Atmospheric pressure plasma preconditioning reduces oxygen and glucose deprivation induced human neuronal SH-SY5Y cells apoptosis by activating protective autophagy and ROS/AMPK/mTOR pathway.

Reactive oxygen species (ROS)/reactive nitrogen species (RNS) exert a "double edged" effect on the occurrence and development of ischemic stroke. We previously indicate that atmospheric pressure plasma (APP) shows a neuroprotective effect in vitro based on the ROS/RNS generations. However, the mechanism is still unknown. In this work, SH-SY5Y cells were treated with oxygen and glucose deprivation (OGD) injuries for stimulating the ischemic stroke pathological injury process. A helium APP was used for SH-SY5Y cell treatment for evaluating the neuroprotective impacts of APP preconditioning against OGD injuries with the optimized parameters. During the preconditioning, APP significantly raised the extracellular and intracellular ROS/RNS production. As a result, APP preconditioning increased SH-SY5Y cell autophagy by elevating LC3-II/LC3-I ratio and autophagosome formation. Meanwhile, APP preconditioning reduced cell apoptosis caused by OGD with the increased APP treatment time, which was abolished by pretreatment with autophagy inhibitor 3-methyladenine (3-MA). The ROS scavenger N-acetyl-L-cysteine (NAC) alone or combined with NO scavenger carboxy-PTIO abolished the APP preconditioning induced SH-SY5Y autophagy and the cytoprotection, whereas the NO scavenger alone did not. In addition, we observed the elevated phosphorylation of AMP-activated protein kinase (AMPK) and decreased phosphorylation of mammalian target of rapamycin (mTOR) in APP treated SH-SY5Y cells. This effect was attenuated by AMPK inhibitor Compound C (CC), the ROS scavenger NAC and autophagy inhibitor 3-MA. Furthermore, the cytoprotective effect of APP was preliminarily confirmed in the rats of middle cerebral artery occlusion (MCAO) model. Results showed that APP inhalation by rats during MCAO process could improve neurological functions, reduce cell apoptosis in brain tissues and decrease cerebral infarct volume. Our data suggested that ROS produced by APP preconditioning played a vital role in the neuroprotective effect of SH-SY5Y cells against OGD injuries by activating autophagy and ROS/AMPK/mTOR pathway.

Cai's herbal tea enhances mitochondrial autophagy of type 1 diabetic mellitus ß cells through the AMPK/mTOR pathway and alleviates inflammatory response.

BACKGROUND: This study investigates the therapeutic mechanisms of Cai's Herbal Tea in Type 1 Diabetes Mellitus (T1DM) mice, focusing on its effects on mitochondrial change and autophagy via the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway. METHODS: The composition of Cai's Herbal Tea was analyzed by Ultra-High Performance Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry (UHPLC-Q/TOF-MS). C57BL/6 mice and Min6 pancreatic beta cells were divided into control, diabetic mellitus (DM)/high glucose (HG), and treatment groups (low, medium, and high doses of Cai's Tea, and Metformin). Key physiological parameters, pancreatic islet health, Min6 cell morphology, viability, and insulin (INS) secretion were assessed. Small Interfering RNA-AMPK (si-AMPK) was utilized to confirm the pathway involvement. RESULTS: Cai's Herbal Tea improved body weight, pancreatic islet pathological injury, and INS secretion whereas reduced total triglycerides, fasting blood sugar, and Interferon gamma (INF-γ) in T1DM mice, particularly at higher doses. In Min6 cells, Cai's Tea mitigated HG-induced damage and proinflammatory response, enhancing cell viability and INS secretion. Notably, it reduced swelling and improved cristae structure in treated groups of mitochondria and promoted autophagy via the AMPK-mTOR pathway, evidenced by increased LC3II/LC3I and P-AMPK/AMPK ratios, and decreased P-mTOR/mTOR and P62 expressions in pancreatic islet ß-cells. Furthermore, these effects were converted by si-AMPK interference. CONCLUSION: Cai's Herbal Tea exhibits significant therapeutic efficacy in T1DM mice by improving mitochondrial health and inducing autophagy through the AMPK-mTOR pathway in pancreatic islet ß-cells. These findings highlight its potential as a therapeutic approach for T1DM management.

Baicalin alleviates angiotensin II-induced cardiomyocyte apoptosis and autophagy and modulates the AMPK/mTOR pathway.

As a main extraction compound from Scutellaria baicalensis Georgi, Baicalin exhibits various biological activities. However, the underlying mechanism of Baicalin on hypertension-induced heart injury remains unclear. In vivo, mice were infused with angiotensin II (Ang II; 500 ng/kg/min) or saline using osmotic pumps, followed by intragastrically administrated with Baicalin (5 mg/kg/day) for 4 weeks. In vitro, H9C2 cells were stimulated with Ang II (1 µM) and treated with Baicalin (12.5, 25 and 50 µM). Baicalin treatment significantly attenuated the decrease in left ventricular ejection fraction and left ventricular fractional shortening, increase in left ventricular mass, left ventricular systolic volume and left ventricular diastolic volume of Ang II infused mice. Moreover, Baicalin treatment reversed 314 differentially expressed transcripts in the cardiac tissues of Ang II infused mice, and enriched multiple enriched signalling pathways (including apoptosis, autophagy, AMPK/mTOR signalling pathway). Consistently, Baicalin treatment significantly alleviated Ang II-induced cell apoptosis in vivo and in vitro. Baicalin treatment reversed the up-regulation of Bax, cleaved-caspase 3, cleaved-caspase 9, and the down-regulation of Bcl-2. Meanwhile, Baicalin treatment alleviated Ang II-induced increase of autophagosomes, restored autophagic flux, and down-regulated LC3II, Beclin 1, as well as up-regulated SQSTM1/p62 expression. Furthermore, autophagy inhibitor 3-methyladenine treatment alleviated the increase of autophagosomes and the up-regulation of Beclin 1, LC3II, Bax, cleaved-caspase 3, cleaved-caspase 9, down-regulation of SQSTM1/p62 and Bcl-2 expression after Ang II treated, which similar to co-treatment with Baicalin. Baicalin treatment reduced the ratio of p-AMPK/AMPK, while increased the ratio of p-mTOR/mTOR. Baicalin alleviated Ang II-induced cardiomyocyte apoptosis and autophagy, which might be related to the inhibition of the AMPK/mTOR pathway.

PMAIP1 regulates autophagy in osteoblasts via the AMPK/mTOR pathway in osteoporosis.

Osteoporosis (OP) is a highly prevalent disorder characterized by low bone mass that severely reduces patient quality of life. Although numerous treatments for OP have been introduced in clinic, many have side effects and high costs. Therefore, there is still an unmet need for optimal solutions. Here, raw signal analysis was used to identify potential high-risk factors for OP, and the biological functions and possible mechanisms of action (MOAs) of these factors were explored via gene set enrichment analysis (GSEA). Subsequently, molecular biological experiments were performed to verify and analyze the discovered risk factors in vitro and in vivo. PMAIP1 was identified as a potential risk factor for OP and significantly suppressed autophagy in osteoblasts via the AMPK/mTOR pathway, thereby inhibiting the proliferation and differentiation of osteoblasts. Furthermore, we constructed an ovariectomy (OVX) model of OP in rats and simultaneously applied si-PMAIP1 for in vivo interference. si-PMAIP1 upregulated the expression of LC3B and p-AMPK and downregulated the expression of p-mTOR, and these effects were reversed by the autophagy inhibitor. Micro-CT revealed that, si-PMAIP1 significantly inhibited the development of osteoporosis in OVX model rats, and this therapeutic effect was attenuated by treatment with an autophagy inhibitor. This study explored the role and mechanism of PMAIP1 in OP and demonstrated that PMAIP1 may serve as a novel target for OP treatment.

Ghrelin Induces the Production of Hypothalamic NPY Through the AMPK-mTOR Pathway to Alleviate Cancer-induced Bone Pain.

BACKGROUND/AIM: Cancer-induced bone pain (CIBP) is one of the most common symptoms of bone metastasis of tumor cells. The hypothalamus may play a pivotal role in the regulation of CIBP. However, little is known about the exact mechanisms. MATERIALS AND METHODS: First, we established a CIBP model to explore the relationship among hypothalamic ghrelin, NPY and CIBP. Then, we exogenously administered NPY and NPY receptor antagonists to investigate whether hypothalamic NPY exerted an antinociceptive effect through binding to NPY receptors. Finally, we exogenously administered ghrelin to investigate whether ghrelin alleviated CIBP by inducing the production of hypothalamic NPY through the AMPK-mTOR pathway. Body weight, food intake and behavioral indicators of CIBP were measured every 3 days. Hypothalamic ghrelin, NPY and the AMPK-mTOR pathway were also measured. RESULTS: The expression of hypothalamic ghrelin and NPY was simultaneously decreased in cancer-bearing rats, which was accompanied by CIBP. Intracerebroventricular (i.c.v.) administration of NPY significantly alleviated CIBP in the short term. The antinociceptive effect of NPY was reversed with the i.c.v. administration of the Y1R and Y2R antagonists. The administration of ghrelin activated the AMPK-mTOR pathway and induced hypothalamic NPY production to alleviate CIBP. This effect of ghrelin on NPY and antinociception was reversed with the administration of a GHS-R1α antagonist. CONCLUSION: Ghrelin could induce the production of hypothalamic NPY through the AMPK-mTOR pathway to alleviate CIBP, which can provide a novel therapeutic mechanism for CIBP.

Enriched oxygen improves age-related cognitive impairment through enhancing autophagy.

Age-related cognitive impairment represents a significant health concern, with the understanding of its underlying mechanisms and potential interventions being of paramount importance. This study aimed to investigate the effects of hyperbaric oxygen therapy (HBOT) on cognitive function and neuronal integrity in aged (22-month-old) C57BL/6 mice. Male mice were exposed to HBOT for 2 weeks, and spatial learning and memory abilities were assessed using the Morris water maze. We employed transcriptome sequencing and Gene Ontology (GO) term enrichment analysis to examine the effects of HBOT on gene expression profiles, with particular attention given to synapse-related genes. Our data indicated a significant upregulation of postsynapse organization, synapse organization, and axonogenesis GO terms, likely contributing to improved cognitive performance. Moreover, the hyperphosphorylation of tau, a hallmark of many neurodegenerative diseases, was significantly reduced in the HBO-treated group, both in vivo and in vitro. Transmission electron microscopy revealed significant ultrastructural alterations in the hippocampus of the HBOT group, including an increase in the number of synapses and the size of the active zone, a reduction in demyelinated lesions, and a decreased number of "PANTHOS." Furthermore, Western blot analyses confirmed the upregulation of PSD95, BDNF, and Syn proteins, suggesting enhanced synaptic plasticity and neurotrophic support. Moreover, HBOT increased autophagy, as evidenced by the elevated levels of Beclin-1 and LC3 proteins and the reduced level of p62 protein. Finally, we demonstrated that HBOT activated the AMPK-mTOR signaling pathway, a critical regulator of autophagy. Notably, our findings provide novel insights into the mechanisms by which HBOT ameliorates age-related cognitive impairment, suggesting the potential therapeutic value of this approach.

Asperuloside alleviates cyclophosphamide-induced myelosuppression by promoting AMPK/mTOR pathway-mediated autophagy.

Cyclophosphamide (CTX) is a common anticancer chemotherapy drug, and myelosuppression is the most common serious side effect. Asperuloside (ASP), the active component of Hedyotis diffusa Willd., may have the effect of ameliorating chemotherapy-induced myelosuppression. This study aimed to explore the effect and possible mechanism of ASP on CTX-induced myelosuppression. Male SPF C57BL/6 mice were randomly divided into five groups: control group, CTX (25 mg/kg) group, CTX + granulocyte-macrophage-colony stimulating factor (GM-CSF) (5 µg/kg) group, CTX + high-dose ASP (50 mg/kg) group and CTX + low-dose ASP (25 mg/kg) group, with six mice in each group. The body weight of mice was monitored every other day, the hematopoietic progenitor cell colony number was measured by colony forming unit, and the relevant blood indicators were detected. Femoral bone marrow was observed by hematoxylin-eosin, C-kit expression was detected by immunohistochemistry, and autophagy and adenine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway protein expressions were detected by immunohistochemistry and western blotting (WB). Then the AMPK inhibitor dorsomorphin was used to interfere with AMPK/mTOR pathway. Results showed that ASP significantly increased the body weight of CTX-induced mice, increased the number of hematopoietic progenitor cells, the expression of white blood cells, red blood cells, platelets, GM-CSF, thrombopoietin and erythropoietin in blood, and the expression of C-kit in bone marrow. In addition, ASP further promoted the expression of Beclin1 and LC-3II/I induced by CTX, and regulated the protein expressions in the AMPK/mTOR pathway. The use of dorsomorphin inhibited the alleviation effect of ASP on CTX-induced myelosuppression and the promotion effect of ASP on autophagy. In conclusion, ASP alleviated CTX-induced myelosuppression by promoting AMPK/mTOR pathway-mediated autophagy.

Effects of intermittent cold stimulation on growth performance, meat quality, antioxidant capacity and liver lipid metabolism in broiler chickens.

Intermittent cold stimulation (ICS) enhances broilers' resistance to cold stress. Nonetheless, further research is needed to investigate the underlying mechanisms that enhance cold stress resistance. A total of 160 one-day-old male Ross 308 broilers were randomly divided into 2 groups (CC and CS5), with the CC group managing temperature according to the standard for broiler growth stages, while the CS5 group were subjected to cold stimulation at a temperature 3℃ lower than the CC group for 5 h, every 2 d from 15 to 35 d. Sampling was conducted at 36 d (36D), 50 d (50D) and after acute cold stress for 24 h (Y24). First, we examined the effects of ICS on broiler growth performance, meat quality, antioxidant capacity, and lipid metabolism. The results demonstrated that ICS enhanced the performance of broilers to a certain degree. Specifically, the average weight gain in the CS5 group was significantly higher than that of the CC group, and the feed conversion ratio significantly decreased compared to CC at 4 W and 6 W (P ≤ 0.05). Compared with the CC group, cold stimulation significantly reduced drip loss, shearing force, and yellowness (a* value) of chicken meat, while significantly increased redness (b* value) (P ≤ 0.05). At Y24, the levels of T-AOC and GSH-PX in the serum of the CS5 group were significantly higher than those of the CC group, while the level of MDA was significantly lower (P ≤ 0.05). The content of TG, FFA, and VLDL in the serum of the CS5 group was significantly elevated, whereas the level of TC and HDL was significantly lower (P ≤ 0.05). In addition, we further explored whether AMPK-mTOR pathway is involved in the regulation of changes in lipid metabolism and the possible regulatory mechanisms downstream of the signaling pathway. The results showed that ICS significantly upregulated the expression levels of AMPK mRNA and protein in the liver of the CS5 group at 36D and Y24, while significantly down-regulating mTOR (P ≤ 0.05). Compared with the CC group, ICS significantly down-regulated the mRNA expression levels of lipid synthesis and endoplasmic reticulum stress-related genes (SREBP1c, FAS, SCD, ACC, GRP78 and PERK) at 36D and Y24, while significantly up-regulating the mRNA expression levels of lipid decomposition and autophagy-related genes (PPAR and LC3) (P ≤ 0.05). In addition, at Y24, the protein expression levels of endoplasmic reticulum stress-related genes (GRP78) in the CS5 group were significantly lower, while autophagy-related genes (LC3 and ATG7) were significantly higher (P ≤ 0.05). ICS can affect meat quality and lipid metabolism in broilers, and when broilers are subjected to acute cold stress, broilers trained with cold stimulation have stronger lipid metabolism capacity.

Jiangqi Pingxiao formula regulates dendritic cell apoptosis in an autophagy-dependent manner through the AMPK/mTOR pathway in a murine model of OVA-induced asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Allergic asthma is a recurring respiratory condition that typically manifests during childhood or adolescence. It is characterized by a dominant type II immune response triggered by the identification and capturing of inhaled allergens by dendritic cells (DCs). Jiangqi Pingxiao Formula (JQPXF), a prescription medicine used for the treatment of pediatric asthma, has been clinically proven to be both safe and effective. However, its mechanism of action in the treatment of asthma has not been fully been fully elucidated. Recent research suggests that several natural compounds have the potential to target dendritic cells (DCs) and alleviate ovalbumin (OVA)-induced asthma, which may also be found within JQPXF. AIM OF THE STUDY: This study aimed to elucidate the effect of JQPXF on OVA-induced asthma model and its molecular mechanism targeting DCs. MATERIALS AND METHODS: The main constituents of JQPXF were analyzed by ultra performance liquid chromatography (UPLC). An asthma model was established by OVA. Hematoxylin-eosin staining and measurement of respiratory function was used to evaluate the treatment effect of JQPXF on asthmatic mice. Cytokine (IL-5, IL-13 and IgE) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was employed to evaluate inflammatory cell infiltration (T helper 2 cells and DCs) in vivo and DC survival in vivo and vitro. Western blot and immunofluorescence were used to verify the molecular mechanisms. RESULTS: The results suggest that JQPXF can ameliorate pathological conditions and improve lung function in asthmatic mice, as well as the Th2 cells. Treatment with JQPXF significantly reduced the number of DCs and increased the number of Propidium iodide+ (PI) DCs. Furthermore, JQPXF upregulated protein levels of the pro-apoptotic factors Cleaved-caspase-3 and Bax, while downregulating the anti-apoptotic factor Bcl-2. Simultaneously, JQPXF increased autophagy levels by facilitating p62 degradation and promoting translation from LC3B I to LC3B II of DCs in vitro, as well as reducing the integrated optical density (IOD) of p62 within the CD11c-positive area in the lung. 3-Methyladenine (3-MA) was used to block autophagic flux and the apoptotic effect of JQPXF on DCs was abolished in vitro, with the number of DCs decreased by JQPXF being reversed in vivo. We further investigated the upstream key regulator of autophagy, the AMPK/mTOR pathway, and found that JQPXF increased AMPK phosphorylation while decreasing mTOR phosphorylation levels. Additionally, we employed Compound C (CC) as an AMPK inhibitor to inhibit this signaling pathway, and our findings revealed that both autophagic flux and apoptotic levels in DCs were abolished in vitro. CONCLUSIONS: In summary, we have demonstrated that JQPXF could alleviate type II inflammation in an asthmatic model by promoting the apoptosis of DCs through an autophagy-dependent mechanism, achieved by regulating the AMPK/mTOR signaling pathway.

Atorvastatin Calcium Ameliorates Cognitive Deficits Through the AMPK/Mtor Pathway in Rats with Vascular Dementia.

AIM: In this study, the protective effects of atorvastatin calcium (AC) on nerve cells and cognitive improvement in vivo and in vitro were investigated by establishing cell models and vascular dementia (VD) rat models. BACKGROUND: VD is a neurodegenerative disease characterized by cognitive deficits caused by chronic cerebral hypoperfusion. AC has been studied for its potential to cure VD but its efficacy and underlying mechanism are still unclear. OBJECTIVE: The mechanism of action of AC on cognitive deficits in the early stages of VD is unclear. Here, the 2-vessel occlusion (2-VO) model in vivo and the hypoxia/reoxygenation (H/R) cell model in vitro was established to investigate the function of AC in VD. METHODS: The spatial learning and memory abilities of rats were detected by the Morris method. The IL-6, tumour necrosis factor-α (TNF-α), malondialdehyde (MDA) and superoxide dismutase (SOD) in cell supernatant was tested by ELISA kits. After behavioural experiments, rats were anaesthetized and sacrificed, and their brains were extracted. One part was immediately fixed in 4% paraformaldehyde for H&E, Nissl, and immunohistochemical analyses, and the other was stored in liquid nitrogen. All data were shown as mean ± SD. Statistical comparison between the two groups was performed by Student's t-test. A two-way ANOVA test using GraphPad Prism 7 was applied for escape latency analysis and the swimming speed test. The difference was considered statistically significant at p < 0.05. RESULTS: AC decreased apoptosis, increased autophagy, and alleviated oxidative stress in primary hippocampal neurons. AC regulated autophagy-related proteins in vitro by western blotting. VD mice improved cognitively in the Morris water maze. Spatial probing tests showed that VD animals administered AC had considerably longer swimming times to the platform than VD rats. H&E and Nissl staining showed that AC reduces neuronal damage in VD rats. Western blot and qRT-PCR indicated that AC in VD rats inhibited Bax and promoted LC3-II, Beclin-1, and Bcl-2 in the hippocampus region. AC also improves cognition via the AMPK/mTOR pathway. CONCLUSION: This study found that AC may relieve learning and memory deficits as well as neuronal damage in VD rats by changing the expression of apoptosis/autophagy-related genes and activating the AMPK/mTOR signalling pathway in neurons.

Exosomes from adipose-derived stem cells alleviate premature ovarian failure via blockage of autophagy and AMPK/mTOR pathway.

Objective: The objective of this study was to investigate the effects and mechanisms of adipose-derived stem cell-derived exosome (ADSCs-Exo) in treating premature ovarian failure (POF). Methods: We constructed a POF mouse model through intraperitoneal injection of cyclophosphamide, followed by the administration of the autophagy inhibitor 3-methyladenine (3-MA). Pathological injury, follicle stimulating hormone (FSH), malondialdehyde (MDA), reactive oxygen species (ROS), estradiol (E2), superoxide dismutase (SOD), granulosa cell (GC) apoptosis, and autophagy were assessed. Exosomes isolated from ADSCs were used to treat POF in mice. The AMPK-mTOR pathway and its proteins (p-AMPK and p-mTOR) were evaluated. A POF cell model was established using cyclophosphamide-treated human ovarian granulosa-like tumor (KGN) cells. We administered ADSCs-Exo and rapamycin to validate the mechanism of ADSCs-Exo against POF. Results: In POF mice, 3-MA treatment attenuated pathological injuries, decreased FSH, MDA, and ROS levels, and increased E2 and SOD levels. 3-MA treatment also inhibited GC apoptosis and autophagy. ADSCs-Exo alleviated pathological injuries, improved ovarian morphology and function, and reduced oxidative stress in POF mice. ADSCs-Exo inhibited GC apoptosis and autophagy. ADSCs-Exo downregulated the expression of AMPK/mTOR pathway proteins (p-AMPK and p-mTOR). In the POF cell model, ADSCs-Exo and rapamycin inhibited AMPK/mTOR-mediated autophagy. Conclusion: ADSCs-Exo inhibits POF through the inhibition of autophagy and the AMPK/mTOR pathway. This study provides a potential target for the clinical treatment of POF.

3,5-diCQA suppresses colorectal cancer cell growth through ROS/AMPK/mTOR mediated mitochondrial dysfunction and ferroptosis.

3,5-diCQA has been shown to have anti-tumor effect by decreasing cancer cell growth. However, the molecular mechanism by which 3,5-diCQA impacts colorectal cancer (CRC) cells is unknown. This study discovered that 3,5-diCQA had a suppressive effect on CRC cells, mainly in the inhibition of proliferation, migration, and the enhancement of apoptosis in HCT116 and SW480 cells. Additionally, 3,5-diCQA was found to cause cell cycle arrest in CRC cells. Meanwhile, we found that 3,5-diCQA activates the AMPK pathway through the generation of ROS, mediates mitochondrial damage, and reduces mitochondrial aerobic glycolysis and oxidative phosphorylation levels. 3,5-diCQA promoted oxidative damage and ferroptosis in CRC cells. Hence, we added ROS inhibitor NAC and found that the NAC reversed the effects of 3,5-diCQA on proliferation, apoptosis, ROS generation, and ferroptosis in CRC cells. Moreover, 3,5-diCQA was also shown to suppress the development of CRC tumor in a tumor-forming model of nude mice. In conclusion, we found that 3,5-diCQA enhances the oxidative damage and ferroptosis while reducing proliferation and migration of CRC cells, depending on mitochondrial dysfunction caused by the ROS/AMPK/mTOR pathway.

PLCL1 suppresses tumour progression by regulating AMPK/mTOR-mediated autophagy in renal cell carcinoma.

Autophagy has been increasingly recognized as a critical regulatory mechanism in the maintenance of cellular homeostasis. A previous study showed that phospholipase C-like protein 1 (PLCL1) is associated with lipid metabolism in renal cell carcinoma (RCC). However, it is unclear whether PLCL1 regulates autophagy, thereby influencing the progression of RCC. Bioinformatics analysis of five microarray datasets revealed that expression of PLCL1 is decreased in tumours and is positively correlated with prognosis in RCC patients. Three independent public datasets, clinical RCC tissues and RCC cell lines, were validated using real-time qPCR, western blotting and immunohistochemistry. Using wound healing and transwell assays, we observed that elevated PLCL1 levels decreased the migratory distance and the invasive number of 786-O and ACHN cells, but PLCL1 knockdown reversed these changes in 769P cell lines compared to those in controls. The results of flow cytometry analysis indicated that PLCL1 promotes apoptosis. Moreover, transcriptional analysis based on stable overexpression of PLCL1 in 786-O cells revealed that PLCL1 is related to autophagy, and western blotting and autophagic experimental results further verified these findings. Mechanistic investigations confirmed that PLCL1 activates the AMPK/mTOR pathway and interacts with decidual protein induced by progesterone (DEPP). Collectively, our data suggest that PLCL1 functions as a suppressor of RCC progression by activating the AMPK/mTOR pathway, interacting with DEPP, initiating autophagy and inducing apoptosis. PLCL1 may be a promising therapeutic target for the diagnosis and treatment of ccRCC patients.

MiR-182/Sestrin2 affects the function of asthmatic airway smooth muscle cells by the AMPK/mTOR pathway.

Background and Objectives: Asthma is a chronic inflammatory airway disease and brings heavy economic and spiritual burdens to patients' families and the society. Airway smooth muscle cells (ASMCs) afect the development of asthma by secreting cytokines, growth factors, and prostates. The stress-inducing protein, Sestrin2, plays a vital role in antioxidant defense. The aim of this study is to investigate the role of Sestrin2 in asthma and its corresponding molecular mechanism. Materials and Methods: Airway remodeling was induced by construction of asthma rat model. Primary ASMCs were isolated through combining tissue block adherence and enzymatic digestion and identified by immunofluorescence staining. Gene expression was measured by quantitative real-time PCR (qPCR) and western blot (WB) experiments. Cell viability, proliferation, migration, and calcium flow of ASMCs were measured by Cell Counting Kit-8 (CCK-8), 5-ethynyl-deoxyuridine (EdU), Transwell, and Fluo-3AM, respectively. The binding of miR-182 and Sestrin2 3'-untranslated region (3'-UTR) was measured by luciferase reporter system and RNA-binding protein immunoprecipitation (RIP) analysis. Results: Sestrin2 expression was upregulated in asthma rat model and cell model. Overexpression of Sestrin2 enhanced the growth, migration, and calcium flow, and inversely, repression of Sestrin2 was reduced in ASMCs from the asthma group. MiR-182, one of the microRNAs (miRNAs) that possesses the potential to regulate Sestrin2, was downregulated in ASMCs from the asthma group. Further experiments revealed that Sestrin2 was inhibited by miR-182 and that overexpression of Sestrin2 reversed the miR-182-induced inhibition of the cellular progression of ASMCs from the asthma group. This study further investigated the downstream signaling pathway of Sestrin2 and found that increased expression of Sestrin2 activated 5'-adenosine monophosphate-activated protein kinase (AMPK), leading to the inactivation of mammalian target of rapamycin (mTOR) and thus promoting the growth, migration, and calcium flow of ASMCs from the asthma group. Conclusion: This study investigated the role of Sestrin2 for the first time and further dissected the regulatory factor of Sestrin2, ultimately elucidating the downstream signaling pathway of Sestrin2 in asthma, providing a novel pathway, and improving the understanding of the development and progression of asthma.

DPP-4 Inhibitors Suppress Tau Phosphorylation and Promote Neuron Autophagy through the AMPK/mTOR Pathway to Ameliorate Cognitive Dysfunction in Diabetic Mellitus.

Dipeptidyl peptidase-4 (DPP-4) inhibitors have been considered as incretin-based agents that signal through GLP-1R. Our high-throughput RNA sequencing (RNA-seq) and bioinformatics methods indicated that GLP-1R, downregulated in diabetes mellitus (DM), was a potential target of DPP-4 inhibitors, which was further confirmed in DM rats. Thus, this study illuminated the alleviatory mechanism of DPP-4 on cognitive dysfunction in diabetes mellitus (DM), which may be associated with GLP-1R signaling. DM rats were administered with DPP-4 inhibitors, Chloroquine (an autophagy inhibitor), Exendin 9-39 (a GLP-1R antagonist), or Compound C (a specific inhibitor of AMPK). An in vitro model of DM was induced in rat hippocampal neuronal cell line H19-7 by exposure to high glucose (HG) and high fat (HF), followed by treatment with the above inhibitors and antagonists. It was found that cognitive dysfunction was promoted, and LC3 expression was lowered in DM rats by an autophagy inhibitor. The DPP-4 inhibitors decreased cognitive dysfunction, repressed Tau phosphorylation, and enhanced GLP-1R protein level, LC3 expression, and AMPK and mTOR phosphorylation in DM rats, while GLP-1R antagonist, an autophagy inhibitor, or AMPK inhibitor counteracted these effects. Such effects were also observed in HG/HF-induced neurons. In conclusion, our data elucidated the alleviatory mechanism of DPP-4 inhibitors in the cognitive dysfunction of DM rats via the AMPK/mTOR pathway.

Lycium barbarum polysaccharide inhibits ischemia-induced autophagy by promoting the biogenesis of neural stem cells-derived extracellular vesicles to enhance the delivery of miR-133a-3p.

BACKGROUND: Neural stem cell-derived extracellular vesicles (NSC-EVs) mediated endogenous neurogenesis determines a crucial impact on spontaneous recovery after stroke. Here, we checked the influence of Lycium barbarum polysaccharide (LBP) on the biogenesis of NSC-EVs and then focused on studying mechanisms of LBP in ameliorating ischemic stroke outcome. METHODS: LBP was prepared to precondition NSCs and isolate EVs. MCAO models and primary NSCs were administrated to evaluate the therapeutic effect. RT-PCR, western blot, flow cytometry, and immunofluorescence techniques were performed to explore the mechanism. RESULTS: LBP pretreatment increased the production of NSC-EVs and improved the neuroprotective and recovery effects of NSC-EV in ischemic stroke mice. LBP-pretreated NSC-EV in a dose-dependent manner substantially reduced neuronal death compared with NSC-EV. Screening of the signaling cascade involved in the interaction between NSC-EV and neurons revealed that AMPK/mTOR signaling pathway inhibited autophagic activity in neurons receiving either treatment paradigm. NSC-EVs but not EVs collected from NSCs pretreated with the anti-miR-133a-3p oligonucleotide reduced cell death, whereas the anti-oligonucleotide promoted autophagy activity and cell death by modulating AMPK/mTOR signaling in OGD-induced primary neurons. CONCLUSION: LBP activated AMPK/mTOR signaling pathway by increasing the enrichment and transfer of miR-133a-3p in NSC-EVs to inhibit stroke-induced autophagy activity.

Glutathione Peroxidase 3 induced mitochondria-mediated apoptosis via AMPK /ERK1/2 pathway and resisted autophagy-related ferroptosis via AMPK/mTOR pathway in hyperplastic prostate.

BACKGROUND: Benign prostatic hyperplasia (BPH) is a common disease in elderly men, mainly resulted from an imbalance between cell proliferation and death. Glutathione peroxidase 3 (GPX3) was one of the differentially expressed genes in BPH identified by transcriptome sequencing of 5 hyperplastic and 3 normal prostate specimens, which had not been elucidated in the prostate. This study aimed to ascertain the mechanism of GPX3 involved in cell proliferation, apoptosis, autophagy and ferroptosis in BPH. METHODS: Human prostate tissues, GPX3 silencing and overexpression prostate cell (BPH-1 and WPMY-1) models and testosterone-induced rat BPH (T-BPH) model were utilized. The qRT-PCR, CCK8 assay, flow cytometry, Western blotting, immunofluorescence, hematoxylin and eosin, masson's trichrome, immunohistochemical staining and transmission electron microscopy analysis were performed during in vivo and in vitro experiments. RESULTS: Our study indicated that GPX3 was localized both in the stroma and epithelium of prostate, and down-regulated in BPH samples. Overexpression of GPX3 inhibited AMPK and activated ERK1/2 pathway, thereby inducing mitochondria-dependent apoptosis and G0/G1 phase arrest, which could be significantly reversed by MEK1/2 inhibitor U0126 preconditioning. Moreover, overexpression of GPX3 further exerted anti-autophagy by inhibiting AMPK/m-TOR and up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4, mitochondrial GPX4 and cytoplasmic GPX4) to antagonize autophagy-related ferroptosis. Consistently, GPX3 deficiency generated opposite changes in both cell lines. Finally, T-BPH rat model was treated with GPX3 indirect agonist troglitazone (TRO) or GPX4 inhibitor RAS-selective lethal 3 (RSL3) or TRO plus RSL3. These treatments produced significant atrophy of the prostate and related molecular changes were similar to our in vitro observations. CONCLUSIONS: Our novel data manifested that GPX3, which was capable of inducing apoptosis via AMPK/ERK1/2 pathway and antagonizing autophagy-related ferroptosis through AMPK/m-TOR signalling, was a promising therapeutic target for BPH in the future.

Trpm2 deficiency in microglia attenuates neuroinflammation during epileptogenesis by upregulating autophagy via the AMPK/mTOR pathway.

Epilepsy is one of the most common neurological disorders. Neuroinflammation involving the activation of microglia and astrocytes constitutes an important and common mechanism in epileptogenesis. Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, non-selective cation channel that plays pathological roles in various inflammation-related diseases. Our previous study demonstrated that Trpm2 knockout exhibits therapeutic effects on pilocarpine-induced glial activation and neuroinflammation. However, whether TRPM2 in microglia and astrocytes plays a common pathogenic role in this process and the underlying molecular mechanisms remained undetermined. Here, we demonstrate a previously unknown role for microglial TRPM2 in epileptogenesis. Trpm2 knockout in microglia attenuated kainic acid (KA)-induced glial activation, inflammatory cytokines production and hippocampal paroxysmal discharges, whereas Trpm2 knockout in astrocytes exhibited no significant effects. Furthermore, we discovered that these therapeutic effects were mediated by upregulated autophagy via the adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway in microglia. Thus, our findings highlight an important deleterious role of microglial TRPM2 in temporal lobe epilepsy.

CangFu Daotan decoction improves polycystic ovarian syndrome by downregulating FOXK1.

Objective: Polycystic ovarian syndrome (PCOS) is a prevalent gynecologic disorder, often associated with abnormal follicular development. Cangfu Daotan decoction (CFD) is a traditional Chinese medicine formula that is effective in alleviating PCOS clinically, but the specific mechanism remains unclear. Forkhead box K1 (FOXK1) is associated with cellular function. This study aimed to explore the effects of CFD and FOXK1 on PCOS.Methods: High-fat diet and letrozole were combined to establish PCOS rat models. Next, primary GCs were extracted from those PCOS rats. Then, GC cells were transfected with si-FOXK1 or oe-FOXK1. CFD-contain serum was prepared, and experiments were conducted to investigate the regulation of FOXK1 by CFD.Results: FOXK1 was highly expressed in GCs of PCOS rats. Further investigation revealed that FOXK1 overexpression resulted in inhibition of proliferation and DNA synthesis, along with promotion of apoptosis and autophagy in GCs. Additionally, it was found that FOXK1 promoted the expressions of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway-related proteins. Interestingly, treatment with CFD reversed all the effects of FOXK1 overexpression in GCs. Conclusion: This study demonstrated that CFD exerted a protective role in PCOS by inhibiting FOXK1, which provided a research basis for the application of CFD in PCOS, and suggested that FOXK1 is a novel therapeutic target in PCOS treatment.

COTE-1 promotes the proliferation and invasion of small cell lung cancer by regulating autophagy activity via the AMPK/mTOR signaling pathway.

BACKGROUND: COTE-1 has been found to promote the proliferation and invasion of non-small cell lung cancer. However, the mechanism of COTE-1 in SCLC is still unclear. Exploring the role of COTE-1 in SCLC is expected to provide a potential target for the prognosis and treatment of SCLC. METHODS: The expression of COTE-1 and ki-67 was detected by immunohistochemical staining. PCR detected COTE-1 expression level. Cell proliferation activity was detected by CCK8 assay. A wound healing test detected cell migrative ability. Transwell invasion assay detected cell invasive ability. The numbers of autophagosomes were observed by transmission electron microscopy. WB detected the expression levels of autophagy-related proteins and AMPK/mTOR pathway-related proteins. The effect of COTE-1 expression level on the proliferation of SCLC tumor tissues was investigated by establishing a mouse SCLC xenograft tumor model. RESULTS: The expression of COTE-1 in SCLC tissues and cells was higher than that in normal tissues and cells. In SCLC cells with high COTE-1 expression, the expression level of autophagy proteins was notably increased, the number of intracellular autophagosomes increased, and the proliferative activity, migration and invasion abilities were enhanced. COTE-1 promotes autophagy, proliferation, and invasion of SCLC cells under nutrient deprivation by activating the AMPK/mTOR signaling pathway. Activation of autophagy by COTE-1 promotes the proliferation and development of xenograft tumors in a mouse model of SCLC. CONCLUSION: COTE-1 promotes the proliferation, migration and invasion of small cell lung cancer by mediating autophagy based on the AMPK/mTOR pathway.