Levels Of Autophagy-related Proteins Research Articles

PSPH induces cell autophagy and promotes cell proliferation and invasion in the hepatocellular carcinoma cell line Huh7 via the AMPK/mTOR/ULK1 signaling pathway.

Phosphoserine phosphatase (PSPH), a key enzyme of the l-serine synthesis pathway, has been involved in cancer progression and survival. However, limited evidence revealed the PSPH influence on hepatocellular carcinoma (HCC). Herein, we observed that PSPH expression was upregulated in both HCC tissues and cell lines, which was determined by western blotting. TCGA database showed that the PSPH protein levels were significantly upregulated and affected patient survival rates in HCC. Then gain- and loss-of-function manipulations were performed by transfection with a pcDNA-PSPH expression vector or a specific short interfering RNA against PSPH in Huh7 cells. Huh7 cell proliferation, stemness, invasion, and apoptosis were assessed by using CCK-8 test, colony formation assay, Transwell assay, and Flow cytometry analysis, respectively, and levels of autophagy-related proteins were detected by using western blotting. The results showed that PSPH could induce Huh7 cell autophagy, promote cell proliferation and invasion, and inhibit apoptosis. The knockdown of PSPH could inhibit Huh7 cell proliferation, invasion, and autophagy. Furthermore, PSPH activated Liver kinase B1 (LKB1) and TGF beta-activated kinase 1 (TAK1), affected the adenosine 5'-monophosphate-activated protein kinase (AMPK)/mTOR/ULK1 signaling pathway, but could not activate calcium/calmodulin-dependent protein kinase kinase (CaMKK) in Huh7 cells. Inhibition of either LKB1, TAK1, or AMPK could eliminate the effect of PSPH overexpression on Huh7 cell behaviors. However, inhibition of CaMKK could not influence the effect of PSPH overexpression on Huh7 cell behaviors. In conclusion, PSPH could induce autophagy, promote proliferation and invasion, and inhibit apoptosis in HCC cells via the AMPK/mTOR/ULK1 signaling pathway.

Long noncoding RNA H19 act as a competing endogenous RNA of Let-7g to facilitate IEC-6 cell migration and proliferation via regulating EGF.

Intestinal mucosal injury is one of the most significant complications of burns. In our previous study, it was found that autophagy could alleviate burn-induced intestinal injury, but the underlying mechanisms are still unclear. Irregular expression of long noncoding RNAs (lncRNAs) is present in many diseases, including burns. However, the relationship between lncRNAs and intestinal mucosal injury requires further elucidation. In this study, we established a burn mice model and detected the expression level of autophagy-related proteins. Then, H19 content after autophagy intervention was tested in vitro and in vivo. The interaction of H19 with Let-7g and that of Let-7g with epidermal growth factor (EGF) were verified by dual-luciferase reporter assays. We found that the expression of the autophagy-associated proteins LC3-II and Beclin-1 was raised in the intestinal tract of the burn mice model. Similarly, the transfection of H19 raised autophagy levels. H19 was elevated after autophagy intervention in vitro and in vivo. H19 overexpression was able to promote IEC-6 cell migration and proliferation. Let-7g was suppressed by the overexpression of H19 and the combination of Let-7g mimic was able to abolish the physiological effect of H19. Moreover, the suppression of Let-7g increased the expression of EGF protein, which heightened IEC-6 cell migration and proliferation. Besides this, dual-luciferase assays revealed that Let-7g was a direct target of H19 as well as the EGF gene. Taken together, autophagy-mediated H19 increases in mouse intestinal tract after severe burnand functions as a sponge to Let-7g to regulate EGF, which suggests that H19 serves as a potential therapeutic target and biomarker for intestinal mucosal injury after burns.

Mechanism of autophagy regulating chemoresistance in esophageal cancer cells

The current study aimed to explore the mechanism of autophagy-regulating chemoresistance in esophageal cancer (EC) cells. Methods: 45 cases of esophageal cancer cell tissue and 25 cases of adjacent normal tissue excised in the surgical resection were collected from the tumor pathology department of our hospital from March to November 2017. The above cancer cells and paracancerous cells were cultured according to the cell culture procedures. The autophagy was induced by cisplatin in human esophageal cancer EC9706 cells line. The effect of autophagy on the survival of EC9706 cells was observed by autophagy inhibitor 3-MA. Cell viability was also measured by cell counting kit-8 (CCK-8). Apoptosis and cell cycle were detected by flow cytometry. Furthermore, monodansylcadaverine (MDC) was used to detect autophagy. Western blot was applied to determine the molecular changes during treatment. Diketopyrrolopyrrole (DPP) is able to inhibit cell proliferation, induce cell death and cell cycle arrest in the S phase. In addition, autophagy was activated through PI3K-III pathway. Results: 3-MA inhibitor plus 10% fetal bovine serum were added for culture, and the cell culture temperature and humidity were the best conditions. There were few autophagic vesicles in the stationary cells, where their brightness was weakened. There were more and brighter green fluorescent particles in the DPP group without a 3-MA inhibitor, indicating that autophagic parameters actually exist in this process. The apoptosis rate of DDP-induced cell death was not found to be the best, but was higher than that of the control group (P<0.05). The combination of DDP and 3-MA had a more obvious catalytic effect on apoptosis, and the apoptosis rate was much higher than that of single DDP (P<0.05), indicating that DDP was capable of inducing significant apoptosis after inhibiting autophagy. The combination of DDP and 3-MA had an obvious catalytic effect on apoptosis, and the apoptosis rate was higher than that of DDP alone (P < 0.05), suggesting that DDP could significantly improve the ability to induce apoptosis after inhibiting autophagy. The expression level of autophagy-related proteins was also detected by Western blotting. Our findings indicated that autophagy may be a self-protective mechanism of esophageal cancer cells induced by DDP, and its inhibition may be a new strategy for adjuvant chemotherapy in esophageal cancer.

CKIP-1 augments autophagy in steatotic hepatocytes by inhibiting Akt/mTOR signal pathway

Nonalcoholic fatty liver disease (NAFLD), which is characterized by aberrant accumulation of intrahepatic triglycerides and lipid droplets (LDs) in the liver cells, is becoming increasingly prevalent at an alarming rate worldwide. LDs can be consumed by either hydrolysis or autophagy, which is shown to be of importance in the regulation of hepatic lipid metabolism. We have shown that deficiency of pleckstrin homology domain-containing casein kinase 2 interacting protein-1 (CKIP-1), a scaffold protein that interacts with various proteins in multiple signal pathways, in mice aggravates high-fat diet induced fatty liver. However, its underlying mechanisms remain largely unknown. In this study, we found that the mRNA and protein levels of CKIP-1 decreased dramatically in steatotic HepG2 cells induced by oleic acid (OA) treatment. Coincidently, hepatic autophagy was also dynamically regulated in steatotic HepG2 cells. In addition, overexpression of CKIP-1 activated autophagy by suppression of Akt/mTOR signaling, which in turn reduced lipid accumulation. Moreover, these phenomena were reversed in CKIP-1-shRNA transfected steatotic hepatocytes. To further evaluate the potential role of CKIP-1 in autophagy, we determined the level of autophagy related proteins in CKIP-1 knockout mice. These results supported our findings in vitro. In summary, we found CKIP-1 to be a positive regulator of hepatic autophagy and a promising therapeutic target for treatment of NAFLD.

Galuteolin Inhibited Autophagy for Neuroprotection Against Transient Focal Cerebral Ischemia in Rats.

Galuteolin, a Chinese herbal medicine, purified from Lonicera Japonica. In this study, we aimed to investigate the neuroprotective effect of galuteolin against cerebral ischemia/reperfusion (I/R) injury. We administered galuteolin or galuteolin and rapamycin to rats which had middle cerebral artery occlusion/reperfusion (MCAO/R). A series of characterizations were carried out to monitor the outcomes of galuteolin in I/R rats regarding the infarct volumes, neurological deficits, and brain water, as well as its effect on neuroprotection and autophagy. It was found that galuteolin significantly reduced the infarct volume, brain water content, and the neurological deficits in a dose-dependent manner. Neuron damages were decreased in the hippocampal carotid artery 1 pyramidal layer by galuteolin. The expression levels of neuron-specific enolase (NSE) increased after galuteolin treatment. Galuteolin significantly decreased the expression levels of autophagy-related proteins. In addition, galuteolin decreased rapamycin-related neuron damages and activations of autophagy in I/R rats. Our data suggested that galuteolin can inhibit ischemic brain injuries through the regulation of autophagy-related indicators in I/R.

Associations between Huwe1 and autophagy in rat cerebral neuron oxygen‑glucose deprivation and reperfusion injury.

Autophagy and the ubiquitin proteasome system (UPS) are two major protein degradation pathways involved in brain ischemia. Autophagy can compensate for UPS impairment-induced cellular dysfunction. HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1 (Huwe1), an E3 ubiquitin ligase, serves critical roles in nervous system plasticity, regeneration and disease. However, the role of Huwe1 in autophagy in brain ischemia/reperfusion (I/R) injury remains unknown. The aim of the present study was to investigate the crosstalk between autophagy and the UPS in brain ischemia. The present study established an oxygen-glucose deprivation and reperfusion (OGD/R) model in rat primary cortex neurons in vitro. Lentiviral interference was used to silence the expression of Huwe1. An autophagy promoter (rapamycin), an autophagy inhibitor (wortmannin) and a JNK pathway inhibitor (SP600125) were also used in the current study. Cellular autophagy-related proteins, including Beclin-1, autophagy related (ATG) 7, ATG5, ATG3 and microtubule associated protein 1 light chain 3 α, and apoptosis-related proteins, such as P53, cleaved caspase 3, Bax and Bcl2, were detected via western blotting and immunocytochemistry. Neuronal apoptosis was evaluated using a TUNEL assay. The results demonstrated that silencing Huwe1 increased the expression levels of autophagy-related proteins at 24 h after OGD/R. Treatment with a JNK inhibitor or cotreatment with Huwe1 shRNA significantly increased autophagy. Rapamycin increased apoptosis under OGD/R conditions. However, treatment with Huwe1 shRNA decreased the number of TUNEL-positive cells at 24 h after OGD/R. Cotreatment with Huwe1 shRNA and wortmannin alleviated neuronal apoptosis under OGD/R conditions compared with cotreatment with DMSO. Collectively, the present results suggested that silencing Huwe1 was accompanied by a compensatory induction of autophagy under OGD/R conditions. Furthermore, the JNK pathway may be a key mediator of the interaction between Huwe1 and autophagy in response to UPS impairment.

Zinc provides neuroprotection by regulating NLRP3 inflammasome through autophagy and ubiquitination in a spinal contusion injury model.

AimSpinal cord injury (SCI) is a serious disabling injury worldwide, and the excessive inflammatory response it causes plays an important role in secondary injury. Regulating the inflammatory response can be a potential therapeutic strategy for improving the prognosis of SCI. Zinc has been demonstrated to have a neuroprotective effect in experimental spinal cord injury models. In this study, we aimed to explore the neuroprotective effect of zinc through the suppression of the NLRP3 inflammasome.MethodAllen's method was used to establish an SCI model in C57BL/6J mice. The Basso Mouse Scale (BMS), Nissl staining were employed to confirm the protective effect of zinc on neuronal survival and functional recovery in vivo. Western blotting (WB), immunofluorescence (IF), and enzyme‐linked immunosorbent assay (ELISA) were used to detect the expression levels of NLRP3 inflammasome and autophagy‐related proteins. Transmission electron microscopy (TEM) was used to confirm the occurrence of zinc‐induced autophagy. In vitro, lipopolysaccharide (LPS) and ATP polarized BV2 cells to a proinflammatory phenotype. 3‐Methyladenine (3‐MA) and bafilomycin A1 (BafA1) were chosen to explore the relationship between the NLRP3 inflammasome and autophagy. A coimmunoprecipitation assay was used to detect the ubiquitination of the NLRP3 protein.ResultsOur data showed that zinc significantly promoted motor function recovery after SCI. In vivo, zinc treatment inhibited the protein expression level of NLRP3 while increasing the level of autophagy. These effects were fully validated by the polarization of BV2 cells to a proinflammatory phenotype. The results showed that when 3‐MA and BafA1 were applied, the promotion of autophagy by zinc was blocked and that the inhibitory effect of zinc on NLRP3 was reversed. Furthermore, co‐IP confirmed that the promotion of autophagy by zinc also activated the protein expression of ubiquitin and suppressed high levels of NLRP3.ConclusionZinc provides neuroprotection by regulating NLRP3 inflammasome through autophagy and ubiquitination after SCI.

Protective effects of quercetin and crocin in the kidneys and liver of obese Sprague-Dawley rats with Type 2 diabetes: Effects of quercetin and crocin on T2DM rats.

Quercetin and crocin are the main active constituents of Eucommia and Gardenia species, respectively. This study was conducted to explore the effects of quercetin and crocin on fat reduction and renal fibrosis and the relationship of these compounds with autophagy. First, a model of high-fat diet- and streptozotocin-induced type 2 diabetes was established and then subjected model animals to 8 weeks of metformin, quercetin and crocin gavage. Then, a high glucose-induced rat mesangial cells (RMCs) model was established, and these cells were cocultured with quercetin and crocin. The results showed that quercetin and crocin can decrease fasting blood glucose levels, reduce fat accumulation in the liver, alleviate renal fibrosis, and reduce blood lipid levels. Quercetin and crocin increased autophagy-related protein (LC3, Atg5, Beclin-1 and p-AMPK) levels in the liver and decreased autophagy-related protein (LC3, Atg5, Beclin-1 and p-AMPK) levels in the kidneys. Moreover, quercetin and crocin inhibited the excessive proliferation of RMCs induced by high-glucose (HG) conditions, decreased autophagy-related protein (LC3, Atg5, Beclin-1 and p-AMPK) levels, and decreased TGF-β1 expression. Importantly, cotreatment with quercetin and crocin had a more significant effect than treatment with either compound alone. These results suggest that combined administration of quercetin and crocin can more significantly reduce blood glucose/lipid levels and improve renal fibrosis than administration of either compound alone and that AMPK-dependent autophagy might be involved in this process. Eucommia ulmoides Oliv. and Gardenia could be developed as drugs for Type 2 diabetes treatment.

VPS53 Suppresses Malignant Properties in Colorectal Cancer by Inducing the Autophagy Signaling Pathway.

BackgroundMany studies found that VPS53, one of the subunits of the golgi-associated retrograde protein (GARP) complexes, was aberrantly expressed in human diseases.AimThis study investigated the functions and molecular mechanisms of VPS53 in colorectal cancer (CRC).MethodsExpression and correlation of Beclin 1 and VPS53 were analyzed by RT-qPCR and Pearson’s correlation in CRC tissues, and VPS53 expression was also determined in CRC cells. The changes of proliferation, migration, invasion, apoptosis, and autophagy of CRC cells were examined by a succession of functional experiments including CCK-8, flow cytometry, transwell assay, and electron microscopy. The levels of autophagy related proteins were evaluated by Western blotting analysis.ResultsRT-qPCR results found that VPS53 was downregulated in CRC tissues and cells, and Beclin 1 expression was also decreased in CRC tissues. There was a positive correlation between VPS53 and Beclin 1. Functional results showed that overexpression of VPS53 could suppress proliferation, migration, and invasion, and accelerate apoptosis and autophagy of CRC cells. Also, VPS53 could upregulate Beclin 1 and LC3BII, suggesting the inductive effect of VPS53 on CRC cell autophagy. Furthermore, it was found that the autophagy inhibitor (Inhb) could attenuate the inhibition of VPS53 on CRC progression.ConclusionVPS53 repressed CRC progression by regulating the autophagy signaling pathway, suggesting that VPS53 might be a promising therapeutic target for CRC.

Long non-coding RNA XIST confers aggressive progression via miR-361-3p/STX17 in retinoblastoma cells.

Retinoblastoma (RB) is a frequent intraocular tumor in children. Long-non-coding RNA X inactive specific transcript (XIST) has been reported to participate in the RB process, while its potential role remains largely unknown. The expression patterns of XIST, microRNA (miR)-361-3p, and Syntaxin 17 (STX17) were determined using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry, and transwell assays were employed to reckon cell viability, apoptosis, and mobility in RB cells, respectively. Besides, the levels of STX17 and autophagy-related proteins were detected utilizing Western blot. Dual-Luciferase reporter assay was implemented to evaluate the interaction between miR-361-3p and XIST or STX17, and the role of XIST in tumor growth was analyzed through xenograft tumor model. The expression levels of XIST and STX17 were higher in RB tissues and cells, but miR-361-3p was downregulated. Loss of XIST was inversely connected with aggressive characteristics, showing as the curb of cell proliferation, migration, invasion, autophagy, and enhancement of apoptosis in RB cells. Also, the deficiency of XIST caused the decrease of tumor growth in vivo. Meanwhile, miR-361-3p inhibitor partially rescued XIST detection-mediated cell behaviors in vitro. Similarly, miR-361-3p mimic-mediated suppressive effect on aggressive phenotypes was abolished after overexpression of STX17 in RB cells. Mechanically, XIST was a sponge of miR-361-3p to regulate STX17. XIST functioned as an oncogenic lncRNA via miR-361-3p/STX17 axis in the progression of RB, which might provide a promising theoretical basis for the clinical therapy of RB.

Lipoxin A4 Inhibits NLRP3 Inflammasome Activation in Rats With Non-compressive Disc Herniation Through the JNK1/Beclin-1/PI3KC3 Pathway.

BackgroundNon-compressive disc herniation is induced by an inflammatory response from the nucleus pulposus tissue and nerve roots. Lipoxins (LXs) are important endogenous anti-inflammatory mediators in the body, helping to inhibit neutrophil recruitment and stimulate autophagy in monocytes and macrophages. Here, we investigated the molecular mechanisms underlying the effects of exogenous lipoxin administration on rats with non-compressive disc herniation.MethodA non-compressive disc herniation model was established in rats. Fifty rats were randomly divided into: sham group, model group, PI3K inhibitor (LY294002) group, lipoxin A4 group (LXA4), and PI3K inhibitor and lipoxin A4 group (LY294002 + LXA4). Similar groupings were established for rat spinal neurons. Changes in the mechanical pain threshold and thermal pain threshold were monitored at different times. The expression of proinflammatory and anti-inflammatory mediators was assessed by ELISA, while immunohistochemistry was employed to measure the expression levels of NLRP3 and p-JNK1. The expression levels of autophagy-related proteins were measured by western blot.ResultsIn vivo, the pain threshold was markedly decreased in the model group at each time point examined compared with that in sham group. LY294002 treatment further reduced the pain threshold. After LXA4 injection, the pain threshold was significantly increased, and the effect of LY294002 was significantly weakened (p < 0.05). The levels of proinflammatory cytokines were increased in rats with non-compressive disc herniation, and these levels were further increased by LY294002 treatment (p < 0.05). However, treatment with LXA4 significantly reduced the levels of these proinflammatory cytokines in the model group (p < 0.05). The opposite effect was observed for anti-inflammatory mediators. The expression of NLRP3 was largely increased in the model group compared with that in the sham group (p < 0.05). Treatment with LY294002 also increased the NLRP3 expression level, while the administration of LXA4 elicited the opposite effect. Furthermore, western blot analysis showed that the expression of autophagy-related proteins was greatly decreased in the model group, whereas it was significantly increased in the LXA4 group (p < 0.05). The in vitro results were consistent with the outcomes observed in vivo.ConclusionsThese data suggested that LXA4 inhibited NLRP3 activation in rats with non-compressive disc herniation by regulating the JNK1/beclin-1/PI3KC3 pathway.

Lnc RNA ZFAS1 regulates the proliferation, apoptosis, inflammatory response and autophagy of fibroblast-like synoviocytes via miR-2682-5p/ADAMTS9 axis in rheumatoid arthritis.

Backgrounds: Rheumatoid arthritis (RA) is a frequent autoimmune disease. Emerging evidence indicated that ZNFX1 antisense RNA1 (ZFAS1) participates in the physiological and pathological processes in RA. However, knowledge of ZFAS1 in RA is limited, the potential work pathway of ZFAS1 needs to be further investigated.Methods: Levels of ZFAS1, microRNA (miR)-2682-5p, and ADAM metallopeptidase with thrombospondin type 1 motif 9 (ADAMTS9) were estimated using quantitative real-time polymerase chain reaction (qRT-PCR) assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to explore the ability of cell proliferation in fibroblast-like synoviocytes (FLS-RA). Cell apoptosis was measured via flow cytometry. Also, levels of ADAMTS9, apoptosis-related proteins, cleaved-caspase-3 (active large subunit), and autophagy-related proteins were identified adopting Western blot. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the productions of inflammatory cytokines. Beside, the interrelation between miR-2682-5p and ZFAS1 or ADAMTS9 was verified utilizing dual-luciferase reporter assay.Results: High levels of ZFAS1 and ADAMTS9, and a low level of miR-2682-5p were observed in RA synovial tissues and FLS-RA. Knockdown of ZFAS1 led to the curbs of cell proliferation, inflammation, autophagy, and boost apoptosis in FLS-RA, while these effects were abolished via regaining miR-2682-5p inhibition. Additionally, the influence of miR-2682-5p on cell phenotypes and inflammatory response were eliminated by ADAMTS9 up-regulation in FLS-RA. Mechanically, ZFAS1 exerted its role through miR-2682-5p/ADAMTS9 axis in RA.Conclusion: ZFAS1/miR-2682-5p/ADAMTS9 axis could modulate the cell behaviors, inflammatory response in FLS-RA, might provide a potential therapeutic target for RA treatment.

NaSH increases SIRT1 activity and autophagy flux through sulfhydration to protect SH-SY5Y cells induced by MPP~+

ABSTRACT Parkinson’s disease (PD) is one of the most prevailing aging diseases around the world. The present study was to investigate the potential effect of hydrogen sulfide (H2S) and silent mating type information regulation 2 homolog 1 (SIRT1) in MPP~+ induced SH-SY5Y cells and its underlying mechanisms in PD. SH-SY5Y cells were induced by MPP~+ and treated with the H2S donor NaHS to detect the effect of H2S on the molecular behaviors of MPP~+ induced SH-SY5Y cells. NaHS reduced the apoptosis rate and expressions of MDA, 4-HNE and p62, while increased cell viability, autophagy flux and expressions of LC3 II/I and Beclin1 in MPP~+ induced SH-SY5Y cells. Then, levels of autophagy-related proteins and inflammation-related proteins (TNF-α, IL-Iβ) were detected, indicating that Chloroquine and Sirtinol reversed the protective effect of H2S on SH-SY5Y cells induced by MPP~+. We further explored the particular function of H2S, SH-SY5Y cells treated with MPP~+, NaHS chloroquine, and SIRT1 inhibitor (Sirtinol). The results showed that H2S increased SIRT1 expression and sulfhydration. Finally, a PD mouse model verified the above results. In a word, H2S ameliorated SIRT1 activity through acceleration of SIRT1 sulfhydration to increase the autophagy flux and attenuate damage of SH-SY5Y cells induced by MPP~+. H2S and SIRT1 activator might be a target in the treatment of PD patients.

Isoquercitrin Delays Denervated Soleus Muscle Atrophy by Inhibiting Oxidative Stress and Inflammation.

Although denervated muscle atrophy is common, the underlying molecular mechanism remains unelucidated. We have previously found that oxidative stress and inflammatory response may be early events that trigger denervated muscle atrophy. Isoquercitrin is a biologically active flavonoid with antioxidative and anti-inflammatory properties. The present study investigated the effect of isoquercitrin on denervated soleus muscle atrophy and its possible molecular mechanisms. We found that isoquercitrin was effective in alleviating soleus muscle mass loss following denervation in a dose-dependent manner. Isoquercitrin demonstrated the optimal protective effect at 20 mg/kg/d, which was the dose used in subsequent experiments. To further explore the protective effect of isoquercitrin on denervated soleus muscle atrophy, we analyzed muscle proteolysis via the ubiquitin-proteasome pathway, mitophagy, and muscle fiber type conversion. Isoquercitrin significantly inhibited the denervation-induced overexpression of two muscle-specific ubiquitin ligases—muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), and reduced the degradation of myosin heavy chains (MyHCs) in the target muscle. Following isoquercitrin treatment, mitochondrial vacuolation and autophagy were inhibited, as evidenced by reduced level of autophagy-related proteins (ATG7, BNIP3, LC3B, and PINK1); slow-to-fast fiber type conversion in the target muscle was delayed via triggering expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α); and the production of reactive oxygen species (ROS) in the target muscle was reduced, which might be associated with the upregulation of antioxidant factors (SOD1, SOD2, NRF2, NQO1, and HO1) and the downregulation of ROS production-related factors (Nox2, Nox4, and DUOX1). Furthermore, isoquercitrin treatment reduced the levels of inflammatory factors—interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)—in the target muscle and inactivated the JAK/STAT3 signaling pathway. Overall, isoquercitrin may alleviate soleus muscle atrophy and mitophagy and reverse the slow-to-fast fiber type conversion following denervation via inhibition of oxidative stress and inflammatory response. Our study findings enrich the knowledge regarding the molecular regulatory mechanisms of denervated muscle atrophy and provide a scientific basis for isoquercitrin as a protective drug for the prevention and treatment of denervated muscle atrophy.

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.