Eleven new sesquiterpenoids from peeled stems of Syringa pinnatifolia / 中国中药杂志

The peeled stems of Syringa pinnatifolia(SP) is a representative Mongolian folk medicine with the effects of anti-depression, heat clearance, pain relief, and respiration improvement. It has been clinically used for the treatment of coronary heart disease, insomnia, asthma, and other cardiopulmonary diseases. As part of the systematic study on pharmacological substances of SP, 11 new sesquiterpenoids were isolated from the terpene-containing fractions of the ethanol extract of SP by liquid chromatography-mass spectrometry(LC-MS) and proton nuclear magnetic resonance(~1H-NMR) guided isolation methods. The planar structures of the sesquiterpenoids were identified by MS, 1D NMR, and 2D NMR data analysis, and were named pinnatanoids C and D(1 and 2), and alashanoids T-ZI(3-11), respectively. The structure types of the sesquiterpenoids included pinnatane, humulane, seco-humulane, guaiane, carryophyllane, seco-erimolphane, isodaucane, and other types. However, limited to the low content of compounds, the existence of multiple chiral centers, the flexibility of the structure, or lack of ultraviolet absorption, the stereoscopic configuration remained unresolved. The discovery of various sesquiterpenoids enriches the understanding of the chemical composition of the genus and species and provides references for further analysis of pharmacological substances of SP.

"Component-target-efficacy" network analysis and experimental verification of Qingkailing Oral Preparation / 中国中药杂志

This study aims to explore the mechanism of Qingkailing(QKL) Oral Preparation's heat-clearing, detoxifying, mind-tranquilizing effects based on "component-target-efficacy" network. To be specific, the potential targets of the 23 major components in QKL Oral Preparation were predicted by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction. The target genes were obtained based on UniProt. OmicsBean and STRING 10 were used for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the targets. Cytoscape 3.8.2 was employed for visualization and construction of "component-target-pathway-pharmacological effect-efficacy" network, followed by molecular docking between the 23 main active components and 15 key targets. Finally, the lipopolysaccharide(LPS)-induced RAW264.7 cells were adopted to verify the anti-inflammatory effect of six monomer components in QKL Oral Preparation. It was found that the 23 compounds affected 33 key signaling pathways through 236 related targets, such as arachidonic acid metabolism, tumor necrosis factor α(TNF-α) signaling pathway, inflammatory mediator regulation of TRP channels, cAMP signaling pathway, cGMP-PKG signaling pathway, Th17 cell differentiation, interleukin-17(IL-17) signaling pathway, neuroactive ligand-receptor intera-ction, calcium signaling pathway, and GABAergic synapse. They were involved in the anti-inflammation, immune regulation, antipyretic effect, and anti-convulsion of the prescription. The "component-target-pathway-pharmacological effect-efficacy" network of QKL Oral Preparation was constructed. Molecular docking showed that the main active components had high binding affinity to the key targets. In vitro cell experiment indicated that the six components in the prescription(hyodeoxycholic acid, baicalin, chlorogenic acid, isochlorogenic acid C, epigoitrin, geniposide) can reduce the expression of nitric oxide(NO), TNF-α, and interleukin-6(IL-6) in cell supernatant(P<0.05). Thus, the above six components may be the key pharmacodynamic substances of QKL Oral Preparation. The major components in QKL Oral Prescription, including hyodeoxycholic acid, baicalin, chlorogenic acid, isochlorogenic acid C, epigoitrin, geniposide, cholic acid, isochlorogenic acid A, and γ-aminobutyric acid, may interfere with multiple biological processes related to inflammation, immune regulation, fever, and convulsion by acting on the key protein targets such as IL-6, TNF, prostaglandin-endoperoxide synthase 2(PTGS2), arachidonate 5-lipoxygenase(ALOX5), vascular cell adhesion molecule 1(VCAM1), nitric oxide synthase 2(NOS2), prostaglandin E2 receptor EP2 subtype(PTGER2), gamma-aminobutyric acid receptor subunit alpha(GABRA), gamma-aminobutyric acid type B receptor subunit 1(GABBR1), and 4-aminobutyrate aminotransferase(ABAT). This study reveals the effective components and mechanism of QKL Oral Prescription.

Integrated Metagenomics and UPLC-Q-TOF-MS/MS to Explore the Mechanism of Dexamethasone on Pneumonia in Rats / 中山大学学报(医学科学版)

ObjectiveUsing multi-omics technology， we conducted the present study to determine whether dexamethasone has therapeutic effect on pneumonia rats through the regulation of intestinal flora and metabolites. MethodsTotally 18 Sprague-Dawley rats were randomly divided into 3 groups （n = 6 each）： Control group， Model group and Dexamethasone （Dex） group. Lipopolysaccharide （LPS） was continuously injected intraperitoneally into rats at a dose of 4 mg/kg for 7 days to induce pneumonia except the Control group. Then the Dex group was given Dex at a dose of 2 mg/kg via oral gavage for 12 days， and both the other two groups received continuously equal volume of sterile PBS buffer for 12 days. On the 19th day， lung， plasma， feces and intestinal contents of rat were collected. Hematoxylin-eosin （H&E） staining and Bio-plex suspension chip system were applied to evaluate the effect of Dex on pneumonia. Furthermore， metagenomic sequencing and UPLC-Q-TOF-MS/MS technology were employed to determine the intestinal flora and metabolites of rats， respectively. ResultsH&E staining results showed that the lung tissue of the Model group was infiltrated with inflammatory cells， the alveolar septum was increased， alveolar hemorrhage， and histological lesions were less severe in Dex group than in the model group. The levels of 3 inflammatory cytokines including TNF-α （P < 0.000 1）， IL-1α （P = 0.009 6） and IL-6 （P < 0.000 1） in the Model group were increased compared with the Control group， while Dex treatment reduced the levels of the three inflammatory factors. Taken together， Dex treatment effectively reversed the features of pneumonia in rats. Metagenomic analysis revealed that the intestinal flora structure of the three groups of rats was changed. In contrast with the Model group， an increasing level of the Firmicutes and an elevated proportion of Firmicutes/Bacteroidetes were observed after Dex treatment. Dex-treated rats possessed notably enrichment of Bifidobacterium， Lachnospiraceae and Lactobacillus. Multivariate statistical analysis showed a great separation between Model group and Dex group， indicating metabolic profile changes. In addition， 69 metabolites （P < 0.05） were screened， including 38 up-regulated in the Model group and 31 elevated in the Dex group， all of which were mainly involved in 3 metabolic pathways： linoleic acid metabolism， tryptophan metabolism and primary bile acid biosynthesis. ConclusionsIn summary， we demonstrate the beneficial effects of Dex on the symptoms of pneumonia. Meanwhile， integrated microbiome-metabolome analysis reveals that Dex improves LPS-induced pneumonia in rats through regulating intestinal flora and host metabolites. This study may provide new insights into the mechanism of Dex treatment of pneumonia in rats.

Gene Mutants and Their Clinical Characteristics of G6PD Deficiency Among Children in Luzhou Area / 中国实验血液学杂志

OBJECTIVE@#To study the gene mutants of G6PD deficiency and their clinical featuers among children in Luzhou area.@*METHODS@#732 children with suspected G6PD deficiency in Luzhou area from March 2017 to July 2019 were selected, which were examined for G6PD enzyme activity and gene mutation. The G6PD enzyme activity was detected by ultraviolet rate quantification, and the gene mutation was detected by melting curve analysis-based PCR assay, and the clinical characteristics of different mutants when acute hemolysis happens were analyzed.@*RESULTS@#387 positive specimens were detected in 732 specimens, among which the gene mutation and the enzyme activity decrease was found in specimens 326, 49 specimens showed gene mutation but without the enzyme activity decrease, and 12 specimens without gene mutation but with the enzyme activity decrease. Among 375 positive samples with gene mutation, c.1376G＞T, c.1388G＞A, c.1024C＞T and c.95A＞G were the most common. The enzyme activity of c.1376G＞T and c.1388G＞A was statistically significantly different with c.1024C＞T. The most common incentives of acute hemolysis was broad bean, the reticulocyte count was statistically significantly different among c.1376G＞T, c.1388G＞A and c.95A＞G. The hemoglobin level of c.1376G＞T was statistically significantly different from with c.95A＞G. Moreover, c.1376G＞T, c.1388G＞A was lower than c.1024 C＞T. When acute hemolysis occurs, the reticulocyte count and hemoglobin changes were different between different mutation types, while the patients age, hospitalization time, blood transfusion, total bilirubin, and urine color recovery time of the patients were not statistically different.@*CONCLUSION@#The common mutants of G6PD deficiency among children in Luzhou area are c.1376G＞T, and c.1388G＞A, c.1024C＞T. Favism is the most common clinical manifestation of G6PD deficiency.

Analysis of in Vivo and in Vitro Metabolites of Coptisine in Rats by HPLC-MS/MS / 中国实验方剂学杂志

Objective::To investigate in vivo and in vitro metabolites of coptisine and their metabolic pathways. Method::SD rats were given coptisine by single gavage (dose of 25 mg·kg-1). Urine and feces from 0 h to 48 h, bile from 0 h to 24 h, and plasma and brain tissue samples at 0.25, 1, 2 h after administration were collected.In vitro metabolism was incubated with rat liver microsomes and intestinal flora.The metabolites were analyzed and identified by the high-resolution HPLC-MS/MS technique.The liquid chromatography separation was carried out on ZORBAX SB-C18 column (4.6 mm×150 mm, 5 μm) with acetonitrile-0.1% formic acid solution as the mobile phase for gradient elution, the flow rate was 1.0 mL·min-1, and column temperature was 25 ℃.The mass spectra were obtained in positive and negative ion mode with electrospray ionization (ESI), the scanning range was m/z 50-1 200.The relative molecular weight was determined according to the quasi-molecular ion peaks.The structures of metabolites were elucidated by comparing the data with literature data, including main ion peaks, UV spectrum and HPLC retention time information. Result::A total of 17 metabolites were identified in each sample, including 11 phase Ⅰ metabolites and 6 phase Ⅱ metabolites.The pathways to these metabolites were hydroxylation, demethylation, dehydrogenation, sulfation and glucuronide conjugation. Conclusion::Coptisine can produce metabolic reaction of phase Ⅰ and phase Ⅱ in rat, and metabolites are predominantly present in urine, and the main metabolic site is liver.Coptisine is poorly absorbed and rarely metabolized in gastrointestinal tract, so it is mostly excreted through feces by prototype.This experiment can provide material basis for the pharmacodynamics and pharmacology of coptisine.

Simultaneous determination of 11 components in Naofuqing Capsule by HPLC-MS/MS / 中草药

Objective: To develop a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous determination of 11 components including eleutheroside E, 2,3,5,4’-tetera-hydroxystilbene-2-O-β-D-glucoside (stilbene glycoside), isofraxidin, rosmarinic acid, notoginsenoside R1, salvianolic acid B, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rd, and emodin in Naofuqing Capsule. Methods: The analysis was performed on an Agilent Poroshell 120 EC-C18 column (75 mm × 4.6 mm, 2.7 μm), and the mobile phase consisted of water containing 0.1% formic acid and acetonitrile with gradient elution at the flow rate of 0.4 mL/min. The column temperature was maintained at 35 ℃. The MS detection for the 11 tested components was performed in negative ion multiple reaction monitoring mode. Results: Good linear relationship were observed in the test range for 11 components, and the recoveries of eleutheroside E, stilbene glycoside, isofraxidin, rosmarinic acid, notoginsenoside R1, salvianolic acid B, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside Rd, and emodin were 106.4%, 104.2%, 99.9%, 102.3%, 104.1%, 98.3%, 108.9%, 103.6%, 105.8%, 101.9%, and 104.2%, respectively. The contents of 10 batches of the 11 components were in the ranges of 0.274-0.310, 1.579-1.642, 0.093-0.099, 0.331-0.352, 1.115-1.229, 1.663-1.870, 4.884-5.173, 0.691-0.762, 2.974-3.358, 1.053-1.493, and 0.100-0.115 mg/g, respectively. Conclusion: The established method is sensitive, stable, and rapid. It is suitable for the simultaneous determination of multiple components in Naofuqing Capsule.

In vitro and In vivo effects of six Coptidis alkaloids on liver microsomes UGTs and UGT1A1 activities in rats and mice / 中国中药杂志

In the present study, the effects of six Coptidis alkaloids (berberine, epiberberine, coptisine, jatrorrhizine, palmatine and magnoflorine) on liver microsomes UGTs and UGT1A1 activities in rats and mice were investigated in vitro and in vivo to study the mechanism of metabolic drug-drug interactions of Coptidis Rhizoma with other drugs. In vitro rat and mice liver microsomal incubation systems combined with UDPGA were applied, as well as mice liver microsomes after administration of six Coptidis alkaloids. 4-Nitrophenol and β-estradiol were selected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC, respectively. According to the in vitro rat study, berberine, epiberberine, coptisine and jatrorrhizine significantly inhibited rat liver microsome UGTs activity, particularly epiberberine showed the strongest inhibition. UGT1A1 activity was lowly inhibited by jatrorrhizine, with IC₅₀ at about 227 μmol•L⁻¹, whereas coptisine and magnoflorine significantly activated UGT1A1. According to the in vitro mice study, berberine, coptisine, jatrorrhizine and palmatine significantly inhibited mice liver microsome UGTs activity, and the six alkaloids all significantly activated UGT1A1. According to the in vivo mice study, UGTs activity was significantly activated only in berberine group, while UGT1A1 activity was significantly activated only in jatrorrhizine group. In conclusion, the effects of Coptidis alkaloids on UGT activity showed significant differences in species and between in vitro and in vivo. Meanwhile, the changes in structures of Coptidis alkaloids also have a big impact on UGT activity, which may be one of the causes for the drug-drug interactions between Coptidis Rhizoma and other drugs.

In vitro effects of Genkwa Flos chloroform extract on activity of human liver microsomes UGTs and UGT1A1 / 中国中药杂志

To predict the mechanism of liver injury induced by Genkwa Flos, we investigated the effect of chloroform extract on UGTs and UGT1A1 activities of the liver microsomes in rat and human. In the present study, 4-nitrophenol(4-NP) and β-estradiol were elected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC. The results showed that there were 1.00% of apigenin, 6.40% of hydroxygenkwanin and 18.38% of genkwanin in chloroform extract; and total diterpene mass fraction was 31.40%. Compared with the control group, chloroform extract could significantly inhibit the activity of UGTs in rat liver microsomes(RLM) system, while the inhibitory effect was not obvious in human liver microsomes(HLM) system. UGT1A1 activity was inhibited by chloroform extract in rat liver microsomes and human liver microsomes (based on genkwanin, IC₅₀=8.76, 10.36 μmol•L⁻¹). The inhibition types were non-competitive inhibition(RLM) and uncompetitive inhibition(HLM). In conclusion, the results indicated that chloroform extract showed different inhibitory effects on UGTs and UGT1A1 activity, which may be one of the mechanisms of liver injury induced by Genkwa Flos.

Inhibitory effect of different species of hydroxygenkwanin on UGTs and UGT1A1 activities / 中国中药杂志

To predit the mechanism of metabolic drug-drug interactions of hydroxygenkwanin with other drugs, we investigated the inhibition inhibitory effect of hydroxygenkwanin on UGTs and UGT1A1 activities of different liver microsomes. In the present study, 4-nitrophenol (4-NP) and β-estradiol were elected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC, respectively. The results showed that, hydroxygenkwanin significantly inhibited UGTs activity in rat, mouse and human liver microsomes. UGT1A1 activity was inhibited by hydroxygenkwanin to varying degrees, with IC₅₀ about 190, 10.93, 20.07, 76.31 μmol•L⁻¹ in mouse liver microsome(MLM), rat liver microsome (RLM) and recombinant UGT1A1, and human liver microsome (HLM), respectively. The inhibition types were competitive inhibition (RLM, HLM) and linear mixed-typed linear inhibition (recombinant UGT1A1). The order for the inhibitory intensity was RLM>rUGT1A1>HLM>MLM. In conclusion, hydroxygenkwanin has an inhibitory effect on UGTs and UGT1A1 activities of different liver microsomes, with differences in species, indicating its potential drug interactions based on UGT1A1 enzyme. This study aims to provide a reliable experimental basis for its further research and development of hydroxygenkwanin, and provide theoretical reference for the clinic drug combination research.

Effect of human cytomegalovirus infection on the expression of hoxc4 and hoxc6 genes in the proliferation of lymphocytic progenitor cells / 中国实验血液学杂志

The objective of this study was to observe the expression of hoxc4 and hoxc6 genes in the process of differentiation of hematopoietic stem cell (HSC) to colony forming unit-T Lymphocyte (CFU-TL) in vitro. and to explore the possible mechanism of HCMV-induced maldevelopment of human cord blood CFU-TL on genetic level through effecting the differentiation progress by human cytomegalovirus (HCMV) with and/or all-trans retinoic acid (ATRA), Normal CFU-TL culture was used as blank control. After detection with MTT, mRNA expression levels in the human cord blood CFU-TL hoxc4 and hoxc6 genes following HCMV infection and ATRA treatment were detected by fluorogenic quantitative reserve transcription polymerize chain reaction (FQ-RT-PCR) method. HCMV of 10(6) plaque formation unit (PFU)/ml was diluted to 0.1 ml 10(5) PFU/ml and added into the infected group. The results showed that the expression of hoxc4 and hoxc6 genes in the differentiation process increased slightly on day 3, and were up to the most on day 7 (p < 0.05), while became lower on day 12 respectively in normal group, HCMV group and ATRA group. Compared with the expression of hoxc6, the expression of hoxc4 was obviously higher in each group (p < 0.05). Compared with the expression of hoxc4 and hoxc6 genes in normal group, the expressions of hoxc4 and hoxc6 in ATRA group were up-regulated remarkably (p < 0.05), while the expressions of hoxc4 and hoxc6 in group HCMV were down-regulated (p < 0.05). It is concluded that the regular expression of hoxc4 and hoxc6 genes mRNA appeared in each group. A positive co-relationship exits between hoxc4/hoxc6 genes and lymphocytic progenitor hematopoiesis. Compared with the expression of hoxc6 gene, the expression of hoxc4 gene is obviously higher in each group. HCMV can down-regulate the expression of hoxc4 and hoxc6 genes and lead to suppression effect on cell morphology, which confirms that the normal hematopoietic lineage determination and maturation rely on the stable and consistent expression of homeobox gene. At the same condition, ATRA (6 x 10(-8) mol/L at 60 nmol/ml) can up-regulate hoxc4 and hoxc6 genes expression. ATRA can up-regulate the expression of hoxc4 and hoxc6 genes.

Association of aldosterone synthase gene -344 T/C polymorphism with early renal damage in Han nationality with essential hypertension / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate whether the -344T/C polymorphism of aldosterone synthase gene is associated with early renal damage in Han nationality with essential hypertension in Shandong province.</p><p><b>METHODS</b>Plasma aldosterone concentration and urinary albumin excretion were measured with radioimmunoassays in 225 patients with essential hypertension, and hypertensives were classified as hypertension with normal albuminuria or hypertension with microalbuminuria according to urinary albumin excretion during 24 hours. -344T/C polymorphism of aldosterone synthase gene was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in controls and hypertensives.</p><p><b>RESULTS</b>No significant differences were found in genotype distribution among groups of control, primary hypertension with normal albuminuria and hypertension with microalbuminuria. The C allele frequency in hypertension with microal buminuria group was significantly higher than that in control and hypertension with normal albuminuria group (P < 0.05). In hypertensive patients, plasma aldosterone concentration and urinary albumin excretion of TC+CC genotypes were significantly higher than that of TT genotype ( P< 0.05).</p><p><b>CONCLUSION</b>These results suggest that -344T/C polymorphism of aldosterone synthase gene may be associated with early renal damage in Han nationality with essential hypertension, C allele may be a genetic factor susceptible to renal damage in hypertensives.</p>

Expression of HOXB6 Gene on Differentiation of Hematopoietic Stem Cells and Intervention Research / 实用儿科临床杂志

Objective To observe the expression level of HOXB6 gene on the differentiation and proliferation of hematopoietic stem cells(HSC) to colony forming unit-granulocyte-monocyte(CFU-GM),erythroid progenitor(CFU-E) and colony forming unite-T-lymphocyt(CFU-TL).And the proliferation procress was affected by all-transretinoic acid(ATRA).Methods 1.By the colony culture in vitro,the impact of ATRA on the CFU-GM,CFU-E,CFU-TL colony formation were surveyed;the expressions of HOXB6 gene were observed on the differentiation procress of HSC to CFU-GM,CFU-E and CFU-TL affected by ATRA on the 3rd,7th,12th.2.Real-time fluorogenic quantitative reserve transcription-polymerize chain reaction(FQ-RT-PCR) method was used to explore the possible mechanism of ATRA up-regulated to human cord blood CFU-GM,CFU-E and CFU-TL in gentic level.Results 1.HOXB6 gene had a regulatory function in the differentiation procress of hematopoiesis.During the differentiation and proliferation of HSC to colony forming CFU-GM or CFU-E,CFU-TL in vitro,the expressions of HOXB6 gene were significant positive.2.Compared with the expressions of CFU-GM and CFU-TL HOXB6 mRNA on day 3,the quantity of HOXB6 mRNA was obviously higher on day 7 and lower on day 12,respectively in each group.Compared with the expression of CFU-E HOXB6 mRNA on day 3,the quantity of HOXB6 mRNA was obviously higher on day 12 in each group.3.Compared with the HOXB6 gene of control group,the expressions of HOXB6 gene of group ATRA were up-regulated remarkable.Conclusions 1.HOXB6 gene has a regulatory function in the differentiation procress of hematopoiesis.HOXB6 gene plays an important role in the progress which can be associated with the regulating effect of HOXB6 gene on the differentiation and proliferation of CFU-GM,CFU-E and CFU-TL.2.During the differentiation and proliferation of HSC,the expression of HOXB6 gene is regular in time.3.Low concentration of ATRA(60 ?mol?L-1) can up-regulate the expression of HOXB6 gene,which confirms the theory that the normal hematopoie-tic lineage determination and maturation rely on the stable and consistent expression of HOXB6 gene.