Ancistrocladus tectorius Extract Inhibits Obesity by Promoting Thermogenesis and Mitochondrial Dynamics in High-Fat Diet-Fed Mice.

Root extracts of Ancistrocladus tectorius (AT), a shrub native to China, have been shown to have antiviral and antitumor activities, but the anti-obesity effects of AT aerial parts, mainly the leaves and stems, have not been investigated. This study is the first to investigate the anti-obesity effects and molecular mechanism of AT 70% ethanol extract in 3T3-L1 adipocytes and high-fat diet (HFD)-fed C57BL/6J mice. Treatment with AT extract inhibited lipid accumulation in 3T3-L1 cells and decreased the expression of adipogenesis-related genes. AT extract also upregulated the mRNA expression of genes related to mitochondrial dynamics in 3T3-L1 adipocytes. AT administration for 12 weeks reduced body weight and organ weights, including liver, pancreas, and white and brown adipose tissue, and improved plasma profiles such as glucose, insulin, homeostasis model assessment of insulin resistance, triglyceride (TG), and total cholesterol in HFD-fed mice. AT extract reduced HFD-induced hepatic steatosis with levels of liver TG and lipogenesis-related genes. AT extract upregulated thermogenesis-related genes such as Cidea, Pgc1α, Ucp1, Prdm16, Adrb1, and Adrb3 and mitochondrial dynamics-related genes such as Mff, Opa1, and Mfn2 in brown adipose tissue (BAT). Therefore, AT extract effectively reduced obesity by promoting thermogenesis and the mitochondrial dynamics of BAT in HFD-fed mice.

Antarctic Krill Oil from Euphausia superba Ameliorates Carrageenan-Induced Thrombosis in a Mouse Model.

FJH-KO obtained from Antarctic krill, especially Euphausia superba, has been reported to contain high amounts of omega-3 polyunsaturated fatty acids (n-3 PUFA) and to exhibit anticancer and anti-inflammatory properties. However, its antithrombotic effects have not yet been reported. This study aimed to investigate the antithrombotic effects of FJH-KO in carrageenan-induced thrombosis mouse models and human endothelial cells. Thrombosis was induced by carrageenan injection, whereas the mice received FJH-KO pretreatment. FJH-KO attenuated carrageenan-induced thrombus formation in mouse tissue vessels and prolonged tail bleeding. The inhibitory effect of FJH-KO was associated with decreased plasma levels of thromboxane B2, P-selectin, endothelin-1, ß-thromboglobulin, platelet factor 4, serotonin, TNF-α, IL-1ß, and IL-6. Meanwhile, FJH-KO induced plasma levels of prostacyclin I2 and plasminogen. In vitro, FJH-KO decreased the adhesion of THP-1 monocytes to human endothelial cells stimulated by TNF-α via eNOS activation and NO production. Furthermore, FJH-KO inhibited the expression of TNF-α-induced adhesion molecules such as ICAM-1 and VCAM-1 by suppressing the NF-κB signaling pathway. Taken together, our study demonstrates that FJH-KO protects against carrageenan-induced thrombosis by regulating endothelial cell activation and has potential as an antithrombotic agent.

Puerarin attenuates hepatic steatosis via G-protein-coupled estrogen receptor-mediated calcium and SIRT1 signaling pathways.

Puerarin, the major bioactive ingredient isolated from the root of Pueraria lobata (Willd.), attenuates body weight gain and reduces lipid levels in high-fat diet-induced obese mice; however, the underlying mechanism responsible for regulating lipid metabolism remains unclear. This study investigated the molecular mechanism(s) underlying the role of puerarin in regulating lipogenesis and lipolysis in human HepG2 cells. In this study, puerarin strongly inhibited the expression of fatty acid synthase (FASN) and sterol regulatory element binding protein 1c (SREBP-1c). Moreover, puerarin significantly induced the expression of adipose triglyceride lipase (ATGL), which is responsible for triacylglycerol hydrolase activity in cells. Puerarin enhanced 5' AMP-activated protein kinase (AMPK) activity, which is a central regulator of hepatic lipid metabolism. Furthermore, this AMPK activation could be mediated by sirtuin 1 (SIRT1) and calcium signaling pathways involved in G protein-coupled estrogen receptor (GPER) signaling. GPER blockage significantly reversed the effect of puerarin on lipid accumulation and the related signaling pathways. Docking studies showed that puerarin could bind in the GPER in a similar manner as GPER agonist G1. Our results suggest that puerarin can improve hepatic steatosis by activating GPER; it's signaling cascade sequentially induced calcium and SIRT1 signaling pathways. Thus, puerarin may be a potential therapeutic agent for the treatment of non-alcoholic fatty liver disease.

Immune-Enhancing Effect of Submerged Culture of Ceriporia lacerata Mycelia on Cyclophosphamide-Induced Immunosuppressed Mice and the Underlying Mechanisms in Macrophages.

The white-rot fungi Ceriporia lacerata is used in bioremediation, such as lignocellulose degradation, in nature. Submerged cultures and extracts of C. lacerata mycelia (CLM) have been reported to contain various active ingredients, including ß-glucan and extracellular polysaccharides, and to exert anti-diabetogenic properties in mice and cell lines. However, the immunostimulatory effects have not yet been reported. This study aimed to identify the immunomodulatory effects, and underlying mechanisms thereof, of submerged cultures of CLM using RAW264.7 macrophages and cyclophosphamide (CTX)-induced immunosuppression in mice. Compared to CTX-induced immunosuppressed mice, the spleen and thymus indexes in mice orally administered CLM were significantly increased; body weight loss was alleviated; and natural killer (NK) cytotoxicity, lymphocyte proliferation, and cytokine (tumor necrosis factor [TNF]-α, interferon [IFN]-γ, and interleukin [IL]-2) production were elevated in the serum. In RAW264.7 macrophages, treatment with CLM induced phagocytic activity, increased the production of nitric oxide (NO), and promoted mRNA expression of the immunomodulatory cytokines TNF-α, IFN-γ, IL-1ß, IL-6, IL-10, and IL-12. In addition, CLM increased the inducible NO synthase (iNOS) concentration in macrophages, similar to lipopolysaccharide (LPS) stimulation. Mechanistic studies showed that CLM induced the activation of the NF-κB, PI3k/Akt, ERK1/2, and JNK1/2 pathways. Moreover, the phosphorylation of NF-κB and IκB induced by CLM in RAW264.7 cells was suppressed by specific MAPKs and PI3K inhibitors. Further experiments with a TLR4 inhibitor demonstrated that the production of TNF-α, IL-1ß, and IL-6 induced by CLM was decreased after TLR4 was blocked. Overall, CLM protected against CTX-induced adverse reactions by enhancing humoral and cellular immune functions, and has potential as an immunomodulatory agent.

Effect of Porcine Whole Blood Protein Hydrolysate on Slow-Twitch Muscle Fiber Expression and Mitochondrial Biogenesis via the AMPK/SIRT1 Pathway.

Skeletal muscle is a heterogeneous tissue composed of a variety of functionally different fiber types. Slow-twitch type I muscle fibers are rich with mitochondria, and mitochondrial biogenesis promotes a shift towards more slow fibers. Leucine, a branched-chain amino acid (BCAA), regulates slow-twitch muscle fiber expression and mitochondrial function. The BCAA content is increased in porcine whole-blood protein hydrolysates (PWBPH) but the effect of PWBPH on muscle fiber type conversion is unknown. Supplementation with PWBPH (250 and 500 mg/kg for 5 weeks) increased time to exhaustion in the forced swimming test and the mass of the quadriceps femoris muscle but decreased the levels of blood markers of exercise-induced fatigue. PWBPH also promoted fast-twitch to slow-twitch muscle fiber conversion, elevated the levels of mitochondrial biogenesis markers (SIRT1, p-AMPK, PGC-1α, NRF1 and TFAM) and increased succinate dehydrogenase and malate dehydrogenase activities in ICR mice. Similarly, PWBPH induced markers of slow-twitch muscle fibers and mitochondrial biogenesis in C2C12 myotubes. Moreover, AMPK and SIRT1 inhibition blocked the PWBPH-induced muscle fiber type conversion in C2C12 myotubes. These results indicate that PWBPH enhances exercise performance by promoting slow-twitch muscle fiber expression and mitochondrial function via the AMPK/SIRT1 signaling pathway.

Preclinical safety assessment of a therapeutic human papillomavirus DNA vaccine combined with intravaginal interleukin-7 fused with hybrid Fc in female rats.

This study was conducted to establish the toxicological profile of combination treatment with therapeutic HPV DNA vaccines (GX-188E) and the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (IL-7hyFc). GX-188E was administered intramuscularly by electroporation with or without IL-7hyFc intravaginally once per 2 weeks for 8 weeks (five times) in female Sprague-Dawley rats. Because up-regulation of immune responses and migration of antigen-specific T cells in cervicoviginal tissue were predicted as therapeutic effects, we distinguished adverse effects from therapeutic effects based on the severity of the systemic immune response, reversibility of lymphoid tissue changes, target tissue damage, and off-target immune responses. We observed that the number of neutrophils was increased, and the number of lymphocytes was decreased in the blood. Further, myofiber degeneration, necrosis, fibroplasia, and cell infiltration were observed at the GX-188E administration site. These changes were fully or partially recovered over a 4-week period. Analysis of lymphocytes in spleen revealed that CD4+ T cells and total T cells decreased in rats treated with GX-188E in combination with a high dose of IL-7hyFc (1.25 mg/animal). However, these changes were not considered adverse because they were transient and may have been related to electroporation-mediated DNA delivery or the local migration of lymphocytes induced by IL-7. Therefore, the potential toxicity of the combination of GX-188E and IL-7hyFc treatment was comparable to that of GX-188E treatment alone, and the no observed adverse effect level for GX-188E with IL-7hyFc was considered as 320 µg/animal for GX-188E and 1.25 mg/animal for IL-7hyFc.

Influence of o,p'-DDT on MUC5AC expression via regulation of NF-κB/AP-1 activation in human lung epithelial cells.

o,p'-Dichlorodiphenyltrichloroethane (o,p'-DDT) is a representative endocrine disruptor, and exposure to o,p'-DDT may produce immune disorders and inflammation, leading to various diseases such as cancer. Chronic airway inflammation is characterized by excessive mucus secretion resulting in chronic obstructive pulmonary disease (COPD). Mucin 5AC  (MUC5AC), one of the mucus genes, plays an important role in mucus secretion and inflammation in the airways. The aim of this study was to examine the effects of o,p'-DDT on the regulation of MUC5AC expression in human lung epithelial A549 cell line. o,p'-DDT increased mRNA levels and the promoter activity of MUC5AC. Transient transfection with mutation promoter constructs of MUC5AC demonstrated that nuclear factor kappa-b (NF-κB) and activator protein 1(AP-1) response elements were essential for the consequences of o,p'-DDT on MUC5AC expression. In addition, o,p'-DDT induced phosphorylation of ERK, JNK, p38, and Akt, which are involved in the regulation of MUC5AC expression. It is noteworthy that inhibitors of NF-κB, AP-1, Akt, and MAPKs blocked enhanced o,p'-DDT-induced MUC5AC mRNA expression. Data indicate that o,p'-DDT increase in NF-κB, and AP-1 transcriptional activation-dependent MUC5AC expression is associated with stimulation of Akt and MAPK signaling pathways in A549 cells.

Effects of tobacco compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the expression of epigenetically regulated genes in lung carcinogenesis.

Cigarette smoking is a major cause of lung cancer. Although tobacco smoking-induced genotoxicity has been well established, there is apparent lack of abundance functional epigenetic effects reported On cigarette smoke-induced lung carcinogenesis. The aim of this study was to determine effects of intratracheal administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) utilizing target gene expression DNA methylation patterns in lung tissues of mice following twice weekly for 8 weeks treatment. An unbiased approach where genomic regions was undertaken to assess early methylation changes within mouse pulmonary tissues. A methylated-CpG island recovery assay (MIRA) was performed to map the DNA methylome in lung tissues, with the position of methylated DNA determined using a Genome Analyzer (MIRA-SEQ). Alterations in epigenetic-regulated target genes were confirmed with quantitative reverse transcription-PCR, which revealed 35 differentially hypermethylated genes including Cdkn1C, Hsf4, Hnf1a, Cdx1, and Hoxa5 and 30 differentially hypomethylated genes including Ddx4, Piwi1, Mdm2, and Pce1 in NNK-exposed lung tissue compared with controls. The main pathway of these genes for mediating biological information was analyzed using the Kyoto Encyclopedia of Genes and Genomes database. Among them, Rssf1 and Mdm2 were closely associated with NNK-induced lung carcinogenesis. Taken together, our data provide valuable resources for detecting cigarette smoke-induced lung carcinogenesis.

Metallothionein 3 Promotes Osteoblast Differentiation in C2C12 Cells via Reduction of Oxidative Stress.

Metallothioneins (MTs) are intracellular cysteine-rich proteins, and their expressions are enhanced under stress conditions. MTs are recognized as having the ability to regulate redox balance in living organisms; however, their role in regulating osteoblast differentiation is still unclear. In this research, we found that the expression of MT3, one member of the MT protein family, was specifically upregulated in the differentiation process of C2C12 myoblasts treated with bone morphogenetic protein 4 (BMP4). Transfection with MT3-overexpressing plasmids in C2C12 cells enhanced their differentiation to osteoblasts, together with upregulating the protein expression of bone specific transcription factors runt-related gene 2 (Runx2), Osterix, and distal-less homeobox 5 (Dlx5). Additionally, MT3 knockdown performed the opposite. Further studies revealed that overexpression of MT3 decreased reactive oxygen species (ROS) production in C2C12 cells treated with BMP4, and MT3 silencing enhanced ROS production. Treating C2C12 cells with antioxidant N-acetylcysteine also promoted osteoblast differentiation, and upregulated Runx2/Osterix/Dlx5, while ROS generator antimycin A treatment performed the opposite. Finally, antimycin A treatment inhibited osteoblast differentiation and Runx2/Osterix/Dlx5 expression in MT3-overexpressing C2C12 cells. These findings identify the role of MT3 in osteoblast differentiation and indicate that MT3 may have interesting potential in the field of osteogenesis research.

Impressic Acid Attenuates the Lipopolysaccharide-Induced Inflammatory Response by Activating the AMPK/GSK3ß/Nrf2 Axis in RAW264.7 Macrophages.

Inflammatory diseases are caused by excessive inflammation from pro-inflammatory mediators and cytokines produced by macrophages. The Nrf2 signaling pathway protects against inflammatory diseases by inhibiting excessive inflammation via the regulation of antioxidant enzymes, including HO-1 and NQO1. We investigated the anti-inflammatory effect of impressic acid (IPA) isolated from Acanthopanax koreanum on the lipopolysaccharide (LPS)-induced inflammation and the underlying molecular mechanisms in RAW264.7 cells. IPA attenuated the LPS-induced production of pro-inflammatory cytokines and reactive oxygen species, and the activation of the NF-κB signaling pathway. IPA also increased the protein levels of Nrf2, HO-1, and NQO1 by phosphorylating CaMKKß, AMPK, and GSK3ß. Furthermore, ML385, an Nrf2 inhibitor, reversed the inhibitory effect of IPA on LPS-induced production of pro-inflammatory cytokines in RAW264.7 cells. Therefore, IPA exerts an anti-inflammatory effect via the AMPK/GSK3ß/Nrf2 signaling pathway in macrophages. Taken together, the findings suggest that IPA has preventive potential for inflammation-related diseases.

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKß/AMPK Signaling Pathway in Human Endothelial Cells.

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca2+ concentration and phosphorylation of Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß), AMP-activated protein kinase (AMPK), and Ca2+/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca2+ concentration and phosphorylation of CaMKII, CaMKKß, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKß inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca2+/CaMKII and CaM/CaMKKß/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.

Impressic Acid Ameliorates Atopic Dermatitis-Like Skin Lesions by Inhibiting ERK1/2-Mediated Phosphorylation of NF-κB and STAT1.

Impressic acid (IPA), a lupane-type triterpenoid from Acanthopanax koreanum, has many pharmacological activities, including the attenuation of vascular endothelium dysfunction, cartilage destruction, and inflammatory diseases, but its influence on atopic dermatitis (AD)-like skin lesions is unknown. Therefore, we investigated the suppressive effect of IPA on 2,4-dinitrochlorobenzene (DNCB)-induced AD-like skin symptoms in mice and the underlying mechanisms in cells. IPA attenuated the DNCB-induced increase in the serum concentrations of IgE and thymic stromal lymphopoietin (TSLP), and in the mRNA levels of thymus and activation regulated chemokine(TARC), macrophage derived chemokine (MDC), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) in mice. Histopathological analysis showed that IPA reduced the epidermal/dermal thickness and inflammatory and mast cell infiltration of ear tissue. In addition, IPA attenuated the phosphorylation of NF-κB and IκBα, and the degradation of IκBα in ear lesions. Furthermore, IPA treatment suppressed TNF-α/IFN-γ-induced TARC expression by inhibiting the NF-κB activation in cells. Phosphorylation of extracellular signalregulated protein kinase (ERK1/2) and the signal transducer and activator of transcription 1 (STAT1), the upstream signaling proteins, was reduced by IPA treatment in HaCaT cells. In conclusion, IPA ameliorated AD-like skin symptoms by regulating cytokine and chemokine production and so has therapeutic potential for AD-like skin lesions.

Viburnum stellato-tomentosum Extract Suppresses Obesity and Hyperglycemia through Regulation of Lipid Metabolism in High-Fat Diet-Fed Mice.

The potential biological activities of Viburnum stellato-tomentosum (VS), a plant mainly found in Costa Rica, have yet to be reported. Supplementation of VS extract for 17 weeks significantly decreased body weight gain, fat weight, fasting glucose, insulin, homeostasis model assessment of insulin resistance (HOMA-IR), and triglyceride levels in high-fat diet (HFD)-fed C57BL/6J mice. The molecular mechanisms underlying the anti-obesity and glucose-lowering effects of VS extract were investigated. VS extract suppressed adipocyte hypertrophy by regulating lipogenesis-related CCAAT/enhancer-binding protein α (C/EBPα) and insulin sensitivity-related peroxisome proliferator-activated receptor γ (Pparg) expression in adipose tissue (AT) and hepatic steatosis by inhibiting C/EBPα and lipid transport-related fatty acid binding protein 4 (FABP4) expression. VS extract enhanced muscular fatty acid ß-oxidation-related AMP-activated protein kinase (AMPK) and PPARα expression with increasing Pparg levels. Furthermore, VS extract contained a much higher content of amentoflavone (AMF) (29.4 mg/g extract) compared to that in other Viburnum species. AMF administration decreased Cebpa and Fabp4 levels in the AT and liver, as well as improved insulin signaling-related insulin receptor substrate 1 (Irs1) and glucose transporter 1 (Glut1) levels in the muscle of HFD-fed mice. This study elucidated the in vivo molecular mechanisms of AMF for the first time. Therefore, VS extract effectively diminished obesity and hyperglycemia by suppressing C/EBPα-mediated lipogenesis in the AT and liver, enhancing PPARα-mediated fatty acid ß-oxidation in muscle, and PPARγ-mediated insulin sensitivity in AT and muscle.

Mitochondrial carnitine palmitoyltransferase 2 is involved in Nε-(carboxymethyl)-lysine-mediated diabetic nephropathy.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease in the world. Advanced glycation end products (AGEs) are thought to be involved in the pathogenesis of DN via multifactorial mechanisms including the generation of oxidative stress and overproduction of various growth factors and cytokines. AGEs are heterogeneous cross-linked sugar-derived proteins, and Nε-(carboxymethyl)-lysine (CML)-conjugated BSA is a major component of AGEs. However, the proteins involved in DN induction by CML have never been reported. Herein, we investigated specific protein regulators of AGE-mediated DN via proteomic analysis of streptozotocin (STZ)-induced diabetic mice kidneys. We identified 937, 976, and 870 proteins in control, STZ, and STZ + CML-BSA samples, respectively. Bioinformatics analysis identified several CML-mediated proteins potentially involved in kidney damage, activation of fatty acid oxidation (FAO), and mitochondrial dysfunction. Furthermore, we identified the CML-specific differential protein carnitine palmitoyltransferase 2 (CPT2), related to FAO. To confirm the effect of CPT2 and the CML-mediated mechanism, human renal tubular HK-2 cells were treated with CML-BSA and cpt2 siRNA, and examined for FAO-mediated fibrosis and mitochondrial dysfunction. CML-BSA and CPT2 knockdown induced fibrosis-related gene expression and damage to mitochondrial membrane potential. Moreover, CPT2 overexpression recovered CML-induced fibrosis-related gene expression. Based on these results, a decrease in CML-induced CPT2 expression causes mitochondrial FAO damage, leading to renal fibrosis and DN.

Integration of transcriptomics, proteomics and metabolomics identifies biomarkers for pulmonary injury by polyhexamethylene guanidine phosphate (PHMG-p), a humidifier disinfectant, in rats.

Polyhexamethylene guanidine phosphate (PHMG-p) was used as a humidifier disinfectant in Korea. PHMG induced severe pulmonary fibrosis in Koreans. The objective of this study was to elucidate mechanism of pulmonary toxicity caused by PHMG-p in rats using multi-omics analysis. Wistar rats were intratracheally instilled with PHMG-p by single (1.5 mg/kg) administration or 4-week (0.1 mg/kg, 2 times/week) repeated administration. Histopathologic examination was performed with hematoxylin and eosin staining. Alveolar macrophage aggregation and granulomatous inflammation were observed in rats treated with single dose of PHMG-p. Pulmonary fibrosis, chronic inflammation, bronchiol-alveolar fibrosis, and metaplasia of squamous cell were observed in repeated dose group. Next generation sequencing (NGS) was performed for transcriptome profiling after mRNA isolation from bronchiol-alveoli. Bronchiol-alveoli proteomic profiling was performed using an Orbitrap Q-exactive mass spectrometer. Serum and urinary metabolites were determined using 1H-NMR. Among 418 differentially expressed genes (DEGs) and 67 differentially expressed proteins (DEPs), changes of 16 mRNA levels were significantly correlated with changes of their protein levels in both single and repeated dose groups. Remarkable biological processes represented by both DEGs and DEPs were defense response, inflammatory response, response to stress, and immune response. Arginase 1 (Arg1) and lipocalin 2 (Lcn2) were identified to be major regulators for PHMG-p-induced pulmonary toxicity based on merged analysis using DEGs and DEPs. In metabolomics study, 52 metabolites (VIP > 0.5) were determined in serum and urine of single and repeated-dose groups. Glutamate and choline were selected as major metabolites. They were found to be major factors affecting inflammatory response in association with DEGs and DEPs. Arg1 and Lcn2 were suggested to be major gene and protein related to pulmonary damage by PHMG-p while serum or urinary glutamate and choline were endogenous metabolites related to pulmonary damage by PHMG-p.

Identification of Potent CD19 scFv for CAR T Cells through scFv Screening with NK/T-Cell Line.

CD19 is the most promising target for developing chimeric-antigen receptor (CAR) T cells against B-cell leukemic cancer. Currently, two CAR-T-cell products, Kymriah and Yescarta, are approved for leukemia patients, and various anti-CD19 CAR T cells are undergoing clinical trial. Most of these anti-CD19 CAR T cells use FMC63 single-chain variable fragments (scFvs) for binding CD19 expressed on the cancer cell surface. In this study, we screened several known CD19 scFvs for developing anti-CD19 CAR T cells. We used the KHYG-1 NK/T-cell line for screening of CD19 scFvs because it has advantages in terms of cell culture and gene transduction compared to primary T cells. Using our CAR construct backbone, we made anti-CD19 CAR constructs which each had CD19 scFvs including FMC63, B43, 25C1, BLY3, 4G7, HD37, HB12a, and HB12b, then made each anti-CD19 CAR KHYG-1 cells. Interestingly, only FMC63 CAR KHYG-1 and 4G7 CAR KHYG-1 efficiently lysed CD19-positive cell lines. In addition, in Jurkat cell line, only these two CAR Jurkat cell lines secreted IL-2 when co-cultured with CD19-positive cell line, NALM-6. Based on these results, we made FMC63 CAR T cells and 4G7 CAR T cells from PBMC. In in vitro lysis assay, 4G7 CAR T cells lysed CD19-positive cell line as well as FMC63 CAR T cells. In in vivo assay with NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, 4G7 CAR T cells eradicated NALM-6 as potently as FMC63 CAR T cells. Therefore, we anticipate that 4G7 CAR T cells will show as good a result as FMC63 CAR T cells for B-cell leukemia patients.

Stimulatory effects of platycodin D on osteoblast differentiation.

Previous studies have suggested that platycodin D is implicated in bone biology and ameliorates osteoporosis development. Platycodin D repressed the osteoclast activity and enhanced bone mineral density in the mouse model. However, the effects of platycodin D on osteoblast differentiation have not been elucidated yet. In C3H10T1/2 cells, platycodin D upregulated osteogenic markers including alkaline phosphatase (ALP), bone sialoprotein, and collagen type 1 alpha 1, and transcription factors, such as Runx2 and osterix, subsequently enhancing the bone mineralization. In a molecular mechanism study, platycodin D induced ß-catenin nuclear accumulation by upregulating GSK3ß phosphorylation. Furthermore, platycodin D upregulated the ALP activity and enhanced the mineralization process in osteoblast cells via the sirtuin 1/ß-catenin pathways. Taken together, these results suggested that platycodin D could be an effective therapeutic compound against osteoporosis because of its regulatory effects during the osteoblast differentiation.

Impressic Acid, a Lupane-Type Triterpenoid from Acanthopanax koreanum, Attenuates TNF-α-Induced Endothelial Dysfunction via Activation of eNOS/NO Pathway.

Atherosclerosis is one of the most reported diseases worldwide, and extensive research and trials are focused on the discovery and utilizing for novel therapeutics. Nitric oxide (NO) is produced mainly by endothelial nitric oxide synthase (eNOS) and it plays a key role in regulating vascular function including systemic blood pressure and vascular inflammation in vascular endothelium. In this study hypothesized that Impressic acid (IPA), a component isolated from Acanthopanax koreanum, acts as an enhancer of eNOS activity and NO production. IPA treatment induced eNOS phosphorylation and NO production, which was correlated with eNOS phosphorylation via the activation of JNK1/2, p38 MAPK, AMPK, and CaMKII. In addition, the induction of eNOS phosphorylation by IPA was attenuated by pharmacological inhibitor of MAPKs, AMPK, and CaMKII. Finally, IPA treatment prevented the adhesion of TNF-α-induced monocytes to endothelial cells and suppressed the TNF-α-stimulated ICAM-1 expression via activation of NF-κB, while treatment with L-NAME, the NOS inhibitor, reversed the inhibitory effect of IPA on TNF-α-induced ICAM-1 expression via activation of NF-κB. Taken together, these findings show that IPA protects against TNF-α-induced vascular endothelium dysfunction through attenuation of the NF-κB pathway by activating eNOS/NO pathway in endothelial cells.

WY-14643 Regulates CYP1B1 Expression through Peroxisome Proliferator-Activated Receptor α-Mediated Signaling in Human Breast Cancer Cells.

Human cytochrome P450 1B1 (CYP1B1)-mediated biotransformation of endobiotics and xenobiotics plays an important role in the progression of human breast cancer. In this study, we investigated the effects of WY-14643, a peroxisome proliferator-activated receptor α (PPARα) agonist, on CYP1B1 expression and the related mechanism in MCF7 breast cancer cells. We performed quantitative reverse transcription-polymerase chain reaction, transient transfection, and chromatin immunoprecipitation to evaluate the effects of PPARα on peroxisome proliferator response element (PPRE)-mediated transcription. WY-14643 increased the protein and mRNA levels of CYP1B1, as well as promoter activity, in MCF-7 cells. Moreover, WY-14643 plus GW6471, a PPARα antagonist, significantly inhibited the WY-14643-mediated increase in CYP1B1 expression. PPARα knockdown by a small interfering RNA markedly suppressed the induction of CYP1B1 expression by WY-14643, suggesting that WY-14643 induces CYP1B1 expression via a PPARα-dependent mechanism. Bioinformatics analysis identified putative PPREs (-833/-813) within the promoter region of the CYP1B1 gene. Inactivation of these putative PPREs by deletion mutagenesis suppressed the WY-14643-mediated induction of CYP1B1 promoter activation. Furthermore, WY-14643 induced PPARα to assume a form capable of binding specifically to the PPRE-binding site in the CYP1B1 promoter. Our findings suggest that WY-14643 induces the expression of CYP1B1 through activation of PPARα.

Curcumin Ameliorates Benzo[a]pyrene-Induced DNA Damages in Stomach Tissues of Sprague-Dawley Rats.

Benzo[a]pyrene (BaP) is a well-known carcinogen formed during the cooking process. Although BaP exposure has been implicated as one of the risk factors for lung cancer in animals and humans, there are only limited data on BaP-induced gastrointestinal cancer. Therefore, this study investigated the protective effects of curcumin on BaP-induced DNA damage in rat stomach tissues. BaP (20 mg/kg/day) and curcumin (50, 100, or 200 mg/kg) were administered daily to Sprague-Dawley rats by oral gavage over 30 days. Curcumin was pre-administered before BaP exposure. All rats were euthanized, and liver, kidney, and stomach tissues were removed at 24 h after the last treatment. We observed that aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose levels were significantly reduced in rats treated with high dose co-administration of curcumin (200 mg/kg) compared to BaP alone. The expression levels of cytochrome P450 (CYP) 1A1 and CYP1B1 were significantly increased in the liver of rats treated with BaP. However, co-administration of curcumin (200 mg/kg) with BaP markedly reduced CYP1A1 expression in a dose-dependent manner. Furthermore, plasma levels of BaP-diolepoxide (BPDE) and BaP metabolites were significantly reduced by co-administration of curcumin (200 mg/kg). Additionally, co-administration of curcumin (200 mg/kg) with BaP significantly reduced the formation of BPDE-I-DNA and 8-hydroxydeoxy guanosine (8-OHdG) adducts in the liver, kidney, and stomach tissues. The inhibition of these adduct formations were more prominent in the stomach tissues than in the liver. Overall, our observations suggest that curcumin might inhibit BaP-induced gastrointestinal tumorigenesis and shows promise as a chemopreventive agent.