Low intensity physical exercise attenuates cardiac remodeling and myocardial oxidative stress and dysfunction in diabetic rats

We evaluated the effects of a low intensity aerobic exercise protocol on cardiac remodeling and myocardial function in diabetic rats. Wistar rats were assigned into four groups: sedentary control (C-Sed), exercised control (C-Ex), sedentary diabetes (DM-Sed), and exercised diabetes (DM-Ex). Diabetes was induced by intraperitoneal injection of streptozotocin. Rats exercised for 9 weeks in treadmill at 11 m/min, 18 min/day. Myocardial function was evaluated in left ventricular (LV) papillary muscles and oxidative stress in LV tissue. Statistical analysis was given by ANOVA or Kruskal-Wallis. Echocardiogram showed diabetic groups with higher LV diastolic diameter-to-body weight ratio and lower posterior wall shortening velocity than controls. Left atrium diameter was lower in DM-Ex than DM-Sed (C-Sed: 5.73 ± 0.49; C-Ex: 5.67 ± 0.53; DM-Sed: 6.41 ± 0.54; DM-Ex: 5.81 ± 0.50 mm; P < 0.05 DM-Sed vs C-Sed and DM-Ex). Papillary muscle function was depressed in DM-Sed compared to C-Sed. Exercise attenuated this change in DM-Ex. Lipid hydroperoxide concentration was higher in DM-Sed than C-Sed and DM-Ex. Catalase and superoxide dismutase activities were lower in diabetics than controls and higher in DM-Ex than DM-Sed. Glutathione peroxidase activity was lower in DM-Sed than C-Sed and DM-Ex. Conclusion. Low intensity exercise attenuates left atrium dilation and myocardial oxidative stress and dysfunction in type 1 diabetic rats.

Sodium nitrite prevents both reductions in circulating nitric oxide and hypertension in 7-day lead-treated rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alpha(2)-adrenergic receptor distribution and density within the nucleus tractus solitarii of normotensive and hypertensive rats during development

The nucleus tractus solitarii (NTS), located in the brainstem, is one of the main nuclei responsible for integrating different signals in order to originate a specific and orchestrated autonomic response. Antihypertensive drugs are well known to stimulate alpha(2)-adrenoceptor (alpha(2R)) in brainstem cardiovascular regions to induce reduction in blood pressure. Because alpha(2R) impairment is present in several models of hypertension, the aim of the present study was to investigate the distribution and density of alpha(2R) binding within the NTS of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats during development (1,15,30 and 90 day-old) by an in vitro autoradiographical study. The NTS shows heterogeneous distribution of alpha(2R) in dorsomedial/dorsolateral, subpostremal and medial/intermediate subnuclei. Alpha(2R) increased from rostral to caudal dorsomedial/dorsolateral subnuclei in 30 and 90 day-old SHR but not in WKY. Alpha(2R) decreased from rostral to caudal subpostremal subnucleus in 15, 30 and 90 day-old SHR but not in WKY. Medial/intermediate subnuclei did not show any changes in alpha(2R) according to NTS levels. Furthermore, alpha(2R) are decreased in SHR as compared with WKY in all NTS subnuclei and in different ages. Surprisingly, alpha(2R) impairment was also found in pre-hypertensive stages, specifically in subpostremal subnucleus of 15 day-old rats. Finally, alpha(2R) decrease from 1 to 90 day-old rats in all subnuclei analyzed. This decrease is different between strains in rostral dorsomedial/dorsolateral and caudal subpostremal subnuclei within the NTS. In summary, our results highlight the importance of alpha(2R) distribution within the NTS regarding the neural control of blood pressure and the development of hypertension. (C) 2011 Elsevier B.V. All rights reserved.

Bacterial translocation in rats nonfunctioning diverted distal colon

To investigate whether the alterations of the diverted colon segment mucosa, evidenced in fecal colitis, would be able to alter Bacterial Translocation (BT). Methods: Sixty-two Wistar male rats ranging from 220 to 320 grams of weight, were divided in two groups: A (Colostomy) and B (Control), with 31 animals each one. In group A, all animals underwent end colostomy, one stoma, in ascending colon; and in the 70th POD was injected in five rats, by rectal route – diverted segment - 2ml of a 0.9% saline solution in animals (A1 subgroup); in eight it was inoculated, by rectal route, 2ml of a solution containing Escherichia coli ATCC 25922 (American Type Culture Collection), in a concentration of 108 Colony Forming Unit for milliliters (CFU/ml) - A2 Subgroup; in ten animals the same solution of E. coli was inoculated, in a concentration of 1011 CFU/ml (A3 Subgroup); and in eight it was collected part of the mucus found in the diverted distal colonic segment for neutral sugars and total proteins dosage (A4 subgroup). The animals from the group B underwent the same procedures of group A, but with differences in the colostomy confection. In rats from subgroups A1, A2, A3, B1, B2, and B3 2ml of blood were aspirated from the heart, and fragments from mesenteric lymphatic nodule, liver, spleen, lung and kidney taken for microbiological analysis, after their death. This analysis consisted of evidencing the presence of E. coli ATCC 25922 CFU. Mann-Whitney and ANOVA Tests were applied as analytic techniques for association of variables. Results: The occurrence of BT was evidenced only in those animals in which inoculated concentration of E. coli ATCC 25922, reached levels of 1011CFU/ml, i.e. in Subgroups A3 and B3, although, being significantly greater (80%) in those animals without colostomy (subgroup B3) when compared to the ones with colostomy (20%) from the subgroup A3 (P <0.05). Lung, liver and mesenteric lymphatic nodules were the tissues with larger percentile of bacterial recovery, so much in subgroup A3, as in B3. Blood culture was considered positive in 60% of the animals from subgroup B3 and in 10% of those from subgroup A3 (p <0.05). There was greater concentration of neutral sugars, in subgroup A4 - mean 27.3mg/ml -, than in subgroup B4 - mean 8.4mg/ml - (P <0.05). Conclusion: The modifications in the architecture of intestinal mucosa in colitis following fecal diversion can cause alterations in the intestinal barrier, but it does not necessarily lead to an increased frequency of BT

Bacterial translocation in rats nonfunctioning diverted distal colon

To investigate whether the alterations of the diverted colon segment mucosa, evidenced in fecal colitis, would be able to alter Bacterial Translocation (BT). Methods: Sixty-two Wistar male rats ranging from 220 to 320 grams of weight, were divided in two groups: A (Colostomy) and B (Control), with 31 animals each one. In group A, all animals underwent end colostomy, one stoma, in ascending colon; and in the 70th POD was injected in five rats, by rectal route – diverted segment - 2ml of a 0.9% saline solution in animals (A1 subgroup); in eight it was inoculated, by rectal route, 2ml of a solution containing Escherichia coli ATCC 25922 (American Type Culture Collection), in a concentration of 108 Colony Forming Unit for milliliters (CFU/ml) - A2 Subgroup; in ten animals the same solution of E. coli was inoculated, in a concentration of 1011 CFU/ml (A3 Subgroup); and in eight it was collected part of the mucus found in the diverted distal colonic segment for neutral sugars and total proteins dosage (A4 subgroup). The animals from the group B underwent the same procedures of group A, but with differences in the colostomy confection. In rats from subgroups A1, A2, A3, B1, B2, and B3 2ml of blood were aspirated from the heart, and fragments from mesenteric lymphatic nodule, liver, spleen, lung and kidney taken for microbiological analysis, after their death. This analysis consisted of evidencing the presence of E. coli ATCC 25922 CFU. Mann-Whitney and ANOVA Tests were applied as analytic techniques for association of variables. Results: The occurrence of BT was evidenced only in those animals in which inoculated concentration of E. coli ATCC 25922, reached levels of 1011CFU/ml, i.e. in Subgroups A3 and B3, although, being significantly greater (80%) in those animals without colostomy (subgroup B3) when compared to the ones with colostomy (20%) from the subgroup A3 (P <0.05). Lung, liver and mesenteric lymphatic nodules were the tissues with larger percentile of bacterial recovery, so much in subgroup A3, as in B3. Blood culture was considered positive in 60% of the animals from subgroup B3 and in 10% of those from subgroup A3 (p <0.05). There was greater concentration of neutral sugars, in subgroup A4 - mean 27.3mg/ml -, than in subgroup B4 - mean 8.4mg/ml - (P <0.05). Conclusion: The modifications in the architecture of intestinal mucosa in colitis following fecal diversion can cause alterations in the intestinal barrier, but it does not necessarily lead to an increased frequency of BT

Thyroxine and reserpine-induced changes in metabolic profiles of rat urine and the therapeutic effect of Liu Wei Di Huang Wan detected by UPLC-HDMS

The promise of metabonomics, a new "omics" technique, to validate Chinese medicines and the compatibility of Chinese formulas has been appreciated. The present study was undertaken to explore the excretion pattern of low molecular mass metabolites in the male Wistar-derived rat model of kidney yin deficiency induced with thyroxine and reserpine as well as the therapeutic effect of Liu Wei Di Huang Wan (LW) and its separated prescriptions, a classic traditional Chinese medicine formula for treating kidney yin deficiency in China. The study utilized ultra-performance liquid chromatography/electrospray ionization synapt high definition mass spectrometry (UPLC/ESI-SYNAPT-HDMS) in both negative and positive electrospray ionization (ESI). At the same time, blood biochemistry was examined to identify specific changes in the kidney yin deficiency. Distinct changes in the pattern of metabolites, as a result of daily administration of thyroxine and reserpine, were observed by UPLC-HDMS combined with a principal component analysis (PCA). The changes in metabolic profiling were restored to their baseline values after treatment with LW according to the PCA score plots. Altogether, the current metabonomic approach based on UPLC-HDMS and orthogonal projection to latent structures discriminate analysis (OPLS-DA) indicated 20 ions (14 in the negative mode, 8 in the positive mode, and 2 in both) as "differentiating metabolites".

Simultaneous determination by UPLC-ESI-MS of scoparone, capillarisin, rhein, and emodin in rat urine after oral administration of Yin Chen Hao Tang preparation

A simple, sensitive, and validated method was developed for simultaneous determination of scoparone, capillarisin, rhein, and emodin in rat urine by ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (UPLC-MS). The urinary samples were analyzed on an Acquity UPLC BEH C18 1.7 microm 2.1x50 mm column. Scoparone, capillarisin, rhein, and emodin in rat urine were simultaneously analyzed with good separation. The lower limits of detection were 6.0, 9.0, 7.0, and 3.0 ng/mL, and the lower limits of quantification were 20.0, 33.0, 24.0, and 12.0 ng/mL for scoparone, capillarisin, rhein, and emodin, respectively. The intra- and inter-day precisions (RSD) were less than 9%. The intra- and inter-accuracies were found to be in the range of 94.14-104.54% for scoparone, 101.72-107.34% for capillarisin, 95.24-103.59% for rhein, and 101.32-107.82% for emodin at three concentration levels. The absolute recoveries for scoparone, capillarisin, rhein, and emodin were not less than 77.0%. The developed method has been applied to determine scoparone, capillarisin, rhein, and emodin in rat urine after oral administration of Yin Chen Hao Tang preparation, a traditional Chinese medicine formulation widely used in China for treatment of jaundice and liver disorders.

Development of a rapid and validated method for investigating the metabolism of scoparone in rat using ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry

Scoparone (6,7-dimethoxycoumarin) is known to have a wide range of pharmacological properties. In this study, a rapid and validated ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (UPLC/ESI-QTof-MS) method was developed to investigate the metabolism of scoparone in rat for the first time. The new method reduced the sample handling and analytical time by three- to six-fold, and the detection limit by five- to 1000-fold, compared to published methods. Far more metabolites were detected and identified compared to published data, which were preliminarily identified as scopoletin, isoscopoletin, isofraxidin, and fraxidin, respectively, when subjected to tandem mass spectrometry analyses. It is found that the metabolic trajectory of scoparone in rat focused on phase I metabolism which is obviously different from published results, and revealed a wide range of pharmacological properties of scoparone partly attributed to the bioactivities of its metabolites.

Protective effects of sweroside on human MG-63 cells and rat osteoblasts

Herbal Fructus Corni is a folk medicine with a long history of safe use for treating osteoporosis in postmenopausal women or elderly men in Asia. Sweroside is a bioactive herbal ingredient isolated from Fructus Corni, which has been widely used for the treatment of osteoporosis in traditional Chinese medicine (TCM). Unfortunately, the working mechanisms of this compound are difficult to determine and thus remain unclear. The aim of the study was performed to determine the potential molecular mechanism of the anti-osteoporotic effect of sweroside on the human MG-63 cells and rat osteoblasts. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to observe the effect of sweroside on cell proliferation. The activity of alkaline phosphatase (ALP) and the amount of osteocalcin were also assayed the cell differentiation. Sweroside significantly increased the proliferation of human MG-63 cells and rat osteoblasts (P<0.01). It increased the activity of ALP, and osteocalcin was also elevated in response to sweroside (P<0.05). Of note, flowcytometer assay showed that sweroside can attenuate and inhibit apoptosis. Sweroside has a direct osteogenic effect on the proliferation and differentiation of cultured human MG-63 cells and rat osteoblasts in vitro. These data will help in understanding the molecular mechanisms of therapeutic efficacy of sweroside, and highlight insights into drug discovery. In the current study, sweroside has been suggested to be a promising osteoporosis therapeutic natural product.

Pharmacokinetics of isofraxidin in rat plasma after oral administration of the extract of acanthopanax senticosus using HPLC with solid phase extraction method

High-performance liquid chromatography coupled with solid phase extraction method was developed for determination of isofraxidin in rat plasma after oral administration of Acanthopanax senticosus extract (ASE), and pharmacokinetic parameters of isofraxidin either in ASE or pure compound were measured. The HPLC analysis was performed on a Dikma Diamonsil RP(18) column (4.6 mm x 150 mm, 5 microm) with the isocratic elution of solvent A (acetonitrile) and solvent B (0.1% aqueous phosphoric acid, v/v) (A : B = 22 : 78) and the detection wavelength was set at 343 nm. The calibration curve was linear over the range of 0.156-15.625 microg/ml. The limit of detection was 60 ng/ml. The intra-day precision was 5.8%, and the inter-day precision was 6.0%. The recovery was 87.30+/-1.73%. When the dosage of ASE is equal to pure compound caculated by the amount of isofraxidin, it has been found to have two maximum concentrations in plasma while the pure compound only showed one peak in the plasma concentration-time curve. The determined content of isofraxidin in plasma after oral administration of ASE is the total contents of free isofraxidin and its precursors in ASE in vitro. The pharmacokinetic characteristics of ASE showed the priority of the extract and the properities of traditional Chinese medicine.

Simultaneous determination of 6,7-dimethylesculetin and geniposide in rat plasma and its application to pharmacokinetic studies of Yin Chen Hao Tang preparation

A method for the rapid and simultaneous determination of 6,7-dimethylesculetin (CAS 120-08-1) and geniposide (CAS 24512-63-8) in rat plasma has been developed, using validated high performance liquid chromatography (HPLC) with solid phase extraction (SPE). The HPLC analysis was performed on a commercially available column (200 mm x 4.6 mm, 5 microm) with acetonitrile-methanol-0.1% aqueous formic acid as mobile phase and the UV detection at 343 nm and 238 nm for 6,7-dimethylesculetin and geniposide, respectively. The calibration curves for 6,7-dimethylesculetin and geniposide were linear over the range 0.4-25.6 microg/mL and 1.12-71.68 microg/mL, respectively. The lower limits of quantitation were 0.40 microg/ mL and 1.12 microg/mL, and the lower limits of detection were 0.06 microg/mL and 0.09 microg/ mL, respectively. The intra-day and inter-day precision for 6,7-dimethylesculetin and geniposide were < 5%, whereas the absolute recovery percentages were > 74%. A successful application of the developed HPLC analysis was demonstrated for the pharmacokinetic study of a Traditional Chinese Medicine formula of Yin Chen Hao Tang preparation.

Analysis of the constituents in the rat plasma after oral administration of Yin Chen Hao Tang by UPLC/Q-TOF-MS/MS

A UPLC/Q-TOF-MS/MS method for analyzing the constituents in rat plasma after oral administration of Yin Chen Hao Tang (YCHT), a traditional Chinese medical formula, has been established. The UPLC/MS fingerprints of the samples were established first in vitro and in vivo, with 45 compounds in YCHT and 21 compounds in rat plasma after oral administration of YCHT were detected. Of the 45 detected compounds in vitro, 30 were identified, and all of the 21 compounds detected in rat plasma were identified either by comparing the retention time and mass spectrometry data with that of reference compounds or by mass spectrometry analysis and retrieving the reference literatures. Of the identified 21 compounds in rat plasma, 19 were the original form of compounds absorbed from the 45 detected compounds in vitro, 2 were the metabolites of the compounds existed in YCHT. It is concluded that a rapid and validated method has been developed based on UPLC-MS/MS, which shows high sensitivity and resolution that is more suitable for identifying the bioactive constituents in plasma after oral administration of Chinese herbal medicines, and provides helpful chemical information for further pharmacology and active mechanism research on the Chinese medical formula.

Pharmacokinetics of cimifugin in rat plasma after oral administration of the extract of Saposhnikovia divaricatae root : Determination of cimifugin by high performance liquid chromatography coupled with solid phase extraction

High performance liquid chromatography (HPLC) coupled with the solid phase extraction method was developed for determining cimifugin (a coumarin derivative; one of Saposhnikovia divaricatae's constituents) in rat plasma after oral administration of Saposhnikovia divaricatae extract (SDE), and the pharmacokinetics of cimifugin either in SDE or as a single compound was investigated. The HPLC analysis was performed on a commercially available column (4.6 mm x 200 mm, 5 pm) with the isocratic elution of solvent A (Methanol) and solvent B (Water) (A:B=60:40) and the detection wavelength was set at 250 nm. The calibration curve was linear over the range of 0.100-10.040 microg/mL. The limit of detection was 30 ng/mL. At the rat plasma concentrations of 0.402, 4.016, 10.040 microg/mL, the intra-day precision was 6.21%, 3.98%, and 2.23%; the inter-day precision was 7.59%, 4.26%, and 2.09%, respectively. The absolute recovery was 76.58%, 76.61%, and 77.67%, respectively. When the dosage of SDE was equal to the pure compound calculated by the amount of cimifugin, it was found to have two maximum peaks while the pure compound only showed one peak in the plasma concentration-time curve. The pharmacokinetic characteristics of SDE showed the superiority of the extract and the properties of traditional Chinese medicine.

Comparative study on the protective effects of Yinchenhao Decoction against liver injury induced by alpha-naphthylisothiocyanate and carbon tetrachloride

OBJECTIVE: To optimize the animal model of liver injury that can properly represent the pathological characteristics of dampness-heat jaundice syndrome of traditional Chinese medicine. METHODS: The liver injury in the model rat was induced by alpha-naphthylisothiocyanate (ANIT) and carbon tetrachloride (CCl(4) ) respectively, and the effects of Yinchenhao Decoction (, YCHD), a proved effective Chinese medical formula for treating the dampness-heat jaundice syndrome in clinic, on the two liver injury models were evaluated by analyzing the serum level of alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP), malondialchehyche (MDA), total bilirubin (T-BIL), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) as well as the ratio of liver weight to body weight. The experimental data were analyzed by principal component analytical method of pattern recognition. RESULTS: The ratio of liver weight to body weight was significantly elevated in the ANIT and CCl(4) groups when compared with that in the normal control (P<0.01). The contents of ALT and T-BIL were significantly higher in the ANIT group than in the normal control (P<0.05,P<0.01), and the levels of AST, ALT and ALP were significantly elevated in CCl(4) group relative to those in the normal control P<0.01). In the YCHD group, the increase in AST, ALT and ALP levels was significantly reduced (P<0.05, P<0.01), but with no significant increase in serum T-BIL. In the CCl(4) intoxicated group, the MDA content was significantly increased and SOD, GSH-PX activities decreased significantly compared with those in the normal control group, respectively (P<0.01). The increase in MDA induced by CCl(4) was significantly reduced by YCHD P<0.05). CONCLUSION: YCHD showed significant effects on preventing liver injury progression induced by CCl(4), and the closest or most suitable animal model for damp-heat jaundice syndrome may be the one induced by CCl(4).

A rapid and sensitive UPLC-ESI MS method for analysis of isofraxidin, a natural antistress compound, and its metabolites in rat plasma

Isofraxidin is one of the main bioactive constituents in the root of Acanthopanax senticosus, which has antifatigue, antistress, and immuno-accommondating effects. In this study, an ultraperformance LC (UPLC)-ESI MS method was developed for analyzing isofraxidin and its metabolites in rat plasma. The analysis was performed on a UPLC coupled with ESI MS (quadropole MS tandem TOF MS). The lower LOD (LLOD) for isofraxidin was 0.25 ng/mL, the intraday precision was less than 10%, the interday precision was less than 10%, and the extraction recovery was more than 80%. Isofraxidin and two metabolites (M1 and M2) were detected in rat plasma after oral administration of isofraxidin, and the molecular polarities of M1 and M2 were both increased compared to isofraxidin. The metabolites were identified as 5,6-dihydroxyl-7-methoxycoumarin and 5-hydroxyl-6,7-dimethoxycoumarin when subjected to parent ion spectra, product ion spectra, and extract mass and element composition analyses.