Autophagy Agonist Research Articles

1,25(OH)2D3 improves SD rats high-altitude pulmonary edema by inhibiting ferroptosis and ferritinophagy in alveolar epithelial cells.

This study investigates the protective effects and potential mechanisms of 1,25(OH)2D3 against high-altitude pulmonary edema (HAPE). Hypoxia-induced rats were administered 1,25(OH)2D3 for 24, 48, and 72hours, and we observed lung tissue injury and pulmonary edema. Immunohistochemistry (IHC) and Western blot analyses were employed to analyze the expression of markers associated with ferroptosis and ferritinophagy in rat lungs. Metabolomics analysis was conducted to investigate changes in serum lipid metabolites. We validated the mechanism of action of 1,25(OH)2D3 in type II alveolar epithelial cells induced by hypoxia. Our results demonstrated that hypoxic exposure significantly altered sodium-water transport in the lungs, leading to edema formation. The degree of pulmonary edema was most pronounced at 48hours of hypoxi. Treatment with 1,25(OH)2D3 improved lung function and reduced the degree of pulmonary edema in hypoxic rats. Hypoxia-induced increases in 4-HNE and MDA levels in the lungs, along with iron accumulation, were observed. Hypoxia also resulted in elevated levels of NCOA4, LC3Ⅱ, and FTH1 proteins in the lungs. Furthermore, treatment with 1,25(OH)2D3 significantly inhibited ferroptosis and ferritinophagy in the lungs after hypoxia. The levels of lipid metabolites, such as L-Aspartic acid and L-Fucose, were significantly elevated in the serum of hypoxic rats. After 1,25(OH)2D3 treatment, these levels exhibited a significant reduction. In hypoxic type II alveolar epithelial cells, 1,25(OH)2D3 improved hypoxia-induced sodium-water transport, ferroptosis, and ferritinophagy, which were reversed by the autophagy agonist Rapamycin.By modulating ferroptosis and ferritinophagy, 1,25(OH)2D3 mitigated the deleterious effects of hypoxia on pulmonary function.

Dexmedetomidine regulates the anti-oxidation and autophagy of adipose-derived stromal cells under H2O2-induced oxidative stress through Nrf2/p62 pathway and improves the retention rate of autologous fat transplantation.

To investigate the protective mechanism of dexmedetomidine (DEX) on adipose-derived stromal cells (ADSCs) under oxidative stress model and its promotion effect on the retention rate of adipose granule transplantation by in vitro and in vivo experiments. The experiment was divided into control group, model group (ADSCs + H2O2+normal serum), DEX group (ADSCs + H202+DEX drug-containing serum), autophagy agonist group (ADSCs + H2O2+rapamycin (RAP)+normal serum), RAP + DEX group (ADSCs + H2O2+normal serum), RAP + DEX drug-containing serum), autophagy inhibitor group (ADSCs + H2O2+chloroquine (CQ)+normal serum), CQ + DEX group (ADSCs + H2O2+CQ + DEX drug-containing serum). HO-1, GSH-PX, SOD and CAT in ADSCs under oxidative stress model were measured. ROS fluorescence intensity and apoptosis ratio were detected. Expression of Nrf2, LC3-II/LC3-I and p62 were detected. In vivo, fat mixed with ADSCs or DEX -pretreated ADSCs was implanted subcutaneously in the lower back region of nude mice. Fat grafts were collected and analyzed at 2-, 4-, 6-, and 8-weeks post-transplantation. DEX pretreatment could reduce the expression of p62 to enhance the autophagy level of ADSCs under oxidative stress model. Additionally, cotransplantation of DEX-pretreated ADSCs with fat improved the long-term texture of fat grafts. DEX increased the fat graft survival and angiogenesis.

Monocrotaline-mediated autophagy via inhibiting PI3K/AKT/mTOR pathway induces apoptosis in rat hepatocytes.

Monocrotaline (MCT), a major pyrrolizidine alkaloid, is well-known for its high liver toxicity. Dysregulation of autophagy induced apoptosis can lead to various liver diseases, including those induced by chemical compounds. Therefore, we aim to explore whether autophagy might serve as a potential strategy for addressing liver apoptosis caused by MCT. In primary rat hepatocytes (PRHs), MCT significantly increased the number of autophagosomes and the expression levels of LC3II, Becline-1, and Atg5, while it decreased the expression of p62 in a concentration-dependent manner at doses of 100, 200, 300, and 400μM. Western blot assays revealed MCT inhibited the phosphorylation levels of the PI3K/AKT/mTOR pathway. To elucidate the role of autophagy in mediating MCT-induced apoptosis, we further pretreated PRHs with the autophagy agonist Rapamycin and the inhibitors Bafilomycin A1 and Chloroquine, respectively, and assessed the apoptosis of PRHs induced by MCT. The results displayed that Rapamycin increased the apoptosis rate and the expression of cleaved caspase-3, whereas Bafilomycin A1 and Chloroquine reduced the apoptosis and the expression of cleaved caspase-3 in PRHs. This study confirms that autophagy enhances PRHs apoptosis induced by MCT. In summary, this study demonstrates that MCT-induced autophagy via inhibition of the PI3K/AKT/mTOR pathway can lead to apoptosis in PRHs.

CH25H Promotes Autophagy and Regulates the Malignant Progression of Laryngeal Squamous Cell Carcinoma Through the PI3K-AKT Pathway.

Laryngeal squamous cell carcinoma (LSCC) is a type of cancer of the respiratory tract that often presents with subtle symptoms at the early stage and is susceptible to recurrence and metastasis. To find out key regulatory genes involved in LSCC development, we downloaded LSCC-related sequencing datasets for bioinformatics analysis. WGCNA was performed on GSE142083 and differential analysis was conducted on GSE51985 and TCGA-HNSC. Intersectiongenes were taken from the above three datasets. To confirm the function of genes, we overexpressed and knocked down genes in cells and treated them with autophagy agonist Rapamycin and PI3K-AKT pathway inhibitor. At the cellular level, the expression of CH25H, autophagy-related proteins (LC3 I, LC3 II, p62, and Beclin 1), and PI3K-AKT pathway-related proteins (PI3K, AKT, and p-AKT) were assessed via Western blot; the mRNA level of CH25H was evaluated through qRT-PCR; the cell activity was examined by CCK8; the apoptosis was assessed through flow cytometry; and the cell migration and invasion were assessed through wound healing and Transwell assays. Through bioinformatics analysis, we screened 7 genes (CH25H, NELL2, STC2, TMEM158, ZIC2, HOXD11, and HOXD10). Ultimately, CH25H was selected for follow-up experiments. By detecting CH25H expression in human immortalized keratinocytes (HaCaT) and LSCC cells (Tu-686, SNU899, and AMC-HN-8), it was found out that CH25H expression was higher in HaCaT cells than in LSCC cells. To elucidate the role of CH25H in LSCC development, we overexpressed CH25H in Tu-686 cells and downregulated its expression in AMC-HN-8 cells. CH25H was revealed to reduce the proliferation, activity, invasion, and migration of LSCC cells while increasing their apoptosis levels. Significant changes were also observed in the expressions of autophagy- and PI3K-AKT pathway-related proteins. To further investigate the roles of autophagy and the PI3K-AKT pathway in LSCC development, we respectively employed autophagy agonists and inhibitors targeting the PI3K-AKT pathway to intervene the cells, and found that CH25H regulated the PI3K-AKT pathway to promote autophagy, thus enhancing the apoptosis of LSCC cells. We further investigated CH25H's impact on tumor growth, autophagy, and the PI3K-AKT pathway at the animal level and found that CH25H promoted autophagy of LSCC cells and inhibited the PI3K-AKT pathway, and ultimately inhibiting the progression of LSCC. In summary, CH25H promotes autophagy and affects the malignant progression of LSCC through the PI3K-AKT pathway.

Protective effects and regulatory mechanisms of Platycodin D against LPS-Induced inflammatory injury in BEAS-2B cells

Platycodin D (PLD), a major bioactive component of triterpene saponins found in Platycodon grandiflora, is renowned for its anti-inflammatory and antioxidant properties. This study aims to explore the protective effects and regulatory mechanisms of PLD in an LPS-induced inflammation injury model of BEAS-2B cells. Initially, PLD was identified from Platycodon grandiflora extracts utilizing UPLC-Q-TOF-MS/MS technology. The effects of PLD on the viability, morphology, ROS levels, and inflammatory factors of LPS-induced BEAS-2B cells were then investigated. The results showed that PLD significantly alleviated LPS-induced oxidative stress and inflammatory injury. Further analysis revealed that PLD positively influenced apoptosis levels, mitochondrial morphology, and related gene expression, indicating its potential to mitigate LPS-induced apoptosis and alleviate mitochondrial dysfunction. Using molecular docking technology, we predicted the binding sites of PLD with mitochondrial autophagy protein. Gene expression levels of autophagy-related proteins were measured to determine the impact of PLD on mitochondrial autophagy. Additionally, the study examined whether the mitochondrial autophagy agonists rapamycin (RAPA) could modulate the upregulation of inflammasome-related factors NLRP3 and Caspase-1 in LPS-induced BEAS-2B cells. This was done to evaluate the regulator mechanisms of PLD in pulmonary inflammatory injury. Our findings suggest that PLD’s mechanism of action involves the regulation of mitochondrial autophagy, which in turn modulates inflammatory responses.

Autophagy aggravates multi-walled carbon nanotube-induced ferroptosis by suppressing PGC-1 dependent-mitochondrial biogenesis in lung epithelial cells

Multi-walled carbon nanotube (MWCNT) induced respiratory toxicity has become a growing concern, with ferroptosis emerging as a novel mechanism implicated in various respiratory diseases. However, whether ferroptosis is involved in MWCNT-elicited lung injury and the underlying molecular mechanisms warrant further exploration. In this study, we found that MWCNT-induced ferroptosis is autophagy-dependent, contributing to its cellular toxicity. Inhibiting of autophagy by pharmacological inhibitors 3-MA or ATG5 gene knockdown significantly attenuated MWCNT-induced ferroptosis, concomitant with rescued mitochondrial biogenesis. Rapamycin, the autophagy agonist, exacerbated the mitochondrial damage and MWCNT-induced ferroptosis. Moreover, lentivirus-mediated overexpression of PGC-1α inhibited ferroptosis, while inhibition of PGC-1α aggravated ferroptosis. In summary, our study unveils ferroptosis as a novel mechanism underlying MWCNT-induced respiratory toxicity, with autophagy promoting MWCNT-induced ferroptosis by hindering PGC-1α-dependent mitochondrial biogenesis.

Impact of acupuncture on ischemia/reperfusion injury: Unraveling the role of miR-34c-5p and autophagy activation

We have previously reported that the expression of miR-34c-5p was up-regulated during acupuncture treatment in the setting of a cerebral ischemia/reperfusion injury (CIRI), indicating that miR-34c-5p plays an important role in healing from a CIRI-induced brain injury. This study sought to evaluate the effects of acupuncture on miR-34c-5p expression and autophagy in the forward and reverse directions using a rat focal cerebral ischemia/reperfusion model. After 120 minutes of middle cerebral artery occlusion and reperfusion, rats were treated with acupuncture at the "Dazhui" (DU20), "Baihui" (DU26) and "Renzhong" (DU14) points. Neurologic function deficit score, cerebral infarct area ratio, neuronal apoptosis and miR-34c-5p expression were evaluated 72 hr after treatment. The autophagy agonist RAPA and the antagonist 3MA were used to evaluate the neuro protective effects of autophagy-mediated acupuncture. We found that acupuncture treatment improved autophagy in the brain tissue of CIRI rats. Acupuncture reversed the negative effects of 3MA on CIRI, and acupuncture combined with RAPA further enhanced autophagy. We also found that acupuncture could increase miR-34c-5p expression in hippocampal neurons after ischemia/reperfusion. Acupuncture and a miR-34c agomir were able to enhance autophagy, improve neurologic deficits, and reduce the cerebral infarct area ratio and apoptosis rate by promoting the expression of miR-34c-5p. Silencing miR-34c resulted in a significantly reduced activating effect of acupuncture on autophagy and increased apoptosis, neurologic deficit symptoms, and cerebral infarct area ratio. This confirms that acupuncture can upregulate miR-34c-5p expression, which is beneficial in the treatment of CIRI.

Naringin inhibits cisplatin resistance of ovarian cancer cells by inhibiting autophagy mediated by the TGF-β2/smad2 pathway.

Resistance to cisplatin (DDP) in patients with ovarian cancer (OC) poses a great challenge to improving the quality of life of patients. Past reports have revealed that naringin can induce apoptosis of OC cells and delay the occurrence of drug resistance in OC cells. However, the molecular role by which naringin inhibits DDP resistance in OC has not been definitively proven by researchers. The objective of this study is to investigate the effect of naringin on DDP resistance in OC cells and the specific mechanism of naringin mediating autophagy. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were selected to evaluate the role of naringin or DDP on the proliferation and apoptosis of human OC cells (SKOV3/A2780). The protein levels of Sequestosome 1 (SQSTM1/p62, hereinafter referred to as p62), microtubule-associated protein 1 light chain 3 (LC3), transforming growth factor-β2 (TGF-β2) and SMAD family member 2 (smad2) were detected with Western blotting assay. Immunofluorescence assay was also used to evaluate the level of LC3 in different groups of cells. Besides, functional analyses were performed in vivo. Naringin was shown to promote DDP sensitivity and apoptosis of human OC DDP-resistant cell line (SKOV3/A2780-DDP cells). Significantly increased p62 expression and reduced LC3 expression were found in naringin-treated cells. The autophagy agonist, rapamycin, reversed the effect of naringin on the resistance of SKOV3-DDP cells to DDP. Naringin inhibited levels of TGF-β2/smad2 pathway-related proteins, and regulated autophagy in SKOV3-DDP cells. In vivo experiments demonstrated that injection of naringin inhibited DDP resistance and autophagy in mice xenograft model. In summary, naringin inhibits DDP resistance in OC cells by inhibiting autophagy mediated by the TGF-β2/smad2 pathway.

Enhanced antibacterial properties of enteric glial cells attenuate intestinal inflammation through the GABABR-mediated autophagy pathway

Enterotoxigenic Escherichia coli (ETEC) infection is a severe threat to global public health because of its high morbidity and mortality among children and infants. Enteric glial cells (EGCs) are involved in host–bacteria communication. However, the mechanisms through which EGCs interact with ETEC remain unclear. We attempted to assess whether γ-aminobutyric acid type B receptor (GABABR) activation participated in EGC autophagy during Escherichia coli K88 (ETECK88) infection. Alterations in autophagy and EGC activity were observed in the intestines of the ETECK88-infected mice, and similar results were obtained from experiments in which the EGCs were directly infected with ETECK88. EGC pretreatment with specific autophagy agonists significantly decreased the inflammatory response and bacterial burden, whereas pretreatment with inhibitors had the opposite effect. Interestingly, in EGCs, GABABR activation notably increased Beclin 1 and LC3 levels and autophagosome and autolysosome numbers, thus promoting autophagy activation and enhancing antimicrobial responses against ETECK88 infection. Furthermore, GABABR defense was mediated via myeloid differentiation factor 88 (MyD88) signaling in EGCs, which was proven to be based on the inhibition or overexpression of MyD88. Notably, comparable results of GABABR activation in vivo were observed in response to ETECK88, implicating this as a defense mechanism that reinforced antibacterial activity to alleviate intestinal inflammation in mice. Our study revealed previously unappreciated roles for GABABR in linking EGC antibacterial autophagy to strengthen host defense against ETECK88 infection, thus identifying GABABR as an important target for the treatment of infective enteritis.

Circadian rhythm disruption-mediated downregulation of Bmal1 exacerbates DSS-induced colitis by impairing intestinal barrier.

Circadian rhythm disruption (CRD) is thought to increase the risk of inflammatory bowel disease. The deletion of Bmal1, a core transcription factor, leads to a complete loss of the circadian rhythm and exacerbates the severity of dextran sodium sulfate (DSS)-induced colitis in mice. However, the underlying mechanisms by which CRD and Bmal1 mediate IBD are still unclear. We used a CRD mouse model, a mouse colitis model, and an in vitro model of colonic epithelial cell monolayers. We also knocked down and overexpressed Bmal1 in Caco-2 cells by transfecting lentivirus in vitro. The collected colon tissue and treated cells were assessed and analyzed using immunohistochemistry, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction, western blot, flow cytometry, transmission electron microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling staining. We found that CRD mice with downregulated Bmal1 expression were more sensitive to DSS-induced colitis and had more severely impaired intestinal barrier function than wild-type mice. Bmal1-/- mice exhibited more severe colitis, accompanied by decreased tight junction protein levels and increased apoptosis of intestinal epithelial cells compared with wild-type mice, which were alleviated by using the autophagy agonist rapamycin. Bmal1 overexpression attenuated Lipopolysaccharide-induced apoptosis of intestinal epithelial cells and impaired intestinal epithelial cells barrier function in vitro, while inhibition of autophagy reversed this protective effect. This study suggests that CRD leads to the downregulation of Bmal1 expression in the colon, which may exacerbate DSS-induced colitis in mice, and that Bmal1 may serve as a novel target for treating inflammatory bowel disease.

Ethyl pyruvate alleviates pulmonary arterial hypertension via PI3K-Akt signaling.

Pulmonary arterial hypertension (PAH) is a pathophysiological syndrome that is extremely difficult to manage, and there is currently no effective treatment. We want to elucidate the therapeutic effect of ethyl pyruvate (EP) on PAH and its possible mechanism. Pulmonary artery endothelial cells (PAECs) were cultured in conventional low-oxygen environments, and cellular proliferation was monitored after treatment with EP. Expression of p-PI3K/Akt, LC3-II, and Beclin-1 was detected by Western blot. After hyperkinetic PAH rabbits' models were treated with EP, hemodynamic data were collected. Right ventricular hypertrophy and pulmonary vascular remodeling were evaluated. Expression of p-PI3K/Akt, LC3-II, and Beclin-1 protein was also detected after using autophagy inhibitor and agonists. We found that EP could inhibit PAECs proliferation. After EP treatment, expression of p-PI3K/Akt was upregulated in vitro and in vivo. LC3-II and Beclin-1 were inhibited and their expression was lower after autophagy inhibitor was given, while after administration of autophagy agonists, their expression was higher than that in the EP alone group. Besides, EP attenuated PAH, and right ventricular hypertrophy and pulmonary vascular remodeling were also reversed. EP can reduce PAH and reverse vascular remodeling which is associated with inhibition of autophagy in PAECs based on PI3K-Akt signaling pathway. The results of this study can provide surgical opportunities for patients with severe PAH caused by congenital heart disease in clinical cardiovascular surgery.

Low shear stress-induced blockage of autophagic flux impairs endothelial barrier and facilitates atherosclerosis in mice

Atherosclerosis preferentially occurs in areas with low shear stress (LSS) and oscillatory flow. LSS has been demonstrated to correlate with the development of atherosclerosis. The sphingosine 1-phosphate receptor 1 (S1PR1), involving intravascular blood flow sensing, regulates vascular development and vascular barrier function. However, whether LSS affects atherosclerosis via regulating S1PR1 remains incompletely clear. In this study, immunostaining results of F-actin, β-catenin, and VE-cadherin indicated that LSS impaired endothelial barrier function in human umbilical vein endothelial cells (HUVECs). Western blot analysis showed that LSS resulted in blockage of autophagic flux in HUVECs. In addition, autophagy agonist Rapamycin (Rapa) antagonized LSS-induced endothelial barrier dysfunction, whereas autophagic flux inhibitor Bafilomycin A1 (BafA1) exacerbated it, indicating that LSS promoted endothelial barrier dysfunction by triggering autophagic flux blockage. Notably, gene expression analysis revealed that LSS downregulated S1PR1 expression, which was antagonized by Rapa. Selective S1PR1 antagonist W146 impaired endothelial barrier function of HUVECs under high shear stress (HSS) conditions. Moreover, our data showed that expression of GAPARAPL2, a member of autophagy-related gene 8 (Atg8) proteins, was decreased in HUVECs under LSS conditions. Autophagic flux blockage induced by GAPARAPL2 knockdown inhibited S1PR1, aggravated endothelial barrier dysfunction of HUVECs in vitro, and promoted aortic atherosclerosis in ApoE−/− mice in vivo. Our study demonstrates that autophagic flux blockage induced by LSS downregulates S1PR1 expression and impairs endothelial barrier function. GABARAPL2 inhibition is involved in LSS-induced autophagic flux blockage, which impairs endothelial barrier function via downregulation of S1PR1.

Ginsenoside Rg_1 ameliorates OGD/R-induced PC12 cell injury by inhibiting autography via IRE1-JNK-CHOP pathway

This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12) cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP) signaling pathway. An OGD/R model was established in PC12 cells, and PC12 cells were randomly classified into control, model, OGD/R+GRg_1(0.1, 1, 10 μmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+3,5-dibromosalicylaldehyde(DBSA, IRE1 inhibitor) groups. Except the control group, the other groups were subjected to OGD/R treatment, i.e., oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h. Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Apoptosis was detected by Hoechst 33342 staining, and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC) assay. Western blot was employed to determine the expression of autophagy-related proteins(Beclin1, LC3-Ⅱ, and p62) and the pathway-related proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently increased the viability of PC12 cells and down-regulated the expression of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, compared with the model group. Furthermore, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 μmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity, up-regulated protein levels of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, decreased relative cell survival rate, and down-regulated protein level of p62. The 3-MA, 4-PBA, and DBSA groups exerted the opposite effects. Taken together, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.

AMPK/MTOR/TP53 Signaling Pathway Regulation by Calcitonin Gene-Related Peptide Reduces Oxygen-Induced Lung Damage in Neonatal Rats through Autophagy Promotion.

Our previous studies indicated that calcitonin gene-related peptide (CGRP) alleviates hyperoxia-induced lung injury and suggested the possible involvement of autophagy in this process. Herein, we aimed to further explore the potential involvement of tumor protein p53 (TP53) and autophagy in the mode of action of CGRP against hyperoxia-induced lung injury in vitro and in vivo. The study conducted tests on type II alveolar epithelial cells (AECII) and rats that were subjected to hyperoxia treatment or combined treatment of hyperoxia with CGRP, CGRP inhibitor, rapamycin (an autophagy agonist), 3-methyladenine (3-MA, an autophagy inhibitor), TP53 silencing/inhibitor (pifithrin-α), or expression vector/activator (PRIMA-1 (2,2-bis(hydroxymethyl)-3-quinuclidinone)) and their corresponding controls. We found that oxidative stress, apoptosis, and autophagy were all increased by hyperoxia treatment in vitro. However, treating AECII cells with CGRP reversed hyperoxia-induced oxidative stress and apoptosis but further promoted autophagy. In addition, the combined treatment with rapamycin or TP53 silencing with CGRP promoted the effect of CGRP, while contrary results were obtained with combined therapy with 3-MA or TP53 overexpression. In vivo, the number of hyperoxia-induced autophagosomes was promoted in the lung tissue of neonatal rats. Furthermore, hyperoxia increased the expression levels of AMP-activated protein kinase (AMPK) alpha 1 (also known as protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1)) but inhibited TP53 and mechanistic target of rapamycin (MTOR); these expression trends were regulated by CGRP treatment. In conclusion, we showed that CGRP can attenuate hyperoxia-induced lung injury in neonatal rats by enhancing autophagy and regulating the TP53/AMPK/MTOR crosstalk axis.

REDUCED CX43 EXPRESSION INDUCES AUTOPHAGY THROUGH ACTIVATION OF THE AMPK-MTOR-ULK1 SIGNALING PATHWAY IN THE COMMON BILE DUCT LIGATION RAT HEART.

Backgrounds: This study aimed to investigate the relationship between Cx43 expression and autophagy mediated by the AMPK-mTOR-Ulk1 signaling pathway in jaundice heart. Methods: In this study, a jaundice model was established in common bile duct ligation (CBDL) rats. Cardiac injury was assessed using various methods including myocardial injury indicators, echocardiography, transmission electron microscopy, hematoxylin and eosin staining, Masson staining, immunohistochemical analyses, and immunofluorescence staining. We investigated the regulatory relationship between Cx43, autophagy, and the AMPK-mTOR-ULK pathway in vivo by administering autophagy agonists (Rapa), autophagy inhibitors (3-MA), and Cx43 inhibitors (Gap 26). In vitro , we observed the relationship between autophagy and the AMPK-mTOR-ULK1 pathway in cells by exposing them to the AMPK inhibitor Compound C and the AMPK activator AICAR. Results: We found that CBDL induced autophagy through the AMPK-mTOR-ULK pathway, leading to the inhibition of myocardial dysfunction. Rapamycin pretreatment with CBDL3d exhibited a protective effect against myocardial injury and promoted autophagy. In contrast, 3-MA had no impact. Pretreatment with rapamycin at CBDL2w enhanced autophagy and aggravated cardiac injury; however, inhibition of autophagy using 3-MA attenuated cardiac injury. Cell viability was enhanced by AMPK inhibitors and inhibited by AMPK agonists. In addition, we observed that increased autophagy led to decreased Cx43 expression, which negatively affected cardiac function. Conclusions: CBDL induces myocardial injury in rats and activates autophagy through the AMPK-mTOR-ULK pathway, resulting in decreased Cx43 protein levels. A moderate increase in early autophagy in CBDL can improve cardiac injury, while late inhibition of autophagy can reduce myocardial injury.

The interaction of endorepellin and neurexin triggers neuroepithelial autophagy and maintains neural tube development

Heparan sulfate proteoglycan 2 (HSPG2) gene encodes the matrix protein Perlecan, and genetic inactivation of this gene creates mice that are embryonic lethal with severe neural tube defects (NTDs). We discovered rare genetic variants of HSPG2 in 10% cases compared to only 4% in controls among a cohort of 369 NTDs. Endorepellin, a peptide cleaved from the domain V of Perlecan, is known to promote angiogenesis and autophagy in endothelial cells. The roles of enderepellin in neurodevelopment remain unclear so far. Our study revealed that endorepellin can migrate to the neuroepithelial cells and then be recognized and bind with the neuroepithelia receptor neurexin in vivo. Through the endocytic pathway, the interaction of endorepellin and neurexin physiologically triggers autophagy and appropriately modulates the differentiation of neural stem cells into neurons as a blocker, which is necessary for normal neural tube closure. We created knock-in (KI) mouse models with human-derived HSPG2 variants, using sperm-like stem cells that had been genetically edited by CRISPR/Cas9. We realized that any HSPG2 variants that affected the function of endorepellin were considered pathogenic causal variants for human NTDs given that the severe NTD phenotypes exhibited by these KI embryos occurred in a significantly higher response frequency compared to wildtype embryos. Our study provides a paradigm for effectively confirming pathogenic mutations in other genetic diseases. Furthermore, we demonstrated that using autophagy inhibitors at a cellular level can repress neuronal differentiation. Therefore, autophagy agonists may prevent NTDs resulting from failed autophagy maintenance and neuronal over-differentiation caused by deleterious endorepellin variants.

Melatonin attenuates diabetic cardiomyopathy by increasing autophagy of cardiomyocytes via regulation of VEGF-B/GRP78/PERK signaling pathway

AimsDiabetic cardiomyopathy (DCM) is a major cause of mortality in patients with diabetes, and the potential strategies for treating DCM are insufficient. Melatonin (Mel) has been shown to attenuate DCM, however, the underlying mechanism remains unclear. The role of vascular endothelial growth factor-B (VEGF-B) in DCM is little known. In present study, we aimed to investigate whether Mel alleviated DCM via regulation of VEGF-B and explored its underlying mechanisms.Methods and resultsWe found that Mel significantly alleviated cardiac dysfunction and improved autophagy of cardiomyocytes in type 1 diabetes mellitus (T1DM) induced cardiomyopathy mice. VEGF-B was highly expressed in DCM mice in comparison with normal mice, and its expression was markedly reduced after Mel treatment. Mel treatment diminished the interaction of VEGF-B and Glucose-regulated protein 78 (GRP78) and reduced the interaction of GRP78 and protein kinase RNA -like ER kinase (PERK). Furthermore, Mel increased phosphorylation of PERK and eIF2α, then up-regulated the expression of ATF4. VEGF-B−/− mice imitated the effect of Mel on wild type diabetic mice. Interestingly, injection with Recombinant adeno-associated virus serotype 9 (AAV9)-VEGF-B or administration of GSK2656157 (GSK), an inhibitor of phosphorylated PERK abolished the protective effect of Mel on DCM. Furthermore, rapamycin, an autophagy agonist displayed similar effect with Mel treatment; while 3-Methyladenine (3-MA), an autophagy inhibitor neutralized the effect of Mel on high glucose-treated neonatal rat ventricular myocytes.ConclusionsThese results demonstrated that Mel attenuated DCM via increasing autophagy of cardiomyocytes, and this cardio-protective effect of Mel was dependent on VEGF-B/GRP78/PERK signaling pathway.

Unravelling the role of autophagy in human dental pulp.

Autophagy is an evolutionarily conserved intracellular catabolic process that recycles and degrades proteins, organelles and pathogens. It is an endogenous defence mechanism regulating multiple cellular pathways like apoptosis, inflammation, immune response and pathogen clearance and acts as a modulator of pathogenesis. This article highlights the emerging role of autophagy in inflammation and regeneration of human dental pulp. It emphasizes exploring autophagy and autophagy agonists as potential targets for the development of novel therapeutic interventions.

Q-switched 1064 nm Nd: YAG laser restores skin photoageing by activating autophagy by TGFβ1 and ITGB1.

Excessive ultraviolet B ray (UVB) exposure to sunlight results in skin photoageing. Our previous research showed that a Q-switched 1064 nm Nd: YAG laser can alleviate skin barrier damage through miR-24-3p. However, the role of autophagy in the laser treatment of skin photoageing is still unclear. This study aims to investigate whether autophagy is involved in the mechanism of Q-switched 1064 nm Nd: YAG in the treatment of skin ageing. Invitro, primary human dermal fibroblast (HDF) cells were irradiated with different doses of UVB to establish a cell model of skin photoageing. Invivo, SKH-1 hairless mice were irradiated with UVB to establish a skin photoageing mouse model and irradiated with laser. The oxidative stress and autophagy levels were detected by western blot, immunofluorescence and flow cytometer. String was used to predict the interaction protein of TGF-β1, and CO-IP and GST-pull down were used to detect the binding relationship between TGFβ1 and ITGB1. Invitro, UVB irradiation reduced HDF cell viability, arrested cell cycle, induced cell senescence and oxidative stress compared with the control group. Laser treatment reversed cell viability, senescence and oxidative stress induced by UVB irradiation and activated autophagy. Autophagy agonists or inhibitors can enhance or attenuate the changes induced by laser treatment, respectively. Invivo, UVB irradiation caused hyperkeratosis, dermis destruction, collagen fibres reduction, increased cellular senescence and activation of oxidative stress in hairless mice. Laser treatment thinned the stratum corneum of skin tissue, increased collagen synthesis and autophagy in the dermis, and decreased the level of oxidative stress. Autophagy agonist rapamycin and autophagy inhibitor 3-methyladenine (3-MA) can enhance or attenuate the effects of laser treatment on the skin, respectively. Also, we identified a direct interaction between TGFB1 and ITGB1 and participated in laser irradiation-activated autophagy, thereby inhibiting UVB-mediated oxidative stress further reducing skin ageing. Q-switched 1064 nm Nd: YAG laser treatment inhibited UVB-induced oxidative stress and restored skin photoageing by activating autophagy, and TGFβ1 and ITGB1 directly incorporated and participated in this process.

Capsaicin Attenuates LPS-Induced Acute Lung Injury by Inhibiting Inflammation and Autophagy Through Regulation of the TRPV1/AKT Pathway.

Acute lung injury (ALI) is a severe pulmonary disease characterized by damage to the alveoli and pulmonary blood vessels, leading to severe impairment of lung function. Studies on the effect of capsaicin (8-methyl-N-geranyl-6-nonamide, CAP) on lipopolysaccharide (LPS)-induced ALI in bronchial epithelial cells transformed with Ad12-SV40 2B (BEAS-2B) are still limited. This study aimed to investigate the effect and specific mechanism by which CAP improves LPS-induced ALI. The present study investigated the effect of CAP and the potential underlying mechanisms in LPS-induced ALI in vitro and vivo via RNA sequencing, Western blotting (WB), quantitative real-time reverse transcription PCR (qRT‒PCR), enzyme-linked immunosorbent assay (ELISA), and transmission electron microscopy (TEM). The TRPV1 inhibitor AMG9810 and the AKT agonist SC79 were used to confirm the protective effect of the TRPV1/AKT axis against ALI. The autophagy agonist rapamycin (Rapa) and the autophagy inhibitors 3-methyladenine (3-MA) and bafilomycin A1 (Baf-A1) were used to clarify the characteristics of LPS-induced autophagy. Our findings demonstrated that CAP effectively suppressed inflammation and autophagy in LPS-induced ALI, both in vivo and in vitro. This mechanism involves regulation by the TRPV1/AKT signaling pathway. By activating TRPV1, CAP reduces the expression of P-AKT, thereby exerting its anti-inflammatory and inhibitory effects on pro-death autophagy. Furthermore, prior administration of CAP provided substantial protection to mice against ALI induced by LPS, reduced the lung wet/dry ratio, decreased proinflammatory cytokine expression, and downregulated LC3 expression. Taken together, our results indicate that CAP protects against LPS-induced ALI by inhibiting inflammatory responses and autophagic death through the TRPV1/AKT signaling pathway, presenting a novel strategy for ALI therapy.