A novel chromone-based as a potential inhibitor of ULK1 that modulates autophagy and induces apoptosis in colon cancer.

Aim: Chromones are promising for anticancer drug development. Methods & results: 12 chromone-based compounds were synthesized and tested against cancer cell lines. Compound 8 showed the highest cytotoxicity (LC50 3.2 µM) against colorectal cancer cells, surpassing 5-fluorouracil (LC50 4.2 µM). It suppressed colony formation, induced cell cycle arrest and triggered apoptotic cell death, confirmed by staining and apoptosis markers. Cell death was accompanied by enhanced reactive oxygen species formation and modulation of the autophagic machinery (autophagy marker light chain 3B (LC3B); adenosine monophosphate-activated protein kinase (AMPK); protein kinase B (PKB); UNC-51-like kinase (ULK)-1; and ULK2). Molecular docking and dynamic simulations revealed that compound 8 directly binds to ULK1. Conclusion: Compound 8 is a promising lead for autophagy-modulating anti-colon cancer drugs.

[Box: see text].

Therapeutic Relevance of Inducing Autophagy in ß-Thalassemia.

The ß-thalassemias are inherited genetic disorders affecting the hematopoietic system. In ß-thalassemias, more than 350 mutations of the adult ß-globin gene cause the low or absent production of adult hemoglobin (HbA). A clinical parameter affecting the physiology of erythroid cells is the excess of free α-globin. Possible experimental strategies for a reduction in excess free α-globin chains in ß-thalassemia are CRISPR-Cas9-based genome editing of the ß-globin gene, forcing "de novo" HbA production and fetal hemoglobin (HbF) induction. In addition, a reduction in excess free α-globin chains in ß-thalassemia can be achieved by induction of the autophagic process. This process is regulated by the Unc-51-like kinase 1 (Ulk1) gene. The interplay with the PI3K/Akt/TOR pathway, with the activity of the α-globin stabilizing protein (AHSP) and the involvement of microRNAs in autophagy and Ulk1 gene expression, is presented and discussed in the context of identifying novel biomarkers and potential therapeutic targets for ß-thalassemia.

Promotion and Mechanism of Acupotomy on Chondrocyte Autophagy in Knee Osteoarthritis Rabbits.

OBJECTIVE: To explore the effect of acupotomy intervention on autophagy of chondrocytes in rabbits with knee osteoarthritis (KOA), and to determine the possible mechanisms of acupotomy to alleviate cartilage degeneration. METHODS: The modified Videman method was used to construct a KOA rabbit model. After modeling, 40 rabbits were randomly divided into 4 groups by a random number table: control; KOA (model); KOA + acupotomy (acupotomy), and KOA + sham acupotomy (sham), 10 in each group. After a 3-week treatment course, the knee joint activity was determined by the modified Lequesne MG index. Hematoxylin-eosin staining staining was used to examine the morphological changes of chondrocytes. Autophagy of chondrocytes was observed by transmission electron microscopy. The surface morphology of cartilage tissue was observed by scanning electron microscope. The mRNA and protein levels of AMP kinase/mammalian target of rapamycin/Unc-51 (AMPK/mTOR/ULK1) signal pathway key proteins, autophagy-related factor Beclin-1 and microtubule-associated protein 1A/1B light chain 3 (LC3) in rabbit knee cartilage were assessed by real-time fluorescence quantitative polymerase chain reaction and Western blot, respectively. RESULTS: The modified Lequesne MG score of acupotomy group was significantly lower than that of model group (P<0.05). Pathological results showed that chondrocyte autophagy decreased and cartilage surface was rough in the model group, which recovered after acupotomy treatment. The mRNA expressions of AMPK, ULK1, Beclin-1 and the protein levels of p-AMPK, p-ULK1, Beclin-1, and LC3 II/LC3 I were decreased in the model group, while the mRNA and protein expressions of mTOR were increased (P<0.01). However, acupotomy treatment reversed these abnormal changes (P<0.05). CONCLUSIONS: Acupotomy could effectively up-regulate the expressions of AMPK, ULK1 and Beclin1, reduce the expression of mTOR, promote autophagy, and alleviate joint degeneration. Acupotomy is a promising complementary and alternative therapy for KOA.

Bone mesenchymal stem cells improve cholestatic liver fibrosis by targeting ULK1 to regulate autophagy through PI3K/AKT/mTOR pathway.

Cholestatic liver disease (CLD) is a severe disease, which can progress to liver cirrhosis, even liver cancer. Hepatic stellate cells (HSCs) activation plays a crucial role in CLD development. Bone mesenchymal stem cells (BMSCs) treatment was demonstrated to be beneficial in liver diseases. However, the therapeutic effect and mechanism of BMSCs on CLD are poorly known. In the present study, we investigated the therapeutic effects and underlying mechanisms of BMSCs transplantation in mouse models of bile duct ligation-induced cholestatic liver fibrosis (CLF). The results revealed that BMSCs significantly improved liver function and reduced the formation of fibrosis after portal vein transplantation. Mechanistically, after coculturing BMSCs and HSCs, we identified that BMSCs alleviated starvation-induced HSCs activation. Further, BMSCs inhibited HSCs activation by decreasing autophagy, and PI3K/AKT/mTOR pathway was involved in the regulation. More importantly, ULK1 is identified as the main autophagy-related gene regulated by BMSCs in HSCs autophagy. Overexpression of ULK1 reversed the suppression of HSCs autophagy by BMSCs. Collectively, our results provide a theoretical basis for BMSCs targeting ULK1 to attenuate HSCs autophagy and activation and suggest that BMSCs or ULK1 may be an alternative therapeutic approach/target for the treatment of CLF.

FIP200 Phosphorylation Regulates Late Steps in Mitophagy.

Mitophagy is a specific type of autophagy responsible for the selective elimination of dysfunctional or superfluous mitochondria, ensuring the maintenance of mitochondrial quality control. The initiation of mitophagy is coordinated by the ULK1 kinase complex, which engages mitophagy receptors via its FIP200 subunit. Whether FIP200 performs additional functions in the subsequent later phases of mitophagy beyond this initial step and how its regulation occurs, remains unclear. Our findings reveal that multiple phosphorylation events on FIP200 differentially control the early and late stages of mitophagy. Furthermore, these phosphorylation events influence FIP200's interaction with ATG16L1. In summary, our results highlight the necessity for precise and dynamic regulation of FIP200, underscoring its importance in the progression of mitophagy.

Low selenium and T-2 toxin may be involved in the pathogenesis of Kashin-Beck disease by affecting AMPK/mTOR/ULK1 pathway mediated autophagy.

Kashin-Beck disease (KBD) is an endemic, environmentally associated cartilage disease. Previous studies have shown that the environmental suspected pathogenic factors of KBD, T-2 toxin and low selenium, are involved in the regulation of inflammation, oxidative stress and autophagy in some tissues and organs. In cartilage diseases, the level of cellular autophagy determines the fate of the chondrocytes. However, whether autophagy is involved in KBD cartilage lesions, and the role of low selenium and T-2 toxins in KBD cartilage injury and autophagy are still unclear. This work took the classical AMPK/mTOR/ULK1 autophagy regulatory pathway as the entry point to clarify the relationship between the environmental suspected pathogenic factors and chondrocyte autophagy. Transmission electron microscopy was used to observe the autophagy of chondrocytes in KBD patients. qRT-PCR and western blot were used to analyze the expression of AMPK/mTOR/ULK1 pathway and autophagy markers. The rat model of KBD was established by low selenium and T-2 toxin, the autophagy in rat cartilage was detected after 4- and 12-week interventions. Chondrocyte autophagy was found in KBD, and the AMPK/mTOR/ULK1 pathway was down-regulated. In the rat model, the pathway showed an up-regulated trend when low selenium and T-2 toxin, were treated for a short time or low concentration, and autophagy level increased. However, when low selenium and T-2 toxin were treated for a long time or at high concentrations, the pathway showed a down-regulated trend, and the autophagy level was reduced and even defective. In conclusion, in the process of KBD cartilage lesion, chondrocyte autophagy level may increase in the early stage, and decrease in the late stage with the progression of lesion. Low selenium and T-2 toxins may affect autophagy by AMPK/mTOR/ULK1 pathway.

Rotenone activates the LKB1-AMPK-ULK1 signaling pathway to induce autophagy and apoptosis in rat thoracic aortic endothelial cells.

BACKGROUND: The specific mechanism by which rotenone impacts thoracic aortic autophagy and apoptosis is unknown. We aimed to investigate the regulatory effects of rotenone on autophagy and apoptosis in rat thoracic aortic endothelial cells (RTAEC) via activation of the LKB1-AMPK-ULK1 signaling pathway and to elucidate the molecular mechanisms of rotenone on autophagy and apoptosis in vascular endothelial cells. METHODS: In vivo, 60 male SD rats were randomly selected and divided into 5 groups: control (Con), DMSO, 1, 2, and 4 mg/kg groups, respectively. After 28 days of treatment, histopathological and ultrastructural changes in each group were observed using HE and transmission electron microscopy; Autophagy, apoptosis, and LKB1-AMPK-ULK1 pathway-related proteins were detected by Western blot; Apoptosis levels in the thoracic aorta were detected by TUNEL. In vitro, RTAEC were cultured and divided into control (Con), DMSO, 20, 100, 500, and 1000 nM groups. After 24 h of intervention, autophagy, apoptosis, and LKB1-AMPK-ULK1 pathway-related factors were detected by Western blot and qRT-PCR; Flow cytometry to detect apoptosis levels; Autophagy was inhibited with 3-MA and CQ to detect apoptosis levels, and changes in autophagy, apoptosis, and downstream factors were detected by the AMPK inhibitor CC intervention. RESULTS: Gavage in SD rats for 28 days, some degree of damage was observed in the thoracic aorta and heart of the rotenone group, as well as the appearance of autophagic vesicles was observed in the thoracic aorta. TUNEL analysis revealed higher apoptosis in the rotenone group's thoracic aorta; RTAEC cultured in vitro, after 24 h of rotenone intervention, showed increased ROS production and significantly decreased ATP production. The flow cytometry data suggested an increase in the number of apoptotic RTAEC. The thoracic aorta and RTAEC in the rotenone group displayed elevated levels of autophagy and apoptosis, and the LKB1-AMPK-ULK1 pathway proteins were activated and expressed at higher levels. Apoptosis and autophagy were both suppressed by the autophagy inhibitors 3-MA and CQ. The AMPK inhibitor CC reduced autophagy and apoptosis in RTAEC and suppressed the production of the AMPK downstream factors ULK1 and P-ULK1. CONCLUSIONS: Rotenone may promote autophagy in the thoracic aorta and RTAEC by activating the LKB1-AMPK-ULK1 signaling pathway, thereby inducing apoptosis.

Unexpected roles for AMPK in the suppression of autophagy and the reactivation of MTORC1 signaling during prolonged amino acid deprivation.

AMPK promotes catabolic and suppresses anabolic cell metabolism to promote cell survival during energetic stress, in part by inhibiting MTORC1, an anabolic kinase requiring sufficient levels of amino acids. We found that cells lacking AMPK displayed increased apoptotic cell death during nutrient stress caused by prolonged amino acid deprivation. We presumed that impaired macroautophagy/autophagy explained this phenotype, as a prevailing view posits that AMPK initiates autophagy (often a pro-survival response) through phosphorylation of ULK1. Unexpectedly, however, autophagy remained unimpaired in cells lacking AMPK, as monitored by several autophagic readouts in several cell lines. More surprisingly, the absence of AMPK increased ULK1 signaling and MAP1LC3B/LC3B lipidation during amino acid deprivation while AMPK-mediated phosphorylation of ULK1 S555 (a site proposed to initiate autophagy) decreased upon amino acid withdrawal or pharmacological MTORC1 inhibition. In addition, activation of AMPK with compound 991, glucose deprivation, or AICAR blunted autophagy induced by amino acid withdrawal. These results demonstrate that AMPK activation and glucose deprivation suppress autophagy. As AMPK controlled autophagy in an unexpected direction, we examined how AMPK controls MTORC1 signaling. Paradoxically, we observed impaired reactivation of MTORC1 in cells lacking AMPK upon prolonged amino acid deprivation. Together these results oppose established views that AMPK promotes autophagy and inhibits MTORC1 universally. Moreover, they reveal unexpected roles for AMPK in the suppression of autophagy and the support of MTORC1 signaling in the context of prolonged amino acid deprivation. These findings prompt a reevaluation of how AMPK and its control of autophagy and MTORC1 affect health and disease.

IL-33 Accelerates Chronic Atrophic Gastritis through AMPK-ULK1 Axis Mediated Autolysosomal Degradation of GKN1.

Chronic atrophic gastritis (CAG) is a complex disease characterized by atrophy and inflammation in gastric mucosal tissue, especially with high expression of interleukins. However, the interaction and mechanisms between interleukins and gastric mucosal epithelial cells in CAG remain largely elusive. Here, we elucidate that IL-33 stands out as the predominant inflammatory factor in CAG, and its expression is induced by H. pylori and MNNG through the ROS-STAT3 signaling pathway. Furthermore, our findings reveal that the IL-33/ST2 axis is intricately involved in the progression of CAG. Utilizing phosphoproteomics mass spectrometry, we demonstrate that IL-33 enhances autophagy in gastric epithelial cells through the phosphorylation of AMPK-ULK1 axis. Notably, inhibiting autophagy alleviates CAG severity, while augmentation of autophagy exacerbates the disease. Additionally, ROS scavenging emerges as a promising strategy to ameliorate CAG by reducing IL-33 expression and inhibiting autophagy. Intriguingly, IL-33 stimulation promotes GKN1 degradation through the autolysosomal pathway. Clinically, the combined measurement of IL-33 and GKN1 in serum shows potential as diagnostic markers. Our findings unveil an IL-33-AMPK-ULK1 regulatory mechanism governing GKN1 protein stability in CAG, presenting potential therapeutic targets for its treatment.

Metformin mitigates adipogenesis of fibro-adipogenic progenitors after rotator cuff tears via activating mTOR/ULK1-mediated autophagy.

Muscular fatty infiltration is a common issue after rotator cuff tears (RCTs), which impair shoulder function. Females suffer a higher prevalence and a more severe degree of muscular fatty infiltration after RCT when compared with males, with the underlying mechanisms remaining unclear. Fibro-adipogenic progenitors (FAPs) are the primary source of muscular fatty infiltration following RCT. Our findings disclose that gender-specific disparities in muscular fatty infiltration are linked to mTOR/ULK1-mediated autophagy of FAPs. Decreased autophagic activity contributes to adipogenic differentiation in female FAPs after RCT. Furthermore, metformin could enhance mTOR/ULK1-mediated autophagic processes of FAPs, thereby alleviating fatty infiltration and improving shoulder functionality after RCT. Together, our study reveals that gender differences in muscular fatty infiltration arise from distinct autophagic activities. Metformin could be a promising noninvasive intervention to ameliorate muscular fatty infiltration of RCT.NEW & NOTEWORTHY The current study demonstrated that gender-specific disparities in muscular fatty infiltration are attributed to mTOR/ULK1-mediated autophagy of FAPs. Decreased autophagic activity contributes to adipogenic differentiation in female FAPs after RCT. Moreover, metformin could enhance mTOR/ULK1-mediated autophagic processes of FAPs, thereby alleviating fatty infiltration and improving shoulder functionality after RCT. Therefore, metformin could be a promising noninvasive intervention to ameliorate muscular fatty infiltration of RCT.

13-Oxyingenol-dodecanoate inhibits the growth of non-small cell lung cancer cells by targeting ULK1.

Lung cancer is the leading cause of cancer deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers. Euphorbia kansui yielded 13-oxyingenol-dodecanoate (13OD), an ingenane-type diterpenoid, which had a strong cytotoxic effect on NSCLC cells. The underlying mechanism and potential target, however, remained unknown. The study found that 13OD effectively inhibited the cell proliferation and colony formation of NSCLC cells (A549 and H460 cells), with less toxicity in normal human lung epithelial BEAS-2B cells. Moreover, 13OD can cause mitochondrial dysfunction, and apoptosis in NSCLC cells. Mechanistically, the transcriptomics results showed that differential genes were mainly enriched in the mTOR and AMPK signaling pathways, which are closely related to cellular autophagy, the related indicators were subsequently validated. Additionally, bafilomycin A1 (Baf A1), an autophagy inhibitor, reversed the mitochondrial damage caused by 13OD. Furthermore, the Omics and Text-based Target Enrichment and Ranking (OTTER) method predicted ULK1 as a potential target of 13OD against NSCLC cells. This hypothesis was further confirmed using molecular docking, the cellular thermal shift assay (CETSA), and Western blot analysis. Remarkably, ULK1 siRNA inhibited 13OD's toxic activity in NSCLC cells. In line with these findings, 13OD was potent and non-toxic in the tumor xenograft model. Our findings suggested a possible mechanism for 13OD's role as a tumor suppressor and laid the groundwork for identifying targets for ingenane-type diterpenoids.

Sea cucumber plasmalogen enhance lipophagy to alleviate abnormal lipid accumulation induced by high-fat diet.

Sea cucumber phospholipids, including the plasmalogen (PlsEtn) and plasmanylcholine (PakCho), have been shown to play a regulatory role in lipid metabolism disorders, but their mechanism of action remains unclear. Therefore, high-fat diet (HFD) and palmitic acid were used to establish lipid accumulation models in mice and HepG2 cells, respectively. Results showed that PlsEtn can reduce lipid deposition both in vivo and in vitro. HFD stimulation abnormally activated lipophagy through the phosphorylation of the AMPK/ULK1 pathway. The lipophagy flux monitor revealed abnormalities in the fusion stage of lipophagy. Of note, only PlsEtn stimulated the dynamic remodeling of the autophagosome membrane, which was indicated by the significantly decreased LC3 II/I ratio and p62 level. In all experiments, the effect of PlsEtn was significantly higher than that of PakCho. These findings elucidated the mechanism of PlsEtn in alleviating lipid accumulation, showed that it might be a lipophagy enhancer, and provided new insights into the high-value utilization of sea cucumber as an agricultural resource.

Hydrogen-rich solution alleviates acute radiation pneumonitis by regulating oxidative stress and macrophages polarization.

This study was aimed to investigate the effect of hydrogen-rich solution (HRS) on acute radiation pneumonitis (ARP) in rats. The ARP model was induced by X-ray irradiation. Histopathological changes were assessed using HE and Masson stains. Inflammatory cytokines were detected by ELISA. Immunohistochemistry and flow cytometry were performed to quantify macrophage (CD68) levels and the M2/M1 ratio. Western blot analysis, RT-qPCR, ELISA and flow cytometry were used to evaluate mitochondrial oxidative stress injury indicators. Immunofluorescence double staining was performed to colocalize CD68/LC3B and p-AMPK-α/CD68. The relative expression of proteins associated with autophagy activation and the adenosine 5'-monophosphate-activated protein kinase/mammalian target of rapamycin/Unc-51-like kinase 1 (AMPK/mTOR/ULK1) signaling pathway were detected by western blotting. ARP decreased body weight, increased the lung coefficient, collagen deposition and macrophage infiltration and promoted M1 polarization in rats. After HRS treatment, pathological damage was alleviated, and M1 polarization was inhibited. Furthermore, HRS treatment reversed the ARP-induced high levels of mitochondrial oxidative stress injury and autophagy inhibition. Importantly, the phosphorylation of AMPK-α was inhibited, the phosphorylation of mTOR and ULK1 was activated in ARP rats and this effect was reversed by HRS treatment. HRS inhibited M1 polarization and alleviated oxidative stress to activate autophagy in ARP rats by regulating the AMPK/mTOR/ULK1 signaling pathway.

NRSN2 promotes the malignant behavior of HPV-transfected laryngeal carcinoma cells through AMPK/ULK1 pathway mediated autophagy activation.

Neurensin-2 (NRSN2) performs a pro-carcinogenic function in multiple cancers. However, the function of NRSN2 in HPV-infected laryngeal carcinoma (LC) remains unclear. HPV transfection was performed in LC cells. The mRNA and protein levels were monitored using RT-qPCR, immunoblotting, and IF. Cell viability and proliferation were found using the CCK-8 assay and Edu staining. Cell invasion, migration, and apoptosis were probed using the Transwell, wound healing, and flow cytometry, respectively. The autophagosome was observed using TEM. NRSN2 was overexpressed in HPV-transfected LC cells. Inhibition of NRSN2 restrained the autophagy and malignant behavior of HPV-transfected LC cells. Meanwhile, the inhibition of AMPK/ULK1 pathway limited the increased autophagy of HPV-transfected LC cells caused by NRSN2 overexpression. Furthermore, NRSN2 knockdown inhibits autophagy by suppressing AMPK/ULK1 pathway, thereby restraining the malignant behavior of HPV-transfected LC cells. Our research confirmed that HPV transfection increased the autophagy and malignant behavior of LC cells by regulating the NRSN2-mediated activation of the AMPK/ULK1 pathway, offering a new target for cure of LC.

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.

[Tetrahydropalmatine inhibiting mitophagy through ULK1/FUNDC1 pathway to alleviate hypoxia/reoxygenation injury in H9c2 cells].

This study explored the specific mechanism by which tetrahydropalmatine(THP) inhibited mitophagy through the UNC-51-like kinase 1(ULK1)/FUN14 domain containing 1(FUNDC1) pathway to reduce hypoxia/reoxygenation(H/R) injury in H9c2 cells. This study used H9c2 cells as the research object to construct a cardiomyocyte H/R injury model. First, a cell viability detection kit was used to detect cell viability, and a micro-method was used to detect lactate dehydrogenase(LDH) leakage to evaluate the protective effect of THP on H/R injury of H9c2 cells. In order to evaluate the protective effect of THP on mitochondria, the chemical fluorescence method was used to detect intracellular reactive oxygen species, intramitochondrial reactive oxygen species, mitochondrial membrane potential, and autophagosomes, and the luciferin method was used to detect intracellular adenosine 5'-triphosphate(ATP) content. Western blot was further used to detect the ratio of microtubule-associated protein 1 light chain 3(LC3) membrane type(LC3-Ⅱ) and slurry type(LC3-Ⅰ) and activated cleaved caspase-3 expression level. In addition, ULK1 expression level and its phosphorylation degree at Ser555 site, as well as the FUNDC1 expression level and its phosphorylation degree of Ser17 site were detected to explore its specific mechanism. The results showed that THP effectively reduced mitochondrial damage in H9c2 cells after H/R. THP protected mitochondria by reducing the level of reactive oxygen species in cells and mitochondria, increasing mitochondrial membrane potential, thereby increasing cellular ATP production, enhancing cellular activity, reducing cellular LDH leakage, and finally alleviating H/R damage in H9c2 cells. Further studies have found that THP could reduce the production of autophagosomes, reduce the LC3-Ⅱ/LC3-Ⅰ ratio, and lower the expression of the apoptosis-related protein, namely cleaved caspase-3, indicating that THP could reduce apoptosis by inhibiting autophagy. In-depth studies have found that THP could inhibit the activation of the ULK1/FUNDC1 pathway of mitophagy and the occurrence of mitophagy by reducing the phosphorylation degree of ULK1 at Ser555 and FUNDC1 at Ser17. The application of ULK1 agonist BL-918 reversely verified the effect of THP on reducing the phosphorylation of ULK1 and FUNDC1. In summary, THP inhibited mitophagy through the ULK1/FUNDC1 pathway to reduce H/R injury in H9c2 cells.

Chrysin Inhibits TAMs-Mediated Autophagy Activation via CDK1/ULK1 Pathway and Reverses TAMs-Mediated Growth-Promoting Effects in Non-Small Cell Lung Cancer.

The natural flavonoid compound chrysin has promising anti-tumor effects. In this study, we aimed to investigate the mechanism by which chrysin inhibits the growth of non-small cell lung cancer (NSCLC). Through in vitro cell culture and animal models, we explored the impact of chrysin on the growth of NSCLC cells and the pro-cancer effects of tumor-associated macrophages (TAMs) and their mechanisms. We observed that M2-TAMs significantly promoted the growth and migration of NSCLC cells, while also markedly activating the autophagy level of these cells. Chrysin displayed a significant inhibitory effect on the growth of NSCLC cells, and it could also suppress the pro-cancer effects of M2-TAMs and inhibit their mediated autophagy. Furthermore, combining network pharmacology, we found that chrysin inhibited TAMs-mediated autophagy activation in NSCLC cells through the regulation of the CDK1/ULK1 signaling pathway, rather than the classical mTOR/ULK1 signaling pathway. Our study reveals a novel mechanism by which chrysin inhibits TAMs-mediated autophagy activation in NSCLC cells through the regulation of the CDK1/ULK1 pathway, thereby suppressing NSCLC growth. This discovery not only provides new therapeutic strategies for NSCLC but also opens up new avenues for further research on chrysin.

Sea Cucumber Plasmalogen Regulates the Lipid Profile in High-Fat Diet Mouse Liver via Lipophagy.

The sea cucumber plasmalogen PlsEtn has been shown to be associated with various chronic diseases related to lipid metabolism. However, the mechanism is unclear. Therefore, the present study used the sea cucumber plasmanylcholine PakCho as a structural contrast to PlsEtn and assessed its effect in 8 week high-fat diet (HFD)-fed mice. The lipidomic approach based on high-resolution mass spectrometry combined with molecular biology techniques was used to evaluate the mechanism of PlsEtn. The results showed that both PlsEtn and PakCho significantly inhibited an increase in mouse body weight and liver total triglyceride and total cholesterol levels caused by HFD. In addition, oil red O staining demonstrated that lipid droplets stored in the liver were degraded. Meanwhile, untargeted lipidomic experiments revealed that total lipids (increased by 42.8 mmol/mg prot; p < 0.05), triglycerides (increased by 38.9 mmol/mg prot; p < 0.01), sphingolipids (increased by 1.5 mmol/mg prot; p < 0.0001), and phospholipids (increased by 2.5 mmol/mg prot; p < 0.05) were all significantly elevated under HFD. PlsEtn resolved lipid metabolism disorders by alleviating the abnormal expression of lipid subclasses. In addition, five lipid molecular species, PE (18:1/20:4), PE (18:1/20:3), PE (18:1/18:3), TG (16:0/16:0/17:0), and TG (15:0/16:0/18:1), were identified as the biomarkers of HFD-induced lipid metabolism disorders. Finally, lipophagy-associated protein expression analysis showed that HFD abnormally activated lipophagy via ULK1 phosphorylation and PlsEtn alleviated lipophagy disorder through lysosomal function promotion. In addition, PlsEtn performed better than PakCho. Taken together, the current study results unraveled the mechanism of PlsEtn in alleviating lipid metabolism disorder and offered a new theoretical foundation for the high-value development of sea cucumber.

Targeting the TRAF3-ULK1-NLRP3 regulatory axis to control alveolar macrophage pyroptosis in acute lung injury.

Acute lung injury (ALI) is a serious condition characterized by damage to the lungs. Recent research has revealed that activation of the NLRP3 inflammasome in alveolar macrophages, a type of immune cell in the lungs, plays a key role in the development of ALI. This process, known as pyroptosis, contributes significantly to ALI pathogenesis. Researchers have conducted comprehensive bioinformatics analyses and identified 15 key genes associated with alveolar macrophage pyroptosis in ALI. Among these, NLRP3 has emerged as a crucial regulator. This study further reveal that the ULK1 protein diminishes the expression of NLRP3, thereby reducing the immune response of alveolar macrophages and mitigating ALI. Conversely, TRAF3, another protein, is found to inhibit ULK1 through a process called ubiquitination, leading to increased activation of the NLRP3 inflammasome and exacerbation of ALI. This TRAF3-mediated suppression of ULK1 and subsequent activation of NLRP3 are confirmed through various in vitro and in vivo experiments. The presence of abundant M0 and M1 alveolar macrophages in the ALI tissue samples further support these findings. This research highlights the TRAF3-ULK1-NLRP3 regulatory axis as a pivotal pathway in ALI development and suggests that targeting this axis could be an effective therapeutic strategy for ALI treatment.