Study on serum pharmacodynamic material basis of Gegen Qinlian Decoction in treatment of ulcerative colitis based on UHPLC-Q-Orbitrap-MS / 中国中药杂志

This study established a mouse model of ulcerative colitis and explored the serum transitional components of Gegen Qinlian Decoction by UHPLC-Q-Orbitrap-MS. Based on the exact relative molecular weight and MS/MS spectrum, 55 prototype components and 59 metabolites were identified from the model group, while 18 prototype components and 35 metabolites from the control group. The prototype components in serum were mainly flavonoids and the characteristic components of the model group were alkaloids. Glucuronidation, sulfonation, and glycosylation have been confirmed to be the main metabolic types in vivo. The results of comparative analysis of differences indicated that puerarin, baicalin, wogonoside, wogonin, chrysin, oroxylin A, berberine, berberrubine, and palmatine were the characteristic components in model state, which at the same time, were confirmed by pharmacological studies to be the serum pharmacodynamic material basis of Gegen Qinlian Decoction in the treatment of ulcerative colitis. This study has provided reference for explaining the metabolic transformation pattern and mechanism of action of Gegen Qinlian Decoction in vivo.

Molecular mechanism of Qishen Yiqi Dripping Pills in treating myocardial ischemia:a study based on HIF-1 signaling pathway / 中国中药杂志

Qishen Yiqi Dripping Pills(QSYQ) are used clinically to treat various myocardial ischemic diseases, such as angina pectoris, myocardial infarction, and heart failure; however, the molecular mechanism of QSYQ remains unclear, and the scientific connotation of traditional Chinese medicine(TCM) compatibility has not been systematically explained. The present study attempted to screen the critical pathway of QSYQ in the treatment of myocardial ischemia by network pharmacology and verify the therapeutic efficacy with the oxygen-glucose deprivation(OGD) model, in order to reveal the molecular mechanism of QSYQ based on the critical pathway. The key targets of QSYQ were determined by active ingredient identification and target prediction, and underwent pathway enrichment analysis and functional annotation with David database to reveal the biological role and the critical pathway of QSYQ. Cell counting Kit-8(CCK-8), lactate dehydrogenase(LDH), and Western blot tests were launched on high-content active ingredients with OGD cell model to reveal the molecular mechanism of QSYQ based on the critical pathway. The results of network pharmacology indicated that QSYQ, containing 18 active ingredients and 82 key targets, could protect cardiomyocytes by regulating biological functions, such as nitric oxide biosynthesis, apoptosis, inflammation, and angiogenesis, through TNF signaling pathway, HIF-1 signaling pathway, PI3 K-Akt signaling pathway, etc. HIF-1 signaling pathway was the critical pathway. As revealed by CCK-8 and LDH tests, astragaloside Ⅳ, salvianic acid A, and ginsenoside Rg_1 in QSYQ could enhance cell viability and reduce LDH in the cell supernatant in a concentration-dependent manner(P<0.05). As demonstrated by the Western blot test, astragaloside Ⅳ significantly down-regulated the protein expression of serine/threonine-protein kinase(Akt1) and hypoxia-inducible factor 1α(HIF-1α) in the HIF-1 signaling pathway, and up-regulated the protein expression of vascular endothelial growth factor A(VEGFA). Salvianic acid A significantly down-regulated the protein expression of upstream phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3 CA) and downstream HIF-1α of Akt1. Ginsenoside Rg_1 significantly down-regulated the expression of HIF-1α protein and up-regulated the expression of VEGFA. The therapeutic efficacy of QSYQ on myocardial ischemia was achieved by multiple targets and multiple pathways, with the HIF-1 signaling pathway serving as the critical one. The active ingredients of QSYQ could protect cardiomyocytes synergistically by regulating the targets in the HIF-1 signaling pathway to inhibit its expression.

Molecular Mechanism of Salvia miltiorrhiza Quality Formation Based on Single Nucleotide Polymorphism of Functional Genes / 中国实验方剂学杂志

Objective:To investigate the relationship between the single nucleotide polymorphism（SNP）of function genes and effective components of <italic>Salvia miltiorrhiza</italic> and the molecular mechanism of specific quality formation of <italic>S. miltiorrhiza</italic>. Method:The fingerprints of components in <italic>S. miltiorrhiza</italic> from eight different habitats and varieties were obtained by high-performance liquid chromatography （HPLC）. The full-length cDNA of three functional genes<italic> </italic>acetyl-CoA C-acetyltransferase（<italic>SmAACT</italic>），4-diphosphocytidyl-2-C-methyl-<italic>D</italic>-erythritol kinase（<italic>SmCMK</italic>） and isopentenyl diphosphate isomerase（<italic>SmIPPI</italic>） in tanshinone metabolic pathway were amplified by polymerase chain reaction（PCR），cloned， and sequenced，followed by bioinformatics analysis. Result:The full-length cDNA sequences of three functional genes <italic>SmAACT</italic>，<italic>SmCMK</italic>， and <italic>SmIPPI</italic> in tanshinone metabolic pathway were obtained from 23 strains of <italic>S. miltiorrhiza</italic> from eight different habitats and varieties. As revealed by the analysis of SNP and amino acid polymorphisms of three functional genes，18，16， and 14 SNP sites were found respectively. HPLC results showed the samples from Beijing，Hubei，Shandong （No. SDB），Shanxi，Henan， and Shandong （No. SDZ） were clustered into one branch，and those from Hebei and Inner Mongolia were clustered into another branch， which suggested that the variation trend of <italic>S. miltiorrhiza</italic> components had little correlation with geographical distance，but the variety was a critical factor for the quality. Conclusion:There was an obvious genetic differentiation trend in <italic>S. miltiorrhiza</italic> from different habitats，and different origin-specific genotypes were formed. The molecular mechanism of the formation of the specific quality of <italic>S. miltiorrhiza</italic> from different habitats was discussed，which laid a foundation for the stability and effectiveness of clinical medication，and guided the breeding of excellent varieties of <italic>S. miltiorrhiza</italic>.

Research and development thought on biopharmaceutics classification system of Chinese materia medica / 中国中药杂志

The biopharmaceutics classification system( BCS) is a scientific framework or method for classifying drugs based on drug solubility and permeability,which can be used to provide drug bioavailability-absorption correlation analysis. Based on the characteristics of multi-component and multi-target of traditional Chinese medicine( TCM) as well as the concept,method and technology of BCS,the research group proposed biopharmaceutics classification system of Chinese materia medica( CMMBCS) and carried out research and data accumulation of classical prescriptions. Based on the previous research results,further development ideas under the CMMBCS concept and framework were further proposed in this study. In the course of research,the influence of the intermediate links of the complex interactions of the multi-component environment was omitted,and the component absorption studies on the main clinical effects of prescription ingredients were directly concerned,or the components and data were reversely extracted from the aspects of metabolism,pharmacodynamic pathways and absorption principles. Studies were conducted from two aspects( single component and compound prescription) to comprehensively evaluate the absorption properties of TCM compound. In the research path,the different ways in which Chinese medicine could exert its efficacy were fully considered,and CMMBCS classification and establishment rules were clarified mainly by focusing on the absorption pathway into the blood. Specifically,the network pharmacology and molecular docking technology were used to screen the compound index components of TCM; the absorption rules were studied by the physiologically based pharmacokinetic models and the absorption parameters of CMMBCS were calculated by reverse reasoning. Then the CMMBCS classification of TCM prescription was corrected by studying the efficacy or absorption pathway. In this paper,the theoretical framework and research methodology of CMMBCS were systematically improved based on the establishment of CMMBCS basic theory,the supplementary of drug-oriented research ideas and the application of modern mature Chinese medicine methodology.

Simultaneous quantification and consistency analysis of 19 representative components in Longshengzhi Capsules by HPLC-QQQ/MS / 中国中药杂志

Longshengzhi capsule consisting of 12 herbs is widely used in clinically treating cerebral ischemia during recovery period.In this study,in order to investigate the consistency of different batches of Longshengzhi capsules,a high performance liquid chromatography coupled to triple quadrupole mass spectrometry method(HPLC-QQQ/MS) was developed for the determination of 19 representative components in Longshengzhi Capsules within 9 min. Methodology validation indicated this method was simple,rapid,accurate,highly sensitive and reproducible,and it could be used for the content determination of components in Longshengzhi Capsules. The consistency analysis results showed that paeoniflorin and calycosin-7-glucoside in Longshengzhi Capsules had the highest content; RSD value of total content of 19 compounds was 5. 2% and the RSD value of main compounds such as astragaloside and calycosin-7-glucoside was all less than 15%,reflecting good consistency among different batches. This study has provided a scientific method and basis for the quality control and consistency evaluation of Longshengzhi Capsules.

Hydroxysafflor yellow A repairing the metabolic disturbances of early atherosclerosis based on fatty acid profiling / 药学学报

Atherosclerosis (AS) is a complex metabolic syndrome that seriously harms human health, and its occurrence and development are directly related to the metabolic disturbances of free fatty acids (FFA). In this study, macrophage-derived foam cells were established as the model of early AS. Therefore, the metabolic disturbances of FFA in ox-LDL induced foamy macrophages were analyzed using target metabolomics. Then the effect of hydroxysafflor yellow A (HSYA) on regulating FFA was also explored. The quantitative analysis of 27 fatty acids was obtained within 20 min based on dynamic MRM mode. Thirteen metabolic biomarkers of macrophage-derived foam cells were identified using multivariate statistical analysis. It was found that upregulation of total SFA and downregulation of C12:0, C14:0, C18:1, total MUFA were the typical metabolic features in macrophage-derived foam cells. Furthermore, HSYA displayed obvious repairing effect on C12:0, C14:0 and C18:1, which were involved in de novo fatty acid biosynthesis pathway. Oleoyl-(acyl-carrier-protein) hydrolase (OLAH), as the key enzyme in de novo fatty acid biosynthesis pathway, may be a drug target of HSYA.

Studies on constituents of Polygonum multiflorum extract and based on UPLC-Q-TOF-MS / 中国中药杂志

To explore the drug-induced constituents of Polygonum multiflorum extract (PM). This study was the first to study the drug-induced constituents in target organ liver. Agilent MassHunter qualitative analysis software and Metabolite ID software were applied for the analysis of retention time, exact relative molecular mass, primary and secondary mass spectrum information based on ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and targeted-MS/MS. By comparison with literature and standards, a total of 5 prototypes and 6 metabolites were identified or tentatively elucidated from the liver samples. In addition, the drug-induced constituents in plasma and PM were also analyzed in this study and 8 prototypes and 19 metabolites were detected from the plasma samples, while 30 compounds were detected from the extract of PM. Emodin oxidative acetylation metabolites, hydroxyl methylation metabolites, carboxylation glucuronidation metabolites and ketone glucuronidation metabolites in this study were first reported. Through the comparative analysis between the and constituents of PM, the study preliminarily revealed the drug-induced constituents (prototypes and metabolites) in liver and clarified the transfer process and transmutation rules of constituents in PM, blood and liver, which would further deepen our understanding on constituents of PM .

Knowledge Discovery-oriented Biomedical Literature Information Retrieval and Visualization Design / 医学信息学杂志

The paper discusses influence and effect of information retrieval process of biomedical literature and visualization design on user knowledge discovery,from the aspects of input and output,retieval process and information positioning,it introduces the interaction model of literature information retrieval process and analyzes approach of literature information visualization design,including color and font,statistical chart,list of categories and labels,layer structure and network pictures,etc.

One-step real-time fluorescence quantitative PCR for detecting WT1 mRNA expression in leukemia / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a one-step real-time quantitative RT-PCR assay for detecting the expression of WT1 mRNA, which allows detection of the minimal residue disease and prognostic prediction in leukemic patients.</p><p><b>METHODS</b>WT1 gene fragment was amplified from the RNAs extracted from K562 cells using one-step RT-PCR. The quantitative standard were constructed by pMD 18-T vector cloning, and a Taqman-MGB fluorescent probe and a pair of primers were used to establish the one-step real-time fluorescence quantitative RT-PCR assay for WT1 gene detection. The sensitivity, repeatability and stability of this assay were evaluated and verified.</p><p><b>RESULTS</b>The sensitivity of this assay reached the 10(-4) level. The standard template of 1.0 x 10(6)-1.0 x 10(2) copies/ml were amplified by the one-step real-time fluorescence quantitative RT-PCR assay , and the Ct value was strongly correlated (r=0.998) to the logarithm of the initial template concentration. The repetition Ct value and both the inter-tube and inter-batch coefficients of variation (CV%) were less than 8%.</p><p><b>CONCLUSION</b>The one-step real-time fluorescence quantitative RT-PCR assay has good sensitivity, repeatability and specificity, and the one-step completion of the reverse transcription and PCR processes may reduce the operational complexities and the possibility of contamination.</p>

Expressions of hypoxia inducible factor-1alpha and insulin-like growth factor-II in gastric carcinoma: correlation with angiogenesis and prognosis / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate relationship between expressions of hypoxia inducible factor-1alpha (HIF-1alpha) and insulin-like growth factor-II (IGF-II) as well as their correlation with angiogenesis and prognosis in gastric carcinoma.</p><p><b>METHODS</b>HIF-1alpha and IGF-II mRNA expression were analyzed using in situ hybridization, microvessel density (MVD) was determined by anti-CD34 immunostaining in 118 cases gastric carcinoma.</p><p><b>RESULTS</b>The positive rates of HIF-1alpha mRNA and IGF-II mRNA were 49.2% and 47.4%, respectively. In stage T3-T4 cases, positive rates of HIF-1alpha and IGF-II mRNA expression, the frequencies of vessel invasion, lymph node and distant metastasis were significantly higher than those in stage T1-T2 cases. The mean MVD in stage T3-T4 tumors, vessel invasion, lymph node metastasis and distant metastasis were significantly more frequent than those in stage T1-T2 tumors. The mean MVD in tumors with positive HIF-1alpha and IGF-II mRNA expression was significantly higher than those in tumors without HIF-1alpha and IGF-II mRNA expression. The expression of HIF-1alpha was positively correlated with IGF-II mRNA. There were positive correlations between MVD and expression of HIF-1alpha and IGF-II mRNA, respectively. The mean survival time and 5-year survival rate in cases with positive HIF-1alpha and VEGF expression and MVD value >/= 41.5 were significantly shorter than those in cases with negative HIF-1alpha and VEGF expression and MVD value < 41.5.</p><p><b>CONCLUSIONS</b>HIF-1alpha and IGF-II play an important role in tumor invasion and metastasis, especially in tumor angiogenesis. So test of the expression of HIF-1alpha and IGF-II may act as a useful index of treatment and prognosis.</p>

mRNA expression of syndecan-1 and heparanase in relation to tumor progression and prognosis of gastric carcinoma / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate mRNA expression of syndecan-1 and heparanase in gastric carcinoma, and their correlation with the growth-pattern, invasion, metastasis and prognosis of gastric carcinoma.</p><p><b>METHODS</b>In situ hybridization technique was used to examine mRNA expression of syndecan-1 and heparanase in 118 specimens of gastric carcinoma.</p><p><b>RESULTS</b>The positive rates of syndecan-1 mRNA and heparanase mRNA were 42.4% and 55.9%, respectively. The expression of syndecan-1 mRNA and heparanase mRNA were related to tumor invasion depth (chi(2) = 32.95, P = 0.001; chi(2) = 23.19, P = 0.001), vessel invasion (chi(2) = 46.22, P = 0.001; chi(2) = 33.78, P = 0.001), lymph node (chi(2) = 28.62, P = 0.001; chi(2) = 25.43, P = 0.001) and distant metastasis (chi(2) = 63.30, P = 0.001; chi(2) = 65.76, P = 0.001), and syndecan-1 mRNA positive expression was related to tumor size (chi(2) = 6.25, P = 0.012). There was a negative relationship between Syndecan-1 mRNA and heparanase mRNA expression (r = -0.844, P = 0.001). The mean survival time of cases with low expression of syndecan-1 mRNA was significantly shorter than that of cases with high expression (r = 36.48, P = 0.001), and meanwhile, the mean survival time of heparanase mRNA positive cases was significantly shorter than that of cases with negative expression (r = 34.41, P = 0.001).</p><p><b>CONCLUSIONS</b>The mRNA expression of syndecan-1 and heparanase can predict the invasion and metastasis of gastric carcinoma, and can be used as markers of prognosis of gastric carcinoma.</p>

Real-time fluorescent quantitative PCR for detection of peripheral blood T-cell receptor excision circles / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop and optimize real-time fluorescence quantitative PCR (FQ-PCR) with the housekeeping gene RAG2 as cell number control to quantify T-cell receptor excision circle (TREC) in the peripheral blood.</p><p><b>METHODS</b>The real-time PCR system for amplifying TREC and RAG2 genes was established on the basis of ABI 7000 apparatus using Golden Taq system, designed primers, TaqMan-MGB probes and optimized buffer. PCR conditions were optimized with standard samples of TREC plasmid.</p><p><b>RESULTS</b>The amplification with the primer pair T(3) and T(4) was more efficient than that with T(1) and T(2). More specific and efficient amplification in FQ-PCR was achieved using TaqMan-MGB probes as compared with general Taq-Man probes. Golden Taq was more effective than general Taq in improving the specificity and decreasing the artifact, and 95 degrees C; for 10 min, 95 degrees C; for 5 s, and 53 degrees C; for 30 s for a total of 40 cycles using ABI7000 was found as the optimized thermal parameter setting.</p><p><b>CONCLUSION</b>An optimized real-time PCR protocol for detecting TREC in peripheral blood mononuclear cells is established.</p>

Establishment of a real time quantitative-PCR assay for detection of TCR VgammaI-Jgamma gene rearrangement in acute lymphoblastic leukemia patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To improve the techniques for minimal residual disease (MRD) detection in acute lymphoblastic leukemia (ALL).</p><p><b>METHODS</b>A real time quantitative PCR method was established for quantifying the clonal TCRVgammaI-Jgamma gene rearrangement in 36 ALL patients.</p><p><b>RESULTS</b>The sensitivity of the established real time quantitative PCR was at 10(-4) level. The amount of TCRVgammaI-Jgamma gene rearrangement in newly diagnosed group, complete remission (CR) group and post hematopoietic stem cell transplantation (HSCT) group was (7.38 +/- 6.65) x 10(-2), (1.02 +/- 1.08) x 10(-2) and (3.89 +/- 5.65) x 10(-3) level, respectively. and the amount in newly diagnosed group was higher than that in CR group and HSCT group (P = 0.001). The MRD level of ALL patients in CR group was higher than that in HSCT group (P = 0.022). MRD can be detected in 6 ALL patients after HSCT, 2 of them with low MRD level (< 1 x 10(-3)) survived long disease-free survival, the other 4 with high MRD level relapsed within one year.</p><p><b>CONCLUSION</b>The established real time quantitative PCR assay is simple, rapid, sensitive and specific. Use of this assay to evaluate MRD in the remission ALL cases is helpful for prognosis prediction.</p>