Protein Expression Levels Of Beclin-1 Research Articles

Curcumin synergistically augments the chemotherapeutic activity of Doxorubicin in prostate cancer cells

Objective Prostate cancer is one of the most commonly diagnosed cancer types in men and many people die every year due to recurring or acquiring aggressive forms of prostate cancer. Numerous chemotherapeutics such as paclitaxel and doxorubicin are commonly used in the treatment of prostate cancer. However, acquired resistance to chemotherapeutics and broad systemic side effects substantially limit their efficacy. Curcumin is one of the most examined phytochemicals of the herbal remedy turmeric. Herein, we aimed to investigate the synergistic capability of curcumin on doxorubicin in prostate cancer cells. Method The human adenocarcinoma cell line LNCaP was used in cell culture studies. Cell viability was examined by WST-1 assay. The protein expression levels of Beclin1, p62/SQSTM1, LC3-I/II, Hrd1, gp78, polyubiquitin, PERK, eIF2, phospho-(Ser51) eIF2, IRE1, XBP-1s, PARP-1, caspase-3, AR, PSA, c-Myc, E-cadherin, N-cadherin and VEGF-A were investigated by immunoblotting assay. Results Our data indicated that co-administration of curcumin with doxorubicin significantly improved the cytotoxic effect of doxorubicin in LNCaP cells. Also, the combination of curcumin and doxorubicin reduced the autophagic flux and remarkably induced endoplasmic reticulum-associated-degradation (ERAD) and unfolded protein response (UPR) signaling. Also, activation of apoptotic proteins PARP-1 and caspase-3 were strongly enhanced by combined treatment in a dose-dependent manner. Moreover, combined treatment markedly decreased AR, PSA, c-Myc and VEGF-A levels. Additionally, the epithelial-mesenchymal transition (EMT) was reduced by decreasing N-cadherin and increasing E-cadherin protein levels. Conclusion Present data strongly suggest that curcumin synergistically improves the anti-cancer features of doxorubicin in prostate cancer cells.

Semaphorin‑3A alleviates cardiac hypertrophy by regulating autophagy.

Cardiac hypertrophy, characterized by cardiomyocyte enlargement, is an adaptive response of the heart to certain hypertrophic stimuli; however, prolonged hypertrophy results in cardiac dysfunction and can ultimately cause heart failure. The present study evaluated the role of semaphorin-3A (Sema3A), a neurochemical inhibitor, in cardiac hypertrophy, utilizing an isoproterenol (ISO) induced H9c2 cell model. Cells were stained with rhodamine-phalloidin to assess the cell surface area and reverse transcription-quantitative PCR was performed to quantify mRNA expression levels of Sema3A, brain natriuretic factor (BNF) and β-myosin heavy chain (β-MHC). The protein expression levels of the autophagy-related proteins light chain 3 (LC3), p62 and Beclin-1, and the Akt/mTOR signaling pathway associated proteins Akt, phosphorylated (p)-Akt, mTOR, p-mTOR, 4E-binding protein 1 (4EBP1) and p-4EBP1 were semi-quantified using western blotting. Rapamycin, a canonical autophagy inducer, was administered to H9c2 cells to elucidate the regulatory mechanism of Sema3A. The results indicated significantly increased cell surface area and elevated BNF and β-MHC mRNA expression levels, increased LC3II/I ratio and Beclin-1 protein expression levels and significantly decreased p62 protein expression levels after treatment of H9c2 cardiomyocytes with ISO for 24 h. Sema3A overexpression improved ISO-induced hypertrophy in H9c2 cells, indicated by decreased cell surface area and reduced BNF and β-MHC mRNA expression levels. Moreover, Sema3A overexpression inhibited ISO-induced autophagy in H9c2 cells, indicated by decreased LC3II/I ratio and Beclin-1 protein expression levels and increased p62 protein expression levels. The autophagy activator rapamycin partially inhibited the protective effect of Sema3A on ISO-induced hypertrophy. Sema3A overexpression suppressed the decrease of the protein expression levels of p-Akt, mTOR and their downstream target 4EBP1, which is induced by ISO. Collectively, these results suggested Sema3A prevented ISO-induced cardiac hypertrophy by inhibiting autophagy via the Akt/mTOR signaling pathway.

β-asarone improves cognitive impairment and alleviates autophagy in mice with vascular dementia via the cAMP/PKA/CREB pathway

BackgroundVascular dementia (VD) is the second most common type of dementia after Alzheimer's disease. β-asarone, a major component of Acorus tatarinowii Schott, is important in neurodegenerative and neurovascular diseases. Studies have confirmed that β-asarone can mitigate autophagy and reduce damage in hypoxic cells. We also reported that β-asarone improves learning and memory. This study further clarifies whether β-asarone attenuates cerebral ischaemic injury by acting through the cAMP/PKA/CREB pathway in VD model mice. MethodsHere, genes and potential pathways that may be targeted by β-asarone for the treatment of transient cerebral ischaemia (TCI) and cognitive impairment (CI) were obtained using network pharmacology. The two-vessel occlusion method was used to establish the VD model. The Morris water maze test was used to evaluate the effects on memory. Then, the protein levels of mitofusin-2 (Mfn2), brain-derived neurotrophic factor (BDNF), optic atrophy 1 (OPA1), cyclic adenosine monophosphate (cAMP), myelin basic protein (MBP), matrix metalloproteinase-9 (MMP9) and neuron specific enolase (NSE) were determined by ELISA. The levels of superoxide dismutase (SOD) and malonaldehyde (MDA) were measured using commercial kits. Then, qRT-PCR was employed to investigate the expression of the candidate genes screened from the protein-protein interaction (PPI) network. Furthermore, the expression of the autophagy-related proteins Beclin-1, (microtubule-associated protein light chain 3) LC3, p62, postsynaptic density protein 95 (PSD95), protein kinase A (PKA), pPKA, cyclic-AMP response binding protein (CREB), and pCREB was determined by western blotting. The expression of autophagy-related proteins, PSD95 and translocase of outer mitochondrial membrane 20 (TOM20) was determined by immunofluorescence analyses. ResultsThe network pharmacological analysis showed 234 targets related to β-asarone, 1,118 genes related to TCI and 2,039 genes associated with CI. Our results confirm that β-asarone treatment not only alleviated brain damage in the VD model by improving mitochondrial and synaptic function, reducing neuronal injury and upregulating the expression of antioxidants but also effectively improved the cognitive behaviour of VD model mice. Moreover, β-asarone downregulated VD-induced RELA and CCND1 mRNA expression. In addition, we validated that β-asarone increased the phosphorylation of PKA and CREB and upregulated cAMP protein expression. The results showed that the cAMP/PKA/CREB signalling pathway was upregulated. Moreover, β-asarone administration decreased the protein expression levels of Beclin-1 and LC3 and increased the expression levels of p62 in VD model mice. Conclusionsβ-asarone inhibits Beclin-1-dependent autophagy and upregulates the cAMP/PKA/CREB signalling pathway to attenuate mitochondrial and synaptic damage from cerebral ischaemia and improve learning and cognitive abilities in VD model mice.

Fluorofenidone alleviates liver fibrosis by inhibiting hepatic stellate cell autophagy via the TGF-β1/Smad pathway: implications for liver cancer.

Liver fibrosis is a key stage in the progression of various chronic liver diseases to cirrhosis and liver cancer, but at present, there is no effective treatment. This study investigated the therapeutic effect of the new antifibrotic drug fluorofenidone (AKF-PD) on liver fibrosis and its related mechanism, providing implications for liver cancer. The effects of AKF-PD on hepatic stellate cell (HSC) autophagy and extracellular matrix (ECM) expression were assessed in a carbon tetrachloride (CCl4)-induced rat liver fibrosis model. In vitro, HSC-T6 cells were transfected with Smad2 and Smad3 overexpression plasmids and treated with AKF-PD. The viability and number of autophagosomes in HSC-T6 cells were examined. The protein expression levels of Beclin-1, LC3 and P62 were examined by Western blotting. The Cancer Genome Atlas (TCGA) database was used for comprehensively analyzing the prognostic values of SMAD2 and SMAD3 in liver cancer. The correlation between SMAD2, SMAD3, and autophagy-related scores in liver cancer was explored. The drug prediction of autophagy-related scores in liver cancer was explored. AKF-PD attenuated liver injury and ECM deposition in the CCl4-induced liver fibrosis model. In vitro, the viability and number of autophagosomes in HSCs were reduced significantly by AKF-PD treatment. Meanwhile, the protein expression of FN, α-SMA, collagen III, Beclin-1 and LC3 was increased, and P62 was reduced by the overexpression of Smad2 and Smad3; however, AKF-PD reversed these effects. SMAD2 and SMAD3 were hazardous factors in liver cancer. SMAD2 and SMAD3 correlated with autophagy-related scores in liver cancer. Autophagy-related scores could predict drug response in liver cancer. AKF-PD alleviates liver fibrosis by inhibiting HSC autophagy via the transforming growth factor (TGF)-β1/Smadpathway. Our study provided some implications about how liver fibrosis was connected with liver cancer by SMAD2/SMAD3 and autophagy.

Role of PI3K/Akt/mTOR pathway-mediated macrophage autophagy in affecting the phenotype transformation of lung fibroblasts induced by silica dust exposure.

The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy. Autophagy plays a key role in the formation of silicosis fibrosis. The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis. This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy. The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h. Macrophages were exposed to different concentrations (0, 25, 50, 100, 200, 400 μg/mL) and different times (0, 6, 12, 24, 48 h) of SiO2 dust suspension. The survival rate of macrophages was measured by cell counting kit-8 (CCK-8) method. Enzyme linked immunosorbent assay (ELISA) was used to measure the contents of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) in the cell supernatant. The co-culture system of macrophages and HFL-1 cells was established by transwell. A blank control group, a SiO2 group, a LY294002 group, a SC79 group, a LY294002+SiO2 group, and a SC79+SiO2 group were set up in this experiment. Macrophages in the LY294002+SiO2 group were pretreated with LY294002 (PI3K inhibitor) for 18 hours, and macrophages in the SC79+SiO2 group were pretreated with SC79 (Akt activator) for 24 hours, and then exposed to SiO2 (100 μg/mL) dust suspension for 12 hours. The expression of microtubule-associated protein 1 light chain 3 (LC3) protein in macrophages was detected by the immunofluorescence method. The protein expressions of PI3K, Akt, mTOR, Beclin-1, LC3 in macrophages, and collagen III (Col III), α-smooth muscle actin (α-SMA), fibronectin (FN), matrix metalloproteinase-1 (MMP-1), tissue metalloproteinase inhibitor-1 (TIMP-1) in HFL-1 cells were measured by Western blotting. After the macrophages were exposed to SiO2 dust suspension of different concentrations for 12 h, the survival rates of macrophages were gradually decreased with the increase of SiO2 concentration. Compared with the 0 μg/mL group, the survival rates of macrophages in the 100, 200, and 400 μg/mL groups were significantly decreased, and the concentrations of TGF-β1 and TNF-α in the cell supernatant were obviously increased (all P<0.05). When 100 μg/mL SiO2 dust suspension was applied to macrophages, the survival rates of macrophages were decreased with the prolonged exposure time. Compared with the 0 h group, the survival rates of macrophages were significantly decreased (all P<0.05), the concentrations of TGF-β1 and TNF-α in the cell supernatant were significantly increased, and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6, 12, 24, and 48 h groups (all P<0.05). Immunofluorescence results demonstrated that after exposure to SiO2 (100 μg/mL) dust for 12 h, LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group (P<0.05). Compared with the blank control group, the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated in the SiO2 group (all P<0.05). Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were decreased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were down-regulated (all P<0.05) in the LY294002+SiO2 group. Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were increased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated (all P<0.05) in the SC79+SiO2 group. Silica dust exposure inhibits the PI3K/Akt/mTOR pathway, increases autophagy and concentration of inflammatory factors in macrophages, and promotes the phenotype transformation of HFL-1 cells into myofibroblasts. The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure, and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.

Modulation of autophagy in the rats with acute intracerebral hemorrhage by acupuncture based on JNK pathway

To investigate the effects of acupuncture on JNK pathway and autophagy level in rats with intracerebral hemorrhage （ICH） and explore the partial mechanism of acupuncture against ICH. SD rats were randomly divided into blank group, model group and acupuncture group. Each group was divided into Day 1, Day 3 and Day 7 subgroups respectively, with 5 rats in each group. The autologous blood injection was adopted to duplicate rat model of ICH. In the acupuncture group, the needle was inserted from "Baihui" （GV20） towards "Qubin" （GB7） on the affected side, stimulating for 30 min each time, once daily； the same acupuncture technique was opera-ted in each subgroup for 1, 3 and 7 days, separately. Using Bederson scale, the neurological deficit was evaluated in each group. Western blot was adopted to detect the protein expression levels of Beclin1, LC3Ⅰ/Ⅱ, phosphorylated c-Jun amino-terminal kinase （p-JNK） and the phosphorylated （p）-c-Jun around hematoma lesion of the brain tissue of rats in each group. After treatment, the neurological deficit score of rats in the model group was higher than that of the blank group at each time point （P<0.05）, and the score of the acupuncture group started declining since the 3rd day of treatment when compared with the model group （P<0.05）. At each time point, compared with the blank group, the protein expression levels of LC3Ⅰ/Ⅱ, Beclin1, p-c-Jun and p-JNK was increased （P<0.01）. Compared with the model group, the protein expression level of LC3Ⅰ/Ⅱ was reduced （P<0.05）； the protein expression levels of Beclin1, p-c-Jun and p-JNK was increased （P<0.05, P<0.01） on day 3 and 7 in the acupuncture group. Acupuncture can activate the JNK pathway in the brain tissue of rats with ICH and increase the level of autophagy, thereby improving the neurological function of the rats with ICH.

Sinularin Exerts Anti-cancer Effects by Inducing Oxidative Stress-mediated Ferroptosis, Apoptosis, and Autophagy in Prostate Cancer Cells.

Prostate cancer is the second-leading cause of cancer death in men. Sinularin is a soft coralsderived natural compound that has anticancer activity in many cancer cells. However, the pharmacological action of sinularin in prostate cancer is unclear. The aim of the study is to examine the anticancer effects of sinularin in prostate cancer cells. We explored the anticancer effects of sinularin on the prostate cancer cell lines, PC3, DU145, and LNCaP, by MTT, Transwell assay, wound healing, flow cytometry, and western blotting. Sinularin inhibited the cell viability and colony formation of these cancer cells. Furthermore, sinularin inhibited testosterone-induced cell growth in LNCaP cells by downregulating the protein expression levels of androgen receptor (AR), type Ⅱ 5α-reductase, and prostate-specific antigen (PSA). Sinularin significantly attenuated the invasion and migration ability of PC3 and DU145 cells, with or without TGF-β1 treatment. Sinularin inhibited epithelialmesenchymal transition (EMT) in DU145 cells after 48 h of treatment by regulating the protein expression levels of Ecadherin, N-cadherin, and vimentin. Sinularin induced apoptosis, autophagy, and ferroptosis by regulating the protein expression levels of Beclin-1, LC3B, NRF2, GPX4, PARP, caspase-3, caspase-7, caspase-9, cleaved-PARP, Bcl-2, and Bax. Moreover, intracellular reactive oxygen species (ROS) were increased but glutathione was decreased after sinularin treatment in PC3, DU145 and LNCaP cells. Sinularin regulated the androgen receptor signaling pathway and triggered apoptosis, autophagy, and ferroptosis in prostate cancer cells. In conclusion, the results indicated that sinularin may be a candidate agent for human prostate cancer and need further study for being applied to human.

Long-term copper exposure caused hepatocytes autophagy in broiler via miR-455-3p-OXSR1 axis

Copper (Cu) is a common environmental pollutant which has been identified to cause toxic effects on animal bodies. MicroRNAs (miRNAs) are a type of non-coding RNAs involved in the regulation of various cellular activities including autophagy, but the potential regulatory mechanisms after excess Cu intake are still uncertain. Our previous study has prompted that Cu exposure reduced liver miR-455-3p levels. Herein, miR-455-3p was found to be an important molecule in the regulation of Cu-induced autophagy in vivo and in vitro. Histopathology observation of liver tissue indicated that Cu-induced severe hepatic damage including cellular swelling and vacuolization. Meanwhile, excessive Cu exposure not only heighten the mRNA and protein expression levels of Beclin1, Atg5, LC3Ⅰ and LC3Ⅱ, but also decreased miR-455-3p levels. In vitro experiment, Cu-induced autophagy can be attenuated by miR-455-3p overexpression. Additionally, oxidative stress-responsive 1 (OXSR1) was identified as a direct downstream target of miR-455-3p by dual luciferase reporter assays. Moreover, knockdown of OXSR1 can attenuate the autophagy induced by Cu treatment and the miR-455-3p inhibitor. Overall, the miR-455-3p-OXSR1 axis works as a regulator of autophagy under Cu stress, which provides a basis for further revealing the mechanism of chronic Cu poisoning.

Adiponectin Protects Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Suppressing Autophagy.

Adiponectin is a cytokine produced by adipocytes and acts as a potential cardioprotective agent and plays an important role in myocardial ischemia/reperfusion injury. In a myocardial hypoxia/reoxygenation model using neonatal rat ventricular myocytes, we investigated the contribution of adiponectin-mediated autophagy to its cardioprotective effects. Cardiomyocytes were exposed to hypoxia/reoxygenation pretreated with or without adiponectin in the presence of absence of rapamycin. Cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Western blotting assay was used to determine the expression levels of microtubule-associated proteins 1A/1B light chain 3B (LC3B), adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), p62/sequestosome 1, unc-51 like autophagy activating kinase 1 (ULK1), and Beclin-1. Autophagosome formation was detected by monodansylcadaverine staining. We found that hypoxia induced a time dependent decline in cardiomyocyte viability, and increase in autophagy and reoxygenation further augmented hypoxia-induced autophagy induction and consequently reduced cell viability. Adiponectin treatment alleviated hypoxia/reoxygenation-induced cellular damage and autophagy in cardiomyocytes. Adiponectin treatment also attenuated hypoxia/reoxygenation-promoted cardiomyocyte autophagy even in the presence of another autophagy stimulator rapamycin in part by inhibiting vacuolar hydron-adenosine triphosphatase. Additionally, autophagy suppression by adiponectin during hypoxia/reoxygenation was associated with the attenuated phosphorylation of AMPK and ULK1, augmented phosphorylation of mTOR, and the reduced protein expression levels of Beclin-1 in cardiomyocytes. Taken together, these results suggest that adiponectin protects ischemia/reperfusion-induced cardiomyocytes by suppressing autophagy in part through AMPK/mTOR/ULK1/Beclin-1 signaling pathway.

The natural medicinal fungus Huaier promotes the anti-hepatoma efficacy of sorafenib through the mammalian target of rapamycin-mediated autophagic cell death.

Sorafenib (SOR) is currently the first line molecular targeting agent for advanced liver cancer therapy. Unfortunately, the insensitivity of liver cancer patients to SOR relatively limits its effectiveness. Huaier (HUA), a natural medicinal parasitic fungus found on the Sophora japonica Linn., has been widely employed as an adjuvant medication for numerous malignancies due to its potent anti-tumoral properties. This studyaims to elucidate the enhancing therapeutic efficacy of HUA on SOR treatment in hepatocellular carcinoma (HCC) cells and mouse models. The CCK-8, clone formation, flow cytometry, immunofluorescence, transmission electron microscopy, western blot, bioinformaticanalysis, and xenografttumorassays were performed to evaluate the synergistic anti-hepatoma efficacy and mechanisms of HUA-SOR combination treatment on HCC cells. The results revealed combination treatment further inhibited proliferation, promoted apoptosis, enhanced autophagy of HCC cells, and suppressed the growth of transplanted tumors in mice, compared with either HUA or SOR treatment alone. For Hep3B and Huh7 cells, the optimal synergistic doses of HUA in combination with SOR were 8 mg/mL + 4 μM and 4 mg/mL + 2 μM, with combination index values of 0.646 and 0.588, respectively. Additionally, the underlying mechanisms might be related to biological processes that are mediated by mammalian target of rapamycin (mTOR). The combination treatment downregulated the protein expression levels of p-mTOR, p-p70S6K, p62, and upregulated the protein expression levels of Beclin-1 and LC3B-II. The mTOR activator MHY1485 attenuated the effect of HUA-SOR combination by inhibiting autophagy, suggesting HUA may potentiate the sensitivity of HCC cells to SOR by partially inducing mTOR-mediated autophagic cell death. These findings might provide a rationale experimental foundation for clinical applications of HUA with SOR.

Lactate inhibits osteogenic differentiation of human periodontal ligament stem cells via autophagy through the MCT1-mTOR signaling pathway.

Periodontal ligament stem cells (PDLSCs) play a crucial role in periodontal bone regeneration. Lactate used to be considered a waste product of glucose metabolism. In recent years, a few pieces of evidence revealed its roles in regulating the osteogenic differentiation of stem cells, but the standpoints were controversial. This study aims to investigate the effects and the mechanisms of lactate on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). The hPDLSCs were treated with different concentrations of lactic acid and lactate to differentiate the effects of the acidic PH and ion lactate. Proliferation and cytotoxicity were measured by Cell Counting Kit-8 (CCK8) assay and Live/Dead assay. The osteogenic differentiation of hPDLSCs was analyzed by alizarin red staining, alkaline phosphatase (ALP) staining, and then osteogenic proteins and genes were measured by western blot and reverse transcription-quantitative PCR (qRT-PCR). To investigate the potential signaling pathways, MCT1 inhibitor, G-protein inhibitors, and rapamycin were used, and then autophagy-related proteins and osteogenic proteins were measured by western blot. The inhibition of lactic acid on the osteogenic differentiation of hPDLSCs was more significant than lactate at the same concentration. Lactate inhibited the expression of ALP which can be rescued by Gα inhibitor. Alizarin red staining, the protein expression levels of osteocalcin (OCN), osteoprotegerin (OPN), osterix (OSX), and beclin1, LC3-II/LC3-I were decreased by lactate and partly rescued by MCT1 inhibitor. Rapamycin restored the protein expression levels of beclin1, LC3-II/LC3-I and OCN, OPN, OSX under the high lactate conditions. Lactate inhibits the expression of ALP via Gα subunit signaling, and inhibits mineralized nodules formation and the expression of osteogenic-related proteins via reducing autophagy through the MCT1-mTOR signaling pathway.

Yifei sanjie Pills Alleviate Chemotherapy-Related Fatigue by Reducing Skeletal Muscle Injury and Inhibiting Tumor Growth in Lung Cancer Mice.

Chemotherapy-related fatigue (CRF), one of the most severe adverse effects observed in cancer patients, has been theoretically related to oxidative stress, and antioxidant treatment might be one of the most valuable therapeutic approaches. However, there are still few effective pharmacological therapies. Yifei Sanjie pills (YFSJ), a classical formula used to treat lung cancer as complementary and alternative medicine, have been proved to alleviate CRF of lung cancer patients in clinical practices. However, the underlying mechanisms have not been clarified. In this study, our data showed that YFSJ alleviated CRF presented as reversing the decline of swimming time and locomotor activity induced by cisplatin (DDP). Moreover, YFSJ significantly reduces the accidence of mitophagy and mitochondrial damage and reduces apoptosis in skeletal muscle tissues caused by DDP. It probably works by decreasing the oxidative stress, inhibiting the activation of the AMPK/mTOR pathway, decreasing protein expression levels of Beclin1 and other autophagy-related proteins, and attenuating the activation of Cytochrome c (cyto. C), Cleaved Caspase-9 (c-Casp 9), and other apoptosis-related proteins. Furthermore, YFSJ enhanced DDP sensitivity by specifically promoting oxidative stress and activating apoptosis and autophagy in the tumor tissues of mice. It was also found that YFSJ reduced the loss of body weight caused by DDP, reversed the ascent of serum concentrations of alanine aminotransferase (ALT), aminotransferase (AST), and creatinine (CREA), increased the spleen index, and prolonged the survival time of mice. Taken together, these results revealed that YFSJ could alleviate CRF by reducing mitophagy and apoptosis induced by oxidative stress in skeletal muscle; these results also displayed the effects of YFSJ on enhancing chemotherapy sensitivity, improving quality of life, and prolonging survival time in lung cancer mice received DDP chemotherapy.

Involvement of Mitophagy in Primary Cultured Rat Neurons Treated with Nanoalumina.

The aim of this study was to explore the influence of the neurotoxicity of nanoalumina on primarily cultured neurons. Normal control, particle size control, aluminum, micron-alumina, and nanoalumina at 50-nm and 13-nm particle sizes were included as subjects to evaluate the level of apoptosis, necrosis, and autophagy in primarily cultured neurons and further explore the mitophagy induced by nanoalumina. The results demonstrated that nanoalumina could induce neuronal cell apoptosis, necrosis, and autophagy, among which autophagy was the most notable. When the autophagy inhibitor was added to the nanoalumina-treated group, it significantly downregulated the protein expression levels of Beclin-1 and LC3II/LC3. Observation under a transmission electron microscope and a fluorescence microscope revealed mitophagy characteristics induced by nanoalumina. Additionally, the neurotoxicological effects induced by nanoalumina were more significant than those induced by aluminum and in a particle size-dependent manner.

6-Gingerol suppresses cell viability, migration and invasion via inhibiting EMT, and inducing autophagy and ferroptosis in LPS-stimulated and LPS-unstimulated prostate cancer cells.

6-Gingerol is a bioactive compound isolated from Zingiber officinale. 6-Gingerol has been shown to have anticancer effects in numerous types of cancer cell. The mechanisms underlying the anticancer effect of 6-Gingerol in prostate cancer requires investigation. In the present study, the effect on cell viability of 6-Gingerol on LNCaP, PC3 and DU145 prostate cancer cells were determined using the MTT and colony formation assays. 6-Gingerol significantly inhibited cell migration, adhesion and invasion in LPS-stimulated and LPS-unstimulated prostate cancer cells. Furthermore, these changes were accompanied by alterations in the protein expression levels of epithelial-mesenchymal transition biomarkers, including E-cadherin, N-cadherin, Vimentin and zonula occludens-1. 6-Gingerol also induced autophagy by significantly increasing LC3B-II and Beclin-1 protein expression levels in prostate cancer cells. Combining 6-Gingerol with LY294002, an autophagy inhibitor, significantly increased cell survival in DU145 cells. Furthermore, 6-Gingerol significantly decreased the protein expression levels of glutathione (GSH) peroxidase 4 and nuclear factor erythroid 2-related factor 2 in prostate cancer cells. Reactive oxygen species (ROS) levels were significantly increased but GSH levels were decreased following 6-Gingerol treatment in prostate cancer cells. Co-treatment with the ferroptosis inhibitor, ferrostatin-1, significantly increased cell viability and significantly decreased ROS levels in 6-Gingerol-treated cells. These results suggested that 6-Gingerol may have inhibited prostate cell cancer viability via the regulation of autophagy and ferroptosis. In addition, 6-Gingerol inhibited cell migration, adhesion and invasion via the regulation of EMT-related protein expression levels in LPS-stimulated and LPS-unstimulated prostate cancer cells. In conclusion, 6-Gingerol may induce protective autophagy, autophagic cell death and ferroptosis-mediated cell death in prostate cancer cells. These findings may provide a strategy for the treatment and prevention of prostate cancer.

Galangin inhibits neointima formation induced by vascular injury via regulating the PI3K/AKT/mTOR pathway.

Aims: The proliferation and migration of vascular smooth muscle cells (VSMCs) play vital roles in the pathological process of neointima formation after vascular injury. Galangin, an extract of the ginger plant galangal, is involved in numerous biological activities, including inhibiting the proliferation and migration of tumor cells, but its effect on VSMCs is unknown. This study focused on the role and mechanism of galangin in the neointima formation induced by vascular injury. Methods and results: In this study, we found that galangin restrained the PDGF-BB-induced proliferation, migration and phenotypic switching of VSMCs in a concentration-dependent manner. In vivo, we established a model of carotid artery balloon injury in rats, followed by intragastric administration of galangin (40 mg kg-1 day-1 or 80 mg kg-1 day-1) for 14 or 28 consecutive days. Then, the degree of neointima hyperplasia was evaluated by H&E staining, and the level of relevant protein expression was assessed by immunofluorescence and western blotting. In vitro, we isolated and grew primary rat aortic smooth muscle cells, which were treated with PDGF-BB and different doses of galangin, and then CCK-8 assay, wound healing assay, transwell assay, western blotting and immunofluorescence assays were performed. We found that galangin significantly inhibited PDGF-BB-induced proliferation, migration, and phenotypic switching of VSMCs and promoted autophagy in VSMCs in vitro, and galangin significantly inhibited neointimal hyperplasia after the common carotid artery balloon injury in rats. In terms of mechanisms, galangin inhibited the PI3K/AKT/mTOR pathway, thereby suppressing VSMC's switch from a contractile to a synthetic phenotype, inhibiting VSMC proliferation, migration and phenotypic switching and upregulating the Beclin1 protein expression levels and the ratio of LC3BII/I, promoting VSMC autophagy, and thereby inhibiting neointimal hyperplasia after vascular injury. Conclusion: Our study suggests that galangin inhibits neointimal hyperplasia after vascular injury by inhibiting smooth muscle cell proliferation, migration and phenotypic switching and by promoting autophagy, and that galangin may be a promising drug for the prevention and treatment of vascular restenosis after PCI.

Dynamic changes of autophagy during hypertrophic scar formation and the role of autophagy intervention

BackgroundThe role of autophagy in the formation of hypertrophic scars (HS) remains unclear. This study aimed to explore the role and potential mechanism of autophagy during the development of HS. MethodsRNA and protein expression levels of Beclin-1, p62, and LC3II in normal skin tissues and HS specimens from different patients were examined. Autophagy inducers and inhibitors were used to cure established HS in rabbit ears, and the expression of Beclin-1, p62, and LC3II at the RNA and protein level was determined. Lastly, the effects of autophagy inducers and inhibitors on HS development were analyzed. ResultsCompared to normal skin tissues, the expression of LC3Ⅱ and Beclin-1 was higher (P<0.05), while that of p62 was lower (P<0.05) in HS tissues. In addition, the LC3II/LC3I ratio was increased during HS formation, and the altered expression of the three proteins stabilized after one year. Administration of autophagy inducers enhanced the formation of HS as well as the expression levels of LC3II and Beclin-1 but decreased p62 expression. Meanwhile, administration of autophagy inhibitors increased the expression of LC3II, Beclin-1, and p62, along with reduced HS formation. ConclusionAutophagic activity increased during HS initiation and subsequent stabilization. In addition, autophagy inhibitors were able to inhibit HS formation by suppressing autophagy, whereas autophagy inducers promoted scar hyperplasia by enhancing autophagy.

Potential protective effect of hesperidin on hypoxia/reoxygenation-induced hepatocyte injury.

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.

Effect of advanced glycation end products on autophagic ability in osteoblasts.

Excessive production of AGEs in diabetic patients will affect the normal function of osteoblasts, and this process may be related to autophagy of osteoblasts. This study aims to explore the effect of advanced glycation end products (AGEs) on autophagic activity during osteogenic differentiation in rat bone marrow mesenchymal stem cells (BMSCs). BMSCs were isolated and cultured in vitro, treated with different concentrations (0, 50, 100, 200, and 400 mg/L) of AGEs for different time (3, 6, 12, 24, 48, and 72 h). The proliferation activity was detected by CCK-8 method. The mRNA and protein expression levels of Beclin1 and LC3 in cells were detected by real-time PCR and Western blotting, respectively.The autophagic vacuoles were observed under the transmission electron microscope. The cells were treated with autophagy promoter rapamycin or autophagy inhibitor 3MA. After 7 days of osteogenic induction, we performed alkaline phosphatase (ALP) staining and real-time PCR to detect the mRNA expression levels of osteogenesis-related genes. In the low-concentration groups, the proliferation activity in BMSCs was increased (P<0.01), the mRNA and protein expressions of autophagy-related genes LC3 and Beclin1 were increased (both P<0.01). The number of autophagosome also was increased. In the high-concentration groups, the results were just the opposite. In the low-concentration groups, the ALP staining was deeper than that of the 0 mg/L AGEs group, and the mRNA expressions of the osteogenic related genes were increased (P<0.01). But the results were reversed in the presence of autophagy inhibitor 3MA. In the high-concentration groups, the ALP staining was lighter than that of the 0 mg/L AGEs group, and the mRNA expressions of the osteogenic related genes were decreased (P<0.01). After the addition of the autophagy promoter rapamycin, the results were reversed. Low concentration of AGEs can enhance the proliferative activity of BMSCs and promote osteogenic differentiation by accelerating autophagy. High concentration of AGEs can suppress the proliferation of BMSCs and inhibit osteogenic differentiation by reducing autophagy.

Beclin-1/LC3-II dependent macroautophagy was uninfluenced in ischemia-challenged vascular endothelial cells

Autophagy has been extensively studied and occurs in many biological settings. However, a question remains as to whether ischemia enhances Beclin-1/LC3-II-dependent macroautophagy in vascular endothelial cells, as has been previously thought. Furthermore, the effect of the level of autophagy on cell or skin flap survival still requires elucidation. We created a lethal ischemia model in human umbilical vascular endothelial cells (HUVECs), performed quantitative proteomics and bioinformatics analyses, and verified the autophagic status and effect both in vitro and in vivo. The significantly upregulated proteins encoded by autophagy-related genes (ATGs) included ATG2A, ATG3, ATG4B, ATG5, ATG7, ATG9A, ATG12, ATG16, and ATG101. The significantly enhanced lysosomal proteins were cathepsin B, cathepsin D, lysosome-associated membrane protein 1 (LAMP1), and LAMP2. However, the differentially expressed proteins excluded Beclin-1, microtubule-associated protein light chain 3 (LC3)-I, and LC3-II. Western blot analyses verified that the protein expression levels of Beclin-1, LC3-I, and LC3-II were neither upregulated nor downregulated in ischemia-challenged HUVECs. The autophagic status was not enhanced by rapamycin in ischemic HUVECs but appeared to be inhibited by chloroquine. Our in vivo study on rats showed that a downregulation in autophagic status jeopardized skin flap survival. In conclusion, Ischemia neither enhanced nor inhibited Beclin-1/LC3-II-dependent canonical macroautophagy both in vitro and in vivo, in contradiction to previous studies. An appropriate autophagic homeostasis can minimize cell or skin flap damage.

Rapamycin Inhibits Glioma Cells Growth and Promotes Autophagy by miR-26a-5p/DAPK1 Axis.

BackgroundGlioma is a common intracranial malignant tumor with high rates of invasiveness and mortality. This study aimed to investigate the mechanism of rapamycin in glioma.MethodsU118-MG cells were treated with and without rapamycin in vivo and then collected for RNA sequencing. Differentially expressed miRNAs (DEMs) were screened and verified. MiR-26a-5p was selected for functional verification, and the target gene of miR-26a-5p was identified. The effects of miR-26a-5p on cell proliferation, cell cycle, apoptosis, and autophagy were also investigated.ResultsIn total, 58 up-regulated and 41 down-regulated DEMs were identified between rapamycin-treated and untreated U118-MG cells. MiR-26-5p levels were up-regulated in U118-MG cells treated with 12.5 μM rapamycin, and death-associated protein kinase 1 (DAPK1) expression, a direct miR-26a-5p target gene, was down-regulated. Rapamycin substantially inhibited cell proliferation and cell percentage in the S phase and promoted cell apoptosis; miR-26a-5p inhibitor increased cell proliferation and cell cycle and decreased cell apoptosis; DAPK1 overexpression further induced cell proliferation, increased the cell number in the S phase, and inhibited apoptosis in glioma cells. Notably, rapamycin increased the autophagy-related Beclin1 protein expression levels and the LC3 II/I ratio.ConclusionRapamycin exerts anti-tumor effects by promoting autophagy in glioma cells, which was dependent on the miR-26a-5p/DAPK1 pathway activation by rapamycin.