Curcumin protects against aging-related stress and dysfunction through autophagy activation in rat brain.

BACKGROUND: Curcumin (Curcuma longa) is a well-known medicinal plant that induces autophagy in various model species, helping maintain cellular homeostasis. Its role as a caloric restriction mimetic (CRM) is being investigated. This study explores the potential of curcumin (CUR), as a CRM, to provide neuroprotection in D galactose induced accelerated senescence model of rats through modulation of autophagy. For six weeks, male rats received simultaneous supplementation of D-gal (300 mg/kg b.w., subcutaneously) and CUR (200 mg/kg b.w., oral). METHOD AND RESULTS: The oxidative stress indices, antioxidants, and electron transport chain complexes in brain tissues were measured using standard methods. Reverse transcriptase-polymerase chain reaction (RT-PCR) gene expression analysis was used to evaluate the expression of autophagy, neuroprotection, and aging marker genes. Our results show that curcumin significantly (p ≤ 0.05) enhanced the level of antioxidants and considerably lowered the level of oxidative stress markers. Supplementing with CUR also increased the activity of electron transport chain complexes in the mitochondria of aged brain tissue, demonstrating the antioxidant potential of CUR at the mitochondrial level. CUR was found to upregulate the expression of the aging marker gene (SIRT-1) and the genes associated with autophagy (Beclin-1 and ULK-1), as well as neuroprotection (NSE) in the brain. The expression of IL-6 and TNF-α was downregulated. CONCLUSION: Our findings demonstrate that CUR suppresses oxidative damage brought on by aging by modulating autophagy. These findings imply that curcumin might be beneficial for neuroprotection in aging and age-related disorders.

A study on the mechanism of Beclin-1 m6A modification mediated by catalpol in protection against neuronal injury and autophagy following cerebral ischemia.

OBJECTIVE: Catalpol (CAT) has various pharmacological activities and plays a protective role in cerebral ischemia. It has been reported that CAT played a protective role in cerebral ischemia by upregulaing NRF1 expression. Bioinformatics analysis reveals that NRF1 can be used as a transcription factor to bind to the histone acetyltransferase KAT2A. However, the role of KAT2A in cerebral ischemia remains to be studied. Therefore, we aimed to investigate the role of CAT in cerebral ischemia and its related mechanism. METHODS: In vitro, a cell model of oxygen and glucose deprivation/reperfusion (OGD/R) was constructed, followed by evaluation of neuronal injury and the expression of METTL3, Beclin-1, NRF1, and KAT2A. In vivo, a MCAO rat model was prepared by means of focal cerebral ischemia, followed by assessment of neurological deficit and brain injury in MCAO rats. Neuronal autophagy was evaluated by observation of autophagosomes in neurons or brain tissues by TEM and detection of the expression of LC3 and p62. RESULTS: In vivo, CAT reduced the neurological function deficit and infarct volume, inhibited neuronal apoptosis in the cerebral cortex, and significantly improved neuronal injury and excessive autophagy in MCAO rats. In vitro, CAT restored OGD/R-inhibited cell viability, inhibited cell apoptosis, LDH release, and neuronal autophagy. Mechanistically, CAT upregulated NRF1, NRF1 activated METTL3 via KAT2A transcription, and METTL3 inhibited Beclin-1 via m6A modification. CONCLUSION: CAT activated the NRF1/KAT2A/METTL3 axis and downregulated Beclin-1 expression, thus relieving neuronal injury and excessive autophagy after cerebral ischemia.

BECN1 mRNA expression in breast cancer tissue; significant correlation to tumor grade.

Breast cancer (BC) is a heterogenous disease with multiple pathways implicated in its development, progression, and drug resistance. Autophagy, a cellular process responsible for self-digestion of damaged organelles, had been recognized as eminent player in cancer progression and chemotherapeutic resistance. The haploinsufficiency of Beclin 1 (BECN1), autophagy protein, is believed to contribute to cancer pathogenesis and progression. In our study, we investigated the expression of BECN1 in a BC female Egyptian patient cohort, as well as its prognostic role through evaluating its association with disease free survival (DFS) after 2 years follow up and association of tumor clinicopathological features. Twenty frozen female BC tissue samples and 17 adjacent normal tissue were included and examined for the expression levels of BECN1. Although the tumor tissues showed lower expression 0.73 (0-8.95) than their corresponding normal tissues 1.02 (0.04-19.59), it was not statistically significant, p: 0.463. BECN1 expression was not associated with stage, nodal metastasis or tumor size, p:0.435, 0.541, 0.296, respectively. However, statistically significant negative correlation was found between grade and BECN1 mRNA expression in the studied cases, p:0.028. BECN1 expression had no statistically significant association with DFS, P = 0.944. However, we observed that triple negative (TNBC) cases had significantly lower DFS rate than luminal BC patients, p: 0.022, with mean DFS 19.0 months, while luminal BC patients had mean DFS of 23.41 months. Our study highlights the potential role of BECN1 in BC pathogenesis, showing that BECN1 expression correlates with poorer differentiation of BC, indicating its probable link with disease aggressiveness. DFS two years follow up showed that TNBC subtype remains associated with less favorable prognosis.

OPN silencing reduces hypoxic pulmonary hypertension via PI3K-AKT-induced protective autophagy.

Hypoxic pulmonary hypertension (HPH) is a pulmonary vascular disease primarily characterized by progressive pulmonary vascular remodeling in a hypoxic environment, posing a significant clinical challenge. Leveraging data from the Gene Expression Omnibus (GEO) and human autophagy-specific databases, osteopontin (OPN) emerged as a differentially expressed gene, upregulated in cardiovascular diseases such as pulmonary arterial hypertension (PAH). Despite this association, the precise mechanism by which OPN regulates autophagy in HPH remains unclear, prompting the focus of this study. Through biosignature analysis, we observed significant alterations in the PI3K-AKT signaling pathway in PAH-associated autophagy. Subsequently, we utilized an animal model of OPNfl/fl-TAGLN-Cre mice and PASMCs with OPN shRNA to validate these findings. Our results revealed right ventricular hypertrophy and elevated mean pulmonary arterial pressure (mPAP) in hypoxic pulmonary hypertension model mice. Notably, these effects were attenuated in conditionally deleted OPN-knockout mice or OPN-silenced hypoxic PASMCs. Furthermore, hypoxic PASMCs with OPN shRNA exhibited increased autophagy compared to those in hypoxia alone. Consistent findings from in vivo and in vitro experiments indicated that OPN inhibition during hypoxia reduced PI3K expression while increasing LC3B and Beclin1 expression. Similarly, PASMCs exposed to hypoxia and PI3K inhibitors had higher expression levels of LC3B and Beclin1 and suppressed AKT expression. Based on these findings, our study suggests that OPNfl/fl-TAGLN-Cre effectively alleviates HPH, potentially through OPN-mediated inhibition of autophagy, thereby promoting PASMCs proliferation via the PI3K-AKT signaling pathway. Consequently, OPN emerges as a novel therapeutic target for HPH.

TRIM27-Induced Protective Autophagy: A Novel Therapeutic Approach for Pneumonia.

BACKGROUND: Pneumonia is a prevalent respiratory ailment involving complex physiological and pathological mechanisms. The tripartite motif containing 27 (TRIM27) plays a crucial role in regulating inflammation mechanisms. Therefore, the purpose of this study is to further explore the therapeutic potential of TRIM27 in pneumonia, based on its regulatory mechanisms in inflammation and autophagy. METHODS: This study established a mouse pneumonia animal model through lipopolysaccharide (LPS) administration, designating it as the LPS model group. Subsequently, adenovirus-mediated TRIM27 overexpression was implemented in the animals of the LPS model group, creating the TRIM27 treatment group. After a 7-day treatment period, lung tissues from the mice were collected. Various techniques, including immunohistochemistry, quantitative reverse transcription PCR (RT-qPCR), western blot, enzyme-linked immunosorbent assay (ELISA), and electron microscopy were utilized to analyze the impact of TRIM27 overexpression on inflammatory factors, oxidative stress, autophagy, and inflammatory processes in pulmonary tissues. Finally, an in vitro LPS cell model was established, and the effects of TRIM27 overexpression and autophagy inhibition on inflammatory cytokines and autophagosomes in LPS-induced inflammatory cells were examined through RT-qPCR and immunofluorescence techniques. RESULTS: The research findings demonstrate a significant reduction in the elevated levels of interleukin-6 (IL-6), IL-1ß, and Tumor necrosis factor-alpha (TNF-α) induced by LPS with TRIM27 overexpression (p < 0.01). Conversely, the autophagy inhibitor 3-Methyladenine (3-MA) diminished the effects induced by TRIM27 overexpression. Moreover, TRIM27 overexpression enhanced the expression of Microtubule-associated protein 1A/1B light chain 3 (LC3) II/I and Beclin-1 proteins in mice subjected to LPS stimulation (p < 0.01), while reducing the expression of the p62 protein (p < 0.01). The addition of 3-MA, however, decreased Beclin-1 expression and inhibited autophagy (p < 0.01). Additionally, TRIM27 overexpression decreased the expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cleaved caspase-1, IL-1ß, and Gasdermin D N-terminal fragment (GSDMD-N) proteins in LPS-stimulated mice (p < 0.05). TRIM27 overexpression also decreased the levels of malondialdehyde (MDA), Activating Transcription Factor 6 (ATF6), and C/EBP-homologous protein (CHOP), while increasing the levels of superoxide dismutase (SOD) and glutathione (GSH) in mice exposed to LPS (p < 0.01). CONCLUSION: The induction of TRIM27 overexpression emerges as a potential and effective pneumonia treatment. The underlying mechanism may involve inducing protective autophagy, thereby reducing oxidative stress and cell pyroptosis.

Beyond self­eating: Emerging autophagy­independent functions for the autophagy molecules in cancer (Review).

Autophagy is a conserved catabolic process that controls organelle quality, removes misfolded or abnormally aggregated proteins and is part of the defense mechanisms against intracellular pathogens. Autophagy contributes to the suppression of tumor initiation by promoting genome stability, cellular integrity, redox balance and proteostasis. On the other hand, once a tumor is established, autophagy can support cancer cell survival and promote epithelial­to­mesenchymal transition. A growing number of molecules involved in autophagy have been identified. In addition to their key canonical activity, several of these molecules, such as ATG5, ATG12 and Beclin­1, also exert autophagy­independent functions in a variety of biological processes. The present review aimed to summarize autophagy­independent functions of molecules of the autophagy machinery and how the activity of these molecules can influence signaling pathways that are deregulated in cancer progression.

Endothelial HIFα/PDGF-B to smooth muscle Beclin1 signaling sustains pathological muscularization in pulmonary hypertension.

Mechanisms underlying maintenance of pathological vascular hypermuscularization are poorly delineated. Herein, we investigated retention of smooth muscle cells (SMCs) coating normally unmuscularized distal pulmonary arterioles in pulmonary hypertension (PH) mediated by chronic hypoxia with or without Sugen 5416, and reversal of this pathology. With hypoxia in mice or culture, lung endothelial cells (ECs) upregulated hypoxia-inducible factor 1α (HIF1-α) and HIF2-α, which induce platelet-derived growth factor B (PDGF-B), and these factors were reduced to normoxic levels with re-normoxia. Re-normoxia reversed hypoxia-induced pulmonary vascular remodeling, but with EC HIFα overexpression during re-normoxia, pathological changes persisted. Conversely, after establishment of distal muscularization and PH, EC-specific deletion of Hif1a, Hif2a, or Pdgfb induced reversal. In human idiopathic pulmonary artery hypertension, HIF1-α, HIF2-α, PDGF-B, and autophagy-mediating gene products, including Beclin1, were upregulated in pulmonary artery SMCs and/or lung lysates. Furthermore, in mice, hypoxia-induced EC-derived PDGF-B upregulated Beclin1 in distal arteriole SMCs, and after distal muscularization was established, re-normoxia, EC Pdgfb deletion, or treatment with STI571 (which inhibits PDGF receptors) downregulated SMC Beclin1 and other autophagy products. Finally, SMC-specific Becn1 deletion induced apoptosis, reversing distal muscularization and PH mediated by hypoxia with or without Sugen 5416. Thus, chronic hypoxia induction of the HIFα/PDGF-B axis in ECs is required for non-cell-autonomous Beclin1-mediated survival of pathological distal arteriole SMCs.

Infectious bronchitis virus (IBV) triggers autophagy to enhance viral replication by activating the VPS34 complex.

Autophagy plays an important role in the lifecycle of viruses. However, there is currently a lack of systematic research on the relationship between Infectious Bronchitis Virus (IBV) and autophagy. This study aims to investigate the impact of IBV on autophagy and the role of autophagy in viral replication. We observed that IBV infection increased the expression of microtubule-associated protein 1 light chain 3, a marker of autophagy, decreased the expression of sequestosome 1, and led to elevated intracellular LC3 puncta levels. These findings suggest that IBV infection activates the autophagic process in cells. To investigate the impact of autophagy on the replication of IBV, we utilized rapamycin as an autophagy activator and 3-methyladenine as an autophagy inhibitor. Our results indicate that IBV promotes viral replication by inducing autophagy. Further investigation revealed that IBV induces autophagosome formation by inhibiting the mTOR-ULK1 pathway and activating the activity of vacuolar protein sorting 34 (VPS34), autophagy-related gene 14, and the Beclin-1 complex. VPS34 plays a crucial role in this process, as inhibiting VPS34 protein activity enhances cell proliferation after IBV infection. Additionally, inhibiting VPS34 significantly improves the survival rate of IBV-infected chicks, suppresses IBV replication in the kidney, and alleviates tracheal, lung, and kidney damage caused by IBV infection. In summary, IBV infection can induce autophagy by modulating the mTOR/ULK1 signaling pathway and activating the VPS34 complex, while autophagy serves to promote virus replication.

Correlation between inflammatory factors, autophagy protein levels, and infection in granulation tissue of diabetic foot ulcer.

OBJECTIVE: To observe the expression of inflammatory factors and autophagy-related proteins in granulation tissue of diabetic foot ulcer (DFU) patients and analyze their relationship with infection. METHODS: This is a retrospective cohort study. One hundred and fifty-two patients with DFU in our hospital from July 2020 to March 2022 were selected as the DFU group, including 98 cases in infection stage group and 54 cases in infection control group. The patients were further graded as the mild (51 cases), the moderate (65 cases), and the severe infection group (36 cases) according to the Wagner grading criteria. Sixty-seven patients with foot burns during the same period were selected as the control group. The distribution of pathogenic bacteria on the ulcer surface was examined using fully automated bacterial analyzer. The expression of inflammatory factors (procalcitonin [PCT], tumor necrosis factor-α [TNF-α], and interleukin-6 [IL-6]) was valued by real-time fluorescence quantitative PCR (qRT-PCR). Protein expression was measured by immunohistochemistry (IHC). The correlation was analyzed by Pearson. RESULTS: The surface infection of DFU patients was mostly induced by gram-negative and gram-positive bacteria, with Pseudomonas aeruginosa predominating among the Gram-negative bacteria and Staphylococcus aureus among the gram-positive bacteria. The infection stage group had higher content of PCT, TNF-α, and IL-6 and lower content of Beclin-1 and LC3 than the infection control group (p < .001). The levels of PCT, TNF-α, and IL-6 in the DFU patients with cardiovascular events were higher than those in the nonoccurrence group (p < .001). Glycated hemoglobin in patients with DFU was positively correlated with PCT, TNF-α, and IL-6 levels (p < .05), and negatively correlated with Beclin-1 and LC3 levels (p < .001). CONCLUSION: P. aeruginosa and S. aureus were predominant bacterial in DFU infections. Inflammatory factor and autophagy protein expression were closely correlated with the degree of infection.

Arabidopsis BECLIN1-induced autophagy mediates reprogramming in tapetal programmed cell death by altering the gross cellular homeostasis.

In flowering plants, the tapetum degeneration in post-meiotic anther occurs through developmental programmed cell death (dPCD), which is one of the most critical and sensitive steps for the proper development of male gametophytes and fertility. Yet the pathways of dPCD, its regulation, and its interaction with autophagy remain elusive. Here, we report that high-level expression of Arabidopsis autophagy-related gene BECLIN1 (BECN1 or AtATG6) in the tobacco tapetum prior to their dPCD resulted in developmental defects. BECN1 induces severe autophagy and multiple cytoplasm-to-vacuole pathways, which alters tapetal cell reactive oxygen species (ROS)-homeostasis that represses the tapetal dPCD. The transcriptome analysis reveals that BECN1- expression caused major changes in the pathway, resulting in altered cellular homeostasis in the tapetal cell. Moreover, BECN1-mediated autophagy reprograms the execution of tapetal PCD by altering the expression of the key developmental PCD marker genes: SCPL48, CEP1, DMP4, BFN1, MC9, EXI1, and Bcl-2 member BAG5, and BAG6. This study demonstrates that BECN1-mediated autophagy is inhibitory to the dPCD of the tapetum, but the severity of autophagy leads to autophagic death in the later stages. The delayed and altered mode of tapetal degeneration resulted in male sterility.

[Effect of Bushen Huoxue（） recipe on autophagy of ovariectomized rat chondrocytes based on Akt/mTOR signaling pathway].

OBJECTIVE: To investigate whether Bushen Huoxue recipe can protect articular cartilage by regulating Akt/mTOR signaling pathway to promote the autophagy of chondrocytes in ovariectomized rats. METHODS: Among 30 SPF 12-week-old female SD rats weighing （247.0±7.0） g, 6 were randomly selected as the blank control group, and the remaining rats were randomly divided into model group, BSHXR-L group, BSHXR-M group and BSHXR-H group, with 6 rats in each group. The protective effect of Bushen Huoxue recipe on articular cartilage injury in rats was determined by visual observation score, muscovine O-solid green staining and immunohistochemistry. The expression of autophagy related proteins was detected by Western-blot, and the relative expression of Akt, mTOR and downstream autophagy genes was detected by qPCR. RESULTS: After modeling, BSHXR （L, M, H） groups could alleviate the histological damage of cartilage. Immunohistochemistry showed that the expression of Collagen-Ⅱand Aggrecan gradually increased, and the expression of MMP-13 gradually decreased, and the differences between BSHXR-M and BSHXR-H groups and model group were statistically significant （P<0.05）. The results of Western-blot showed that the autophagy pathway proteins p-Akt/Akt and p-mTOR/mTOR were inhibited in the BSHXR（L, M, H） groups, and the expressions of downstream proteins Beclin-1 and LC3Ⅱwere gradually increased, while p62 was gradually decreased, showing a dose effect. QPCR results showed that BSHXR（L, M, H） groups could promote the relative expression of Beclin-1 and LC3ⅡmRNA, and inhibit the relative expression of p62, Akt, mTOR mRNA, and the differences were statistically significant compared with model group （P<0.05）. CONCLUSION: Bushen Huoxue recipe can enhance the cartilage autophagy response by inhibiting the Akt/mTOR signaling pathway, and then protect the cartilage.

Interleukin-22 Alleviates Caerulein-Induced Acute Pancreatitis by Activating AKT/mTOR Pathway.

BACKGROUND: Acute pancreatitis (AP) is one of the most common acute abdominal disorders; due to the lack of specific treatment, the treatment of acute pancreatitis, especially serious acute pancreatitis (SAP), is difficult and challenging. We will observe the changes of Interleukin -22 levels in acute pancreatitis animal models, and explore the mechanism of Interleukin -22 in acute pancreatitis. OBJECTIVE: This study aims to assess the potential protective effect of Interleukin -22 on caerulein-induced acute pancreatitis and to explore its mechanism. METHODS: Blood levels of amylase and lipase and Interleukin -22 were assessed in mice with acute pancreatitis. In animal model and cell model of caerulein-induced acute pancreatitis, the mRNA levels of P62 and Beclin-1 were determined using PCR, and the protein expression of P62, LC3-II, mTOR, AKT, p-mTOR, and p-AKT were evaluated through Western blot analysis. RESULTS: Interleukin -22 administration reduced blood amylase and lipase levels and mitigated tissue damage in acute pancreatitis mice model. Interleukin -22 inhibited the relative mRNA levels of P62 and Beclin-1, and the Interleukin -22 group showed a decreased protein expression of LC3-II and P62 and the phosphorylation of the AKT/mTOR pathway. Furthermore, we obtained similar results in the cell model of acute pancreatitis. CONCLUSION: This study suggests that Interleukin -22 administration could alleviate pancreatic damage in caerulein-induced acute pancreatitis. This effect may result from the activation of the AKT/mTOR pathway, leading to the inhibition of autophagy. Consequently, Interleukin -22 shows potential as a treatment.

Alternative autophagy dampens UVB-induced NLRP3 inflammasome activation in human keratinocytes.

Sunlight exposure results in an inflammatory reaction of the skin commonly known as sunburn, which increases skin cancer risk. In particular, the ultraviolet B (UVB) component of sunlight induces inflammasome activation in keratinocytes to instigate the cutaneous inflammatory responses. Here, we explore the intracellular machinery that maintains skin homeostasis by suppressing UVB-induced inflammasome activation in human keratinocytes. We found that pharmacological inhibition of autophagy promoted UVB-induced NLRP3 inflammasome activation. Unexpectedly, however, gene silencing of Atg5 or Atg7, which are critical for conventional autophagy, had no effect, whereas gene silencing of Beclin1, which is essential not only for conventional autophagy but also for Atg5/Atg7-independent alternative autophagy, promoted UVB-induced inflammasome activation, indicating an involvement of alternative autophagy. We found that damaged mitochondria were highly accumulated in UVB-irradiated keratinocytes when alternative autophagy was inhibited, and they appear to be recognized by NLRP3. Overall, our findings indicate that alternative autophagy, rather than conventional autophagy, suppresses UVB-induced NLRP3 inflammasome activation through the clearance of damaged mitochondria in human keratinocytes and illustrate a previously unknown involvement of alternative autophagy in inflammation. Alternative autophagy may be a new therapeutic target for sunburn and associated cutaneous disorders.

Melittin as an Activator of the Autophagy and Unfolded Protein Response Pathways in Colorectal HCT116 Cell Line.

Background: The potential anticancer effect of melittin has motivated scientists to find its exact molecular mechanism of action. There are few data on the effect of melittin on the UPR and autophagy as two critical pathways involved in tumorigenesis of colorectal and drug resistance. This study aimed to investigate the effect of melittin on these pathways in the colorectal cancer (CRC) HCT116 cells. Methods: MTT method was carried out to assess the cytotoxicity of melittin on the HCT116 cell line for 24, 48, and 72 h. After selecting the optimal concentrations and treatment times, the gene expression of autophagy flux markers (LC3-ßII and P62) and UPR markers (CHOP and XBP-1s) were determined using qRT-PCR. The protein level of autophagy initiation marker (Beclin1) was also determined by Western blotting. Results: MTT assay showed a cytotoxic effect of melittin on the HCT116 cells. The increase in LC3-ßII and decrease in P62 mRNA expression levels, along with the elevation in the Beclin1 protein level, indicated the stimulatory role of melittin on the autophagy. Melittin also significantly enhanced the CHOP and XBP-1s expressions at mRNA level, suggesting the positive role of the melittin on the UPR activation. Conclusion: This study shows that UPR and autophagy can potentially be considered as two key signaling pathways in tumorigenesis, which can be targeted by the BV melittin in the HCT116 cells. Further in vivo evaluations are recommended to verify the obtained results.

Upregulation of miR-499a Targets Beclin1 to Modulate Astrocyte Autophagy and Promote Ischemic Stroke.

OBJECTIVE: To investigate the clinical significance of miR-499a expression in the serum of ischemic stroke patients and its potential mechanism in regulating astrocytes to promote ischemic stroke. METHODS: Serum samples from 99 ischemic stroke patients and 99 healthy individuals were collected and analyzed for miR-499a expression through RT-PCR. Statistical analysis was performed to compare the expression differences between the two groups, and correlation between miR-499a expression and clinical pathological indices in stroke patients was analyzed. MiR-499a mimic, inhibitor, and negative control vectors were constructed and transfected into astrocyte SVGp12 cells. Afterward, miR-499a expression was validated by RT-PCR, cell viability was assessed by CCK8 assay, and apoptosis was detected using flow cytometry. The binding sites of miR-499a and Beclin1 were predicted by the Target-scan database and confirmed by dual luciferase assay. After overexpressing Beclin1, co-transfection with miR-499a mimic or negative control was conducted to observe the reverse effect of miR-499a mimic on Beclin1 overexpression. RESULTS: MiR-499a was significantly upregulated in the stroke group (p<0.001), it was positively correlated with TC (Total Cholesterol), LDL-C (Low-density lipoprotein cholesterol), and APO-A1 (Apolipoprotein A1) (R2>0.3, p<0.001). MiR-499a mimics promoted cell viability while inhibiting apoptosis of astrocytes. MiR-499a targeted Beclin 1 and inhibited its mRNA and protein expression, as well as the expression of autophagy-related proteins LC-3 and p62. MiR-499a could reverse the impact of Beclin1 overexpression on SVGp12 astrocyte proliferation and apoptosis. CONCLUSION: Serum miR-499a in stroke patients may serve as a potential diagnostic indicator. MiR-499a-mediated inhibition of Beclin 1, subsequently leading to suppression of astrocytic autophagy and viability, may represent a pivotal mechanism underlying its promotion of IS.

BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.

Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology.

A mechanistic study of Anwei decoction intervention in a rat model of gastric intestinal metaplasia through the endoplasmic reticulum stress - Autophagy pathway.

OBJECTIVE: To investigate the mechanism of Anwei decoction (AWD) intervention on gastric intestinal metaplasia (GIM) using a rat model through the endoplasmic reticulum stress-autophagy pathway. METHODS: Gastric intestinal metaplasia was induced in rats using 1-methyl-3-nitro-1-nitrosoguanidine. The experiment included a normal control group, a model group, and low-, medium- and high-dose AWD groups. The specificity of intestinal epithelial cells was determined for model establishment and drug efficacy by detecting the protein expression of markers such as MUC2, VILLIN and CDX2 through western blotting (WB). The effects of AWD on endoplasmic reticulum stress and autophagy were evaluated by measuring the mRNA and protein expression levels of endoplasmic reticulum stress markers (PEPK, ATF6, CHOP and caspase-12) and autophagy markers (LC3Ⅱ and Beclin-1) using reverse transcription polymerase chain reaction and the WB method. Furthermore, the ultrastructure of gastric mucosal cells and autophagosome status were observed using transmission electron microscopy. RESULTS: Compared with the model group, the AWD-treated rats exhibited significant improvement in body weight (P < 0.01), reduced protein expression of the intestine epithelial cell-specific markers MUC2, VILLIN, CDX2 and KLF4 (P < 0.01 for all) and increased SOX2 protein expression (P < 0.01). In addition, AWD suppressed the mRNA and protein expression of endoplasmic reticulum stress markers PEPK and ATF6 (P < 0.01 for all) and promoted the mRNA and protein expression of autophagy and apoptosis markers CHOP, caspase-12, LC3Ⅱ and Beclin-1 (P < 0.01 for all). CONCLUSION: Anwei decoction effectively inhibits the further progression of GIM and prevents the occurrence of gastric mucosal carcinogenesis.

VHL suppresses autophagy and tumor growth through PHD1-dependent Beclin1 hydroxylation.

The Von Hippel-Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α.

Comparative Analysis of Autophagy and Apoptosis in Disc Degeneration: Understanding the Dynamics of Temporary-Compression-Induced Early Autophagy and Sustained-Compression-Triggered Apoptosis.

The purpose of this study was to investigate the initiation of autophagy activation and apoptosis in nucleus pulposus cells under temporary compression (TC) and sustained compression (SC) to identify ideal research approaches in intervertebral disc degeneration. Various techniques were used: radiography (X-ray), magnetic resonance imaging (MRI), transmission electron microscope (TEM), H&E staining, Masson's trichrome staining, immunohistochemistry (IHC) (LC3, beclin-1, and cleaved caspase-3), and real-time polymerase chain reaction (RT-qPCR) for autophagy-related (beclin-1, LC3, and P62) and apoptosis-related (caspase-3 and PARP) gene expression analysis. X-ray and MRI revealed varying degrees of disc degeneration, ranging from moderate to severe in both groups. The severity was directly linked to compression duration, with SC resulting in notably severe central NP cell degeneration. Surprisingly, TC also caused similar, though less severe, degeneration. Elevated expression of LC3 and beclin-1 was identified after 6 weeks, but it notably declined after 12 weeks. Central NP cells in both groups exhibited increased expression of cleaved caspase-3 that was positively correlated with the duration of SC. TC showed fewer apoptotic markers compared to SC. LC3, beclin-1, and P62 mRNA expression peaked after 6 weeks and declined after 12 weeks in both groups. Cleaved caspase-3 and PARP expression peaked in SC, positively correlating with longer compression duration, while TC showed lower levels of apoptosis gene expression. Furthermore, TEM results revealed different events of the autophagic degradation process after 2 weeks of compression. TCmay be ideal for studying early triggered autophagy-mediated degeneration, while SC may be ideal for studying late or slower-triggered apoptosis-mediated degeneration.

Lipopolysaccharide inhibits osteoblast formation and receptor activator of nuclear factor-κB ligand degradation via autophagy inhibition.

Lipopolysaccharide (LPS) and Receptor Activator of Nuclear Factor-κB Ligand (RANKL) are the two important factors causing bone loss, which is an important pathogenesis for osteoporosis. However, the relationship between LPS and RANKL is not yet clear. LPS can be involved in the weakened osteoblast formation as an autophagy regulator, and osteoblasts and their precursors are the source cells for RANKL production. Our study aimed to explore the relationship between autophagy changes and RANKL production during LPS-regulated osteoblasts. Our results showed that LPS inhibited autophagy (LC3 conversion and autophagosome formation) and enhanced the protein and mRNA expression of RANKL in MC3T3-E1 osteoblast precursor line. Autophagy upregulation with Rapamycin over BECN1 overexpression rescued LPS-inhibited osteoblast formation and -promoted RANKL protein production in MC3T3-E1 cells. In vivo experiments supported that damaged bone mass, bone microstructure, osteoblastic activity (ALP and P1NP production by ELISA assays) and enhanced RANKL production by LPS administration were partially rescued by Rapamycin application. In conclusion, LPS can inhibit autophagy in osteoblast precursors, thereby inhibiting osteoblast formation and RANKL autophagic degradation.