) can enhance the phagocytosis ability of THP-1 to erythrocytes and promote the NLRP3 activation in THP-1 cells, while the autophagy inducer rapamycin inhibits the phagocytosis ability of THP-1 to RBCs and downregulates the NLRP3 activation. Results showed that autophagy and phagocytosis may be dynamic balance processes that can provide sufficient nutrition and energy to cells. Choosing NLRP3 as a target may regulate the phagocytic ability and the degree of autophagy in the meantime. These findings may be a potential strategy for regulating the clearance rate of phagocytes to aged RBCs and the secretion of proinflammatory cytokines to ensure transfusion safety.

Mechanism of autophagy induced by low concentrations of chlorantraniliprole in silk gland, Bombyx mori

Chlorantraniliprole (CAP) is widely used in the control of agricultural pests, and its residues can affect the formation of silkworm (Bombyx. mori) cocoon easily. To accurately evaluate the toxicity of CAP to silkworms and clarify the mechanism of its effect on silk gland function, we proposed a novel toxicity evaluation method based on the body weight changes after CAP exposure. We also analyzed the Ca2+-related ATPase activity, characterized energy metabolism and transcriptional changes about the autophagy key genes on the downstream signaling pathways. The results showed that after a low concentration of CAP exposed for 96 h, there were CAP residues in the silk glands of B. mori, the activities of Ca2+-ATPase and Ca2+-Mg2+-ATPase decreased significantly (P ≤ 0.01), and the activation of AMPK-related genes AMPK-α and AMPK-β were up-regulated by 6.39 ± 0.02-fold and 12.33 ± 1.06-fold, respectively, reaching a significant level (P ≤ 0.01)). In addition, the autophagy-related genes Atg1, Atg6, Atg5, Atg7, and Atg8 downstream AMPK were significantly up-regulated at 96 h (P ≤ 0.05). The results of immunohistochemistry and protein expression assay for autophagy marker Atg8 further confirmed the occurrence of autophagy. Overall, our results indicate that CAP exposure leads to autophagy in the silk gland of B. mori and affects their physiological functions, which provides guidance for the evaluation of toxicity of low concentration environmental CAP residues to insects.

Boost therapy of hepatocellular carcinoma by amplifying vicious cycle between mitochondrial oxidative stress and endoplasmic reticulum stress via biodegradable ultrasmall nanoparticles and old drug

Despite advances in early diagnosis and treatment of hepatocellular carcinoma (HCC), HCC shows a very high mortality and development of innovative drugs and treatment methods are highly desirable. Herein, we report an innovative therapy of HCC through the formation and amplification of vicious cycle between mitochondrial oxidative stress and ER stress by joint use of biodegradable ultrasmall Cu2-xSe nanoparticles (CS NPs) and alcohol-abuse drug disulfiram (DSF). The CS NPs can alleviate the tumor hypoxia and generate reactive oxygen species (ROS) through their Fenton-like reaction with endogenous H2O2 in tumor. Their released copper ions can react with DSF to in situ form active drug CuET to significantly suppress the growth of HCC and exhibit excellent anti-tumor effect. We reveal the novel anti-tumor mechanism of the formed CuET complex, which can immobilize nuclear protein localization protein 4 (NPL4) to inhibit the proteasome system to induce endoplasmic reticulum (ER) stress and increase the burden of Ca2+ ions in mitochondria, leading to mitochondrial oxidative stress and the burst of ROS. The mitochondrial oxidative stress produces less ATP to induce the occurrence of autophagy, and the generated ROS further strengthens the ER stress. The formation of such vicious cycle between mitochondrial oxidative stress and ER stress significantly enhances the severe apoptosis of tumor cells and notable decrease in tumor size, which has been demonstrated in both subcutaneous and orthotopic HCC models. Our work provides insights into repurposing of metabolic nanoparticles and old drugs for new applications, demonstrates the great potential of this strategy in anti-tumor therapy.

Novel Selenoesters as a Potential Tool in Triple-Negative Breast Cancer Treatment.

Simple SummaryBreast cancer ranks at the forefront of all malignancies worldwide, and the numerous incidences and mortality rate associated with it are very burdensome to the health care system. Consequently, there is a constant need for new effective drugs with anticancer activity. There have been reports of highly cytotoxic effects of selenium compounds against cancer cells for some time. Hence, our team decided to evaluate the anticancer activity of novel selenoesters in MCF-7 and MDA-MB-231 breast cancer cells. Our results reveal that these compounds are cytotoxic at very low micromolar concentrations, which is associated with the induction of apoptosis and autophagy and arrest of breast cancer cells in the S or G2/M phase of the cell cycle. The obtained results are promising and show that selenium-containing compounds are worth more attention, as they show considerable potential for future candidates as anticancer agents.Disturbing cancer statistics, especially for breast cancer, are becoming a rationale for the development of new anticancer therapies. For the past several years, studies have been proving a greater role of selenium in the chemoprevention of many cancers than previously considered; hence, a trend to develop compounds containing this element as potential agents with anticancer activity has been set for some time. Therefore, our study aimed to evaluate the anticancer activity of novel selenoesters (EDA-71, E-NS-4) in MCF-7 and MDA-MB-231 human breast cancer cells. The assays evaluating proliferation and cell viability, and flow cytometer analysis of apoptosis/autophagy induction, changes in mitochondrial membrane potential, disruption of cell cycle phases, and protein activity of mTOR, NF-κB, cyclin E1/A2, and caspases 3/7, 8, 9, 10 were performed. The obtained results indicate that the tested selenoesters are highly cytotoxic and exhibit antiproliferative activity at low micromolar doses (<5 µM) compared with cisplatin. The most active compound—EDA-71—highly induces apoptosis, which proceeds via both pathways, as evidenced by the activation of all tested caspases. Furthermore, we observed the occurrence of autophagy (↓ mTOR levels) and cell cycle arrest in the S or G2/M phase (↓ cyclin E1, ↑ cyclin A2).

Perfluorooctanoic acid (PFOA) exposure affects early embryonic development and offspring oocyte quality via inducing mitochondrial dysfunction.

Perfluorooctanoic acid (PFOA) is a synthetic perfluorinated compound that is extensively used as an integral surfactant in commercial production. Owing to its hydrophilicity and persistence, PFOA can accumulate in living organisms and induce severe disease in animals and humans. It has been reported that PFOA exposure can affect ovarian function and induce reproductive toxicity; however, the effects and potential mechanism of PFOA exposure during gestation on early embryonic development and offspring remain unclear. This study found that PFOA exposure in vitro disrupted spindle assembly and chromosome alignment during the first cleavage of early mouse embryos, which impacted early embryonic cleavage and blastocyst formation. Moreover, PFOA exposure caused mitochondrial dysfunction and oxidative stress by inducing aberrant Ca2+ levels, liquid drops(LDs), and mitochondrial membrane potential in the 2-cell stage. Furthermore, we found that PFOA exposure resulted in DNA damage, autophagy, and apoptosis in 2-cell stage by inhibiting SOD2 function. Gestational exposure to PFOA significantly increased ovarian apoptosis and disrupted follicle development in F1 offspring. In addition, oocyte maturation competence was decreased in F1 offspring. Finally, single-cell transcriptome analysis revealed that PFOA-induced oocyte deterioration was caused by mitochondrial dysfunction and apoptosis in the F1 offspring. In summary, our results indicated that gestational exposure to PFOA had potential toxic effects on ovarian function and led to a higher incidence of meiotic defects in F1 female offspring.

Autophagy and its consequences for platelet biology

Autophagy, the continuous recycling of intracellular building blocks, molecules, and organelles is necessary to preserve cellular function and homeostasis. In this context, it was demonstrated that autophagy plays an important role in megakaryopoiesis, the development and differentiation of hematopoietic progenitor cells into megakaryocytes. Furthermore, in recent years, autophagic proteins were detected in platelets, anucleate cells generated by megakaryocytes, responsible for hemostasis, thrombosis, and a key cell in inflammation and host immune responses. In the last decade studies have indicated the occurrence of autophagy in platelets. Moreover, autophagy in platelets was subsequently demonstrated to be involved in platelet aggregation, adhesion, and thrombus formation.Here, we review the current knowledge about autophagy in platelets, its function, and clinical implications. However, at the advent of platelet autophagy research, additional discoveries derived from evolving work will be required to precisely define the contributions of autophagy in platelets, and to expand the ever increasing physiologic and pathologic roles these remarkable and versatile blood cells play.

Amlexanox-modified platinum(IV) complex triggers apoptotic and autophagic bimodal death of cancer cells

Platinum(IV) prodrugs c,c,t-[PtCl2(NH3)2(OH)(amlexanox)] (MAP) and c,c,t-[PtCl2(NH3)2(amlexanox)2] (DAP) were synthesized by reacting amlexanox with oxoplatin and characterized by NMR, HR-MS, HPLC, and elemental analysis. The complexes could be reduced to platinum(II) species and amlexanox to exert antitumor activity. Generally, MAP was more potent than DAP and cisplatin towards various human cancer cell lines; particularly, it was active in cisplatin-resistant Caov-3 ovarian cancer and A549/DDP lung cancer cells. MAP induced serious damage to DNA, remarkable change in mitochondrial morphology, decrease in mitochondrial membrane potential, release of cytochrome c from mitochondria, and up-regulation of pro-apoptotic protein Bax in Caov-3 cells, thereby leading to evident apoptosis. Meanwhile, MAP markedly promoted the autophagic flux, including affecting the expression of microtubule-associated protein light chain 3 (LC3) and autophagy adaptor protein p62 in Caov-3 cells, with an increase in the ratio of LC3-II/LC3-I and a decrease in p62, thus trigging the occurrence of autophagy. The MAP-induced bimodal cell death mode is uncommon for platinum complexes, which presents a new possibility to invent anticancer drugs with unique mechanism of action.

GFAT1-linked TAB1 glutamylation sustains p38 MAPK activation and promotes lung cancer cell survival under glucose starvation

Reprogrammed cell metabolism is deemed as one of the hallmarks of cancer. Hexosamine biosynthesis pathway (HBP) acts as an “energy sensor” in cells to regulate metabolic fluxes. Glutamine-fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme of HBP, is broadly found with elevated expression in human cancers though its exact and concrete role in tumorigenesis still remains unknown and needs further investigation. P38 mitogen-activated protein kinase (MAPK) is an important component of stress-signaling pathway and plays a critical role in cell fate decision, whereas the underlying mechanism of its activation under nutrient stress also remains elusive. In this study, we show that glucose deprivation induces the interaction of GFAT1 with transforming growth factor β-activated kinase 1 binding protein 1 (TAB1) in a TAB1 S438 phosphorylation-dependent manner. Subsequently, the binding of GFAT1 to TAB1 facilitates TTLL5–GFAT1–TAB1 complex formation, and the metabolic activity of GFAT1 for glutamate production further contributes to TTLL5-mediated TAB1 glutamylation. In consequence, TAB1 glutamylation promotes the recruitment of p38α MAPK and thus drives p38 MAPK activation. Physiologically, GFAT1-TAB1-p38 signaling promotes autophagy occurrence and thus protects tumor cell survival under glucose deficiency. Clinical analysis indicates that both GFAT1 and TAB1 S438 phosphorylation levels correlate with the poor prognosis of lung adenocarcinoma patients. These findings altogether uncover an unidentified mechanism underlying p38 MAPK signaling regulation by metabolic enzyme upon nutrient stress and provide theoretical rationality of targeting GFAT1 for cancer treatment.

Effects of Macroautophagy and Mitophagy on the Pathogenicity of Fusarium graminearum.

Fusarium graminearum is the main pathogen of Fusarium head blight (FHB), which causes huge economic losses every year. In this study, an attempt was made to control FHB from the point of view of the physiological behavior of the pathogen itself. Autophagic inhibitors and activators were used, and the pathogenicity-related indices of F.graminearum were measured. The results showed that under nitrogen-rich conditions, macroautophagy inhibition and activation greatly reduced the mycelium weight to 0.28 and 0.25 g/ml at 24 h, which were 17.82 and 24.77% lower than that of the control treatment, respectively. Mitophagy inhibition also significantly decreased the mycelium weight (P < 0.05). Conidial yield was found to be affected by factors related to autophagy occurrence. It was found that both autophagy inhibition and activation could reduce the conidiation of F.graminearum. The toxin contents in wheat medium of macroautophagy activation treatments were 0.678, 0.190, 0.402, and 0.195 μg/g when cultured for 8 and 24h under 0% N and 100% N conditions, respectively, which were significantly higher than those of the control treatments (P < 0.05). The infection length was measured to characterize the infectivity of F.graminearum, and we found that the length was short under macroautophagy activation conditions. However, mitophagy did not seem to affect the infectivity of F.graminearum. In summary, the above results indicate that macroautophagy and mitophagy inhibition could reduce the pathogenicity of F.graminearum, which may provide a new perspective for management of plant fungal diseases.

Selenium deficiency aggravates bisphenol A-induced autophagy in chicken kidney through regulation of nitric oxide and adenosine monophosphate activated protein kinase/mammalian target of rapamycin signaling pathway.

Bisphenol A (BPA), a phenolic compound, is harmful to humans and animals as its residue in the water threatens multiple organs, especially the kidney. Low selenium (Se) diets are consumed in many regions of the world, and poor Se status has exacerbating effect on toxicity of several environmental chemicals. Here, we described the discovery path of Se deficiency aggravation on autophagy in BPA treated chicken kidney through regulating nitric oxide (NO) and adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathways. The actual dietary Se intake for chickens was 0.30 mg/kg in control group and 0.03 mg/kg in Low-Se group, and BPA exposure concentration for chickens was 0.05 g/kg. Chicken embryo kidney (CEK) cells were used in vitro and the BPA exposure concentration for CEK cells was 150 nM. We found that BPA significantly increased levels of NO and inducible nitric oxide synthase, activated AMPK/mTOR signaling pathways, thereby triggering p62/LC3/Beclin1 signaling, resulting in formations of autophagosome and autolysosome, and finally stimulating autophagy in the chicken kidney. Additionally, Se deficiency promoted the occurrence of autophagy in BPA-treated kidneys. Altogether, our findings showed that Se deficiency exacerbates BPA-induced renal autophagy in chickens via regulation of NO and AMPK/mTOR signaling pathways. These findings will improve our understandings of the mechanisms of nephrotoxicity of BPA and detoxification by Se in chickens. In addition, further work is required to determine if Se status of exposed populations needs to be considered in future epidemiological assessments.

Difenoconazole causes cardiotoxicity in common carp (Cyprinus carpio): Involvement of oxidative stress, inflammation, apoptosis and autophagy

Difenoconazole, a commonly used broad-spectrum triazole fungicide, is widely applied to fish culture in paddy fields. Due to its high chemical stability, low biodegradability, and easy transfer, difenoconazole persists in aquatic systems, raising public awareness of environmental threats. Difenoconazole causes cardiotoxicity in carp, however, the potential mechanisms of difenoconazole-induced cardiotoxicity remain unclear. Here, common carp were exposed to difenoconazole, and cardiotoxicity was evaluated by measuring the creatine kinase (CK) and the lactate dehydrogenase (LDH) in the serum. Cardiac pathological injury was determined by HE staining. The content and expression of oxidative stress indicators were detected using biochemical kits and qPCR analysis. Changes in inflammation-related cytokines were examined by qPCR. Apoptosis levels were assessed by TUNEL assay and qPCR. The occurrence of autophagy was measured by western blotting detection of autophagy flux LC3II/LC3I, and autophagy regulatory pathways were detected using qPCR. The results showed that difenoconazole exposure induced cardiotoxicity accompanied by obviously elevated LDH and CK levels and caused myocardial fibers to swell and inflammatory cells to increase. Elevated peroxide MDA and reduced transcriptional and activity levels of the antioxidant enzymes CAT, SOD and GSH-Px were dependent on the Nrf2/Keap-1 pathway. Moreover, the proinflammatory cytokines IL-1β, IL-6, and TNF-α were upregulated, iNOS activity was enhanced, whereas the anti-inflammatory cytokines TGF-β1 and IL-10 were downregulated after exposure to difenoconazole. Moreover, apoptosis was observed in the TUNEL assay and mediated through the p53/Bcl-2/Bax-Caspase-9 mitochondrial pathway. Furthermore, difenoconazole increased the autophagy markers LC3II, ATG5 and p62 and regulated them through the PI3K/AKT/mTOR pathway. Altogether, this study demonstrated that difenoconazole exposure caused common carp cardiotoxicity, which is regulated by oxidative stress, inflammation, apoptosis and autophagy, providing central data for toxicological risk assessment of difenoconazole in the ecological environment.

Melatonin protects against maternal diabetes-associated meiotic defects by maintaining mitochondrial function

Maternal diabetes has been widely reported to adversely affect oocyte quality. Although various molecules and pathways may be involved in this process, strategies to prevent maternal diabetes-induced deterioration of oocyte quality remain unexplored. Melatonin is synthesized by the pineal gland and has been shown to have beneficial effects on oocyte quality owing to its antioxidative function. In the present study, we found that the exposure of oocytes of diabetic mice to melatonin, in vitro, alleviated aberrant oocyte maturation competence. Notably, melatonin supplementation attenuated defects in spindle organization and chromosome alignment by mediating the expression of TPX2 and pericentrin localization. Importantly, melatonin eliminated the accumulation of reactive oxygen species and increased the cytosolic Ca2+ levels in diabetic oocytes by maintaining mitochondrial function. Moreover, the occurrence of autophagy and apoptosis was reversed in diabetic oocytes after melatonin exposure via decreased LC3β expression. Collectively, our findings provide evidence that melatonin supplementation can protect oocytes from maternal diabetes-related meiotic defects and poor egg quality, providing a potential strategy for improving oocyte quality in assisted reproductive technologies.

Abstract 5340: GATA3-VMP1 axis induces trastuzumab resistance via promoting autophagy in HER2-positive breast cancer cells

Abstract Trastuzumab is the most important agent for HER-2 positive breast cancer both in neoadjuvant/adjuvant and metastatic settings. However, primary or acquired resistance to trastuzumab often compromises the efficacy and impairs patients’ survival. Vacuole Membrane Protein-1(VMP1), a transmembrane protein located in the endoplasmic reticulum membrane, has been reported to induce chemotherapy resistance by promoting autophagy. Our preliminary study found that the expression of GATA Binding Protein 3(GATA3) and VMP1 are positively correlated in tissue samples from HER-2 positive breast cancer patients. GATA3 and VMP1 were significantly increased in both protein and mRNA levels in trastuzumab-resistant breast cancer cell lines as compared to the sensitive counterparts. Furthermore, the expression autophagy signature protein P62 significantly decreased in trastuzumab-resistant cell lines, while the expression of autophagy activation protein ATG5 increased, indicating a close relationship between autophagy and trastuzumab resistance. In tissue samples from patients treated with neoadjuvant regiments containing trastuzumab, the expression of VMP1 significantly increased in the non-pCR (non-pathological complete response) group than the pCR group. Overexpression of VMP1 in wild-type breast cancer cells decreased their sensitivity to trastuzumab. On the contrary, when VMP1 was knocked-down in trastuzumab-resistance cells, the sensitivity of cell lines to trastuzumab was increased. Moreover, overexpressed GATA3 in wild-type HER-2 positive cell lines, the expression of VMP1 increased as well as the expression of ATG5, the signature protein of autophagy, suggesting that the transcription factor GATA3 may play a role in the transcriptional activation of VMP1. The promoter sequence was predicted, and it was found that GATA3 regulatory core sequence "TGATA" existed in front of the transcription start site from -327bp to -322bp, indicating that GATA3 may transcriptionally activate the expression of VMP1 and promote the occurrence of autophagy. TCGA (The Cancer Genome Atlas) correlation analysis showed that, both VMP1 and GATA3 are positively correlated with classical autophagy related factors ATG2B, ATG7 and BCL1, indicating VMP1 and GATA3 may be involved in the positive regulation of autophagy in HER-2 positive breast cancer. Collectively, our results suggest that GATA3 transcriptionally activates VMP1 to promote autophagy, resulting in the induction of trastuzumab resistance in HER-2 positive breast cancer. Further mechanistic study on autophagy in contribution to trastuzumab resistance is warranted. Citation Format: hao-yu Lin, Yu-cui Gu, Zhen-hao Wang, Kai-yuan Huang, He-xing Sun, De Zeng, Yuan-ke Liang. GATA3-VMP1 axis induces trastuzumab resistance via promoting autophagy in HER2-positive breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5340.

Benzene exposure causes structural and functional damage in rat ovaries: occurrence of apoptosis and autophagy.

Studies to date have provided evidence for damage that can occur from hydrocarbon benzene on different tissues/organs. However, little is known regarding the possible influence of this hydrocarbon on female reproduction. In this study, female Wistar rats were treated with low (2000ppm), middle (4000ppm), and high (8000ppm) doses of benzene by inhalation for 30min daily for 28days. Benzene exposure adversely affected ovarian function and structure by inducing histopathological changes and altering reproductive steroid hormone release. In addition, benzene-exposed ovaries exhibited increased TMR red fluorescent signals at middle and high doses, revealing significant apoptosis. Interestingly, the investigation of the autophagic protein marker LC3 showed that this protein significantly increased in all benzene-treated ovaries, indicating the occurrence of autophagy. Moreover, ovaries from benzene-treated groups exhibited differential regulation of several specific genes involved in ovarian folliculogenesis and steroidogenesis, including the INSL3, CCND1, IGF-1, CYP17a, LHR, ATG5, and GDF9 genes.

The role of TNF-α/TNFR1 inhibiting autophagy of M1-type Kupffer cells in liver injury in trichloroethylene-sensitized mice

Objective: To detect the expression levels of M1-type polarization and autophagy-related indicators in the liver of trichloroethylene (TCE) -sensitized mice, and to explore the role of liver tumor necrosis factor-α (TNF-α) and tumor necrosis factor receptor 1 (TNFR1) in regulating M1-type Kupffer cells autophagy in liver injury in TCE-sensitized mice. Methods: In November 2019, according to simple random grouping, 45 SPF grade BALB/c female mice (6-8 weeks old) were divided into 4 groups: blank control group (n=5) , solvent control group (n=5) , TCE treatment group (n=18) , TCE+R7050 (inhibitor) treatment group (n=17) . Transdermally sensitized mice, 24 h after the last challenge, the mice were divided into TCE sensitized group and TCE non-sensitized group according to the skin reaction score. The livers of mice were harvested, and the pathological changes of the livers were observed under light and electron microscopes. Western blotting was used to detect the expressions of TNF-α, TNFR1 and autophagy-related indexes. The expression of inducible nitric oxide synthase (iNOS) , a marker of M1-type Kupffer cells, was detected by immunohistochemistry, and the occurrence of autophagy in M1-type Kupffer cells was detected by immunofluorescence double-labeling method. Results: The sensitization rate of TCE treatment group was 38.9% (7/18) , and TCE+R7050 treatment group was 35.3% (6/17) , with no significant difference between the two groups (P=1.000) . Compared with the blank control group, mice in the TCE sensitized group had abnormal liver ocytes, obvious liver injury, reduced mitochondria and broken endoplasmic reticulum. Western blotting results showed that the expressions of TNF-α and TNFR1 protein in the liver of the mice in the TCE sensitized group increased, the expression of iNOS protein in M1-type Kupffer cells increased, and the expressions of autophagic microtubule-associated protein 1 light-chain 3 (LC3B) and Beclin1 protein were decreased (P<0.05) . The results of immunohistochemistry showed that iNOS was not significantly expressed in the blank control group and solvent control group, and a small amount of expression was found in the TCE non-sensitized group, the positive staining area was obvious in TCE sensitized group, and the expression of iNOS was significantly increased (P<0.05) . Immunofluorescence results showed that the iNOS protein levels in the blank control group, solvent control group and TCE non-sensitized group were lower, and only partially colocalized with P62; the colocalization of iNOS with P62 in the TCE sensitized group was significantly increased. Conclusion: TNF-α/TNFR1 signaling pathway may promote liver injury in TCE-sensitized mice by inhibiting autophagy of M1-type Kupffer cells.

Microwave radiation induces neuronal autophagy through miR-30a-5p/AMPKα2 signal pathway

The potential health hazards of microwaves have attracted much more attention. Our previous study found that 2856 MHz microwave radiation damaged synaptic plasticity and activated autophagy in neurons. However, the mechanisms underlying microwave-induced autophagy were still unclear. In the present study, we established neuronal damage models by exposing rat hippocampal neurons and rat adrenal pheochromocytoma (PC12) cell-derived neuron-like cells to 30 mW/cm2 microwaves, which resulted in miR-30a-5p (‘miR-30a’ for short) down-regulation and autophagy activation in vivo and in vitro. Bioinformatics analysis was conducted, and Beclin1, Prkaa2, Irs1, Pik3r2, Rras2, Ddit4, Gabarapl2 and autophagy-related gene 12 (Atg12) were identified as potential downstream genes of miR-30a involved in regulating autophagy. Based on our previous findings that microwave radiation could lead to abnormal energy metabolism in neurons, Prkaa2, encoding adenosine 5′-monophosphate-activated protein kinase (AMPK) α2 (AMPKα2, an important catalytic subunit of energy sensor AMPK), was selected for further analysis. Dual-luciferase reporter assay results showed that Prkaa2 was a downstream gene of miR-30a. Moreover, microwave radiation increased the expression of AMPKα2 and the phosphorylation of AMPKα (Thr172) both in vivo and in vitro. The transfection of PC12 cells with miR-30a mimics increased miR-30a levels, reduced AMPKα2 expression, suppressed AMPKα (Thr172) phosphorylation, and inhibited autophagy occurrence in neuron-like cells. Importantly, miR-30a overexpression abolished microwave-activated autophagy and inhibited microwave-induced AMPKα2 up-regulation and AMPKα (Thr172) phosphorylation. In conclusion, microwave radiation promoted the occurrence of autophagy in neurons through the miR-30a/AMPKα2 signal pathway.

VDR promotes nucleus pulposus cell mitophagy as a protective mechanism against oxidative stress injury

Intervertebral disk degeneration (IDD) is a common aging disease. Excessive apoptosis of nucleus pulposus (NP) cells has been widely considered a main contributor to IDD. Emerging science has shown that autophagy plays a protective role against apoptosis under oxidative stress. Vitamin D receptor (VDR) is a steroid hormone receptor that can regulate autophagy. The purpose of this study was to clarify whether VDR alleviates IDD by promoting autophagy. H2O2 stimulation was used to establish oxidative stress conditions. Initially, the expression level of VDR in human degenerative NP tissues was measured by immunohistochemistry. In addition, the CRISPR-dCas9-VPR system and siRNA were utilized to upregulate or downregulate VDR and Parkin expression, respectively. Autophagic and apoptotic markers were determined by Western blotting and RT–qPCR. Transmission electron microscopy was used to monitor the occurrence of autophagy in rat NP cells. VDR expression was downregulated in human degenerative NP tissues and H2O2-stimulated rat NP cells, indicating a negative correlation between VDR expression and IDD. VDR overexpression promoted mitophagy and prevented apoptosis and mitochondrial injury under oxidative stress. Additionally, mitophagy inhibition by 3-MA abolished the protective effect of VDR activation in vitro. Furthermore, VDR activation promoted mitophagy via the PINK1/Parkin pathway in H2O2-treated NP cells. This study demonstrates that VDR activation ameliorates oxidative damage and decreases NP cell apoptosis by promoting PINK1/Parkin-dependent mitophagy, indicating that VDR may serve as a promising therapeutic target in the management of IDD.

Autophagy Induced by BCL2-Related ceRNA Network Participates in the Occurrence of COPD.

PurposeChronic obstructive pulmonary disease (COPD) is a predominant cause of mortality worldwide. Autophagy, which depends on a lysosomal degradation pathway, plays an essential role in the occurrence of COPD. The aim of our study was to identify the potential function of autophagy and construct a BCL2-related competing endogenous RNA (ceRNA) network that induces autophagy in COPD.MethodsBlood sample data from GSE31568, GSE24709, and GSE61741 were collected from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs in COPD and controls were identified via GEO2R. Transcription factors were obtained from FunRich. DIANA, miRDB, miRTarBase, and TargetScan were used to predict target genes of miRNAs. Autophagy genes were collected from the Human Autophagy Database (HADb). The GSE151052 dataset was used to identify autophagy-related differentially expressed genes in tissues. Functional enrichment and protein–protein interaction (PPI) network analyses were conducted via Metascape and the STRING network. Spearman correlation analysis was used to analyze the relationship between autophagy-related differentially expressed genes and lung function. The BCL2-related ceRNA network was modeled by Cytoscape.ResultsWe obtained 41 differentially expressed miRNAs and 10 significantly different transcription factors. We identified 19 autophagy-related differentially expressed genes that were significantly different (P<0.05) in tissue samples. The most significant enrichment in Metascape was an autophagy item, which further confirmed autophagy participation in the occurrence of COPD. PPI network analysis found four genes (BCL2, BECN1, MAPK8, and ITPR1), among which BCL2 was correlated with both FEV1/FVC and FEV1 prediction. Finally, the BCL2-related ceRNA network was constructed to clarify the interaction of RNAs and occurrence of autophagy, including 18 miRNAs and 65 lncRNAs.ConclusionWe identified 19 autophagy-related differentially expressed genes that participated in COPD; among them, BCL2 was correlated with lung function, and a BCL2-related ceRNA network was constructed, which further revealed the potential mechanism of autophagy involvement in COPD.

Effect of Sirt3 on retinal pigment epithelial cells in high glucose through Foxo3a/ PINK1-Parkin pathway mediated mitophagy

Sirt3 is closely associated with mitophagy. This study aimed to investigate the effect and potential mechanism of Sirt3 on mitophagy in retinal pigment epithelium (RPE) in a high glucose environment. The expression levels of Sirt3, Foxo3a, PINK1, Parkin and LC3B in RPE subjected to high-glucose (HG, 30 mM D-glucose) conditions were detected by RT-PCR and western blotting. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining was used to detect the level of reactive oxygen species (ROS) in RPE treated with HG. MitoTracker and LysoTracker probes were used to label mitochondria and lysosomes, respectively, to observe the occurrence of autophagy. Sirt3-dependent regulation of mitophagy through the Foxo3a/PINK1-Parkin pathway was further investigated by virus transfection-mediated Sirt3 overexpression and PINK1 silencing. The effect of Sirt3 overexpression on apoptosis was detected by flow cytometry. The Sirt3 expression was decreased, the Foxo3a/PINK1-Parkin pathway was inhibited, intracellular ROS level was increased, and mitophagy was attenuated in RPE under HG condition. Sirt3 overexpression activated the Foxo3a/PINK1-Parkin signaling pathway and mitophagy, and inhibited cell apoptosis. Silencing PINK1 inhibited the effect of Sirt3 overexpression on mitophagy. In summary, Sirt3 can activate mitophagy through the Foxo3a/PINK1-Parkin pathway and reduce HG-induced apoptosis of RPE. This study provides a new direction to understand the pathogenesis and develop a potential therapeutic target for diabetic retinopathy.

Transcriptional and Post-Transcriptional Regulation of Autophagy.

Autophagy is a widely conserved process in eukaryotes that is involved in a series of physiological and pathological events, including development, immunity, neurodegenerative disease, and tumorigenesis. It is regulated by nutrient deprivation, energy stress, and other unfavorable conditions through multiple pathways. In general, autophagy is synergistically governed at the RNA and protein levels. The upstream transcription factors trigger or inhibit the expression of autophagy- or lysosome-related genes to facilitate or reduce autophagy. Moreover, a significant number of non-coding RNAs (microRNA, circRNA, and lncRNA) are reported to participate in autophagy regulation. Finally, post-transcriptional modifications, such as RNA methylation, play a key role in controlling autophagy occurrence. In this review, we summarize the progress on autophagy research regarding transcriptional regulation, which will provide the foundations and directions for future studies on this self-eating process.