Stevioside Ameliorates Palmitic Acid-Induced Abnormal Glucose Uptake via the PDK4/AMPK/TBC1D1 Pathway in C2C12 Myotubes.

BACKGROUND: Stevioside (SV) with minimal calories is widely used as a natural sweetener in beverages due to its high sweetness and safety. However, the effects of SV on glucose uptake and the pyruvate dehydrogenase kinase isoenzyme (PDK4) as an important protein in the regulation of glucose metabolism, remain largely unexplored. In this study, we used C2C12 skeletal muscle cells that was induced by palmitic acid (PA) to assess the effects and mechanisms of SV on glucose uptake and PDK4. METHODS: The glucose uptake of C2C12 cells was determined by 2-NBDG; expression of the Pdk4 gene was measured by quantitative real-time PCR; and expression of the proteins PDK4, p-AMPK, TBC1D1 and GLUT4 was assessed by Western blotting. RESULTS: In PA-induced C2C12 myotubes, SV could significantly promote cellular glucose uptake by decreasing PDK4 levels and increasing p-AMPK and TBC1D1 levels. SV could promote the translocation of GLUT4 from the cytoplasm to the cell membrane in cells. Moreover, in Pdk4-overexpressing C2C12 myotubes, SV decreased the level of PDK4 and increased the levels of p-AMPK and TBC1D1. CONCLUSION: SV was found to ameliorate PA-induced abnormal glucose uptake via the PDK4/AMPK/TBC1D1 pathway in C2C12 myotubes. Although these results warranted further investigation for validation, they may provide some evidence of SV as a safe natural sweetener for its use in sugar-free beverages to prevent and control T2DM.

Engineered Exosomes with Growth Differentiation Factor-15 Overexpression Enhance Cardiac Repair After Myocardial Injury.

Background: Cardiac repair remains a thorny issue for survivors of acute myocardial infarction (AMI), due to the regenerative inertia of myocardial cells. Cell-free therapies, such as exosome transplantation, have become a potential strategy for myocardial injury. The aim of this study was to investigate the role of engineered exosomes in overexpressing Growth Differentiation Factor-15 (GDF-15) (GDF15-EVs) after myocardial injury, and their molecular mechanisms in cardiac repair. Methods: H9C2 cells were transfected with GDF-15 lentivirus or negative control. The exosomes secreted from H9C2 cells were collected and identified. The cellular apoptosis and autophagy of H2O2-injured H9C2 cells were assessed by Western blotting, TUNEL assay, electron microscopy, CCK-8 and caspase 3/7 assay. A rat model of AMI was constructed by ligating the left anterior descending artery. The anti-apoptotic, pro-angiogenic effects of GDF15-EVs treatment, as well as ensuing functional and histological recovery were evaluated. Then, mRNA sequencing was performed to identify the differentially expressed mRNAs after GDF15-EVs treatment. Results: GDF15-EVs inhibited apoptosis and promoted autophagy in H2O2 injured H9C2 cells. GDF15-EVs effectively decreased the infarct area and enhanced the cardiac function in rats with AMI. Moreover, GDF15-EVs hindered inflammatory cell infiltration, inhibited cell apoptosis, and promoted cardiac angiogenesis in rats with AMI. RNA sequence showed that telomerase reverse transcriptase (TERT) mRNA was upregulated in GDF15-EVs-treated H9C2 cells. AMPK signaling was activated after GDF15-EVs. Silencing TERT impaired the protective effects of GDF15-EVs on H2O2-injured H9C2 cells. Conclusion: GDF15-EVs could fulfil their protective effects against myocardial injury by upregulating the expression of TERT and activating the AMPK signaling pathway. GDF15-EVs might be exploited to design new therapies for AMI.

Progranulin promotes regulatory T cells plasticity by mitochondrial metabolism through AMPK/PGC-1α pathway in ARDS.

As the aging population increases, the focus on elderly patients with acute respiratory distress syndrome (ARDS) is also increasing. In this article, we found progranulin (PGRN) differential expression in ARDS patients and healthy controls, even in young and old ARDS patients. Its expression strongly correlates with several cytokines in both young and elderly ARDS patients. PGRN has comparable therapeutic effects in young and elderly mice with lipopolysaccharide-induced acute lung injury, manifesting as lung injury, apoptosis, inflammation, and regulatory T cells (Tregs) differentiation. Considering that Tregs differentiation relies on metabolic reprogramming, we discovered that Tregs differentiation was mediated by mitochondrial function, especially in the aged population. Furthermore, we demonstrated that PGRN alleviated the mitochondrial damage during Tregs differentiation through the AMPK/PGC-1α pathway in T cells. Collectively, PGRN may regulate mitochondria function to promote Tregs differentiation through the AMPK/PGC-1α pathway to improve ARDS.

AMPK activation enhances osteoblast differentiation on a titanium disc via autophagy.

PURPOSE: The acquisition of osseointegration during implant therapy is slower and poorer in patients with diabetes compared with healthy persons. The serum concentration of adiponectin in patients with type II diabetes is lower than that of healthy persons via the suppression of AMP-activated protein kinase (AMPK). Therefore, we hypothesized that the AMPK activation enhances bone formation around implants, resulting in the improved acquisition of osseointegration. The purpose of this study was to evaluate the impact of AMPK activation on osteoblast differentiation and its mechanism of downstream signaling on titanium disc (Ti). METHODS: Confluent mouse pre-osteoblasts (MC3T3-E1) cells (1 × 105 cells/well) were cultured with BMP-2 for osteoblast differentiation, in the presence or absence AICAR, an AMPK activator. We examined the effects of AMPK activation on osteoblast differentiation and the underlying mechanism on a Ti using a CCK8 assay, a luciferase assay, quantitative RT-PCR, and western blotting. RESULTS: Although the proliferation rate of osteoblasts was not different between a Ti and a tissue culture polystyrene dish, the addition of AICAR, AMPK activator slightly enhanced osteoblast proliferation on the Ti. AICAR enhanced the BMP-2-dependent transcriptional activity on the Ti, leading to upregulation in the expression of osteogenesis-associated molecules. AICAR simultaneously upregulated the expression of autophagy-associated molecules on the Ti, especially LC3-II. AdipoRon, an adiponectin receptor type1/type2 activator activated AMPK, and upregulated osteogenesis-associated molecules on Ti. CONCLUSIONS: AMPK activation enhances osteoblast differentiation on a Ti via autophagy, suggesting that it promotes the acquisition of osseointegration during implant therapy.

Seawater pearl hydrolysate inhibits photoaging via decreasing oxidative stress, autophagy and apoptosis of Ultraviolet B-induced human skin keratinocytes.

BACKGROUND: Ultraviolet (UV) is the main reason to cause photoaging skin which not only hinders beauty, brings the patients with psychological burden, but also pathologically leads to the occurrence of tumors in skin. OBJECTIVE: This study goes into the inhibitory effect and mechanism of seawater pearl hydrolysate (SPH) to address human skin keratinocytes photoaging induced by UVB. METHODS: The photoaging model of Hacat cell was constructed by UVB irradiation, the levels of oxidative stress, apoptosis, aging, autophagy and autophagy-related protein and signal pathway expression were assessed to characterize the inhibitory effect and mechanism of SPH on photoaging Hacat cell. RESULTS: Seawater pearl hydrolysate significantly accelerated (p < 0.05) the activities of superoxide dismutase, catalase, and glutathione peroxidase, and markedly reduced (p < 0.05) the contents of reactive oxygen species (ROS), malondialdehyde, protein carbonyl compound and nitrosylated tyrosine protein, aging level, apoptosis rate in Hacat cell induced by 200 mJ cm-2 UVB after 24 and 48 h of culture; high dose SPH significantly raised (p < 0.05) relative expression level of p-Akt, p-mTOR proteins, and markedly decreased (p < 0.05) relative expression level of LC3II protein, p-AMPK, and autophagy level in Hacat cell induced by 200 mJ cm-2 UVB, or in combination with the intervention of PI3K inhibitor or AMPK overexpression after 48 h of culture. CONCLUSION: Seawater pearl hydrolysate can effectively inhibit 200 mJ cm-2 UVB-induced photoaging of Hacat cells. The mechanism indicates removing the excessive ROS through increasing the antioxidation of photoaging Hacat cells. Once redundant ROS is eliminated, SPH works to reduce AMPK, increase PI3K-Akt pathway expression, activate mTOR pathway to lowdown autophagy level, and as a result, inhibit apoptosis and aging in photoaging Hacat cells.

Differential G Protein-Coupled Estrogen Receptor-1 Regulation of Counter-Regulatory Transmitter Marker and 5'-AMP-Activated Protein Kinase Expression in Ventrolateral versus Dorsomedial Ventromedial Hypothalamic Nucleus.

INTRODUCTION: The ventromedial hypothalamic nucleus (VMN) is an estrogen receptor (ER)-rich structure that regulates glucostasis. The role of nuclear but not membrane G protein-coupled ER-1 (GPER) in that function has been studied. METHODS: Gene silencing and laser-catapult microdissection/immunoblot tools were used to examine whether GPER regulates transmitter and energy sensor function in dorsomedial (VMNdm) and/or ventrolateral (VMNvl) VMN counter-regulatory nitrergic and γ-Aminobutyric acid (GABA) neurons. RESULTS: Intra-VMN GPER siRNA administration to euglycemic animals did not affect VMNdm or -vl nitrergic neuron nitric oxide synthase (nNOS), but upregulated (VMNdm) or lacked influence on (VMNvl) GABA nerve cell glutamate decarboxylase65/67 (GAD) protein. Insulin-induced hypoglycemia (IIH) caused GPER knockdown-reversible augmentation of nNOS, 5'-AMP-activated protein kinase (AMPK), and phospho-AMPK proteins in nitrergic neurons in both divisions. IIH had dissimilar effects on VMNvl (unchanged) versus VMNdm (increased) GABAergic neuron GAD levels, yet GPER knockdown affected these profiles. GPER siRNA prevented hypoglycemic upregulation of VMNvl and -dm GABA neuron AMPK without altering pAMPK expression. CONCLUSIONS: Outcomes infer that GPER exerts differential control of VMNdm versus -vl GABA transmission during glucostasis and is required for hypoglycemic upregulated nitrergic (VMNdm and -vl) and GABA (VMNdm) signaling. Glycogen metabolism is reported to regulate VMN nNOS and GAD proteins. Data show that GPER limits VMNvl glycogen phosphorylase (GP) protein expression and glycogen buildup during euglycemia but mediates hypoglycemic augmentation of VMNvl GP protein and glycogen content; VMNdm glycogen mass is refractory to GPER control. GPER regulation of VMNvl glycogen metabolism infers that this receptor may govern local counter-regulatory transmission in part by astrocyte metabolic coupling.

IL-17A is involved in the hyperplasia of blood vessels in local lesions of psoriasis by inhibiting autophagy.

OBJECTIVE: Increased angiogenesis is a pathological feature of psoriasis, but the pathomechanisms of angiogenesis in psoriasis are not clear. Interleukin-17A (IL-17A) is the major effect factor in the pathogenesis of psoriasis. Our results showed that IL-17A can promote angiogenesis and cause endothelial cell inflammation. Autophagy plays an important role not only in regulating inflammation, but also in regulating angiogenesis. Whether angiogenesis in psoriasis is related to autophagy remains unclear. In this study, we treated human umbilical vein endothelial cells (HUVECs) with IL-17A to simulate increased angiogenesis to study whether increased angiogenesis in psoriasis is related to autophagy. METHODS AND RESULTS: Our results showed that treatment of HUVECs with IL-17A significantly increased angiogenesis and expression levels of mRNA for multiple proinflammatory cytokines (CCL20, IL-8, CCL2, IL-6, and IL-1ß) and, while decreasing intracellular levels of nitric oxide (NO) and NO synthase (NOS) activity. Moreover, IL-17A inhibited autophagy as shown that IL-17A significantly increased expression levels of LC3II and p62 proteins. Induction of autophagy ameliorated IL-17A-mediated inflammatory response and inhibited angiogenesis, accompanied by increased p-AMPKα(Thr172) and p-ULK1(Ser555) expression, and decreased p-mTOR(Ser2448) and p-ULK1(Ser757) expression. Furthermore, inhibition of either AMPK or lysosomal acidification completely overrode autophagy-induced changes in angiogenesis and NOS activity. Finally, induction of autophagy decreased apoptosis and caspase-3 activity in IL-17A-treated HUVECs. CONCLUSIONS: These results showed that IL-17A is involved in angiogenesis and inflammatory response by inhibiting autophagy through AMPK signaling pathway, suggesting that autophagy may be a new therapeutic target for psoriasis.

Manganese Enhances the Osteogenic Effect of Silicon-Hydroxyapatite Nanowires by Targeting T Lymphocyte Polarization.

Biomaterials encounter considerable challenges in extensive bone defect regeneration. The amelioration of outcomes may be attainable through the orchestrated modulation of both innate and adaptive immunity. Silicon-hydroxyapatite, for instance, which solely focuses on regulating innate immunity, is inadequate for long-term bone regeneration. Herein, extra manganese (Mn)-doping is utilized for enhancing the osteogenic ability by mediating adaptive immunity. Intriguingly, Mn-doping engenders heightened recruitment of CD4+ T cells to the bone defect site, concurrently manifesting escalated T helper (Th) 2 polarization and an abatement in Th1 cell polarization. This consequential immune milieu yields a collaborative elevation of interleukin 4, secreted by Th2 cells, coupled with attenuated interferon gamma, secreted by Th1 cells. This orchestrated interplay distinctly fosters the osteogenesis of bone marrow stromal cells and effectuates consequential regeneration of the mandibular bone defect. The modulatory mechanism of Th1/Th2 balance lies primarily in the indispensable role of manganese superoxide dismutase (MnSOD) and the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). In conclusion, this study highlights the transformative potential of Mn-doping in amplifying the osteogenic efficacy of silicon-hydroxyapatite nanowires by regulating T cell-mediated adaptive immunity via the MnSOD/AMPK pathway, thereby creating an anti-inflammatory milieu favorable for bone regeneration.

Vitamin D plays a protective role in osteoarthritis by regulating AMPK/mTOR signalling pathway to activate chondrocyte autophagy.

OBJECTIVES: The deletion of chondrocyte autophagy seems to play a key role in the pathogenesis of osteoarthritis (OA). Patients with OA often have vitamin D (VD) deficiency, and VD supplementation can improve pain and alleviate the progression of joint structures in patients. In this study, we aimed to investigate whether VD could enhance autophagy by activating the adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signalling pathway and protect against OA. METHODS: In this study, the levels of target proteins and genes were examined by western blot and qRT-PCR. Apoptotic cells were detected using TUNEL staining. Characteristics of autophagy were observed by LysoTracker red staining, mRFP-GFP-LC3 adenovirus transfection, and transmission electron microscopy. siRNA-mediated AMPK and mTOR knockdown were used to investigate the role of the AMPK/ mTOR signalling pathway in VD-induced autophagy. Haematoxylin and eosin and safranin-O/fast green staining were used detect cartilage alterations. RESULTS: We suggested that VD significantly reduced chondrocyte death and alleviated extracellular matrix degradation. Further studies showed that VD promoted the expression of the autophagy-related protein LC3II through the AMPK/mTOR signalling pathway in chondrocytes, activated lysosome activity, promoted the formation of autophagy-associated lysosomes, which played a crucial role in the degradation of intracellular organelles and maintained homeostasis. The anti-apoptotic effect of VD on chondrocytes was associated with the activation of autophagy. The group of AMPK-normal and mTOR-knockdown in the presence of VD inhibited chondrocyte apoptosis by promoting autophagy. CONCLUSIONS: This study highlights that VD can activate chondrocyte autophagy through the AMPK/mTOR signalling pathway.

Mitigation of cisplatin-induced nephrotoxicity by chelidonic acid in Wistar rats.

INTRODUCTION: Cisplatin, an anti-cancer drug is used to treat a wide range of solid tumors. Nevertheless, nephrotoxicity is the major adverse effect that restricts its clinical application. The present study focuses on the effect of chelidonic acid in cisplatin-induced nephrotoxicity. METHODS: Wistar rats were injected with cisplatin (5 mg/kg, intraperitoneally (i.p.), once in a week for 4 weeks) and chelidonic acid (10, 20, and 40 mg/kg, per oral (p.o.) for 4 weeks). Body weight, urine, biochemical, and oxidative stress parameters were performed to evaluate the effect of chelidonic acid in cisplatin-induced nephrotoxicity in rats. Pro-inflammatory cytokines and nuclear factor erythroid 2-related factor 2 (Nrf2) concentrations were determined. Expression of phospho-AMP activated protein kinase (phospho-AMP) and hypoxia-inducible factor 1-alpha (HIF-1α) was studied with western blot. Haematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome staining were used to study kidney tissues. RESULTS: Relative kidney weight and urine output were significantly increased in cisplatin-administered rats. Whereas, albumin, and creatinine concentration were decreased, and treatment with chelidonic acid reverses these deleterious effects of cisplatin significantly. Kidney functions were improved by chelidonic acid treatment with a reduction in tumor necrosis factor-alpha (TNF-α), Interleukin-6 (IL-6), and transforming growth factor-beta (TGF-ß1) concentration. The oxidative stress was decreased as compared to the cisplatin group. Furthermore, Nrf2 was significantly increased by chelidonic acid treatment. Chelidonic acid treatment significantly increased the expression of phospho-AMPK and HIF-1α in kidney tissue. Histopathological studies revealed that chelidonic acid reduced kidney damage. CONCLUSION: The findings showed that chelidonic acid increases phospho-AMPK and HIF-1α in the kidney tissue and significantly lowers the inflammatory cytokines, thus it is an effective molecule for providing protection against cisplatin-induced nephrotoxicity.

Lactobacillus plantarum and Bifidobacterium longum Alleviate Liver Injury and Fibrosis in Mice by Regulating NF-κB and AMPK Signaling.

In a preliminary study, live biotherapeutic products (LBPs) Lactobacillus plantarum LC27 and Bifidobacterium longum LC67 inhibited the secretion of alanine transaminase (ALT) and aspartate transaminase (AST) in LPS-stimulated HepG2 cells, while Escherichia coli K1 (Ec) increased ALT and ALT secretion. Therefore, we examined the effects of LC27 and LC67 on LPS-induced liver injury and fibrosis in mice and the correlation between their biomarkers in cell and animal experiments. Orally administered LC27 or LC67 significantly decreased blood ALT, AST, γ-glutamyl transferase (γGTP), TNF-α, triglyceride (TG), total cholesterol (TCh), total bile acid, and LPS levels, liver TNF-α, toll-like receptor-4 gene (Tlr4), α-smooth muscle actin (αSMA), and collagen-1 expression and αSMA+GFAP+ and NF-κB+F4/80+ cell populations, and colonic Tlr4, TNF-α, and IL-6 expression and NF-κB-positive cell population in LPS-treated mice. Furthermore, they increased AMPKa phosphorylation in the liver and colon. However, Ec increased the expression of TNF-α and IL-6 in blood, liver, and colon. The suppression of LPS-stimulated ALT and AST secretion in HepG2 cells by LBPs was positively correlated with their ameliorating effects on LPS-induced blood γGTP, ALT, and AST levels and liver αSMA and collagen-1 expression in mice. Based on these findings, LC27 and LC67 may improve liver injury and fibrosis by regulating NF-κB and AMPK signaling pathway and a protocol that can assay the inhibitory activity of LBPs on LPS-induced ALT and AST secretion in HepG2 may be useful for guessing their antihepatitic effects in the in vivo experiments.

Bone Mesenchymal Stem Cells Origin Exosomes are Effective Against Sepsis-Induced Acute Kidney Injury in Rat Model.

Introduction: The incidence and mortality rates of sepsis-induced acute kidney injury (SAKI) remain high, posing a substantial healthcare burden. Studies have implicated a connection between the development of SAKI and inflammation response, apoptosis, and autophagy. Moreover, evidence suggests that manipulating autophagy could potentially influence the prognosis of this condition. Notably, exosomes derived from bone mesenchymal stem cells (BMSCs-Exo) have exhibited promise in mitigating cellular damage by modulating pathways associated with inflammation, apoptosis, and autophagy. Thus, this study aims to investigate the influence of BMSCs-Exo on SAKI and the potential mechanisms that drive this impact. Methods: The SAKI model was induced in HK-2 cells using lipopolysaccharide (LPS), while rats underwent cecal ligation and puncture (CLP) to simulate the condition. Cell viability was assessed using the CCK-8 kit, and kidney damage was evaluated through HE staining, blood urea nitrogen (BUN), and serum creatinine (SCr) measurements. Inflammatory-related RNAs and proteins were quantified via qPCR and ELISA, respectively. Apoptosis was determined through apoptosis-related protein levels, flow cytometry, and TUNEL staining. Western blot analysis was utilized to measure associated protein expressions. Results: In vivo, BMSCs-Exo ameliorated kidney injury in CLP-induced SAKI rats, reducing inflammatory cytokine production and apoptosis levels. Fluorescence microscope observed the absorption of BMSCs-Exo by renal cells following injection via tail vein. In the SAKI rat kidney tissue, there was an upregulation of LC3-II/LC3-I, p62, and phosphorylated AMP-activated protein kinase (p-AMPK) expressions, indicating blocked autophagic flux, while phosphorylated mammalian target of rapamycin (p-mTOR) expression was downregulated. However, BMSCs-Exo enhanced LC3-II/LC3-I and p-AMPK expression, concurrently reducing p62 and p-mTOR levels. In vitro, BMSCs-Exo enhanced cell viability in LPS-treated HK-2 cells, and exerted anti-inflammation and anti-apoptosis effects which were consistent with the results in vivo. Similarly, rapamycin (Rapa) exhibited a protective effect comparable to BMSCs-Exo, albeit partially abrogated by 3-methyladenine (3-MA). Conclusion: BMSCs-Exo mitigate inflammation and apoptosis through autophagy in SAKI, offering a promising avenue for SAKI treatment.

[Protective effect of metformin on pulmonary fibrosis caused by paraquat through activating AMP-activated protein kinase pathway].

OBJECTIVE: To observe whether metformin (MET) inhibits transforming growth factor-ß1 (TGF-ß1)/Smad3 signaling pathway by activating adenosine activated protein kinase (AMPK), so as to alleviate the pulmonary fibrosis caused by paraquat (PQ) poisoning in mice. METHODS: Male C57BL/6J mice were randomly divided into the Control group, PQ poisoning model group (PQ group), MET intervention group (PQ+MET group), AMPK agonist group (PQ+AICAR group), and AMPK inhibitor group (PQ+MET+CC group), according to a random number table method. A mouse model of PQ poisoning was established by one-time peritoneal injection of 1 mL PQ solution (20 mg/kg). The Control group was injected with the same volume of normal saline. After 2 hours of modeling, the PQ+MET group was given 2 mL of 200 mg/kg MET solution by gavage, the PQ+AICAR group was given 2 mL of 200 mg/kg AICAR solution by intraperitoneal injection, the PQ+MET+CC group was given 2 mL of 200 mg/kg MET solution by gavage and then 1 mL complex C (CC) solution (20 mg/kg) was intraperitoneally injected, the Control group and PQ group were given 2 mL of normal saline by gavage. The intervention was given once a day for 21 consecutive days. The 21-day survival rate of ten mice in each group was calculated, and the lung tissues of remaining mice were collected at 21 days after modeling. The pathological changes of lung tissues were observed under light microscope after hematoxylin-eosin (HE) staining and Masson staining, and the degree of pulmonary fibrosis was evaluated by Ashcroft score. The content of hydroxyproline in lung tissue and oxidative stress indicators such as malondialdehyde (MDA) and superoxide dismutase (SOD) were detected. The protein expressions of E-cadherin, α-smooth muscle actin (α-SMA), phosphorylated AMPK (p-AMPK), TGF-ß1 and phosphorylated Smad3 (p-Smad3) in lung tissue were detected by Western blotting. RESULTS: Compared with the Control group, the 21 days survival rate was significantly reduced, lung fibrosis and Ashcroft score were significantly increased in PQ group. In addition, the content of hydroxyproline, MDA and the protein expressions of α-SMA, TGF-ß1 and p-Smad3 in lung tissue were significantly increased, while the activity of SOD and the protein expressions of E-cadherin and p-AMPK were significantly decreased in PQ group. Compared with the PQ group, the 21 days survival rates of mice were significantly improved in the PQ+MET group and PQ+AICAR group (70%, 60% vs. 20%, both P < 0.05). The degree of pulmonary fibrosis and the Ashcroft score were significantly reduced (1.50±0.55, 2.00±0.63 vs. 6.67±0.52, both P < 0.05). The content of hydroxyproline and MDA in lung tissue, as well as α-SMA, TGF-ß1 and p-Smad3 protein expressions were significantly reduced [hydroxyproline (mg/L): 2.03±0.11, 3.00±0.85 vs. 4.92±0.65, MDA (kU/g): 2.06±1.48, 2.10±1.80 vs. 4.06±1.33, α-SMA/GAPDH: 0.23±0.06, 0.16±0.06 vs. 1.00±0.09, TGF-ß1/GAPDH: 0.28±0.03, 0.53±0.05 vs. 0.92±0.06 p-Smad3/GAPDH: 0.52±0.04, 0.69±0.06 vs. 1.11±0.10, all P < 0.05], SOD activity and the protein expressions of E-cadherin and p-AMPK were significantly increased [SOD (µmol/g): 39.76±1.35, 33.03±1.28 vs. 20.08±1.79, E-cadherin/GAPDH: 0.91±0.08, 0.72±0.08 vs. 0.26±0.04, p-AMPK/GAPDH: 0.62±0.04, 0.60±0.01 vs. 0.20±0.04, all P < 0.05]. However, these protective effects of MET were inhibited by the addition of AMPK inhibitor CC solution. CONCLUSIONS: MET can effectively alleviate the degree of pulmonary fibrosis in mice poisoned with PQ, and its mechanism may be related to the activation of AMPK and inhibition of TGF-ß1/Smad3 signaling pathway, which can be inhibited by AMPK inhibitor CC.

Bisdemethoxycurcumin alleviates LPS-induced acute lung injury via activating AMPKα pathway.

OBJECTIVE: Inflammation and oxidative stress contribute to the pathogenesis of acute lung injury (ALI), and subsequently result in rapid deterioration in health. Considering the indispensable role of bisdemethoxycurcumin (BDMC) in inflammation and oxidative stress, the present study aims to examine the effect of BDMC on sepsis-related ALI. METHODS: C57BL/6 mice were administered with BDMC (100 mg/kg) or an equal volume of vehicle, and then injected with lipopolysaccharides (LPS) to induce ALI. We assessed the parameters of lung injury, inflammatory response and oxidative stress in lung tissues. Consistently, the macrophages with or without BDMC treatment were exposed to LPS to verify the effect of BDMC in vitro. RESULTS: BDMC suppressed LPS-induced lung injury, inflammation and oxidative stress in vivo and in vitro. Mechanistically, BDMC increased the phosphorylation of AMPKα in response to LPS stimulation, and AMPK inhibition with Compound C almost completely blunted the protective effect of BDMC in LPS-treated mice and macrophages. Moreover, we demonstrated that BDMC activated AMPKα via the cAMP/Epac pathway. CONCLUSION: Our study identifies the protective effect of BDMC against LPS-induced ALI, and the underlying mechanism may be related to the activation of cAMP/Epac/AMPKα signaling pathway.

Unraveling the mechanism of quercetin alleviating perfluorooctane sulfonate-induced apoptosis in grass carp (Ctenopharyngodon idellus) hepatocytes: AMPK/mTOR-mediated mitophagy.

Exposure to persistent new organic pollutants in the environment often leads to high mortality and causes serious economic losses to the aquaculture industry. Currently, perfluorooctane sulfonate (PFOS) is persistent and bio-accumulative in the environment, causing potential risks to aquatic ecosystems, but its toxicity mechanism to aquatic organisms is still unclear. As a natural flavonoid compound, quercetin (QU) has many biological activities such as anti-oxidation, anti-inflammatory, anti-apoptosis and immune regulation. Whether it can be used as a candidate medicine to alleviate PFOS toxicity needs to be further explored. Therefore, in this study, we treated (Ctenopharyngodon idellus) grass carp hepatocytes (L8824) with PFOS (200 µM) and/or QU (60 µM) for 24 h. The results showed that PFOS significantly increased the release of LDH and active oxygen (ROS) in L8824 cells, and led to the decrease of mitochondrial membrane potential (ΔΨm) and ATP content, the increase of mitochondrial ROS, the disorder of mitochondrial dynamics, and the initiation of Bcl-2/Bax-mediated apoptosis. Surprisingly, QU can alleviate the above PFOS-induced grass carp hepatocyte toxicity. In addition, in order to further explore the protective mechanism of QU, we used the molecular docking to predict the binding site between QU and AMPK, and found that there was a high binding capacity between QU and AMPK. In addition, we used Compound C (CC) and 3-Methyladenine (3-MA) to intervene. The results showed that CC and 3-MA intervention aggravated mitochondrial dysfunction and apoptosis factor expression in the QU+PFOS group. These data indicate that PFOS induces oxidative stress, mitochondrial dysfunction, and apoptosis. The regulation of AMPK/mTOR mediated mitophagy by QU may be a new therapeutic strategy to alleviate the hepatotoxicity of PFOS grass carp. This study provides theoretical basis and reference for exploring the toxic mechanism and biological toxic effects of PFOS, and provides a scheme for improving the economic benefits of aquaculture.

Polystyrene nanoplastic exposure induces excessive mitophagy by activating AMPK/ULK1 pathway in differentiated SH-SY5Y cells and dopaminergic neurons in vivo.

BACKGROUND: Microplastics and nanoplastics (MNPs) are emerging environmental contaminants detected in human samples, and have raised concerns regarding their potential risks to human health, particularly neurotoxicity. This study aimed to investigate the deleterious effects of polystyrene nanoplastics (PS-NPs, 50 nm) and understand their mechanisms in inducing Parkinson's disease (PD)-like neurodegeneration, along with exploring preventive strategies. METHODS: Following exposure to PS-NPs (0.5-500 µg/mL), we assessed cytotoxicity, mitochondrial integrity, ATP levels, and mitochondrial respiration in dopaminergic-differentiated SH-SY5Y cells. Molecular docking and dynamic simulations explored PS-NPs' interactions with mitochondrial complexes. We further probed mitophagy's pivotal role in PS-NP-induced mitochondrial damage and examined melatonin's ameliorative potential in vitro. We validated melatonin's intervention (intraperitoneal, 10 mg/kg/d) in C57BL/6 J mice exposed to 250 mg/kg/d of PS-NPs for 28 days. RESULTS: In our in vitro experiments, we observed PS-NP accumulation in cells, including mitochondria, leading to cell toxicity and reduced viability. Notably, antioxidant treatment failed to fully rescue viability, suggesting reactive oxygen species (ROS)-independent cytotoxicity. PS-NPs caused significant mitochondrial damage, characterized by altered morphology, reduced mitochondrial membrane potential, and decreased ATP production. Subsequent investigations pointed to PS-NP-induced disruption of mitochondrial respiration, potentially through interference with complex I (CI), a concept supported by molecular docking studies highlighting the influence of PS-NPs on CI. Rescue experiments using an AMPK pathway inhibitor (compound C) and an autophagy inhibitor (3-methyladenine) revealed that excessive mitophagy was induced through AMPK/ULK1 pathway activation, worsening mitochondrial damage and subsequent cell death in differentiated SH-SY5Y cells. Notably, we identified melatonin as a potential protective agent, capable of alleviating PS-NP-induced mitochondrial dysfunction. Lastly, our in vivo experiments demonstrated that melatonin could mitigate dopaminergic neuron loss and motor impairments by restoring mitophagy regulation in mice. CONCLUSIONS: Our study demonstrated that PS-NPs disrupt mitochondrial function by affecting CI, leading to excessive mitophagy through the AMPK/ULK1 pathway, causing dopaminergic neuron death. Melatonin can counteract PS-NP-induced mitochondrial dysfunction and motor impairments by regulating mitochondrial autophagy. These findings offer novel insights into the MNP-induced PD-like neurodegenerative mechanisms, and highlight melatonin's protective potential in mitigating the MNP's environmental risk.

Corilagin attenuates airway inflammation and collagen deposition in ovalbumin-induced asthmatic mice.

OBJECTIVE: To investigate the effects of corilagin on inflammation and collagen deposition in ovalbumin (OVA)-induced asthma mouse model and uncover the mechanism. METHODS: We constructed a mouse model of OVA-induced asthma. Enzyme-linked-immunosorbent serologic assays were conducted to detect the effects of corilagin on cytokines and Immunoglobulin E (IgE) production. Hematoxylin and eosin staining was used to show pathological features in lung tissues. Masson trichrome assay was used to examine collagen deposition. In addition, the lung function was detected by mouse lung function apparatus. Immunoblot was used to confirm the mechanism. RESULTS: Corilagin alleviates OVA-induced cytokine and IgE production. In addition, corilagin alleviates OVA-induced pathological changes and collagen deposition in lung tissues. Corilagin also suppressed airway resistance and lung function in mice. Mechanically, corilagin activated the adenosine monophosphate-activated protein kinase (AMPK) pathway in lung tissues. CONCLUSION: Corilagin attenuates airway inflammation and collagen deposition in OVA-induced asthmatic mice via AMPK pathway.

GPNMB Ameliorates Neuroinflammation Via the Modulation of AMPK/NFκB Signaling Pathway After SAH in Mice.

Glycoprotein non-metastatic melanoma protein B (GPNMB) got its name from the first discovery in a cell line of non-metastatic melanoma. Later studies found that GPNMB is widely expressed in various tissues and cells of the human body, most abundant in neural tissue, epithelial tissue, bone tissue, and monocyte-macrophage system. GPNMB has been shown to have anti-inflammatory effects in a variety of neurological diseases, however, it has not been reported in subarachnoid hemorrhage (SAH). Male CD-1 mice were used and intra-arterial puncture method was applied to establish the SAH model. Exogenous recombinant GPNMB (rGPNMB) was injected intracerebroventricularly 1 h after SAH. SAH grading, brain edema and blood-brain barrier (BBB) integrity were quantified, and neurobehavioral tests were performed to evaluate the effect of GPNMB on the outcome. Dorsomorphin, the selective inhibitor on AMPK was introduced to study the downstream signaling through which the GPNMB works. Furthermore, western blot, immunofluorescence staining and ELISA were utilized to confirm the signaling. After SAH, GPNMB expression increased significantly as a result of the inflammatory response. GPNMB was expressed extensively in mouse microglia, astrocytes and neurons. The administration of rGPNMB could alleviate brain edema, restore BBB integrity and improve the neurological outcome of mice with SAH. GPNMB treatment significantly magnified the expression of p-AMPK while p-NFκB, IL-1ß, IL-6 and TNF-α were suppressed; in the meantime, the combined administration of GPNMB and AMPK inhibitor could decrease the intensity of p-AMPK and reverse the quantity of p-NFκB and the above inflammatory cytokines. GPNMB has the potential of ameliorating the brain edema and neuroinflammation, protecting the BBB and improving the neurological outcome, possibly via the AMPK/NFκB signaling pathway.

[Platelet-rich Plasma Induces M2 Macrophage Polarization via Regulating AMPK Singling Pathway].

OBJECTIVE: To investigate the role of platelet-rich plasma (PRP) in inducing the M2 macrophage polarization via regulating AMPK singling pathway. METHODS: The expressions of M1 marker CD11c and M2 marker CD206 in macrophages of blank control group, LPS group, LPS+PRP group, and LPS+PRP+Compound C group were detected by flow cytometry. Western blot was used to observe the effects of PRP on the expression of AMPK-mTOR signaling pathway-related proteins at different times (12 h, 18 h and 24 h) after LPS treatment. RNA interference technology was used to silence the expression of AMPK in macrophages, and the expression of TGF-ß protein was subsequently examined by Western blot. RESULTS: LPS significantly reduced the expression of CD206 and increased the expression of CD11c (P <0.05). After the addition of PRP, the expression of CD206 was significantly increased (P <0.05), while the expression of CD11c was significantly decreased (P <0.05). Compared with LPS group, PRP treatment significantly increased the expressions of p-AMPK and p-ULK1 proteins at 12 h, 18 h and 24 h, while significantly decreased the expression of p-mTOR protein (P <0.05). After the addition of AMPK inhibitor Compound C, the expression of CD206 was significantly reduced (P <0.05) and the expression of CD11c was significantly increased compared with LPS+PRP group (P <0.05). After silencing the expression of AMPK in macrophages, the promotion effect of PRP on TGF-ß was significantly reduced (P <0.05). CONCLUSION: PRP can stimulate the transformation of macrophages to M2 type via AMPK signalling pathway.

Anxiolytic effect of antidiabetic metformin is mediated by AMPK activation in mPFC inhibitory neurons.

Diabetic patients receiving the antidiabetic drug metformin have been observed to exhibit a lower prevalence of anxiety disorders, yet the precise mechanism behind this phenomenon is unclear. In our study, we found that anxiety induces a region-specific reduction in AMPK activity in the medial prefrontal cortex (mPFC). Concurrently, transgenic mice with brain-specific AMPK knockout displayed abnormal anxiety-like behaviors. Treatment with metformin or the overexpression of AMPK restored normal AMPK activity in the mPFC and mitigated social stress-induced anxiety-like behaviors. Furthermore, the specific genetic deletion of AMPK in the mPFC not only instigated anxiety in mice but also nullified the anxiolytic effects of metformin. Brain slice recordings revealed that GABAergic excitation and the resulting inhibitory inputs to mPFC pyramidal neurons were selectively diminished in stressed mice. This reduction led to an excitation-inhibition imbalance, which was effectively reversed by metformin treatment or AMPK overexpression. Moreover, the genetic deletion of AMPK in the mPFC resulted in a similar defect in GABAergic inhibitory transmission and a consequent hypo-inhibition of mPFC pyramidal neurons. We also generated a mouse model with AMPK knockout specific to GABAergic neurons. The anxiety-like behaviors in this transgenic mouse demonstrated the unique role of AMPK in the GABAergic system in relation to anxiety. Therefore, our findings suggest that the activation of AMPK in mPFC inhibitory neurons underlies the anxiolytic effects of metformin, highlighting the potential of this primary antidiabetic drug as a therapeutic option for treating anxiety disorders.