Levels Of Autophagy-related Proteins Research Articles

MiR-506-3p regulates autophagy and proliferation in post-burn skin fibroblasts through post-transcriptionally suppressing Beclin-1 expression.

MicroRNAs (miRNAs) is involved in diverse biological processes of cells including dermal fibroblasts that contributed to wound healing and resulted in keloid scarring. MiR-506-3p has been identified as a tumor suppressor or oncogene in fibroblasts of various cancers, while the role of miR-506-3p in regulating functions of post-burn dermal fibroblasts is poorly known. In this study, miR-506-3p was confirmed to be significantly downregulated in burned tissues and heat-stimulated dermal fibroblasts. Expression levels of autophagy-related proteins suggested thermal stimulus promoting the autophagy in dermal fibroblasts. Then, miR-506-3p inhibition enhanced cell proliferation and cell cycle process in dermal fibroblasts after thermal stimulus, whereas overexpression of miR-506-3p showed the opposite effect. Western blot assay showed that inhibition of miR-506-3p resulted in the upregulation of the expression levels of LC3-II, ATG5, and structural protein collagen I, as well as the downregulation of p62. Marker proteins of intermolecular cross-links in collagen synthesis, including hydroxylysylpyridinoline (HP), lysinepyridine (LP), and lysyl hydroxylase 2 (LH2), were increased by miR-506-3p overexpression and decreased by miR-506-3p inhibition. Moreover, transfection with miR-506-3p mimic suppressed the proliferation and autophagy in heat-stimulated dermal fibroblasts in a dose-dependent manner. Subsequently, dual luciferase reporter gene assay demonstrated that Beclin-1 was a direct target of miR-506-3p, and reintroduction of Beclin-1 could antagonize the suppressive effect of miR-506-3p overexpression on fibroblast proliferation, autophagy, and the intermolecular cross-links in collagen synthesis. Taken together, our findings showed that miR-506-3p regulated autophagy and proliferation in post-burn skin fibroblasts through post-transcriptionally suppressing Beclin-1 expression.

Neuregulin 4 alleviates hepatic steatosis via activating AMPK/mTOR-mediated autophagy in aged mice fed a high fat diet

Neuregulin 4 (Nrg4) is a brown fat-enriched endocrine factor that exerts beneficial metabolic effects on insulin resistance and hepatic steatosis. Autophagy is a mechanism that is essential for preventing hepatic steatosis. The aim of this study was to explore whether Nrg4 ameliorates hepatic steatosis by inducing autophagy. Aged C57BL/6 mice were maintained on a high fat diet with or without Nrg4 intervention for 3 months. Lipid accumulation in the liver was investigated. Autophagy related protein levels along with related signaling pathways that regulate autophagy were evaluated. In addition, the effects of Nrg4 on autophagy were also determined in cultured L-02 cells. Nrg4 decreased high-fat induced intrahepatic lipid content both in vivo and in vitro. Autophagy level in the liver also decreased in obese mice and Nrg4 intervention reactivated autophagy. Further, Nrg4 intervention was found to have activated autophagy via the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. Moreover, when the AMPK/mTOR pathway was suppressed or autophagy was inhibited, the beneficial effects of Nrg4 intervention on hepatic steatosis were diminished. These results indicated that Nrg4 intervention attenuated hepatic steatosis by promoting autophagy in the liver of aged obese mice. Additionally, Nrg4 induced autophagy via the AMPK/mTOR signaling pathway.

Age-Related Maintenance of the Autophagy-Lysosomal System Is Dependent on Skeletal Muscle Type

The skeletal muscle plays an important role in maintaining whole-body mechanics, metabolic homeostasis, and interorgan crosstalk. However, during aging, functional and structural changes such as fiber integrity loss and atrophy can occur across different species. A commonly observed hallmark of aged skeletal muscle is the accumulation of oxidatively modified proteins and protein aggregates which point to an imbalance in proteostasis systems such as degradation machineries. Recently, we showed that the ubiquitin-proteasomal system was impaired. Specifically, the proteasomal activity, which was declining in aged M. soleus (SOL) and M. extensor digitorum longus (EDL). Therefore, in order to understand whether another proteolytic system would compensate the decline in proteasomal activity, we aimed to investigate age-related changes in the autophagy-lysosomal system (ALS) in SOL, mostly consisting of slow-twitch fibers, and EDL, mainly composed of fast-twitch fibers, from young (4 months) and old (25 months) C57BL/6JRj mice. Here, we focused on changes in the content of modified proteins and the ALS. Our results show that aged SOL and EDL display high levels of protein modifications, particularly in old SOL. While autophagy machinery appears to be functional, lysosomal activity declines gradually in aged SOL. In contrast, in old EDL, the ALS seems to be affected, demonstrated by an increased level of key autophagy-related proteins, which are known to accumulate when their delivery or degradation is impaired. In fact, lysosomal activity was significantly decreased in old EDL. Results presented herein suggest that the ALS can compensate the high levels of modified proteins in the more oxidative muscle, SOL, while EDL seems to be more prone to ALS age-related alterations.

Human umbilical cord MSC-derived hepatocyte growth factor enhances autophagy in AOPP-treated HK-2 cells.

Mesenchymal stem cell (MSC) transplantation may serve as an important treatment modality in chronic kidney disease (CKD); however, the underlying mechanisms remain unclear. Advanced oxidation protein products (AOPP) have been demonstrated to induce renal tubular epithelial cell (RTEC) injury via autophagy inhibition. Therefore, the present study was performed to investigate the role of human umbilical cord-derived MSCs (hUC-MSCs) in RTEC autophagy. AOPP-treated HK-2 cells were co-cultured with hUC-MSCs or treated with recombinant humanized hepatocyte growth factor (HGF). Western blotting was used to detect the levels of autophagy-and PI3K/AKT/mTOR signaling pathway-related proteins, and immunofluorescence staining was used to detect the levels of autophagy-related proteins. The HGF protein levels in HK-2 cells and the hUC-MSC co-culture system were measured. The cells were subsequently treated with tivantinib, an HGF competitive inhibitor, and the levels of autophagy-related proteins were detected. Microtubule-associated protein 1 light chain 3B (LC3B) II/LC3B I (LC3II/LC3I) and beclin 1 protein levels were increased, while p62, PI3K, phosphorylated (p)-AKT and the p-mTOR protein levels were decreased in AOPP-treated HK-2 cells co-cultured with hUC-MSC, compared with the group treated with AOPP only. Furthermore, HGF expression was increased in AOPP-treated HK-2 cells co-cultured with hUC-MSC, compared with the group treated with AOPP alone. When HGF activity was inhibited using tivantinib, these effects on LC3II/LC3I, beclin 1, p62, PI3K, p-AKT, and p-mTOR expression were partially reversed. Furthermore, the effects of tivantinib were reversed by Ly294002. In conclusion, the present study revealed that hUC-MSCs partially reversed AOPP-mediated inhibition of autophagy in HK-2 cells via secretion of HGF, indicating that hUC-MSCs may serve as a potential therapy for preventing the progression of CKD.

Long noncoding RNA LINC00511 regulates the proliferation, apoptosis, invasion and autophagy of trophoblast cells to mediate pre-eclampsia progression through modulating the miR-31-5p/homeobox protein A7 axis.

Pre-eclampsia (PE) is the usual complication during pregnancy. Long noncoding RNAs are essential regulatory factors in many diseases. Nevertheless, the role of LINC00511 in the development of PE has not been fully elucidated. The expression of LINC00511, homeobox protein A7 (HOXA7) and miR-31-5p was determined by quantitative real-time polymerase chain reaction. The levels of HOXA7 protein and autophagy-related proteins were measured by western blot analysis. Besides, cell proliferation was evaluated using cell counting kit 8 and colony formation assays. The apoptosis and invasion of cells were detected via flow cytometry and transwell assay, respectively. Further, the interaction between miR-31-5p and LINC00511 or HOXA7 was confirmed by dual-luciferase reporter assay. The LINC00511 and HOXA7 expression levels were decreased in placental tissues of PE patients, and the expression levels of both were positively correlated. LINC00511 knockdown suppressed proliferation, invasion and autophagy, while enhanced apoptosis in trophoblast cells. Meanwhile, the elevated HOXA7 expression promoted proliferation, invasion, autophagy, and inhibited the apoptosis of trophoblast cells. Besides, overexpression of HOXA7 also could reverse the effect of LINC00511 knockdown on the biological function of trophoblast cells. Further experiments confirmed that miR-31-5p could be sponged by LINC00511 and could target HOXA7. Also, miR-31-5p mimic could invert the promoting effect of LINC00511 overexpression on the biological function of trophoblast cells. LINC00511 expression was crucial for maintaining the normal function of trophoblast cells, and the decreased its expression might promote the progress of PE, which might provide some theoretical strategies for reducing the development of PE.

Ulinastatin attenuates lipopolysaccharide-induced cardiac dysfunction by inhibiting inflammation and regulating autophagy.

Ulinastatin exerts protective effects against lipopolysaccharide (LPS)-induced cardiac dysfunction. Autophagy has been demonstrated to serve an important role in sepsis-induced cardiomyopathy; however, whether ulinastatin has an anti-autophagic effect in sepsis requires further investigation. The present study aimed to determine the protective effects of ulinastatin on cardiac dysfunction and its role in autophagy during sepsis. C57BL/6J mice were randomly divided into a control, LPS and LPS + ulinastatin group, the survival status of the mice was observed every 6 h and the survival rate at each time point was calculated for 7 days. Furthermore, JC-1 dye and ELISAs were used to analyze the mitochondrial membrane potential (MMP) and serum cardiac troponin I (cTnI) levels, respectively. Western blotting and ELISAs were used to measure the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6. In addition, the cardiac ultrastructure and the number of autophagosomes formed were visualized using transmission electron microscopy, and the pathological changes in the myocardial tissues were analyzed using hematoxylin & eosin staining. Finally, the expression levels of autophagy-related proteins were analyzed using western blotting and immunofluorescence staining. The current study indicated that ulinastatin significantly improved the survival rate of septic mice. It was suggested that ulinastatin may protect against LPS-induced myocardium injury through its anti-inflammatory activity, as decreased cTnI levels, increased MMP and decreased expression levels of TNF-α and IL-6 were all observed following ulinastatin treatment. Furthermore, the number of autophagosomes formed, and the expression levels of microtubule-associated protein light chain 3 and Beclin 1 were significantly decreased following ulinastatin treatment. It was further observed that ulinastatin suppressed LPS-induced autophagosome formation, as indicated by the accumulation of sequestosome 1/p62, and the elimination of lysosome-associated membrane glycoprotein 1. In conclusion, the results of the present study suggested that ulinastatin treatment may improve survival and exert a protective effect over LPS-induced cardiac dysfunction. Furthermore, this protective effect may be associated with its anti-inflammatory and anti-autophagic activity.

SATB2 knockdown decreases hypoxia-induced autophagy and stemness in oral squamous cell carcinoma.

Increasing evidence has suggested that special AT-rich sequence-binding protein 2 (SATB2) may be involved in the progression of numerous types of human cancer; however, the biological function of SATB2 in oral squamous cell carcinoma (OSCC) occurrence and progression remains relatively unknown. The present study aimed to investigate the potential role of SATB2 in the regulation of biological characteristics of OSSC during hypoxia. The expression of SATB2 in SCC9 cells was knocked down using small interfering RNA. Western blotting was used to determine the protein expression levels of SATB2, autophagy-related proteins microtubule-associated protein light chain (LC)3-I/II and Beclin-1, and stemness markers such as Oct-4 (POU class 5 homeobox 1), Sox-2 (SRY-box 2) and Nanog (nanog homeobox). Transmission electron microscopy and monodansylcadaverine staining were used to detect the presence of autophagosomes. Furthermore, the self-renewal capacity of cells was analyzed using colony forming assays; the cell proliferative, migratory and invasive ability were evaluated using CCK-8, wound healing and Transwell assays, respectively; and the cell cycle distribution and rate of apoptosis were detected using flow cytometry. The expression levels of SATB2, autophagy-related proteins and stemness markers were significantly increased in SCC9 cells following hypoxic treatment. Meanwhile, the genetic knockdown of SATB2 inhibited hypoxia-mediated autophagy by decreasing the expression levels of Beclin-1, and preventing the conversion of LC3-I to LC3-II and the accumulation of autophagosomes. The knockdown of SATB2 also inhibited the hypoxia-induced colony-forming ability and the expression of stemness markers. Functionally, it also inhibited the proliferative, migratory and invasive abilities of SCC9 cells, while inducing apoptosis and cell cycle arrest under hypoxia. In conclusion, the present study suggested that SATB2 may function as an oncogene in OSCC cells, and targeting SATB2 may be a potential therapeutic strategy for the treatment of OSCC.

Different training intensities induced autophagy and histopathology appearances potentially associated with lipid metabolism in wistar rat liver

IntroductionAerobic training has a beneficial effect on enhancing liver functions. Autophagy might potentially play a role in preventing excessive lipid accumulation, regulating oxidative stress, and inflammation in the liver. ObjectiveTo investigate the potential linking role of autophagy-related gene expressions and protein levels with histopathology changes in Wistar rat livers after treadmill training under different intensities. Methods20 rats were divided into 4 groups (control, low intensity, moderate intensity, and high intensity). 8 weeks of treadmill training was conducted with a frequency of 5 days per week, for a duration of 30 min per day. Liver histopathology was studied using hematoxylin-eosin, and oil red O staining. RNA and protein from the liver tissues were extracted to examine the autophagy-related gene (LC3, p62) and protein levels (Beclin, ATG5, LC3, p62). The gene expressions of CPT1a, CD36, FATP 2,3,5, GLUT2, and FGF21 were also studied. ResultsDifferent intensities of training might potentially modulate autophagy-related gene expressions in rat livers. LC3 and p62 mRNA expressions in moderate and high intensities decreased compared to control. Beclin, ATG5, and LC3 protein level increased compared to control, while p62 protein level decreased compared to control. Whereas for the other genes, we found an increase in CPT1a, but we did not observed any changes in the expression of the other genes. Interestingly, autophagy-related gene expressions might be correlated with the changes of sinusoidal dilatation, cloudy swelling, inflammation, and lipid droplets of the liver tissues. ConclusionModerate and high intensities of training induce autophagy activity, combined with a shift in metabolic zonation in liver that might be potentially correlated with lipophagy. Our results showed the potential interplay role between autophagy and liver histopathology appearances as a part of the adaptation process to training.

Hyperoside alleviates epilepsy-induced neuronal damage by enhancing antioxidant levels and reducing autophagy

Ethnopharmacological relevanceHypericum perforatum L. (genus Hypericum, family Hypericaceae), a plant commonly used in traditional Chinese medicine, is believed to confer a wide range of benefits, including fever reduction, detoxification, calming, and pain relief via decoctions of its stems and leaves. Hyperoside (HYP), a natural compound extracted from Hypericum perforatum L., has been shown to demonstrate a wide array of bioactivities including antioxidative, anti-inflammatory, and anti-apoptotic effects. In this study, we investigated the effects of HYP on epilepsy-induced neuronal damage in mice and the associated regulatory factors. Aim of the studyThis study examined the potential therapeutic use of HYP for the treatment of neuronal damage in a mouse model of epilepsy and explored the relationships of the potential neuroprotective effects of HYP pretreatment with antioxidant levels and autophagy. Materials and methodsICR mice were randomly divided into six groups: sham group, sham-HYP group, KA group, KA-HYP group, KA-HYP-DDC group and KA-CQ group. Immunohistochemical staining was used to assess changes in NeuN, IBA-1, and GFAP expression in the CA3 region of the hippocampus. Immunofluorescence staining was used to assess the effects of HYP on the number of autophagosomes that accumulated in neurons in the hippocampal CA3 region. The levels of SOD1, SOD2, LC3I/II, Beclin1, and PI3K/AKT and MAPK signaling-related proteins were detected by Western blot. ResultsPretreatment with 50 mg/kg HYP protected against epilepsy-induced neuronal damage in the hippocampal CA3 region. Additionally, HYP enhanced antioxidant levels and reduced the levels of autophagy-related proteins via the PI3K/AKT and MAPK pathways. ConclusionHYP protected the hippocampal CA3 region against epilepsy-induced neuronal damage via enhancing antioxidant levels and reducing autophagy. The mechanism of action may be related to the maintenance of antioxidant levels and the suppression of autophagy via the PI3K/Akt and MAPK pathways.

Glucocorticoids Enhanced Osteoclast Autophagy Through the PI3K/Akt/mTOR Signaling Pathway.

Autophagy is an evolutionarily conserved dynamic process and present in variety of cells at basal levels to maintain homeostasis and to promote cell survival in response to stresses. The early bone loss with excessive glucocorticoids (GCs) was reported to be related with the extension of the life span of osteoclasts. However, the connection between GCs induced bone loss and osteoclast autophagy remains to be elucidated. Autophagy was detected in a Dexamethasone (Dex) induced osteoporotic mice model and primary osteoclast cultures by autophagosome detection kit, and autophagy-related proteins were assayed by Western blotting and Immunostaining. The bone morphology was examined by micro-CT and TRAP staining. The trabecular bone micro-architecture was deteriorated, and the osteoclast number and spread area were increased in the Dex-treated mice compared with the control group (P < 0.01). Meanwhile, autophagy in pre-osteoclasts was increased in mice under Dex administration evidenced by the increased number of autophagosome and up-regulation of autophagy-related protein levels. Further, the enhanced autophagy under Dex treatment was verified in primary cultured osteoclasts, as shown by the increased levels of Beclin 1 and LC3-II/LC3-I and the autophagy complex formation members including Atg1, Atg13, and Atg7. However, the expressions of PI3K, p-Akt and p-mTOR in primary cultured osteoclasts were inhibited under Dex induced autophagy. Using the selective PTEN inhibitor SF1670 to activate the PI3K/Akt/mTOR pathway reversed this osteoclast autophagy under Dex treatment. Our study suggests that osteoclast autophagy was enhanced in glucocorticoids induced bone loss, and the PI3K/Akt/mTOR signaling pathway mediated the increased autophagy in primary cultured osteoclasts under glucocorticoids treatment.

Autophagic dysfunction contributes to α-synuclein accumulation in dopaminergic neurons induced by paraquat

Objective: To investigate the effects of Paraquat on autophagy level in SH-SY5Y cell and the mechanism of abnormal aggregation of α-synuclein. Methods: Human neuroblastoma cell (SH-SY5Y cell) was used as model of dopaminergic neurons in vitro. The cells were treated with different concentrations of PQ (0, 18.75, 37.5, 75, 150, 300, 600 μmol/L) for 24 hours, and induced by 150 μmol/L PQ for 0, 12, 24, 36, 48, 60, 72, 96 hours to detect the relative survival rate of cells and determine dose/time-effect relationship. The cells were treated with concentration of 0, 75, 150, 300, 600 μmol/L PQ for 24 hours, and induced by 150 μmol/L PQ for different hours to detect intracellular LDH activity. The expression levels of autophagy-related proteins(LC3I, LC3II, Beclin1 , Vps34 and p62) and α-synuclein were detected by Western blot. The gene expression level of α-synuclein was assayed by Real-time quantitative PCR. The expression level of α-synuclein was also evaluated by immunofluorescence. The cells were pretreated with 100 nmol/L autophagy inducer rapamycin (RAPA) for 6 hours. The expression levels of autophagy-related proteins and α-synuclein were detected by Western blot. Results: CCK8 assay showed PQ induced cell survival rate decrease in a time and dose dependent manner; Compared with control group, the activity of LDH in the cell supernatant increased significantly after PQ exposure (P<0.05) ; Western blot analysis showed the ratio of autophagy-related protein LC3II/LC3I, Beclin1 and Vps34 protein expression were significantly lower after PQ treatment while the expression of p62 protein was higher (P<0.05) ; The protein and gene expression of α-synuclein were increased significantly after PQ treatment (P <0.05) ; Immunofluorescence showed the fluorescence intensity of α- synuclein in cells was significantly enhanced (P <0.05) . Compared with PQ group, the expression levels of autophagy-related proteins LC3II/LC3I and Beclin1 were significantly increased whlie α-synuclein protein level was decreased after RAPA induction (P<0.05) . Similarly, the IF result showed the fluorescence signal of α- synuclein significantly decreased after RAPA induction (P<0.05) . Conclusion: Paraquat induced autophagy dysfunction in SH-SY5Y cells, which leads to an abnormal aggregation of α-synuclein.

Effect of sublingual immunotherapy on inflammatory factors and autophagy in patients with allergic rhinitis

Objective:To observe the effect of sublingual immunotherapy on the expression of serum inflammatory factors IL-4 and TNF-α as well as autophagy-associated protein LC3 in nasal mucosa in patients with allergic rhinitis（AR）. Method:Forty patients with AR were randomly divided into SLIT group（n=20） and control group（n=20）, the SLIT group received a 2-year intervention with a standardized dust mite vaccine SLIT in combination with conventional drugs, the control group received placebo and conventional drug treatment. Blood samples and inferior turbinate mucosa were collected of both groups before and after the treatment; the clinical symptoms, signs and medication scores of the two groups before and after treatment were analyzed; the expressions of serum IL-4 and TNF-α were detected by ELISA before and after treatment; the expression of autophagy-related protein LC3 was detected by Western blot. Result:There were no significant differences in the pre-treatment signs, symptoms, medication scores, age, gender, serum IL-4, TNF-α, and LC3 expression between the SLIT group and the control group（P>0.05）. After a 2-year treatment, the symptom scores the of SLIT group were significantly improved compared with the control group; serum levels of IL-4 and TNF-α were significantly decreased in the SLIT group; the expression of autophagy-related protein LC3 in the SLIT group was significantly lower than that in the control group, and the difference was statistically significant（P<0.05）. Conclusion:SLIT combined with conventional drug therapy is more effective in improving the symptoms of AR patients than conventional drug therapy. SLIT can reduce the inflammation level and expression of autophagy-related proteins in AR patients to a certain extent.

Dexmedetomidine Protects Against Lipopolysaccharide-Induced Acute Kidney Injury by Enhancing Autophagy Through Inhibition of the PI3K/AKT/mTOR Pathway.

BackgroundAcute kidney injury (AKI) is often secondary to sepsis. Previous studies suggest that damaged mitochondria and the inhibition of autophagy results in AKI during sepsis, but dexmedetomidine (DEX) alleviates lipopolysaccharide (LPS)-induced AKI. However, it is uncertain whether the renoprotection of DEX is related to autophagy or the clearance of damaged mitochondria in sepsis-induced AKI.MethodsIn this study, AKI was induced in rats by injecting 10 mg/kg of LPS intraperitoneally (i.p.). The rats were also pretreated with DEX (30 μg/kg, i.p.) 30 min before the injection of LPS. The structure and function of kidneys harvested from the rats were evaluated, and the protein levels of autophagy-related proteins, oxidative stress levels, and apoptosis levels were measured. Further, atipamezole (Atip) and 3-Methyladenine (3-MA), which are inhibitors of DEX and autophagy, respectively, were administered before the injection of DEX to examine the protective mechanism of DEX.ResultsPretreatment with DEX ameliorated kidney structure and function. DEX decreased the levels of blood urea nitrogen (BUN) and creatinine (Cre), urine kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), reactive oxygen species (ROS), and apoptosis proteins (such as cleaved caspase-9 and cleaved caspase-3). However, DEX upregulated the levels of autophagy and mitophagy proteins, such as Beclin-1, LC3 II and PINK1. These results suggest that DEX ameliorated LPS-induced AKI by reducing oxidative stress and apoptosis and enhancing autophagy. To promote autophagy, DEX inhibited the phosphorylation levels of PI3K, AKT, and mTOR. Furthermore, the administration of Atip and 3-MA inhibitors blocked the renoprotection effects of DEX.ConclusionsHere, we demonstrate a novel mechanism in which DEX protects against LPS-induced AKI. DEX enhances autophagy, which results in the removal of damaged mitochondria and reduces oxidative stress and apoptosis in LPS-induced AKI through the α2-AR and inhibition of the PI3K/AKT/mTOR pathway.

Impaired autophagic flux contributes to muscle atrophy in obesity by affecting muscle degradation and regeneration

Increased proteolytic activity has been widely associated with skeletal muscle atrophy. However, elevated proteolysis is also critical for the maintenance of intracellular homeostasis. In this study, we aimed to investigate the significance of autophagy in obesity-induced muscle atrophy and clarify the mechanism involved. First, high-fat diet (HFD)-fed rats were administered vehicle or chloroquine (CQ), an autophagy inhibitor, and we found that HFD inhibited autophagic flux and reduced myofiber size and function in rats. Additionally, the expression levels of MyoD were decreased whereas those of Atrogin-1 were increased in rats fed a HFD. Sustained autophagy inhibition by CQ exacerbated HFD-induced muscular damage and changes in the expression of Atrogin-1 and MyoD. Similar effects were reproduced in vitro in myotubes, which exhibited increased levels of autophagy-related proteins, but the resultant autophagic flux was reduced following exposure to palmitic acid (PA)-conditioned medium. Moreover, PA significantly decreased MyoD levels and induced Atrogin-1 expression, leading to progressive myotube atrophy; this phenomenon was aggravated by CQ but alleviated by the autophagy activator rapamycin. Taken together, these in vivo and in vitro findings suggest that autophagic flux is blocked in skeletal muscle of individuals with high lipid, and autophagy mediates high lipid-induced muscle atrophy by affecting muscle degradation and regeneration.

Cardiac protection of functional chicken-liver hydrolysates on the high-fat diet induced cardio-renal damages via sustaining autophagy homeostasis.

Cardio-renal syndrome (CRS) is an integrative problem related to chronic malnutrition, obesity, etc. Amino acids and peptides are regarded as protective and essential for tissues. Pepsin-digested chicken liver hydrolysates (CLHs), which are made from the byproducts of the poultry industry, are amino-acid based and of animal origin, and may be protective against the myocardial and renal damage induced by a high-fat diet (HFD). Our results showed that CLHs contain large quantities of anserine, taurine, and branched-chain amino acids (BCAAs), and supplementing the diet with CLHs reduced (P < 0.05) weight gain, liver weight, peri-renal fat mass / adipocyte-area sizes, serum total cholesterol (TC), aspartate aminotransferase (AST), and low-density lipoprotein cholesterol (LDLC) levels in HFD-fed mice but increased (P < 0.05) serum high-density lipoprotein cholesterol (HDLC) levels. By histological analyses, CLHs alleviated (P < 0.05) renal lipid deposition and fibrosis, as well as cardiac fibrosis and inflammation of HFD-fed mice. Meanwhile, increased (P < 0.05) inflammatory and fibrotic cytokines levels in the myocardia of the HFD-fed mice were downregulated (P < 0.05) by CLH supplementation. Regarding autophagy-related protein levels, protective effects of CLHs on the myocardia against HFD feeding may result from the early blockade of the autophagy pathway to prevent autophagosome accumulation. Functional CLHs could be a novel food ingredient as a cardio-renal protective agent against a high-fat dietary habit in a niche market. © 2020 Society of Chemical Industry.

Matrine reverses the drug resistance of K562/ADM cells to ADM and VCR via promoting autophagy.

BackgroundThis study aimed to investigate the effects of matrine (MAT) on the drug resistance of K562/ADM cells.MethodsK562/S and K562/ADM cells were treated with different concentrations of vincristine (VCR), adriamycin (ADM), and/or MAT for 48 h. The cell viability was determined by MTT assay, and the drug-resistant multiple and drug-resistant reversal multiple were calculated based on half-inhibitory concentration (IC50). The cell apoptosis, as well as cell cycle were detected by flow cytometry. The autophagy was evaluated by transmission electron microscope and GFP-LC3 staining. Autophagy-related protein levels of LC3 II, and P62 were detected by Western blot.ResultsMAT reduced the viability of K562/S and K562/ADM cells in a dose-dependent manner, and 0.5 mg/mL was identified as the non-cytotoxic concentration. MAT eliminated the drug resistance of K562/ADM cells to ADM and VCR, and the drug resistance reversal multiple was 10.12 and 4.91, respectively. Chloroquine (CQ), a lysosomal inhibitor significantly reversed the inhibitory effect of MAT on the viability of K562/ADM cells. In addition, MAT induced the apoptosis of K562/ADM cells through arresting the cell cycle at G0/G1 phase in a dose-dependent manner. MAT also increased the autophagic vacuoles, the LC3+ punctate fluorescence, as well as the LC3 II protein level, and decreased P62 protein level in K562/ADM cells in a dose-dependent manner.ConclusionsMAT reversed the drug resistance of K562/ADM cells to ADM and VCR through promoting autophagy.

Effects of Cadmium and/or Lead on Autophagy and Liver Injury in Rats.

Exposure to cadmium (Cd) and lead (Pb) can induce liver damage. However, the effects of the combined exposure to Cd and Pb on liver function have not been fully clarified. In the present study, we investigated the liver function in rats co-exposed to Cd and Pb. A total of 24 female SD rats were divided into 4 groups as follows: control group (DDW), Cd group (50mg/l Cd), Pb group (300mg/l Pb), Pb + Cd group (300mg/l + 50mg/l Cd). Following 12weeks of continuous exposure, the results showed a large accumulation of Cd and Pb in the liver. The Liver weight and Liver coefficient were decreased, as well as liver structure and function was destroyed. In addition, Pb + Cd group exhibited additional pathological alterations. Moreover, the indices of oxidative stress and related trace elements were detected following treatment. The results showed that the single treatment of Pb or Cd and the combined Cd and Pb treatment could upregulate the contents of antioxidant enzymes and related trace elements. We further examined the expression levels of autophagy-related proteins and mRNAs, and we found that the single treatment of Pb or Cd and the combined Cd and Pb treatment could upregulate the expression of levels of autophagy-related proteins and mRNAs (Atg5, Atg7, Beclin-1, p62, and LC3). Transmission electron microscopy revealed the presence of autophagosomes in the exposed groups. All the results indicated that Cd and Pb may affect the level of oxidative stress and autophagy in hepatocytes, whereas the combination of Cd and Pb showed a tendency of escalation compared with the single treatment group.

Effect of electroacupuncture on relieving central post-stroke pain by inhibiting autophagy in the hippocampus

IntroductionTo explore the underlying mechanism of electroacupuncture (EA) treatment on central post-stroke pain (CPSP), and provide basic evidence for the EA treatment on CPSP. MethodsFirstly, 40 male SD rats were successfully established with a model of CPSP, under the intervention of different EA frequencies (2 Hz and 15 Hz) and fluoxetine (5 ml/kg and 0.4 mg/ml), whose brain tissue was then removed for paraffin-embedded sectioning; secondly, LPS induced the primary brain cells in the hippocampus to cause inflammation model which were added NS398 (inhibitor of COX-2) and DKK-1 (inhibitor of β-catenin) later. The lesion sites of brain tissue were observed by Nissl staining and Transmission Electron Microscope (TEM) and autophagy-related proteins (LC3B, p62, LAMP-1), COX-2 and β-catenin were detected by Western Blot and immunohistochemical staining. Finally, the correlation between LC3B, COX-2, and β-catenin was calculated by multispectral quantification. Results(1) In the EA group (15 Hz), the number of Nissl bodies increased, autophagy-related protein LC3B-Ⅱ/Ⅰ, LAMP-1, COX-2, and β-catenin was lowly expressed, p62 was highly expressed; (2) COX-2, β-catenin and LC3B are positively correlated with each other (COX-2 & β-catenin: r = 0.923; COX-2 & LC3B: r = 0.818; β-catenin & LC3B: r = 0.801); (3) Nissl bodies of primary brain cells of the hippocampus under LPS were like animal experiments; after addition of DKK-1, high expression of β-catenin and COX-2 induced by LPS was significantly down-regulated, and LC3B-II/I was significantly down-regulated, and p62 protein only had up-regulation trend; after addition of NS398, COX-2 and LC3B-II/I was significantly down-regulated. ConclusionEA may inhibit autophagy in the hippocampus by reducing β-catenin/COX-2 protein expression and effectively alleviating CPSP. Significance StatementPrevious studies have found that EA can reduce the expression of NK-1R in damaged rats by inhibition of COX-2 and β-catenin loops, which controls the activation of glial cells in the damaged area and the apoptosis of neuronal cells, and alleviated pain. In the male SD rat model, we evaluated this effect that EA inhibits autophagy in the hippocampus by reducing β-catenin/COX-2 protein expression in the brain tissue. In addition, we assessed expression levels of autophagy-related proteins and genes on the inflammatory primary brain cells model. From the experiment, we found EA may inhibit autophagy in the hippocampus by reducing β-catenin/COX-2 protein expression. These findings provide a foundation for the interpretation of the mechanism of EA on relieving CPSP in clinical practice.

Resveratrol induces SIRT1-Dependent autophagy to prevent H2O2-Induced oxidative stress and apoptosis in HTR8/SVneo cells

IntroductionPre-eclampsia (PE) is a serious complication of pregnancy, and the likely pathogenic basis of early onset PE are placental dysfunction and increased oxidative stress. Resveratrol (RES) is a potent antioxidant which has shown beneficial effects in many diseases. The aim of this study was to investigate the protective effects of RES against oxidative stress-induced damage in trophoblasts, and elucidate the potential mechanisms. MethodsWe established an in vitro model of oxidative stress by exposing the human first-trimester extravillous trophoblast cell line HTR8/SVneo to H2O2. The level of oxidative stress was reflected by ROS, MDA and SOD. The viability of cells was determined by the MTS assay. Apoptosis was detected using Annexin V-FITC staining and flow cytometry. Levels of SIRT1(sirtuin 1) and autophagy-related proteins (LC3, Beclin-1, p62) were detected by western blot. Autophagosomes were observed by transmission electron microscopy (TEM). ResultsPre-treatment with RES significantly ameliorated H2O2-induced cytotoxicity, morphological damage, oxidative stress and apoptosis. Mechanistically, RES restored the levels of SIRT1 and autophagy-related proteins including LC3-II, Beclin-1 and p62 that were dysregulated by H2O2. Blocking autophagy by 3-methyladenine (3-MA) completely abolished the protective effects of RES, as did knocking down SIRT1. ConclusionRES may protect human trophoblasts against H2O2-induced oxidative stress by activating SIRT1-dependent autophagy, and therefore has therapeutic potential in PE.

Inhibition of Autophagy Signaling via 3-methyladenine Rescued Nicotine-Mediated Cardiac Pathological Effects and Heart Dysfunctions.

Rationale: Cigarette smoking is a well-established risk factor for myocardial infarction and sudden cardiac death. The deleterious effects are mainly due to nicotine, but the mechanisms involved and theranostics remain unclear. Thus, we tested the hypothesis that nicotine exposure increases the heart sensitivity to ischemia/reperfusion injury and dysfunction, which can be rescued by autophagy inhibitor.Methods: Nicotine or saline was administered to adult rats via subcutaneous osmotic minipumps in the absence or presence of an autophagy inhibitor, 3-methyladenine (3-MA). After 30 days of nicotine treatment, the rats underwent the cardiac ischemia/reperfusion (I/R) procedure and echocardiography analysis, and the heart tissues were isolated for molecular biological studies.Results: Nicotine exposure increased I/R-induced cardiac injury and cardiac dysfunction as compared to the control. The levels of autophagy-related proteins including LC3 II, P62, Beclin1, and Atg5 were upregulated in the reperfused hearts isolated from nicotine-treated group. In addition, nicotine enhanced cardiac and plasma ROS production, and increased the phosphorylation of GSK3β (ser9) in the left ventricle tissues. Treatment with 3-MA abolished nicotine-mediated increase in the levels of autophagy-related proteins and phosphorylation of GSK3β, but had no effect on ROS production. Of importance, 3-MA ameliorated the augmented I/R-induced cardiac injury and dysfunction in the nicotine-treated group as compared to the control.Conclusion: Our results demonstrate that nicotine exposure enhances autophagy signaling pathway, resulting in development of ischemic-sensitive phenotype of heart. It suggests a potentially novel therapeutic strategy of autophagy inhibition for the treatment of ischemic heart disease.