Uncovering the key pharmacodynamic material basis and possible molecular mechanism of Xiaoke formulation improve insulin resistant through a comprehensive investigation.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoke formulation (XKF) has been utilized in clinical practice for decades in China as a treatment option for mild to moderate type 2 diabetes. However, there is still a need for systematic research to uncover the key pharmacodynamic material basis and mechanism of XKF. AIM OF THE STUDY: Aim of to investigate the distribution and metabolism of XKF in normal and insulin resistant (IR) mice were different, and elucidate its key pharmacodynamic material basis and mechanism of action. MATERIALS AND METHODS: Ultra performance liquid chromatography/time of flight mass spectrometry technology was employed to investigate the differences in XKF absorption, distribution, and metabolism between normal and IR mice across blood, liver, feces, and urine samples. Further, network pharmacology was used to predict target proteins and their associated signaling pathways. Then, molecular docking was utilized to validate the activity of key pharmacodynamic components and targets. Finally, IR HepG2 cells were used to detect the glucose consumption under the action of key pharmacodynamic material basis. In addition, the expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and phospho-protein kinase B (p-AKT) was determined using western blotting. RESULTS: The study demonstrates significant distinctions in plasma and liver number and abundance of alkaloids, organic acids, flavonoids, iridoids and saponins between normal and IR mice when XKF was administered. Further analysis has shown that the representative components of XKF, including berberine, chlorogenic acid, calycosin, swertiamarin and astragaloside IV have significantly different metabolic pathways in plasma and liver. Prototypes and metabolites of these components were rarely detected in the urine and feces of mice. According to the network pharmacological analysis, these differential components are predicted to improve IR by targeting key factors such as SRC, JUN, HRAS, NOS3, FGF2, etc. Additionally, the signaling pathways involved in this process include PI3K-AKT pathway, GnRH signaling pathway, and T cell receptor signaling pathway. In addition, in vitro experiments indicate that berberine and its metabolites (berberine and demethyleneberine), chlorogenic acid and its metabolites (3-O-ferulic quinic acid and 5-O-ferulic quinic acid), calycosin and swertiamarin could improve IR in IR-HepG2 cells by elevating the expression of PI3K and AKT, leading to an increase in glucose consumption. CONCLUSION: The key pharmacodynamic material basis of XKF, such as berberine and its metabolites (berberrubine and demethyleneberberine), chlorogenic acid and its metabolites (3-O-feruloylquinic acid and 5-O-feruloylquinic acid), calycosin and swertiamarin influence the glucose metabolism disorder of IR-HepG2 cells by regulating the PI3K/AKT signalling pathway, leading to an improvement in IR.

Quantitative analysis of aberrant fatty acid composition of zebrafish hepatic lipids induced by organochlorine pesticide using stable isotope-coded transmethylation and gas chromatography-mass spectrometry.

Organochlorine pesticides have been extensively used worldwide for agricultural purposes. Due to their resistance to metabolism, a major public health concern has been raised. Aberrant hepatic lipid composition has been a hallmark of many liver diseases associated with exposure to various toxins and chemicals. And thus lots of efforts have been focused on the development of analytical techniques that can rapidly and quantitatively determine the changes in fatty acid composition of hepatic lipids. In this work, changes in fatty acid composition of hepatic lipids in response to DDT (dichlorodiphenyltrichloroethane) exposure were quantitatively analyzed by a gas chromatography-mass spectrometric approach based on stable isotope-coded transmethylation. It has been quantitatively demonstrated that polyunsaturated fatty acids including C20:3n3, C20:4n6, and C22:6n3 decrease in response to DDT exposure. However, saturated long chain fatty acids including C16:0, C18:0, as well as monounsaturated long chain fatty acid C18:1n9 consistently increase in a DDT-concentration-dependent manner. In particular, much higher changes in the level of hepatic C16:0 and C18:0 for male fish were observed than that for female fish. These experimental results are in accordance with qualitative histopathological analysis that revealed liver morphological alterations. The stable isotope-coded mass spectrometric approach provides a reliable means for investigating hepatotoxicity associated with fatty acid synthesis, desaturation, mitochondrial beta-oxidation, and lipid mobilization. It should be useful in elucidation of hepatotoxic mechanisms and safety assessment of environmental toxins.

Comparative analysis of S-fatty acylation of gel-separated proteins by stable isotope-coded fatty acid transmethylation and mass spectrometry.

Covalent attachment of palmitic acid or other fatty acids to the thiol groups of cysteine residues of proteins through reversible thioester bonds has an important role in the regulation of diverse biological processes. We describe here the development of a mass spectrometry protocol based on stable isotope-coded fatty acid transmethylation (iFAT) for qualitative and comparative analysis of protein S-fatty acylation under different experimental conditions. In this approach, cellular proteins extracted from different cell states are separated by SDS-PAGE and then the gel is stained with either Coomassie blue or Nile red for improved sensitivity. Protein bands are excised and then an in-gel stable iFAT procedure is performed. The fatty acid methyl esters resulting from derivatization with d0- and d3-methanol are identified by mass spectrometry. By measuring the intensities of labeled and unlabeled fragment ion pairs of fatty acid methyl esters, the levels of S-fatty acylation in different cells or tissues can be compared. This approach has been applied to monitor the changes of S-fatty acylation of zebrafish liver proteome in response to environmental dichlorodiphenyltrichloroethane exposure. Compared with the approach using metabolic incorporation of radioactive fatty acid analogs, it is not only simple and effective but also eliminates the hazards of handling radioactive isotopes.

Typing of unknown microorganisms based on quantitative analysis of fatty acids by mass spectrometry and hierarchical clustering.

Rapid identification of unknown microorganisms of clinical and agricultural importance is not only critical for accurate diagnosis of infections but also essential for appropriate and prompt treatment. We describe here a rapid method for microorganisms typing based on quantitative analysis of fatty acids by iFAT approach (Isotope-coded Fatty Acid Transmethylation). In this work, lyophilized cell lysates were directly mixed with 0.5M NaOH solution in d3-methanol and n-hexane. After 1 min of ultrasonication, the top n-hexane layer was combined with a mixture of standard d0-methanol derived fatty acid methylesters with known concentration. Measurement of intensity ratios of d3/d0 labeled fragment ion and molecular ion pairs at the corresponding target fatty acids provides a quantitative basis for hierarchical clustering. In the resultant dendrogram, the Euclidean distance between unknown species and known species quantitatively reveals their differences or shared similarities in fatty acid related pathways. It is of particular interest to apply this method for typing fungal species because fungi has distinguished lipid biosynthetic pathways that have been targeted for lots of drugs or fungicides compared with bacteria and animals. The proposed method has no dependence on the availability of genome or proteome databases. Therefore, it is can be applicable for a broad range of unknown microorganisms or mutant species.

Chemical and genetic probes for analysis of protein palmitoylation.

Reversible protein palmitoylation is one of the most important posttranslational modifications that has been implicated in the regulation of protein signaling, trafficking, localizing and enzymatic activities in cells and tissues. In order to achieve a precise understanding of mechanisms and functions of protein palmitoylation as well as its roles in physiological processes and disease progression, it is necessary to develop techniques that can qualitatively and quantitatively monitor the dynamic protein palmitoylation in vivo and in vitro. This review will highlight recent advances in both chemical and genetic encoded probes that have been developed for accurate analysis of protein palmitoylation, including identification and quantification of acyl moieties and palmitoylated proteins, localization of amino acid residues on which acyl moieties are attached, and imaging of cellular distributions of palmitoylated proteins. The role of major techniques of fluorescence microscopy and mass spectrometry in facilitating the analysis of protein palmitoylation will also be explored.

Effects of co-existed proteins on measurement of pesticide residues in blood by gas chromatography-mass spectrometry.

Accurate measurement of pesticides in biological fluids such as blood is important for quantifying environmental exposures. Beyond sample enrichment and separation, the method presented here is focused on studies of interactions between pesticides and co-existed proteins. It was experimentally demonstrated that entrapped or adsorbed pesticide residues within the folded native structures of proteins were poorly recovered using direct solvent extraction solely. We described here an effective approach termed Enzymatic Digestion-Organic Solvent Extraction (eDOSE) that utilizes the enzymatic approach to disrupt the folded structures of proteins and release entrapped or adsorbed pesticide residues. In this approach, samples were first reduced, alkylated, tryptically digested and then diluted 10 times before the subsequent extraction using an n-hexane solution. Resultant pesticide residues were determined by capillary gas chromatography coupled with a mass spectrometer. Mean recoveries of the 5 organophosphorus pesticides pre-spiked in fish blood including diazinon, parathion-methyl, malathion, parathion-ethyl and ethion were 85%, 95%, 84%, 103%, and 43% respectively using eDOSE strategy but only 24%, 45%, 40%, 27%, and 29% respectively using direct solvent extraction approach. The eDOSE approach was effective for demonstrating the critical role of folded native structure of serum albumin in adsorption of exogenous chemicals. It provides an alterative means for denaturation of proteins when the target analytes are not stable in acidic solution or entrapped within the protein aggregates caused by organic solvents such as acetone that have been applied for protein denaturation. The eDOSE approach should be able to combine with other advanced techniques of enrichment and separation for more efficient and accurate measurement of target compounds present in the context of complex biological systems. This approach can provide wide applications to the analysis of a variety of small molecules including environmental pesticide residues and metabolites as well as other toxins present in cells, tissues and biofluids.