Expression Of Autophagy Proteins Research Articles

The Mitochondrial-Targeted Peptide, SS31, Protects Murine 661W Cells from Oxidative Damage via Induction of Autophagy.

The goal of this study was to examine the impact of the mitochondrial-targeted antioxidant peptide, SS31, and its role in promoting autophagy in cone photoreceptor 661W cells that were subjected to oxidative damage. To do so, we examined the viability of 661W cells in the presence of increasing concentrations of H₂O₂ with or without SS31 pre-treatment using the MTT assay and by expression of autophagy and apoptosis-associated proteins LC3-II/I, P62, and caspase-3. Autophagy was evaluated by fluorescence microscopy in cells stained with monodansyl cadaverine (MDC). Autophagy was induced with rapamycin (Rap) and inhibited with bafamycin A1 (bafA1) followed by examination of Reactive oxygen species (ROS) levels in target 661W cells by fluorescence microscopy and flow cytometry. Annexin V/PI staining was used to evaluate the rate of apoptosis and mRNA sequencing (mRNA-seq) analysis (Illumina platform) was performed on H₂O₂-exposed 661W cells treated with SS31. Among our results, we observed a substantial and concentration-dependent decrease in 661W cell viability in response to H₂O₂-exposure; production of ROS, autophagy and apoptosis were induced at 8 h in response to exposure to 100 μM of H₂O₂. Pre-treatment with 100 nM SS31 resulted in significant attenuation of H₂O₂-mediated cytotoxicity, together with reduced ROS production and enhanced autophagy observed in response to oxidative stress. Both Rap and bafA1 were used to modulate SS31-mediated autophagy; the impact of Rap was similar to that of SS31. By contrast, administration of bafA1 counteracted autophagy induced by SS31. Furthermore, mRNAseq analysis revealed that SS31 promoted significant alterations in gene expression in 661W cells and suggested that autophagy was induced via the mTORC1-mediated signaling. In conclusion, our results indicate that exposure to H₂O₂ resulted in reduced 661W cell viability via mechanisms associated with oxidative damage, apoptosis, and autophagy. Notably, we demonstrated that pre-treatment with SS31 protects 661W cells from H₂O₂-induced oxidative damage that may result in part from induction of autophagy via mTORC1-mediated signaling pathways.

Inhibition of excessive mitophagy by N-acetyl-L-tryptophan confers hepatoprotection against Ischemia-Reperfusion injury in rats

In order to investigate the mechnism of hepatoprotective of N-acetyl-L-tryptophan (L-NAT) against ischemia-reperfusion (I/R) injury, the effects of L-NAT were investigated in hepatic ischemia-reperfusion injury (HIRI) models both in vitro and in vivo, which were made by BRL cells and Sprague-Dawley (SD) rats, respectively. The cell viability of hepatocyte was assessed by cell counting kit-8 (CCK-8) staining. The activation of autophagy was detected by electron microscopy (EM), quantitative real-time PCR (qRT-PCR), Western blotting and immunofluorescence. The activation of mitophagy was determined by the change of autophagy related protein, change of mitochondrial structure and function, co-location of autophagy protein and MitoTracker. Results showed that the morphological structures of hepatocytes were changed significantly after HIRI, and the cell viability of hydrogen peroxide (H2O2)-induced BRL cells was decreased. Autophagy markers Beclin1, microtubule associated protein 1 light chain 3-II (LC3-II) and autophagy related protein-7 (ATG-7) were highly expressed and the expression of SQSTM1 (P62) was decreased after HIRI, which suggested that autophagy of hepatocytes was activated after I/R. The reduction of ATP, mitochondrial DNA (mtDNA) and the mitochondrial transmembrane potential (ΔΨm) after H2O2-induced revealed that function of mitochondrial had also undergone significant changes. The increased expression of autophagy protein, destructure of mitochondria and mitochondrial dysfunction, the increased co-location of Beclin1 and MitoTracker induced by H2O2 implied the excessive mitophagy. The expression of the autophagy protein was increased by 3-Methyladenine (3-MA), providing another piece of evidence. Importantly, all changes were restored by L-NAT pretreament. In conclusion, the present findings demonstrate that excessive mitophagy involved in the process of HIRI and L-NAT may protect hepatocytes against HIRI by inhibiting activation of mitophagy and improving the structure and function of mitochondria.

Endurance exercise-induced expression of autophagy-related protein coincides with anabolic expression and neurogenesis in the hippocampus of the mouse brain.

Autophagy and neurogenesis play a pivotal role in maintaining cellular homeostasis of neurons in the brain. Endurance exercise (EXE) serves as a potent regulator of both autophagy and neurogenesis in the hippocampus of the brain; however, the underlying molecular mechanisms of the dual expression remains unclear. Thus, we examined the signaling pathways of EXE-induced autophagy and neurogenesis-associated protein expression in the hippocampus. C57BL/6 male mice (10 weeks old) were randomly divided into two groups: control group (n = 10) and EXE group (EXE, n = 10). Our results showed that EXE increased expression of autophagy-related protein [LC3 II, BECLIN1, autophagy-related 7 (ATG7), p62, LAMP2, CATHEPSIN L and transcription factor EB] in the presence of anabolic signaling expression (AKT-mammalian target of rapamycin-ribosomal S6 kinase). Intriguingly, long-term EXE-mediated neurogenesis in the hippocampus was observed despite the downregulated expressions of canonical neurotrophic factors (e.g. brain-derived neurotrophic factor, glial cell line-derived neurotrophic factors and nerve growth factor); instead, upregulation of neuregulin-1 (NRG1)-mediated signaling cascades (e.g. NRG1-extracellular signal-regulated kinase-ribosomal s6 kinase-cyclic adenosine mono-phosphate response element-binding protein) were associated with EXE-induced hippocampal neurogenesis and synaptic plasticity. Our data, for the first time, show that EXE-mediated expression of autophagy-related protein coincides with anabolic expression and that NRG1 is involved in EXE-mediated neurogenesis and synaptic plasticity. Taken together, this study provides a novel mechanism of hippocampal autophagy and neurogenesis, which may provide potential insight into developing therapeutic neuroprotective strategies.

Diurnal Rhythmicity of Autophagy Is Impaired in the Diabetic Retina.

Retinal homeostasis is under both diurnal and circadian regulation. We sought to investigate the diurnal expression of autophagy proteins in normal rodent retina and to determine if this is impaired in diabetic retinopathy. C57BL/6J mice and Bio-Breeding Zucker (BBZ) rats were maintained under a 12h/12h light/dark cycle and eyes, enucleated over a 24 h period. Eyes were also collected from diabetic mice with two or nine-months duration of type 1 diabetes (T1D) and Bio-Breeding Zucker diabetic rat (BBZDR/wor rats with 4-months duration of type 2 diabetes (T2D). Immunohistochemistry was performed for the autophagy proteins Atg7, Atg9, LC3 and Beclin1. These autophagy proteins (Atgs) were abundantly expressed in neural retina and endothelial cells in both mice and rats. A differential staining pattern was observed across the retinas which demonstrated a distinctive diurnal rhythmicity. All Atgs showed localization to retinal blood vessels with Atg7 being the most highly expressed. Analysis of the immunostaining demonstrated distinctive diurnal rhythmicity, of which Atg9 and LC3 shared a biphasic expression cycle with the highest level at 8:15 am and 8:15 pm. In contrast, Beclin1 revealed a 24-h cycle with the highest level observed at midnight. Atg7 was also on a 24-h cycle with peak expression at 8:15 am, coinciding with the first peak expression of Atg9 and LC3. In diabetic animals, there was a dramatic reduction in all four Atgs and the distinctive diurnal rhythmicity of these autophagy proteins was significantly impaired and phase shifted in both T1D and T2D animals. Restoration of diurnal rhythmicity and facilitation of autophagy protein expression may provide new treatment strategies for diabetic retinopathy.

Autophagy and Heat Shock Protein 70 Expression During Acute Heat Stress in Isosmotic and Hyperosmotic Conditions in Peripheral Blood Mononuclear Cells from Young Adults: Preliminary Data

Heat‐stress induced dehydration is associated with elevations in extracellular osmolality. To counteract the resultant osmotic stress, cells employ several stress response systems including the heat shock protein (HSP) and autophagy pathways. HSPs refold damaged proteins, whereas autophagy acts to degrade damaged cellular components, thereby promoting cellular survival. Accumulating evidence indicates a coordinated response between the HSP and autophagy systems, whereby both systems are activated under conditions of heat stress. However, it remains unknown whether elevated extracellular osmolality augments the HSP and autophagic response to whole‐body heat stress in humans. Thus, the purpose of this study was to examine changes in HSP (HSP70) and autophagy resulting from elevated intravascular osmolality during acute heat exposure. We hypothesized that both autophagy and HSP70 protein expression would be greater under hyperosmotic stress compared to isosmotic conditions. On separate days (separated by ~1 week), six young men (mean [SD]; 27 [5] years) received a 90‐min intravenous infusion with either isosmotic (ISO; 0.9% NaCl; 0.200 mL·kg−1·min−1) or hyperosmotic (HYP; 3.0% NaCl; 0.125 mL·kg−1·min−1) saline, followed by passive whole‐body heating to ~0.8°C above baseline esopha‐‐‐geal temperature using a water perfused suit. Serum osmolality was determined via freeze‐point depression from blood samples at baseline and at the end of passive heating. Peripheral blood mononuclear harvested at these time‐points were analyzed via Western blotting to assess changes in HSP70 protein and proteins indicative of autophagic activity including microtubule associated protein 1 light chain 3 beta‐II (LC3‐II) and p62/sequesterome‐1 (p62). All values were normalized to β‐actin and reported as fold changes relative to baseline. Data are presented as means and standard deviations (SD) and were compared between‐conditions at the end of passive heating using paired (2‐tailed) t‐tests (α=0.05). At the end of heating, serum osmolality was higher during HYP when compared to ISO (298 [SD 3] vs. 291 [SD 3]; p<0.001). During passive heating, LC3‐II was increased (2.20 [SD 2.12] vs. 1.62 [SD 1.47]; p=0.04) and p62 protein was reduced (0.47 [SD 0.33] vs. 1.20 [SD 0.56]; p=0.01) in HYP compared to ISO, indicating greater autophagic activation during HYP. Despite this, HSP70 protein was lower under HYP conditions following heating than ISO (0.72 [SD 0.24] vs. 1.20 [SD 0.19]; p=0.04). Contrary to our hypothesis, these preliminary findings indicate greater reliance on the autophagic response rather than simultaneous activation of HSP70 during acute heat exposure with super‐imposed hyperosmotic stress.Support or Funding InformationNational Science and Engineering Research Council of Canada

Autophagy, Mitophagy and MicroRNA Expression in Chronic Hepatitis C and Autoimmune Hepatitis

Although the role of autophagy has been implicated in several forms of chronic hepatitis, it is still not fully understood. Active autophagy eliminates damaged molecules and organelles (such as mitochondria) by lysosomal degradation. In the present study, we aimed to examine and compare autophagy activity in chronic hepatitis C (CHC) and autoimmune hepatitis (AIH) by detecting the expression of autophagy (LC3 and p62) and mitochondrium-related (TOMM20) proteins, as well as the levels of selected microRNAs (miR-101, -155, -204 and − 224) known to be involved in the regulation of autophagy. In addition, the expression levels were related to pathohistological parameters. Liver biopsy samples, including 45 CHC and 18 AIH cases, were immunohistochemically stained for LC3, p62 and TOMM20 and the expression of miRNAs was determined using real-time PCR. We found elevated LC3 and p62 in AIH samples as compared with CHC ones, indicating an activated autophagy that is impaired in AIH as no degradation of p62 seemed to occur. Moreover, p62 showed strong correlation with necroinflammatory grades in the AIH group. The observed elevated levels of TOMM20 and p62 suggest a less efficient elimination of damaged mitochondria in AIH as opposed to CHC, in which autophagy seems to have a more active function. The level of miR-101 was increased in case of CHC as compared with AIH, however, miR-155, -204 and 224 resulted in no expressional. Furthermore, miR-224 level correlated with steatosis and miR-155 expression with fibrosis stage in CHC. In conclusion, dissimilar autophagic activity was observed in CHC and AIH, suggesting a close association between impaired autophagy and severity of necroinflammation. This impairment may not be regulated by the analyzed miRNAs. Nevertheless, miR-224 and − 155 seem to be associated with CHC progression.

Antihelminthic Niclosamide Induces Autophagy and Delayed Apoptosis in Human Non-small Lung Cancer Cells In Vitro and In Vivo.

Niclosamide is an antihe-minthic drug that has shown cytotoxic effects on non-small cell lung carcinoma (NSCLC) cells. However, the exact mechanisms underlying the anti-tumour activity of niclosamide in NSCLC cancer cells remains to be defined. The aim of this study was to evaluate the antitumor activity of niclosamide in human A549 and CL1-5 non-small cell lung cancer cells using in vitro and in vivo. We investigated the effects of niclosamide on cell viability, apoptosis, the mitochondrial membrane potential (MMP; Δϕm), and autophagy and apoptosis-related protein expression in human A549 and CL1-5 non-small cell lung cancer cells. Niclosamide induced mainly caspase-independent apoptosis through apoptosis-inducible factor (AIF) translocation to the nucleus upon mitochondria damage. Moreover, niclosamide-induced autophagy may act as adaptive response against apoptosis. AMPK/AKT/mTOR pathway were involved in niclosamide-induced cell death and autophagy in response to ATP depletion. Furthermore, niclosamide efficiently suppressed tumor growth and induce autophagy in vivo. Niclosamide induced apoptosis by activating the intrinsic and caspase-independent pathway in human A549 and CL1-5 non-small cell lung cancer cells. Therefore, niclosamide is a potential candidate for anti-NSCLC therapy.

带状疱疹患者外周血CD4+ T淋巴细胞及皮损组织中自噬水平的检测

Objective To observe changes in expression of autophagy proteins in peripheral CD4+ T lymphocytes and the epidermis of skin lesions, as well as generation of autophagy vesicles in epidermal cells in skin lesions of patients with herpes zoster, and to explore the relationship between varicella-herpes zoster virus (VZV) infection and autophagy. Methods Totally, 35 patients with herpes zoster were enrolled from Department of Dermatology, General Hospital of Southern Theater Command of PLA between December 2017 and December 2018, including 20 males and 15 females. Their age ranged from 18 to 79 (59.23 ± 9.27) years, pain duration was 5.14 ± 2.28 days, and lesion duration (from the onset of the lesion to the clinic visit) was 3.45 ± 1.77 days. Flow cytometry was performed to determine the expression of autophagy proteins including microtubule-associated protein 1 light chain 3B (LC3B) , Beclin-1 and p62 in peripheral blood CD4+ T lymphocytes of these patients. Thirty healthy adults served as control group. Lesional skin tissues were obtained from 12 patients with herpes zoster, and perilesional normal skin tissues of the same patient served as the control. Immunohistochemical study was conducted to determine the expression of autophagy proteins LC3B, Beclin-1 and p62 in epidermal tissues, and transmission electron microscopy to observe the generation of autophagy vesicles in epidermal cells. Two independent-sample t-test was carried out for intergroup comparison. Results The expression rates of autophagy proteins LC3B and Beclin-1 in peripheral CD4+ T lymphocytes were significantly higher in the herpes zoster group (61.23% ± 7.61%, 35.84% ± 4.22%, respectively) than in the control group (36.56% ± 4.27%, 15.34% ± 1.89%, respectively; t = 15.75, 24.56 respectively, both P < 0.01) , while the expression rate of p62 (5.75% ± 0.67%) was significantly lower in the herpes zoster group than in the control group (10.03% ± 1.15%, t = 18.65, P < 0.01) . Among the 12 patients with herpes zoster, the expression levels of LC3B and Beclin-1 in the epidermis were significantly higher in the skin lesions than in the perilesional normal skin tissues (t = 2.86, 4.58, P < 0.05) , but the expression level of p62 was significantly lower in the skin lesions than in the perilesional normal skin tissues (t = 2.43, P < 0.05) . Transmission electron microscopy showed formation of autophagy vesicles containing virus particles in epidermal cells in the skin lesions of 12 patients with herpes zoster, and vesicle counts were significantly higher in the skin lesions than in perilesional normal skin tissues (t = 9.67, P < 0.01) . Conclusion The autophagy level was elevated in peripheral CD4+ T lymphocytes and epidermis of skin lesions of patients with herpes zoster. Key words: Herpes zoster; Herpesvirus 3, human; Autophagy; CD4-positive T-lymphocytes; Host-pathogen interactions; LC3B; Beclin-1; p62

Delayed effects of sulfur mustard on autophagy suppression in chemically-injured lung tissue

BackgroundAutophagy is an intracellular hemostasis mechanism, responding to extracellular or intracellular stresses. Sulfur mustard (SM) induces cellular stress. Iranian soldiers exposed to SM gas, during the Iraq-Iran war, suffer from delayed complications even 30 years after exposure. In this study, for exploring the SM effect on autophagy pathway, gene and protein expression of autophagy markers are evaluated in the lung of SM-exposed people. Methods52 FFPE lung tissues of SM-exposed people and 33 lung paraffin blocks of non-exposed patients to SM were selected. LC3 and Beclin-1 mRNA expressions were evaluated by QRT-PCR. LC3-B protein and LC3II/LC3I proteins ratio were detected by Immunohistochemistry and immunoblotting method. The collected data were analyzed in SPSS, and P value ≤ 0.05 was considered significant. ResultsLC3 gene expression in SM-exposed subjects (median CT value = 4.97) increased about 4 fold compared with the control group (median CT value = 0.46, P = 0.025). Beclin-1 mRNA expression had not significant difference between two groups. After adjusting the confounding variables such as drug usage, LC3-B protein (P = 0.041) and LC3II/LC3I ratio (P = 0.044) were found significantly lower in the lung cells of SM-exposed group. ConclusionUpon exposure to SM gas, the lung cells are affected by acute cellular stress such as oxidative stress. The study results show that LC3 mRNA level increases in these patients, but, surprisingly, LC3-B protein via unknown mechanism has been down-regulated. N-acetyl cysteine and salbutamol drugs could induce the autophagy, and help to reduce the SM effects and improve the clinical condition of SM-injured patients.

Salidroside induces apoptosis and protective autophagy in human gastric cancer AGS cells through the PI3K/Akt/mTOR pathway

Salidroside, a natural active ingredient extracted from Rhodiola rosea, has been shown to exert antitumor activity against breast cancer Dong Young et al. [1], colon cancer Sun et al. [2] and bladder cancer Tian et al. [3]. However, the effect of salidroside on apoptosis and autophagy in gastric cancer remains unclear. In our research, we observed the biological effect of salidroside on human gastric cancer AGS cells. Our results demonstrated that salidroside inhibited the growth of AGS cells both in vivo and in vitro and exerted a proapoptotic effect on AGS cells as confirmed by flow cytometry, Hoechst staining and western blot analysis. Additionally, we found that salidroside decreased the phosphorylation of PI3K and Akt and that pretreatment with the PI3K/Akt agonist IGF-1 could weaken the proapoptotic effect of salidroside. Interestingly, the exposure of AGS cells to salidroside induced autophagy as indicated by transmission electron microscopy, mRFP-GFP-LC3 transfection and western blot analysis, suggesting that salidroside promoted autophagy in gastric cancer AGS cells. Furthermore, treatment with the autophagy inhibitor chloroquine enhanced salidroside-induced cell apoptosis, indicating that the autophagy mediated by salidroside may protect AGS cells from death. Additionally, we found that salidroside decreased the level of p-mTOR protein in a concentration-dependent manner and that pretreatment with IGF-1 decreased the expression of autophagy proteins, suggesting that salidroside induced autophagy through the PI3K/Akt/mTOR pathway. The above findings indicate that salidroside inhibited the growth of gastric cancer and induced apoptosis and protective autophagy through the PI3K/Akt/mTOR pathway. In summary, our study provides novel insights regarding the activity of salidroside against gastric cancer and contributes to the clinical application of salidroside combined with autophagy inhibitors as a chemotherapeutic strategy for human gastric cancer.

The Application and Analytical Pathway of Dexmedetomidine in Ischemia/Reperfusion Injury.

Ischemia/reperfusion cerebral injury can cause serious damage to nerve cells. The injured organelles are cleared by autophagy eventually, which is critical for cell survival. Dexmedetomidine is neuroprotective in various ischemia/reperfusion models. Mitochondrial calcium uniporter (MCU) is the most important channel of mitochondrial Ca2+ influx into mitochondria, where Ca2+ has a potential effect on mitochondrial autophagy. However, the role of MCU in the changes of mitophagy and autophagy caused by dexmedetomidine is unknown. In this study, we constructed an in vitro I/R model by subjecting the oxygen and glucose deprivation/reperfusion model to SH-SY5Y cells to mimic the cerebral I/R injury. We found that postconditioning with dexmedetomidine and 3-methyladenine (3MA, an autophagy inhibitor) increased the cell survival meanwhile reduced the production of autophagic vesicles and the expression of LC3 and Beclin 1. This process also increased the expression of BCL-2, P62, and TOM20. After applied with spermine (MCU-specific agonist), the expression of autophagy proteins by dexmedetomidine was reversed, and the same changes were also observed in immunofluorescence. The results of our study suggested that dexmedetomidine can inhibit MCU and reduce excessive mitophagy and autophagy for conferring protection against I/R injury.

Sleep Deprivation Modifies Noise-Induced Cochlear Injury Related to the Stress Hormone and Autophagy in Female Mice.

A lack of sleep is linked with a range of inner ear diseases, including hearing loss and tinnitus. Here, we used a mouse model to investigate the effects of sleep deprivation (SD) on noise vulnerability, and explored the mechanisms that might be involved in vitro, focusing particularly corticosterone levels and autophagic flux in cells. Female BALB/c mice were divided into six groups [control, acoustic trauma (AT)-alone, 1 day (d) SD-alone, 1d SD pre-AT, 5d SD-alone, and 5d SD pre-AT]. Cochlear damage was then assessed by analyzing auditory brainstem response (ABR), and by counting outer hair cells (OHCs) and the synaptic ribbons of inner hair cells (IHCs). In addition, we measured levels of serum corticosterone and autophagy protein expression in the basilar membranes by ELISA kits, and western blotting, respectively. We found that SD-alone temporarily elevated ABR wave I amplitude, but had no permanent effect on hearing level or IHC ribbon numbers. Combined with AT, the number of synaptic ribbons in the 1d SD pre-AT group was significantly higher than that in the AT-alone group, whereas the 5d SD pre-AT group showed more severe synaptopathy, and a greater loss of OHCs after 2 weeks than the other experimental groups exposed to noise. Correspondingly, the levels of corticosterone in the AT-alone group were higher than those of the 1d SD pre-AT group, but lower than those of the 5d SD pre-AT group. The 1d SD pre-AT group showed a marked elevation in the expression of microtubule-associated protein 1 light chain 3B (LC3B), whereas the AT-alone group exhibited only a mild increase. In contrast, the levels of LC3B did not change in the 5d SD pre-AT group. Experiments with HEI-OC-1 cells and cochlear basilar membrane cultures showed that high-concentrations of dexamethasone, and the inhibition of autophagy, aggravated cellular apoptosis induced by oxidative stress. In conclusion, noise-induced synaptopathy and hair cell loss can be mitigated by preceding 1d SD, but will be aggravated by preceding 5d SD. These findings may be attributable to corticosterone levels and the extent of autophagy.

NT-3 Promotes Oligodendrocyte Proliferation and Nerve Function Recovery After Spinal Cord Injury by Inhibiting Autophagy Pathway

BackgroundSpinal cord injury (SCI) is a serious medical problem, leading to lifelong disability and increasing the health burden worldwide. Traditional treatments have limited effects on neuronal function recovery. Previous studies showed that neurotrophin-3 (NT-3) promoted oligodendrocyte survival and improved neuronal functional recovery after SCI. However, the mechanism by which NT-3 promotes oligodendrocyte survival after SCI remains unclear, which limits its application. Materials and methodsA total of 75 female Sprague–Dawley rats were randomly divided into three groups: the NS group, NT-3 group, and NT-3 + rapamycin group. After successful modeling, the spinal cord specimens were taken at the corresponding time points. Western blot was used to detect autophagy-related proteins and Olig1 protein expression and combined with pathology, immunohistochemistry, flow cytometry, and other methods to detect the proliferation of oligodendrocytes after NT-3 application. ResultsNT-3 was found to significantly promote the recovery of motor function by Basso–Beattie–Bresnahan scores analysis in the rat SCI model. Furthermore, intraspinal administration of NT-3 could downregulate the expression of Beclin-1 in oligodendrocytes, indicating that NT-3 could inhibit excessive autophagy of oligodendrocytes after SCI. The effects of NT-3 on oligodendrocyte survival could be blocked by an autophagy activator rapamycin. ConclusionsThis study found that NT-3 could promote the recovery of motor function after SCI in rats. The underlying reason may be that NT-3 inhibits the expression of autophagy proteins in oligodendrocytes and promotes oligodendrocyte proliferation. This study provided evidence for the future clinical application of NT-3 in SCI patients.

The Anticancer Activity of Lycium barbarum Polysaccharide by Inhibiting Autophagy in Human Skin Squamous Cell Carcinoma Cells In Vitro and In Vivo

Objective. This study is aimed at investigating the effects of Lycium barbarum polysaccharide (LBP) on the proliferation and apoptosis of human cutaneous squamous cell carcinoma A431 cells in vitro and in vivo via its regulation on autophagy. Methods. In vitro experiment: A431 cells were treated with different concentrations of LBP, and cell viability was measured by the CCK8 method. Flow cytometry was used to detect the cell apoptosis rate. The expression of Ki67, PCNA, cl-caspase-3, Bcl-2, and LC3II and the phosphorylation status of JNK and ERK1/2, as well as the effect of SP600125 cotreatment on the expression of autophagy and apoptosis-associated proteins, were determined via Western blot. In vivo experiment: a transplanted tumor model was established by subcutaneous injection of A431 cells to the nude mice. 50 mg/kg LBP was injected into the mice intraperitoneally; the survival rate of mice, volume, and weight of tumor were determined on the 30th day. The expression of Ki67 and MMP-2 proteins was measured by immunohistochemistry. Results. LBP at concentrations of 400 μg/ml and above was significantly cytotoxic to A431 cells, whereas, within the dose range of 50 μg/ml~200 μg/ml, LBP significantly inhibited the expression of Ki67 and PCNA proteins, promoted the expression of cl-caspase-3, inhibited the expression of Bcl-2 protein, downregulated the expression of autophagy marker LC3II, and reduced the phosphorylation of ERK1/2, whereas the level of JNK phosphorylation was upregulated. At the same time, the regulation of Beclin1, LC3II, Bcl-2, and cl-caspase-3 by LBP was effectively reversed by the cotreatment of SP600125. In addition, LBP increased the survival rate of transplanted nude mice, reduced tumor volume and weight, and downregulated the expression of Ki67 and MMP-2. Conclusion. LBP can induce apoptosis of A431 cells by inhibiting autophagy and can inhibit tumor growth in vivo.

Sevoflurane attenuates brain damage through inhibiting autophagy and apoptosis in cerebral ischemia‑reperfusion rats.

The present study aimed to investigate the effects of sevoflurane post-conditioning in a rat brain cerebral ischemia-reperfusion (I/R) model and examine its possible mechanism. Rats were randomly divided into six groups: Sham control group (Sham), I/R group, sevoflurane group (Se), Toll-like receptor-4 (TLR4) inhibitor group (Tak-242), nuclear factor (NF)-κB inhibitor group (QNZ) and Sevoflurane post-conditioning combined with TLR4-NF-κB signaling pathway inhibitor group (Se + Tak-242). Morris water maze test and tetrazolium chloride staining were used to investigate the I/R injury. The nerve cell apoptosis and autophagy in cortical tissue were detected by TUNEL and transmission electron microscopy, respectively. The expression of TLR4 protein in cortical tissue was observed by immunohistochemical staining. The expression of autophagy and apoptotic associated proteins in cortical tissues and the activity of TLR4-NF-κB signaling pathway were assayed by western blot analysis. Sevoflurane post-conditioning improved the learning and memory dysfunction caused by cerebral I/R injury. The cerebral infarction area, nerve cell apoptosis and formation of autophagic vacuoles were reduced after sevoflurane administration. The expression of light chain 3II/I, Beclin-1, Bad and Cleaved-Caspase-3 proteins were inhibited and the expression of Bcl-2 protein was upregulated after sevoflurane administration. Sevoflurane post-conditioning also inhibited the TLR4 protein and NF-κB phosphorylation, and increased inhibitor of kBα phosphorylation. The treatment effect of Tak-242 and QNZ groups were not significantly different compared with the Se group (P>0.05), and the Se + Tak-242 group had the best results. The present study demonstrated that sevoflurane post-conditioning could protect middle cerebral artery occlusion-induced brain injury rats by inhibiting autophagy and apoptosis, and that its mechanism is related to the TLR4-NF-κB signaling pathway.

MTOR Signal Pathway Inhibition Promote the Cytotoxicity of Chemotherapy on SHI-1 Cells

Acute myeloid leukemia (AML) is a malignant hematologic disorder. Although current chemotherapy could improve the survival rate of AML patients, some patient would suffer from relapse and had bad therapeutic response. Nowadays, many studies have found that the abnormal expression of mTOR signal pathway is closely related to the occurrence and progression of AML, but the roles of mTOR inhibitor in AML is not elucidated. So, in this paper, we would investigate whether rapamycin, a mTOR inhibitor, could enhance the cytotoxicity of chemotherapy. Rapamycin or chemotherapy (adriamycin plus cytarabine) could inhibit the proliferation and induce apoptosis of SHI-1 cells (the proliferation inhibition rate and apoptosis ratio were 35.4%±2.94% and 13.33%±0.74% respectively in rapamycin group; Chemotherapy group: 49.9%±4.11%, 13.2%±1.11%). When rapamycin were combined with chemotherapy, the proliferation inhibition rate and apoptosis ratio were significantly increased to 60.3%±3.36% and 30%±1.74% respectively (p&lt;0.05) (Fig.1A, Fig.1B). Western blotting shown that chemotherapy could enhance the expression of Bax, Beclin-1 and ATG5, and inhibit the expression of Bcl-2, caspase-3, nevertheless chemotherapy didn't influence the expression of proteins in the mTOR signal pathway. When mTOR signal pathway were inhibited by rapamycin, the proteins of mTOR signal pathway such as mTOR, p-mTOR, P70S6K1, p-P70S61, 4EBP1, p-4EBP1, and the apoptosis related proteins Bcl-2 and caspase3 of SHI-1 cells were decreased, otherwise, the expression of autophagy-related proteins LC3B/II, Beclin-1, ATG5 and apoptosis-related proteins Bax increased. Rapamycin combined with chemotherapy could significantly inhibit the expression of proteins in mTOR signal pathway and enhance the expression of autophagy and apoptosis-related proteins of SHI-1 cells than that treated by rapamycin alone (Fig.1C). In order to explore whether mTOR pathway inhibition could enhance the cytotoxicity of chemotherapy in AML animal model. 1×107 SHI-1 cells were inoculated into NPG mice via tail vein to construct systemic leukemia NPG mice. On the 19th day after inoculation, the ratio of human CD45 and 33 double positive cells in peripheral blood of NPG mice was 1.3%, then NPG mice were randomly divided into four groups, control group didn't had treatment, chemotherapy group was treated by adriamycin (1mg/kg) once every other day for three times and cytarabine (100mg/kg) once a day for five days, rapamycin group was treated by rapamycin (10mg/kg) once a day for five days, and combination group was treated by combination chemotherapy and rapamycin. On the first day (24th day)after treatment, the ratio of human CD33 and CD45 double positive cells in peripheral blood, bone marrow and spleen of NPG mice in each treatment group were: control group (20.3%, 23.6%, 15.6%), chemotherapy group (13.6%, 14.1%, 5.50%), rapamycin group (2.03%, 2.68%, 3.31%), combined group (0.308%, 2.49%, 1.10%) (Fig. 1D). The weight of spleen in NPG mice of combination group were significantly lighter than the weight of spleen of NPG mice of other three groups. Pathological immunohistochemistry showed that the infiltration of human CD45 positive cells in spleen of mice in the combination group was significantly lower than that of the other three groups (Fig. 1E). The median survival time of NPG mice in the control group, chemotherapy group, rapamycin group and combination group were 30.5 days, 35.0 days, 34.0 days and 39.0 days, respectively. The survival time of NPG mice in combination group was significantly longer than that in chemotherapy and rapamycin group (P&lt;0.05), while there was no significant difference between chemotherapy group and rapamycin group (P&gt;0.05) (Fig. 1F). When NPG mice were dead, more neoplasms were found in the NPG mice in control group, the treatment of rapamycin or chemotherapy could significantly decrease the degree of extramedullary infiltration of SHI-1 cells in NPG mice, and fewer neoplasms were found in the NPG mice of combination group(Fig.1G). In conclusion, our studies verified that mTOR signal pathway inhibition by rapamycin could enhance the cytotoxicity of chemotherapy via inhibiting mTOR signal pathway, promoting cell autophagy and inducing apoptosis. mTOR signal pathway may be a potential target for the treatment of AML. Figure 1 Disclosures No relevant conflicts of interest to declare.

Emerging protective roles of shengmai injection in septic cardiomyopathy in mice by inducing myocardial mitochondrial autophagy via caspase-3/Beclin-1 axis.

Sepsis, a life-threatening systemic syndrome related to inflammatory response, usually accompanied by major organ dysfunctions. The aim of the present study was to elucidate the role by which Shengmai injection (SMI) acts to septic cardiomyopathy. Initially, the induced mice with septic cardiomyopathy were treated with SMI or normal saline (NS) with oe-caspase-3, and HL-1 cells were treated with oe-Beclin-1 and oe-caspase-3 and then cultured with lipopolysaccharide (LPS). Subsequently, we measured the cardiac troponin I (cTnI) level, and expression of mitochondrial autophagy protein (parkin and pink1) and myocardial cell autophagy-related proteins (LC3-II and LC3-I). Additionally, we identified the cleavage of Beclin-1 by caspase-3 and detected the changes of mitochondrial membrane potential, level of reactive oxygen species (ROS), and apoptosis of myocardial cells in myocardial tissues of mice. It has been demonstrated that SMI contributed to the increase of myocardial mitochondrial autophagy, reduction of cTnI level, and elevation of mitochondrial membrane potential in septic cardiomyopathy mice. Both in vitro and in vivo experiments showed that caspase-3 promoted cleavage of Beclin-1 and release of ROS, whereas repressed lipopolysaccharide (LPS)-induced mitochondrial autophagy. Furthermore, the facilitation of myocardial mitochondrial autophagy and protection of myocardial mitochondria by SMI through inhibition of cleavage Beclin-1 by caspase-3 in septic cardiomyopathy mice were also proved by in vivo experiments. Taken together, SMI could protect myocardial mitochondria by promoting myocardial mitochondrial autophagy in septic cardiomyopathy via inhibition of cleavage of Beclin-1 by caspase-3. Our study demonstrates that SMI could represent a novel target for treatment of septic cardiomyopathy.

CCAAT/enhancer-binding protein beta (C/EBPβ) knockdown reduces inflammation, ER stress, and apoptosis, and promotes autophagy in oxLDL-treated RAW264.7 macrophage cells.

Atherosclerosis is associated with deregulated cholesterol metabolism and formation of macrophage foam cells. CCAAT/enhancer-binding protein beta (C/EBPβ) is a transcription factor, and its inhibition has recently been shown to prevent atherosclerosis development and foam cell formation. However, whether C/EBPβ regulates inflammation, endoplasmic reticulum (ER) stress, and apoptosis, in macrophage foam cells and its underlying molecular mechanism remains unknown. Here, we investigated the effect of C/EBPβ knockdown on proteins and genes implicated in inflammation, ER stress, apoptosis, and autophagy in macrophage foam cells. RAW264.7 macrophage cells were transfected with control and C/EBPβ-siRNA and then treated with nLDL and oxLDL. Key proteins and genes involved in inflammation, ER stress, apoptosis, and autophagy were analyzed by western blot and qPCR. We found that short interfering RNA (siRNA)-mediated knockdown of C/EBPβ attenuated atherogenic lipid-mediated induction of proteins and genes implicated in inflammation (P-NFkB-p65, NFkB-p65, and TNFα), ER stress (ATF4 and ATF6), and apoptosis (CHOP, caspase 1, 3, and 12). Interestingly, C/EBPβ knockdown upregulated the expression of autophagy proteins (LC3A/B-II, ATG5) and genes (LC3B, ATG5) but decreased the mammalian target of rapamycin (mTOR) protein phosphorylation and mTORC1 gene expression in oxLDL-loaded RAW264.7 macrophage cells. More importantly, treatment with rapamycin (inhibitor of mTOR) increased expression of proteins implicated in autophagy and cholesterol efflux in oxLDL-loaded RAW 264.7 macrophage cells. The present results suggest that C/EBPβ inactivation regulates macrophage foam cell formation in atherogenesis by reducing inflammation, ER stress, and apoptosis and by promoting autophagy and inactivating mTOR.

Role of Different Salmonella enterica Serotypes in THP-1 Macrophage Derived Foam Cell Autophagy and Inflammatory Response

Infected aortic aneurysm is a rare but life-threatening condition. Intimal injury, especially in arteries with atherosclerotic plaques, is vulnerable to Salmonella species. In this study, THP-1 macrophage-derived foam cells were used to mimic the atherosclerotic plaques. We investigated the role of different Salmonella serotype infections in foam cell autophagy and inflammatory response. Three different serotypes of Salmonella enterica-Typhimurium, Enteritidis, and Choleraesuis - with or without a virulence plasmid were used in this study. The foam cell model was established and observed under confocal microscopy. The expression of autophagy and inflammasome-related proteins after Salmonella infection was detected by Western blotting, and the secretion of the proinflammatory cytokines interleukin-1β and IL-18 was detected using ELISA. The plasmid-bearing S. enteritidis OU7130- and plasmid-less S. choleraesuis OU7266-infected foam cells both showed a lower expression of beclin-1 at 0.5 h post-infection (hpi) and p62 at 2 hpi as well as a higher expression of caspase-1 at 2 hpi than other infected cells. Moreover, infection of OU7266 induced the highest IL-1β and IL-18 production among the tested strains. In conclusion, the virulence plasmid of S. enteritidis OU7130 induced foam cell inflammatory responses, whereas that of S. choleraesuis played an opposite role. The different serotype of Salmonella may impact the infected aortic aneurysms through regulating inflammatory balance of the macrophage and foam cells in the atherosclerotic plaque.

Short Call Abstracts

Short Call Abstracts