Plasma metabolic biomarkers for syndrome of phlegm and blood stasis in hyperlipidemia and atherosclerosis

OBJECTIVE:To explore the plasma metabolite profiles in patients with the syndrome of phlegm and blood stasis in hyperlipidemia and atherosclerosis(As),and to search for the metabolic biomarkers of the syndrome.METHODS:The plasma metabolite profiles of 31 patients with the syndrome of phlegm and blood stasis in hyperlipidemia and As,6 patients with syndromes without phlegm and blood stasis,and 10 healthy subjects were analyzed by gas chromatography-mass spectrometry(GC-MS).Partial least squares-discriminant analyses(PLS-DA) were used to carry out the pattern-recognition analyses of the data.The plasma metabolic biomarkers of patients were obtained by variable importance plot value(VIP value) and Student’s t-test.The structures of biomarkers were defined by the National Institute of Standards andTechnology(NIST) database.RESULTS:PLS-DA score plots of plasma metabolomes did not show overlap between the phlegm-blood stasis syndrome group and syndromes without phlegm and blood stasis group,whereas significant differences in the concentrations in the plasma of 5 metabolites were found(P< 0.05).They were identified as urine,isoleucine,glucuronic acid,palmitic acid and glycerol by searching in NIST database.The concentrations of four metabolites in the plasma of patients with syndrome of phlegm and blood stasis were higher than those with syndromes without phlegm and blood stasis,whereas the glycerol concentration was lower.CONCLUSION:Compared with patients with syndromes without phlegm and blood stasis,five metabolites showed abnormal levels in patients with the syndrome of phlegm and blood stasis.These metabolites could be diagnostic and prognostic biomarkers.     

Changes in expression of adrenomedullin in the myocardium of streptozotocin-induced diabetic rats

Background Adrenomedullin is a potent vasodilating peptide and involved in many cardiovascular diseases. However, whether adrenomedullin is involved in the pathogenesis of diabetic cardiomyopathy is still unknown. Our aim was to characterize the expression pattern of adrenomedullin in the myocardium of streptozotocin-induced diabetic rats. Methods The weight, blood glucose, and urine glucose of 20 streptozotocin-induced diabetic rats were measured before and after model induction in the diabetic and control groups. The alteration of the adrenomedullin expression was explored in the left ventricular myocardium in both groups by immunohistochemistry. Changes in heart ultrastructure were also analyzed by using hemotoxylin and eosin staining and transmission electron microscopy. All data were analyzed by the independent samples ttest. Results The data of weight, blood glucose, and urine glucose had no significant difference between the control and the diabetic groups before animal model induction. Four weeks after the induction of diabetes, the differences between the two groups in weight, blood glucose, and urine glucose were distinct. When compared with the control group, the diabetic group showed ultrastructural changes including hypertrophy, fibrosis, myofibrillar disarrangements, mitochondrial disruption, and increase in nuclear membrane invaginations. A significant decrease of adrenomedullin expression was also observed in cardiac myocytes of the diabetic rats （P〈0.01）. Conclusions Our study provides experimental evidence that hyperglycemia could damage cardiac myocytes. Down-regulation of cardioprotective peptide adrenomedullin in the myocardium of streptozotocin-induced diabetic rats may contribute to the diabetic cardiomyopathy and left ventricular dysfunction.     

Prediction of response of collagen-induced arthritis rats to methotrexate: An 1H-NMR-based urine metabolomic analysis

Over one half the patients with rheumatoid arthritis (RA) are being treated with methotrexate (MTX). Although well proven, the efficacy of MTX varies in individual patients. This study examined the metabolic biomarkers that can be used to predict the therapeutic effect of MTX by using metabolomic analysis. Rats were immunized with collagen to rapidly cause collagen-induced arthritis (CIA) and then treated with 0.1 mg/kg MTX for 4 weeks. The clinical signs and the histopathological features of CIA were observed to evaluate the therapeutic effects. Urine samples of CIA rats were collected, and analyzed by using 600 M 1H-nuclear magnetic resonance (1H-NMR) for spectral binning after the therapy. The urine spectra were divided into spectral bins, and 20 endogenous metabolites were assigned by Chenomx Suite. Multivariate analyses were performed to identify the spectral pattern of endogenous metabolites related to MTX therapy. The results showed that the clustering of the spectra of the urine samples from the responsive rats (n=20) was different from that from the non-responsive rats (n=11). Multivariate analysis showed difference in metabolic profiles between the responsive and non-responsive rats by using partial least squares-discrimination analysis (PLS-DA) (R2=0.812, Q2=0.604). In targeted profiling, 13 endogenous metabolites (uric acid, taurine, histidine, methionine, glycine, etc.) were selected as putative biomarkers for predicting therapeutic response to MTX. It was suggested that 1H-NMR-based metabolomic analysis can be used to predict the therapeutic effect of MTX, and several metabolites were found to be related to the therapeutic effects of MTX.     

Biogenic Amines as Biomarkers for Neurotoxicity

There has been a surge of interest over the past several years in the use of neurochemical endpoints to contribute to our understanding of the mechanism of action of neurotoxicants. In our present presentation, two biogenic amine systems were selected as examples of biomarkers for neurotoxicity.To investigate these neurochemical endpoints ,two prototype neurotoxicants were evaluated in experimental animals.One agent,reserpine, was used to assess developmental neurotoxicity and administered prenatally ,while the other ,MDMA, was used in the adult animal.The neurochemical biomarkers measured were dopamine,serotonin,and their metabolite(DOPAC and 5-HIAA) concentrations by HPLC/EC and dopamine receptor binding by radioligand receptor techniques.A review of the background, experimental design,and results are presented in this article.Our findings indicate that components of the biogenic amine systems can be used as sensitive neurochemical biomarkers of neurotoxicity.These neurochemical biomarkers can be correlated with neuropathological and behavioral biomarkers to aid in the understanding of mechanisms of neurotoxicity.     

Establishment of an Infected Necrotizing Pancreatitis Model by Retrograde Pancreatic Duct Injection of Sodium Taurocholate and E. coli in Rats

A stable and reliable infected necrotizing pancreatitis （INP） model in rats was established in order to study the pathophysiological mechanism and pathological development role of INP and explore the new therapeutic methods for the diseases. Forty-six SD rats were randomly divided into 5 groups. The animals in group A received the injection of 5% sodium taurocholate into the pancreatic duct and those in group B underwent that of E. coli into the pancreatic duct. The rats in groups C, D and E were subjected to the injection of 5% sodium taurocholate in combination with different concentrations of E. coli （10^3, 10^4, 10^5/mE, respectively） into the pancreatic duct. The dose of injection was 0.1 mL/100 g and the velocity of injection was 0.2 mL/min in all the 5 groups. Eight h after the injection, the survival rate of animals was recorded and the surviving rats were killed to determine the serum content of amylase and perform pathological examination and germ cultivation of the pancreatic tissue. The results showed that acute necrotizing pancreatitis model was induced by injection of 5% sodium taurocholate into the pancreatic duct. The positive rate of germ cultivation in group A was 12.5%. The acute necrotizing pancreatitis model was not induced by injection of E. coli into the pancreatic duct and the positive rate of germ cultivation in group B was 0. The INP model was established in groups C to E. The positive rate of germ cultivation was 60%, 100% and 100% and 8-h survival-rate 100%, 100% and 70% in groups C, D and E, respectively. It was concluded that a stable and reliable model of INP was established by injection of 5% sodium taurocholate in combination with 10^4/mL E. coli into the pancreatic duct with a dose of 0.1 mL/100 g and a velocity of 0.2 mL/min. The pathogenesis of INP might be that the hemorrhage and necrosis of pancreatic tissue induced by sodium taurocholate results in weakness of pancreatic tissue in fighting against the germs. Meanwhile, the necrotic pancreatic tissue provides a good p     

改良补肾活血方对实验性骨关节炎兔子模型的控制代谢及抗氧化作用

Objective: To observe the metabolic, regulatory and anti-oxidative effects of modified Bushen Huoxue Decoction (BSHXD, 补肾活血方), a Chinese herbal medicine for Kidney (Shen)-reinforcement and blood-activation, on an osteoarthritis (OA) rabbit model. Methods: A rabbit model for knee joint OA was established by the classic Hulth''s method. The OA model rabbits were randomized into 5 groups: the model control group, the positive control group treated with glucosamine sulfate, and the three BSHXD treated groups treated respectively with low, moderate, and high doses of BSHXD. In addition, a normal control group and a sham-operated group were set up. Experimental animals were sacrificed after a 7-week treatment, and pathological changes in cartilaginous tissue were estimated using the Mankin criteria. Hydroxyproline (Hyp) and malonaldehyde (MDA) contents in blood serum and urine, as well as superoxide dismutase (SOD) activity and nitric oxide (NO) content in blood serum and knee joint synovial homogenates were detected. Results: Mankin scoring showed insignificant statistical differences between the various treatment groups (P>0.05), but all were better than the model control group (P<0.05). Serum and urinary contents of Hyp and MDA as well as serum and synovial levels of NO were significantly lower, but the SOD activity in blood serum and synovial tissue was higher in the BSHXD treated groups than in the model group (P<0.01); the effect of BSHXD was dose-dependent to some extent. Conclusion: The modified BSHXD shows an effect of improving cartilage metabolism in experimental rabbits with OA, and possesses osteo-chondric protective effects in antagonizing peroxidation injury.     

Biomarkers of Aging：Changes in Circadian Rhythms Related to the Modulation of Metabolic Output

Twenty-four hour (circadian) rhythmicity is an important component of biological variability associated with studies relating to biomarkers of aging.Chronobiological testing techniques must be utilized because(1) many variables that are related to the modulation of metabolic output vary dramatically at different times of the day;(2) various experimental variable and treatment groups must be synchronized with environmental cues that control circadian rhythms;and (3) multiple circadian variables may interact together to modulate the rate of aging.The rhythm for physiological factors such as whole animal metabolic output,body temperature,heart rate,urine flow ,potassium, etc ,were found to be dissociated or altered by the senescence process,behavioral variables such as spontaneous activity, wheel running ,feeding and drinking,verbal performance ,as well as sleep-wakefulness rhythms,seem to be accurate predictors of biological age.Ciercadian rhythms for a variety of enzymes of intermediary metabolism which are directly associated with energy metabolism have been well documented.These well-defined rhythms of enzyme activity have also been shown to degenerate with aging .Rhythms tend to lose amplitude as activity falls with age and as a general loss of regulation(especially time of day where maximal activity might be found) of activity across the 24-h span occurs.As with behavioral variables,changes in enzyme rhythms apper to accurately predict aging.Generally speaking, the loss of temporal organization with age, characterized by decreased circadian amplitude, loose internal synchronization, and poor response to extemal environmental time queues, is associated with poor health states and decreased longevity .Temporal rhythms for whole animal parameters are highly correlated with molecular events, such as regulation of cellular metabolism.DNA repair, and gene expression .Automated data acquisition and process control systems will be required for future chronobiological studies to develop biomarkers of aging.     

Effects of Carvedilol on Cardiomyocyte Apoptosis and Gene Expression In Vivo after Ischemia-Reperfusion in Rats

The effects of carvedilol on cardiomyocyte apoptosis and expression of bcl-2, bax genesfollowing ischemia (0.5 h) and reperfusion (48 h) in vivo and the possible biological mechanism ofcarvedilol inhibiting cardiomyocyte apoptosis were studied. The left anterior descending artery inWistar rats were ligated to establish ischemia-reperfusion (I/R) models. The model animals were di-vided into two groups: I/R group, the model rats not subject to other treatments except ischemia-reperfusion (n=8); carvedilol-treated group (n=8), I/R model rats treated with carvedilol. Eightrats in the sham-operated group were subjected to only experimental open operation. The number ofapoptotic cardiomyocyte was determined by TUNEL staining. Immunohistochemistry and in situ hy-bridization histochemistry (ISHH) were used to detect the expression of bcl-2 and bax genes. Imageprocessing system was used to quantitatively dispose the positive metric substances of both immuno-histochemistry and ISHH through the average optical den     

Effect of Poria cocos Hydroethanolic Extract on Treating Adriamycin-Induced Rat Model of Nephrotic Syndrome

Objective: To evaluate the effect of Poria cocos(Schw.) Wolf hydroethanolic extract(PHE) against nephrotic syndrome(NS) in rats and to identify the potential active components from PHE. Methods: The high content compounds were isolated and purified by using column chromatography followed by preparative highperformance liquid chromatography(p-HPLC). Forty male Wistar rats with adriamycin(ADR)-induced NS were randomly divided into 5 groups, 8 in each group: model control group, positive control group(with prednisone treatment), PHE low-dose group, PHE middle-dose group and PHE high-dose group. Another 8 rats were recruited as vehicle control group. All rats received the intragastric administration of corresponding drugs or saline for 30 days. During the experimental period, rats' behavior and appearance were observed and recorded daily, and their body weights were recorded weekly. After treatment, 24-h urine samples were collected to evaluate the urine protein and urine creatinine(Ucr); then the rats were sacrificed to collect carotid blood and to determine the levels of serum total protein(TP), albumin(Alb), globulin(Glo), total cholesterol(TC) and cytokine interlukin-4(IL-4). Results: Six acidic components were isolated and identified from the PHE section: pachymic acid, 15α-hydroxydehydrotumulosic acid, trametenolic acid, dehydropachymic acid, 3β-hydroxy-lanosta-7,9(11), 24-trien-21-oic-acid and dehydroeburicoic acid. Compared with the model control group, the urine protein content were significantly decreased in the PHE treatment groups and positive control group(P0.05), especially PHE middle-dose group(P0.01). The Ucr values and serum levels of TP, Glo, TC and IL-4 in PHE low-and middle-dose groups were also presented obvious recover tendency as compared with the model control group(P0.05 or P0.01). However, positive control group and all PHE groups indicated no significant therapeutic effect on raising Alb value, although PHE low-and middle-dose treatment groups showed better outcomes than positive control group(P0.05). Conclusions: PHE showed an encouraging therapeutic effect against ADR-induced NS in a rat model. PHE might be a group of effective substances for the treatment of NS.     

Thinking path and method for establishing the disease/Chinese medicine syndrome conjugated pattern of a mammary hyperplasia animal model

Mammary hyperplasia (MHP) is the most commonly encountered mammary disease in women at the child-bearing stage. Especially, atypical hyperplasia which belongs to the precancerous category, is the disease for class Ⅰ prevention of breast cancer. Therefore, advancing the clinical efficacy of MHP treatment is of critical importance. Chinese medicine (CM) and drugs show a peculiar effect in this field; the clinical or experimental researches concerning MHP treatment by CM compounds or patent drugs have been increasing gradually in recent years, but the thinking paths and methods for establishing the MHP animal model are divergent. Particularly, the disease/CM syndrome conjugated model (D/S model) has rarely been studied. For this reason, the pathogenetic mechanism, the establishment of an animal disease model, as well as the thinking paths and methods for establishing the D/S model of MHP are discussed and summarized preliminarily in this paper by the authors. This could provide a new way of thinking and method for creating the MHP model in modern medicine.     

Effect of Qingfei Mixture (清肺合剂) on Pediatric Mycoplasma Pneumoniae Pneumonia with Phlegm Heat Obstructing Fei (Lung) Syndrome

Objective: To explore the effect and mechanism of Qingfei Mixture(清肺合剂), a Chinese medicine, in treating mycoplasma pneumonia(MP) in MP patients and rat model. Methods: A total of 46 MP children with phlegm heat obstructing Fei(Lung) syndrome were randomly assigned to two groups by the method of random number table, with 23 children in each group. The control group was treated with intravenous infusion of azithromycin; the treatment group received intravenous infusion of azithromycin and oral administration of Qingfei Mixture. The treatment course was 7 days. Major symptoms and minor symptoms were observed and scored before and after treatments. A rat model of MP was also established. A total of 120 wistar rats were randomly divided into 5 groups: a normal group, infection group, Qingfei Mixture treatment group, azithromycin treatment group, and Qingfei Mixture + azithromycin treatment group. Each group contained 24 rats, from which every 6 were euthanatized 1, 3, 7 and 14 days after infection. MP DNA in pulmonary tissue homogenates was detected using real-time fluorescence quantitative polymerase chain reaction. Pathology was assessed after hematoxylin(HE) staining and lung tissue pathology scores were determined in pulmonary tissue. Transmission electron microscopic detection and electronic image analysis were performed on lung tissue 3 days after infection. Interleukin(IL)-17 was detected in serum using enzymelinked immunosorbent assay(ELISA) 7 days after infection. Results: In the clinical study, both control and the treatment group showed improved results on removing symptoms of phlegm heat syndrome compared to the control group(P0.05). In animal experiments, On the 7th day after MP infection, as detected by electron microscopy, the pulmonary capillary basement membranes of the azithromycin + Qingfei Mixture treatment group were much thinner than those of the azithromycin or Qingfei mixture treatment groups(P0.05). The level of serum IL-17 in the azithromycin + Qingfei Mixture treatment group was lower than that in the azithromycin or Qingfei Mixture groups(P0.01). Conclusion: Both Qingfei Mixture and azithromycin have therapeutic effects on mycoplasma pneumoniae pneumonia, but the combination of both agents had the greatest effect.     

Applied research on serum protein fingerprints for prediction of Qi deficiency syndrome and phlegm and blood stasis in patients with non-small cell lung cancer

OBJECTIVE:This study screened serum tumor biomarkers by surface enhanced laser desorption/ionization time-of-flight mass spectrometry(SELDI-TOF-MS) to establish a subset which could be used for the prediction of Qi deficiency syndrome and phlegm and blood stasis in patients with non-small cell lung cancer;and as diagnostic model of Chinese medicine.METHODS:Serum samples from 63 lung cancer patients with Qi deficiency syndrome and phlegm and blood stasis,and 28 lung cancer patients with non-Qi deficiency syndrome and phlegm and blood stasis were analyzed using SELDI-TOF-MS with a PBS II-C protein chip reader.Protein profiles were generated using immobilized metal affinity capture(IMAC3) protein chips.Differentially-expressed proteins were screened.Protein peak clustering and classification analyses were performed using Biomarker Wizard and Biomarker Pattern software packages,respectively.RESULTS:A total of 268 effective protein peaks were detected in the 1,000-10,000 Da molecular range for the 15 serum proteins screened(P<0.05).The decision tree model was M 2284.97,with a sensitivity of 96.2% and a specificity of 66.7%.CONCLUSION:SELDI-TOF-MS techniques,combined with a decision tree model,can help identify serum proteomic biomarkers related to Qi deficiency syndrome and phlegm and blood stasis in lung cancer patients;and the predictive model can be used to discriminate between Chinese medicine diagnostic models of disease.     

曾耀明 郑伟伟 余维微 陈余圣 林上助

Objective: To observe the level of serum CGRP and 5-hydroxytryptamine in animals model of irritable bowel syndrome treated with sheng yang yi wei decoction.Methods: 70 SPF SD rats were divided randomly into normal control group,model group,dicetel group,sheng yang yi wei decoction group(low dosage,moderate dosage,high dosage).The rat model of irritable bowel syndrome was made by binding stress and clasping their tails.group.Changes of diarrhea rate were observed in these groups．Radioimmunoassay was used to detect the level of CGRP and 5-HT in plasma．Results：The diarrhea rates in group were decreased after treatment,and had the statistics significance in comparison with model group and dicetel group(P<0.05)．The contents of 5-HT in plasma in model group were higher than those in normal control group,while the levels of CGRP in model group were slighly lower than those in normal control group(P<0.05).After treatment,the contents of 5-HT in plasma in dicetel group and sheng yang yi wei decoction group were markedly decreased than those in model group(P<0.05),while the levels of CGRP had no statistics significance among all groups(P>0.05).Conclusions: Sheng yang yi wei decoction can markly alleviate the symptom of diarrhea,inhibit 5-HT synthesis and secretion in animal model of irritable bowel syndrome,while it does not effect the levels of CGRP.     

Effect of scraping therapy on Interleukin-1 in serum of rats with lumbar disc herniation

OBJECTIVE:To explore the effect of scraping therapy on the Interleukin-1 (IL-1) levels of rats with lumbar disc herniation (LDH). METHODS: Fifty male rats were devided into a blank group (A), a sham operation group (B), a model group (C), a scraping group (D), and a drug group (E). The rats in the group B were treated with sham operation, and groups C, D and E were made into the LDH model by operation. After operation group C were treated with no interventions, D were given scraping and E were fed with azathioprine Then the IL-1 levels of different groups were detected by enzyme-linked immuno sorbent assay method. And the transplanted coccygeal vertebra discs were observed by pathological section. RESULTS: The IL-1 levels in the groups C, D, and E were significantly higher than those in the groups A and B (all P<0.01), which proved the operationwas successful.The IL-1 levels in the groups D and E at different periods had statistical significance (F= 414.158, P<0.01). The treatment periods and interventions have interation (F=46.613, P<0.01). Multiple comparison results showed that the IL-1 levels in the groups D and E was significantly lower than that in the group C (P<0.01), while the IL-1 levels between the groups D and E had no statistical significance (P>0.05). Moreover, pathological section indicated that immuno-inflammatory response was hardly found in coccygeal vertebra discs in the groups A and B, while local immuno-inflammatory responses of the groups D and E were much lighter than that of the group C. CONCLUSION: Scraping therapy could inhibit the immuno-inflammatory responses in the rats with LDH caused by transplantation of autologous nu cleus pulposus.     

Effect of Poria cocos Hydroethanolic Extract on Treating Adriamycin-Induced Rat Model of Nephrotic Syndrome

Objective: To evaluate the effect of Poria cocos (Schw.) Wolf hydroethanolic extract (PHE) against nephrotic syndrome (NS) in rats and to identify the potential active components from PHE. Methods: The high content compounds were isolated and purified by using column chromatography followed by preparative high-performance liquid chromatography (p-HPLC). Forty male Wistar rats with adriamycin (ADR)-induced NS were randomly divided into 5 groups, 8 in each group: model control group, positive control group (with prednisone treatment), PHE low-dose group, PHE middle-dose group and PHE high-dose group. Another 8 rats were recruited as vehicle control group. All rats received the intragastric administration of corresponding drugs or saline for 30 days. During the experimental period, rats'' behavior and appearance were observed and recorded daily, and their body weights were recorded weekly. After treatment, 24-h urine samples were collected to evaluate the urine protein and urine creatinine (Ucr); then the rats were sacrificed to collect carotid blood and to determine the levels of serum total protein (TP), albumin (Alb), globulin (Glo), total cholesterol (TC) and cytokine interlukin-4 (IL-4). Results: Six acidic components were isolated and identified from the PHE section: pachymic acid, 15α-hydroxydehydrotumulosic acid, trametenolic acid, dehydropachymic acid, 3β-hydroxy-lanosta-7,9(11), 24-trien-21-oic-acid and dehydroeburicoic acid. Compared with the model control group, the urine protein content were significantly decreased in the PHE treatment groups and positive control group (P<0.05), especially PHE middle-dose group (P<0.01). The Ucr values and serum levels of TP, Glo, TC and IL-4 in PHE low- and middle-dose groups were also presented obvious recover tendency as compared with the model control group (P<0.05 or P<0.01). However, positive control group and all PHE groups indicated no significant therapeutic effect on raising Alb value, although PHE low- and middle-dose treatment groups showed better outcomes than positive control group (P>0.05). Conclusions: PHE showed an encouraging therapeutic effect against ADR-induced NS in a rat model. PHE might be a group of effective substances for the treatment of NS.     

STUDY ON INFLAMMATORY CELLS IN BALF OF SMOKE-INDUCED CHRONIC BRONCHITIS RAT MODEL

Objective To establish a smoke-induced chronic bronchitis rat model and evaluate the patho-logical change semi-quantitatively, and study the characteristics of the inflammatory cells in the bronchoalveolar lav-age fluid (BALF) in various stages. Methods Chronic bronchitis sequential rat model was established by passively inhaling smoke mixture. Experiments were performed in 30 young male Sprague-Dawley rats, which comprised 5 groups in random, i.e.,4 chronic bronchitis model groups and 1 control group. After stained with hematoxylin and eosin, the specimens were studied by semi-quantitative method to evaluate the morphologic changes in various stages. Meanwhile, the inflammatory cells of the BALF and the activity of myeloperoxidase (MPO) of lung tissue were analysed. Results During the process of the chronic bronchitis, the pathologic score was increasing as time went on, and the typical morphologic changes of chronic bronchitis emerged in the group 7 weeks. The total number of inflammatory cells in BAL