Aim To prepare prostate cancer exosomes containing melittin and observe their uptake by prostate cancer cells. Methods Cells treated with starvation for different time were screened for exosome extraction. Exosomes from PC-3 cells were extracted by ultracentrifugation, and the extracted particles were examined by transmission electron microscopy, nanoparticle tracking analyzer(NTA), and Western blot. Melittin exosome system was prepared by repeated freeze-thaw method, incubation at room temperature as well as electroporation, and the size of encapsulation efficiency was measured by centrifugation. A high-performance liquid chromatography(HPLC)method was applied to assay the content of melittin exosomes(exo-mel). Fluorescence inverted microscopy was employed to evaluate the uptake of melittin exosomes by PC-3 cells, DU145 cells as well as LNCaP cells. Results The results of starvation treatment showed that 24 h starvation treatment was the optimal time point. TEM results showed that the exosomes were round or oval in shape with a distinct membranous structure, and the diameter was around 100 nm. The reagent protein concentration for NTA analysis of exosomes was 0.222 g·L-1. The results of Western blot for the marker proteins of exosomes showed that Alix and CD63 were positively expressed, which indicated that the exosomes could be obtained by starvation culture of PC-3 cells and ultracentrifugation. The results of entrapment efficiency showed that the entrapment efficiency of electroporation method was 17.51% ± 2.39%, that of repeated freeze-thaw method was 11.46% ± 1.02%, and that of room temperature incubation method was 3.93% ± 2.44%. The encapsulation efficiency of electroporation was the highest with significant difference(P<0.05). The uptake assay showed that PC-3 cells could efficiently take up exo-mel in a time-dependent manner, and DU145 cells and LNCaP cells also could take up exo-mel over time. Conclusions Exosomes can be accessed by starvation treatment and high-speed centrifugation, and the prostate cancer melittin exosome system prepared by electroporation method could be taken up by prostate cancer cells.

The development of chemoresistance which results in a poor prognosis often renders current treatments for colorectal cancer(CRC).In this study,we identified reduced microvessel density(MVD)and vascular immaturity resulting from endothelial apoptosis as therapeutic targets for overcoming chemoresistance.We focused on the effect of metformin on MVD,vascular maturity,and endothelial apoptosis of CRCs with a non-angiogenic phenotype,and further investigated its effect in overcoming chemoresistance.In situ transplanted cancer models were established to compare MVD,endothelial apoptosis and vascular maturity,and function in tumors from metformin-and vehicle-treated mice.An in vitro co-culture system was used to observe the effects of metformin on tumor cell-induced endothelial apoptosis.Transcriptome sequencing was performed for genetic screening.Non-angiogenic CRC developed inde-pendently of angiogenesis and was characterized by vascular leakage,immaturity,reduced MVD,and non-hypoxia.This phenomenon had also been observed in human CRC.Furthermore,non-angiogenic CRCs showed a worse response to chemotherapeutic drugs in vivo than in vitro.By suppressing endo-thelial apoptosis,metformin sensitized non-angiogenic CRCs to chemo-drugs via elevation of MVD and improvement of vascular maturity.Further results showed that endothelial apoptosis was induced by tumor cells via activation of caspase signaling,which was abrogated by metformin administration.These findings provide pre-clinical evidence for the involvement of endothelial apoptosis and subsequent vascular immaturity in the chemoresistance of non-angiogenic CRC.By suppressing endothelial apoptosis,metformin restores vascular maturity and function and sensitizes CRC to chemotherapeutic drugs via a vascular mechanism.

In this study, UPLC-Q-Exactive-MS/MS was used to rapidly analyze the chemical constituents of Meconopsis quintupli-nervia, and the anti-liver fibrosis mechanism of M. quintuplinervia was preliminarily analyzed by network pharmacology, molecular docking, and cell experiments. The chemical constituents of M. quintuplinervia were identified according to the information of MS~1 and MS~2, as well as the data in the literature and databases. SwissTargetPrediction and TargetNet were used to predict the potential targets. The targets related to liver fibrosis were collected from GeneCards and OMIM. The protein-protein interaction(PPI) network was constructed by STRING. Cytoscape 3.6.1 was used to construct and analyze the "constituent-target-disease" network to obtain key targets and their corresponding constituents in the network. DAVID 6.8 was used for GO analysis and KEGG signaling pathway enrichment analysis. Finally, the preliminary verification was carried out by molecular docking and cell experiments. As a result, 106 chemical constituents were identified from M. quintuplinervia, including 66 flavonoids, 16 alkaloids, 18 phenolic acids, 1 anthocyanin, and 5 other constituents. Among them, 3 constituents were identified as potential new compounds, and 59 constituents were reported in M. quintuplinervia for the first time. Network pharmacology analysis showed that M. quintuplinervia presumably acted on AKT1, SRC, JUN, EGFR, STAT3, HSP90 AA1, MAPK3, and other core targets through luteolin, isorhamnetin, quercetin, apigenin, kaempferide, amurine, 2-methylflavinantine, allocryptopine, the multi and other active compounds, thereby regulating the PI3 K/AKT signaling pathway, pathways in cancer, proteoglycans in cancer, FoxO signaling pathway, and other pathways to exert anti-liver fibrosis effects. M. quintuplinervia extract(MQE) could significantly down-regulate PI3 K and AKT protein levels in the HSC-T6 cell model induced by TGF-β1, suggesting that MQE may have the ability to regulate the PI3 K/AKT signaling pathway. The findings of this study indicated that the anti-liver fibrosis effect of M. quintuplinervia had multi-constituent, multi-target, and multi-pathway characteristics, which may provide a scientific basis for the research on the pharmacodynamic materials, action mechanism, and quality markers of M. quintupli-nervia.
Tandem Mass Spectrometry ; Molecular Docking Simulation ; Network Pharmacology ; Proto-Oncogene Proteins c-akt ; Papaveraceae ; Liver Cirrhosis ; Drugs, Chinese Herbal/pharmacology*

Objective: To evaluate the immunogenicity of group A+C meningococcal polysaccharide conjugate vaccine in infants under 2 years old. Methods: From March 2017 to June 2018, 1 932 healthy infants in Biyang County, Henan Province, who were not vaccinated with meningococcal meningitis vaccine and whose axillary temperature was ≤37.0 ℃, were recruited as participants. The 3 months and 6-11 months old infants were allocated to the experiment group and the control group in a ratio of 1∶1. Infants aged 12-23 months were allocated to the 1-dose group, the 2-dose group and the control group in a ratio of 1∶1∶1, with 276 infants in each group. The infants in the experiment group were intramuscularly injected with freeze-dried group A+C meningococcal polysaccharide conjugate vaccine to be evaluated, and infants in the control group received intramuscular injection of commercially available freeze-dried group A+C meningococcal conjugate vaccine. The venous blood of infants was collected 30 days before the first dose and after the last dose of inoculation, and the antibody seroconversion of each group was determined and compared. Results: The completion rate of immunogenicity study was 95.2% (1 839/1 932). Before inoculation, there was no statistical difference in the geometric mean titer and positive rate of group A+C antibodies between the experiment group and the control group in 3 months and 6-11 months old infants (all P values >0.05). The geometric mean titers and positive rate of group A antibodies in the 1-dose group were higher than those in the control group (all P values <0.05), but there was no statistical difference between the 2-dose group and the control group (all P values >0.05) in infants aged 12-23 months. After inoculation, the differences (95%CI) in the positive conversion rate of group A+C antibodies between the experiment group and the control group were -0.12% (-6.01%-5.77%) and 0.82% (-4.23%-5.86%) in the 3 months old infants. At the age of 6-11 months, the differences were 6.75% (1.71%-11.79%) and -4.32% (-8.73%-0.08%), respectively. At the age of 12-23 months, the differences were 1.02% (-3.80%-5.83%) and -4.40% (-7.79%- -1.01%) in the 2-dose group and -7.22% (-12.90%- -1.54%) and -18.61% (-23.75%- -13.46%) in the 1-dose group, respectively. The geometric mean titers of group A+C antibodies in the 3 months old infants were 48.50 and 63.12, respectively, which had no significant difference from the control group (43.02 and 57.99, respectively) (both P values <0.05). The geometric mean titers of group A+C antibodies in the 6-11 months and 12-23 months old infants were 84.09 and 92.51 (2-dose group), which were higher than those in the corresponding control group (43.10 and 61.83, respectively) (all P values <0.001). Conclusion: Group A+C meningococcal conjugate vaccine has good immunogenicity in infants under 2 years old.
Humans ; Infant ; Child, Preschool ; Meningococcal Vaccines ; Vaccines, Conjugate ; Vaccination ; Neisseria meningitidis ; Polysaccharides ; Antibodies, Bacterial

OBJECTIVES: To study the clinical features of severe meconium aspiration syndrome (MAS) and early predicting factors for the development of severe MAS in neonates with meconium-stained amniotic fluid (MSAF). METHODS: A total of 295 neonates who were hospitalized due to Ⅲ° MSAF from January 2018 to December 2019 were enrolled as subjects. The neonates were classified to a non-MAS group (n=199), a mild/moderate MAS group (n=77), and a severe MAS group (n=19). A retrospective analysis was performed for general clinical data, blood gas parameters, infection indicators, and perinatal clinical data of the mother. The respiratory support regimens after birth were compared among the three groups. The receiver operating characteristic (ROC) curve and multivariate logistic regression analysis were used to investigate predicting factors for the development of severe MAS in neonates with MSAF. RESULTS: Among the 295 neonates with MSAF, 32.5% (96/295) experienced MAS, among whom 20% (19/96) had severe MAS. Compared with the mild/moderate MAS group and the non-MAS group, the severe MAS group had a significantly lower 5-minute Apgar score (P<0.05) and a significantly higher blood lactate level in the umbilical artery (P<0.05). Compared with the non-MAS group, the severe MAS group had a significantly higher level of interleukin-6 (IL-6) in peripheral blood at 1 hour after birth (P<0.017). In the severe MAS group, 79% (15/19) of the neonates were born inactive, among whom 13 underwent meconium suctioning, and 100% of the neonates started to receive mechanical ventilation within 24 hours. Peripheral blood IL-6 >39.02 pg/mL and white blood cell count (WBC) >30.345×10⁹/L at 1 hour after birth were early predicting indicators for severe MAS in neonates with MSAF (P<0.05). CONCLUSIONS Meconium suctioning cannot completely prevent the onset of severe MAS in neonates with MSAF. The neonates with severe MAS may develop severe respiratory distress and require mechanical ventilation early after birth. Close monitoring of blood lactate in the umbilical artery and peripheral blood IL-6 and WBC at 1 hour after birth may help with early prediction of the development and severity of MAS.
Amniotic Fluid/chemistry* ; Female ; Humans ; Infant, Newborn ; Interleukin-6 ; Lactates ; Meconium ; Meconium Aspiration Syndrome/diagnosis* ; Pregnancy ; Retrospective Studies

Objective:To study the soil physical and chemical properties， microorganisms， and metabolites in different culture environments of Gastrodia elata， so as to provide scientific basis for subsequent cultivation of G. elata in multiple environments. Method:The tubersphere soil of G. elata cultured in different environments was collected for analyzing the soil nutrients， microbial numbers， and metabolite differences using the agrochemical method， plate-count method， and gas chromatography-time-of-flight mass spectrometry （GC-TOF-MS）-based non-targeted metabonomic approach. Result:The analysis of soil physical and chemical properties revealed the highest soil moisture， pH， available potassium， and available phosphorus in the spinney and the highest electrical conductivity， total nitrogen， alkali-hydrolyzable nitrogen， and organic matter in the pinewood. As demonstrated by the quantitative analysis of soil microorganisms， the cultivable microorganisms were bacteria， actinomycetes， and fungi， with the bacterial population and total microbial biomass in the spinney and the number of fungi and actinomycetes in the barren slope detected to be the largest. The ratio of bacteria to fungi （B/F value） in the pinewood was the highest， while that in the barren slope was the lowest. The results of metabonomic research demonstrated that the compositions and quantities of soil metabolites in the spinney （Z group）， pinewood （S group）， and barren slope （HD group） varied. Through comparisons between S and Z groups， between HD and Z groups， as well as between HD and S groups by orthogonal partial least squares discriminant analysis （OPLS-DA）， 18， 35， and 24 differential metabolites were separately screened out， and the Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis yielded 5， 9， and 13 metabolic pathways. There existed a significant causal relationship of the soil physical and chemical properties and microbial numbers with the metabolites. Conclusion:The soil physical and chemical properties， microbial numbers， and metabolite changes differed significantly in different culture environments of G. elata， which were sorted by the suitability in a descending order as follows： spinney > pinewood >barren slope. The soil physical and chemical properties and microbial numbers are the crucial factors driving changes in soil metabolites， suggesting that regulating the soil physical and chemical characteristics and microbial characteristics in the culture environment is an important mechanism for maintaining the G. elata-soil-microbial symbiotic system.

Objective:Chemical constituents in hypoglycemic effective fractions of Longan Folium were isolated and identified by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry （UPLC-Q-Orbitrap HRMS） to clarify the hypoglycemic substance basis of Longan Folium. Method:Chemical constituents in hypoglycemic effective fractions of Longan Folium were isolated on a Thermo Hypersil GOLD C₁₈ column （2.1 mm×100 mm， 1.9 μm）， the mobile phase was 0.1% formic acid acetonitrile solution and 0.1% formic acid solution （containing and 10 mmol ammonium acetate） for gradient elution. HRMS was operated in the positive and negative ion modes with the scanning range of m/z 100-1 500. Result:The secondary fragment ion information of target compounds was selected and compared with the compounds reported in the databases and related literature to further confirm these compounds. Nine compounds were identified in the ethanol fraction of Longan Folium， including cynaroside， kaempferol， quercitrin， luteolin， shikimic acid， citric acid， L-tyrosine， adenosine and nicotinamide. A total of 11 compounds were determined in the ethyl acetate fraction （cynaroside， quercitrin， kaempferol， luteolin， shikimic acid， gallic acid， protocatechuic acid， adenosine， nicotinamide， L-phenylalanine and scopoletin）， and 10 compounds were identified in the n-butanol fraction （cynaroside， kaempferol-3-O-rutinoside， kaempferol， astragalin， luteolin， citric acid， gallic acid， adenosine， nicotinamide and 5-hydroxymethylfurfural）. And five common compounds were identified in these three hypoglycemic effective fractions. Conclusion:The established UPLC-Q-Orbitrap HRMS can quickly identify chemical constituents in three hypoglycemic effective fractions of Longan Folium， their main chemical constituents are flavonoids and their glycosides， organic acids and nitrogen-containing compounds， which provides technical support and scientific evidence for the study on pharmacodynamic material basis and quality control of Longan Folium.

Objective:The aim of this study was to research the relationship between HPLC fingerprint and anti-inflammatory effect of Zhideke granules， and the substance basis of its anti-inflammatory effect was preliminary explored. Method:The fingerprint of 10 batches of Zhideke granules were determined by HPLC， the mobile phase was consisted of methanol-0.2% phosphoric acid solution for gradient elution with a detection wavelength of 254 nm. Similarity analysis， hierarchical cluster analysis （HCA）， principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） were used to evaluate the quality difference between batches of Zhideke granules. The correlation analysis between the common peaks and the inhibition rates of Zhideke granules on ear swelling and cotton ball granuloma in mice was carried out by partial least squares （PLS）， and the peaks greatly contributing to the anti-inflammatory effect were screened out. Result:There were 31 common peaks in the HPLC fingerprint of Zhideke granules. The similarities of 10 batches samples were ≥0.992. The HCA and PCA analysis results were consistent， and the samples were divided into 3 categories. Combined with the OPLS-DA results， 15 components were the main markers affecting the differences of different batches of samples. Different batches of Zhideke granules differed in anti-inflammatory effect. The chromatographic peaks being positively correlated with the anti-inflammatory effect were mainly from Belamcandae Rhizoma and Scutellariae Radix， Chromatographic peaks 3， 6， 19， 27-30 had significant contribution to anti-inflammatory effect， of which peaks 28 and 30 were irisflorentin and wogonin. Conclusion:HPLC fingerprint combined with chemical pattern recognition method can provide a reference for systematic evaluation of the overall quality of Zhideke granules. Zhideke granules has a certain inhibitory effect on acute and chronic inflammation in mice， and the anti-inflammatory effect is the result of the combined action of various ingredients， while Belamcandae Rhizoma and Scutellariae Radix have significant significance for the anti-inflammatory effect.