[Metabolomic study on urine of chronic inflammation rats treated with Buyang Huanwu Decoction based on UPLC-Q-TOF-MS].

The study investigated the effect of Buyang Huanwu Decoction(BYHWD) on endogenous biomarkers in the urine of rats with chronic inflammation induced by lipopolysaccharide(LPS) using ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS), aiming to elucidate the molecular mechanism underlying the therapeutic effect of BYHWD on chronic inflammation from a metabolomics perspective. Male SD rats were randomly divided into a normal group, a model group, and low-, medium-, and high-dose BYHWD groups(7.5, 15, and 30 g·kg~(-1)). The model group and BYHWD groups received tail intravenous injection of LPS(200 µg·kg~(-1)) on the first day of each week, followed by oral administration of BYHWD once a day for four consecutive weeks. Urine samples were collected at the end of the administration period, and UPLC-Q-TOF-MS was used to analyze the metabolic profiles of the rat urine in each group. Multivariate statistical analysis methods such as principal component analysis(PCA), partial least squares-discriminant analysis(PLS-DA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to analyze the effect of BYHWD on endogenous metabolites. One-way ANOVA and variable importance for the projection(VIP) were used to screen for potential biomarkers related to chronic inflammation. The identified biomarkers were subjected to pathway and enrichment analysis using MetaboAnalyst 5.0. A total of 25 potential biomarkers were screened and identified in the rat urine in this experiment. Compared with the normal group, the model group showed significant increases in the levels of 14 substances(P<0.05) and significant decreases in the levels of 11 substances(P<0.05). BYHWD was able to effectively reverse the trend of most endogenous biomarkers. Compared with the model group, BYHWD significantly down-regulated 13 biomarkers(P<0.05) and up-regulated 10 biomarkers(P<0.05). The metabolic products were mainly related to the biosynthesis of pantothenic acid and coenzyme A, tryptophan metabolism, retinol metabolism, and propionate metabolism. BYHWD has therapeutic effect on chronic inflammation induced by LPS, which may be related to its ability to improve the levels of endogenous metabolites, enhance the body's anti-inflammatory and antioxidant capabilities, and restore normal metabolic activity.

Metabolomics of the anti-inflammatory effect of Pueraria lobata and Pueraria lobata var. Thomsonii in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria lobata (Willd.) Ohwi and Pueraria lobata var. Thomsonii (Benth.) Maesen are essential medicinal and edible homologous plants widely cultivated in Asian countries. Therefore, P. lobata and P. thomsonii are widely used in the food, health products and pharmaceutical industries and have significant domestic and international market potential and research value. P. lobata and P. thomsonii have pharmacological effects in the clinic, such as antipyretic, analgesic, anti-inflammatory and antioxidant effects. These plants are commonly used in the treatment of inflammatory diseases and other related diseases. However, the potential mechanisms of the anti-inflammatory effects of P. lobata and P. thomsonii have not been elucidated. AIM OF THE STUDY: This study aimed to confirm the anti-inflammatory effects of P. lobata and P. thomsonii on inflammatory model diseases and to investigate the mechanism of their anti-inflammatory effects from the perspective of plasma metabolomics. MATERIALS AND METHODS: First, P. lobata and P. thomsonii were identified by high‒performance liquid chromatography (HPLC). Second, we established the following three inflammation models: an acute inflammation model of auricular swelling in mice induced by xylene, an acute inflammation model of foot swelling in rats induced by carrageenan gum, and a chronic inflammation model of cotton ball granuloma in rats. Then we examined the weight and swelling rate of auricular swelling in mice; the residence time, contact area, and mean contact pressure in rats on the gait meter; and the weight of granulomas in rats and the content of IL-1ß and TNF-α in plasma to investigate the anti-inflammatory pharmacodynamics of P. lobata and P. thomsonii. Third, we used LC‒MS‒based plasma metabolomics techniques to obtain potential biomarkers of P. lobata and P. thomsonii related to inflammation. Then, the potential biomarkers were enriched by MetaboAnalyst and KEGG metabolomics analysis tools to obtain metabolic pathways related to inflammation. Finally, we tested the indicators of COX-2, 5-LOX, GSH, GSSG and γ⁃GCL in rat plasma from the granuloma model by enzyme-linked immunosorbent assays (ELISAs) to verify the inflammation-related metabolic pathway. RESULTS: The experimental results showed that P. lobata and P. thomsonii could reduce the swollen weight and swelling rate of the auricle in mice, and could increase the residence time, contact area and mean contact pressure in rats on the gait meter. Moreover, P. lobata and P. thomsonii could inhibit the growth of granulomas and reduce the content of IL-1ß and TNF-α in plasma in rats. The above results preliminarily verified that P. lobata and P. thomsonii have different anti-inflammatory effects. We identified eighteen plasma biomarkers associated with P. lobata and sixteen plasma biomarkers related to P. thomsonii in regulating inflammation by a plasma metabolomics analysis. The following two major metabolic pathways were further screened and enriched: arachidonic acid metabolism and glutathione metabolism. Then we noted that P. lobata and P. thomsonii could reduce the COX-2, 5-LOX and GSSG levels and increase the GSH, GSH/GSSG and γ⁃GCL levels based on the ELISA results, which demonstrated that P. lobata and P. thomsonii affect the anti-inflammatory mechanism through arachidonic acid metabolism and glutathione metabolism. CONCLUSIONS: The results of this study further elucidate the anti-inflammatory mechanism of action of P. lobata and P. thomsonii, providing a scientific basis for developing new drugs for the treatment of inflammation-related diseases and laying a foundation for the development of herbal resources, such as P. lobata and P. thomsonii.

Timing of Surgery and Treatment Options for Congenital Ptosis in Children: A Narrative Review of the Literature.

Congenital ptosis has varying degrees of impact on the visual development or psychological health of a child depending on its severity. Some controversies and misconceptions remain regarding the management of congenital ptosis in children. Particularly, the accurate diagnosis of the severity of congenital ptosis in younger children, assessment of the visual developmental status of the child, optimal timing of surgery, and treatment choice are still issues in clinical practice that need to be explored. This report presents a comprehensive review of these aspects of the correction of congenital ptosis to provide a valuable reference for clinical practice. Our review shows that currently used surgical procedures for ptosis may result in over- or under-correction to varying degrees. The differences may be due to the physical condition and age of the child and the degree of cooperation during preoperative examination and assessment, resulting in inaccurate results. Alternatively, intraoperative swelling and bleeding may lead to errors in the values recorded by the surgeon. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Research on the Material Basis and Mechanism of Kudzu Root in Preventing and Treating Cerebral Ischemia based on Network Pharmacology.

BACKGROUND: It has been shown that Kudzu root has significant pharmacological effects such as improving microcirculation, dilating coronary arteries, and increasing cerebral and coronary blood flow, but its material basis and mechanism of action are not clear. OBJECTIVE: The aim of this study was to investigate the mechanism of action of Kudzu root in the prevention and treatment of cerebral ischemia (CI) through network pharmacology combined with animal experiments. METHODS: The components of kudzu root were screened by using the Chemistry Database, Chinese Academy of Science. Linpinski's five rules were used to perform pharmacophore-like analysis to obtain the active ingredients of Kudzu root. The Swiss Target Prediction Service database was used to predict the potential protein targets of kudzu root components associated with CI. An active ingredient-target network was constructed by using Cytoscape 3.6.0. A rat model of middle cerebral artery occlusion (MCAO) was established, then the main targets and signaling pathways predicted were verified by observing the area of cerebral infarction and Western blot experiments. RESULTS: In total, 84 major active compounds and 34 targets included gerberoside, belonging to the isoflavone class, gallic acid, amino acid class, 4-Methylphenol, phenolic class, and quercetin, and flavonoid class (Flavonoids). The targets covered were proteins related to excitatory amino acids and calcium overload, including Excitatory amino acid transporter 2 (SLC1A2), Glutamate receptor ionotropic, kainate 1 (GRIK1), Glutamate receptor ionotropic, NMDA 1 (GRIN1), Glutamate receptor 2(GRIA2), Calcium/calmodulin-dependent protein kinase II (CaMKII), Neuronal nitric oxide synthase(nNOS). Glutamatergic energy is prominent, and calcium transport across the membrane is central to the network and occupies an important position. CONCLUSION: Kudzu root can significantly reduce neurological damage in rats with CI, and also significantly reduce the rate of cerebral infarction. It is worth noting that Kudzu root can prevent and treat CI by reducing excitatory amino acid toxicity and improving calcium overload.

Traditional Chinese Medicine Constitution Discrimination Model Based on Metabolomics and Random Forest Decision Tree Algorithm.

Constitution refers to the comprehensive and relatively stable characteristics of the genetic or acquired morphological structure, physiological function, and psychological state in the process of human individual life. A special metabolomics data processing method is established to find the unique m/z value of each constitution. Combined with the random forest decision tree algorithm, the discrimination model of 9 constitutions in traditional Chinese medicine is constructed, and the model is verified and tested. The test results show that the classification accuracy of each constitution is higher than 80%, indicating that the model can well identify nine constitutions of traditional Chinese medicine. The classification accuracy is related to the difficulty of distinguishing between constitutions. In a word, this study provides a fast and accurate method to distinguish the constitution of traditional Chinese medicine, provides an objective representation for the classification and judgment of clinical constitution of traditional Chinese medicine, and provides a scientific basis for the modernization of traditional Chinese medicine.

Metabonomic Study on the Plasma of High-Fat Diet-Induced Dyslipidemia Rats Treated with Ge Gen Qin Lian Decoction by Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry.

Gegen Qinlian decoction (GGQLD) has a definite effect on T2DM in clinic, and it has the effect of lowering blood sugar, improving insulin resistance, and improving the blood lipid level of rats with dyslipidemia, but the intervention mechanism of GGQLD on dyslipidemia has not been clarified. The changes in endogenous metabolites in the plasma of high-fat diet-induced dyslipidemia rats treated with Ge Gen Qin Lian Decoction (GGQLD) were studied to elucidate the therapeutic effects and mechanism of action of GGQLD in dyslipidemia. Based on ultrahigh-performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS), the metabolic profiles of rat serum samples were collected. The rat model of dyslipidemia was induced by a 60% fat-fed high-fat diet. After feeding the rats with a high-fat diet for 4 weeks, dyslipidemia appeared. After 5 weeks of GGQLD (14.85 g kg-1) administration, the metabonomics of rats' plasma samples in the normal group, model group, and administration group were analyzed. Mass profiler professional (MPP), SIMCA-P 14.1, and Graphpad prism 6.0 software were used combined with METLIN biological database and human metabolite database HMDB to screen and identify endogenous biomarkers. Metaboanalyst 4.0 software was used by combining with HMDB and KEGG databases; the enrichment and metabolic pathway of biomarkers were analyzed to explore the metabolic mechanism of dyslipidemia rats induced by high-fat diet and the intervention mechanism of Gegen Qinlian decoction. After 5 weeks of administration of GGQLD, the levels of serum TC and TG were significantly decreased (P < 0.05, P < 0.01), while HDL-C and LDL-C were not significantly affected. After administration, the food intake of rats in the administration group decreased gradually, and the change trend of body weight gradually slowed down. The metabonomics of rat plasma samples results showed that 23 potential biomarkers including α-linolenic acid, arachidonic acid, and lysophosphatidylcholine were significantly changed in positive ion mode. Studies have shown that GGQLD has a significant lipid-lowering effect on dyslipidemia rats induced by a high-fat diet, and its preventive mechanism is related to tryptophan metabolism, fatty acid biosynthesis, α-linolenic acid metabolism, arachidonic acid, and glycerophosphatidyl metabolism pathway.

Simultaneous Determination of Ten Bioactive Components from Shenling Baizhu San in Rat Plasma by UHPLC-MS/MS: Application to a Comparative Pharmacokinetic Study in Normal and Two Models of Ulcerative Colitis Rats.

Shenling Baizhu San, a traditional formula, has a long history of treating spleen asthenic diarrhea by invigorating the spleen and dispelling dampness in China. A rapid and accurate UHPLC-MS/MS method was developed and fully validated for the simultaneous determination of ten active constituents in rat plasma: panaxadiol, ginsenoside Rg1, atractylenolide I, atractylenolide III, pachymic acid, neferine, nuciferine, diosgenin, platycodin D, and isoliquiritigenin. The plasma samples were pretreated by the protein precipitation method with acetonitrile. The analytes and puerarin (internal standard) were determined with high selectivity and sensitivity (LLOQ, 0.31-0.68 ng·mL-1) within 10 minutes. The validation parameters, including intra-/interday precisions, accuracy, recovery, matrix effect, and stability, were within acceptable ranges. The validated method was successfully applied to the pharmacokinetics study of ten components in normal and two rat models of ulcerative colitis (i.e., spleen deficiency with dampness retention-ulcerative colitis (SDDR-UC) rats and pure-ulcerative colitis (P-UC) rats). The pharmacokinetic parameters were significantly different among the three groups of rats. Overall, the absorption of the components was shown as follows: normal group > SDDR-UC group > P-UC group. The study could provide a scientific basis for further studies on pharmacokinetics and clinical differential application of SDDR-UC and P-UC patients.

[Study on mechanisms of anti-Alzheimer's disease action of absorbed components of Gardeniae Fructus based on network pharmacology].

Gardeniae Fructus has the traditional effects of promoting intelligence and inducing resuscitation, but its mechanism is unclear. In this study, the relationship between Gardeniae Fructus's traditional effect of promoting intelligence and inducing resuscitation and anti-Alzheimer's disease effect was taken as the starting point to investigate the anti-Alzheimer's disease mechanism of the major absorbed components in Gardeniae Fructus by the network pharmacology method. The network pharmacology research model of "absorbed composition-target-pathway-disease" was adopted. In this study, the active components screening and target prediction technology were used to determine the active components and targets of Gardeniae Fructus in treatment of Alzheimer's disease. The enrichment pathway and biological process of Gardeniae Fructus were studied by using the bioinformatics annotation database(DAVID), and the results of molecular docking validation network analysis were used to elaborate the mechanism of Gardeniae Fructus in treatment of Alzheimer's disease. It was found that 35 absorbed components of Gardeniae Fructus not only regulated 48 targets such as cholines-terase(BCHE) and carbonic anhydrase 2(CA2), but also affected 11 biological processes(e.g. transcription factor activity, nuclear receptor activity, steroid hormone receptor activity, amide binding and peptide binding) and 7 metabolic pathways(MAPK signaling pathway, Alzheimer disease and estrogen signaling pathway, etc.). Molecular docking results showed that more than 60% of the active components could be well docked with key targets, and the relevant literature also showed that the active components could inhibit the MAPK1 expression of key targets, indicating a high reliability of results. These results indicated that Gardeniae Fructus may play its anti-Alzheimer's disease action via a "multi-ingredients-multi-targets and multi-pathways" mode, providing a scientific basis for further drug research and development.

[Research on network pharmacology of Mongolian medicine Cymbaria in treatment of type 2 diabetes].

The network pharmacology was used to explore the potential active ingredients and action mechanisms of Mongolian medicine Cymbaria in the treatment of type 2 diabetes. According to the literatures collected, Cymbaria component database was established to define important active ingredients and key targets for the anti-hyperglycemic effect to predict action mechanism by active ingredient screening and target prediction techniques. Molecular docking predicted binding activity of main active components with key targets in Cymbaria, then verified the action mechanism in vitro. The Cymbaria component database contained 177 chemical components, 90 chemical structures were confirmed, including 34 chemical components with effective targets. According to the prediction results from network pharmacology, 61 biological processes were significantly affected, such as fatty acid metabolism including PPARs signaling pathway, protein kinase activity and insulin signal pathway. Moreover, the key target proteins were Akt1 and TNFα and quercetin, luteolin and catalpol were the main active ingredients of Cymbaria. Molecular docking prediction showed that luteolin, quercetin and catalpol had a strong binding activity with Akt1; luteolin had strong binding activity but quercetin and catalpol had a certain binding activity with TNFα. Furthermore, catalpol showed hypoglycemic effects in vitro, which up-regulated p-Akt(Ser473)/Akt, PPARα and PPARδ levels and reduced FABP4 expression to regulate glycose and lipid metabolism for improving insulin sensitivity. The network pharmacology predicted that the hypoglycemic effect of Cymbaria was mainly related to anti-inflammatory and lipid regulation with a multi-component, multi-target manner. It provided a scientific view of hypoglycemic effect and action mechanism of Cymbaria for further study.

Gegen Qinlian Decoction Coordinately Regulates PPARγ and PPARα to Improve Glucose and Lipid Homeostasis in Diabetic Rats and Insulin Resistance 3T3-L1 Adipocytes.

Gegen Qinlian Decoction (GQD), a well-documented traditional Chinese Medicine (TCM) formula, was reported with convincing anti-diabetic effects in clinical practice. However, the precise antidiabetic mechanism of GQD remains unknown. In this study, the anti-hyperglycemic and/or lipid lowering effects of GQD were demonstrated in high-fat diet with a low dose of streptozotocin induced diabetic Sprague-Dawley rats and insulin resistance (IR)-3T3-L1 adipocytes. GQD treatment increased expression and activity levels of both PPARγ and PPARα in adipocytes, which transcriptionally affected an ensemble of glucose and lipid metabolic genes in vivo and in vitro. The results clearly indicated that GQD treatment intervened with multiple pathways controlled by concomitantly downstream effects of adipocytic PPARγ and PPARα, to influence two opposite lipid pathways: fatty acid oxidation and lipid synthesis. Antagonist GW9662 decreased the mRNA expression of Pparγ and target genes Adpn and Glut4 whereas GW6471 decreased the mRNA expression of Pparα and target genes Cpt-1α, Lpl, Mcad, Lcad, Acox1, etc. Nuclear location and activity experiments showed that more PPARγ and PPARα shuttled into nuclear to increase its binding activities with target genes. GQD decreased the phosphorylation level of ERK1/2 and/or CDK5 to elevate PPARγ and PPARα activities in IR-3T3-L1 adipocytes through post-translational modification. The increase in p-p38MAPK and SIRT1 under GQD treatment may be attributed to partially reduce PPARγ adipogenesis activity and/or activate PPARα activity. Compared with the rosiglitazone-treated group, GQD elevated Cpt-1α expression, decreased diabetic biomarker Fabp4 expression, which produced an encouraging lipid profile with triglyceride decrease partially from combined effects on upregulated adipocytic PPARγ and PPARα activities. These results suggested that GQD improved diabetes by intervening a diverse array of PPARγ and PPARα upstream and downstream signaling transduction cascades, which jointly optimized the expression of target gene profiles to promote fatty acid oxidation and accelerate glucose uptake and utilization than PPARγ full agonist rosiglitazone without stimulating PPARα activity. Thus, GQD showed anti-diabetic/or antihyperglycemic effects, partially through regulating adipocytic PPARα and PPARγ signaling systems to maintaining balanced glucose and lipid metabolisms. This study provides a new insight into the anti-diabetic effect of GQD as a PPARα/γ dual agonist to accelerate the clinical use.

Screening cyclooxygenase-2 inhibitors from Andrographis paniculata to treat inflammation based on bio-affinity ultrafiltration coupled with UPLC-Q-TOF-MS.

The cyclooxygenase-2 (COX-2) is a key enzyme in the synthesis of prostaglandins, its inhibitors are effective for the treatment of inflammation. In this study, an analytical method based on bio-affinity ultrafiltration coupled with ultra performance liquid chromatography and quadrupole-time-of-flight mass spectrometry (BAUF-UPLC-Q-TOF-MS) was established for rapidly screening and identifying COX-2 ligands. Meanwhile, the specificity of the method was verified by denatured COX-2 and inactive compound. Next, the biological activity of ligands screened was proved by enzyme inhibition assay. The preferred binding modes for these COX-2 inhibitors were then determined by molecular docking. Finally, network pharmacology was used to explore the pathways involved anti-inflammatory effects. As a result, five COX-2 inhibitors were selected in the extract of Andrographis Herba (AH), including andrographolide (1), 14-deoxy-11,12-didehydroandrographiside (2), andrographidine E (3), andrographidine D (4), and deoxyandrographolide (5). Among them, compounds 2, 3, 4 and 5 were reported to have COX-2 inhibitory activity for the first time. The result of COX-2 inhibition assay showed that compound 3 had an IC50 of 19 µM, compounds 2 and 5 had an IC50 of >200 µM. And each ligand could bind to multiple amino acid residues of COX-2 based on molecular docking. At last, combined with network pharmacology, these ligands could exert anti-inflammatory effects through three pathways related to COX-2, arachidonic acid metabolism, synthesis of prostaglandins, and prostanoid ligand receptors. The method established in the study could be used to rapidly screen other enzyme inhibitors in complex mixtures, and help to understand the mechanism of action.

Dendrobium officinale Polysaccharides Inhibit 1-Methyl-2-Nitro-1-Nitrosoguanidine Induced Precancerous Lesions of Gastric Cancer in Rats through Regulating Wnt/ß-Catenin Pathway and Altering Serum Endogenous Metabolites.

Dendrobium officinale is a herb in traditional Chinese medicine where D. officinale polysaccharides (DOP) are the main active ingredient. This study aimed at evaluating DOP efficiency at inhibiting 1-Methyl-2-nitro-1-nitrosoguanidine (MNNG) induced precancerous lesions of gastric cancer (PLGC) in rats through the Wnt/b-catenin pathway and analyzing the variations of serum endogenous metabolites. PLGC was established in male Sprague-Dawley (SD) rats by administering 150 µg/mL MNNG in drinking water for 7 months and giving 0.1 mL of 10% NaCl once weekly during the initial 20 weeks. Treatment with DOP inhibited the progress of PLGC through decreasing the expression of ß-catenin by immunohistochemical analysis. The futher study indicated DOP downregulated gene expression of Wnt2ß, Gsk3ß, PCNA, CyclinD1, and ß-catenin, as well as protein expression of Wnt2ß, PCNA, and ß-catenin. On the other hand, there were nine endogenous metabolites identified after the DOP treatment. Among these, the most significant one is betaine because of its strong antioxidant activity, leading to an anti-tumor effect. DOP can inhibit MNNG-induced PLGC models via regulating Wnt/ß-catenin pathway and by changing endogenous metabolites.

[Compound traditional Chinese medicine in treatment of diabetes].

Diabetes is a chronic disorder of glucose metabolism characterized by elevated blood glucose levels. With the improvement in living standards and the changes in lifestyle,T2 DM incidence has a dramatic increase in the past decades,bringing a series of public health problems. In the research and development field of diabetic drugs,T1 DM drugs are mainly long-acting sustained-release insulin preparations,whereas T2 DM drugs mainly are based on single target. T2 DM drugs usually have a good anti-hyperglycemic effect,but with side effects for long-term administration. Therefore,the research and development of hypoglycemic drugs focus on formula drugs with multi-component,multi-target and multi-pathway effects. In the similar principle of action,traditional Chinese medicine formula has achieved a good efficacy in the treatment of diabetes,with mild anti-hyperglycemic effects and multiple-component synergistic effects in intervening the pathogenesis of diabetes,including additive effect,synergy effect and toxicity scattering effect. This article mainly reviews clinical trials and mechanisms of action of traditional Chinese medicine formula for the treatment of diabetes,so as to provide a reference to the rational and effective clinical application of traditional Chinese medicine formula in treating diabetes.

A Comprehensive and Rapid Quality Evaluation Method of Traditional Chinese Medicine Decoction by Integrating UPLC-QTOF-MS and UFLC-QQQ-MS and its Application.

Decoction is one of the oldest forms of traditional Chinese medicine and it is widely used in clinical practice. However, the quality evaluation and control of traditional decoction is a challenge due to the characteristics of complicated constituents, water as solvent, and temporary preparation. ShenFu Prescription Decoction (SFPD) is a classical prescription for preventing and treating many types of cardiovascular disease. In this article, a comprehensive and rapid method for quality evaluation and control of SFPD was developed, via qualitative and quantitative analysis of the major components by integrating ultra-high-performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry and ultra-fast-performance liquid chromatography equipped with triple quadrupole mass spectrometry. Consequently, a total of 39 constituents were tentatively identified in qualitative analysis, of which 21 compounds were unambiguously confirmed by comparing with reference substances. We determined 13 important constituents within 7 min by multiple reaction monitoring. The validated method was applied for determining five different proportion SFPDs. It was found that different proportions generated great influence on the dissolution of constituents. This may be one of the mechanisms for which different proportions play different synergistic effects. Therefore, the developed method is a fast and useful approach for quality evaluation of SFPD.

[Gegen Qinlian decoction activates PPARγ to ameliorate adipocytic insulin resistance in diabetic SD rats and IR-3T3-L1 adipocytes].

To investigate the effects of Gegen Qinlian decoction(GQD) in improving adipocytic insulin resistance(IR) and explore its related molecular mechanism. Diabetic rats models were induced by high glucose and high-fat diet with a small dose of streptozotocin, and after GQD treatment for 3 months, blood biochemical indexes such as fasting blood-glucose(FBG), insulin, glycosylated serum protein(GSP) and HOMA-IRI were detected and assessed. After the total RNA was extracted from the adipose tissue of diabetic SD rats, PPARγ, ADPN, GLUT4, GLUT2, ACACA and ACACB mRNA expression levels were separately detected by qPCR. Then, stable IR-3T3-L1 adipocyte model was built with 1 µmol•L⁻¹ dexamethasone. After the cell viability was detected by CCK-8 assay, 5%, 10% and 15% GQD-containing serum(GQD-CS) were respectively used to treat IR-3T-L1 adipocytes for 24 h. The contents of glucose, nonesterified fatty acid(NEFA) and adiponectin in cell culture supernatants were separately detected whereas the intracellular triglyceride(TG) contents of IR-3T3-L1 adipocytes were also measured. The ADPN, PPARγ and GLUT4 mRNA and protein expression levels were respectively detected by qPCR and Western blot in IR-3T3-L1 adipocytes. Results showed that GQD significantly decreased fasting blood glucose, insulin and GSP(P<0.01), and down-regulated HOMA-IRI(P<0.05) after the high-fat diet/streptozotocin-induced diabetic SD rats were treated for three months, with a good hypoglycemic effect. Moreover, PPARγ, ADPN, GLUT4, GLUT2, ACACA and ACACB mRNA expression levels were significantly elevated in the adipose tissue of GQD-treated diabetic SD rats. The 5%, 10% and 15% GQD-CS significantly increased glucose consumption of IR-3T3-L1 adipocytes at 24 h treatment(P<0.01), significantly decreased the intracellular TG content (P<0.01), and down-regulated NEFA to a certain extent but not significantly. Moreover, GQD-CS significantly up-regulated GLUT4 and ADPN expression. The results indicated that GQD could activate PPARγ to ameliorate adipocytic insulin resistance in the diabetic SD rats and IR-3T3-L1 adipocytes.

Metabonomic study on the plasma of streptozotocin-induced diabetic rats treated with Ge Gen Qin Lian Decoction by ultra high performance liquid chromatography-mass spectrometry.

Changes in endogenous metabolites in the plasma of streptozotocin (STZ)-induced diabetic rats treated with Ge Gen Qin Lian Decoction (GGQLD) were studied. The endogenous compounds in plasma were detected using ultra high performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS). Rats were divided into three groups: control, model, and administration (4.95g crude drug/kg body weight). After the final administration, plasma samples from the three groups were analyzed using metabonomics. The three sample groups could be clearly distinguished. The administration group exhibited a distinct return to the levels of phytosphingosine and dihydrosphingosine of the control group according to the principal component analysis score, and the corresponding biomarkers were defined. Significant changes in endogenous metabolites, such as dihydrosphingosine, phytosphingosine, cholylglycine, and pantothenic acid, were identified in STZ-induced diabetic rats. These biochemical changes are associated with the metabolism of sphingolipids, fats, and acetyl coenzyme-A, which could be useful to further investigate the characteristics of STZ-induced diabetes mellitus and the therapeutic mechanism of action of GGQLD. This metabonomic analysis could provide a useful starting point toelucidate the therapeutic effects and mechanism of action of GGQLD in diabetes mellitus.

[Effects of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin-resistant adipocytes].

Adipocytokines are closely associated with insulin resistance (IR) in adipose tissues, and they are more and more seriously taken in the study of the development of diabetes. This experiment was mainly to study the effect of berberine on mRNA expression levels of PPARγ and adipocytokines in insulin resistant adipocytes, and investigate the molecular mechanism of berberine in enhancing insulin sensitization and application advantages of droplet digital PCR (ddPCR). ddPCR absolute quantification analysis was taken in this experiment to simply and intuitively determine the appropriate reference genes. ddPCR and quantitative Real-time PCR (qPCR) were used to compare the effect of different doses of berberine (10, 20, 50, 100 µmol•L⁻¹) on mRNA expression levels of PPARγ, adiponectin, resistin and leptin in IR 3T3-L1adipocytes. Antagonist GW9662 was added to study the inherent correlation between PPARγ and adiponectin mRNA expression levels. ddPCR results showed that the expression level of ß-actin in adipocytes was stable, and suitable as reference gene for normalization of quantitative PCR data. Both of ddPCR and qPCR results showed that, as compared with IR models, the mRNA expression levels of adiponectin were decreased in the treatment with berberine (10, 20, 50, 100 µmol•L⁻¹) in a dose-dependent manner (P<0.01); the expression of PPARγ was decreased by 20, 50, 100 µmol•L⁻¹ berberine in a dose-dependent manner in qPCR assay (P<0.01) and decreased only by 50 and 100 µmol•L⁻¹ berberine in ddPCR assay (P<0.05). PPARγ specific antagonist GW9662 intervention experiment showed that adiponectin gene expression was directly relevant with PPARγ (P<0.05). ddPCR probe assay showed that various doses of berberine could significantly reduce mRNA expression levels of resistin and leptin (P<0.01) in a dose-dependent manner. In conclusion, berberine enhanced insulin sensitization effect not by up-regulating adiponect in expression of transcriptional level in PPARγ-dependent manner, but may by the elevated multimerization of adiponectin in the posttranslational regulation level. Berberine down-regulated the resistin and leptin expression levels, which could alleviate lipolysis and improve IR in adipocytes. ddPCR provided better sensitivity and linear range than qPCR, with obvious technical advantages for the detection of low abundance expression of target genes.

[Kudzu root (Ge-Gen) regulates on glucose and lipid metabolism to ameliorating insulin resistance on 3T3-L1 adipocytes].

This study aimed to explore the mechanism of Chinese traditional medicine, Kudzu root(Chinese name：Ge-Gen; Latin name： Puerariae Lobatae Radix) how to improving insulin resistance (IR) through the regulation of the glucose and lipid metabolism in the IR-3T3-L1 adipocytes. After the 3T3-L1 mouse preadipocytes were differentiated into mature adipocytes, IR model(IR-3T3-L1) was built with 1 µmol•L-1 dexamethasone treatment for 96 h. IR adipocytes were treated with different concentrations (5%,10% and 15%) of Ge-Gen containing serum (GG-CS)for 12 h or 24 h, whereas rosiglitazone group as positive control in this study. The glucose contents in cell culture supernatants were detected by glucose oxidase assay and the intracellular triglyceride (TG) contents were measured by glycerol phosphate oxidase assay respectively.The mRNA expression levels of PPARγ, ADPN, GLUT4, LPL, FABP4 and FASn gene were determined by real-time quantitative PCR(qPCR).Results showed that IR-3T3-L1 adipocytes significantly increased glucose consumption (P<0.01)and decreased TG contents (P<0.01) as compared with the normal control group, the glucose consumption significantly increased with the treatment of GG-CS (P<0.01) by dose-dependent and time-dependent manners,whereas the intracellular TG content was sigificantly decreased (P<0.01) by dose-dependent manner.qPCR analysis revealed that 10% and 15% GG-CS significantly up-regulated the mRNA expression level of PPARγ, ADPN and GLUT4 (P<0.01) with the same dose-dependent manner,whereas the GLUT4 mRNA expression was showed similar expression pattern with the treatment of 10% and 15% GG-CS (P<0.01).We also detected the mRNA expression levels of several important lipid-metabolizing enzymes such as LPL, FASn and FABP4 by PPARγ regulation. 15% GG-CS elevated LPL mRNA expression (P<0.05);10% and 15% GG-CS enhanced the FASn mRNA expression (P<0.01), whereas 5%,10% and 15% GG-CS down-regulated FABP4 mRNA expression (P<0.01). Together, our results indicated that GG could regulate the glucose and lipid metabolism to ameliorate IR with multi-target manners in 3T3-L1 adipocytes.

[Study of metabonomics on pharmacological action appraisal Rhizoma coptidis in rats].

OBJECTIVE: HPLC-MS/MS-based metabonomics method was used to find the possible biomarker of Rhizoma Coptidis in rat urine. METHOD: Sprague-Dawley rats were successively administrated 7 g x kg(-1) aqueous extract of Rhizoma Coptidis for 30 days, urine were collected by metabolism cages and detected by using the HPLC-MS-MS. All dates were analyzed by the principal component analysis (PCA) through using the SIMCA-P 10.0 software. RESULT: The PCA demonstrated that the metabolome between treated group and control group had difference in rat urine sample after of 22 days administrated, for treated group 169 kinds of biomarkers were found including oxalacetic acid, malic acid, 2-ketoglutaric acid, NE, arachidonic acid, 5-HIAA and other compounds, the result was consistent with pharmacological effects of R. coptidis, such as antiinflammatory, inhibiting biosynthesis of CA biosynthesis, anticentral nerve and energy metabolism inhibition. CONCLUSION: Metabonomics may be available in pharmacological action evaluation of drugs.