Ginseng-derived nanoparticles induce skin cell proliferation and promote wound healing.

Background: Past studies suggested that ginseng extracts and ginseng-derived molecules exerted significant regulatory effects on skin. However, no reports have described the effects of ginseng-derived nanoparticles (GDNPs) on skin cell proliferation and wound healing. In this study, we investigated whether GDNPs regulate the proliferation of skin cells and promote wound healing in a mouse model. Methods: GDNPs were separated and purified via differential centrifugation and sucrose/D2O gradient ultracentrifugation. GDNP uptake, cell proliferation and cell cycle progression were measured by confocal microscopy, CCK-8 assay and flow cytometry, respectively. Cell migration and angiogenic effects were assessed by the wound scratch assay and tube formation assay, respectively. ELISA was used to detect extracellular matrix secretion. The relevant signaling pathway was confirmed by western blotting. The effects of GDNPs on skin wound healing were assessed by wound observation, HE staining, and western blotting. Results: GDNPs possessed the essential features of exosomes, and they were accumulated by skin cells. Treatment with GDNPs notably enhanced the proliferation of HaCaT, BJ and HUVECs. GDNPs also enhanced the migration in HaCaT cells and HUVECs and angiogenesis in HUVECs. GDNPs increased the secretion of MMP-1, fibronectin-1, elastin-1, and COL1A1 in all three cell lines. GDNPs regulated cell proliferation through the ERK and AKT/ mTOR pathways. Furthermore, GDNPs facilitated skin wound healing and decreased inflammation in a mouse skin wound model. Conclusion: GDNPs can promote skin wound healing through the ERK and AKT/mTOR pathways. GDNPs thus represent an alternative treatment for chronic skin wounds.

Ginseng root extract attenuates inflammation by inhibiting the MAPK/NF-κB signaling pathway and activating autophagy and p62-Nrf2-Keap1 signaling in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C.A. Meyer is a type of herbal plant that has frequently been used in many Asian countries to treat a variety of diseases. Ginseng is considered to exhibit anti-inflammatory and anti-oxidative pharmacological effects. However, the specific mechanism is still not entirely understood. AIM OF THE STUDY: In this study, we investigated if ginseng extract could attenuate inflammation and oxidative stress in RAW264.7 cells and in dextran sulfate sodium (DSS)-induced colitis mouse model. MATERIALS AND METHODS: RAW264.7 cells and LPS were used to develop inflammatory and oxidative cell models. C57/6J male mice and DSS were used to construct the animal models. O2-, mitochondria number, and mitochondrial membrane potential were analyzed using fluorescent probes and fluorescence microscopy. Reactive oxygen species and nitric oxide generation were detected with probes and microplate readers. The secreted amounts of inflammatory cytokines were measured by enzyme-linked immunosorbent assay kits. Protein expression levels in the cytoplasm and nucleus were measured by western blotting analyses. Quantitative real-time PCR (qRT-PCR) was used to determine the changes in mRNA levels. Autophagosome accumulation was analyzed by transmission electron microscopy. A p62-specific siRNA was used to evaluate the effect of p62 on the anti-oxidative function of ginseng root extract (GRE). Asperuloside and SP600125 were used to confirm the involvement of the MAPK/NF-κB signaling pathway. RESULTS: We performed a systematic analysis of the anti-inflammatory, anti-oxidative, and autophagy regulatory mechanisms of GRE in LPS-treated RAW264.7 cells. GRE considerably reduced the levels of nitric oxide, TNF-α, and IL-6 secreted by LPS-treated cells. GRE treatments dose-dependently upregulated IL-10 mRNA levels and decreased IL-6 and IL-1ß mRNA levels in LPS-treated cells. Similar to the NF-κB and JNK inhibitors, GRE treatment significantly inhibited NF-κB activity and phosphorylation of MAPKs (JNK, ERK-1/2, and p38). Additionally, GRE treatment remarkably decreased LPS-triggered reactive oxygen species production and mitochondrial dysfunction by motivating Nrf2 nuclear translocation by enhancing phosphorylated p62. Knockdown of p62 resulted in the loss of GRE anti-oxidative ability. Autophagy was strongly induced by GRE via the Akt-mTOR signaling pathway, relieving excessive oxidation, mitochondrial dysfunction, and inflammation, while enhancing Beclin-1, LC3 II, and Atg7 protein expression. Furthermore, GRE alleviated the degree of injury, inflammatory cytokine production, and regulated the relative signaling pathway in DSS-induced colitis. CONCLUSIONS: GRE can exert both anti-inflammatory and anti-oxidative functions by targeting the MAPK/NF-κB and p62-Nrf2-Keap1 pathways, as well as autophagy, in vitro and vivo.

20(S)-Ginsenoside Rh2-induced apoptosis and protective autophagy in cervical cancer cells by inhibiting AMPK/mTOR pathway.

20(S)-Ginsenoside Rh2 (GRh2) has various biological activities including anticancer effects. However, no reports have investigated the connection between autophagy and apoptosis in HeLa cells treated with 20(S)-GRh2. In this study, we found that 20(S)-GRh2 suppressed proliferation and induced apoptosis in HeLa cells by activating the intrinsic apoptotic pathway and causing mitochondrial dysfunction. 20(S)-GRh2 enhanced cell autophagy through promoting the phosphorylation of AMPK, depressed the phosphorylation of AKT, and suppressed mTOR activity. Furthermore, treatment with the autophagy inhibitor 3-methyladenine (3-MA) enhanced 20(S)-GRh2-induced apoptosis, while the autophagy inducer rapamycin promoted cell survival. Moreover, the apoptosis inhibitor Z-VAD-FMK significantly restrained the apoptosis and autophagy induced by 20(S)-GRh2 in HeLa cells. We found that 20(S)-ginsenoside Rh2-induced protective autophagy promotes apoptosis of cervical cancer cells by inhibiting AMPK/mTOR pathway.

Total ginsenosides induce autophagic cell death in cervical cancer cells accompanied by downregulation of bone marrow stromal antigen-2.

Ginsenosides are important active components in Panax ginseng. In the present study, total ginsenosides (TGNs) were demonstrated to enhance autophagy by promoting acidic vacuole organelle formation, recruitment of enhanced green fluorescent protein-microtubule-associated protein light chain 3 and expression of autophagy-related factors in cervical cancer cell lines. TGN markedly increased the expression of p62 at the transcriptional level, but decreased p62 protein expression in the presence of actinomycin D. The autophagic regulatory effect was reversible. TGN (≤120 µg/ml) did not affect the proliferation of cervical cancer cells under normal culture conditions, but markedly inhibited the growth of serum-deprived cells. Treatment with an inhibitor of autophagy (3-methyladenine) impaired TGN-induced cell death. This suggested that TGN caused autophagic cell death. In addition, western blot analysis demonstrated that the protein level of bone marrow stromal antigen-2 (BST-2) was downregulated by TGN. Upregulation of BST-2 reduced cell death. The results of the combined actions of various monomeric ginsenosides in TGN provide the molecular basis to develop TGN as a promising candidate for cancer therapy.

Microphysiological systems in early stage drug development: Perspectives on current applications and future impact.

Microphysiological systems (MPS) are making advances to provide more standardized and predictive physiologically relevant responses to test articles in living tissues and organ systems. The excitement surrounding the potential of MPS to better predict human responses to medicines and improving clinical translation is overshadowed by their relatively slow adoption by the pharmaceutical industry and regulators. Collaboration between multiorganizational consortia and regulators is necessary to build an understanding of the strengths and limitations of MPS models and closing the current gaps. Here, we review some of the advances in MPS research, focusing on liver, intestine, vascular system, kidney and lung and present examples highlighting the context of use for these systems. For MPS to gain a foothold in drug development, they must have added value over existing approaches. Ideally, the application of MPS will augment in vivo studies and reduce the use of animals via tiered screening with less reliance on exploratory toxicology studies to screen compounds. Because MPS support multiple cell types (e.g. primary or stem-cell derived cells) and organ systems, identifying when MPS are more appropriate than simple 2D in vitro models for understanding physiological responses to test articles is necessary. Once identified, MPS models require qualification for that specific context of use and must be reproducible to allow future validation. Ultimately, the challenges of balancing complexity with reproducibility will inform the promise of advancing the MPS field and are critical for realization of the goal to reduce, refine and replace (3Rs) the use of animals in nonclinical research.

Knockdown of p62/sequestosome enhances ginsenoside Rh2-induced apoptosis in cervical cancer HeLa cells with no effect on autophagy.

p62/sequestosome is a multifunctional adaptor protein that participates in a wide variety of cellular processes. 20(S)-Ginsenoside Rh2 (G-Rh2) has various biological effects, including anticancer activity. We found that G-Rh2 can induce apoptosis and autophagy in HeLa cells. G-Rh2 significantly enhanced the transcriptional level of p62. A siRNA was constructed to knock down p62 and assess its effect on apoptosis induced by G-Rh2. p62 protein levels were successfully downregulated in cells transfected with the p62-specific siRNA. Silencing of p62 further decreased cell viability while also enhancing cell apoptosis, reactive oxygen species generation, the ratio of Bax to Bcl-2, and the cleavage of PARP. p62 knockdown decreased expression levels of Nrf2. Moreover, silencing of p62 had no significant effect on autophagy induced by G-Rh2. These results suggest that combining G-Rh2 treatment with inhibition of p62 may be a potential treatment strategy for cervical cancer.

Implementation of a Human Renal Proximal Tubule on a Chip for Nephrotoxicity and Drug Interaction Studies.

Proximal tubule epithelial cells (PTEC) are susceptible to drug-induced kidney injury (DIKI). Cell-based, two-dimensional (2D) in vitro PTEC models are often poor predictors of DIKI, probably due to the lack of physiological architecture and flow. Here, we assessed a high throughput, 3D microfluidic platform (Nephroscreen) for the detection of DIKI in pharmaceutical development. This system was established with four model nephrotoxic drugs (cisplatin, tenofovir, tobramycin and cyclosporin A) and tested with eight pharmaceutical compounds. Measured parameters included cell viability, release of lactate dehydrogenase (LDH) and N-acetyl-ß-d-glucosaminidase (NAG), barrier integrity, release of specific miRNAs, and gene expression of toxicity markers. Drug-transporter interactions for P-gp and MRP2/4 were also determined. The most predictive read outs for DIKI were a combination of cell viability, LDH and miRNA release. In conclusion, Nephroscreen detected DIKI in a robust manner, is compatible with automated pipetting, proved to be amenable to long-term experiments, and was easily transferred between laboratories. This proof-of-concept-study demonstrated the usability and reproducibility of Nephroscreen for the detection of DIKI and drug-transporter interactions. Nephroscreen it represents a valuable tool towards replacing animal testing and supporting the 3Rs (Reduce, Refine and Replace animal experimentation).

Loss of CLN3, the gene mutated in juvenile neuronal ceroid lipofuscinosis, leads to metabolic impairment and autophagy induction in retinal pigment epithelium.

Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease) is a lysosomal storage disease characterized by progressive blindness, seizures, cognitive and motor failures, and premature death. JNCL is caused by mutations in the Ceroid Lipofuscinosis, Neuronal 3 (CLN3) gene, whose function is unclear. Although traditionally considered a neurodegenerative disease, CLN3 disease displays eye-specific effects: Vision loss not only is often one of the earliest symptoms of JNCL, but also has been reported in non-syndromic CLN3 disease. Here we described the roles of CLN3 protein in maintaining healthy retinal pigment epithelium (RPE) and normal vision. Using electroretinogram, fundoscopy and microscopy, we showed impaired visual function, retinal autofluorescent lesions, and RPE disintegration and metaplasia/hyperplasia in a Cln3 ~ 1 kb-deletion mouse model [1] on C57BL/6J background. Utilizing a combination of biochemical analyses, RNA-Seq, Seahorse XF bioenergetic analysis, and Stable Isotope Resolved Metabolomics (SIRM), we further demonstrated that loss of CLN3 increased autophagic flux, suppressed mTORC1 and Akt activities, enhanced AMPK activity, and up-regulated gene expression of the autophagy-lysosomal system in RPE-1 cells, suggesting autophagy induction. This CLN3 deficiency induced autophagy induction coincided with decreased mitochondrial oxygen consumption, glycolysis, the tricarboxylic acid (TCA) cycle, and ATP production. We also reported for the first time that loss of CLN3 led to glycogen accumulation despite of impaired glycogen synthesis. Our comprehensive analyses shed light on how loss of CLN3 affect autophagy and metabolism. This work suggests possible links among metabolic impairment, autophagy induction and lysosomal storage, as well as between RPE atrophy/degeneration and vision loss in JNCL.

Phenotypic Characterization of Targeted Knockdown of Cyclin-Dependent Kinases in the Intestinal Epithelial Cells.

Cyclin-dependent kinases (CDKs) are serine/threonine kinases that regulate cell cycle and have been vigorously pursued as druggable targets for cancer. There are over 20 members of the CDK family. Given their structural similarity, selective inhibition by small molecules has been elusive. In addition, collateral damage to highly proliferative normal cells by CDK inhibitors remains a safety concern. Intestinal epithelial cells are highly proliferative and the impact of individual CDK inhibition on intestinal cell proliferation has not been well studied. Using the rat intestinal epithelial (IEC6) cells as an in vitro model, we found that the selective CDK4/6 inhibitor palbociclib lacked potent anti-proliferative activity in IEC6 relative to the breast cancer cell line MCF7, indicating the absence of intestinal cell reliance on CDK4/6 for cell cycle progression. To further illustrate the role of CDKs in intestinal cells, we chose common targets of CDK inhibitors (CDK 1, 2, 4, 6, and 9) for targeted gene knockdown to evaluate phenotypes. Surprisingly, only CDK1 and CDK9 knockdown demonstrated profound cell death or had moderate growth effects, respectively. CDK2, 4, or 6 knockdowns, whether single, double, or triple combinations, did not have substantial impact. Studies evaluating CDK1 knockdown under various cell seeding densities indicate direct effects on viability independent of proliferation state and imply a potential noncanonical role for CDK1 in intestinal epithelial biology. This research supports the concept that CDK1 and CDK9, but not CDKs 2, 4, or 6, are essential for intestinal cell cycle progression and provides safety confidence for interphase CDK inhibition.

20(S)-Ginsenoside Rg3 Promotes HeLa Cell Apoptosis by Regulating Autophagy.

20(S)-Ginsenoside Rg3 (GRg3) has various bioactivities including anti-cancer effects and inhibition of autophagy. However, no reports have investigated the appearance of autophagy or the connection between autophagy and apoptosis in HeLa cells treated with 20(S)-GRg3. Cell viability was measured by CCK-8 (cell counting kit-8) assays. Apoptosis and the cell cycle were analyzed by Hoechst 33342 staining and flow cytometry. Apoptotic pathways were examined by ROS (reactive oxygen species) determination and rhodamine 123 assays. Western blot analysis was used to determine changes in protein levels. Autophagy induction was monitored by acidic vesicular organelle staining and EGFP-LC3 transfection. 20(S)-GRg3 inhibited autophagy of cells in a starved state, making it impossible for cells to maintain a steady state through autophagy, and then induced apoptosis. 20(S)-GRg3 blocked the late stage of autophagy (fusion of lysosomes and degradation of autophagic lysosomes), including a decrease in acidic vesicular organelle fluorescence, increased LC3 I-II conversion, accumulation of EGFP-LC3 fluorescence, GFP-mRFP-LC3 red-green fluorescence ratio, degradation of the substrate p62, and loss of the balance between autophagy and apoptosis, which induced apoptosis. ROS increased, the mitochondrial membrane potential decreased, apoptotic inducer AIF was released from mitochondria, and nuclear transfer occurred, triggering a series of subsequent apoptotic events. Autophagy inducer rapamycin inhibited the apoptosis induced by 20(S)-GRg3, whereas autophagy inhibitor BA1 promoted apoptosis induced by 20(S)-GRg3. Therefore, 20(S)-GRg3 promoted HeLa cell apoptosis by regulating autophagy. In the autophagic state, 20(S)-GRg3 can be used as a novel autophagy inhibitor in synergy with tumor-blocking therapies such as chemotherapy, which supports its application in the medical field.

Intestinal Toxicity in Rats Following Administration of CDK4/6 Inhibitors Is Independent of Primary Pharmacology.

Recently three different cyclin-dependent kinase 4 and 6 (CDK4/6) dual inhibitors were approved for the treatment of breast cancer (palbociclib, ribociclib, and abemaciclib), all of which offer comparable therapeutic benefits. Their safety profiles, however, are different. For example, neutropenia is observed at varying incidences in patients treated with these drugs; however, it is the most common adverse event for palbociclib and ribociclib, whereas diarrhea is the most common adverse event observed in patients treated with abemaciclib. To understand the mechanism of diarrhea observed with these drugs and in an effort to guide the development of safer drugs, we compared the effects of oral administration of palbociclib, ribociclib, and abemaciclib on the gastrointestinal tract of rats using doses intended to produce comparable CDK4/6 inhibition. Rats administered abemaciclib, but not palbociclib or ribociclib, had fecal alterations, unique histopathologic findings, and distinctive changes in intestinal gene expression. Morphologic changes in the intestine were characterized by proliferation of crypt cells, loss of goblet cells, poorly differentiated and degenerating enterocytes with loss of microvilli, and mucosal inflammation. In the jejunum of abemaciclib-treated rats, downregulation of enterocyte membrane transporters and upregulation of genes associated with cell proliferation were observed, consistent with activation of the Wnt pathway and downstream transcriptional regulation. Among these CDK4/6 inhibitors, intestinal toxicity was unique to rats treated with abemaciclib, suggesting a mechanism of toxicity not due to primary pharmacology (CDK4/6 inhibition), but to activity at secondary pharmacologic targets.

Proteome-wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors.

Patients with non-small cell lung cancers that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR whereby the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may affect drug activity and safety.

Lysosomal adaptation: How cells respond to lysosomotropic compounds.

Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside lysosomes (lysosomotropic). How cells respond to the lysosomal stress associated with lysosomotropism is not well characterized. Our goal is to assess the lysosomal changes and identify the signaling pathways that involve in the lysosomal changes. Eight chemically diverse lysosomotropic drugs from different therapeutic areas were subjected to the evaluation using the human adult retinal pigmented epithelium cell line, ARPE-19. All lysosomotropic drugs tested triggered lysosomal activation demonstrated by increased lysosotracker red (LTR) and lysosensor green staining, increased cathepsin activity, and increased LAMP2 staining. However, tested lysosomotropic drugs also prompted lysosomal dysfunction exemplified by intracellular and extracellular substrate accumulation including phospholipid, SQSTM1/p62, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and opsin. Lysosomal activation observed was likely attributed to lysosomal dysfunction, leading to compensatory responses including nuclear translocation of transcriptional factors TFEB, TFE3 and MITF. The adaptive changes are protective to the cells under lysosomal stress. Mechanistic studies implicate calcium and mTORC1 modulation involvement in the adaptive changes. These results indicate that lysosomotropic compounds could evoke a compensatory lysosomal biogenic response but with the ultimate consequence of lysosomal functional impairment. This work also highlights a pathway of response to lysosomal stress and evidences the role of TFEB, TFE3 and MITF in the stress response.

Protection of curcumin against amyloid-ß-induced cell damage and death involves the prevention from NMDA receptor-mediated intracellular Ca2+ elevation.

Alzheimer's disease (AD) is one of the common neurodegenerative diseases and amyloid-ß (Aß) is thought to be a key molecule contributing to AD pathology. Recently, curcumin is supposed to be beneficial to AD treatment. This study investigates the inhibitory effects of curcumin on Aß-induced cell damage and death involving NMDA receptor-mediated intracellular Ca(2+) elevation in human neuroblastoma SH-SY5Y cells. Cells were impaired significantly in Aß-damaged group compared with the control group, and cell viability was decreased while the released LDH from the cytosol was increased. Curcumin promotes cell growth and decreases cell impairment induced by Aß. Curcmin attenuates Aß-induced elevation of the ratio of cellular glutamate/γ-aminobutyric acid (GABA) with a concentration-dependent manner. Curcumin inhibits Aß-induced increase of cellular Ca(2+) and depresses Aß-induced phosphorylations of both NMDA receptor and cyclic AMP response element-binding protein (CREB) and activating transcription factor 1 (ATF-1). These results indicated that curcumin inhibits Aß-induced neuronal damage and cell death involving the prevention from intracellular Ca(2+) elevation mediated by the NMDA receptor.

Contribution of membrane trafficking perturbation to retinal toxicity.

The retina is a highly structured tissue that is formed by layers containing 7 different cell types. The photoreceptor cell is a specialized type of neuron in the retina that is capable of absorbing and converting light into electrophysiological signals. There is a constant renewal process for photoreceptors consisting of intermittent shedding of the distal tips of the photosensitive outer segment and subsequent phagocytosis (uptake, degradation and recycling) by retinal pigmented epithelial (RPE) cells. This rebuilding process is essential for vision and the survival of photoreceptors and RPE cells. Drugs with a basic moiety have the potential to accumulate in the lysosome and impair its functions including the phagocytosis process, which could hinder clearance of outer segments and ultimately induce retinopathy. To determine the prevalence of this cellular mechanism in retinal toxicity, a collection of proprietary compounds associated with retinal toxicity were subjected to a battery of in vitro tests using the human adult retinal pigmented epithelium cell line, ARPE-19. The tests included a phagocytosis assay, and lysosomal and autophagosomal staining. The compounds that induced retinopathy clustered in the basic and lipophilic region, which drives lysosomal sequestration. This accumulation coincided with phagocytosis inhibition and an increase in autophagosome staining, suggesting a blockage of the membrane trafficking process. A correlation between the physicochemical properties and in vitro lysosomal pathway effects was established. These data reveal the importance of physicochemical properties of compounds and lysosome accumulation as a potential mechanism for drug-induced retinopathy and demonstrate the usefulness of in vitro screening in predicting this liability.

Chitooligosaccharides attenuate Cu2+-induced cellular oxidative damage and cell apoptosis involving Nrf2 activation.

Alzheimer's disease (AD) is one of the common neurodegenerative diseases. Increase of labile copper pool plays an important role in the pathogenesis of AD. Nrf2(NF-E2-related factor-2)-ARE (antioxidant response element) signaling is an important intracellular manner to defend against oxidative stress. In this study, we used SH-SY5Y cells as a model of neuron to test the effect of chitooligosaccharides (COSs) on Cu(2+)-induced oxidative damage. SH-SY5Y cells were treated with different concentrations of COSs (100-800 mg/L) before incubated with Cu(2+). Cell viability and cell damage and apoptosis were assessed. Both extracellular H(2)O(2) and intracellular ROS were measured and the relative levels of Nrf2, phosphorylated Nrf2, and HO-1 were analyzed by Western blotting, and further HO-1 mRNA was relatively quantified by real-time quantitative PCR. The results indicated that Cu(2+)-induced decrease of cell viability and increase of LDH release. In cell-free solution, COSs alone or with Cu(2+) cannot scavenge O(2)(-); however, COSs downregulate the levels of cellular oxidative stress and activated Caspase-3 induced by Cu(2+). Further, the levels of pSer40-Nrf2 protein and both the transcription and the translation of HO-1 gene are dramatically increased in COSs-protective group compared with Cu(2+) damage group. Therefore, these results indicate that Nrf2 activation might be involved in the protection of COSs against Cu(2+)-induced cellular oxidative damage. COSs contribute to the attenuation of oxidative damage and could be used as a nutritional agent for AD treatment.

Endoplasmic reticulum stress as a novel neuronal mediator in Alzheimer's disease.

Alzheimer's disease (AD) is one of the most common types of progressive dementias. The typical neuropathological changes in AD include extracellular senile plaques, intracellular neurofibrillary tangles, and loss of neurons. The pathogenetic mechanism of this disease is not comprehensively understood yet. Recently, endoplasmic reticulum stress (ER stress) has been considered as a potential event involved in AD development. Some AD-related factors, such as misfolded protein and Ca(2+) depletion, could disrupt the homeostasis of ER lumen. In AD, the aggregated amyloid-beta peptide (Abeta) could induce ER stress in an assembly dependent way. The presenilin has been identified as a Ca(2+) channel. Mutations of presenilin could change the balance of Ca(2+) in ER lumen and thus disrupts the ER homeostasis. Furthermore, the ER stress could lead to cellular disorders like inflammation. Through activating the expression of inflammatory factors, ER stress triggers inflammatory response in AD pathology. Herein, we reviewed the recent progress of ER stress-induced unfolded protein response (UPR) and the roles of ER stress in AD pathological process.

Estimation of high risk pregnancy contributing to perinatal morbidity and mortality from a birth population-based regional survey in 2010 in China.

BACKGROUND: Neonatal mortality reduction in China over past two decades was reported from nationwide sampling surveys, however, how high risk pregnancy affected neonatal outcome is unknown. The objective of this study was to explore relations of pregnancy complications and neonatal outcomes from a regional birth population. METHODS: In a prospective, cross-sectional survey of complete birth population-based data file from 151 level I-III hospitals in Huai'an region in 2010, pregnancy complications were analyzed for perinatal morbidity and mortality in association with maternal and perinatal characteristics, hospital levels, mode of delivery, newborn birth weight and gestational age, using international definition for birth registry and morbidities. RESULTS: Pregnancy complications were found in 10% of all births, in which more than 70% were delivered at level II and III hospitals associated with higher proportions of fetal and neonatal death, preterm birth, death at delivery and congenital anomalies. High Cesarean section delivery was associated with higher pregnancy complications, and more neonatal critical illnesses. The pregnancy complications related perinatal morbidity and mortality in level III were 2-4 times as high as in level I and II hospitals. By uni- and multi-variate regression analysis, impact of pregnancy complications was along with congenital anomalies and preterm birth, and maternal child-bearing age and school education years contributing to the prevalence. CONCLUSIONS: This survey revealed variable links of pregnancy complications to perinatal outcome in association with very high Cesarean section deliveries, which warrants investigation for causal relations between high risk pregnancy and neonatal outcome in this emerging region.

The contribution of lysosomotropism to autophagy perturbation.

Autophagy refers to the catabolic process in eukaryotic cells that delivers cytoplasmic material to lysosomes for degradation. This highly conserved process is involved in the clearance of long-lived proteins and damaged organelles. Consequently, autophagy is important in providing nutrients to maintain cellular function under starvation, maintaining cellular homeostasis, and promoting cell survival under certain conditions. Several pathways, including mTOR, have been shown to regulate autophagy. However, the impact of lysosomal function impairment on the autophagy process has not been fully explored. Basic lipophilic compounds can accumulate in lysosomes via pH partitioning leading to perturbation of lysosomal function. Our hypothesis is that these types of compounds can disturb the autophagy process. Eleven drugs previously shown to accumulate in lysosomes were selected and evaluated for their effects on cytotoxicity and autophagy using ATP depletion and LC3 assessment, respectively. All eleven drugs induced increased staining of endogenous LC3 and exogenous GFP-LC3, even at non toxic dose levels. In addition, an increase in the abundance of SQSTM1/p62 by all tested compounds denotes that the increase in LC3 is due to autophagy perturbation rather than enhancement. Furthermore, the gene expression profile resulting from in vitro treatment with these drugs revealed the suppression of plentiful long-lived proteins, including structural cytoskeletal and associated proteins, and extracellular matrix proteins. This finding indicates a retardation of protein turnover which further supports the notion of autophagy inhibition. Interestingly, upregulation of genes containing antioxidant response elements, e.g. glutathione S transferase and NAD(P)H dehydrogenase quinone 1 was observed, suggesting activation of Nrf2 transcription factor. These gene expression changes could be related to an increase in SQSTM1/p62 resulting from autophagy deficiency. In summary, our data indicate that lysosomal accumulation due to the basic lipophilic nature of xenobiotics could be a general mechanism contributing to the perturbation of the autophagy process.

Mechanistic investigation of imatinib-induced cardiac toxicity and the involvement of c-Abl kinase.

The Bcr-abl tyrosine kinase inhibitor imatinib mesylate is the frontline therapy for chronic myeloid leukemia. Imatinib has been reported to cause congestive heart failure and left ventricular contractile dysfunction in patients and cardiomyopathy in rodents, findings proposed to be associated with its pharmacological activity. To investigate the specific role of Abelson oncogene 1 (c-Abl) in imatinib-induced cardiac toxicity, we performed targeted gene inhibition of c-Abl by RNA interference in neonatal cardiomyocytes (NCMs). Suppression of c-Abl did not lead to cytotoxicity or induction of endoplasmic reticulum (ER) stress. To further dis associate c-Abl from imatinib-induced cardiac toxicity, we designed imatinib structural analogs that do not have appreciable c-Abl inhibition in NCMs. The c-Abl inactive analogs induced cytotoxicity and ER stress, at similar or greater potencies and magnitudes as imatinib. Furthermore, combining c-Abl gene silencing with imatinib and analogs treatment did not significantly shift the cytotoxicity dose response curves. Imatinib and analogs were shown to accumulate in lysosomes, likely due to their physicochemical properties, and disrupt autophagy. The toxicity induced by imatinib and analogs can be rescued by bafilomycin A pretreatment, demonstrating the involvement of lysosomal accumulation in cardiac toxicity. The results from our studies strongly suggest that imatinib induces cardiomyocyte dysfunction through disruption of autophagy and induction of ER stress, independent of c-Abl inhibition.