Drug Response of Patient-Derived Lung Cancer Cells Predicts Clinical Outcomes of Targeted Therapy.

Intratumor heterogeneity leads to different responses to targeted therapies, even within patients whose tumors harbor identical driver oncogenes. This study examined clinical outcomes according to a patient-derived cell (PDC)-based drug sensitivity test in lung cancer patients treated with targeted therapies. From 487 lung cancers, 397 PDCs were established with a success rate of 82%. In 139 PDCs from advanced non-small-cell lung cancer (NSCLC) patients receiving targeted therapies, the standardized area under the curve (AUC) values for the drugs was significantly correlated with their tumor response (p = 0.002). Among 59 chemo-naive EGFR/ALK-positive NSCLC patients, the PDC non-responders showed a significantly inferior response rate (RR) and progression-free survival (PFS) for the targeted drugs than the PDC responders (RR, 25% vs. 78%, p = 0.011; median PFS, 3.4 months [95% confidence interval (CI), 2.8-4.1] vs. 11.8 months [95% CI, 6.5-17.0], p < 0.001). Of 25 EGFR-positive NSCLC patients re-challenged with EGFR inhibitors, the PDC responder showed a higher RR than the PDC non-responder (42% vs. 15%). Four patients with wild-type EGFR or uncommon EGFR-mutant NSCLC were treated with EGFR inhibitors based on their favorable PDC response to EGFR inhibitors, and two patients showed dramatic responses. Therefore, the PDC-based drug sensitivity test results were significantly associated with clinical outcomes in patients with EGFR- or ALK-positive NSCLC. It may be helpful for predicting individual heterogenous clinical outcomes beyond genomic alterations.

Molecular Subtypes and Tumor Microenvironment Characteristics of Small-Cell Lung Cancer Associated with Platinum-Resistance.

Although molecular subtypes of small-cell lung cancer (SCLC) have been proposed, their clinical relevance and therapeutic implications are not fully understood. Thus, we aimed to refine molecular subtypes and to uncover therapeutic targets. We classified the subtypes based on gene expression (n = 81) and validated them in our samples (n = 87). Non-SCLC samples were compared with SCLC subtypes to identify the early development stage of SCLC. Single-cell transcriptome analysis was applied to dissect the TME of bulk samples. Finally, to overcome platinum resistance, we performed drug screening of patient-derived cells and cell lines. Four subtypes were identified: the ASCL1+ (SCLC-A) subtype identified as TP53/RB-mutated non-SCLC representing the early development stage of SCLC; the immune activation (SCLC-I) subtype, showing high CD8+/PD-L1+ T-cell infiltration and endothelial-to-mesenchymal transition (EndMT); the NEUROD1 (SCLC-N) subtype, which showed neurotransmission process; and the POU2F3+ (SCLC-P) subtype with epithelial-to-mesenchymal transition (EMT). EndMT was associated with the worst prognosis. While SCLC-A/N exhibited platinum sensitivity, the EndMT signal of SCLC-I conferred platinum resistance. A BET inhibitor suppressed the aggressive angiogenesis phenotype of SCLC-I. We revealed that EndMT development contributed to a poor outcome in SCLC-I. Moreover, heterogenous TME development facilitated platinum resistance. BET inhibitors are novel candidates for overcoming platinum resistance.

Ataxia-Telangiectasia Mutated Is Involved in Autolysosome Formation.

Ataxia-telangiectasia mutated (ATM), a master kinase of the DNA damage response (DDR), phosphorylates a multitude of substrates to activate signaling pathways after DNA double-strand breaks (DSBs). ATM inhibitors have been evaluated as anticancer drugs to potentiate the cytotoxicity of DNA damage-based cancer therapy. ATM is also involved in autophagy, a conserved cellular process that maintains homeostasis by degrading unnecessary proteins and dysfunctional organelles. In this study, we report that ATM inhibitors (KU-55933 and KU-60019) provoked accumulation of autophagosomes and p62 and restrained autolysosome formation. Under autophagy-inducing conditions, the ATM inhibitors caused excessive autophagosome accumulation and cell death. This new function of ATM in autophagy was also observed in numerous cell lines. Repression of ATM expression using an siRNA inhibited autophagic flux at the autolysosome formation step and induced cell death under autophagy-inducing conditions. Taken together, our results suggest that ATM is involved in autolysosome formation and that the use of ATM inhibitors in cancer therapy may be expanded.

Patient-derived cell-based pharmacogenomic assessment to unveil underlying resistance mechanisms and novel therapeutics for advanced lung cancer.

BACKGROUND: A pharmacogenomic platform using patient-derived cells (PDCs) was established to identify the underlying resistance mechanisms and tailored treatment for patients with advanced or refractory lung cancer. METHODS: Drug sensitivity screening and multi-omics datasets were acquired from lung cancer PDCs (n = 102). Integrative analysis was performed to explore drug candidates according to genetic variants, gene expression, and clinical profiles. RESULTS: PDCs had genomic characteristics resembled with those of solid lung cancer tissues. PDC molecular subtyping classified patients into four groups: (1) inflammatory, (2) epithelial-to-mesenchymal transition (EMT)-like, (3) stemness, and (4) epithelial growth factor receptor (EGFR)-dominant. EGFR mutations of the EMT-like subtype were associated with a reduced response to EGFR-tyrosine kinase inhibitor therapy. Moreover, although RB1/TP53 mutations were significantly enriched in small-cell lung cancer (SCLC) PDCs, they were also present in non-SCLC PDCs. In contrast to its effect in the cell lines, alpelisib (a PI3K-AKT inhibitor) significantly inhibited both RB1/TP53 expression and SCLC cell growth in our PDC model. Furthermore, cell cycle inhibitors could effectively target SCLC cells. Finally, the upregulation of transforming growth factor-ß expression and the YAP/TAZ pathway was observed in osimertinib-resistant PDCs, predisposing them to the EMT-like subtype. Our platform selected XAV939 (a WNT-TNKS-ß-catenin inhibitor) for the treatment of osimertinib-resistant PDCs. Using an in vitro model, we further demonstrated that acquisition of osimertinib resistance enhances invasive characteristics and EMT, upregulates the YAP/TAZ-AXL axis, and increases the sensitivity of cancer cells to XAV939. CONCLUSIONS: Our PDC models recapitulated the molecular characteristics of lung cancer, and pharmacogenomics analysis provided plausible therapeutic candidates.

Anti-adhesive effect of chondrocyte-derived extracellular matrix surface-modified with poly-L-lysine.

After an injury, soft tissue structures in the body undergo a natural healing process through specific phases of healing. Adhesions occur as abnormal attachments between tissues and organs through the formation of blood vessels and/or fibrinous adhesions during the regenerative repair process. In this study, we developed an adhesion-preventing membrane with an improved physical protection function by modifying the surface of chondrocyte-derived extracellular matrices (CECM) with anti-adhesion function. We attempted to change the negative charge of the CECM surface to neutral using poly-L-lysine (PLL) and investigated whether it blocked fibroblast adhesion to it and showed an improved anti-adhesion effect in animal models of tissue adhesion. The surface of the membrane was modified with PLL coating (PLL 10), which neutralized the surface charge. We confirmed that the surface characteristics except for the potential difference were maintained after the modification and tested cell attachment in vitro. Adhesion inhibition was identified in a peritoneal adhesion animal model at 1 week and in a subcutaneous adhesion model for 4 weeks. Neutralized CECM (N-CECM) suppressed fibroblast and endothelial cell adhesion in vitro and inhibited abdominal adhesions in vivo. The CECM appeared to actively inhibit the infiltration of endothelial cells into the injured site, thereby suppressing adhesion formation, which differed from conventional adhesion barriers in the mode of action. Furthermore, the N-CECM remained intact without degradation for more than 4 weeks in vivo and exerted anti-adhesion effects for a long time. This study demonstrated that PLL10 surface modification rendered a neutral charge to the polymer on the extracellular matrix surface, thereby inhibiting cell and tissue adhesion. Furthermore, this study suggests a means to modify extracellular matrix surfaces to meet the specific requirements of the target tissue in preventing post-surgical adhesions.

Sensitivity to tumor development by TALEN-mediated Trp53 mutant genes in the susceptible FVB/N mice and the resistance C57BL/6 mice.

BACKGROUND: This study was undertaken to compare the sensitivities of mice strains during tumor induction by transcription activator-like effector nucleases (TALEN)-mediated Trp53 mutant gene. Alterations of their tumorigenic phenotypes including survival rate, tumor formation and tumor spectrum, were assessed in FVB/N-Trp53em2Hwl/Korl and C57BL/6-Trp53em1Hwl/Korl knockout (KO) mice over 16 weeks. RESULTS: Most of the physiological phenotypes factors were observed to be higher in FVB/N-Trp53em2Hwl/Korl KO mice than C57BL/6-Trp53em1Hwl/Korl KO mice, although there were significant differences in the body weight, immune organ weight, number of red blood cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total bilirubin (Bil-T) and glucose (Glu) levels in the KO mice relative to the wild type (WT) mice. Furthermore, numerous solid tumors were also observed in various regions of the surface skin of FVB/N-Trp53em2Hwl/Korl KO mice, but were not detected in C57BL/6-Trp53em1Hwl/Korl KO mice. The most frequently observed tumor in both the Trp53 KO mice was malignant lymphoma, while soft tissue teratomas and hemangiosarcomas were only detected in the FVB/N-Trp53em2Hwl/Korl KO mice. CONCLUSIONS: Our results indicate that the spectrum and incidence of tumors induced by the TALEN-mediated Trp53 mutant gene is greater in FVB/N-Trp53em2Hwl/Korl KO mice than C57BL/6-Trp53em1Hwl/Korl KO mice over 16 weeks.

PTX-3 Secreted by Intra-Articular-Injected SMUP-Cells Reduces Pain in an Osteoarthritis Rat Model.

Mesenchymal stem cells (MSCs) are accessible, abundantly available, and capable of regenerating; they have the potential to be developed as therapeutic agents for diseases. However, concerns remain in their further application. In this study, we developed a SMall cell+Ultra Potent+Scale UP cell (SMUP-Cell) platform to improve whole-cell processing, including manufacturing bioreactors and xeno-free solutions for commercialization. To confirm the superiority of SMUP-Cell improvements, we demonstrated that a molecule secreted by SMUP-Cells is capable of polarizing inflammatory macrophages (M1) into their anti-inflammatory phenotype (M2) at the site of injury in a pain-associated osteoarthritis (OA) model. Lipopolysaccharide-stimulated macrophages co-cultured with SMUP-Cells expressed low levels of M1-phenotype markers (CD11b, tumor necrosis factor-α, interleukin-1α, and interleukin-6), but high levels of M2 markers (CD163 and arginase-1). To identify the paracrine action underlying the anti-inflammatory effect of SMUP-Cells, we employed a cytokine array and detected increased levels of pentraxin-related protein-3 (PTX-3). Additionally, PTX-3 mRNA silencing was applied to confirm PTX-3 function. PTX-3 silencing in SMUP-Cells significantly decreased their therapeutic effects against monosodium iodoacetate (MIA)-induced OA. Thus, PTX-3 expression in injected SMUP-Cells, applied as a therapeutic strategy, reduced pain in an OA model.

Fabrication of decellularized meniscus extracellular matrix according to inner cartilaginous, middle transitional, and outer fibrous zones result in zone-specific protein expression useful for precise replication of meniscus zones.

Meniscus is a fibrocartilage composite tissue with three different microstructual zones, inner fibrocartilage, middle transitional, and outer fibrous zone. We hypothesized that decellularized meniscus extracellular matrix (DMECM) would have different characteristics according to zone of origin. We aimed to compare zone-specific DMECM in terms of biochemical characteristics and cellular interactions associated with tissue engineering. Micronized DMECM was fabricated from porcine meniscus divided into three microstructural zones. Characterization of DMECM was done by biochemical and proteomic analysis. Inner DMECM showed the highest glycosaminoglycan content, while middle DMECM showed the highest collagen content among groups. Proteomic analysis showed significant differences among DMECM groups. Inner DMECM showed better adhesion and migration potential to meniscus cells compared to other groups. DMECM resulted in expression of zone-specific differentiation markers when co-cultured with synovial mesenchymal stem cells (SMSCs). SMSCs combined with inner DMECM showed the highest glycosaminoglycan in vivo. Outer DMECM constructs, on the other hand, showed more fibrous tissue features, while middle DMECM constructs showed both inner and outer zone characteristics. In conclusion, DMECM showed different characteristics according to microstructural zones, and such material may be useful for zone-specific tissue engineering of meniscus.

Anti-inflammatory effects of Capparis ecuadorica extract in phthalic-anhydride-induced atopic dermatitis of IL-4/Luc/CNS-1 transgenic mice.

CONTEXT: The natural products derived from Capparis ecuadorica H.H. Iltis (Capparaceae) could have great potential for anti-inflammation since they inhibited the inflammatory response in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. OBJECT: This study investigated the anti-inflammatory effects and related mechanism of methanol extract of C. ecuadorica leaves (MCE) during atopic dermatitis (AD) responses. MATERIALS AND METHODS: Alterations in the phenotypical markers for AD, luciferase signal, iNOS-mediated COX-2 induction pathway, and inflammasome activation were analysed in non-Tg (n = 5) and 15% phthalic anhydride (PA) treated IL-4/Luc/CNS-1 transgenic (Tg) HR1 mice (n = 5 per group), subsequent to treatment with acetone-olive oil (AOO), vehicle (DMSO) and two dose MCE (20 and 40 mg/kg) three times a week for 4 weeks. RESULTS: MCE treatment reduced the intracellular ROS level (48.2%), NO concentration (7.1 mmol/L) and inflammatory cytokine expressions (39.1%) in the LPS-stimulated RAW264.7 cells. A significant decrease was detected for ear thickness (16.9%), weight of lymph node (0.7 mg), IgE concentration (1.9 µg/mL), and epidermal thickness (31.8%) of the PA + MCE treated Tg mice. MCE treatment induced the decrease of luciferase signal derived from the IL-4 promoter and the recovery of the IL-4 downstream regulator cytokines. PA + MCE treated Tg mice showed decreasing infiltration of mast cells (42.5%), iNOS-mediated COX-2 induction pathway, MAPK signalling pathway and inflammasome activation in the ear tissue. CONCLUSIONS: These findings provide the first evidence that MCE may have great potential to suppress chemical-induced skin inflammation through the suppression of IL-4 cytokine and the iNOS-mediated COX-2 induction pathway, and activation of inflammasome.

Protective role of fermented mulberry leave extract in LPS­induced inflammation and autophagy of RAW264.7 macrophage cells.

Mulberry leaves have antioxidant activity and anti­inflammatory effects in several types of cells. However, the efficacy of mulberry leaves fermented with Cordyceps militaris remains unknown. Therefore, the present study aimed to investigate whether the ethanol extracts of mulberry leaves fermented with C. militaris (EMfC) can prevent lipopolysaccharide (LPS)­induced inflammation and autophagy in macrophages. To achieve this, RAW264.7 cells pretreated with three different dose of EMfCs were subsequently stimulated with LPS, and examined for alterations in the regulatory factors of inflammatory responses and key parameters of the autophagy signaling pathway. EMfC treatment inhibited the generation of reactive oxidative species; however, significant activity was observed for 2,2­diphenyl­1­picrylhydrazyl (DPPH) radical scavenging (IC50=579.6703 mg/ml). Most regulatory factors in inflammatory responses were significantly inhibited following treatment with EMfC, without any significant cellular toxicity. EMfC­treated groups exhibited marked suppression of nitrogen oxide (NO) levels, mRNA expression levels of iNOS/COX­2, levels of all inflammatory cytokines (TNF­α, IL­1ß and IL­6) and phosphorylation of MAPK members, as well as recovery of cell cycle progression. Furthermore, similar effects were observed in the LPS­induced autophagy signaling pathway of RAW264.7 cells. The expression levels of microtubule­associated protein 1A/1B­light chain 3 (LC3) and Beclin exhibited a dose­dependent decrease in the EMfC+LPS­treated groups compared with in the Vehicle+LPS­treated group, whereas the phosphorylation of PI3K and mTOR were enhanced in a dose­dependent manner in the same groups. Overall, the results of the present study provide evidence that exposure to EMfC protects against LPS­induced inflammation and autophagy in RAW264.7 cells. These results indicated that EMfC is a potential candidate for treatment of inflammatory diseases.

Deletion of NKX3.1 via CRISPR/Cas9 Induces Prostatic Intraepithelial Neoplasia in C57BL/6 Mice.

Several techniques have been employed for deletion of the NKX3.1 gene, resulting in developmental defects of the prostate, including alterations in ductal branching morphogenesis and prostatic secretions as well as epithelial hyperplasia and dysplasia. To investigate whether the CRISPR/Cas9-mediated technique can be applied to study prostate carcinogenesis through exon I deletion of NKX3.1 gene, alterations in the prostatic intraepithelial neoplasia (PIN) and their regulatory mechanism were observed in the prostate of NKX3.1 knockout (KO) mice produced by the CRISPR/Cas9-mediated NKX3.1 mutant gene, at the ages of 16 and 24 weeks. The weight of dorsal-lateral prostate (DLP) and anterior prostate (AP) were observed to be increased in only the 24 weeks KO mice, although morphogenesis was constant in all groups. Obvious PIN 1 and 2 lesions were frequently detected in prostate of the 24 weeks KO mice, as compared with the same age wild type (WT) mice. Ki67, a key indicator for PIN, was densely stained in the epithelium of prostate in the 24 weeks KO mice, while the expression of p53 protein was suppressed in the same group. Also, both the 16 and 24 weeks KO mice reveal inhibition of the PI3K/AKT/mTOR pathway in the prostate. However, prostate specific antigen (PSA) levels and Bax/Bcl-2 expressions were decreased in the prostate of 16 weeks KO mice, and were increased in only the 24 weeks KO mice. Taken together, the results of the present study provide additional evidence that CRISPR/Cas9-mediated exon 1 deletion of the NKX3.1 gene successfully induces PIN lesions, along with significant alterations of Ki67 expression, EGFR signaling pathway, and cancer-regulated proteins.

Therapeutic Effects of Cold-Pressed Perilla Oil Mainly Consisting of Linolenic acid, Oleic Acid and Linoleic Acid on UV-Induced Photoaging in NHDF Cells and SKH-1 Hairless Mice.

Positive physiological benefits of several plant oils on the UV-induced photoaging have been reported in some cell lines and model mice, but perilla oil collected from the seeds of Perilla frutescens L. has not been investigated in this context. To study the therapeutic effects of cold-pressed perilla oil (CPO) on UV-induced photoaging in vitro and in vivo, UV-induced cellular damage and cutaneous photoaging were assessed in normal human dermal fibroblasts (NHDFs) and HR-1 hairless mice. CPO contained five major fatty acids including linolenic acid (64.11%), oleic acid (16.34%), linoleic acid (11.87%), palmitic acid (5.06%), and stearic acid (2.48%). UV-induced reductions in NHDF cell viability, ROS production, SOD activity, and G2/M cell cycle arrest were remarkably improved in UV + CPO treated NHDF cells as compared with UV + Vehicle treated controls. Also, UV-induced increases in MMP-1 protein and galactosidase levels were remarkably suppressed by CPO. In UV-radiated hairless mice, topical application of CPO inhibited an increase in wrinkle formation, transepidermal water loss (TEWL), erythema value, hydration and melanin index on dorsal skin of UVB-irradiated hairless mice. CPO was observed to similarly suppress UV-induced increases in epidermal thickness, mast cell numbers, and galactosidase and MMP-3 mRNA levels. These results suggest CPO has therapeutic potential in terms of protecting against skin photoaging by regulating skin morphology, histopathology and oxidative status.

Up-Regulation of Superoxide Dismutase 2 in 3D Spheroid Formation Promotes Therapeutic Potency of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

Umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) are accessible, available in abundance, and have been shown to be a promising source that can regenerate cartilage in patients with osteoarthritis or other orthopedic diseases. Recently, a three-dimensional (3D) cell culture system was developed to mimic the naive tissue microenvironment. However, the efficacy of cells generated from the 3D spheroid culture system has not yet been elucidated. In the present study, we demonstrate the changes in superoxide dismutase 2 (SOD2) gene expression, an indicator of oxidative stress, on 3D spheroid MSCs. Moreover, siRNA transfection and neutralizing antibody investigations were performed to confirm the function of SOD2 and E-cadherin. Overall, we found that SOD2 siRNA transfection in the spheroid form of MSCs increases the expression of apoptotic genes and decreases the clearance of mitochondrial reactive oxygen species (ROS). As a result, we confirm that 3D spheroid formation increases E-cadherin and SOD2 expression, ultimately regulating the phosphoinositide 3-kinase (PI3K/pAkt/pNrf2 and pERK/pNrf2 signaling pathway. Additionally, we show that SOD2 expression on 3D spheroid MSCs affects the regeneration rates of destructive cartilage in an osteoarthritic model. We postulate that the impact of SOD2 expression on 3D spheroid MSCs reduces oxidative stress and apoptosis, and also promotes cartilage regeneration.

Development of three-dimensional articular cartilage construct using silica nano-patterned substrate.

Current strategies for cartilage cell therapy are mostly based on the use of autologous chondrocytes. However, these cells have limitations of a small number of cells available and of low chondrogenic ability, respectively. Many studies now suggest that fetal stem cells are more plastic than adult stem cells and can therefore more efficiently differentiate into target tissues. This study introduces, efficiency chondrogenic differentiation of fetal cartilage-derived progenitor cells (FCPCs) to adult cells can be achieved using a three-dimensional (3D) spheroid culture method based on silica nanopatterning techniques. In evaluating the issue of silica nano-particle size (Diameter of 300, 750, 1200 nm), each particle size was coated into the well of a 6-well tissue culture plate. FCPCs (2 x 105 cells/well in 6-well plate) were seeded in each well with chondrogenic medium. In this study, the 300 nm substrate that formed multi-spheroids and the 1200 nm substrate that showed spreading were due to the cell-cell adhesion force(via N-cadherin) and cell-substrate(via Integrin) force, the 750 nm substrate that formed the mass-aggregation can be interpreted as the result of cell monolayer formation through cell-substrate force followed by cell-cell contact force contraction. We conclude that our 3D spheroid culture system contributes to an optimization for efficient differentiation of FCPC, offers insight into the mechanism of efficient differentiation of engineered 3D culture system, and has promise for wide applications in regeneration medicine and drug discovery fields.

Preparation of a Cross-Linked Cartilage Acellular-Matrix Film and Its In Vivo Evaluation as an Antiadhesive Barrier.

In this paper, a cartilage acellular-matrix (CAM) is chosen as a biomaterial for an effective antiadhesive barrier to apply between injured tissue and healthy tissues or organs. CAM is cross-linked using glutaraldehyde to create a cross-linked CAM (Cx-CAM) film. Cx-CAM has higher elastic modulus and toughness and more hydrophobic surface properties than CAM before cross-linking. Small intestinal submucosa (SIS), cross-linked SIS (Cx-SIS) as a negative control, and Seprafilm as a positive control are used in an experiment as adhesion barriers. Human umbilical vein endothelial cells (HUVECs) on SIS, Cx-SIS, or in a culture plate get attached and effectively proliferate for 7 days, but Cx-CAM and Seprafilm allow for little or no attachment and proliferation of HUVECs, thus manifesting antiadhesive and antiproliferative effects. In animals with surgical damage to the peritoneal wall and cecum, Cx-CAM and Seprafilm afford little adhesion and negligible inflammation after seven days, as confirmed by hematoxylin and eosin staining and macrophage staining, in contrast to an untreated-injury model, SIS, or Cx-SIS film. Cx-CAM significantly suppresses the formation of blood vessels between the peritoneal wall and cecum, as confirmed by CD31 staining. Overall, the newly designed Cx-CAM film works well as an antiadhesion barrier and has better anti-tissue adhesion efficiency.

Constipation in Tg2576 mice model for Alzheimer's disease associated with dysregulation of mechanism involving the mAChR signaling pathway and ER stress response.

BACKGROUND: Although constipation has been researched in various neurological disorders, including Parkinson's disease (PD) and spinal cord injury (SCI), the pathological mechanism of this symptom has not been investigated in Alzheimer's disease (AD) associated with loss of nerve cells in the brain. This study was undertaken to gain scientific evidences for a molecular correlation between constipation and AD. METHODS: To understand the etiology, we measured alterations in various constipation parameters, muscarinic acetylcholine receptors (mAChRs) and endoplasmic reticulum (ER) stress response, in 11-month-old Tg2576 transgenic (Tg) mice showing AD-like phenotypes. RESULTS: A high accumulation of amyloid beta (Aß) peptides, a key marker of AD pathology, were detected in the cortex and hippocampus of Tg mice. Furthermore, significant alterations were observed in various constipation parameters including stool weight, histological structure, cytological structure and mucin secretion in Tg2576 mice. Moreover, M2 and M3 expression and the downstream signaling pathways of mAChRs were decreased in the Tg group, as compared with non-Tg (NT) group. Furthermore, activation of ER stress proteins and alteration of ER structure were also detected in the same group. CONCLUSIONS: The results of the present study provide strong novel evidence that the neuropathological constipation detected in Tg2576 mice is linked to dysregulation of the mAChR signaling pathways and ER stress response.

Anti-obesity effect in high-fat-diet-induced obese C57BL/6 mice: Study of a novel extract from mulberry (Morus alba) leaves fermented with Cordyceps militaris.

The therapeutic effects of mulberry (Morus alba) leaves on lipid metabolism, including lipogenesis, lipolysis and hyperlipidemia are widely known, although their fermented products are yet to be applied. To investigate the therapeutic effects of a novel extract of mulberry leaves fermented with Cordyceps militaris (EMfC) on lipid metabolism, the lipid profile of serum, lipid accumulation, lipolytic activity and lipogenesis regulation were measured in high fat diet (HFD)-induced obese C57BL/6 mice treated with EMfC for 12 weeks. Briefly, the concentrations of low-density lipoprotein, triglyceride, total cholesterol and glucose significantly decreased in the serum of the HFD+EMfC treated group when compared with the HFD+Vehicle treated group, while the levels of high-density lipoprotein increased in the HFD+EMfC group. The amount of abdominal fat and the size of adipocytes were significantly lower in the HFD+EMfC treated group when compared with the HFD+Vehicle treated group. The weight and number of lipid droplets of liver tissue exhibited a similar decrease. Furthermore, the mRNA levels of peroxisome proliferator-activated receptor-γ for adipogenesis as well as adipocyte protein 2 and Fas cell surface death receptor for lipogenesis reduced following EMfC treatment for 12 weeks. Phosphorylation of perilipin and hormone-sensitive lipase, and in the adipose triglyceride lipase expression showed a significant increase in the HFD+EMfC treated group. These results indicated that EMfC may prevent fat accumulation in the HFD-induced obese C57BL/6 mice through the inhibition of lipogenesis and by stimulating lipolysis. Thus, the results provide evidence for the potential use of EMfC as an anti-obesity complex in the treatment of obesity.

Synergic Laxative Effects of an Herbal Mixture of Liriope platyphylla, Glycyrrhiza uralensis, and Cinnamomum cassia in Loperamide-Induced Constipation of Sprague Dawley Rats.

Constipation is an acute or chronic illness attributed to various causes, ranging from lifestyle habits to side effects of a disease. To improve the laxative effects of some traditional medicines, herbal mixtures of Liriope platyphylla, Glycyrrhiza uralensis, and Cinnamomum cassia (LGC) were evaluated for their mechanism of action and therapeutic effects in loperamide (Lop)-induced constipated Sprague Dawley rats by examining alterations in excretion parameters, histological structure, mucin secretion, and related protein levels. Food intake and water consumption were constant for all animals. We observed that the Lop+LGC-treated group had significantly greater excretion of stool and urine than was observed in the Lop+Vehicle-treated group. Administration of LGC in the constipation model restored the intestinal transit ratio to normal levels, and increased the number of goblet cells, mucosal layer, and muscle thickness. Mucin secretion was greater in the Lop+LGC-treated group than in the Lop+Vehicle-treated group, and the expression of MUC2 and AQP8 genes were also increased. In addition, reverse transcription polymerase chain reaction and Western blot revealed an increase in the muscarinic acetylcholine receptors (mAChRs) in the Lop+LGC-treated group compared to the Lop+Vehicle-treated group. Furthermore, compared with the Lop+Vehicle-treated group, treatment with LGC reduced the phosphorylation of PKC and PI3K, and expression of Gα protein, but increased levels of IP3. Our results suggest that the traditional herbal mixture of LGC induces a potent laxative effect in Lop-induced constipation through mucosal tissue changes and mucin production. We also demonstrated that the laxative effect of LGC is closely related to the expression of mAChR and its downstream signals, suggesting the possibility of developing a constipation-laxative agent using LGC.

Therapeutic effects of a liquid bandage prepared with cellulose powders from Styela clava tunics and Broussonetia kazinoki bark: Healing of surgical wounds on the skin of Sprague Dawley rats.

Cellulose in different forms has extensively been applied in biomedical treatments, including scaffolding, tissue engineering and tissue formation. To evaluate the therapeutic effects of a liquid bandage (LB) prepared with cellulose powders from Styela clava tunics (SCT) and Broussonetia kazinoki bark (BSLB) for healing cutaneous wounds, the remedial effects of a low concentration (LoBSLB) and a high concentration (HiBSLB) of BSLB on skin regeneration and toxicity in Sprague Dawley rats. Results indicated that the total area of skin involved in the surgical wound was lower in the BSLB­treated group compared with the Vehicle­treated group at days 4­12, although some variations were observed in the HiBSLB­treated group. In addition, the BSLB­treated group showed significantly enhanced width of the re­epithelialization region and epidermal thickness when compared with the Vehicle­treated group. Furthermore, significant stimulation in the expression level of collagen­1 and the signaling pathway of VEGF after topical application of BSLB was indicated. No liver or kidney toxicities were detected for either doses of BSLB. Overall, the results of the present study suggest that BSLB accelerates the process of wound healing in surgical skin wounds of Sprague Dawley rats through stimulation of re­epithelialization and connective tissue formation, without any accompanying significant toxicity.

Dose dependence and durability of the therapeutic effects of Asparagus cochinchinensis fermented extract in an ovalbumin-challenged asthma model.

The butanol extract of Asparagus cochinchinensis roots fermented with Weissella cibaria (BAfW) significantly suppressed the inflammatory response induced by lipopolysaccharide (LPS) treatment in RAW264.7 cells. To investigate the dose dependence and durability of BAfW on the anti-asthma effects, alterations in key parameters were measured in ovalbumin (OVA)-challenged Balb/c mice treated with the different doses of BAfW at three different time points. The number of immune cells, OVA-specific IgE level, thickness of respiratory epithelium and mucus score decreased significantly in a dose-dependent manner in response to treatment with 125 to 500 mg/kg BAfW (P<0.05), although the highest level was detected in the 500 mg/kg treated group. Moreover, the decrease in these parameters was maintained from 24 to 48 h in the 500 mg/kg of BAfW treated group. At 72 h, the effects of BAfW on the number of immune cells, OVA-specific IgE level and thickness of respiratory epithelium partially disappeared. Overall, this study provides the first evidence that the anti-asthma effect of BAfW may reach the maximum level in OVA-challenged Balb/c mice treated with 500 mg/kg and that these effects can last for 48 h.