Emerging evidence that ginseng components improve cognition in subjective memory impairment, mild cognitive impairment, and early Alzheimer's disease dementia.

Ginseng is a traditional herbal medicine used for prevention and treatment of various diseases as a tonic. Recent scientific cohort studies on life prolongation with ginseng consumption support this record, as those who consumed ginseng for more than 5 years had reduced mortality and cognitive decline compared to those who did not. Clinical studies have also shown that acute or long-term intake of ginseng total extract improves acute working memory performance or cognitive function in healthy individuals and those with subjective memory impairment (SMI), mild cognitive impairment (MCI), or early Alzheimer's disease (AD) dementia who are taking AD medication(s). Ginseng contains various components ranging from classical ginsenosides and polysaccharides to more recently described gintonin. However, it is unclear which ginseng component(s) might be the main candidate that contribute to memory or cognitive improvements or prevent cognitive decline in older individuals. This review describes recent clinical contributors to ginseng components in clinical tests and introduces emerging evidence that ginseng components could be novel candidates for cognitive improvement in older individuals, as ginseng components improve SMI cognition and exhibits add-on effects when co-administered with early AD dementia drugs. The mechanism behind the beneficial effects of ginseng components and how it improves cognition are presented. Additionally, this review shows how ginseng components can contribute to SMI, MCI, or early AD dementia when used as a supplementary food and/or medicine, and proposes a novel combination therapy of current AD medicines with ginseng component(s).

Gintonin-Induced Wound-Healing-Related Responses Involve Epidermal-Growth-Factor-like Effects in Keratinocytes.

Epidermal growth factor (EGF) receptor activation and related downstream signaling pathways are known to be one of the major mechanisms of the proliferation and migration of keratinocytes. The heparin-binding EGF-like growth factor (HB-EGF) binds to EGF receptors and stimulates keratinocyte proliferation and migration. Gintonin, a novel ginseng compound, is a lysophosphatidic acid (LPA) receptor ligand. Gintonin has skin-wound-healing effects. However, the underlying mechanisms for these gintonin actions remain unclear. In this study, we aimed to elucidate the involvement of EGFRs in gintonin-induced wound repair in HaCaT keratinocytes. In this study, a water-soluble tetrazolium salt-based assay, a modified Boyden chamber migration assay, and immunoblotting were performed. Gintonin increased EGF receptor activation in HaCaT cells. However, the gintonin-induced phosphorylation of the EGF receptor was markedly reduced via treatment with the LPA inhibitor Ki16425 or the EGF receptor inhibitor erlotinib. Gintonin-enhanced proliferation and migration were blocked by the EGF receptor inhibitors (erlotinib and AG1478). Additionally, gintonin stimulated the expression and release of HB-EGF in HaCaT cells. EGF receptor inhibitors blocked gintonin-enhanced HB-EGF expression. These results indicate that the wound-healing effects of gintonin are closely related to the collaboration between EGF receptor activation and HB-EGF release-mediated downstream signaling pathways.

Transformed cells maintain survival by downregulating autophagy at a high cell confluency.

Autophagy plays a dual role in tumorigenesis by functioning as both a tumor suppressor and promoter, depending on the stage of tumorigenesis. However, it is still unclear at what stage the role of autophagy changes during tumorigenesis. Herein, we investigated the differences in the basal levels and roles of autophagy in five cell lines at different stages of cell transformation. We found that cell lines at higher transformation stages were more sensitive to the autophagy inhibitors, suggesting that autophagy plays a more important role as the transformation progresses. Our ptfLC3 imaging analysis to measure Atg5/LC3-dependent autophagy showed increased autophagic flux in transformed cells compared to untransformed cells. However, the Cyto-ID analysis, which measures Atg5-dependent and -independent autophagic flux, showed high levels of autophagosome formation not only in the transformed cells but also in the initiated cell and Atg5 KO cell line. These results indicate that Atg5-independent autophagy may be more critical in initiated and transformed cell lines than in untransformed cells. Specially, we observed that transformed cells maintained relatively high basal autophagy levels under rapidly proliferating conditions but exhibited much lower basal autophagy levels at high confluency; however, autophagic flux was not significantly reduced in untransformed cells, even at high confluency. In addition, when continuously cultured for 3 weeks without passage, senescent cells were significantly less sensitive to autophagy inhibition than their actively proliferating counterparts. These results imply that once a cell has switched from a proliferative state to a senescent state, the inhibition of autophagy has only a minimal effect. Taken together, our results suggest that autophagy can be differentially regulated in cells at different stages of tumorigenesis under stressful conditions.

Preparation of Red Ginseng Marc-Derived Gintonin and Its Application as a Skin Nutrient.

Ginseng is one of the traditional herbal medicines for tonic. Gintonin is a new material derived from white/red ginseng and its lysophosphatidic acids (LPAs) play as a ligand for G protein-coupled LPA receptors. Korean red ginseng marc (KRGM) is a by-product after the KRG processes. We developed a low-cost/high-efficiency method for KRGM gintonin production. We further studied the KRGM gintonin-mediated anti-skin aging effects under UVB exposure using human dermal fibroblasts (HDFs). KRGM gintonin yield is about 8%. KRGM gintonin contains a high amount of LPA C18:2, lysophosphatidylcholine (LPC), and phosphatidylcholine (PC), which is similar to white ginseng gintonin. KRGM gintonin induced [Ca2+]i transient via LPA1/3 receptors and increased cell viability/proliferation under UVB exposure. The underlying mechanisms of these results are associated with the antioxidant action of KRGM gintonin. KRGM gintonin attenuated UVB-induced cell senescence by inhibiting cellular ß-galactosidase overexpression and facilitated wound healing. These results indicate that KRGM can be a novel bioresource of KRGM gintonin, which can be industrially utilized as new material for skin nutrition and/or skin healthcare.

A novel protocol for batch-separating gintonin-enriched, polysaccharide-enriched, and crude ginsenoside-containing fractions from Panax ginseng.

Background: Ginseng contains three active components: ginsenosides, gintonin, and polysaccharides. After the separation of 1 of the 3 ingredient fractions, other fractions are usually discarded as waste. In this study, we developed a simple and effective method, called the ginpolin protocol, to separate gintonin-enriched fraction (GEF), ginseng polysaccharide fraction (GPF), and crude ginseng saponin fraction (cGSF). Methods: Dried ginseng (1 kg) was extracted using 70% ethanol (EtOH). The extract was water fractionated to obtain a water-insoluble precipitate (GEF). The upper layer after GEF separation was precipitated with 80% EtOH for GPF preparation, and the remaining upper layer was vacuum dried to obtain cGSF. Results: The yields of GEF, GPF, and cGSF were 14.8, 54.2, and 185.3 g, respectively, from 333 g EtOH extract. We quantified the active ingredients of 3 fractions: L-arginine, galacturonic acid, ginsenosides, glucuronic acid, lysophosphatidic acid (LPA), phosphatidic acid (PA), and polyphenols. The order of the LPA, PA, and polyphenol content was GEF > cGSF > GPF. The order of L-arginine and galacturonic acid was GPF >> GEF = cGSF. Interestingly, GEF contained a high amount of ginsenoside Rb1, whereas cGSF contained more ginsenoside Rg1. GEF and cGSF, but not GPF, induced intracellular [Ca2+]i transient with antiplatelet activity. The order of antioxidant activity was GPF > GEF = cGSF. Immunological activities (related to nitric oxide production, phagocytosis, and IL-6 and TNF-α release) were, in order, GPF > GEF = cGSF. The neuroprotective ability (against reactive oxygen species) order was GEF > cGSP > GPF. Conclusion: We developed a novel ginpolin protocol to isolate 3 fractions in batches and determined that each fraction has distinct biological effects.

Paradoxical downregulation of LPAR3 exerts tumor-promoting activity through autophagy induction in Ras-transformed cells.

BACKGROUND: Lysophosphatidic acid receptor 3 (LPAR3) is coupled to Gαi/o and Gα11/q signaling. Previously, we reported that LPAR3 is highly methylated in carcinogen-induced transformed cells. Here, we demonstrate that LPAR3 exhibits malignant transforming activities, despite being downregulated in transformed cells. METHODS: The LPAR3 knockout (KO) in NIH 3 T3 and Bhas 42 cells was established using the CRISPR/Cas9 system. Both RT-PCR and DNA sequencing were performed to confirm the KO of LPAR3. The cellular effects of LPAR3 KO were further examined by WST-1 assay, immunoblotting analysis, transwell migration assay, colony formation assay, wound scratch assday, in vitro cell transformation assay, and autophagy assay. RESULTS: In v-H-ras-transformed cells (Ras-NIH 3 T3) with LPAR3 downregulation, ectopic expression of LPAR3 significantly enhanced the migration. In particular, LPAR3 knockout (KO) in Bhas 42 (v-Ha-ras transfected Balb/c 3 T3) and NIH 3 T3 cells caused a decrease in cell survival, transformed foci, and colony formation. LPAR3 KO led to the robust accumulation of LC3-II and autophagosomes and inhibition of autophagic flux by disrupting autophagosome fusion with lysosome. Conversely, autolysosome maturation proceeded normally in Ras-NIH 3 T3 cells upon LPAR3 downregulation. Basal phosphorylation of MEK and ERK markedly increased in Ras-NIH 3 T3 cells, whereas being significantly lower in LPAR3 KO cells, suggesting that increased MEK signaling is involved in autophagosome-lysosome fusion in Ras-NIH 3 T3 cells. CONCLUSIONS: Paradoxical downregulation of LPAR3 exerts cooperative tumor-promoting activity with MEK activation through autophagy induction in Ras-transformed cells. Our findings have implications for the development of cancer chemotherapeutic approaches.

Increase in the sensitivity to PLX4720 through inhibition of transcription factor EB-dependent autophagy in BRAF inhibitor-resistant cells.

Long-term treatment with oncogenic BRAF inhibitors confers resistance to BRAF inhibitor monotherapy. In this study, a combination treatment strategy with autophagy inhibitors was proposed to increase the sensitivity of BRAF mutant containing A375P melanoma cells that have developed resistance to BRAF inhibitors. We found that the A375P/Multi-drug resistance (A375P/Mdr) cells, which are resistant to both BRAF inhibitors and MEK inhibitors, exhibited a higher basal autophagic flux compared to their parental A375P cells, as determined by tandem mRFP-GFP-tagged LC3 imaging assay and LC3 conversion. In addition, transcription factor EB (TFEB), which acts as a transcription factor regulating the transcription of autophagy-related genes, was much more localized in the nucleus in A375P/Mdr cells than in A375P cells, indicating that the increase in basal autophagic flux was TFEB-dependent. In particular, the overexpression of an activated form of TFEB (TFEBAA) caused a modest increase in PLX4720 resistance in A375P/Mdr cells. Interestingly, treatment with early stage autophagy inhibitors reversed BRAF inhibitor-induced resistance, whereas late autophagy inhibition did not. In contrast, inhibition of ER stress by 4-phenylbutyric acid suppressed basal autophagic flux. Moreover, ER stress inhibition significantly remarkably inhibited the nuclear localization of TFEB, resulting in an increase in the sensitivity of A375P/Mdr cells to PLX4720. Taken together, these results suggest that autophagy may be an important mechanism of acquired resistance to BRAF inhibitors. Thus, targeting autophagy may be suitable for the treatment of tumors resistant to BRAF inhibitor.

Effect of the Gintonin-Enriched Fraction on Glucagon-Like-Protein-1 Release.

Ginseng-derived gintonin reportedly contains functional lysophosphatidic acids (LPAs) as LPA receptor ligands. The effect of the gintonin-enriched fraction (GEF) on in vitro and in vivo glucagon-like protein-1 (GLP-1) secretion, which is known to stimulate insulin secretion, via LPA receptor(s) remains unclear. Accordingly, we examined the effects of GEF on GLP-1 secretion using human enteroendocrine NCI-H716 cells. The expression of several of LPA receptor subtypes in NCI-H716 cells using qPCR and Western blotting was examined. LPA receptor subtype expression was in the following order: LPA6 > LPA2 > LPA4 > LPA5 > LPA1 (qPCR), and LPA6 > LPA4 > LPA2 > LPA1 > LPA3 > LPA5 (Western blotting). GEF-stimulated GLP-1 secretion occurred in a dose- and time-dependent manner, which was suppressed by cAMP-Rp, a cAMP antagonist, but not by U73122, a phospholipase C inhibitor. Furthermore, silencing the human LPA6 receptor attenuated GEF-mediated GLP-1 secretion. In mice, low-dose GEF (50 mg/kg, peroral) increased serum GLP-1 levels; this effect was not blocked by Ki16425 co-treatment. Our findings indicate that GEF-induced GLP-1 secretion could be achieved via LPA6 receptor activation through the cAMP pathway. Hence, GEF-induced GLP secretion via LPA6 receptor regulation might be responsible for its beneficial effects on human endocrine physiology.

Wound Healing Effect of Gintonin Involves Lysophosphatidic Acid Receptor/Vascular Endothelial Growth Factor Signaling Pathway in Keratinocytes.

Gintonin, a novel compound of ginseng, is a ligand of the lysophosphatidic acid (LPA) receptor. The in vitro and in vivo skin wound healing effects of gintonin remain unknown. Therefore, the objective of this study was to investigate the effects of gintonin on wound healing-linked responses, especially migration and proliferation, in skin keratinocytes HaCaT. In this study, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, Boyden chamber migration assay, scratch wound healing assay, and Western blot assay were performed. A tail wound mouse model was used for the in vivo test. Gintonin increased proliferation, migration, and scratch closure in HaCaT cells. It also increased the release of vascular endothelial growth factor (VEGF) in HaCaT cells. However, these increases, induced by gintonin, were markedly blocked by treatment with Ki16425, an LPA inhibitor, PD98059, an ERK inhibitor, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester), a calcium chelator, and U73122, a PLC inhibitor. The VEGF receptor inhibitor axitinib also attenuated gintonin-enhanced HaCaT cell proliferation. Gintonin increased the phosphorylation of AKT and ERK1/2 in HaCaT cells. In addition, gintonin improved tail wound healing in mice. These results indicate that gintonin may promote wound healing through LPA receptor activation and/or VEGF release-mediated downstream signaling pathways. Thus, gintonin could be a beneficial substance to facilitate skin wound healing.

Development and Validation of a Trigger Tool for Identifying Drug-Related Emergency Department Visits.

There are various trigger tools for detecting adverse drug events (ADEs), however, a drug-related emergency department (ED) visit trigger tool (DrEDTT) has not yet been developed. We aimed to develop and validate a DrEDTT with a multi-center cohort. In this cross-sectional study, we developed the DrEDTT consisting of 28 triggers through a comprehensive literature review and three phase expert group discussion. Next, we evaluated the performance of the DrEDTT by applying it to relevant medical records retrieved from four hospitals from January 2016 to June 2016. Two experts performed an in-depth chart review of a 25% of random sample of trigger flagged and unflagged ED visits and a true ADE was determined through causality assessment. Among 66,564 patients who visited the ED for reasons other than traffic accident and trauma during the study period, at least one trigger was found in 21,268 (32.0%) patients. A total of 959 true ADE cases (5.8%) were identified from a randomly selected 25% of ED visit cases. The overall positive predictive value was 14.0% (range: 8.3-66.7%). Sensitivity and specificity of DrEDTT were 77.7% and 70.4%, respectively. In conclusion, this newly developed trigger tool might be helpful to detect ADE-related ED visits.

Differential Sensitivity of Wild-Type and BRAF-Mutated Cells to Combined BRAF and Autophagy Inhibition.

BRAF inhibitors are insufficient monotherapies for BRAF-mutated cancer; therefore, we investigated which inhibitory pathway would yield the most effective therapeutic approach when targeted in combination with BRAF inhibition. The oncogenic BRAF inhibitor, PLX4720, increased basal autophagic flux in BRAF-mutated cells compared to wild-type (WT) BRAF cells. Interestingly, early autophagy inhibition improved the effectiveness of PLX4720 regardless of BRAF mutation, whereas late autophagy inhibition did not. Although ATG5 knockout led to PLX4720 resistance in both WT and BRAF-mutated cells, the MEK inhibitor trametinib exhibited a synergistic effect on PLX4720 sensitivity in WT BRAF cells but not in BRAF-mutated cells. Conversely, the prolonged inhibition of endoplasmic reticulum (ER) stress reduced basal autophagy in BRAF-mutated cells, thereby increasing PLX4720 sensitivity. Taken together, our results suggest that the combined inhibition of ER stress and BRAF may simultaneously suppress both pro-survival ER stress and autophagy, and may therefore be suitable for treatment of BRAF-mutated tumors whose autophagy is increased by chronic ER stress. Similarly, for WT BRAF tumors, therapies targeting MEK signaling may be a more effective treatment strategy. Together, this study presents a rational combination treatment strategy to improve the efficacy of BRAF inhibitors depending on BRAF mutation status.

Comparative associations between anticholinergic burden and emergency department visits for anticholinergic adverse events in older Korean adults: a nested case-control study using national claims data for validation of a novel country-specific scale.

BACKGROUND: Considering the limited generalizability of previous anticholinergic burden scales, the Korean Anticholinergic Burden Scale (KABS) as a scale specific to the Korean population was developed. We aimed to validate the KABS by detecting the associations between high anticholinergic burden, measured with the KABS, and emergency department (ED) visits compared to the pre-existing validated scales in older Korean adults. METHODS: A nested case-control study was conducted using national claims data. The cases included the first anticholinergic ED visits between July 1 and December 31, 2016. Anticholinergic ED visits were defined as ED visits with a primary diagnosis of constipation, delirium, dizziness, fall, fracture, or urinary retention. Propensity score-matched controls were identified. Average daily AB scores during 30 days before the index date were measured. Multivariate logistic regression analyses were performed. RESULTS: In total, 461,034 were included. The highest proportion of those with high AB was identified with KABS (5.0%). Compared with those who had a KABS score of 0, older adults with a score ≥ 3 were at higher risk for overall anticholinergic ED visits (aOR, 1.62, 95% CI, 1.53-1.72), as well as visits for falls/fractures (aOR: 1.54, 95% CI: 1.40-1.69), dizziness (aOR: 1.44, 95% CI: 1.30-1.59), delirium (aOR: 2.96, 95% CI: 2.28-3.83), constipation (aOR: 1.84, 95% CI: 1.68-2.02), and urinary retention (aOR: 2.13, 95% CI: 1.79-2.55). High AB by KABS showed a stronger association with overall anticholinergic ED visits and visits due to delirium and urinary retention than those by other scales. CONCLUSIONS: In conclusion, KABS is superior to pre-existing scales in identifying patients with high AB and predicting high AB-related ED visits in older Korean adults.

Effects of gintonin-enriched fraction on hippocampal gene expressions.

BACKGROUND: Recently, gintonin and gintonin-enriched fraction (GEF) have been isolated from ginseng, a herbal medicine. Gintonin induces [Ca2+]i transition in cultured hippocampal neurons and stimulates acetylcholine release through LPA receptor activation. Oral administration of GEF is linked to hippocampus-dependent cognitive enhancement and other neuroprotective effects; however, effects of its long-term administration on hippocampal gene expression remains unknown. Here, we used next-generation sequence (NGS) analysis to examine changes in hippocampal gene expressions after long-term oral administration of GEF. METHODS: C57BL/6 mice were divided into three groups: control group, GEF50 (GEF 50 mg/kg, p.o.), and GEF100 (GEF 100 mg/kg, p.o.). After 22 days, total RNA was extracted from mouse hippocampal tissues. NGS was used for gene expression profiling; quantitative-real-time PCR and western blot were performed to quantify the changes in specific genes and to confirm the protein expression levels in treatment groups. RESULTS: NGS analysis screened a total of 23,282 genes, analyzing 11-related categories. We focused on the neurogenesis category, which includes four genes for candidate markers: choline acetyltransferase (ChAT) gene, ß3-adrenergic receptor (Adrb3) gene, and corticotrophin-releasing hormone (Crh) gene, and tryptophan 2,3-dioxygenase (Tdo2) gene. Real-time PCR showed a marked overexpression of ChAT, Adrb3, and Crh genes, while reduced expression of Tdo2. Western blot analysis also confirmed increased ChAT and decreased Tdo2 protein levels. CONCLUSION: We found that GEF affects mouse hippocampal gene expressions, associated with memory, cognitive, anti-stress and anti-anxiety functions, and neurodegeneration at differential degree, that might explain the genetic bases of GEF-mediated neuroprotective effects.

Selection of Potent Inhibitors of Soluble Epoxide Hydrolase for Usage in Veterinary Medicine.

The veterinary pharmacopeia available to treat pain and inflammation is limited in number, target of action and efficacy. Inhibitors of soluble epoxide hydrolase (sEH) are a new class of anti-inflammatory, pro-resolving and analgesic drugs being tested in humans that have demonstrated efficacy in laboratory animals. They block the hydrolysis, and thus, increase endogenous concentrations of analgesic and anti-inflammatory signaling molecules called epoxy-fatty acids. Here, we screened a library of 2,300 inhibitors of the sEH human against partially purified feline, canine and equine hepatic sEH to identify inhibitors that are broadly potent among species. Six very potent sEH inhibitors (IC50 < 1 nM for each enzyme tested) were identified. Their microsomal stability was then measured in hepatic extracts from cat, dog and horse, as well as their solubility in solvents suitable for the formulation of drugs. The trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB, 1,728) appears to be the best compromise between stability and potency across species. Thus, it was selected for further testing in veterinary clinical trials of pain and inflammation in animals.

Preparation and evaluation of soluble epoxide hydrolase inhibitors with improved physical properties and potencies for treating diabetic neuropathic pain.

Soluble epoxide hydrolase (sEH), a novel therapeutic target for neuropathic pain, is a largely cytosolic enzyme that degrades epoxy-fatty acids (EpFAs), an important class of lipid signaling molecules. Many inhibitors of sEH have been reported, and to date, the 1,3-disubstituted urea has the highest affinity reported for the sEH among the central pharmacophores evaluated. An earlier somewhat water soluble sEH inhibitor taken to the clinic for blood pressure control had mediocre potency (both affinity and kinetics) and a short in vivo half-life. We undertook a study to overcome these difficulties, but the sEH inhibitors carrying a 1,3-disubstituted urea often suffer poor physical properties that hinder their formulation. In this report, we described new strategies to improve the physical properties of sEH inhibitors with a 1,3-disubstituted urea while maintaining their potency and drug-target residence time (a complementary in vitro parameter) against sEH. To our surprise, we identified two structural modifications that substantially improve the potency and physical properties of sEH inhibitors carrying a 1,3-disubstituted urea pharmacophore. Such improvements will greatly facilitate the movement of sEH inhibitors to the clinic.

Predicting Carcinogenic Mechanisms of Non-Genotoxic Carcinogens via Combined Analysis of Global DNA Methylation and In Vitro Cell Transformation.

An in vitro cell transformation assay (CTA) is useful for the detection of non-genotoxic carcinogens (NGTXCs); however, it does not provide information on their modes of action. In this study, to pursue a mechanism-based approach in the risk assessment of NGTXCs, we aimed to develop an integrated strategy comprising an in vitro Bhas 42 CTA and global DNA methylation analysis. For this purpose, 10 NGTXCs, which were also predicted to be negative through Derek/Sarah structure-activity relationship analysis, were first tested for transforming activity in Bhas 42 cells. Methylation profiles using reduced representation bisulfite sequencing were generated for seven NGTXCs that were positive in CTAs. In general, the differentially methylated regions (DMRs) within promoter regions showed slightly more bias toward hypermethylation than the DMRs across the whole genome. We also identified 13 genes associated with overlapping DMRs within the promoter regions in four NGTXCs, of which seven were hypermethylated and six were hypomethylated. Using ingenuity pathway analysis, the genes with DMRs at the CpG sites were found to be enriched in cancer-related categories, including "cell-to-cell signaling and interaction" as well as "cell death and survival". Moreover, the networks related to "cell death and survival", which were considered to be associated with carcinogenesis, were identified in six NGTXCs. These results suggest that epigenetic changes supporting cell transformation processes occur during non-genotoxic carcinogenesis. Taken together, our combined system can become an attractive component for an integrated approach for the testing and assessment of NGTXCs.

Dose response relationship of cumulative anticholinergic exposure with incident dementia: validation study of Korean anticholinergic burden scale.

BACKGROUND: The dose response relationship of nine-year cumulative anticholinergic exposure and dementia onset was investigated using the Korean version anticholinergic burden scale (KABS) in comparison with the Anticholinergic Cognitive Burden Scale (ACB). We also examined the effect of weak anticholinergics in the prediction of dementia. METHODS: A retrospective case-control study was conducted comprising 86,576 patients after 1:2 propensity score matching using the longitudinal national claims database. For cumulative anticholinergic burden estimation, average daily anticholinergic burden score during the 9 years prior to dementia onset was calculated using KABS and ACB and categorized as minimal, < 0.25; low, 0.25-1; intermediate, 1-2; and high, ≥ 2. Adjusted odds ratio (aOR) between cumulative anticholinergic burden and incident dementia was estimated. RESULTS: Patients with high exposure according to KABS and ACB comprised 3.2 and 3.4% of the dementia cohort and 2.1 and 2.8% of the non-dementia cohort, respectively. Dose-response relationships were observed between anticholinergic burden and incident dementia. After adjusting covariates, compared with minimal exposure, patients with high exposure according to KABS and ACB had a significantly higher risk for incident dementia with aOR of 1.71 (95% confidence interval (CI) 1.55-1.87) and 1.22 (CI 1.12-1.33), respectively. With the exclusion of weak anticholinergics, the association became stronger, i.e., 1.41 (CI 1.14-1.75) with ACB whereas the association became slightly weaker with KABS, i.e., 1.60 (CI 1.38-1.86). CONCLUSION: This study confirmed the dose response relationship for cumulative anticholinergic burden measured using the Korean specific anticholinergic burden scale with incident dementia.

ATG5 knockout promotes paclitaxel sensitivity in drug-resistant cells via induction of necrotic cell death.

Autophagy regulators are often effective as potential cancer therapeutic agents. Here, we investigated paclitaxel sensitivity in cells with knockout (KO) of ATG5 gene. The ATG5 KO in multidrug resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) was generated using the CRISPR/Cas9 technology. The qPCR and LC3 immunoblot confirmed knockout of the gene and protein of ATG5, respectively. The ATG5 KO restored the sensitivity of Ras-NIH 3T3/Mdr cells to paclitaxel. Interestingly, ATG5 overexpression restored autophagy function in ATG5 KO cells, but failed to rescue paclitaxel resistance. These results raise the possibility that low level of resistance to paclitaxel in ATG5 KO cells may be related to other roles of ATG5 independent of its function in autophagy. The ATG5 KO significantly induced a G2/M arrest in cell cycle progression. Additionally, ATG5 KO caused necrosis of a high proportion of cells after paclitaxel treatment. These data suggest that the difference in sensitivity to paclitaxel between ATG5 KO and their parental MDR cells may result from the disparity in the proportions of necrotic cells in both populations. Thus, our results demonstrate that the ATG5 KO in paclitaxel resistant cells leads to a marked G2/M arrest and sensitizes cells to paclitaxel-induced necrosis.

Lysophosphatidic Acid Inhibits Simvastatin-Induced Myocytoxicity by Activating LPA Receptor/PKC Pathway.

Statins such as simvastatin have many side effects, including muscle damage, which is known to be the most frequent undesirable side effect. Lysophosphatidic acid (LPA), a kind of biolipid, has diverse cellular activities, including cell proliferation, survival, and migration. However, whether LPA affects statin-linked muscle damage has not been reported yet. In the present study, to determine whether LPA might exert potential protective effect on statin-induced myocyotoxicity, the effect of LPA on cytotoxicity in rat L6 myoblasts exposed to simvastatin was explored. Viability and apoptosis of rat L6 myoblasts were detected via 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, respectively. Protein expression levels were detected via Western blotting. Simvastatin decreased viability of L6 cells. Such decrease in viability was recovered in the presence of LPA. Treatment with LPA suppressed simvastatin-induced apoptosis in L6 cells. In addition, treatment with LPA receptor inhibitor Ki16425, protein kinase C (PKC) inhibitor GF109203X, or intracellular calcium chelator BAPTA-AM attenuated the recovery effect of LPA on simvastatin-induced L6 cell toxicity. These findings indicate that LPA may inhibit simvastatin-induced toxicity in L6 cells probably by activating the LPA receptor-PKC pathway. Therefore, LPA might have potential as a bioactive molecule to protect muscles against simvastatin-induced myotoxicity.

Prevalence of anticholinergic burden and risk factors amongst the older population: analysis of insurance claims data of Korean patients.

Background Despite growing interest in the negative clinical outcomes of multiple anticholinergic use, limited studies have evaluated anticholinergic burden in the geriatric population nationally. Objective To evaluate the prevalence of high anticholinergic burden using the newly developed Korean Anticholinergic Burden Scale in comparison with previous tools and to identify associated factors. Setting National insurance data from a cross section (20%) of older Koreans (2016). Methods Anticholinergic burden was measured using the Korean scale in comparison to the Anticholinergic Drug Scale, Anticholinergic Cognitive Burden, and Anticholinergic Risk Scale. High anticholinergic burden was defined as a summed score of ≥ 3 for concurrent medications or a dose-standardized average daily score of ≥ 3, using each anticholinergic scale. Main outcomes measured Prevalence and predictors of high anticholinergic burden. Results Data of 1,292,323 patients were analyzed. According to the Korean scale, the prevalence of high anticholinergic burden was 25.5%. This result was similar to that from the Anticholinergic Drug Scale (24.9%) and Anticholinergic Cognitive Burden (22.2%). Factors associated with an increased likelihood of anticholinergic burden include: age, gender (female), high Charlson comorbidity index score, polypharmacy, medical aid beneficiary, co-morbidities (such as schizophrenia, depression, urinary incontinence, and Parkinson's disease), frequent healthcare visits, various healthcare facilities utilized, and predominantly visiting hospital-level facilities. According to the Korean Anticholinergic Burden Scale, the major drugs contributing to the anticholinergic burden were ranitidine, chlorpheniramine, tramadol, and dimenhydrinate. Conclusion This study showed that 1 in 4 older Koreans are exposed to high anticholinergic burden. The predictors identified in this research might assist pharmacists in early interventions for their patients.