AMPK Expression Research Articles

Chaihu and Longgu Muli Decoction demonstrate neuroprotective effects in the rat model of Parkinson's disease with depression by regulating the AMPK/mTOR signaling pathway

BackgroundChaihu and Longgu Muli decoction (CLMD) has been used as a classical prescription of traditional Chinese medicine (TCM) prescription to treat Parkinson's disease with depression (PDD). Nevertheless, the potential mechanisms underlying their neuroprotective effects are not well-understood. PurposeTo evaluate the neuroprotective effects and possible involvement of a molecular signaling pathway of CLMD in a PDD rat model. MethodsAdult male SD rats were randomly divided into six groups: control, model, positive (madopar 0.032 g kg−1 day−1), low dosage (L, 5 g kg−1 day−1), medium dosage (M, 10 g kg−1 day−1) and high dosage (H, 20 g kg−1 day−1) of CLMD. The neuroprotective effects of CLMD were investigated using behavioral tests, immunohistochemistry, immunofluorescence, high-performance liquid chromatography (HPLC), and Nissl staining. Western blotting was used to further investigate the mechanism of action of CLMD. The protein expression of LC3, AMPK, p-AMPK, mTOR, and p-mTOR were examined. ResultsCLMD exhibited a protective effect on dopaminergic neurons by increasing the expression of tyrosine hydroxylase, which was predicted to be decreased by rotenone. CLMD restored behavior impaired by rotenone and increased the degradation of α-Syn aggregation to reduce tyrosine hydroxylase loss. Additionally, it upregulated the expression of LC3, p-AMPK/AMPK, and downregulated the expression of p-mTOR/mTOR. Conclusion: These findings provide evidence that CLMD restored behavior performance, protected dopaminergic neurons and activated autophagy by regulating the AMPK/mTOR signaling pathway in rat model of PDD. CLMD restored behavior and protected dopaminergic neurons by regulating the AMPK/mTOR signaling pathway to induce autophagy and inhibit apoptotic activity.

Anti-Inflammatory Effect of Caffeine on Muscle under Lipopolysaccharide-Induced Inflammation.

Evidence has shown that caffeine administration reduces pro-inflammatory biomarkers, delaying fatigue and improving endurance performance. This study examined the effects of caffeine administration on the expression of inflammatory-, adenosine receptor- (the targets of caffeine), epigenetic-, and oxidative metabolism-linked genes in the vastus lateralis muscle of mice submitted to lipopolysaccharide (LPS)-induced inflammation. We showed that caffeine pre-treatment before LPS administration reduced the expression of Il1b, Il6, and Tnfa, and increased Il10 and Il13. The negative modulation of the inflammatory response induced by caffeine involved the reduction of inflammasome components, Asc and Casp1, promoting an anti-inflammatory scenario. Caffeine treatment per se promoted the upregulation of adenosinergic receptors, Adora1 and Adora2A, an effect that was counterbalanced by LPS. Moreover, there was observed a marked Adora2A promoter hypermethylation, which could represent a compensatory response towards the increased Adora2A expression. Though caffeine administration did not alter DNA methylation patterns, the expression of DNA demethylating enzymes, Tet1 and Tet2, was increased in mice receiving Caffeine+LPS, when compared with the basal condition. Finally, caffeine administration attenuated the LPS-induced catabolic state, by rescuing basal levels of Ampk expression. Altogether, the anti-inflammatory effects of caffeine in the muscle can be mediated by modifications on the epigenetic landscape.

Erythropoietin exerts neuroprotective effect against hypoxic ischemic brain injury in neonatal mice by activating AMPK pathway and upregulating UCP2

Purpose: To investigate the neuroprotective effect of erythropoietin (EPO) against hypoxic-ischemic brain injury in neonatal mice, and the underlying mechanism(s) of action.
 Methods: Sixty neonatal mice were assigned to 3 groups: sham, model and EPO intervention groups, each with 20 mice. Western blot assay was used to determine changes in the protein expressions of AMPK, UCP2 and KLF2. Fluorescence intensity of reactive oxygen species (ROS) was evaluated with flow cytometry.
 Results: The protein expressions of AMPK and KLF2 in the cerebral tissues of EPO intervention mice were significantly up-regulated, relative to model mice (p < 0.05), while UCP2 protein level was also significantly higher in EPO intervention mice than in model mice (p < 0.05). Mean fluorescence intensity of ROS was significantly higher in the cerebral cortex of model mice than in sham mice, but it was down-regulated in the cerebral cortex of mice in EPO intervention group, relative to the model group (p < 0.05).
 Conclusion: EPO exerts neuroprotective effect by activating AMPK pathway, up-regulating protein expression of UCP2, inhibiting production of mitochondrial ROS, and reducing oxidative stress in brain tissue. Thus, EPO has potentials for use in clinical practice as a neuroprotective agent.

Empagliflozin suppresses mitochondrial reactive oxygen species generation and mitigates the inducibility of atrial fibrillation in diabetic rats.

Recent studies have demonstrated that sodium-glucose co-transporter-2 inhibitors (SGLT2-i) reduce the risk of atrial fibrillation (AF) in patients with diabetes mellitus (DM), in which oxidative stress due to increased reactive oxygen species (ROS) contributes to the pathogenesis of AF. We aimed to further investigate this, and examine whether the SGLT2-i empagliflozin suppresses mitochondrial-ROS generation and mitigates fibrosis. A high-fat diet and low-dose streptozotocin treatment were used to induce type-2 DM (T2DM) in Sprague-Dawley rats. The rats were randomly divided into three groups: control, DM, and DM treated with empagliflozin (30 mg/kg/day) for 8 weeks. The mitochondrial respiratory capacity and ROS generation in the atrial myocardium were measured using a high-resolution respirometer. Oxidative stress markers and protein expression related to mitochondrial biogenesis and dynamics as well as the mitochondrial morphology were examined in the atrial tissue. Additionally, mitochondrial function was examined in H9c2 cardiomyoblasts. Atrial tachyarrhythmia (ATA) inducibility, interatrial conduction time (IACT), and fibrosis were also measured. Inducibility of ATA, fibrosis, and IACT were increased in rats with DM when compared to controls, all of which were restored by empagliflozin treatment. In addition, the rats with DM had increased mitochondrial-ROS with an impaired complex I-linked oxidative phosphorylation capacity. Importantly, empagliflozin seemed to ameliorate these impairments in mitochondrial function. Furthermore, empagliflozin reversed the decrease in phosphorylated AMPK expression and altered protein levels related to mitochondrial biogenesis and dynamics, and increased mitochondrial content. Empagliflozin also improved mitochondrial function in H9c2 cells cultured with high glucose medium. These data suggest that empagliflozin has a cardioprotective effect, at least in part, by reducing mitochondrial ROS generation through AMPK signaling pathways in the atrium of diabetic rats. This suggests that empagliflozin might suppress the development of AF in T2DM.

Renoprotective effects of ferulic acid mediated by AMPKα1 against lipopolysaccharide-induced damage.

The kidney is susceptible to lipopolysaccharide (LPS)-induced damage with sepsis, and renal dysfunction is a leading cause of mortality in patients with sepsis. However, the renoprotective effects of ferulic acid (FA) during sepsis and the underlying mechanism remain unclear. This study explored these renoprotective effects using NRK-52E cells and mice with LPS-induced renal damage. The results showed that after LPS challenge, NRK-52E cell viability decreased, whereas lactate dehydrogenase, caspase-3 activity, apoptosis, the release of the inflammatory cytokines, and reactive oxygen species generation increased. Further, the activities of endogenous enzymatic and non-enzymatic antioxidant systems, and energy metabolism were inhibited, mitochondrial membrane potential was lost, mitochondrial permeability transition pores opened, renal blood flow and excretory functions were reduced, and the morphology and ultrastructure of renal tissue were seriously damaged in mice exposed to LPS. FA pretreatment upregulated AMP-activated protein kinase (AMPK) α1 expression and phosphorylation and significantly reversed the aforementioned functional, enzymological, and morphological indexes in vivo and in vitro. However, these renoprotective effects of FA were attenuated by compound C, an AMPK inhibitor. In conclusion, FA pretreatment can upregulate AMPKα1 expression and phosphorylation, inhibit inflammatory cytokine release and oxidative stress, improve mitochondrial function and energy supply, alleviate apoptosis, and ultimately protect renal tissue against LPS damage.

Protopanaxadiol ameliorates NAFLD by regulating hepatocyte lipid metabolism through AMPK/SIRT1 signaling pathway

Non-alcoholic fatty liver disease (NAFLD) has become one of the main chronic liver diseases worldwide. Protopanaxadiol (PPD), an active compound derived from Gynostemma pentaphyllum, has been found able to improve free fatty acid-induced lipid accumulation in hepatocytes. However, the efficacy of PPD on NAFLD and the underlying mechanism remains unknown. In this study, the mice were fed with a high-fat diet for 22 weeks to induce the NAFLD model, and then were treated with PPD by gavage for 8 weeks. Moreover, AML12 and HepG2 cells induced by free fatty acids for 24 h, were treated with different doses of PPD and/or AMPK or SIRT1 inhibitor to explore the pharmacological mechanism of PPD. The results showed that mice with PPD treatment had significantly reduced liver weight and serum aminotransferase levels, less severe hepatosteatosis, and inflammatory cell infiltration in liver tissues when compared with the model mice. PPD also reversed the down-regulated activation of AMPK and SIRT1 expression as well as the change of lipid metabolism-related molecules in the mice liver tissues. Consistently, the in vitro experiments showed the effect of PPD in ameliorating lipid accumulation in hepatocytes. The inhibitor of AMPK or SIRT1 suppressed the AMPK and SIRT1 signaling and markedly diminished the anti-steatosis effect of PPD. In conclusion, our results prove the ameliorating impact of PPD on NAFLD and also reveal the involvement of regulation of AMPK/SIRT1 signaling pathway-mediated lipid metabolism in the underlying mechanism, suggesting PPD as a potential natural compound for the treatment of NAFLD.

2,2',4,4',5,5'-Hexabromophenyl ether (BDE-153) causes abnormal insulin secretion and disorders of glucose and lipid metabolism in mice.

Environmental polybrominated diphenyl ether (PBDE) exposure may be associated with diabetes and obesity. 2,2',4,4',5,5'-Hexabromodiphenyl ether (BDE-153) is one of the most abundant and widely distributed homologs of PBDEs detected in humans. This study investigated the effects of BDE-153 on the expression of adipokines and glucose and lipid metabolism. Adult male C57BL/6 mice were divided into five BDE-153 groups and one control group. After BDE-153 exposure for 4 weeks, the levels of biochemical indexes and the mRNA and protein expression levels of leptin, adiponectin, peroxisome proliferators activated receptors gamma (PPARγ), and AMPKα were measured. The histomorphological changes of liver and pancreas tissues were observed. After BDE-153 exposure, the weight of mice in the medium-high-dose group at different exposure times was lower than that in the control group ( p all <0.05), and the body weight decreased slightly with the increase of the dose of BDE-153. BDE-153 caused the disorder of glucose and lipid metabolism in mice, the weight of liver and pancreas increased, lipid droplets accumulated in liver cells, and the positive rate of insulin staining increased in a dose-dependent manner. BDE-153 also interfered with the expression of PPARγ, AMPKα, and adipokines. The results of restrictive cubic splines (RCS) showed that there were a nonlinear dose-response relationship between the exposure dose of BDE-153 and the expression levels of PPARγ, AMPKα, and adipokines. Our results suggest that BDE-153 may interfere with the expression of adipokines and the secretion of insulin by affecting the expression of PPARγ and AMPKα, which play a key role in glucose and lipid metabolism, leading to the occurrence of glucose and lipid metabolism disorder.

The role of CSE1L silencing in the regulation of proliferation and apoptosis via the AMPK/mTOR signaling pathway in chronic myeloid leukemia

ABSTRACTObjectivesChromosome segregation 1-like (CSE1L) is abundant and strongly expressed in solid tumors. However, the expression and role of CSE1L in chronic myeloid leukemia(CML) remain largely unknown.Materials and methodsThe relative expression levels of CSE1L in bone marrow granulocytes from patients with primary CML and non-hematologic controls were measured by flow cytometry. Cell counting kit-8 analysis, DNA Content Quantitation Assay, and Annexin V-PE/7-AAD staining were applied to assess the effects of CSE1L knockdown on cell proliferation, cell cycle progression, and apoptosis.ResultsElevated expression of CSE1L was detected in bone marrow granulocytes of patients with primary CML. In the CML cell line K562 cells, CSE1L knockdown impaired cell proliferation blocked the cell cycle shift from G0/G1 phase to the S phase, and promoted apoptosis. Knockdown of CSE1L reduced Bcl-2 protein expression and increased Bax protein expression. Meanwhile, knockdown of CSE1L enhanced the expression of phospho-AMPK protein and decreased the expression of phospho-mTOR protein. The expression of total AMPK and mTOR proteins was not affected. In addition, CSE1L expression levels were decreased in imatinib-treated K562 cells.ConclusionsCSE1L plays a pivotal role in K562 cell survival and growth. These functions may be partially dependent on the AMPK/mTOR signaling pathway to achieve. In addition, CSE1L may have had a future impact on the treatment of CML patients.

Astaxanthin promotes mitochondrial biogenesis and antioxidant capacity in chronic high-intensity interval training.

Reactive oxygen and nitrogen species are required for exercise-induced molecular adaptations; however, excessive exercise may cause cellular oxidative distress. We postulate that astaxanthin (ASX) can neutralize oxidative distress and stimulate mitochondrial biogenesis in high-intensity exercise-trained mice. Six-week-old mice (n = 8/group) were treated with ASX (10mg/kg BW) or placebo. Training groups participated in 30min/day high-intensity interval training (HIIT) for6weeks. Gastrocnemius muscle was collected and assayed following the exercise training period. Compared to the HIIT control mice, the ASX-treated HIIT mice reduced malonaldehyde levels and upregulated the expression of Nrf2 and FOXO3a. Meanwhile, the genes NQO1 and GCLC, modulated by Nrf2, and SOD2, regulated by FOXO3a, and GPx4, were transcriptionally upregulated in the ASX-treated HIIT group. Meanwhile, the expression of energy sensors, AMPK, SIRT1, and SIRT3, increased in the ASX-treated HIIT group compared to the HIIT control group. Additionally, PGC-1α, regulated by AMPK and SIRT1, was upregulated in the ASX-treated HIIT group. Further, the increased PGC-1α stimulated the transcript of NRF1 and Tfam and mitochondrial proteins IDH2 and ATP50. Finally, the ASX-treated HIIT mice had upregulations in the transcript level of mitochondrial fusion factors, including Mfn1, Mfn2, and OPA1. However, the protein level of AMPK, SIRT1, and FOXO3a, and the transcript level of Nrf2, NQO1, PGC-1α, NRF1, Mfn1, Mfn2, and OPA1 decreased in the HIIT control group compared to the sedentary control group. Supplementation with ASX can reduce oxidative stress and promote antioxidant capacity and mitochondrial biogenesis during strenuous HIIT exercise in mice.

Dietary β-conglycinin induces intestinal enteritis and affects glycerophospholipid and arginine metabolism in mirror carp (Cyprinus carpio)

The use of soybean meal in aquafeed industry is partially limited by anti-nutritional factors such as β-conglycinin (β-CG). However, the effects of β-CG on mirror carp (Cyprinus carpio) growth performance and intestinal health has not been elucidated. This study investigated the effects of β-CG levels on growth performance, intestine immunity, and intestinal lipid and protein metabolism in mirror carp. Four hundred and fifty mirror carp (4.22 ± 0.03) g were fed a control (CON) diet containing fish meal as the protein source and four experimental diets containing β-CG at 40 (β-CG1), 80 (β-CG2), 120 (β-CG3), and 160 g/kg (β-CG4) replacing the fishmeal, respectively. Each treatment had 30 fish replicated three times and the feeding was done for six weeks. The results showed that weight gain, specific growth rate, survival rate, protein efficiency, feed conversion ratio, and condition factor were not affected by dietary β-CG (P > 0.05). Interestingly, the fish fed on the β-CG diets upregulated significantly the expression of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and eIF4E-binding protein (4E-BP) in the proximal intestine (PI) than those fed on the CON diet (P < 0.05). The fish fed on the β-CG2 diet decreased significantly the expression of transforming growth factor beta 1 (TGFβ1) in the PI compared to those fed on the CON diet. The fish fed on the β-CG diets downregulated significantly the expression of target of rapamycin (TOR) in the PI than those fed on the CON diet. The fish fed on β-CG diets downregulated significantly the expression of AMP-activated protein kinase alpha (AMPK), acetyl-CoA carboxylase (ACC), TOR in the mid intestine (MI) while, claudin11 gene was significantly increased (P < 0.05). In the distal intestine (DI), feeding the fish on the β-CG diets downregulated significantly the expression of AMPK and ACC genes, while increased the expression TNF-α, IL-1β and claudin11 (P < 0.05). Feeding the fish with the β-CG diets mostly altered glycerophospholipid metabolism and arginine biosynthesis in intestinal mucosa. These results suggest that dietary β-CG induces enteritis in different intestinal segments and changes glycerophospholipid metabolism and arginine biosynthesis pathways. Fish farmers using soybean meal in the diets should process the ingredients to reduce β-CG concentration before use.

Protective Effect of Buyang Huanwu Decoction on Cerebral Ischemia/Reperfusion Injury by Inhibiting Autophagy Based on AMPK/UlK1 Signaling Pathway

The aim of the paper is to explore whether AMPK/UlK1 signaling pathway is involved in regulating autophagy and alleviating cerebral ischemia/reperfusion (I/R) injury. On this basis, study the regulatory effect of Buyang Huanwu Decoction (BHD) on AMPK/UlK1 signaling pathway, aiming to clarify the molecular mechanism of BHD in alleviating cerebral I/R injury and the protective effect of BHD containing serum (BHDS) on OGD/R neurons. The research group adopted the following methods: Neurons cultured for 7 days in vitro were randomly divided into normal control group, model group (OGD/R), AMPK activator (AICAR) group (3 mmol/L), Buyang Huanwu Decoction medicated serum (BHDS) group (3%), Buyang Huanwu Decoction medicated serum + AMPK activator (BHDS + AICAR) group. The protein expressions of p-AMPK, AMPK, p-UlK1, UlK1, Beclin-1 and LC3B in neurons were detected by Western blot, the fluorescence intensity and apoptosis rate of neuron autophagosome CYTO-ID staining were detected by flow cytometry, the fluorescence intensity and morphological changes of neuron LC3B were detected by immunofluorescence double labeling method, the morphological changes of neurons were observed by inverted microscope, and the survival rate of neurons was detected by CCK-8 method. Neuron OGD/R model and the optimal concentration of BHDS. The neurons in the model group were subjected to hypoxia and glucose deprivation for 1 h, 2 h, 3 h, 4 h and reoxygenation and glucose deprivation for 24 h. The survival rate of neurons and the release rate of l DH to determine whether the model was successfully established. And the results show that BHDS group, the survival rate of OGD/R neurons was significantly increased, and the apoptosis rate was significantly decreased. Among them, 3% BHDS intervention at the same time of hypoxia glucose deprivation for 3 h and reoxygenation glucose for 24 h was the best condition. In BHDS group, the levels of p-AMPK/AMPK, p-UlK1/UlK1, beclin-1, CYTO-ID and LC3B fluorescence intensity, and the transformation from LC3B-Ⅰ to LC3B-Ⅱ of OGD/R neurons were significantly decreased. So we can draw a conclusion that BHD can inhibit autophagy and alleviate cerebral I/R injury through AMPK/UlK1 signaling pathway.

Acute effects of a single moderate-intensity exercise bout performed in fast or fed states on cell metabolism and signaling: Comparison between lean and obese rats

AimsAlthough the benefits of exercise can be potentiated by fasting in healthy subjects, few studies evaluated the effects of this intervention on the metabolism of obese subjects. This study investigated the immediate effects of a single moderate-intensity exercise bout performed in fast or fed states on the metabolism of gastrocnemius and soleus of lean and obese rats. Main methodsMale rats received a high-fat diet (HFD) for twelve weeks to induce obesity or were fed standard diet (SD). After this period, the animals were subdivided in groups: fed and rest (FER), fed and exercise (30 min treadmill, FEE), 8 h fasted and rest (FAR) and fasted and exercise (FAE). Muscle samples were used to investigate the oxidative capacity and gene expression of AMPK, PGC1α, SIRT1, HSF1 and HSP70. Key findingsIn relation to lean animals, obese animals' gastrocnemius glycogen decreased 60 %, triglycerides increased 31 %; glucose and alanine oxidation decreased 26 % and 38 %, respectively; in soleus, triglycerides reduced 46 % and glucose oxidation decreased 37 %. Exercise and fasting induced different effects in glycolytic and oxidative muscles of obese rats. In soleus, fasting exercise spared glycogen and increased palmitate oxidation, while in gastrocnemius, glucose oxidation increased. In obese animals' gastrocnemius, AMPK expression decreased 29 % and SIRT1 increased 28 % in relation to lean. The AMPK response was more sensitive to exercise and fasting in lean than obese rats. SignificanceExercise and fasting induced different effects on the metabolism of glycolytic and oxidative muscles of obese rats that can promote health benefits in these animals.

Total coumarins of Pileostegia tomentella induces cell death in SCLC by reprogramming metabolic patterns, possibly through attenuating β-catenin/AMPK/SIRT1

BackgroundSmall-cell lung cancer (SCLC) is a high malignant and high energy-consuming type of lung cancer. Total coumarins of Pileostegia tomentella (TCPT) from a traditional folk medicine of Yao minority, is a potential anti-cancer mixture against SCLC, but the pharmacological and molecular mechanism of TCPT remains largely unknown.MethodsScreening of viability inhibition of TCPT among 7 cell lines were conducted by using CCK-8 assays. Anti-proliferative activities of TCPT in SCLC were observed by using colony formation and flow cytometry assays. Morphological changes were observed by transmission electron microscope and Mito-Tracker staining. High Throughput RNA-seq analysis and bio-informatics analysis were applied to find potential targeted biological and signaling pathways affected by TCPT. The mRNA expression of DEGs and protein expression of signalling proteins and metabolic enzymes were verified by qPCR and Western blot assays. Activity of rate-limiting enzymes and metabolite level were detected by corresponding enzyme activity and metabolites kits. Xenograft nude mice model of SCLC was established to observe the in vivo inhibition, metabolism reprogramming and mechanism of TCPT.ResultsTCPT treatment shows the best inhibition in SCLC cell line H1688 rather than other 5 lung cancer cell lines. Ultrastructural investigation indicates TCPT induces mitochondria damage such as cytoplasm shrinkage, ridges concentration and early sight of autolysosome, as well as decrease of membrane potential. Results of RNA-seq combined bio-informatics analysis find out changes of metabolism progression affected the most by TCPT in SCLC cells, and these changes might be regulated by β-catenin/AMPK/SIRT1 axis. TCPT might mainly decline the activity and expression of rate-limiting enzymes, OGDH, PDHE1, and LDHA/B to reprogram aerobic oxidation pattern, resulting in reduction of ATP production in SCLC cells. Xenograft nude mice model demonstrates TCPT could induce cell death and inhibit growth in vivo. Assimilate to the results of in vitro model, TCPT reprograms metabolism by decreasing the activity and expression of rate-limiting enzymes (OGDH, PDHE1, and LDHA/B), and attenuates the expression of β-catenin, p-β-catenin, AMPK and SIRT1 accordance with in vitro data.ConclusionOur results demonstrated TCPT induces cell death of SCLC by reprograming metabolic patterns, possibly through attenuating master metabolic pathway axis β-catenin/AMPK/SIRT1.

Effects of Cirsium setidens extract on gene expression related to apoptosis induction and metastasis inhibition in human breast cancer

Cell apoptosis induction and metastasis suppression in human breast cancer cell, MDA-MB-231, were studied to assess the potential of Cirsium setidens extract (CSE) on breast cancer treatment. Cell viability and metastasis analysis using MDA-MB-231 confirmed CSE significantly inhibited the growth and proliferation of cancer cell without suppressing normal cell growth in the range of 0.25 to 0.5 mg/mL of CSE. To analyze the anticancer effect of CSE, the expressions of major genes related to in the apoptosis, including p53, AMPK-α, Bcl-2, and Bax were analyzed by reverse transcription-polymerase chain reaction. The expressions of AMPK-α, p53, and Bax increased by 45, 43, and 68%, respectively, while the expression of Bcl-2 decreased by 12%, showing that CSE treatment induced apoptosis of MDA-MB-231. The major compound of CSE was identified as rhoifolin, a type of flavone, through analysis using LC-MS/MS. Our findings confirmed that rhoifolin, known to possess a variety of significant biological activities, could be obtained from C. setidens and used as an effective natural compound to suppress breast cancer.

Shikonin promotes hypertrophic scar repair by autophagy of hypertrophic scar-derived fibroblasts.

To investigate the Shikonin (SHI) induce autophagy of hypertrophic scar-derived fibroblasts (HSFs) and the mechanism of which in repairing hypertrophic scar. This study showed that SHI induced autophagy from HSFs and repaired skin scars through the AMPK/mTOR pathway. Alamar Blue and Sirius red were used to identify cell activity and collagen. Electron microscopy, label-free quantitative proteomic analysis, fluorescence and other methods were used to identify autophagy. The differences in the expression of autophagy and AMPK/mTOR pathway-related proteins after SHI treatment were quantitatively analyzed by Western blots. A quantitative real-time polymerase chain reaction assay was used to detect the expression of LC3, AMPK and ULK after adding chloroquine (CQ) autophagy inhibitor. After treatment with SHI for 24 hours, it was found that the viability of HSFs was significantly reduced, the protein expression of LC3-II/LC3-I and Beclin1 increased, while the protein expression of P62 decreased. The expression of phosphorylated AMPK increased and expression of phosphorylated mTOR decreased. After the use of CQ, the cell autophagy caused by SHI was blocked. The key genes LC3 and P62 were then reexamined by immunohistochemistry using a porcine full-thickness burn hypertrophic scar model, and the results verified that SHI could induce autophagy in vivo. These findings suggested that SHI promoted autophagy of HSFs cells, and the potential mechanism may be related to the AMPK/mTOR signal pathway, which provided new insights for the treatment of hypertrophic scars.

Astaxanthin improves fatty acid dysregulation in diabetes by controlling the AMPK-SIRT1 pathway.

Due to the rising prevalence of metabolic disorders, including type 2 diabetes (T2DM), new prevention and treatment strategies are needed. The aim was to examine the effect of astaxanthin (AST) on the major regulatory metabolism pathway SIRT-MAPK and fatty acid (FA) profile of plasma in patients with T2DM. This clinical trial included 68 T2DM patients randomly assigned to receive 10 mg/day of oral AST (n = 34) or placebo (n = 33) for 12 weeks. The expression level of SIRT1, AMPK activity, and the level of fatty acids in the serum were examined. The results showed that AST could modify the serum levels of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA), particularly that of Arachidonic acid, from 11.31±0.35 to 8.52±0.72 %. Also, AST increased the expression and activity levels of SIRT1 and AMPK, respectively. Pearson analysis also revealed a significant association between AMPK activity and Linoleic acid serum (LA) levels (~ -0.604, p~0.013). AST can modify the FA profile of plasma by inducing metabolizing cells to uptake them. Also, it can activate the SIRT-AMPK pathway related to metabolism regulation. See also Figure 1(Fig. 1).

S-38-3: LONG NONCODING RNA P21 ALLEVIATES ENDOTHELIAL PROGENITOR CELLS DAMAGE AND PROMOTES ENDOTHELIAL REPAIR IN HYPERTENSION THROUGH ENHANCING AUTOPHAGY

Objective: Vascular damage of hypertension has been the focus of hypertension treatment, and endothelial progenitor cells (EPCs) play an important role in the repair of vascular endothelial damage. Functional damage and decreased number of EPCs are observed in the peripheral circulation of hypertensive patients, but its mechanism is not yet elucidated. Methods: Peripheral circulating endothelial progenitor cells of hypertensive patients were labeled and detected by flow cytometry. At the same time, the endothelial progenitor cells of hypertensive patients were cultured in vitro to detect their cell function and aging. The rat bone marrow-derived endothelial progenitor cells were isolated and cultured in vitro. After the intervention of AngII, the cell function, autophagy, and cell senescence were detected. PCR and WB were used to measure the expression and phosphorylation of SESN2, AMPK and TSC2. Results: Our results showed that the number of EPCs in hypertensive patients is significantly lower than that of normal population, and the cell function decreases with a higher proportion of EPCs at later stages. A decrease in autophagy is responsible for the senescence and damage of EPCs induced by AngII. Moreover, lncRNA-p21 plays a critical regulator role in the senescence and dysfunction of EPCs. Furthermore, lncRNA-p21 activates SESN2/AMPK/TSC2 pathway by promoting the transcriptional activity of p53 and lncRNA-p21 overexpression enhanced autophagy via AMPK/TSC2/mTOR pathway to protect against AngII-induced EPC damage. EPCs with lncRNA-p21 knockdown showed poor adhesion ability and vascular repair function. These results suggest that lncRNA-p21 enhances autophagy to alleviate endothelial progenitor cells damage and promote endothelial repair in hypertension through SESN2/AMPK/TSC2 pathway. Conclusion: The data provide evidence that a reversal of decreased autophagy serves as the protective mechanism of EPC injury in hypertensive patients, and lncRNA-p21 is a new therapeutic target for vascular endothelial repair in hypertension.

Vitamin D and lactoferrin attenuate stress-induced colitis in Wistar rats via enhancing AMPK expression with inhibiting mTOR-STAT3 signaling and modulating autophagy.

Irritable bowel syndrome (IBS) is a global gastrointestinal disorder closely related to psychological stress exposure and local colonic inflammation. Herein, we investigated the effect of wrap-restraint stress (WRS) on rat behavior, on adenosine monophosphate-activated protein kinase-mammalian/mechanistic target of rapamycin-signal transducer and activator of transcription 3(AMPK-mTOR-STAT3) signaling, and autophagy in colonic mucosa. The impact of chronic administration of vitamin D3 and lactoferrin was compared.Twenty-four male Wistar rats were randomly divided into four groups. Chronic WRS protocol was applied as a rodent model of IBS. Group I: naïve animals, Group II: WRS animals, Group III: WRS-exposed and treated with vitamin D3 (500 IU/kg/day),and Group IV: WRS-exposed and treated with lactoferrin (300 mg/kg/day). In this study, we found that chronic administration of each of vitamin D3 and lactoferrin resulted in a significant increase in social interaction test, interleukin-10, AMPK, optical density of LC3B, goblet cell count and marked decrease in serum cortisol level, STAT3, inflammatory cell count, and optical density of mTOR in comparison to the WRS rats. Our findings suggest that both vitamin D3 and Lactoferrin could augment colonic autophagy through enhanced AMPK expression and inhibition of mTOR-STAT3 signaling, which offers practical insights into their clinical use in the prevention and therapy of IBS. However, lactoferrin intake as a nutritional supplement could be more helpful for stress-induced colitis treatment than vitamin D3.

Melatonin Mitigates Cisplatin-Induced Ovarian Dysfunction via Altering Steroidogenesis, Inflammation, Apoptosis, Oxidative Stress, and PTEN/PI3K/Akt/mTOR/AMPK Signaling Pathway in Female Rats.

Ovarian damage and fertility impairment are major side effects of chemotherapy in pre-menopausal cancer patients. Cisplatin is a widely used chemotherapeutic drug. The present study was designed to assess the ameliorative effects of melatonin as an adjuvant for fertility preservation. Thirty-two adult female Wistar rats were divided randomly into four equal groups: Control, Melatonin, Cisplatin (CP) treated, and CP + Melatonin treated. The cisplatin-treated group showed decreased body and ovarian weights, decreased serum E2 and AMH, increased serum LH and FSH, reduced ovarian levels of SOD, CAT, GSH, and TAC, and increased ovarian MDA. The histopathological examination of the cisplatin-treated group showed deleterious changes within ovarian tissue in the form of damaged follicles and corpus luteum, hemorrhage, and inflammatory infiltrates with faint PAS reaction in zona pellucida, increased ovarian collagen deposition, and marked expression of caspase-3 immune reaction in granulosa and theca cells, stroma, and oocytes. Alongside, there was a significant downregulation in the mRNA expression of steroidogenic enzymes, IL10, AMPK, PI3K, AKT, mTOR, and PTEN, while TGF-β1, IL1β, IL6, TNF-α, NF-Kβ, P53, p38-MAPK, JNK, and FOXO3 mRNA expressions were upregulated in cisplatin-treated rats' ovarian tissue. Coadministration of cisplatin-treated rats with melatonin reversed these changes significantly. In conclusion, melatonin's antioxidant, anti-inflammatory, and anti-apoptotic activities could modulate ovarian disturbances induced by cisplatin and preserve fertility.

Activated AMPK mitigates diabetes-related cognitive dysfunction by inhibiting hippocampal ferroptosis

Clinical and preclinical interest in Type 2 diabetes (T2D)-associated cognitive dysfunction (TDACD) has grown in recent years. However, the precise mechanisms underlying TDACD need to be further elucidated. Ferroptosis was reportedly involved in neurodegenerative diseases and diabetes-related organ injuries; however, its role in TDACD remains elusive. In this study, mice fed with a high-fat-diet combined with streptozotocin (HFD-STZ) were used as a T2D model to assess the role of ferroptosis in cognitive dysfunction. We found that ferroptosis was mainly activated in hippocampal neurons but not in microglia or astrocytes. Accordingly, increased levels of transferrin receptor and decreased levels of ferritin, GPX4, and SLC7A11 were observed in hippocampal neurons. In addition, pre-treatment with liproxstatin-1, a ferroptosis inhibitor, attenuated iron accumulation and oxidative stress response, which resulted in improved cognitive function in the HFD-STZ group. Furthermore, we found that p-AMP-activated protein kinase (AMPK) was decreased in the HFD-STZ group. Pre-treatment with AMPK agonist increased the expression of AMPK and GPX4, but decreased lipocalin 2 (LCN2) in the hippocampus that resulted in improved spatial learning ability in the HFD-STZ group. Taken together, we found that activation of neuronal ferroptosis in the hippocampus contributed to cognitive impairment of HFD-STZ mice. Furthermore, AMPK activation may reduce hippocampal ferroptosis, and consequently improve cognitive performance in diabetic mice.