Benzyl isothiocyanate inhibits TNFα-driven lipolysis via suppression of the ERK/PKA/HSL signaling pathway in 3T3-L1 adipocytes.

Tumor necrosis factor α (TNFα), an inflammatory cytokine, induces lipolysis and increases circulating concentrations of free fatty acids. In addition, TNFα is the first adipokine produced by adipose tissue in obesity, contributing to obesity-associated metabolic disease. Given that benzyl isothiocyanate (BITC) is a well-known anti-inflammatory agent, we hypothesized that BITC can ameliorate TNFα-induced lipolysis and investigated the working mechanisms involved. We first challenged 3T3-L1 adipocytes with TNFα to induce lipolysis, which was confirmed by increased glycerol release, decreased protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and perilipin 1 (PLIN1), and increased phosphorylation of ERK, protein kinase A (PKA), and hormone-sensitive lipase (HSL). However, inhibition of ERK or PKA significantly attenuated the lipolytic activity of TNFα. Meanwhile, pretreatment with BITC significantly ameliorated the lipolytic activity of TNFα; the TNFα-induced phosphorylation of ERK, PKA, and HSL; the TNFα-induced ubiquitination of PPARγ; the TNFα-induced decrease in PPARγ nuclear protein binding to PPAR response element; and the TNFα-induced decrease in PLIN1 protein expression. Our results indicate that BITC ameliorates TNFα-induced lipolysis by inhibiting the ERK/PKA/HSL signaling pathway, preventing PPARγ proteasomal degradation, and maintaining PLIN1 protein expression.

Clinical study of Chinese medicine holographic scraping combined with hot ironing in improving early diabetic retinopathy.

OBJECTIVES: To investigate the clinical effect of holographic scraping combined with Chinese medicine hot ironing on the improvement of early diabetic retinopathy (DR). METHODS: The clinical data of 120 inpatients with diabetes mellitus were retrospectively analyzed. All patients had early retinopathy. According to different treatment methods, the patients were segmented into a scraping group (accepted holographic scrapping), an ironing group (accepted Chinese medicine hot ironing), and a combined treatment group (accepted holographic scraping combined with Chinese medicine hot ironing). The traditional Chinese medicine (TCM) symptom scores, efficacy in TCM symptom relief and fundus symptom relief, quality of life, blood glucose index level, and safety were compared among the three groups. RESULTS: Compared with the scraping group and the ironing group, the TCM symptom scores of the combined treatment group on the 3rd day, 7th day, and 14th day of treatment were decreased; The total effective rates in TCM symptom relief and fundus symptom relief were increased; The scores of four dimensions in QoL of patients increased (all P<0.05); and Fasting blood glucose (FBG), 2 h postprandial blood glucose, and glycosylated hemoglobin were decreased (all P<0.05). There was no significant difference in the distribution of DR grade (I, II, and III) in the combined treatment group compared with the scraping group and ironing group (all P>0.05). The resistance index of the combined group after treatment was lower than that before treatment and lower than that of the hot ironing group and scraping group (all P<0.05). CONCLUSIONS: The application of holographic scraping combined with Chinese medicine hot ironing in the treatment of early DR could alleviate the symptoms of blurred vision and dry eyes. Early intervention for retinopathy with both methods can reduce the disability rate and improve the quality of life of patients, which has a better effect than simple therapy.

Investigating noncovalent squarylium dye-protein interactions by capillary electrophoresis-frontal analysis.

Noncovalent interactions between fluorescent probe molecules and protein analyte molecules, which typically occur with great speed and minimal sample handling, form the basis of many high sensitivity analytical techniques. Understanding the nature of these interactions and the composition of the resulting complexes represents an important area of study that can be facilitated by capillary electrophoresis (CE). Specifically, we will present how frontal analysis (FA) and Hummel-Dreyer (HD) methods can be implemented with CE to determine association constants and stoichiometries of noncovalent complexes of the red luminescent squarylium dye Red-1c with bovine serum albumin (BSA) and beta-lactoglobulin A. By adjusting solution conditions, such as pH or ionic strength, it is possible to selectively modify the binding process. As such, conditions for optimal selectivity for labeling reactions can be established by capillary electrophoresis-frontal analysis (CE-FA) investigations.

Development and validation of a direct LC-MS-MS method to determine the acrolein metabolite 3-HPMA in urine.

A new direct method using liquid chromatography-tandem mass spectrometry has been developed and validated for quantitation of 3-hydroxypropylmercapturic acid (3-HPMA) in urine. The method is fast, simple, and does not require extraction from urine. Analyte was separated on a hydrophilic interaction liquid chromatography column. Severe ion suppression was circumvented by a fast gradient after separation. Assay specificity, linearity, precision, and accuracy met the required FDA/CDER bioanalytical method criteria. Matrix effect and carryover of the assay were assessed. Urine sample storage stability and standard solution stability were also tested. The limit of quantitation was 22.0 ng/mL. The results for 3-HPMA obtained by our method were significantly correlated with results obtained by a contract lab.

Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS.

A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF(2 alpha)-III, 15-epi-iPF(2 alpha)-III, iPF(2 alpha)-VI, and 8,12-iso-iPF(2 alpha)-VI along with the prostaglandin PGF(2 alpha) and 2,3-dinor-iPF(2 alpha)-III, a metabolite of iPF(2 alpha)-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF(2 alpha)-III obtained by our method were significantly correlated with results by an ELISA, although an approximately 2-fold high bias was observed for the ELISA data. For iPF(2 alpha)-III and its metabolite 2,3-dinor-iPF(2 alpha)-III, smokers had significantly higher concentrations than nonsmokers (513 +/- 275 vs. 294 +/- 104 pg/mg creatinine; 3,030 +/- 1,546 vs. 2,046 +/- 836 pg/mg creatinine, respectively). The concentration of iPF(2 alpha)-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents.

Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection.

Two new red luminescent asymmetric squarylium dyes (designated "Red-1c and Red-3") have been shown to exhibit absorbance shifts to longer wavelengths upon the addition of protein, along with a concomitant increase in fluorescence emission. Specifically, the absorbance maxima for Red-1c and Red-3 dyes are 607 and 622 nm, respectively, in the absence of HSA, and 642 and 640 nm in the presence of HSA, making the excitation of their protein complexes feasible with inexpensive and robust diode lasers. Fluorescence emission maxima, in the presence of HSA, are 656 and 644 nm for Red-1c and Red-3, respectively. Because of the inherently low fluorescence of the dyes in their free state, Red-1c and Red-3 were used as on-column labels (that is, with the dye incorporated into the separation buffer), thus eliminating the need for sample derivatization prior to injection and separation. A comparison of precolumn and on-column labeling of proteins with these squarylium dyes revealed higher efficiencies and greater sensitivities for on-column labeling, which, when conducted with a basic, high-salt content buffer, permitted baseline resolution of a mixture of five model proteins. LOD for model proteins, such as transferrin, alpha-lactalbumin, BSA, and beta-lactoglobulin A and B, labeled with these dyes and analyzed by CE with LIF detection (CE-LIF) were found to be dependent upon dye concentration and solution pH, and are as low as 5 nM for BSA. Satisfactory linear relationships between peak height (or peak area) and protein concentration were obtained by CE-LIF for this on-column labeling method with Red-3 and Red-1c.

Fluorimetric studies and noncovalent labeling of protein with the near-infrared dye HITCI for analysis by CE-LIF.

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.

Design, synthesis, and anti-tumor activity of (2-O-alkyloxime-3-phenyl)-propionyl-1-O-acetylbritannilactone esters.

The extracts of Inula britannica have anti-inflammatory, anti-bacterial, anti-hepatitic, and anti-tumor activities. Various sesquiterpene lactones with cytotoxic properties including 1-O-acetylbritannilactone (1) have been isolated from this Chinese medicinal plant. Eight derivatives of 1-O-acetylbritannilactone, (2-O-alkyloxime-3-phenyl)-propionyl-1-O-acetylbritannilactone esters were designed and synthesized. Four of these compounds were tested to show inhibitory activity on the growth of human leukemia HL-60 and cancer Bel-7402 cell lines.

Studies on 1-O-acetylbritannilactone and its derivative, (2-O-butyloxime-3-phenyl)-propionyl-1-O-acetylbritannilactone ester.

The crystal structures of 1-O-acetylbritannilactone 6 isolated from Inula britannica, and its derivative (2-O-butyloxime-3-phenyl) propionyl 1-O-acetylbritannilactone ester 7 are reported. The structure of synthesized derivative 7 was established by spectroscopic methods. Both compounds inhibited the growth of human HL-60, Bel-7402 cell lines and compound 7 had the stronger cytotoxic activity.