Macrophage autophagy has a protective role in the development of atherosclerosis; however, it turns dysfunctional in advanced lesions with an increase in p62/sequestosome-1 protein. Little is known about the role and significance of p62 accumulation in atherosclerosis. The present study investigated the association between p62 expression and the process of foam cell formation. Foam cell models were established through incubation of THP-1-derived macrophages with oxidized low-density lipoprotein, and the process of foam cell formation was detected by Oil red O staining. Furthermore, the dynamic change of p62 expression was detected by western blotting and quantitative polymerase chain reaction. Additionally, using gene silencing techniques, the roles of p62 in foam cells were investigated with ELISA, MTT and flow cytometry. The results indicated that besides serving as a marker of autophagy deficiency, the p62 protein could also mediate inflammation and cytotoxicity in advanced foam cells. Additionally, the implication of p62 in autophagy inhibition and foam cell formation makes it a key atherogenic factor under autophagy-deficient conditions.

Invasiveness and metastatic potential are among the most essential characteristics of malignant tumors. Furthermore, it has been reported that autophagy and invasion are enhanced when tumor cells are grown in adverse conditions, such as nutritional deficiency and starvation. However, the association between autophagy and invasion remains largely unclear. In the present study, Earle's balanced salt solution (EBSS) was used to induce autophagy and an autophagy inhibitor was used to block autophagy. The results of Transwell assays revealed that autophagy inhibition limited the invasiveness of human ovarian cancer cells. Furthermore, the results of invadopodia formation assay indicated that autophagy stimulated invadopodia formation, and the selective autophagy receptor and signaling adaptor, sequestosome-1 (SQSTM1/p62 or simply p62), was closely associated with invadopodia formation in human ovarian cancer SKOV3 cells. The results of western blot analysis indicated that autophagy induced changes in p62 protein levels and p62 then functioned as a negative regulator of extracellular signal-regulated kinase 1/2 (ERK1/2) activity and invadopodia formation. The interaction between autophagy and invasion may thus be a self-protective mechanism for tumor cells in an unfavorable environment of nutritional deficiency, that maintains their survival and leads to increased invasiveness. An exploration of the intrinsic link between autophagy and invasion may provide a novel theoretical basis to reverse the resistance of tumor cells to a nutritional deficient environment.

Hesperidin (HDN) has been reported to have hydrogen radical- and hydrogen peroxide-removal activities and to serve an antioxidant role in biological systems. However, whether HDN protects hepatocytes (HCs) against hypoxia/reoxygenation (H/R)-induced injury remains unknown. The present study aimed to explore the role of HDN in H/R-induced injury. HCs were isolated and cultured under H/R conditions with or without HDN treatment. HC damage was markedly induced under H/R, as indicated by cell viability, supernatant lactate dehydrogenase levels and alanine aminotransferase levels; however, HDN treatment significantly reversed HC injury. Oxidative stress markers (malondialdehyde, superoxide dismutase, glutathioneand reactive oxygen species) were increased markedly during H/R in HCs; however, this effect was significantly attenuated after exposure to HDN. Compared with those of the control group, the mRNA expression levels of IL-6 and TNF-α in HCs and the concentrations of IL-6 and TNF-α in the supernatants increased significantly following H/R, and HDN significantly ameliorated these effects. Western blotting demonstrated that microtubule-associated protein 1 light chain 3α (MAP1LC3A, also known as LC3) and Beclin-1 protein expression levels increased, while sequestosome 1 levels decreased during H/R following exposure to HDN. The number of GFP-LC3 puncta in HCs following exposure to HDN was increased compared with that observed in HCs without HDN exposure under the H/R conditions after bafilomycin A1 treatment. In summary, the present study demonstrated that HDN attenuated HC oxidative stress and inflammatory responses while enhancing autophagy during H/R. HDN may have a potential protective effect on HCs during H/R-induced injury.

The NICE-3 protein serves an oncogenic role in hepatocellular carcinoma, but its role in lung adenocarcinoma (LUAD) remains unknown. The aim of the present study was to investigate the potential role and underlying mechanisms of NICE-3 in LUAD. In the present study, NICE-3 expression in LUAD tissues and its association with patient prognosis were analyzed using datasets from The Cancer Genome Atlas and Gene Express Omnibus. After NICE-3-knockdown with small interfering RNA in LUAD cells, cell proliferation was measured by cell counting, cell cycle was examined by flow cytometry, cell invasion and migration were detected by Transwell assays and autophagic markers LC3 and p62, as well as phosphorylation of S6K and AKT, were determined by western blotting. The results of public database analysis demonstrated that compared with normal lung tissues, NICE-3 expression was increased in LUAD tissues, where high expression levels were associated with a poor prognosis. The results of in vitro experimentation in LUAD cells indicated that NICE-3-knockdown inhibited proliferation, cell cycle, migration and invasion, but enhanced autophagy. Notably, NICE-3-knockdown inhibited AKT/mTORC1 signaling. The present results suggested that NICE-3 may serve an oncogenic role in LUAD via the AKT/mTORC1 signaling pathway and may therefore be a potential therapeutic target for LUAD.

Autophagy has been reported to be widely involved in the pathogenesis of osteoarthritis (OA). Increasing evidence suggested the important role of microRNAs (miRs) in the progression of OA. However, the functional role of miR-17-5p in OA development has remained to be fully elucidated. First, a mouse model of OA was established and the relative level of miR-17-5p was determined using PCR. Safranin O-fast green staining was applied to determine cartilage degeneration. TargetScan software and a dual luciferase reporter assay were applied to determine potential target genes of miR-17-5P. Autophagy measurement was performed using green fluorescent protein-microtubule-associated protein 1 light chain 3 (LC3) dot analysis. The results demonstrated that the relative expression of miR-17-5p was significantly decreased in OA model mice. In addition, the level of miR-17-5p was decreased in SW1353 human chondrosarcoma cells treated with interleukin-1β. Furthermore, autophagy was found to be suppressed in the knee joints of experimental OA model mice. The dual luciferase reporter assay confirmed that p62/sequestosome 1 was a target gene of miR-17-5p. Of note, miR-17-5p inhibitor-induced reduction of LC3 dots was markedly reversed by knockdown of p62 in SW1353 cells. In conclusion, decreased miR-17-5p expression in chondrocytes induced autophagy mainly through suppressing the expression of p62, thereby contributing to OA progression.

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.

Long non-coding RNA (lncRNA) ADAM metallopeptidase with thrombospondin type 1 motif 9 antisense RNA 2 (ADAMTS9-AS2) is involved in various types of cancer, such as ovarian cancer, lung cancer and clear cell renal cell carcinoma. However, the roles of ADAMTS9-AS2 in liver cancer are not completely understood. The present study aimed to determine the functional role of ADAMTS9-AS2 in human liver cancer and investigate the potential underlying molecular mechanisms. The expression levels of ADAMTS9-AS2 and ADAMTS9 were determined following ADAMTS9-AS2 overexpression and knockdown. The results indicated that ADAMTS9-AS2 overexpression and knockdown increased and decreased ADAMTS9 mRNA and protein expression levels, respectively, indicating that alterations in ADAMTS9 expression corresponded with ADAMTS9-AS2 expression. Subsequently, the effects of ADAMTS9-AS2 on liver cancer cell proliferation, migration and invasion were analyzed by performing Cell Counting Kit-8, wound healing and Transwell assays, respectively. The results demonstrated that ADAMTS9-AS2 inhibited liver cancer cell proliferation, migration and invasion. Finally, the effect of ADAMTS9 on PI3K/AKT/mTOR signaling pathway-associated proteins [AKT, phosphorylated-AKT, phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit β (PIK3CB), mTOR and phosphorylated-mTOR], several key autophagy-related proteins [light chain 3-I/II (LC3-I/II), beclin 1 (BECN1) and sequestosome 1 (SQSTM1)] and apoptosis-related proteins (Bax and Bcl-2) was detected via western blotting. The results suggested that ADAMTS9-AS2 downregulated the phosphorylation of AKT and mTOR, the protein expression level of PIK3CB, as well as the expression levels of autophagy protein SQSTM1 and antiapoptotic protein Bcl-2. By contrast, ADAMTS9-AS2 upregulated the expression levels of autophagy proteins LC3-II and BECN1, and the proapoptotic protein Bax. Collectively, ADAMTS9-AS2 inhibited liver cancer cell proliferation, migration and invasion via inhibiting the PI3K/AKT/mTOR signaling pathway. The present study provided a novel insight into the role of ADAMTS9-AS2 in liver cancer.

The aim of the present study was to explore sunitinib-induced autophagic effects and the specific molecular mechanisms involved, in vitro, using PC-3 and LNCaP human prostate cancer cell lines. Cells were exposed to escalating doses of sunitinib treatment and subsequent cell viability and cell cycle analyses were performed to evaluate the inhibitory effect of sunitinib in vitro. Immunofluorescence staining of microtubule associated protein 1A/1B-light chain 3 (LC3) puncta was employed to assess autophagy levels after sunitinib treatment. Western blot analysis was performed to evaluate variations in the levels of LC3, sequestosome-1, extracellular signal regulated kinase 1/2 (ERK1/2), mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (p70S6K) and cleaved caspase-3 proteins. The present study revealed that sunitinib treatment inhibited cell growth and triggered autophagy in a dose-dependent manner in both cell lines. In addition, sunitinib activated ERK1/2 and inhibited mTOR/p70S6K signaling. Sunitinib-induced autophagy was notably reversed by ERK1/2 kinase inhibitor, U0126. Furthermore, inhibition of sunitinib-induced autophagy by 3-methyladenine enhanced apoptosis and exhibited improved cell viability, which indicated that sunitinib induces not only apoptosis but also autophagic cell death in prostate cancer cell lines. These results may lead to an improved understanding of the mechanism of sunitinib's cytotoxic action and may provide evidence that combined sunitinib autophagy-regulating treatment may be of benefit to anti-prostate cancer therapy.

Sepsis increases the risk of developing liver injury. Previous studies have demonstrated that thymoquinone (TQ) exhibits hepatoprotective properties in vivo as well as in vitro. The present study aimed to investigate the underlying mechanisms of the protective effects of TQ against liver injury in septic BALB/c mice. Male BALB/c mice (age, 8 weeks) were randomly divided into four groups, namely, the control, TQ (50 mg/kg/day) treatment, cecal ligation and puncture (CLP), and TQ + CLP groups. CLP was performed following gavage of TQ for 2 weeks. At 48 h post-CLP, the histopathological alterations in the liver tissue (LT) and plasma levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were assessed. The present study evaluated microtubule-associated protein light chain 3 (LC3), sequestosome-1 (p62) and beclin 1 protein expression by western blotting and immunostaining, as well as interleukin (IL)-6, IL-1β, IL-10, monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) mRNA expression by RT-qPCR. The results of the present study indicated that administration of TQ to mice reduced the histological alterations caused by CLP in LT. TQ inhibited the plasma levels of ALT, AST and ALP in the CLP group. TQ significantly inhibited the elevation of p62, IL-1β, IL-6, MCP-1 and TNF-α levels as well as increased the LC3, beclin 1 and IL-10 levels in LT. PI3K expression in the TQ + CLP group was significantly decreased compared with that in the CLP group. TQ treatment effectively modulated the expression levels of p62, LC3, beclin 1, PI3K and proinflammatory cytokines, and may be an important agent for the treatment of sepsis-induced liver injury.

The aim of present study was to evaluate the potential effects of Rhodiola crenulata oral liquid (RCOL) on exhaustive exercise (EE)-induced fatigue in mice. Male Institute of Cancer Research mice from five treatment groups (n=10 per group) were orally administered with sterilized water for the Control and EE groups and/or RCOL at doses of 1.02, 3.03 and 6.06 ml/kg/day, once daily for 2 weeks. Anti-fatigue activity was subsequently evaluated by measuring the levels of creatine kinase (CK), lactic acid (LA), lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and total anti-oxidative capability (T-AOC). Histopathology was assessed using hematoxylin and eosin staining. Ultrastructures of mitochondria were observed by transmission electron microscopy. Energy supply capacity was assessed using citrate synthase (CS), succinate dehydrogenase (SDH), Na+-K+-ATPase, and liver and quadriceps glycogen content assays. Expression levels of mRNA and protein associated with mitophagy in the skeletal muscle were measured by reverse transcription-quantitative PCR and western blotting, respectively. RCOL was observed to markedly inhibit fatigue-induced oxidative stress by increasing the activities of SOD, CAT and T-AOC, whilst reducing the accumulation of LA, CK, LDH and MDA. Histological analysis of the quadriceps femoris tissue suggested increased numbers of muscle fibers in the RCOL groups compared with those in the EE group. RCOL administration was found to reverse EE-induced mitochondrial structural damage and alleviated defects inflicted onto the energy supply mechanism by increasing CS, SDH, Na+-K+-ATPase and glycogen levels. Additionally, RCOL reduced the protein expression of PTEN-induced kinase 1 (PINK1), Parkin, microtubule-associated proteins 1A/1B light chain 3, sequestosome 1 and ubiquitin, whilst lowering the gene expression of PINK1 and Parkin. Taken together, results from the present study clarified the anti-fatigue effect of RCOL, where the underlying mechanism may be associated with increased antioxidant activity, enhanced energy production and the inhibition of mitophagy by suppressing the PINK1/Parkin signaling pathway.

Shikonin has been reported to regulate autophagy via the AMP-activated protein kinase (AMPK)/mTOR signalling pathway and decrease apoptosis in transplanted human umbilical cord mesenchymal stem cells (HUMSCs). In the present study, HUMSCs were exposed to oxygen glucose deprivation (OGD) in vitro for 12 h, and TUNEL fluorescence staining was used to detect apoptosis. Differences in autophagy and AMPK/mTOR pathway-related protein expression following treatment with shikonin were quantitatively analyzed by western blotting. Green fluorescent protein-labelled stem cells were implanted into traumatic brain injury-model mice and the survival of HUMSCs was observed after 7 days. Shikonin increased the number of cells in brain tissue surrounding the contusion 7 days after transplantation. Furthermore, shikonin treatment decreased apoptosis, increased the expression of autophagy-related proteins, increased phosphorylated AMPK expression and downregulated phosphorylated mTOR expression. In addition, the autophagy inhibitor 3-methyladenine attenuated these effects and aggravated apoptosis. Subsequently, shikonin upregulated autophagy and protected HUMSCs in the area surrounding contused brain tissue. Shikonin may regulate autophagy via the AMPK/mTOR signalling pathway and protect transplanted HUMSCs from apoptosis induced by hypoxia/ischemia.

Autophagy serves an important role in amyloid-β (Aβ) metabolism and τ processing and clearance in Alzheimer's disease. The progression of Aβ plaque accumulation and hyperphosphorylation of τ proteins are enhanced by oxidative stress. A hydrogen peroxide (H2O2) injury cell model was established using SH-SY5Y cells. Cells were randomly divided into normal, H2O2 and chlorogenic acid (5-caffeoylquinic acid; CGA) groups. The influence of CGA on cell viability was evaluated using a Cell Counting Kit-8 assay and cell death was assessed using Hoechst 33342 nuclear staining. Autophagy induction and fusion of autophagic vacuoles assays were performed using monodansylcadaverine staining. Additionally, SH-SY5Y cells expressing Ad-mCherry-green fluorescent protein-LC3B were established to detect autophagic flow. LysoTracker Red staining was used to evaluate lysosome function and LysoSensor™ Green staining assays were used to assess lysosomal acidification. The results demonstrated that CGA decreased the apoptosis rate, increased cell viability and improved cell morphology in H2O2-treated SH-SY5Y cells. Furthermore, CGA alleviated the accumulation of autophagic vacuoles, reduced the LC3BII/I ratio and decreased P62 levels, resulting in increased autophagic flux. Additionally, CGA upregulated lysosome acidity and increased the expression levels of cathepsin D. Importantly, these effects of CGA on H2O2-treated SH-SY5Y cells were mediated via the mTOR-transcription factor EB signaling pathway. These results indicated that CGA protected cells against H2O2-induced oxidative damage via the upregulation of autophagosomes, which promoted autophagocytic degradation and increased autophagic flux.