Study of mass spectrometry imaging metabolomics of paclitaxel based on esophageal cancer multicellular tumor spheroids cultured in vitro / 药学学报

Multicellular tumor spheroids (MCTS) can simulate the structure and metabolic characteristics of tumors in vivo, which is of great significance to study the metabolic phenotype of tumor cells and the mechanism of drug intervention. In this study, esophageal cancer MCTS were constructed, and MCTS frozen sections were prepared after treated with different formulations of paclitaxel (PTX) including common PTX injection, PTX liposome and albumin bound PTX. MCTS mass spectrometry imaging analysis method was established by using air flow assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI). The visualization of the permeation and enrichment process of PTX in MCTs after PTX treatment was realized, and the spatially resolved metabolomics of PTX injection group was studied. The results showed that the permeation and enrichment behavior of PTX in MCTs model were related to the formulations. The changes of endogenous metabolites in MCTs of esophageal cancer after treated with PTX injection had temporal and spatial characteristics. The metabolic changes of MCTS during the initial 0-4 hours were dominated by the down-regulation of middle-high polarity metabolites and some lipids in the central region of MCTS, while the metabolic changes of MCTS during 8-72 hours were mainly up-regulated by lipid metabolites in the peripheral region of MCTS. The combination of in vivo tumor-associated MCTs model with label free, highly sensitive and high coverage mass spectrometry imaging technology provided a new method and strategy for the study of pharmacometabolomics.

Metabonomic study of biochemical changes in serum of PCPA-induced insomnia rats after treatment with Suanzaoren Decoction / 中国中药杂志

Suanzaoren Decoction(SZRD) is a classical formula for the clinical treatment of insomnia. This study analyzed the effect of SZRD on endogenous metabolites in insomnia rats based on metabonomics and thereby explored the anti-insomnia mechanism of SZRD. To be specific, DL-4-chlorophenylalanine(PCPA) was used to induce insomnia in rats. Then pathological changes of the liver and brain were observed and biochemical indexes such as 5-hydroxytryptamine(5-HT), dopamine(DA), glutamate(Glu), γ-aminobutyric acid(GABA), and norepinephrine(NE) in the hippocampus and prostaglandin D2(PGD2), tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and IL-6 in the serum of rats were detected. On this basis, the effect of SZRD on PCPA-induced insomnia rats was preliminarily assessed. The metabolic profile of rat serum samples was further analyzed by ultra-performance liquid chromatography-quadrupole-time of flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS). Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were combined with t-test and variable importance in projection(VIP) to identify differential metabolites, and MetaboAnalyst 5.0 was employed for pathway analysis. The results showed that SZRD could improve the pathological changes of brain and liver tissues, increase the levels of neurotransmitters 5-HT, DA, and GABA in hippocampus and the level of PGD2 in hypothalamic-pituitary-adrenal axis(HPA axis), and reduce the levels of IL-1β and TNF-α in serum of insomnia rats. Metabonomics analysis yielded 12 significantly changed potential metabolites: 5-aminovaleric acid, N-acetylvaline, L-proline, L-glutamate, L-valine, DL-norvaline, D(-)-arginine, pyroglutamic acid, 1-methylguanine, L-isoleucine, 7-ethoxy-4-methylcoumarin, and phthalic acid mono-2-ethylhexyl ester(MEHP), which were related with multiple biochemical processes including metabolism of D-glutamine and D-glutamate, metabolism of alanine, aspartate, and glutamate, metabolism of arginine and proline, arginine biosynthesis, glutathione metabolism. These metabolic changes indicated that SZRD can improve the metabolism in insomnia rats by regulating amino acid metabolism.

Identification of Chemical Constituents in Suanzaoren Tang Granules by UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:A comprehensive and in-depth analysis method for identification of chemical constituents in Suanzaoren Tang granules was established. Method:Ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry （UPLC-Q-TOF-MS/MS） was employed with the mobile phase of 0.1% formic acid aqueous solution （A）-acetonitrile （B） for gradient elution （0-8 min， 5%-17%B； 8-10 min， 17%B； 10-11 min， 17%-18%B； 11-12 min， 18%-20%B； 12-17 min， 20%-23%B； 17-22 min， 23%-33%B； 22-30 min， 33%-60%B； 30-32 min， 60%-100%B； 32-36 min， 100%B）， the flow rate of 0.3 mL·min^-1 and electrospray ionization （ESI）. High quality MS/MS data were scanned in positive and negative ion modes with scanning range of <italic>m</italic>/<italic>z</italic> 50-1 500. The local database of the chemical components from different Chinese medicines in Suanzaoren Tang granules was established by SCIEX OS software. Then the chemical components in Suanzaoren Tang granules were characterized by matching with the local database and comparing with the reference substance and literature information. Result:A total of 134 compounds were characterized and identified under positive and negative ion modes， mainly including flavonoids， triterpenoids， phthalides， steroidal saponins， alkaloids and organic phenolic acids. In addition， the sources of Chinese medicines for all compounds identified in Suanzaoren Tang granules were assigned. Among them， 41 were from Ziziphi Spinosae Semen， 11 were from Poria， 22 were from Anemarrhenae Rhizoma， 28 were from Chuanxiong Rhizoma and 35 were from Glycyrrhizae Radix et Rhizoma. Conclusion:The method can be used to identify the chemical constituents in Suanzaoren Tang granules systematically， quickly and accurately， which can provide a new strategy for the rapid and accurate identification of other Chinese patent medicines.

The development of auditory speech perception and spatial hearing abilities within one year after cochlear implantation in preschool prelingual deaf children / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate the development of auditory speech perception and spatial hearing abilities within one year after cochlear implantation in preschool prelingual deaf children and the relationship between the two abilities. Methods: This retrospective study analyzed 31 preschool children with an average age of (2.3±1.2) years. All cases were assessed at pre-implant, 6 months and 12 months post-implant using the Infant-toddler Meaningful Auditory Integration Scale (IT-MAIS), the Meaningful Auditory Integration Scale (MAIS) and the Mandarin Early Speech Perception test (MESP) to evaluate their listening and speech perception abilities, and using the Speech,Spatial,and Other Qualities of Hearing Scale for Parents (SSQ-P) questionnaires to evaluate their speech perception and spatial hearing abilities. SPSS 23.0 was used for the statistical analysis. Results: All children performed better at 6 months and 12 months post-implant with IT-MAIS/MAIS, MESP than pre-implant, and the scoring rate continued to improve, with a significant difference (P<0.01). For the SSQ-P (Speech) and SSQ-P (Spatial) scores, the mean scores of pre-implant were (0.9±0.2) points and (0.8±0.3) points, those of 6 months post-implant were (4.6±0.2) and (2.6±0.3), and 12 months post-implant were (6.2±0.2) and (6.3±0.3), the scores of the two groups were significantly different at pre-implant, 6 months and 12 months post-implant (P<0.01). The growth rate of SSQ-P (Spatial) from pre-implant to 12 months post-implant was 675.3%, and the growth rate from 6 months post-implant to 12 months post-implant was 140.6%, the growth rate showed an significant increase compared with IT-MAIS/MAIS, MESP and SSQ-P (Speech).SSQ-P (Speech) and SSQ-P (Spatial) scores were moderate correlation at 12 months post-implant(r=0.465, P=0.008). Conclusions: Within one year after cochlear implantation, listening, speech perception and spatial hearing abilities of preschool prelingual deaf children could show a comprehensive, continuous and significant progress as the implantation time increasing. The growth rate of spatial hearing is greater than that of speech perception at 12 months post-implant, and the spatial hearing could still show rapid development characteristics after 6 months post-implant.

Comparison of intestinal bacteria and inflammatory cytokine expression in rats with ulcerative colitis after treatment of three regiments / 药学学报

This study was designed to compare intestinal bacteria and inflammatory cytokine expression in rats with ulcerative colitis (UC) after treatment of three regiments, Huang-qin-tang (HQT), Si-shen-wan (SSW), and Tong-xie-yao-fang (TXYF). After approved by Institute of Chinese Materia Medica Ethics Committees in China Academy of Chinese Medical Sciences, UC in rats was induced by using a compound method (trinitrobenzenesulfonic acid plus ethanol). Rats were randomly divided into control, disease, positive control salazosulfapyridine (SASP, 0.5 g·kg-1), HQT (20 g·kg-1), SSW (26 g·kg-1), and TXYF groups (22 g·kg-1). After 7 days of treatment, colonic tissues and the blood were taken for various assays. Damage of colonic tissues was detected by H&E staining. The levels of tumor necrosis factor-ɑ (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8) and prostaglandin E2 (PGE2) in the serum were detected by the enzyme linked immunosorbent assay (ELISA). Total DNA was extracted from stool samples for analyses of 16SMiseqPE300V3-4 segment using high-throughput sequencing. The inflammatory cytokine results showed that compared with the disease group, the content of IL-6, PGE2, TNF-α in SASP group were decreased (P<0.05), with the most significant decrease being the level of IL-8 (P<0.01), whereas the levels of IL-6, IL-8 and TNF-α in HQT group were reduced (P<0.05) and PGE2 content was clearly reduced (P<0.01). The contents of four cytokines in SSW group were decreased, but there was no statistical difference. While the levels of IL-6 and TNF-α in TXYF group were reduced, and the reductions of IL-8 and PGE2 were significant (P<0.05). The results after sequencing showed that microbiome species richness SSW group > HQT group > TXYF group; the similarity between samples TXYF group > SSW group > HQT group; the species of HQT and TXYF group have greater difference when compared to the disease group. The content of beneficial bacteria in the intestine of HQT group > SSW group > TXYF group. Three regiments all have therapeutic effects on UC, manifested by improvements of the signs and mental status of UC rats. However, in terms of inhibition of inflammatory factors IL-6, IL-8, PGE2 and TNF-α, and regulation of intestinal microbiome, the therapeutic effect of HQT was superior than SSW and TXYF.

The effects of Huangqin Tang on oxidative stress and ulcerative colitis in rats through the Nrf2 signal pathway / 药学学报

This study aimed to investigate the effect of Huangqin Tang (HQT) on oxidative stress associated with ulcerative colitis in rats, and to explore its antioxidant mechanism. After approved by Institute of Chinese Materia Medica Ethics Committees in China Academy of Chinese Medical Sciences, the rats were given 2,4,6-trinitrobenzenesulfonic (TNBS)/ethanol mixture to induce the ulcerative colitis (UC), and were randomly divided into normal group, model group, the salazosulfapyridine (SASP) group, and high, middle or low dose (20, 10, 5 g·kg-1) of HQT groups. After 5 days of treatment, the activity of catalase (CAT) from micrococcus lysodeikticus, glutathione peroxidase (GSH-px), myeloperoxidase (MPO), superoxide dismutase (SOD) were detected by biochemical assays. The levels of lipid peroxide (LPO) were detected by ELISA. The positive protein expression of nuclear factor erythroid-2-related factor 2 (Nrf2) were detected by immunohistochemistry method and the downstream antioxidant enzymes of Nrf2 were determined by Western blot analyses. The levels of SOD, CAT and GSH-px activities in the normal group were significantly higher than the model group, while the serum MPO activity in the model group was obviously increased (P<0.05 or P<0.01). Compared with the model group, there was a significant difference in the activity of CAT in the high and middle dose groups of HQT (P<0.05 or P<0.01), the activity of GSH-px in the high, middle and low dose groups of HQT were apparently higher than the model group (P<0.05); The serum levels of LPO in the model group were significantly higher than those in the normal group (P<0.05), while the up-regulating effects on LPO were reversed by the high and middle dose groups of HQT (P<0.05). The expression of Nrf2 in the high-dose group of HQT and SASP group was statistically significant (P<0.05). The results of Western blot showed that compared with the model group, each of the HQT and SASP group could increase the heme oxygenase (HO-1) and NAD[P]H: quinone oxidoreductase 1 (NQO-1) expression in a dose-dependence manner. HQT has significant anti-oxidative stress and obviously improves the signs, mental status and defecation of UC rats. The mechanism of action for HQT maybe related to activate the Nrf2 pathway and increase the expression of Ⅱ phase metabolic enzymes such as HO-1 and NQO-1, reduce the content of LPO and MPO in serum and enhance the activity of SOD, CAT and GSH-px.

Experimental study on the effect of Sishen Wan on intestinal flora in rats with diarrhea-type irritable bowel syndrome / 药学学报

To investigate the effect of Sishen Wan (SSW) on intestinal flora in diarrhea-predominant irritable bowel syndrome (IBS-D) rats and explore the efficacy of this regiment for improving IBS-D, we divided 45 SPF male SD rats randomly into control, disease, SSW, Ershen Wan (ESW) and Wuweizasan (WWZS) groups. The spleen-kidney-yang deficiency type IBS-D rat model was prepared by a composite factor and administered for 14 days. After collecting the feces of the rats, total DNA was extracted from the stool samples. Primers were designed based on the 16S r RNA V3 to V4 regions of the bacteria, and used for high-throughput sequencing with the Illumina Miseq platform. We found that SSW can effectively reduce the diarrhea index (P<0.05) and reduce the high sensitivity of intestinal tract (P<0.05) of IBS-D rats. The principal component analysis (PCA), principal co-ordinates analysis (PCoA) and non-metric multidimensional scale analysis (NMDS) based on the Beta diversity distance showed that there were significant differences in the composition of the gut microbiota among the five groups (P<0.05). The disease group has the lowest in abundance, uniformity and diversity of gut microbiota. Compared with the control group, the disease group showed a significant increase in Proteobacteria, Actinobacteria, Veillonococcus and Mycoplasma (P<0.05), but a significant reduction in Pleaverella (P<0.05). Compared with the disease group, SSW administration caused significant reduction in the Proteobacteria and Mycoplasma (P<0.05), but significant increases of Clostridium, Turicibacter and Romboutsia (P<0.05). Our study shows that SSW has the potential as a therapeutic regiment for treatment of IBS-D due to partial regulation of the intestinal flora. In addition, there is a synergy between ESW and WWZS.

Early effects of low-level long-term occupational chromate exposure on workers'health / 北京大学学报(医学版)

OBJECTIVE@#To explore the effects of low-level long-term occupational exposure to chromate on the health of workers, and the potential biomarkers of early health effects in terms of lung function, immune toxicity and genetic damage.@*METHODS@#A total of 22 chromate contact workers and 44 non-chromate contact workers from an electroplating enterprise with long-term occupational environment monitoring in line with the national standards in Inner Mongolia were investigated. The questionnaire survey was conducted to collect the basic situation, the history of smoking, drinking, diseases and so on. The portable lung function instrument, inductively coupled plasma mass spectrometry and cytokinesis-blocked micronucleus test were performed to measure the chromate contact workers'lung function, whole blood Cr (WB-Cr) and micronuclei frequency (MNF) of peripheral blood lymphocytes respectively. The cytometric bead array was used to detect the levels of IL-1β, IL-6, IL-8, IL-10, IL-12P70 and TNFα in the serum among the two groups. The effects of chromate exposure on the above-mentioned indexes involved biological exposure, lung function, immune response and genetic damage, and their correlation were analyzed with different statistical methods.@*RESULTS@#(1) the average length of service for chromate contact workers was 31 years, and their concentration of WB-Cr was 1.11-4.19 μg/L. They were divided into high and low exposure groups according to the median of 1.72 μg/L. The WB-Cr in the high exposure group (2.17 μg/L) was higher than that in the low exposure group (1.58 μg/L) as well as the reference value of the healthy population (1.74 μg/L, P<0.05); (2) the lung function test showed 10 (45.45%) chromate exposure workers had single or multiple abnormal lung function indexes, among which large airway injury index PEF, and small airway injury indexes MVV and FEF25%-75% were all negatively correlated with WB-Cr (r=-0.53, P<0.05; r=-0.52, P<0.05; r=-0.44, P<0.05); (3) IL-1β, IL-6, IL-8 and TNFα in the serum of chromate contact workers were higher than those in the control group (P<0.05), and there was a positive correlation between TNFα and WB-Cr, and among these cytokines (P<0.05); (4) the average lymphocyte MNF in chromate contact workers was 1.341%, higher than the reference value of the general population (0.436%, P<0.01). Poisson regression analysis showed MNF in thehigh exposure group was higher than that in the low exposure group, OR (95%CI) =1.323 (1.049, 1.669); (5) multiple linear regression analysis showed that the lung function index FEF25%-75% decreased with the increase of TNFα (P<0.05), no significant correlation was found between other cytokines, MNF and lung function indexes.@*CONCLUSION@#Long-term low-level occupational exposure to chromate can cause the decline of lung function, immune inflammatory reaction and genetic damage in workers, in which local or systemic inflammatory response is associated with decreased lung function. Lung function indexes PEF, FEF25%-75% and MVV, serum cytokines IL-1β, IL-6, IL-8, and TNFα, and peripheral blood lymphocyte MNF may be used as early health effects biomarkers of chromate exposure.

Lipoprotein-associated phospholipase A2 as a predictor for severity of coronary atherosclerosis / 第二军医大学学报

Objective: To investigate whether plasma lipoprotein-associated phospholipase A2 (Lp-PLA2) activity correlates with severity and stability of coronary atherosclerosis and other established cardiovascular risk factors. Methods: Coronary angiography (CAG) was performed in 180 hospitalized patients who were suspected as having coronary heart disease (CHD). The severity of pathological changes of the coronary artery was assessed by the number of diseased coronary branches and Gensini's score. According to the results of CAG, the 180 patients were divided into 2 groups: CHD group (n=112) and non-CHD group (n=68). The CHD patients were further divided into subgroups according to the clinical types, the number of diseased coronary branches and Gensini's score. Lp-PLA2 activity, white blood cell (WBC) count, high sensitive C reactive protein (hsCRP), lipids, blood pressure and body mass index were measured. The age, sex and prior medical histories including hypertension, diabetes mellitus and smoking status were obtained before CAG in all patients. Lp-PLA2, WBC count and hsCRP were compared statistically between the subgroups, and correlation coefficients of Lp-PLA2 activity with WBC count, hsCRP and other conventional risk factors for CHD were calculated. Results: Plasma Lp-PLA2 activity in CHD patients was significantly higher than that in controls (P<0.01). No significant difference was found in Lp-PLA2 activity between patients with acute coronary syndrome and stable angina pectoris. Lp-PLA2 activity increased with the increasing number of diseased coronary branches and Gensini's score. Plasma Lp-PLA2 activity was positively correlated with TC and LDL-C, negatively correlated with HDL-C, weakly correlated with WBC count, and not correlated with hsCRP. Conclusion: Plasma Lp-PLA2 activity can be used as a parameter to predict pathological severity of coronary atherosclerosis, though it is not associated with stability of pathological changes of the coronary artery.