OBJECTIVE:To study the inhibitory effects of berberine on EA.hy926 human umbilical vein endothelial cells (EA. hy926 cells) injury induced by particulates with no more than 2.5 μm air aerodynamic diameter in atmospheric (PM2.5),and its p38 mitogen-activated protein kinase(MAPK)signal pathway mechanism. METHODS:PM2.5 samples were collected and hatched EA.hy926 cells with concentrations of 0(blank control),20,200 and 400 mg/L for 24 h. The survival rate and apoptosis rate of cells,contents of IL-6,TNF-α and MDA,activities of SOD and LDH,protein levels of p-p38 MAPK,Bcl-2 and Bcl-2 associated X protein (Bax) were detected. The above indexes of EA.hy926 cells in blank control group,PM2.5 group (200 mg/L PM2.5), p38 MAPK pathway-specific blocker SB203580 group (20 μmol/L SB203580+200 mg/L PM2.5),berberine low-,medium- and high-concentrations groups(5,10,20 μmol/L berberine+200 mg/L PM2.5)were also determined. RESULTS:Compared with blank control,survival rate of cells,SOD activity and Bcl protein decreased after 200,400 mg/L PM2.5 hatched;apoptosis rate of cells, contents of IL-6,TNF-α and MDA,LDH activity,protein levels of p-p38 MAPK and Bax increased (P<0.05),in concentra-tion-dependent manner. Compared with PM2.5 group,survival rate of cells,SOD activity and Bcl-2 protein increased in berberine medium-,high-concentrations groups and SB203580 group;apoptosis rate of cells,contents of IL-6,TNF-α and MDA,LDH ac-tivity,protein levels of p-p38 MAPK and Bax decreased (P<0.05). CONCLUSIONS:Berberine attenuates PM2.5-induced EA. hy926 cells injury via the inhibition of p38 MAPK pathway.

AIM:To investigate the effect and the mechanism of tanshinone ⅡA in attenuating PM2.5-induced human umbilical vein endothelial EA.hy926 cell injury.METHODS:The samples of fine particulate matter (PM2.5) were collected in Guangzhou and made into suspension.Different concentrations (0, 20, 200 and 400 mg/L) of PM2.5 were added to EA.hy926 cells.The viability and apoptosis of EA.hy926 cells, the protein levels of p-p38 MAPK, Bax and Bcl-2 in the EA.hy926 cells, the contents of interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α) and malonaldehyde ( MDA) , and the activity of superoxide dismutase ( SOD) and lactic dehydrogenase ( LDH) in the EA.hy926 cell culture supernatant were measured by MTT assay, flow cytometry, Western blot, ELISA and colorimetry, respectively.Tanshinone ⅡA at different concentrations (5, 10 and 20 μmol/L) or a specific inhibitor of p38 MAPK pathway, SB203580 (20μmol/L) , was added into the EA.hy926 cells to observe the effect of tanshinone ⅡA.RESULTS:Compared with control group, PM2.5 significantly increased the apoptosis, the contents of IL-6, TNF-αand MDA, the activity of LDH, and the protein levels of p-p38 MAPK and Bax/Bcl-2 ratio, but decreased the viability and SOD activity in the EA.hy926 cells (P<0.05).Compared with PM2.5 group, tanshinone IIA significantly decreased the apoptosis, the contents of IL-6, TNF-αand MDA, the activity of LDH, and the protein levels of p-38 MAPK and Bax/Bcl-2 ratio, but increased the viabil-ity and SOD activity in the EA.hy926 cells (P<0.05).CONCLUSION:Tanshinone ⅡA attenuates PM2.5-induced EA. hy926 cell injury via the inhibition of p38 MAPK pathway.

Aim To investigate the protective effect of allicin against EA. hy926 endothelial cell injury in-duced by PM2. 5 and the possible mechanism. Meth-ods The samples of fine particulate matter ( PM2. 5 ) were collected and made into suspension. Different concentrations of PM2. 5 ( 20 , 200 , 400 mg · L-1 ) were added to EA. hy926 cell. The viability and apop-tosis of EA. hy926 cell, the protein levels of p-ERK1/2, Bax and Bcl-2 in the EA. hy926 cell, the contents of tumor necrosis factor-α( TNF-α) , interleukin-6 ( IL-6 ) , and malonaldehyde ( MDA ) , the activities of su-peroxide dismutase ( SOD ) and lactic dehydrogenase ( LDH) in the EA. hy926 cell culture supernatant were measured by MTT assay, flow cytometry, Western blot, enzyme-linked immunosorbent assay ( ELISA ) and colorimetry, respectively. Allicin at different con-centrations(5,20,40 mg·L-1 ) or a specific inhibitor of ERK1/2 signaling pathway PD98059 ( 20 μmol · L-1 ) was added into the EA. hy926 cell to observe the effect of allicin. Results Compared with control group, PM2. 5 significantly increased the apoptosis, the contents of TNF-α, IL-6 and MDA, the activity of LDH, the protein levels of p-ERK1/2 and Bax/Bcl-2 ratio, but decreased the viability and SOD activity in the EA. hy926 cell(P<0. 05). Compared with PM2. 5 group, allicin significantly decreased the apoptosis, the contents of TNF-α, IL-6 and MDA, the activity of LDH, the protein levels of p-ERK1/2 and Bax/Bcl-2 ratio, but increased the viability and SOD activity in the EA. hy926 cell ( P <0. 05 ) . Conclusion Allicin displays a significant protective effect against EA. hy926 endothelial cell injury induced by PM2 . 5 and its mechanism may be related to the attenuations of in-flammation and oxidative stress via the inhibition of ERK1/2 pathway.

A method was developed for the determination of tetrodotoxin in marine organisms by high perfor-mance liquid chromatography-mass spectrometry with immunoaffinity column. The samples were extracted with 1% acetic acid methanol solution and diluted with phosphate buffer at pH 7-8. After cleaned up by immuno-affinity column, the samples were analyzed by LC-MS/MS and quantitatively determined by external standard method. The chromatographic separation was performed on an ACQUITY UPLC BEH Amide column with gradient elution by using acetonitrile and 5 mol/L ammonium acetate solution containing 0. 1% formic acid as mobile phase. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple reaction monitoring mode. Linear ranges of TTX was in the range of 0. 3 -20. 0 μg/L with correlation coeffi-cient more than 0. 997. The quantification limit of the method was 0. 3 μg/kg. The recoveries of standard addition for tetrodotoxin were 88. 7%-102. 3%, and the relative standard deviation was 2. 0%-6. 4%. The method could be used to identify and quantify tetrodotoxin in marine organisms with satisfactory reproducibility and sensitivity.

Objective To observe the curative effect of low molecular heparin for treating secondary high coagulation state in the patients with nephrotic syndrome(NS) .Methods Total 87 cases of NS in our hospital were divided into the conventional treat‐ment group (n=42) and the low molecular heparin treatment group (n=45) .The routine treatment group was given the prednisone treatment and the low molecular heparin treatment group was treated by low molecular heparin combined with prednisone .The re‐lated indicators of blood coagulation before and after treatment were detected and the clinical curative effects in two groups were an‐alyzed .Results The coagulation related indicators in the conventional treatment group had no statistically significant difference be‐tween before and after treatment (P>0 .05) ,the prothrombin time(PT) and activated partial thrombin time(APTT) after treat‐ment in the low molecular heparin treatment group were significantly extended compared with before treatment ,while the concen‐trations of D‐dimer and fibrinogen were significantly decreased and the concentration of antithrombin Ⅲ was markedly increased compared with before treatment ,showing statistically significant differences between the two groups (P<0 .05);the patients of the low molecular heparin group patients had no bleeding after treatment .Conclusion Low molecular heparin combined with predni‐sone can reduce the secondary high condensation state in NS without bleeding and has a significantly clinical effect .

Objective To study the effects of taking clopidogrel on relevant indicators of platelet aggregation function in 138 cases acute coronary syndrome (ACS) .Methods The platelet function analyzer and flow cytometry were adopted to detect the ADP‐induced platelet aggregation rate ,P selectin and activated GP Ⅱ b/ Ⅲ before medication and on 7 d after taking clopidogrel . Results The platelet aggregation rate after taking clopidogrel for 7 continuous d was decreased significantly (P<0 .01);the P se‐lectin level and activated GP Ⅱ b/ Ⅲ a expressed on platelet surface were significantly reduced (P<0 .01) as well .Conclusion Taking clopidogrel could reduce the platelet aggregation significantly in the patients with ACS and has the effect for inhibiting the platelet aggregation .

We studied the immunogenicity of pseudorabies virus gC DNA vaccination by fusing the murine complement C3d receptor binding domain. First, pseudorabies virus gC gene was linked to four copies of C3d receptor binding domain (M284), and then cloned into the vector pcDNA3.1 to construct the recombinant plasmid sgC-M284. Through the experiment of immunized BALB/c mice, we found that the enzyme linked immunosorbent assay (ELISA) antibody titer for sgC-M284 was 17-fold higher than that for sgC alone, and protective rate of mice was augmented from 25% to 88% after lethal dose PrV (316 LD50) challenge. In addition, the IL-4 levels for sgC-M284 immunization approached that for the pseudorabies virus inactivated vaccine. In conclusion, we demonstrated murine C3d receptor binding domain fusion significantly increased Th2-biased immune response by inducing IL-4 production.
Adjuvants, Immunologic ; physiology ; Animals ; Antibody Formation ; immunology ; Binding Sites ; Cloning, Molecular ; Complement C3d ; genetics ; immunology ; Herpesvirus 1, Suid ; genetics ; immunology ; Interleukin-4 ; immunology ; Mice ; Mice, Inbred BALB C ; Pseudorabies Vaccines ; immunology ; Receptors, Complement 3d ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; pharmacology ; Viral Fusion Proteins ; immunology

ObjectiveTo investigate the difference in protein expression between hepatocellular carcinoma (HCC) patients with recurrence and those with good prognosis, the differential expression and regulatory mechanism of miR-152-3p target proteins, and the role of miR-152-3p in the recurrence of HCC. MethodsTMT-labeled proteomic sequencing and RT-PCR were used to measure the expression of proteins and the expression of miR-152-3p in the HCC tissue of six patients with recurrence at 2 years after HCC resection and six patients with good prognosis at 5 years. Six databases were used to analyze the target genes of miR-152-3p, and Gene Ontology, DAVID, and REACTOME databases were used to perform target gene screening, enrichment annotation, and signal transduction pathway enrichment analysis. Gene mutation frequency and survival curve analysis were performed for the target genes of miR-152-3p to verify the role of miR-152-3p target genes in patients with HCC recurrence. The independent samples t-test was used for comparison of continuous data between two groups, and a Kaplan-Meier analysis was performed to investigate the survival rates of liver-related genes. ResultsCompared with the patients with HCC recurrence, the patients with good prognosis after HCC resection had a significantly higher transcriptional expression level of miR-152-3p in HCC tissue (P＜0.05). The results of protein sequencing showed that there were 365 differentially expressed proteins in HCC tissue between the patients with good prognosis and the patients with recurrence, and the analysis of HCC recurrence databases showed that 17 proteins were regulated by miR-152-3p. Further analysis of the signaling pathways showed that the function of the 17 target genes regulated by miR-152-3p was enriched in the translation and regulation of mitochondria and ribosome, and multiple enrichment revealed that six target genes were closely associated with mitochondrial respiratory chain complex, i.e., AKAP1, FOXRED1, MRPL28, MRPL50, SHC1, and STAU1. Gene mutation frequency and survival curve analysis showed that the loss or weakening of the function of mitochondrial respiratory chain-related target proteins seriously affected the prognosis and survival rate of patients. ConclusionThere is a significant difference in the expression of miR-152-3p in HCC tissue between patients with good prognosis and those with recurrence after HCC resection, and miR-152-3p may lead to the recurrence of HCC by regulating the target genes AKAP1, FOXRED1, MRPL28, MRPL50, SHC1, and STAU1, acting on the mitochondrial respiratory chain, and affecting the oxidative respiratory function of cells.

OBJECTIVE To analyze the chemical components and components migrating to the blood of Jianpi huazhuo tiaozhi granules （JHTG）. METHODS SD rats were divided into a control group and a medication group， with 6 rats in each group. The medication group was given JHTG 3 mL. Sixty minutes after medication， the serum samples of the 2 groups were collected， and the chemical components and components migrating to the blood of JHTG were separated by ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） and mass spectrometry data were collected. Combined with the overall scheme of UNIFI natural products， based on the 6400 natural product theory mass spectrometry database， the structure was analyzed and confirmed by literature review and reference substance comparison. RESULTS & CONCLUSIONS A total of 130 components were identified from JHTG， including 3 in Codonopsis Radix， 13 in Nelumbinis Folium， 15 in Poria， 5 in Atractylodis Macrocephalae Rhizoma， 9 in Citri Reticulatae Pericarpium， 1 in Coicis Semen， 19 in Alisma Rhizoma， 24 in Salviae Miltiorrhizae Radix et Rhizoma， 7 in Hordei Fructus Germinatus， 24 in Crataegi Fructus， 2 in Amomi Fructus， and 3 in Aucklandiae Radix. In addition， quercetin and quercetin-3-O-β-D-glucopyranoside， kaempferol and citric acid may originate from Nelumbinis Folium or Crataegi Fructus， while oleanolic acid may originate from Poria or Crataegi Fructus. By comparing the reference substances， 8 components were finally determined （pachymic acid， atractylenolide Ⅱ， alisol A， alisol B， alantolactone， bornyl acetate， salvianolic acid A， salvianolic acid C）. A total of 72 prototype components such as quercetin and kaempferol were identified， mainly including flavonoids， terpenoids， lignans and phenolic acids. A total of 11 metabolites such as （NATCM’s Project of High- dehydroanonaine and 16-O-acetylpachymic acid were level Construction of Key TCM Disciplines）identified， mainly terpenoids. Metabolic pathways include phase Ⅰ metabolic reactions such as dehydrogenation and dehydroxylation， and phase Ⅱ metabolic reactions such as methylation and acetylation.

ObjectiveTo investigate the role and mechanism of DNA repair regulation in the process of hepatocellular carcinoma （HCC） recurrence. MethodsHCC tissue samples were collected from the patients with recurrence within two years or the patients with a good prognosis after 5 years， and the Tandem Mass Tag-labeled quantification proteomic study was used to analyze the differentially expressed proteins enriched in the four pathways of DNA replication， mismatch repair， base excision repair， and nucleotide excision repair， and the regulatory pathways and targets that play a key role in the process of HCC recurrence were analyzed to predict the possible regulatory mechanisms. The independent samples t-test was used for comparison of continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsFor the eukaryotic replication complex pathway， there were significant reductions in the protein expression levels of MCM2 （P=0.018）， MCM3 （P=0.047）， MCM4 （P=0.014）， MCM5 （P=0.008）， MCM6 （P=0.006）， MCM7 （P=0.007）， PCNA （P=0.019）， RFC4 （P=0.002）， RFC5 （P<0.001）， and LIG1 （P=0.042）； for the nucleotide excision repair pathway， there were significant reductions in the protein expression levels of PCNA （P=0.019）， RFC4 （P=0.002）， RFC5 （P<0.001）， and LIG1 （P=0.042）； for the base excision repair pathway， there were significant reductions in the protein expression levels of PCNA （P=0.019） and LIG1 （P=0.042） in the HCC recurrence group； for the mismatch repair pathway， there were significant reductions in the protein expression levels of MSH2 （P=0.026）， MSH6 （P=0.006）， RFC4 （P=0.002）， RFC5 （P<0.001）， PCNA （P=0.019）， and LIG1 （P=0.042） in recurrent HCC tissue. The differentially expressed proteins were involved in the important components of MCM complex， DNA polymerase complex， ligase LIG1， long patch base shear repair complex （long patch BER）， and DNA mismatch repair protein complex. The clinical sample validation analysis of important differentially expressed proteins regulated by DNA repair showed that except for MCM6 with a trend of reduction， the recurrence group also had significant reductions in the relative protein expression levels of MCM5 （P=0.008）， MCM7 （P=0.007）， RCF4 （P=0.002）， RCF5 （P<0.001）， and MSH6 （P=0.006）. ConclusionThere are significant reductions or deletions of multiple complex protein components in the process of DNA repair during HCC recurrence.