Analysis of Brain Absorption Components and Their Distribution of Tianyuan Zhitong Prescription Based on UPLC-Q-TOF-MS and DESI-MSI / 中国实验方剂学杂志

ObjectiveTo investigate the brain absorption components of Tianyuan Zhitong prescription and their distribution based on ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS）， desorption electrospray ionization mass spectrometry imaging（DESI-MSI） and hyperspectral imaging techniques. MethodTen BALB/c mice were randomly divided into blank group（n=3） and administration group（n=7）， the administration group was gavaged with 0.3 mL of Tianyuan Zhitong prescription liquid at a concentration of about 5 g·mL-1 of the raw material， and the blank group was gavaged with an equal volume of normal saline， and the whole brain of the mice were taken for the preparation of tissue homogenates and frozen sections， respectively. The tissue homogenates were qualitatively analyzed by UPLC-Q-TOF-MS for the brain absorption components in positive and negative ion modes， frozen sections were used for imaging to observe the distribution of these components in the brain. Cytoviva dark-field enhancement microscope was used to perform hyperspectral imaging scanning on the brain sections of mice from each group， and the scattered light data of at least 1 000 pixels in the visible-near-infrared（400-1 000 nm） band in the microscopic field of view were collected and average spectrum were created， which were used to compare the components in the brain tissues of mice from the blank and administration groups. ResultA total of 27 brain absorption components of Tianyuan Zhitong prescription were identified by UPLC-Q-TOF-MS， including 10 organic acids， 5 glycosides， 4 alkaloids， 1 phenol， 4 flavonoids， 2 phthalides and 1 other compound， which were mainly derived from Gastrodiae Rhizoma， Chuanxiong Rhizoma， vinegar-processed Corydalis Rhizoma， Ziziphi Spinosae Semen and processed Morindae Officinalis Radix. A total of 14 components were identified by mass spectrometry imaging， of which ferulic acid， tetrahydropalmatine and N-methyl dehydroberberine were mainly distributed in the cerebral cortex， vitamin B5， vemonoic acid and ricinoleic acid were mainly distributed in the hypothalamus， elemicin， octadecenic acid and octadecanoic acid were mainly distributed in the cortex and hypothalamus， while senkyunolide B， ligustilide， linoleic acid， 9，12-octadecadienoyl ethyl ester and spinosin were distributed in most regions of the brain tissues. Hyperspectral imaging showed that in the visible-near-infrared band range， the average spectrum of the brain tissues of mice in the administration group was significantly red-shifted， indicating that there were differences in the physical properties or contents of the chemical components in the brain between mice in the blank group and those in the administration group， and further verified the results of mass spectrometry imaging. ConclusionThrough the combination of UPLC-Q-TOF-MS and imaging techniques， the pharmacodynamic components of Tianyuan Zhitong prescription in the treatment of headache and the regional characteristics in brain tissue are clarified， which can provide reference for the selection of the index components of the research on the quality standard of this prescription and the research on the mechanism of the pharmacological effect.

Molecular cloning and expression mechanism of Mnp65 in Megalobrama amblycephala response to Aeromonas hydrophilia challenge.

p65 is one of the important subunits of the inflammation-related transcription factor NF-κB. In the present study, we cloned and identified the p65 from Megalobrama amblycephala (Mnp65) by homologous cloning and RACE technique. The full-length Mnp65 cDNA consisted of 2331 bp, and included one open reading frame encoding a 604-amino acid putative protein. The protein sequence included a DNA binding motif, a well conserved N-terminal Rel-homology domain (RHD), and a C-terminal IG-like plexins transcription (IPT). Mnp65 was closely related with the other p65 proteins of Cypriniformes and clearly distinct from that of Perciformes and Salmoniformes in terms of sequence homology. Mnp65 homodimer may interact with IκBα in the IPT domain based on the predicted 3D structure of IκBα/Mnp65 complex. Mnp65 was ubiquitously expressed in M. amblycephala tissues, and the highest levels were detected in muscle and liver. Intragastric infection with Aeromonas hydrophila caused respiratory burst and cytokine storm from 8 h to 48 h, showing significantly higher level of respiratory burst activities and significantly high cytokines levels, such as TNF-α, IL-1ß, IL-6, IL-8 etc., compared to 0 h. In addition, the bacterial challenge downregulated the IkBα, and upregulated Mnp65 and TNF-α in the liver. IkBα-Mnp65 was regulated by the negative feedback of cytokine storm, to increase IkBα and decrease Mnp65. Then cytokine storm was relieved at 96 h. Finally, severe intestinal inflammation was observed from 24 h to 48 h after infection, characterized by extensive villous necrosis, epithelial hyperplasia and lymphocyte infiltration, all of which were relieved at 96 h. Taken together, Mnp65 plays a crucial role in the physiological response of teleost fish to bacterial infection.

High glucose affected respiratory burst activity of peripheral leukocyte via G6PD and NOX inhibition in Megalobrama amblycephala.

High glucose levels are known to impair growth and immune function in fish. Here we investigated the role of glucose-6-phosphate dehydrogenase (G6PD) and NADPH oxidase (NOX) in high glucose-associated impairment of leukocyte respiratory burst activity in Megalobrama amblycephala. We cultured peripheral leukocytes isolated from M. amblycephala with media containing no glucose (non-glucose group), 11.1 mmol/L d-glucose (physiologic glucose group), 22.2 mmol/L d-glucose (high-glucose group), or 11.1 mmol/L d-glucose + 100 µmol/L dehydroepiandrosterone (DHEA) (DHEA-treated group). After 24 h, we assayed production of reactive oxygen species (ROS) as a measure of respiratory burst function as well as activity of G6PD and NOX. The high-glucose group and DHEA-treated group showed significantly reduced respiratory burst function, reduced production of ROS, and reduced G6PD and NOX activity at 24 h, compared to the non-glucose and physiologic glucose groups (P < 0.05). The degree of impairment was similar between high-glucose and DHEA-treated groups (P > 0.05). These findings suggest that reduced NADPH availability likely underlies the suppression of respiratory burst function in M. amblycephala leukocytes exposed to high glucose levels.

In Vivo Analysis of miR-34a Regulated Glucose Metabolism Related Genes in Megalobrama amblycephala.

The Megalobrama amblycephala (M. amblycephala) is one of the most important economic freshwater fish in China. The molecular mechanism under the glucose intolerance responses which affects the growth performance and feed utilization is still confused. miR-34a was reported as a key regulator in the glucose metabolism, but how did the miR-34a exert its function in the metabolism of glucose/insulin in M. amblycephala was still unclear. In this study, we intraperitoneally injected the miR-34a inhibitor (80 nmol/100 g body weight) into M. amblycephala (fed with high starch diet, 45% starch) for 12 h, and then analyzed the gene expression profiling in livers by RNA-seq. The results showed that miR-34a expression in M. amblycephala livers was inhibited by injection of miR-34a inhibitor, and a total of 2212 differentially expressed genes (DEGs) were dysregulated (including 1183 up- and 1029 downregulated DEGs). Function enrichment analysis of DEGs showed that most of them were enriched in the peroxisome proliferator-activated receptor (PPAR), insulin, AMP-activated protein kinase (AMPK) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways, which were all associated with the glucose/lipid metabolic and biosynthetic processes. In addition, we examined and verified the differential expression levels of some genes involved in AMPK signaling pathway by qRT-PCR. These results demonstrated that the inhibition of miR-34a might regulate glucose metabolism in M. amblycephala through downstream target genes.

Comparative transcriptome analysis reveals the gene expression profiling in bighead carp (Aristichthys nobilis) in response to acute nitrite toxicity.

OBJECTIVE: Nitrite exposure induces growth inhibition, metabolic disturbance, oxidative stress, organic damage, and infection-mediated mortality of aquatic organism. This study aimed to investigate the mechanism in responses to acute nitrite toxicity in bighead carp (Aristichthys nobilis, A. nobilis) by RNA-seq analysis. METHODS: Bighead carps were exposed to water with high nitrite content (48.63 mg/L) for 72 h, and fish livers and gills were separated for RNA-seq analysis. De novo assembly was performed, and differentially expressed genes (DEGs) between control and nitrite-exposed fishes were identified. Furthermore, enrichment analysis was performed for DEGs to annotate the molecular functions. RESULTS: A total of 406,135 transcripts and 352,730 unigenes were tagged after de novo assembly. Accordingly, 4108 and 928 DEGs were respectively identified in gill and liver in responses to nitrite exposure. Most of these DEGs were up-regulated DEGs. Enrichment analysis showed these DEGs were mainly associated with immune responses and nitrogen metabolism. CONCLUSIONS: We suggested that the nitrite toxicity-induced DEGs were probably related to dysregulation of nitrogen metabolism and immune responses in A. nobilis, particularly in gill.

Effect of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of bighead carp, Aristichthys nobilis.

Nitrite (NO2-) can cause oxidative stress in aquatic animal when it accumulates in the organism, resulting in different toxic effects on fish. In the present study, we investigated the effects of nitrite exposure on the antioxidant enzymes and glutathione system in the liver of Bighead carp (Aristichthys nobilis). Fish [Initial average weight: (180.05 ±â€¯0.092) g] were exposed to 48.634 mg/L nitrite for 96 h, and a subsequent 96 h for the recovery test. Fish livers were collected to assay antioxidant enzymes activity, hepatic structure and expression of genes after 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h of exposure and12 h, 24 h, 48 h, 72 h, 96 h of recovery. The results showed that the activity of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) increased significantly in the early stages of nitrite exposure. The study also showed that nitrite significantly up-regulated the mRNA levels of glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), and glutathione reductase (GR) after 6, 48, and 72 h of exposure respectively. Nitrite also increased the formation of malondialdehyde (MDA), oxidized glutathione (GSSG), and the activity of catalase (CAT). Nitrite was observed to reduce the activity of superoxide dismutase (SOD) and the level of glutathione (GSH). In the recovery test, GSH and the GSSG recovered but did not return to pre-stress levels. The results suggested that the glutathione system played important roles in nitrite-induced oxidative stress in fish. The bighead carp responds to oxidative stress by enhancing the activity of GSH-Px, GST, GR and up-regulating the expression level of GSH-Px, GST, GR, a whilst simultaneously maintaining the dynamic balance of GSH/GSSG. CAT was also indispensable. They could reduce the degree of lipid peroxidation, and ultimately protect the body from oxidative damage.

Identification of Differentially Expressed Micrornas Associate with Glucose Metabolism in Different Organs of Blunt Snout Bream (Megalobrama amblycephala).

Blunt snout bream (Megalobrama amblycephala) is a widely favored herbivorous fish species and is a frequentlyused fish model for studying the metabolism physiology. This study aimed to provide a comprehensive illustration of the mechanisms of a high-starch diet (HSD) induced lipid metabolic disorder by identifying microRNAs (miRNAs) controlled pathways in glucose and lipid metabolism in fish using high-throughput sequencing technologies. Small RNA libraries derived from intestines, livers, and brains of HSD and normal-starch diet (NSD) treated M. amblycephala were sequenced and 79, 124 and 77 differentially expressed miRNAs (DEMs) in intestines, livers, and brains of HSD treated fish were identified, respectively. Bioinformatics analyses showed that these DEMs targeted hundreds of predicted genes were enriched into metabolic pathways and biosynthetic processes, including peroxisome proliferator-activated receptor (PPAR), glycolysis/gluconeogenesis, and insulin signaling pathway. These analyses confirmed that miRNAs play crucial roles in glucose and lipid metabolism related to high wheat starch treatment. These results provide information on further investigation of a DEM-related mechanism dysregulated by a high carbohydrate diet.

Continuously Monitoring of Concentration of Extracellular Ascorbic Acid in Spinal Cord Injury Model / 分析化学

Acute traumatic spinal cord injury (SCI) represents one of the most devastating injuries that afflict the human body. Ascorbic acid ( AA) plays an important role in mammalian central nervous system, especially in SCI. In this study, the change of AA concentration after SCI was investigated by using an on-line electrochemical method integrated with in vivo microdialysis. A microdialysis probe (2 mm in length) was implanted into the spinal cord of an anesthetized rat (Thoracic-10). Microdialysis perfusate (2 μL/ min) was collected in the sample loop of an on-line injector for direct injection onto a glassy carbon electrode which was modified with the heat-treated single-walled carbon nanotubes (SWNTs). Normal ascorbic acid concentration in the extracellular fluids of spinal cords was (26. 17 ± 1. 25) μmol/ L (n =8). The experimental spinal cord injury, induced by a lesion at T-10, significantly increased the extracellular ascorbic acid levels to (53. 24± 1. 95) μmol/ L (n =8). This study provides the experimental evidence on the essential roles of ascorbic acid in spinal cord injuries.

Chronic stress effects of high doses of vitamin D3 on Megalobrama amblycephala.

Dietary vitamin D3 plays an important role in the growth of aquatic animals, but long-term excessive feeding has potential hazards. In this study, Megalobrama amblycephala specimens were fed different experimental diets with 2000 IU/kg or 200,000 IU/kg of vitamin D3 for 90 days, in order to evaluate chronic stress effects of high doses of vitamin D3 on growth, immunity, and structural damage to enterohepatic tissues. The results showed that high doses of vitamin D3 did not have a significant influence on the growth performance of M. amblycephala (P > 0.05), but it significantly reduced the survival rate after infection by Aeromonas hydrophila (P < 0.05). Serum albumin, alkaline phosphatase, and insulin levels, as well as hepatic total antioxidant capacity, were also significantly reduced (P < 0.05). Serum cortisol levels and hepatic heat stress protein 70 expression in M. amblycephala showed that high doses of vitamin D3 significantly inhibit the anti-stress ability of M. amblycephala (P < 0.05). Paraffin tissue sections and electron microscopy showed that high doses of vitamin D3 could cause different degrees of structural damage to enterohepatic tissues of M. amblycephala. Our results indicate that, although M. amblycephala can tolerate high doses of dietary vitamin D3 over a long period, its glycolipid metabolism, immune function, anti-stress function, and resistance to pathogenic infections are adversely affected.

Distribution and virulence gene comparison of Aeromonas strains isolated from diseased fish and water environment.

A total of 71 Aeromonas strains were isolated in the south of Jiangsu Province China in order to analyze the difference ofAeromonas spp. distribution between diseased fish and water environment. The sequence of 16S rDNA and gyrB demonstrated that the 71 Aeromonas isolates could be divided into 4 species, including A. veronii (55), A. hydrophila (11), A. salmonicida (3) and A. media (2). A. veronii was the most common species isolated from fish and water environment. All Aeromonas isolates were screened for three putative virulence genes, aer, hly and alt. hly was the most common gene among three virulence genes.

On correlation between body mass index and lumbar spine average bone mineral density: a study in male patients with osteopenia and those with osteoporosis / 生物医学工程学杂志

From among the patients who visited the Dept. of Rehabilitation of West China Hospital for arthalgia in the period from Aug. 2003 to Dec. 2005, we recruited 566 male patients who were over 40 years of age and did not have hyperostosis in the lumbar spine, and whose T scores were each less than--1. Their ages ranged from 40 to 93 years, and the average age was 62.93 +/- 13.50. The dual-energy X-ray absorptiometry from DMS Company in France was used to measure the bone density of the L2-4 anterior-posterior. The basic data about the subjects, containing the age, height, weight, diabetes mellitus, exercise and smoking or not, were recorded. Then the body mass index were calculated. In accordance to the T score, the subjects were separated into two groups: osteopenia group and osteoporosis group. In comparison of the basic data between groups, BMI of osteoporosis group was significantly higher than that of the osteopenia group, but the number of the subjects who exercised was smaller (P < 0.05). Multiple linear regression analysis revealed that in the case BMI increased by 1 kg/m2, BMD significantly decreased by 0.003 g/cm2 (P = 0.002), and the age negatively correlated with BMD (B = -0.001, P = 0.035). "Exercise or not" was positively related to BMD (B = 0.028, P = 0.000). "Smoking or not" and BMD were not significantly correlated (P = 0.837). In conclusion, increase of BMI, or we may say, increase of fat, would decrease the lumbar spine average BMD in the patients of osteoporosis or osteopenia. Some reports have pointed out that only by increasing BMI with increased amount of muscles, but not with increased amount of fat, would be beneficial to the prevention of osteoporosis. So we concluded that the muscle amount in the subjects should be taken into account when we probe into the relation between BMI and BMD.

Systematic assessment of acupoint stimulating therapy for deglutition disorders after apoplexy / 中国针灸

<p><b>OBJECTIVE</b>To assess the therapeutic effect of acupoint stimulating therapy on deglutition disorders after apoplexy, so as to provide basis for clinical treatment and further study.</p><p><b>METHODS</b>According to Cochrane systematic assessment method of evidence-based medicine, Meta analysis on randomized controlled trials or half randomized controlled trials of acupoint stimulating therapy for deglutition disorders after apoplexy were made.</p><p><b>RESULTS</b>In 37 Chinese literatures, 3 697 patients in total were enrolled, and the results indicated that the effective rate of acupoint stimulating therapy on deglutition disorders after apoplexy was better than that in the control group [RR was 1.38, 95% CI (1.28, 1.49), Z = 8.38, P < 0.01]; and the cured rate also was better than that of the control group [RR was 2.56, 95% CI (2.15, 3.04), Z = 10.70, P < 0.01].</p><p><b>CONCLUSION</b>The therapeutic effect of acupoint stimulating therapy on deglutition disorders after apoplexy is better than that of the control group, but more randomized, double blind, controlled trials with good designs are needed to confirm this result.</p>

Correlation between obesity and female hyperostosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hyperostosis is a common pathological change among people more than 50 years old; it is connected with many risk factors, which are all indefinite. The aim of this study was to prospectively investigate the correlation between obesity and female hyperostosis.</p><p><b>METHODS</b>Totally 4326 females were included in this study and their basic information including their age, stature, body weight, course of disease, symptoms, medical complications, frequency of exercise and smoking, and X-ray and bone mineral density (BMD) examination results, was carefully collected for a statistical analysis. The t test or chi(2) test was used to evaluate the differences between two groups; an analysis of variance (ANOVA) was used to evaluate the differences among several groups; the relationship between hyperostosis and body mass index (BMI), age, medical complications, exercise, average BMD was analyzed using Logistic regression.</p><p><b>RESULTS</b>The incidence rate of hyperostosis in obese patients was higher than that in patients with normal weight (P = 0.000). Obesity was relevant to hyperostotic sites (P = 0.000), and the incidence of hyperostosis in one or several sites of the lumbar vertebrae, knee joints, and other sites was higher in obese patients than in patients with normal weight. There was also a difference in the extent of hyperostosis between these two groups. BMI had positive effects on the incidence and degree of hyperostosis, which were also relative to the sites of hyperostosis, and the BMI of patients without hyperostosis were much lower than those of the patients with hyperostosis in their lumbar vertebrae, knee joints, or multiple sites. Obesity, age, and exercise had positive effects on the incidence of hyperostosis (P = 0.002, 0.000, 0.018).</p><p><b>CONCLUSIONS</b>Obesity is a significant potential stimulant of hyperostosis, especially hyperostosis in knee joints and multiple sites; keeping fit might be an important way to prevent it.</p>

[Analysis of genetic relationship among Chinese native chicken breeds using microsatellites marker].

The allele frequencies, heterozygosity (H), mean heterozygosity, polymorphism information content (PIC) and genetic relationships were studied in Luyuan chicken, Gushi chicken, Tibetan chicken, Baier chicken, Xianju chicken, Chahua chicken, Dagu chicken,Beijing Fatty chicken, Langshan chicken, Henan Game chicken,Taihe Silkies chicken and Xiaoshan chicken using seven microsatellite DNA markers. The results showed that there were differences among allele fequencies of seven microsatellite loci in 12 Chinese native chicken breeds. Among the 12 Chinese native chicken breeds, mean heterozygosity of Luyuan chicken was the highest (0.5929), and that of Chahua chicken was the lowest (0.3514). There were similar results in PIC. By fuzzy cluster analysis was found that the genetic relationship between Taihe Silkies chicken and Henan Game chicken was the nearest, and that between Gushi chicken and the other chicken breeds was the most distant. The 12 Chinese native chicken breeds were divided into three group by fuzzy cluster analysis. The first group included Taihe Silkies chicken, Henan Game chicken, Langshan chicken, Xiaoshan chicken, Dagu chicken, Beijing Fatty chicken and Luyuan chicken, the second group included Chahua chicken, Tibetan chicken, Xianju chicken and Baier chicken, and the third group included Gushi chicken.