Light Chain 3B Research Articles

The Evaluation of Xiaozeng Qianggu Tablets for Treating Postmenopausal Osteoporosis via up-Regulated Autophagy.

Objective Postmenopausal osteoporosis (PMOP) is a common age-associated disease in the life course. Clinically, Xiaozeng Qianggu Tablets (XQT) have a potent therapeutic effect on the PMOP. However, the bioactive components and the mechanism of XQT underlying the PMOP treatment were unclear and it should be explored to discover the scientific connotation in traditional medical practice. Methods The components in XQT were identified by UPLC-Q-TOF/MS. The animal model of PMOP was established by surgical ovariectomy in the female Sprague-Dawley rats. After treatment of XQT, the therapeutic effect was assessed by the determination of bone metabolism biomarkers in serum and histopathological examination. The effect of XQT on the autophagy and bone micro-situation were tested using western blot, RT-qPCR, and transmission electron microscope. Results There were 27 compounds identified in XQT, including catalpol, monotropein, verbascoside, cryptochlorogenic acid, 5,7-dihydroxychromone 7-rutinoside, biorobin, and so on. The bone metabolism markers (alkaline phosphatase, bone alkaline phosphatase, procollagen type I intact N-terminal propeptide, cross-linked carboxy-terminal telopeptide of type I collagen, and tartrate-resistant acid phosphatase) were significantly increased in the PMOP rats and reversed by XQT administration. Moreover, the width of bone trabeculae and the ratio of the area of calcium deposition to bone trabeculae were also improved after treating the middle dose of XQT. Meanwhile, the bone micro-structure was improved by XQT. The mRNA and protein expression of unc-51 like kinase 1, beclin-1, and microtubule-associated protein 1B-light chain 3 in PMOP rats were down-regulated and up-regulated by XQT administration. Conclusions The compounds in XQT, including catalpol, monotropein, verbascoside cryptochlorogenic acid, and so on, were valuable for further pharmacy evaluation. The pathological changes and bone micro-structure were improved by XQT, and the down-regulated autophagy level was also restored, which suggested a potent effect of XQT on treating PMOP, corresponding to its clinic use.

Brain Neurons during Physiological Aging: Morphological Features, Autophagic and Mitochondrial Contribution.

Accumulating data suggest that the brain undergoes various changes during aging. Among them are loss of both white and gray matter, neurons and synapses degeneration, as well as oxidative, inflammatory, and biochemical changes. The above-mentioned age-related features are closely related to autophagy and mitochondria. Therefore, we aimed to reveal the most peculiar morphological features of brain nervous tissue and to characterize the expression of autophagy and mitochondrial immunohistochemical biomarkers in neurons of different human brain zones during aging. Counting the number of neurons as well as Microtubule-associated proteins 1A/1B light chain 3B (LC3B), Heat shock protein 70 (HSP70), Lysosome-associated membrane protein type 2A (LAMP2A), Alpha subunit of ATP synthase (ATP5A), and Parkinson disease protein 7 (DJ1) immunohistochemical staining were performed on FFPE samples of human prefrontal cortex, corpus striatum, and hippocampus obtained from autopsy. Statistical analysis revealed a loss of neurons in the studied elderly group in comparison to the young group. When the expression of macroautophagy (LC3B), chaperon-mediated autophagy (HSP70, LAMP2A), and mitochondrial respiratory chain complex V (ATP5A) markers for the young and elderly groups were compared, the latter was found to have a significantly higher rate of optical density, whilst there was no significance in DJ1 expression. These findings, while preliminary, suggest that both autophagy and mitochondria are involved in neuronal maintenance during aging and could indicate their potential role in adaptive mechanisms that occur in aging.

Effect of moxibustion on autophagy lysosome function mediated by mTOR/TFEB pathway and lncRNA H19 expression in APP/PS1 double transgenic mice

To observe the effect of moxibustion (Moxi) at acupoints of Governor Vessel on autophagy lysosomal function and lncRNA H19 in amyloid precursor protein/presenilin 1 (APP/PS1) double transgenic Alzheimer's disease (AD) mice, so as to explore its underlying mechanisms in relieving AD. Fifty two male APP/PS1 double transgenic AD mice were randomly divided into model, Moxi, Moxi+inhibitor and medication (rapamycin) groups, with 13 mice in each group. Other 13 male C57BL/6J mice of the same age were selected as the control group. The mice of the Moxi group received aconite cake-separated Moxi stimulation at "Baihui" (GV20), "Dazhui"(GV14) and "Fengfu" (GV16), for 15 min, those of the Moxi+inhibitor group received intraperitoneal injection of 3-methyladenine (an inhibitor of PI3K for suppressing autophagy) 1.5 mg· kg-1 · d-1 on the basis of Moxi, and those of the medication group received intraperitoneal injection of rapamycin 2 mg· kg-1 · d-1. The treatment was conducted once daily for 2 weeks. The mouse's learning-memory ability was detected by Morris water maze tests. The hippocampus tissue was sampled for observing the formation of autophagy by using transmission electron microscope, detecting the expression of Aβ_(1-42) protein with immunohistochemical staining, and for detecting the expression levels of long noncoding RNA H19 (lncRNA H19), mammalian target of rapamycin kinase (mTOR), nuclear transcription factor EB (TFEB), Cathepsin D and lysosome associated membrane protein-1 (LAMP1) genes and proteins as well as microtubule associated protein 1 light chain 3B (LC3B)-Ⅱ/LC3B-Ⅰand autophagy protein p62 protein by quantitative real-time PCR and Western blot, respectively. In contrast to the control group, the model group had an evident increase in the escape latency of Morris water maze test, and in the expression levels of Aβ_(1-42) protein, lncRNA H19 mRNA, mTOR mRNA and protein, and p62 protein (P<0.05), and a significant decrease in the expression levels of TFEB, Cathepsin D, LAMP1 mRNAs and proteins and LC3B-Ⅱ/LC3B-Ⅰ (P<0.05). After the treatment and relevant to the model and Moxi+inhibitor groups, both the Moxi and medication groups had an obvious down-regulation in the levels of latency of Morris water maze, expression levels of Aβ_(1-42) protein, lncRNA H19 mRNA, mTOR mRNA and protein, and p62 protein (P<0.05), and a significant up-regulation in the levels of TFEB, Cathepsin D, LAMP1 mRNAs and proteins and LC3B-Ⅱ/LC3B-Ⅰ (P<0.05). Moxi at acupoints of Governor Vessel can improve cognitive function of AD mice, which may be associated with its functions in inhibiting mTOR/TFEB pathway by down-regulating the expression of lncRNA H19, improving autophagy lysosomal function, promoting autophagy and clearing away Aβ1-42 in the hippocampus.

Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy

In synapses that show signs of local apoptosis and mitochondrial stress and undergo neuro-immunological synapse pruning, an increase in the levels of the presynaptic protein, neuronal-specific septin-3 can be observed. Septin-3 is a member of the septin GTPase family with the ability to form multimers and contribute to the cytoskeleton. However, the function of septin-3 remains elusive. Here, we provide evidence that septin-3 is capable of binding the most-studied autophagy protein Atg8 homolog microtubule-associated protein 1 light chain 3B (LC3B), besides another homolog, GABA receptor-associated protein-like 2 (GABARAPL2). Moreover, we demonstrate that colocalization of septin-3 and LC3B increases upon chemical autophagy induction in primary neuronal cells. Septin-3 is accumulated in primary neurons upon autophagy enhancement or blockade, similar to autophagy proteins. Using electron microscopy, we also show that septin-3 localizes to LC3B positive membranes and can be found at mitochondria. However, colocalization results of septin-3 and the early mitophagy marker PTEN-induced kinase 1 (PINK1) do not support that binding of septin-3 to mitochondria is mitophagy related. We conclude that septin-3 correlates with synaptic/neuronal autophagy, binds Atg8 and localizes to autophagic membranes that can be enhanced with chemical autophagy induction. Based on our results, elevated septin-3 levels might indicate enhanced or impeded autophagy in neurons.

Progranulin Preserves Autophagy Flux and Mitochondrial Function in Rat Cortical Neurons Under High Glucose Stress.

Chronic hyperglycemia in type II diabetes results in impaired autophagy function, accumulation of protein aggregates, and neurodegeneration. However, little is known about how to preserve autophagy function under hyperglycemic conditions. In this study, we tested whether progranulin (PGRN), a neurotrophic factor required for proper lysosome function, can restore autophagy function in neurons under high-glucose stress. We cultured primary cortical neurons derived from E18 Sprague-Dawley rat pups to maturity at 10 days in vitro (DIV) before incubation in high glucose medium and PGRN for 24-72 h before testing for autophagy flux, protein turnover, and mitochondrial function. We found that although PGRN by itself did not upregulate autophagy, it attenuated impairments in autophagy seen under high-glucose conditions. Additionally, buildup of the autophagosome marker light chain 3B (LC3B) and lysosome marker lysosome-associated membrane protein 2A (LAMP2A) changed in both neurons and astrocytes, indicating a possible role for glia in autophagy flux. Protein turnover, assessed by remaining advanced glycation end-product levels after a 6-h incubation, was preserved with PGRN treatment. Mitochondrial activity differed by complex, although PGRN appeared to increase overall activity in high glucose. We also found that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3β (GSK3β), kinases implicated in autophagy function, increased with PGRN treatment under stress. Together, our data suggest that PGRN prevents hyperglycemia-induced decreases in autophagy by increasing autophagy flux via increased ERK1/2 kinase activity in primary rat cortical neurons.

Heme oxygenase-1 reduces inflammatory response by inhibiting thioredoxin interacting protein/NOD-like receptor protein 3 inflammasome activation in RAW264.7 cells

To investigate the inhibitory effect and mechanism of heme oxygenase-1 (HO-1) on the inflammatory response of macrophages. Mouse macrophage strain RAW264.7 was cultured in vitro, and the cells in the logarithmic growth phase were used for the experiment. The RAW264.7 cells were divided into four groups. In blank control group, the cells were continuously incubated and received no treatment (cultured at 37 centigrade, 95% air, 5% CO2). In lipopolysaccharide (LPS) model group, 1 mg/L LPS was added to the medium to prepare LPS challenge model. In HO-1 inducer group, the cells were incubated with 30 μmol/L HO-1 inducer hemin for 1 hour, and then 1 mg/L LPS was added for incubation. In HO-1 inhibition group, the cells were incubated with 5 μmol/L HO-1 specific antagonist Zinc protoporphyrin IX (ZnPPIX) for 0.5 hour, and then 1 mg/L LPS was added for incubation. After 48 hours of incubation with LPS, the supernatant of each group was taken, and the protein expressions of HO-1, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), thioredoxin interacting protein (TXNIP), NOD-like receptor protein 3 (NLRP3) and mitochondrial autophagy marker microtubule-associated protein 1 light chain 3B (LC-3B) were detected by Western blotting. The expression of reactive oxygen species (ROS) was detected by immunofluorescence staining. Compared with the blank control group, the cells in the LPS model group had a certain stress response, and autophagy occurred in mitochondria, but the expression of some inflammatory factors was restricted, which was related to the impairment of cell function. The protein expressions of HO-1, IL-1β, LC-3B, ROS were significantly increased, the protein expressions of TNF-α, TXNIP, and NLRP3 were decreased significantly, indicating that the cells were seriously injured after LPS challenge, and the model was successfully established. Compared with the LPS model group, HO-1 protein expression in the HO-1 inducer group was significantly increased (HO-1/GAPDH: 0.31±0.03 vs. 0.22±0.03, P < 0.05), the protein expressions of TNF-α, IL-1β, TXNIP, NLRP3, LC-3B and ROS were significantly inhibited [TNF-α protein (TNF-α/GAPDH): 0.08±0.01 vs. 0.45±0.05, IL-1β protein (IL-1β/GAPDH): 0.50±0.01 vs. 0.82±0.03, TXNIP protein (TXNIP/GAPDH): 0.21±0.02 vs. 0.28±0.02, NLRP3 protein (NLRP3/GAPDH): 0.11±0.01 vs. 0.17±0.02, LC-3B protein (LC-3B/GAPDH): 0.67±0.04 vs. 0.92±0.12, ROS (fluorescence intensity): 80.9±12.5 vs. 94.1±19.5, all P < 0.05], indicating that HO-1 could inhibit inflammatory response and oxidative stress, and reduce mitochondrial autophagy. Antagonizing HO-1 could increase inflammatory response, oxidative stress and mitochondrial autophagy, the inhibitory degree of TNF-α and IL-1β expression was significantly reduced as compared with the HO-1 inducer group [TNF-α protein (TNF-α/GAPDH): 0.26±0.02 vs. 0.08±0.01, IL-1β protein (IL-1β/GAPDH): 0.76±0.01 vs. 0.50±0.01, both P < 0.05], the protein expressions of TXNIP, NLRP3, LC-3B and ROS were significantly increased as compared with the LPS model group [TXNIP protein (TXNIP/GAPDH): 0.43±0.02 vs. 0.28±0.02, NLRP3 protein (NLRP3/GAPDH): 0.24±0.02 vs. 0.17±0.02, LC-3B protein (LC-3B/GAPDH): 1.12±0.07 vs. 0.92±0.12, ROS (fluorescence intensity): 112.0±17.0 vs. 94.1±19.5, all P < 0.05]. HO-1 can reduce the inflammatory response by inhibiting the activation of TXNIP/NLRP3 inflammasome and reducing the release of inflammatory mediators.

Inhibitory effects of LOXL2 knockdown on cellular functions of liver cancer stem cells.

BackgroundLysyl oxidase-like 2 (LOXL2) plays a role in tumor microenvironment formation and metastasis of hepatocellular carcinoma (HCC), which has a high mortality burden. Liver cancer stem cells (LCSCs) are related with the major malignant phenotypes of HCC. The function of LOXL2 in regulation of LCSCs remains unknown.MethodsCD133+HepG2 and CD133+Hep3B cells were sorted by fluorescence-activated cell sorting (FACS) from two human hepatoblastoma cell lines. Spheroid formation, apoptosis, cell cycle, as well as transwell assays were performed upon LOXL2 knockdown in CD133+HepG2 and CD133+Hep3B cells. Protein and mRNA levels were quantified by Western blotting, immunofluorescence and reverse transcription-PCR (RT-PCR).ResultsKnockdown of LOXL2 decreased spheroid formation, migration and invasion (P<0.05), also induced apoptosis (P<0.05) and cell cycle arrest (P<0.05) in CD133+HepG2 and CD133+Hep3B cells. Knockdown of LOXL2 effectively inhibited expression of the anti-apoptosis proteins baculoviral inhibitor of apoptosis protein (IAP) repeat-containing 3 (BIRC3) and murine double minute 2 (MDM2) (P<0.01), as well as autophagy marker microtubule-associated protein 1 light chain 3 B (LC3B) and autophagy gene ATG5 in CD133+HepG2 and CD133+Hep3B cells (P<0.01).ConclusionsThe results revealed that LOXL2 inhibition could reduce the proliferation and expansion of LCSCs, making LOXL2 inhibitors an attractive and novel therapeutic strategy of HCC.

Ophiopogonin D′-induced mitophagy and mitochondrial damage are associated with dysregulation of the PINK1/Parkin signaling pathway in AC16 cells

Shenmai injection (SMI) is a patented traditional Chinese medicine that is extracted from Panax ginseng and Ophiopogon japonicus and is commonly used to treat cardiovascular diseases and tumors. The O. japonicus extract Ophiopogonin D′ (OPD') is highly cardiotoxic. Mitochondria are central to OPD'-induced cardiotoxicity, although the precise mechanisms remain unclear. Excessive mitophagy activation and mitochondrial dysfunction lead to apoptosis, and the PTEN-induced kinase 1(PINK1)/Parkin pathway is critical in regulating mitophagy and mitochondrial function. We investigated the role of the PINK1/Parkin pathway in OPD'-induced mitochondrial damage and cardiotoxicity in AC16 cells. Concentrations of 2 μM OPD' and above inhibited cardiomyocyte viability and increased lactate dehydrogenase (LDH) release in a concentration- and time-dependent manner. OPD' was toxic to cells and mitochondria and increased the rate of apoptosis, triggering pyknosis, decreasing mitochondrial membrane potential (MMP), and decreasing the protein expression of the biogenesis regulator peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α). The increased ratio of microtubule-associated proteins 1 A/1B light chain 3B (LC3-II/LC3-I) in mitochondria indicated that OPD' induced mitophagy. OPD' significantly induced oxidative stress and apoptosis, including increased reactive oxygen species (ROS) generation and decreased nuclear factor erythroid-2 related factor 2 (Nrf2), heme oxygenase-1(HO-1), and B-cell lymphoma 2 (Bcl-2) protein expression. OPD' activated the PINK1/Parkin pathway and promoted PINK1/Parkin translocation to mitochondria. Inhibiting mitophagy attenuated OPD'-induced PINK1/Parkin pathway activation and preserved mitochondrial biogenesis, consequently mitigating OPD'-induced mitochondrial dysfunction and apoptosis. These findings suggest that OPD'-induced cardiomyocyte mitophagy and mitochondrial damage are at least partially mediated by dysregulation of the PINK1/Parkin pathway.

O-038 Role of apoptosis and autophagy in ovarian follicle pool decline from birth to late reproductive age and after gonadotoxic chemotherapeutic treatments

Abstract Study question Which biological mechanisms are responsible for physiological age-dependent and non-physiological ovarian reserve decline throughout a woman’s life? Summary answer Ovarian follicle pool size is modulated by both apoptosis and autophagy, the latter playing a major role in its decline before puberty. What is known already Among different pathways of controlled cell death, apoptosis and autophagy are both involved in follicle death. Apoptosis participtes in eliminating damaged follicles, like those affected by chemotherapy (CHT)-induced DNA damage, but its role in physiological age-related follicle decline is less understood. Autophagy has proved crucial in follicle quiescence maintenance in murine models, but its contribution to human follicle pool modulation is still unclear. Study design, size, duration Eighty-four patients aged between 1 and 35 years, with ovarian tissue stored for histological analyses at the time of cryopreservation (between 2012 and 2021) at the Université Catholique de Louvain (Belgium) were included in retrospective evaluations. Participants/materials, setting, methods Ovarian fragments were used for the following analyses: hematoxylin and eosin staining for follicle count and classification; caspase-3 immunostaining to identify follicle apoptosis; and microtubule-associated proteins 1A/1B light chain 3B (LC3B) immunolabeling to detect follicle autophagy. Ovarian fragments from 9 patients were assigned for transmission electron microscopy to investigate ultrastructural features of oocytes and granulosa cells. All analyses were conducted stratifying patients by age, menarchal status (premenarchal=32; postmenarchal=52), and potentially gonadotoxic CHT before cryopreservation (n = 14). Main results and the role of chance Premenarchal patients had a larger follicle pool in terms of total follicle density (4979.98±4952.16 vs 918.8±903.86, p &amp;lt; 0.0001), but higher rates of morphologically abnormal (8.52±6.48% vs 3.54±4.54%, p &amp;lt; 0.0001) and atretic follicles (15.8±9.14% vs 10.6±9.43%, p = 0.01) than postmenarchal subjects. Apoptosis rates did not change with increasing age (27.94±28.78% in prepubertal subjects and 29.5±33.58% in postpubertal subjects). Autophagic follicles were around 10 times more common in premenarchal compared to postmenarchal subjects (10.21±16.% vs 1.34±4.02%, p &amp;lt; 0.0001), playing a crucial role in age-related follicle decline and elimination of ‘abnormal follicles’, that are rarely seen after menarche. The impact of previous CHT varied according to age. In premenarchal patients with previous CHT, significantly more apoptotic (40.22±40.44% vs 26.79±25.35%, p = 0.04) and fewer abnormal (3.84±3.91% vs 9.83±6.5%, p = 0.02) follicles were detected than in those with no CHT prior to ovarian tissue cryopreservation, suggesting a direct effect on follicle elimination, especially of those with abnormalities. In postmenarchal subjects with previous CHT, lower quiescent follicle rates were observed compared to patients with no CHT before tissue freezing (70.1±36.36% vs 85.89±12.61%, p = 0.04), suggesting accelerated follicle activation and growth. Limitations, reasons for caution The present study could not investigate the impact of specific CHT protocols, since the group of patients with previous CHT was highly heterogeneous. However, these patients could not be excluded a priori, since the effect of follicle pool depletion has not been fully elucidated. Wider implications of the findings This study yields a deeper understanding of follicle pool decline regulation, evidencing for the first time that (i) different cell death pathways are involved in physiological and non-physiological follicle depletion, and (ii) the follicle pool responds differently to gonadotoxic damage according to age and ovarian reserve size. Trial registration number N/A

Long non-coding RNA IGFBP7-AS1 promotes odontogenic differentiation of stem cells from human exfoliated deciduous teeth through autophagy: An in vitro study

ObjectiveIn the present study, we aimed to investigate whether long non-coding RNA (lncRNA) insulin-like growth factor binding protein 7-antisense 1 (IGFBP7-AS1) regulates the odonto-differentiation of stem cells from human exfoliated deciduous teeth (SHED) and its underlying mechanism. DesignReal-time polymerase chain reaction (PCR) and correlation analysis were used to determine the expression of IGFBP7-AS1 during odontogenesis. Alkaline phosphate staining, alizarin red S staining, and real-time PCR in vitro were performed to investigate the effects of IGFBP7-AS1 during odontogenesis. Western blot and immunostaining (with or without chloroquine treatment) were applied to detect the expression of the autophagy-related markers, microtubule-associated proteins 1A/1B light chain 3B (LC3B) and p62. The autophagy inhibitor 3-methyladenine was used to further clarify the effect of autophagy in odonto-differentiation as promoted by IGFBP7-AS1. ResultsThe expression of lncRNA IGFBP7-AS1 is significantly upregulated during odonto-differentiation of SHED and promotes odontogenesis of SHED in vitro. IGFBP7-AS1 promotes autophagy during odontogenesis. ConclusionsIGFBP7-AS1 elicits odontogenic differentiation of SHED through autophagy. Furthermore, IGFBP7-AS1 shows promise as a gene target in the regeneration of dental hard tissue and dental–pulp complex.

Apoptotic and autophagic death union by Thuja occidentalis homeopathic drug in cervical cancer cells with thujone as the bioactive principle

Objective“Multi-targeting” drugs can prove fruitful to combat drug-resistance of multifactorial disease—cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts. MethodsThuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency. ResultsThuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC50) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways—apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC50 of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood–brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk. ConclusionRobust “multi-targeting” anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.

The effect of MiR-497 on the expression of target genes BCL-2 and LC3B on cardiomyocytes injured by hypoxia/reoxygenation.

Myocardial ischemia is easy to cause hypoxia or necrosis of myocardial cells. At present, the performance of various patients is different. Basically, it is mainly caused by chest pain or chest discomfort. Severe patients may die suddenly. Therefore, looking for effective drugs or methods to prevent and treat Cardiomyocyte injury is of great significance for clinical practice, in which the expression of regulatory gene BCL-2 and microtubule-associated protein light chain 3B (LC3B) has a certain effect on hypoxia/reoxygenation injured cardiomyocytes. To this end, the team designed a study on the effect of miR-497 on the expression of target genes BCL-2 and LC3B on cardiomyocytes injured by hypoxia/reoxygenation. In this study, a control group experiment was set up for the study. During the experiment, the cells were treated with hypoxia-reoxygenation and transfected with the corresponding miR-497 treated cells. By detecting apoptosis, the kit was used to detect cell activity and RT-PCR detection. Gene expression levels and other methods are comparatively judged. The results of this study showed that compared with the normal group 14.50±0.78, the viability of cardiomyocytes in the model group was significantly reduced (P<0.01), the amount of NO released by cardiomyocytes was reduced (P<0.01), and the protein expression in cardiomyocytes was significantly reduced (P<0.01). The experimental results of this study prove that miR-497 can alleviate the damage caused by hypoxia-reoxygenation of cardiomyocytes by regulating target genes BCL-2 and LC3B.

Comparative Study of Autophagy in Oxaliplatin-Sensitive and Resistant SNU-C5 Colon Cancer Cells.

Few studies have evaluated the role of autophagy in the development of oxaliplatin (OXT) resistance in colon cancer cells. In this study, we compared the role of autophagy between SNU-C5 colon cancer cells and OXT-resistant SNU-C5 (SNU-C5/OXTR) cells. At the same concentration of OXT, the cytotoxicity of OXT or apoptosis was significantly reduced in SNU-C5/OXTR cells compared with that in SNU-C5 cells. Compared with SNU-C5 cells, SNU-C5/OXTR cells exhibited low levels of autophagy. The expression level of important autophagy proteins, such as autophagy-related protein 5 (Atg5), beclin-1, Atg7, microtubule-associated proteins 1A/1B light chain 3B I (LC3-I), and LC3-II, was significantly lower in SNU-C5/OXTR cells than that in SNU-C5 cells. The expression level of the autophagy-essential protein p62 was also lower in SNU-C5/OXTR cells than in SNU-C5 cells. In SNU-C5/OXTR cells, the production of intracellular reactive oxygen species (ROS) was significantly higher than that in SNU-C5 cells, and treatment with the ROS scavenger N-acetylcysteine restored the reduced autophagy levels. Furthermore, the expression of antioxidant-related nuclear factor erythroid 2-related factor 2 transcription factor, heme oxygenase-1, and Cu/Zn superoxide dismutase were also significantly increased in SNU-C5/OXTR cells. These findings suggest that autophagy is significantly reduced in SNU-C5/OXTR cells compared with SNU-C5 cells, which may be related to the production of ROS in OXT-resistant cells.

Potential Method of Autophagy Imaging with Cationized Gelatin Nanospheres Incorporating Molecular Beacon.

The objective of this research is to develop an imaging method with cationized gelatin nanospheres incorporating molecular beacon (cGNSMB) to visualize an autophagy activity in living cells. Cationized gelatin nanospheres (cGNS) were prepared by the conventional coacervation method, and then molecular beacon (MB) was incorporated into them. The cGNSMB prepared were internalized into cells at a high efficiency. In this study, a starvation medium of serum and amino acids-free was used to induce autophagy. The autophagy activity was confirmed by an immunofluorescence staining for microtubule-associated proteins light chain 3B (LC3B) of an autophagy specific protein. With the autophagy induction time, the number of LC3 fluorescent dots increased, which indicated an increased autophagy activity. As the autophagy-related genes, sequestosome 1 (SQSTM1) and cathepsin F (CTSF), which up-regulate after autophagy induction, were chosen as the targets of cGNSMB. The fluorescence intensity of cGNSMB targeting to SQSTM1 and CTSF increased with the starvation treatment time, which well corresponded with the gene expression results. When applied to cells in different autophagy conditions, the cGNSMB visualized the autophagy activity corresponding with the autophagy condition of cells. From the results obtained, it was concluded that the cGNSMB provide a promising method to visualize the autophagy of cells. The advantage of cGNSMB visualization is to obtain the temporal and spatial information without destroying sample cells.

THE CORRELATION OF AUTOPHAGY AND GLYCOLYSIS MECHANISM TO THE HISTOPATHOLOGICAL GRADING OF PENILE SQUAMOUS CELL CARCINOMA THROUGH EXPRESSION MICROTUBULE-ASSOCIATED PROTEIN 1 LIGHT CHAIN 3B (LC3B) AND GLUCOSE TRANSPORTER-1 (GLUT-1)

Background: Penile cancer incidence is still common in developing countries. Squamous cell carcinoma (SCC) is the dominant histopathological type found in penile cancer. Autophagy and glycolysis play important roles in cancer pathology. LC3B is one of the most widely used autophagy markers and GLUT-1 is an isoform of the glucose transporter family that is expressed in many cancers. Objective: To analyze correlation of autophagy and glycolysis mechanism to the histopathological grading of penile squamous cell carcinoma through expression microtubule-associated protein1 light chain 3B (LC3B) and glucose transporter-1 (GLUT-1). Method: This study is an analysis with a cross sectional approach on 50 samples of paraffin blocks diagnosed as penile squamous cell carcinoma. All samples met the inclusion criteria and were reviewed by 2 pathologists. The results of the immunohistochemical expression of LC3B and GLUT-1 will be assessed for correlation with the histopathological grade of penile SCC. The statistical test used in this study was the Mann Whitney test. Results: A total of 50 samples of penile SCC, obtained grade I as many as 17 samples (34%), grade II 22 samples (44%), and grade III 11 samples (22%). From the results of the immunohistochemical expression of LC3B and GLUT-1 on the histopathological grading of penile SCC, the same results were found. In grade I, 15 samples were positively expressed, and 2 samples were negative. In grade II, all samples were positively expressed, and in grade III, 9 samples were positively expressed and 2 were negative. Conclusion: There was no significant correlation between the histopathological grading of penile SCC with the immunohistochemical expression of LC3B and GLUT-1 (p=0.818).

Autophagic LC3+ calcified extracellular vesicles initiate cartilage calcification in osteoarthritis.

Pathological cartilage calcification plays an important role in osteoarthritis progression but in which the origin of calcified extracellular vesicles (EVs) and their effects remain unknown. Here, we demonstrate that pathological cartilage calcification occurs in the early stage of the osteoarthritis in which the calcified EVs are closely involved. Autophagosomes carrying the minerals are released in EVs, and calcification is induced by those autophagy-regulated calcified EVs. Autophagy-derived microtubule-associated proteins 1A/1B light chain 3B (LC3)–positive EVs are the major population of calcified EVs that initiate pathological calcification. Release of LC3-positive calcified EVs is caused by blockage of the autophagy flux resulted from histone deacetylase 6 (HDAC6)–mediated microtubule destabilization. Inhibition of HDAC6 activity blocks the release of the LC3-positive calcified EVs by chondrocytes and effectively reverses the pathological calcification and degradation of cartilage. The present work discovers that calcified EVs derived from autophagosomes initiate pathological cartilage calcification in osteoarthritis, with potential therapeutic targeting implication.

The Pathological Evaluation of Autophagy-Related Protein (LC3B) and Its Association with the Infiltration of Immune Cells in Glioma

Background:Tumor cell autophagy can influence cellular immunity by participation in the recognition and modification of tumor-related antigens. Objectives:The objective of this study was to evaluate the immunohistochemical expression of the autophagy-related marker; light chain 3B (LC3B) in tumor cells and the assessment of T lymphocytes by a cluster of differentiation 3 (CD3) in gliomas, and to correlate them with the available clinic-pathological variables in glioma patients. Materials and methods:Immunohistochemical staining for LC3B and CD3 was performed on 60 paraffin-embedded glioma tissue. LC3B immunoreactivity score of 0-6 was designated negative, and those scoring 7-12 were considered positive. The median level of CD3 positive T lymphocytes was calculated for both high and low-grade gliomas. In low-grade gliomas, the CD3 expressing T lymphocytes equal to or more than 2.6, were considered positive while in high-grade gliomas, those equal to or more than 16 were considered positive. Results:LC3B expression in tumor cells was detected in 24/60 (40%) of gliomas. Expression of LC3B was significantly more frequent in high-grade gliomas (23/33, 69.7%) compared to low grade ones (1/27, 5%), (p value= 0.000). LC3B expression was correlated with the patients age (P value= 0.047) & histological variants (P value= 0.000). CD3 positive T lymphocytes were significantly more prominent in high-grade gliomas (25/33 , 41.7%) than low-grade ones (2/27, 3.3%), (P value= 0.001). A significant association was noted between CD3 expression and the patients age (P value= 0.003), sex (P value: 0.035) and histological variants (P value= 0.001). LC3B expression in tumor cells was significantly correlated with CD3 positive T lymphocytes (P value: 0.000). Conclusion:Autophagic activity of tumor cells and T lymphocyte infiltrates were reported more in high-grade gliomas compared to low-grade ones, giving high-grade gliomas the chance in autophagy target therapy & immunotherapy.

Role of Autophagy Markers (Light Chain 3B and Beclin-1) in Pathogenicity of Breast Cancer in a Sample of Iraqi Women: A Pilot Study

Background: Breast cancer (BC) is the most common malignancy in women and the second greatest cause of cancer death. There are relatively little data available on the role of autophagy markers in pathogenicity of breast cancer in the Iraqi population. We have therefore investigated the role of Baclin-1 and LC3-B in pathogenicity of breast cancer in Iraqi women. Methods: A case-control study was conducted to assess the role of Bacline-1 and LC3-B in pathogenicity of breast cancer in Iraqi women. The study included 50 patients with BC age matched with 50 healthy individuals served as a control group. The Baclin-1 and LC3-B concentrations were determined by enzyme-linked immunosorbent assay (ELISA) technique. Results: Body mass index (BMI) were significantly increase in patients with BC compared to the controls. Serum Baclin-1 and LC3-B levels in cases were significantly higher (P= 0.001) than those in controls. According to histological tumor characteristics. Grade 2 and stage IIA were significantly higher in BC patients and the tumor grade and stage did not show any significant associations with serum levels of Bacline-1 and LC3-B. No significant correlation between serum levels of Baclin-1 and LC3-B (r = 0.034; P = 0.812). Conclusion: The study indicated that the Beclin-1 and LC3-B my consider a good biomarkers for progression of BC and it could be a therapeutic target to reduce the aggressiveness of disease. Keywords: Breast cancer; Baclin-1; Light Chain3B.

Vitamin D ameliorates asthma-induced lung injury by regulating HIF-1α/Notch1 signaling during autophagy.

Herein, we aimed to determine the effect of vitamin D (Vit D) and underlying mechanisms on asthma‐induced lung injury via regulation of HIF‐1α/Notch1 (hypoxia‐inducible factor 1 alpha/neurogenic locus notch homolog protein 1) signaling during autophagy. We established an asthma mouse model using respiratory syncytial virus (RSV) nasal drop combined with ovalbumin (OVA) atomization. Mice were treated with different Vit D concentrations. Pathological changes and cell apoptosis were examined using hematoxylin–eosin (HE) staining and TUNEL (terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate (dUTP) nick end‐labeling) assay, respectively. Additionally, periodic acid–Schiff (PAS) and Masson's trichrome staining solutions were used to examine changes in lung tissue. Immunofluorescence determined LC 3B (microtubule‐associated protein 1 light chain 3B) expression in lung tissues, whereas western blotting and immunohistochemistry were used to evaluate other proteins, including HIF‐1α and Notch1. Compared with the normal group, the asthma model group exhibited pathological lung tissue deterioration, elevated fibrosis, increased apoptosis cell numbers, and upregulated autophagy. Vitamin D supplementation ameliorated pathological changes and fibrosis in the lung tissue. Furthermore, Vit D treatment significantly suppressed apoptotic cell numbers and autophagy while enhancing the HIF‐1α/Notch1 pathway. Given the HIF‐1α/Notch1 agonistic activity, Vit D treatment inhibited apoptosis cell numbers, which were increased following asthma‐induced upregulation of autophagy. Vitamin D improved asthma‐induced lung tissue injury by suppressing autophagy via regulation of HIF‐1α/Notch1 signaling in vivo.

Selenium alleviates cadmium-induced aging via mitochondrial quality control in the livers of sheep

Cadmium (Cd) is a heavy metal with toxicity that induces mitochondrial dysfunction and aging, and selenium (Se) can alleviate its toxicity. However, the underlying mechanism of Se alleviating Cd-induced aging in sheep livers deserves further study. This study was to explore the protective mechanism of Se on the Cd-induced aging in the livers of sheep. A total of forty-eight sheep weighing about 10 kg were randomly divided into four groups: control group, Se group [0.34 mg Se·kg−1·body weight (BW)], Cd group (1 mg Cd·kg−1·BW), and Se + Cd group (0.34 mg Se·kg−1·BW +1 mg Cd·kg−1·BW). The results showed that Cd caused vacuolization, granule denaturation, and mitochondrial vacuolization in hepatocytes. Furthermore, the levels of catalase (CAT), total superoxide dismutase (T-SOD), glutathione (GSH) and adenosine triphosphate (ATP) in liver mitochondria were down-regulated, but the levels of hydrogen peroxide (H2O2) and malonaldehyde (MDA) were up-regulated under Cd treatment. Besides, the cyclin-dependent kinase inhibitor 1 (P21) immunohistochemistry positive signal and the puncta of immunofluorescence co-locations of E3 ubiquitin ligase Parkin (Parkin)/ cytochrome c oxidase IV (COX IV) and light chain 3B (LC3B)/COX IV were increased under Cd stress. Moreover, Cd exposure decreased the levels of mitochondrial biogenesis and fusion related factors and minichromosome maintenance protein 2 (MCM2), but increased the levels of mitochondrial fission, mitophagy, and cell aging related factors. However, the variations mentioned above caused by Cd were effectively ameliorated by Se co-treatment. In conclusion, Se might alleviate Cd-induced aging via regulating mitochondrial quality control in sheep livers.