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ObjectiveTo investigate whether the whole intestinal microbiota transplantation in Alzheimer's disease （AD） model mice has more significant effects on ileum intestinal microenvironment in normal mice under the guidance of the theory of traditional Chinese medicine that "interior-exterior relationship exists between the heart and small intestine". MethodsThe whole intestinal microbiota of fourteen 6-month-old specific pathogen free male APP/PS1 double-transgenic AD model mice was transplanted into the gut of six normal C57BL/6J mice of the same age and background treated with mixed antibiotics for 14 days. Then， after 14 days of normal rearing， the mice were sacrificed. Next， the pathological changes in the ileum and colon were observed， and the composition and diversity of the ileal and colonic microbiota was analyzed by sequencing. ResultsAfter the whole intestinal microbiota of AD mice was transplanted into normal mice， pathological analysis showed that only the ileum tissue had mucosal damage and crypt gland epithelial cell degeneration， necrosis， and shedding. Moreover， the microbiota analysis found that only the number of genera （P<0.01）， Chao1 index （P<0.01） and Simpson index of ileal microbiota in normal mice decreased （P<0.01）， and the composition of intestinal microbiota was quite similar to that of AD model mice. ConclusionUnder the effect of whole gut microbiota transplantation in AD mice， the diversity and composition of ileal microbiota change more than that of colonic microbiota in normal mice， and at the same time， it results in pathological damage to the ileal mucosa， indicating that the ileal microenvironment may be more closely related to the occurrence and development of AD， which is highly consistent with the traditional Chinese medicine theory of "interior-exterior relationship between heart and small intestine".
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ObjectiveTo explore the effect of Qishengwan on ileal flora during its treatment of Alzheimer's disease （AD） under the guidance of the theory of "interior-exterior relationship between heart and small intestine". MethodThe AD model was established by bilateral intraventricular injection of β-amyloid 1-42 （Aβ1-42）. The rats were then randomly divided into the blank group， sham-operated group， model group， low-， medium-， and high-dose （5.6， 11.2，22.4 g·kg-1·d-1） Qishengwan groups， and donepezil （0.46 mg·kg-1·d-1） group. After medication for 28 successive days， the spatial memory ability of rats was observed in water maze test， and the levels of Aβ1-42， nuclear transcription factor-κB （NF-κB）， tumor necrosis factor-α （TNF-α）， and interleukin-6 （IL-6） in the hippocampus were analyzed by enzyme-linked immunosorbent assay （ELISA）. Additionally， the contents of the ileum were collected and subjected to 16SrRNA-sequencing analysis for figuring out the changes in ileal flora. ResultCompared with the blank group and sham-operated group， the model group exhibited significantly reduced stay time in the target quadrant and number of target quadrant and platform crossings （P<0.05， P<0.01） and elevated Aβ1-42 content in the hippocampus （P<0.01） and central inflammatory factors NF-κB， TNF-α， and IL-6 （P<0.05， P<0.01）. Compared with the model group， Qishengwan at each dose significantly alleviated the impaired spatial memory function （P<0.05， P<0.01）， improved the deposition of Aβ1-42 in the hippocampus of rats （P<0.05， P<0.01）， and reduced the expression of central nervous system inflammatory factors （P<0.05， P<0.01）， thus exerting a good therapeutic effect on AD rats. The 16SrRNA-sequencing analysis results showed that the structure of the ileal flora in the model group was significantly separated from those in the blank group and sham-operated group. The abundance of Lachnospiraceae NK4A136 group was significantly increased （P<0.01）， while that of Escherichia-Shigella was reduced （P<0.05， P<0.01）. Qishengwan at each dose significantly changed the ileal flora structure and regulated the relative abundance of Lachnospiraceae NK4A136 group， Escherichia-Shigella， and Ruminococcaceae. ConclusionQishengwan has a positive therapeutic effect on AD. It can significantly enhance the memory and cognitive abilities in AD rats， which may be related to its regulation of the structure of rat ileal flora and the relative abundance of Lachnospiraceae NK4A136 group， Escherichia-Shigella， and Ruminococcaceae， the attenuation of the central neuroinflammatory response， and the reduction of central Aβ1-42 deposition.
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Objective:To explore the correlation between the efficacy of <italic>Usnea diffracta</italic> in treating atherosclerosis （AS） and the altered microbial flora in rat ileum based on the interior-exterior relationship between heart and small intestine in traditional Chinese medicine （TCM）. Method:Forty-eight SD rats were randomized into a normal group （<italic>n</italic>=8） and a modeling group （<italic>n</italic>=40）. The AS model was established with high-fat diet combined with intraperitoneal injection of vitamin D<sub>3</sub>. The successfully modeled rats were further randomly divided into the model group， positive control （simvastatin， 4 mg·kg<sup>-1</sup>） group， and low- （0.7 g·kg<sup>-1</sup>）， medium- （1.4 g·kg<sup>-1</sup>）， and high-dose （2.8 g·kg<sup>-1</sup>） <italic>U. diffracta</italic> ethanol extract groups， with eight rats in each group. After four weeks of intervention， the blood， aorta， ileum， and ileum content of rats in each group were collected. The levels of serum lipopolysaccharide （LPS）， tumor necrosis factor-<italic>α </italic>（TNF-<italic>α</italic>） and interleukin 6 （IL-6） were measured by enzyme-linked immunosorbent assay （ELISA）， and the pathological changes in rat thoracic aorta was detected by hematoxylin-eosin （HE） staining. Western blot was conducted to determine the protein expression levels of tight junction protein zonula occluden （ZO-1） and Occludin in rat ileum， and the high-throughput 16S rRNA sequencing technology was employed to detect changes in microbial diversity and abundance in rat ileum of each group. Result:Compared with the normal group， the model group exhibited obvious aortic plaque deposition， increased LPS， TNF-<italic>α</italic>， and IL-6 levels （<italic>P</italic><0.01）， but decreased ZO-1 and Occludin protein expression （<italic>P</italic><0.01）. The comparison with the model group revealed that <italic>U. diffracta</italic> significantly ameliorated the aortic plaque deposition of model rats， lowered serum LPS， TNF-<italic>α</italic>， and IL-6 levels （<italic>P</italic><0.05， <italic>P</italic><0.01）， and up-regulated ZO-1 and Occludin protein expression （<italic>P</italic><0.05， <italic>P</italic><0.01）. The abundance of Proteobacteria and Bacteroidetes in the model group changed significantly in contrast to that in the normal group， and the Bacteroidetes/Firmicutes（B/F） value declined （<italic>P</italic><0.05）. Alpha and Beta diversity analysis indicated higher total number of intestinal flora species in the model group， but lower richness and uneven distribution （<italic>P</italic><0.05， <italic>P</italic><0.01）， with a large number of pathogenic bacteria enriched. The ethanol extract of <italic>U. diffracta</italic> significantly increased the B/F value， corrected the structural disorder of microbial flora in ileum， reduced pathogenic bacteria， and increased the relative abundance of probiotics. Conclusion:<italic>U. diffracta</italic> exerts the therapeutic effect against AS possibly by improving the intestinal microbial communities， strengthening the intestinal mucosal barrier function， and reducing the serum LPS and inflammatory factors.
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Objective::To investigate the antihypertensive effect of Tianmu Jiangya powder and its related antihypertensive mechanism by using SHR rats as a model, and protein expressions provide an experimental basis for the clinical application of Tianmu Jiangya powder in the treatment of hypertension. Method::Sixty male SHR rats were randomly divided into six groups according to body weight after one week of adaptive feeding: model group, valsartan group (12 mg·kg-1), captopril group (9 mg·kg-1), hydrochlorothiazide group (6 mg·kg-1), Tianmu Jiangya powder low and high-dose group (0.36, 1.44 g·kg-1), WKY rats were used as the normal group, and the intragastric administration lasted for 16 weeks. Softron BP-2010A intelligent non-invasive blood pressure meter was used to measure the systolic blood pressure (SBP)and heart rate (HR) of rat tail arteries. Adobe Photoshop CS5 software was used to analyze the left auricle and claw fixed selected areas to evaluate the effect on blood stasis syndrome. Vevo 2100 small animal ultrasound imaging system detects left ventricular ejection fraction (LVEF), left ventricular shortening (FS), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular end-systole dimension (LVIDs), left ventricular end-diastole dimension (LVIDd), interventricular septum end-systolic depth (IVSs), and interventricular septum end-diastolic depth (IVSd). Then the rats were sacrificed and the materials were taken (blood, heart, aorta, liver, kidney, tibia), and the weight of heart, liver, kidney and tibia length were measured and recorded. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of the heart and thoracic aorta. Separation of serum and plasma, and determination of nitric oxide (NO) in serum by nitrate reductase method. Radioimmunoassay was used to detect plasma adrenaline/3 methoxyadrenaline (MN), urea (UREA), and uric acid (UA) contents. The expression of nitric oxide synthase(iNOS) and vascular endothelial growth factor(VEGF) protein in thoracic aorta of each group was detected and analyzed by immunohistochemical method. Result::Compared with normal group, the SBP and HR of the rats in model group were significantly increased (P<0.05). The r value of the claw was significantly reduced and the g value was significantly increased at 8 and 16 weeks (P<0.05). LVEF and FS significantly decreased, LVESV, LVIDs, IVSd increased significantly (P<0.05). Heart weight, heart weight /tibia length, liver weight and liver weight /tibia length, plasma of MN, UREA, and UA contents significantly increased, and promoted the expression of iNOS and VEGF proteins in the aortic (P<0.05). Compared with the model group, the Tianmu Jiangya powder administration group could continuously reduce SBP in SHR rats, maintain HR stability (P<0.05), significantly increase the claw of r value, lower the claw of g value(P<0.05). LVEF, FS significantly increased, LVEDV, LVESV, LVIDd and LVIDs significantly decreased (P<0.05), significantly increased serum NO content, decreased liver weight, liver weight/tibia length, plasma MN, UREA, UA content (P<0.05), and down-regulated the expression of iNOS and VEGF protein in the aorta(P<0.05). Conclusion::Tianmu Jiangya powder has a certain antihypertensive effect, and its mechanism may be mainly related to protecting heart function, improving vascular endothelial function, reducing catecholamines and sedative analgesia.
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Objective:Metabolomics was used to analyze the brain tissue samples of model mice with chronic unpredictable mild stress (CUMS) depression, in order to find out the differential metabolites related to depression and to explore the possible antidepressant mechanism of iridoid part of Valerianae Jatamansi Rhizoma et Radix (IEFV). Method:Forty-two Kunming mice were randomly divided into 6 groups, including the normal group, the model group, the fluoxetine group (2.5 mg·kg-1) and the IEFV low, medium, and high dose groups (doses were 5.73, 11.47, 22.94 mg·kg-1, respectively). The behavioral and biochemical indicators of CUMS model mice were used for pharmacodynamic evaluation with IEFV and a positive drug (fluoxetine) as the intervention drugs. Then, the effect of IEFV on endogenous substances of the brain tissue in CUMS model mice were analyzed by nuclear magnetic resonance hydrogen spectrum (1H-NMR) metabolomics, and multivariate statistical analysis was used to identify the differential metabolites and to enrich the metabolic pathways involved in the differential metabolites. Result:After modeling, the immobility time of the model mice increased significantly, their sucrose preference rate and the excitatory neurotransmitters [5-hydroxytryptamine (5-HT) and norepinephrine (NE)] decreased significantly, indicating the success of modeling. The depression was relieved after IEFV administration, mainly manifested by the recovery of the immobility time, sucrose preference rate and the excitatory neurotransmitters (5-HT and NE). Principal component analysis (PCA) of endogenous metabolites in brain tissue showed that the model group could be significantly separated from the normal group, while the IEFV groups and fluoxetine group all showed a trend of deviating from the model group to the normal group, which was consistent with the behavioral results. The results of orthogonal partial least squares discriminant analysis (OPLS-DA) showed that there were 16 different metabolites between the model group and the normal group, including 12 water-soluble metabolites and 4 liposoluble metabolites. Seven potential metabolism pathways were obtained through MetPA analysis, including metabolism of phenylalanine, metabolism of phenylalanine, tyrosine and tryptophan, metabolism of taurine and hypotaurine acid, metabolism of alanine, aspartic acid and glutamic acid, biosynthesis of valine, leucine and isoleucine, metabolism of D-glutamine and D-glutamate and tricarboxylic acid cycle (TCA). IEFV-high dose group could significantly recall 11 differential metabolites. Conclusion:IEFV may play an antidepressant role mainly by affecting energy metabolism, amino acid metabolism and neurotransmitter levels, which provides a reference for further study on the antidepressant mechanism of IEFV.
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Psychiatric diseases represented by depression have gradually become one of the major health problems of people in the fast-paced, high-pressure society. Severe cases can cause suicides, huge harm or disaster to families and the society. Although modern medicine has made great progress in the field of anti-depressant drug therapy, depression still cannot be cured. At the same time, traditional Chinese medicines(TCM) with a definite curative effect, few adverse reactions, and mild efficacy have received increasing attention. TCM valerianae Jatanmansi Rhizoma et Radix has been widely used to alleviate sleep disorder, and its root extract is popularly known as valerian and used as a mild sedative for a long time in European. Tagara takes Valerianae Jatamansi Rhizoma et Radix as the key ingredient for treatment of depression-type insomnia, and is available abroad. It is reported that iridoid, sesquiterpenes, flavonoids or extract from Valerianae Jatamansi Rhizoma et Radix has a superior anti-depression activity in both animal and clinical trials, and the mechanism is mainly related to the regulation of neurotransmitters in the brain, the improvement of the function of the hypothalamic-pituitary-adrenal (HPA) axis, the resistance of free radicals and inflammation, and the neuroprotective effect. However, there is still lack of report on the anti-depression system and in-depth research of Valerianae Jatamansi Rhizoma et Radix. Therefore, it is urgently necessary to systematically collect and summarize the anti-depressant activity and explain the relevant mechanisms, so as to provide reference for the further development of Valerianae Jatamansi Rhizoma et Radix medicinal resources.
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Nerve growth factor (NGF) is mainly secreted by the neuroglia cells, which can exert biological effect through its receptors on the specific target cell surface. NGF is closely related to neurocyte growth, differentiation and apoptosis. As a neurotropic virus, HSV-1 an easily lead to neurocyte, neuroglia cells death or apoptosis. In this study, the U251 human glioma cells were chosen as target cells to study the change of NGF and its receptors in the apoptosis process of HSV-1 infection. Our results showed that U251 cells were permissive to HSV-1 replication. In the apoptosis process of HSV-1 infected U251 cells, the expression of both NGF and P75NTR increased and then decreased, while the expression of TrkA decreased gradually. These result indicated that HSV-1 was able to induce the abnormal expression of NGF and its receptors in U251 cells.
Subject(s)
Humans , Apoptosis , Cell Line, Tumor , Gene Expression , Glioma , Genetics , Metabolism , Virology , Herpes Simplex , Genetics , Metabolism , Virology , Herpesvirus 1, Human , Genetics , Physiology , Nerve Growth Factor , Genetics , Metabolism , Receptor, Nerve Growth Factor , Genetics , Metabolism , Receptor, trkA , Genetics , Metabolism , Virus ReplicationABSTRACT
<p><b>OBJECTIVE</b>To explore the change of endogenic nerve growth factor (NGF) expression in human glioma cells infected with human cytomegalovirus (HCMV).</p><p><b>METHODS</b>U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV.</p><p><b>RESULTS</b>The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group (P < 0.05).</p><p><b>CONCLUSION</b>HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.</p>
Subject(s)
Humans , Actins , Metabolism , Cell Line, Tumor , Cytomegalovirus , Physiology , Cytomegalovirus Infections , Pathology , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic , Glioma , Genetics , Pathology , Virology , Models, Biological , Nerve Growth Factor , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Reproducibility of Results , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
During the infection of host cells, IE2 protein is one of the first and most abundantly expressed products of HCMV genome, which plays an important role in the controlling of cell cycle and apoptosis. But the correlation between expression level and anti-apoptotic activity of IE2 protein is still not clear. In this study, we successfully established a HCMV IE2 protein expression cell line that was controlled by Tet-On system. The effect of IE2 protein on cell apoptosis and the expression of p53 was detected under different condition of induction. Our results showed that the IE2 protein could inhibit cell apoptosis induced by TNF-alpha. Additionally, the anti-apoptotic activity of IE2 protein seemed to be relevant to its expression level. However, we failed to detect any difference of p53 expression between the IE2 protein expression and non-expression cells. These data indicated that the IE2 protein might inhibit cell apoptosis through regulating different signal pathways.
Subject(s)
Humans , Anti-Bacterial Agents , Pharmacology , Apoptosis , Genetics , Doxycycline , Pharmacology , Gene Expression Regulation , Genetics , HeLa Cells , Immediate-Early Proteins , Genetics , Metabolism , Plasmids , Genetics , Trans-Activators , Genetics , Metabolism , Tumor Suppressor Protein p53 , MetabolismABSTRACT
Objective To explore the molecular types of methicillin-resistant Staphylococcus aureus (MRSA) strains present in major hospitals in Qingdao area, using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) methods, trying to find out the epidemiological characteristics of these MRSA isolates. Correlation of the PFGE types with microbiological phenotypes and clinical data was also studied. Methods 360 isolates of MRSA were procured during 2003 to 2007 from major hospitals in Qingdao. PFGE technology was applied to comparatively analyze the chromosomal DNA digested with endonuclease Sma Ⅰ . Comparison of DNA fragments patterns from each MRSA strain and cluster analysis were performed with the Bionumericus version ' 2.0' software. A dendogram was generated using PFGE macrorestriction fragments on gel images. Data was used to predict the possibility of each PFGE type via SPSS software version 11.0, using the variables as predictors including groups on patient's age, gender, source and the site where MRSA was isolated. Antibiotic sensitivity patterns of these MRSA isolates were determined by K-B tests, and a correlation between these patterns and PFGE types was investigated. Housekeeping genes were amplified with PCR and sequenced in representative strains of variant PFGE types to identify their allelic profile. Results 5 types of PFGE patterns (M0-M4) were identified with MI being the predominant and M2 next to it which was significantly correlated to the isolates from wounds. M3 type strains were mainly isolated from ICU wards and there were a few cases complied with M4 type with no correlated variant factors found in this study. A unique pattern of MRSA isolates with its M0 distinct from other types had not been reported. No significant association was found between PFGE individual types,gender or age groups. M1 and M2 types were the major proportional PFGE patterns among different hospitals. No vancomycin-resistant isolates were detected among 360 MRSA strains. No significant association was found between individual antibiotic resistance and specific PFGE types. Data from MLST analysis showed that the aUelic profiles of M1 and M3 type strain had the same ST239 linage which was commonly present in China. For M2 and M4 representative strains, the allelic profiles were ST5 and ST240, respectively. ST45 and ST398 were corresponding to two PFGE patterns clustered as M0 type. Conclusion Nosocomial infection due to MRSA was evenly distributed among different age groups and no gender bias was observed. The PFGE types of MRSA strains isolated in major hospitals in Qingdao were highly correlated with the sources of isolates and ST239 isolate seemed the prevalent and widespread one. Strategies should be designed to further monitor and prevent or minimize the spread of ST5 MRSA isolates and the like, in Qingdao area.
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Human herpes simplex virus 1 (HSV-1) causes facial,ocular,and encephalitic disease and is associated with latent infection and cancer.Here,we developed a means of studying the pathogenesis of HSV-1 infection at the protein level by using the SELDI Protein Chip to detect changes of protein expression in Vero cells cultured in vitro.After infection with HSV-1 and culture for 12,24 or 48 h,cells were harvested and lysed.IMAC3 arrays were applied to SELDI-TOF-MS to detect proteomic differences before and after infection.The chip detected a series of differentially expressed protein peaks.Interestingly,both peaks at 16 912 Da and 17 581 Da corresponded precisely with the molecular mass of ISG 15,which may participate in antiviral activity during the process of infection.Thus,the results we obtained can serve as a basis to study the pathogenesis of HSV-1 and the interaction between the virus and its host.In addition,they can help in the discovery of new therapeutic targets for treatment of HSV-1 infection.
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<p><b>OBJECTIVE</b>To establish a new method of multiplex semi-nested polymerase chain reaction (PCR) to detect pathogens in cerebrospinal fluid (CSF).</p><p><b>METHODS</b>According to the analysis of the conservative and variable regions in bacterial 16S rRNA genes, we designed universal primers for all bacteria and specific primers for most gram-positive and gram-negative bacteria. All primers were added into the same reaction systems successively of a two-step PCR assay to amplify the different bacterial DNA in CSF, and the results were compared with common culture method with sensitivity and the specificity both detected at the same time.</p><p><b>RESULTS</b>Both gram-positive and gram-negative bacteria amplified DNA fragment about 1,032 bp after first-step amplification with universal primers. In the second step, specific fragments of 336 bp and 127 bp were amplified in gram-positive and gram-negative bacteria respectively besides fragments of 1,032 bp; The detection limit for E. coli was 8 cfu/ml. The comparison of 62 CSF samples detected by both multiplex semi-PCR and conventional culture method revealed sensitivity, specificity, positive and negative values of 93.8%, 95.7%, 88.2%, and 97.8% respectively for PCR.</p><p><b>CONCLUSION</b>The result suggested that the multiplex semi-nested PCR we established was sensitive, specific and rapid method for clinical laboratory to detect pathogens in CSF.</p>