Chiral recognition and discrimination studies of tyrosine enantiomers on (-)-18-crown-6-tetracarboxylic acid as a chiral selector by nuclear magnetic resonance spectroscopy and docking simulations.

Considering the substantial significance of chiral biomolecules, such as amino acids, in our daily routines, we performed chiral recognition and discrimination of tyrosine (Tyr) enantiomers on (-)-(18-crown-6)-2,3,11,12-tetracarboxylic acid [(-)-18-C-6-TA] as crown-ether type chiral selector (CS) by nuclear magnetic resonance (NMR) spectroscopy and docking simulations. In this study, successful discrimination of the enantiomers of Tyr was achieved, as evidenced by the proton chemical shift differences (ΔΔδ) of Tyr enantiomers observed in the 1 H NMR spectra with (-)-18-C-6-TA CS. We compared the results of these two techniques with the findings obtained from high performance liquid chromatography (HPLC) investigations. In both NMR and HPLC experimental and docking simulation studies, a stronger interaction between the L-Tyr enantiomer with (-)-18-C-6-TA CS than the D-Tyr was consistently observed. Also, the binding energy differences (ΔΔEL-D ) found in simulation data that correspond to enantioselectivity aligned well with the NMR experimental result.

Momordicae Semen inhibits migration and induces apoptotic cell death by regulating c-Myc and CNOT2 in human pancreatic cancer cells.

Pancreatic cancer(PC) is less common than other cancers; however, it has a poor prognosis. Therefore, studying novel target signaling and anticancer agents is necessary. Momordicae Semen (MS), the seed of Momordica sochinensis Spreng, mainly found in South-East Asia, including China and Bangladesh, is used to treat various diseases because of its anticancer, antioxidant, anti-inflammatory, and antibacterial properties. However, the effect of the MS extract on pancreatic cancer cells remains unknown. In this study investigated whether the MS extract exerted an anti-cancer effect by regulating c-Myc through CNOT2. Cytotoxicity and proliferation were investigated using MTT and colony formation assays. The levels of apoptotic, oncogenic, and migration-associated factors were confirmed using immunoblotting and immunofluorescence. Wound closure was analyzed using a wound healing assay. The chemical composition of the MS methanol extracts was analyzed using liquid chromatography-mass spectrometry. We confirmed that the MS extract regulated apoptotic factors and attenuated the stability of c-Myc and its sensitivity to fetal bovine serum. Furthermore, the MS extract increased apoptosis by regulating c-Myc and CNOT2 expression and enhanced the sensitivity of 5-FU in pancreatic cancer. This study showed that the MS extract is a promising new drug for PC.

Euonymus sachalinensis Induces Apoptosis by Inhibiting the Expression of c-Myc in Colon Cancer Cells.

We hypothesized that Euonymus sachalinensis (ES) induces apoptosis by inhibiting the expression of c-Myc in colon cancer cells, and this study proved that the methanol extract of ES has anticancer effects in colon cancer cells. ES belongs to the Celastraceae family and is well known for its medicinal properties. Extracts of species belonging to this family have been used to treat diverse diseases, including rheumatoid arthritis, chronic nephritis, allergic conjunctivitis, rhinitis, and asthma. However, ES has been targeted because there are currently few studies on the efficacy of ES for various diseases, including cancer. ES lowers cell viability in colon cancer cells and reduces the expression of c-Myc protein. We confirm that the protein level of apoptotic factors such as PARP and Caspase 3 decrease when ES is treated with Western blot, and confirm that DNA fragments occur through TUNEL assay. In addition, it is confirmed that the protein level of oncogenes CNOT2 and MID1IP1 decrease when ES is treated. We have also found that ES enhances the chemo-sensitivity of 5-FU in 5-FU-resistant cells. Therefore, we confirm that ES has anticancer effects by inducing apoptotic cell death and regulating the oncogenes CNOT2 and MID1IP1, suggesting its potential for use in the treatment of colon cancer.

Development of analytical methods for the determination of 3-hydroxy fatty acids and muramic acid as bacterial markers in airborne particles and settled dust.

Inhalation of airborne bacteria in indoor environments is known to be associated with respiratory diseases. Analytical methods for the determination of 3-hydroxy fatty acids (3-OHFAs) and muramic acid (MA) as chemical markers of gram-negative and gram-positive bacteria, respectively, were developed for airborne particle and dust samples in this study. 3-OHFAs as markers of endotoxin were released and esterified during the hydrolysis process under methanolic acid conditions, and their hydrolysates, i.e., 3-OHFA methyl esters, were cleaned up by solid-phase extraction using silica sorbent that provided more effective separation from interferents than polymeric sorbent through elution pattern. The SPE eluent was analyzed by GC-MS/MS measurement after the trimethylsilylation reaction. The recovery of the method ranged from 82.1 % to 103.2 %, with a limit of detection ranging from 0.5 to 1.1 ng/filter and good linearity (R2 > 0.991). For the analysis of MA, muramic acid methyl ester (MAME), a product formed during methanolic hydrolysis, was selected as a specific marker of peptidoglycan. It was the first proposed compound identified and confirmed with MS and MS/MS spectra using high-resolution measurement. In particular, the measurement of MAME providing 12.5 times greater sensitivity than MA with the application of the LC-MS/MS method is one of the notable findings of this study. The recovery by simple liquid extraction was 99.4 % following the removal of the hydrophobic matrix and neutralization with solvent reconstruction. The method displayed a LOD of 0.7 ng/filter and linearity (R2) of 0.997 through a simple pretreatment process. Both developed methods were applied and evaluated by determining 3-OHFAs and MA in airborne particles collected from multipurpose facilities and settled dust in the laboratory and office.

Timosaponin A3 Inhibits Palmitate and Stearate through Suppression of SREBP-1 in Pancreatic Cancer.

Timosaponin A3 (TA3) was demonstrated as a potent anticancer chemical by several studies. Although the effects of inhibiting growth, metastasis, and angiogenesis in various cancer cells were demonstrated through multiple mechanisms, the pharmacological mechanism of TA3 shown in pancreatic cancer (PC) is insufficient compared to other cancers. In this study, we aimed to explore the key molecular mechanisms underlying the growth inhibitory effects of TA3 using PC cells and a xenograft model. First, from the microarray results, we found that TA3 regulated INSIG-1 and HMGCR in BxPC-3 cells. Furthermore, we showed that inhibition of sterol regulatory element-binding protein-1 (SREBP-1) by TA3 reduced the fatty acid synthases FASN and ACC, thereby controlling the growth of BxPC-3 cells. We also tried to find mechanisms involved with SREBP-1, such as Akt, Gsk3ß, mTOR, and AMPK, but these were not related to SREBP-1 inhibition by TA3. In the BxPC-3 xenograft model, the TA3 group had more reduced tumor formation and lower toxicity than the gemcitabine group. Interestingly, the level of the fatty acid metabolites palmitate and stearate were significantly reduced in the tumor tissue in the TA3 group. Overall, our study demonstrated that SREBP-1 was a key transcription factor involved in pancreatic cancer growth and it remained a precursor form due to TA3, reducing the adipogenesis and growth in BxPC-3 cells. Our results improve our understanding of novel mechanisms of TA3 for the regulation of lipogenesis and provide a new approach to the prevention and treatment of PC.

A modified quick, easy, cheap, effective, rugged, and safe method for determination of by-products originating from ozonation of chlorpyrifos and diazinon spiked in cherry tomato and perilla leaf.

In this study, sample pretreatment methods have been developed for the determination of chlorpyrifos, diazinon, and their by-products present in cherry tomato and perilla leaf using liquid chromatography-tandem mass spectrometry. To optimize a quick, easy, cheap, effective, rugged, and safe method, the recoveries at each step were evaluated. The steps improved the recoveries of chlorpyrifos, chlorpyrifos oxon, diazinon, diazoxon, and 2-isopropyl-6-methyl-4-pyrimidinol up to 80% or more by removing interferents, but diethyl phosphate was almost lost during the partition procedure, and the 3,5,6-trichloro-2-pyridinol recovery was below 65%. Therefore, the compounds were evaluated using different solvent compositions based on a quick polar pesticides method; note that 100% methanol showed acceptable extraction results. The optimized method provided method detection limits ranging from 0.03 to 1.22 ng/g and good linearities (R2  > 0.996). The recovery values were between 82.1 and 113.3%. The intra- and interday reproducibility was evaluated to be within 8.6 and 9.9%, respectively. The method was applied to determine the degradation efficiency of chlorpyrifos and diazinon and their by-products formed during plasma treatment.

Silica-coated magnetic nanoparticles activate microglia and induce neurotoxic D-serine secretion.

BACKGROUND: Nanoparticles have been studied for brain imaging, diagnosis, and drug delivery owing to their versatile properties due to their small sizes. However, there are growing concerns that nanoparticles may exert toxic effects in the brain. In this study, we assessed direct nanotoxicity on microglia, the resident macrophages of the central nervous system, and indirect toxicity on neuronal cells exerted by silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)]. METHODS: We investigated MNPs@SiO2(RITC)-induced biological changes in BV2 murine microglial cells via RNA-sequencing-based transcriptome analysis and gas chromatography-mass spectrometry-based intracellular and extracellular amino acid profiling. Morphological changes were analyzed by transmission electron microscopy. Indirect effects of MNPs@SiO2(RITC) on neuronal cells were assessed by Transwell-based coculture with MNPs@SiO2(RITC)-treated microglia. MNPs@SiO2(RITC)-induced biological changes in the mouse brain in vivo were examined by immunohistochemical analysis. RESULTS: BV2 murine microglial cells were morphologically activated and the expression of Iba1, an activation marker protein, was increased after MNPs@SiO2(RITC) treatment. Transmission electron microscopy analysis revealed lysosomal accumulation of MNPs@SiO2(RITC) and the formation of vesicle-like structures in MNPs@SiO2(RITC)-treated BV2 cells. The expression of several genes related to metabolism and inflammation were altered in 100 µg/ml MNPs@SiO2(RITC)-treated microglia when compared with that in non-treated (control) and 10 µg/ml MNPs@SiO2(RITC)-treated microglia. Combined transcriptome and amino acid profiling analyses revealed that the transport of serine family amino acids, including glycine, cysteine, and serine, was enhanced. However, only serine was increased in the growth medium of activated microglia; especially, excitotoxic D-serine secretion from primary rat microglia was the most strongly enhanced. Activated primary microglia reduced intracellular ATP levels and proteasome activity in cocultured neuronal cells, especially in primary cortical neurons, via D-serine secretion. Moreover, ubiquitinated proteins accumulated and inclusion bodies were increased in primary dopaminergic and cortical neurons cocultured with activated primary microglia. In vivo, MNPs@SiO2(RITC), D-serine, and ubiquitin aggresomes were distributed in the MNPs@SiO2(RITC)-treated mouse brain. CONCLUSIONS: MNPs@SiO2(RITC)-induced activation of microglia triggers excitotoxicity in neurons via D-serine secretion, highlighting the importance of neurotoxicity mechanisms incurred by nanoparticle-induced microglial activation.

Simultaneous enantioselective separation method for thyroid hormones using liquid chromatography-tandem mass spectrometry and its applications.

An analytical method for the simultaneous determination of chiral thyroxine and the related iodinated chiral compounds using LC-MS/MS is introduced in this study. D-Thyroid hormones (THs), which are not commercially available, were produced through the racemization reaction of the L-THs in acetic acid solution containing salicylaldehyde. The solution containing D- and L-THs after the reaction was used for optimizing the chiral separation. The D- and L-THs were well separated enantiomerically under isocratic conditions in 70 % acetonitrile containing 0.1 % formic acid on a CROWNPAK® CR-I (+) column, but some peaks, such as those of diiodo-D-tyrosine (D-DIT)/monoiodo-L-tyrosine, diiodo-D-thyronine/diiodo-L-tyrosine and D-thyroxine/triiodo-L-thyronine, overlapped chromatographically, causing misinterpretation in impurity analysis. This was overcome by using the gradient condition providing the best chiral selectivity (α) and resolution (Rs) ranging from 1.14 to 1.37 and from 2.39 to 4.52, respectively. The linearity was above 0.999 and the detection limits ranged from 8.2 to 57.7 ng/mL by the separation method. This method was applied to identify and quantify chiral impurities in authentic standards and pharmaceuticals. As a result, D-enantiomers corresponding to the L-THs standards as well as L-DIT were commonly observed as impurities. In the stability test of DL-thyroxine under acidic conditions for identifying the distribution of chiral products, it was observed that the formation of DIT by hydrolysis increased over time. Additional products formed through esterification, including thyroxine methyl ester and diiodo-tyrosine methyl ester, were newly separated and identified using a C18 column.

Identification and determination of by-products originating from ozonation of chlorpyrifos and diazinon in water by liquid chromatography-mass spectrometry.

The degradation of two organophosphates, chlorpyrifos and diazinon, in water using microplasma equipment to produce ozone and the identification of their products were studied by using liquid chromatography-mass spectrometry. The organophosphates gradually decreased with time and were completely removed after 10 min, and diazinon was degraded at a relatively fast rate compared to chlorpyrifos. The products formed during the process were identified and determined with accurate mass measurements and tandem mass spectrometry spectra, providing reliable structural determination. Chlorpyrifos oxon was formed through the oxidation of chlorpyrifos, followed by the formation of 3,5,6-trichloro-2-pyridinol and diethyl phosphate by hydrolysis. Diazinon formed various products through more complicated degradation processes than those of chlorpyrifos. The major products of diazinon degradation were 2-isopropyl-6-methyl-4-pyrimidinol and diethyl phosphate by hydrolysis after oxidation, exhibiting diazoxon as an intermediate at trace levels. Direct hydrolysis of diazinon also occurred, producing diethyl thiophosphate, which was observed at a low concentration for a transient time and exhibited a less favorable process than sequential oxidation and hydrolysis. The other products, hydroxy diazinons and hydroxy-2-isopropyl-6-methyl-4-pyrimidinols, formed by hydroxylation, were also identified, but they were present in low amounts. Degradation mechanisms of chlorpyrifos and diazinon were proposed with the quantitatively evaluated products.

Apoptosis and G2/M cell cycle arrest induced by a timosaponin A3 from Anemarrhena asphodeloides Bunge on AsPC-1 pancreatic cancer cells.

BACKGROUND: Timosaponin A3 (TA3), one of the active components of spirostanol saponin isolated from A. asphodeloides, is widely used as an anticancer agent in a variety of cancer cell lines. However, the research on the anticancer efficacy is very limited in human pancreatic cancer models. PURPOSE: In this study, we investigated the molecular targets in the active components of A. asphodeloides, which showed anti-cancer effects in human pancreatic cancer cells, and confirmed the pathways involved. STUDY DESIGN: The apoptotic effects of five solvent extracts of A. asphodeloides in human pancreatic cancer cells (AsPC-1) was studied, and the phytochemical leading to their effects identified. Next, we determined whether the phytochemical inhibit STAT3 and ERK1/2, and investigated the pathways involved. METHODS: Five solvent extracts of A. asphodeloides (100  µg/ml, 24  h) was investigated for their cytotoxicity against AsPC-1 cells. The active ingredient of the extract exhibiting the highest toxicity were analyzed by liquid chromatography-mass spectrometry. Next, we studied the mechanism of action of the phytochemical in pancreatic cancer. Cell cycle and annexin V/FITC assays were performed to assess cell growth and apoptosis capacity. The effects on apoptosis and proliferation-related pathways, STAT3, and MAPKs were confirmed at the protein level using immunoblotting. The factors regulated in the pathways were investigated using reverse transcription polymerase chain reaction. RESULTS: The results showed that the ethyl acetate extract of A. asphodeloides (EAA) induced apoptotic and anti-proliferative activities through the STAT3 and MAPKs pathways. We found that TA3, an active component of EAA, inhibits constitutive STAT3 and ERK1/2 proteins. EAA and TA3 decreased the viability of AsPC-1 cells, leading to cell cycle arrest at the sub-G1 and G2/M phases. Moreover, TA3 inhibited the expression of various genes encoding anti-apoptotic (Bcl-2, Bcl-xl), proliferative (Cyclin D1), metastatic (MMP-9), and angiogenic (VEGF-1) proteins. CONCLUSION: The results indicated that TA3, an active phytochemical from A. asphodeloides, could induce apoptosis and suppress cell proliferation by inhibiting the STAT3 and ERK1/2 pathways. Thus, TA3 is a candidate cancer chemotherapeutic agent instead to treat human pancreatic cancer.

Chiral separation of intact amino acids by capillary electrophoresis-mass spectrometry employing a partial filling technique with a crown ether carboxylic acid.

An enantiomeric separation method for underivatized free amino acids (AAs) using a partial filling technique with CE-MS was developed for the determination of D-AAs in vinegars. A typical chiral separation method was performed with different concentrations of (18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6H4) dissolved in water or formic acid as the background electrolyte. Seventeen AAs, excluding proline and asparagine, were separated, showing chiral resolution values (Rs) ranging from 0.5 to 21.0. These results included baseline separations of 11 AAs, the peaks of which were observed as the ions [AA+18C6H4+H]+. The migration order of the chiral AAs was also evaluated, and the L-AAs migrated faster than the counterpart D-AAs except for serine, threonine and methionine when using (+)-18C6H4. To reduce contamination of the ESI source by the nonvolatile chiral selector and improve the ionization efficiency in partial filling technique, the separation zone length was adjusted to 70% of the capillary, which was filled with 30 mM 18C6H4 in water. This method showed a similar separation efficiency as the typical method, and the separated AA peaks were observed as free AA ions, [AA+H]+. The optimized method provided limits of detection (LODs) ranging from 0.07 to 1.03 µg/mL and good linearity (R2 > 0.99) up to 50 µg/mL for DL-AAs. The developed method was utilized to determine DL-AAs in vinegars with a simple pretreatment process. It may be extended to sensitive AA analysis in the determination of minor enantiomeric impurities in the major component.

Saturated fatty acid determination method using paired ion electrospray ionization mass spectrometry coupled with capillary electrophoresis.

A sensitive and selective capillary electrophoresis-mass spectrometry (CE-MS) method for determination of saturated fatty acids (FAs) was developed by using dicationic ion-pairing reagents forming singly charged complexes with anionic FAs. For negative ESI detection, 21 anionic FAs at pH 10 were separated using ammonium formate buffer containing 40% acetonitrile modifier in normal polarity mode in CE by optimizing various parameters. This method showed good separation efficiency, but the sensitivity of the method to short-chain fatty acids was quite low, causing acetic and propionic acids to be undetectable even at 100 mgL-1 in negative ESI-MS detection. Out of the four dicationic ion-pairing reagents tested, N,N'-dibutyl 1,1'-pentylenedipyrrolidium infused through a sheath-liquid ion source during CE separation was the best reagent regarding improved sensitivity and favorably complexed with anionic FAs for detection in positive ion ESI-MS. The monovalent complex showed improved ionization efficiency, providing the limits of detection (LODs) for 15 FAs ranging from 0.13 to 2.88 µg/mL and good linearity (R2 > 0.99) up to 150 µg/mL. Compared to the negative detection results, the effect was remarkable for the detection of short- and medium-chain fatty acids. The optimized CE-paired ion electrospray (PIESI)-MS method was utilized for the determination of FAs in cheese and coffee with simple pretreatment. This method may be extended for sensitive analysis of unsaturated fatty acids.

The therapeutic effects of Yongdamsagan-tang on autoimmune hepatitis models.

Autoimmune hepatitis (AIH) is an immunity disorder that is the result of antibodies in the liver tissue of the patient that are attacked by activated immune cells due to an unknown cause. In this study, we aimed to investigate the anti-inflammatory effect of Yongdamsagan-tang (YST) extracts and confirm effects on autoimmune hepatitis models as the therapeutic agent using the YST extracted by various solvents. YST, a mixture of 11 herbal extracts, is known in traditional Korean medicine as a widely used treatment for inflammatory diseases. We proposed the AIH-condition in vitro model by the addition of recombinant IL-17A and then observed several markers linked to AIH symptoms, including an increase of IL-6 expression, lipid accumulation, and fibrosis. In AIH-condition hepatic cell model, YST reduced IL-6 expression and lipid accumulation caused by treatment of IL-17 combination in hepatocyte cells. Also, YST blocked several activated fibrosis factors including transforming growth factor-ß (TGF- ß1), collagen type 1 (Col-α1(I)), and α-smooth muscle actin (α-SMA) in liver stellate cells. Furthermore, pretreatment with YST protected hepatic damage and reduces histological injury by suppressing apoptosis mediator and inflammatory cytokines expression in concanavalin A (Con A)-induced autoimmune hepatitis mice model. The findings here improve our understanding of YST extracted by 80% ethanol, suggesting that YST can be used as a therapeutic treatment for AIH.

Metabolic Profiling of Liver Tissue in Diabetic Mice Treated with Artemisia Capillaris and Alisma Rhizome Using LC-MS and CE-MS.

Artemisia Capillaris (AC) and Alisma Rhizome (AR) are natural products for the treatment of liver disorders in oriental medicine clinics. Here, we report metabolomic changes in the evaluation of the treatment effects of AC and AR on fatty livers in diabetic mice, along with a proposition of the underlying metabolic pathway. Hydrophobic and hydrophilic metabolites extracted from mouse livers were analyzed using HPLC-QTOF and CE-QTOF, respectively, to generate metabolic profiles. Statistical analysis of the metabolites by PLS-DA and OPLA-DA fairly discriminated between the diabetic, and the AC- and AR-treated mice groups. Various PEs mostly contributed to the discrimination of the diabetic mice from the normal mice, and besides, DG (18:1/16:0), TG (16:1/16:1/20:1), PE (21:0/20:5), and PA (18:0/21:0) were also associated with discrimination by s-plot. Nevertheless, the effects of AC and AR treatment were indistinct with respect to lipid metabolites. Of the 97 polar metabolites extracted from the CE-MS data, 40 compounds related to amino acid, central carbon, lipid, purine, and pyrimidine metabolism, with [Formula: see text] values less than 0.05, were shown to contribute to liver dysregulation. Following treatment with AC and AR, the metabolites belonging to purine metabolism preferentially recovered to the metabolic state of the normal mice. The AMP/ATP ratio of cellular energy homeostasis in AR-treated mice was more apparently increased ([Formula: see text]) than that of AC-treated mice. On the other hand, amino acids, which showed the main alterations in diabetic mice, did not return to the normal levels upon treatment with AR or AC. In terms of metabolomics, AR was a more effective natural product in the treatment of liver dysfunction than AC. These results may provide putative biomarkers for the prognosis of fatty liver disorder following treatment with AC and AR extracts.

Anti-diabetic and anti-obesitic effects of aqueous extracts of Yangkyuksanhwa-tang and its two major compositions on db/db mice.

Type 2 diabetes mellitus (T2DM) is a metabolic syndrome that results from target-tissue resistance to insulin. Obesity is the condition of excess body fat accumulation. T2DM and obesity are both associated with hypertension, hyperlipidemia, and abdominal obesity. In Korean medicine, Yangkyuksanhwa-tang (YKSHT) has been prescribed for patients with T2DM. Oral glucose tolerance tests (OGTT), multiplex assays and hemoglobin A1C (HbA1C) assessments were performed to determine the anti-diabetic effects of YKSHT and two major compositions of YKSHT, Lonicera japonica Thunb. (LJT) and Rehmannia glutinosa (RG) on db/db mice, a rodent model for T2DM. To study the anti-obesitic effects of LJT, RG or YKSHT, blood profiling including the triglycerides (TGs) and the total, LDL and HDL cholesterol levels were measured. In addition, body index measures such as the liver, retroperitoneal and epididymal fat tissues were collected and weighed. Mice treated with RG or YKSHT showed reduced blood glucose levels after stimulating the plasma GLP-1 levels. The multiplex assay results support the weight-controlling effects of the LJT, RG and YKSHT treatments, showing reducing levels of ghrelin and the induction of peptide YY (PYY) secretion. The YKSHT treatment reduced plasma TG levels and increased HDL cholesterol levels. The weights of the liver, retroperitoneal and epididymal fat tissues were reduced after the YKSHT treatment. Hence, we suggest that YKSHT can be utilized for the prevention and treatment of T2DM and obesity simultaneously.

Anti-lipoapoptotic effects of Alisma orientalis extract on non-esterified fatty acid-induced HepG2 cells.

BACKGROUND: Liver steatosis was caused by lipid accumulation in the liver. Alisma orientale (AO) is recognized as a promising candidate with therapeutic efficacy for the treatment of nonalcoholic fatty liver disease (NAFLD). HepG2 hepatocyte cell line is commonly used for liver disease cell model. METHOD: The HepG2 cells were cultured with the NEFAs mixture (oleic and palmitic acids, 2:1 ratio) for 24 h to induce hepatic steatosis. Then different doses of Alisma orientale extract (AOE) was treated to HepG2 for 24 h. Incubated cells were used for further experiments. RESULTS: The AOE showed inhibitory effects on lipid accumulation in the Oil Red O staining and Nile red staining tests with no cytotoxicity at a concentration of 300 µg/mL. Fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1) mRNA and protein expression level were down-regulated after AOE treatment. Bcl-2 associated X protein (Bax) and c-Jun N-terminal kinase (JNK) mRNA expression level were decreased as well as p-JNK (activated form of JNK), Bax, cleaved caspase-9, caspase-3 protein expression level. Anti-apopototic B-cell lymphoma 2 (Bcl-2) protein level increased after AOE treatment. In addition, inflammatory protein expression including p-p65, p65, COX-2 and iNOS were inhibited by AOE treatment. CONCLUSION: The results suggest that AOE has anti-steatosis effects that involve lipogenesis, anti-lipoapoptosis, and anti-inflammation in the NEFA-induced NAFLD pathological cell model.

Antihyperglycemic and Antiobesity Effects of JAL2 on db/db Mice.

Lonicera japonica Thunb. (LJT) and Rehmannia glutinosa Libosch. (RGL) have been used traditionally as a herbal medicine in Korean medicine. Using LC/Q-TOF was performed to profile the two herbal medicines and the mixture of LJR and RGL (JAL2, ratio 1 : 1). We performed oral glucose tolerance test (OGTT) and plasma GLP-1 and insulin secretion by multiplex assays to investigate antidiabetic effects of LJT, RGL, and JAL2 in db/db mice, the mice model of type 2 diabetes mellitus (T2DM). Also, the antiobesity-related factors such as plasma peptide YY (PYY), triglyceride, total cholesterol, HDL, LDL, and weight of liver, epididymal, and retroperitoneal fat tissue were investigated. Through the multiplex assay, it was found that JAL2 treatment more efficiently attenuated high levels of blood glucose by stimulating GLP-1 secretion and reduced LDL concentration and weight of liver and retroperitoneal fat tissue compared to LJT or RGL treated separately. These results suggest that the JAL2 has antidiabetes and antiobesity effects in T2DM mice model.

Faecalibacterium prausnitzii subspecies-level dysbiosis in the human gut microbiome underlying atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a serious global epidemic associated with a modern lifestyle. OBJECTIVE: Although aberrant interactions between gut microbes and the intestinal immune system have been implicated in this skin disease, the nature of the microbiome dysfunction underlying the disease remains unclear. METHODS: The gut microbiome from 132 subjects, including 90 patients with AD, was analyzed by using 16S rRNA gene and metagenome sequence analyses. Reference genomes from the Human Microbiome Project and the KEGG Orthology database were used for metagenome analyses. Short-chain fatty acids in fecal samples were compared by using gas chromatographic-mass spectrometric analyses. RESULTS: We show that enrichment of a subspecies of the major gut species Faecalibacterium prausnitzii is strongly associated with AD. In addition, the AD microbiome was enriched in genes encoding the use of various nutrients that could be released from damaged gut epithelium, reflecting a bloom of auxotrophic bacteria. Fecal samples from patients with AD showed decreased levels of butyrate and propionate, which have anti-inflammatory effects. This is likely a consequence of an intraspecies compositional change in F prausnitzii that reduces the number of high butyrate and propionate producers, including those related to the strain A2-165, a lack of which has been implicated in patients with Crohn disease. CONCLUSIONS: The data suggest that feedback interactions between dysbiosis in F prausnitzii and dysregulation of gut epithelial inflammation might underlie the chronic progression of AD by resulting in impairment of the gut epithelial barrier, which ultimately leads to aberrant TH2-type immune responses to allergens in the skin.

The aglycone of ginsenoside Rg3 enables glucagon-like peptide-1 secretion in enteroendocrine cells and alleviates hyperglycemia in type 2 diabetic mice.

Ginsenosides can be classified on the basis of the skeleton of their aglycones. Here, we hypothesized that the sugar moieties attached to the dammarane backbone enable binding of the ginsenosides to the sweet taste receptor, eliciting glucagon-like peptide-1 (GLP-1) secretion in the enteroendocrine L cells. Using the human enteroendocrine NCI-H716 cells, we demonstrated that 15 ginsenosides stimulate GLP-1 secretion according to the position of their sugar moieties. Through a pharmacological approach and RNA interference technique to inhibit the cellular signal cascade and using the Gαgust(-/-) mice, we elucidated that GLP-1 secreting effect of Rg3 mediated by the sweet taste receptor mediated the signaling pathway. Rg3, a ginsenoside metabolite that transformed the structure through a steaming process, showed the strongest GLP-1 secreting effects in NCI-H716 cells and also showed an anti-hyperglycemic effect on a type 2 diabetic mouse model through increased plasma GLP-1 and plasma insulin levels during an oral glucose tolerance test. Our study reveals a novel mechanism where the sugar moieties of ginsenosides Rg3 stimulates GLP-1 secretion in enteroendocrine L cells through a sweet taste receptor-mediated signal transduction pathway and thus has an anti-hyperglycemic effect on the type 2 diabetic mouse model.

HepG2 Cell Resistance against Camptothecin from a Lysosomal Drug Delivery.

A galactose-appended drug delivery system released camptothecin (CPT) to lysosomes of HepG2 hepatoma cells, resulting in the cell resistance to the anticancer drug. We found that the resistance to CPT is caused by alteration of the drug release from the prodrug in lysosomes, emphasizing that the final delivery locations may critically influence drug efficacy.