OBJECTIVE:To establish a method for simultaneous determination of 5 effective components in Luohua zizhu dry extract. METHODS:UPLC-MS/MS was conducted. The separation was performed on an Zorbax Eclipse Plus C18 column with mo-bile phase of acetonitrile-water(gradient elution)at the flow rate of 0.3 mL/min. The column temperature was set at 40℃and sam-ple size was 2 μL. The analytes were detected in the multiple reaction monitoring(MRM)mode. Nitrogen was used as drying gas and atomized gas. The temperature and flow rate of drying gas were 325 ℃ and 6 L/min. The pressure of atomized gas was 45 psi. The temperature and flow rate of sheath gas were 350℃and 12 L/min. The voltage of capillary were 4000 V(+)and 3500 V(-). The voltage of nozzle was 500 V. RESULTS:The linear ranges of luteoloside,acteoside,quercetin,luteolin and rutin were 0.5048-252.4 ng/mL(r=0.9999),0.7124-356.2 ng/mL(r=0.9990),0.5094-254.7 ng/mL(r=0.9962),0.3030-151.5 ng/mL(r=0.9998) and 0.6022-301.1 ng/mL(r=0.9996),respectively. RSDs of precision,stability and reproducibility tests were all less than 3.0%. The limit of quantitation were 0.42,0.87,0.33,0.12,0.76 ng/mL. The recoveries were 97.99%-101.20%(RSD=1.3%,n=6), 96.50%-101.20%(RSD=1.7%,n=6), 94.81%-99.34%(RSD=1.7%,n=6), 97.54%-100.51%(RSD=1.2%,n=6), 93.37%-98.70%(RSD=1.9%,n=6),respectively. CONCLUSIONS:The method is simple,precise,stable and reproducible, and can be used for simultaneous determination of 5 effective components in Luohua zizhu dry extract.

Objective Wnt signaling plays an important role in the development and progression of renal cell carcinoma (RCC).This study aimed to evaluate the effects of the Wnt signaling inhibitor 15-oxospiramilactone on the proliferation , migration, cell apoptosis, and cycles of the human RCC cell line 786-0, and to investigate the possible mechanisms of this small molecule acting on RCC in ivtro. Methods We treated 786-0 cells with DMSO ( blank control group ) and 15-oxospiramilactone at the concentrations of1.25μmol/L (low 15 -OSL), 2.5μmol/L (medium 15-OSL), and 5μmol/L (high 15-OSL), respectively, for 72 hours.Then we observed the changes in the proliferation and migration of the 786-0 cells by MTT and scratch-wound assay and determined their apopto-sis and cycles by Annexin V-FITC/PI assay and flow cytometry . Results 15-oxospiramilactone significantly inhibited the growth of the 7860-cells, with the IC 50of 1.088 μmol/L at 72 hours, and decreased their migration distance (P<0.05).After 36 hours of treatment, the apoptosis rates of the 786-0 cells in the low, medium, and high 15-OSL groups were (12.17 ±0.56), (18.54 ± 1.07), and (50.74 ±1.28) %, respectively, significantly increased as compared with (7.85 ±0.42) %in the blank control group (P<0.05), and in an obviously concentration-dependent manner.15-oxospiramilactone remarkably reduced the number of cells in the G0/G1 phase and increased that in the G 2/M phase (P<0.05). Conclusion 15-oxospiramilactone can significantly inhibit the pro -liferation and migration and induce the apoptosis of 786-0cells in vitro.It may be a potential anti-RCC agent.

Objectives Tostudytheexpressionchangesofproproteinconvertase1(PC1)incerebral cortex nerve cells and its substrate neuropeptide Y (NPY)after focal cerebral ischemia in mice and to investigatetheeffectofPC1inneuronalischemicinjury.Methods Twenty-fourmaleC57micewere randomly allocated into a sham-operation group,an ischemia-reperfusion 4-or 24-hour group with computer (n=8 in each group). A rat model of middle cerebral artery occlusion was induced by the intraluminal suture method. Western blot and real-time quantitative nucleic acid amplification were used to detect the expression changes of PC1,NPY,and mRNA in mouse cortical neurons. Results (1)Compared with the sham operation group,the expression of PC1 mRNA of ischemic cortex brain tissue at ischemic side in the ischemia-reperfusion 4-hour group increased 2. 66 ± 0. 24 and in the ischemia-reperfusion 24-hour group expressed 2. 07 ± 0. 23 (all P<0. 05). Compared with the sham operation group,the PC1 precursor protein level increased significantly at 4 hours (P<0. 05). There was no significant difference in the 24-hour group (P >0. 05 ). (2 )Compared with the sham operation group,the preproNPY mRNA and protein level increased significantly after reperfusion in the ischemia-reperfusion 4-hour group (P < 0. 05 ),the mRNA expressed 2. 31 ± 0. 27,and the increase of precursor protein level continued until 24 hours. Conclusion TheexpressionofprecursorPC1increasedaftercerebralischemia-reperfusioninmice, thus affecting the processing activity of PC1 ,and resulting in NPY protein,an active substrate of PC1 accumulated with the form of precursors,which may be one of the underlying mechanisms of neuronal ischemic injury.

Objective To study the relationships between tissue damage and the ability of the pancreatic cells to regenerate ,and analyze the alteration of the pancreatic cells regeneration .Methods Sixty rats were divided into two groups :impact group(the pan‐creas was injured by a BIM‐Ⅲ biotical impact machine ,40 rats) and control group(sham operated ,20 rats) .All rats were sacrificed at 6 h ,24 h ,72 h ,7 d after operation .The level of AMS ,LPS in the serum were detected by spectrophotometry ,pancreatic cells re‐generation were examined and analyzed by TUNEL staining and flow cytomertry ,and the Bcl‐2 and Bax expression were measured by Western blot .Results In the impact groups ,LPS was activated later than AMS ,and lasted persistently .The results from TUNEL stain ,flow cytometry and Western blot indicated that pancreatic trauma induces cell death and the compensatory prolifera‐tion of pancreatic cells .The characteristics of pancreatic cells regeneration in the animal model of isolated pancreatic trauma indicate that the proper remedial time is in the first 24h after the pancreatic trauma .Conclusion Detecting AMS and LPS at the same time can help us to determine the exocrine function of pancrease .

ObjectiveTo screen a 12-mer phage display peptide library by the polyclonal antibody (pAb) against the recombinant adhesion protein of Mycoplasma genitalium (rMgPa) in order to obtain the antigenic mimic epitopes of MgPa.MethodsThe purified pAb was used to screen the immunodominant mimic epitopes of MgPa by a random 12-peptide phage display library.Seventy-four recombinant phage clones were randomly selected,and then DNA sequence analysis and computer-based bioinformatics analysis were performed to define the consensus amino acid residues of the mimotopes by MIMOX.The binding specificities of the selected phage-displayed peptides to the purified pAb were confirmed by ELISA,competitive ELISA and Western blot analysis.Results After four rounds of biopanning,a significant enrichment of phages was achieved,the inserts from 74 phage clones distinguished 45 peptides based on the different amino acids sequences.Amongst 45 peptides,36 peptides were ELISA positive and 23 peptides that absorbance values were higher than 1.5 showed high reactivities with pAb and effectively inhibited the binding of pAb to rMgPa.Immunoscreening via phage display peptide library revealed three different mimptopes of adhesion protein of M.genitalium,P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I.Results of bioinformatics analysis by MIMOX demonstrated that S,A,F for cluster 1,A,K,I,T and L for cluster 2,K,S,L,R,D and I for cluster 3,may be the key consensus amino acid residues in the aligned mimotopes,respectively.ConclusionAntigenic mimics on MgPa were successfully identified and the motif P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I may represent the immunodominant mimic epitopes of MgPa.And S,A,F K,I,T,L,R and D may be the key amino acid residues for the epitopes of MgPa.

AIM: To study cellular and molecular mechanisms of cardiac development associated genes ex- pression and its function during early stage cardiomyogenesis. METHODS: (1 ) Mouse embryonic stem cells (ESC) line D3 culture. (2) Inductive culals of ESC differentiated into cardiac myocytes in vitro.(3) Identification of ESC -derived cardiac myocytes: RNA isolation; synthesis of specific primer and RT - PCR; Label of RT - PCR products with [? - 32P] dATP as probes, purifyed by sephadex G - 50 columns, determined the yield of DNA. RNA dot hy- bridization. RESULTS: 80% of ESC differentiated into cardiomyocytes by improved conditional medium. Cardiomy- ocytes contraCted in a synchronous manner. The results of RT - PCR and RNA blot showed that cardiac genes were expressed abundantly and specifically during the early cardiomyogenesis. CONCLUSIONS: ESC were able to be dif- ferentiate into cardiomyocytes. Different concentrations and components of RA, DMSO and FCS affected ESC car- diomyogenesis in de. The optimal result obtained was from the conditional medium, a mixturce of 2 nmol/L retinoic acid (RA), 0.6% dimethyl sulfoxide (DMSO) and 20% fend calf serum (FCS).

Objective Studies on the application of fast track surgery ( FTS) are comparatively limited in urologic proce-dures.This randomized controlled study was to evaluate the impact of FTS on recovery after retroperitoneal laparoscopic adrenalectomy . Methods Eighty patients undergoing retroperitoneal laparoscopic adrenalectomy were randomly assigned to an FTS and a control group of equal number to receive an FTS recovery program and conventional perioperative care , respectively .Comparisons were made between the two groups in the time of the first flatus , first oral nutrition , and first mobilization , the incidence of gastrointestinal tract complica-tions, the time of drainage and transurethral catheterization , the length of postoperative hospital stay , hospitalization expenses , visual analogue scale (VAS) pain scores, and general state of the patients . Results The FTS group, in comparison with the control, showed significantly earlier time of first flatus ([20.6 ±8.3] vs [39.8 ±18.3]h, P<0.05), first oral nutrition ([21.1 ±9.9] vs [51.8 ±16.9]h, P<0.05), and first mobilization ([23.6 ±9.0] vs [55.6 ±18.5]h, P<0.05), markedly shorter time of drain-age ([20.9 ±7.9] vs [70.6 ±18.9]h, P<0.05), transurethral catheterization ([20.2 ±8.3] vs[62.5 ±27.1]h, P<0.05), and postoperative hospital stay ([2.43 ±0.94] vs [5.46 ±1.60] d, P<0.05), remarkably less expenses of hospitalization ([21.7 ± 3.2] vs [28.6 ±6.5] ￥1000, P<0.05), and lower postoperative pain scores at 12 h (0.93 ±0.89 vs 1.80 ±1.38), at 24 h while coughing (1.27 ±0.99 vs 4.65 ±1.33), and at 24 h at rest (0.70 ±0.61 vs 1.40 ±0.84) (P<0.05).The general state score was dramatically higher in the FTS patients than in the control on postoperative day (POD) 1 (6.85 ±1.00 vs 4.28 ±1.11) and POD 2 (8.30 ±0.94 vs 5.53 ±1.24) (P<0.01).No significant differ-ences were observed in the general state of the patients between POD 2 and the baseline (P>0.05), nor in the incidence of gastrointesti-nal tract complications between the FTS and control groups ( P >0.05). Conclusion By improving the general state and accelera-ting the recovery of the patients , FTS can be applied safely and effectively in retroperitoneal laparoscopic adrenalectomy .

Objective To explore whether prenatal stress promotes formation of chronic stress-induced hippocampal amyloid β (Aβ) protein in 6-month-old male offspring mice and its mechanism.Methods The APPswe/PSIdE9 double transgenic mice were divided into 4 groups according to the prenatal stress and offspring mice stress:prenatal control-offspring control group (CC group),prenatal control-chronic offspring stress group (CT group),chronic prenatal stress-offspring control group (TC group),and chronic prenatal stress-chronic offspring stress group (TT group) (n=18).The number of amyloid plaques in brains was checked using Congo red staining.ELISA was used to examine the hippocampus levels ofamyloid-β proteins (Aβ1-42 and Aβ1-40) in the offspring mice;β-site APP-cleaving enzyme 1 (BACE1) activity was detected using fluorospectrophotometry.Additionally,Western blotting were used to observe the expression levels of phosphorylated eukaryotic initiation factor 2α (p-eIF2α),phosphorylated protein kinase R [PKR]-like ER kinase (p-PERK),glucose-regulated protein 78 (Grp78) and β-site BACE1 in the hippocampus.Results As compared with that in the CC group,the number of amyloid plaques in brain in CT,TT and TC groups was increased.The expressions of p-eIF2α,p-PERK,Grp78,BACE1,Aβ1-40 and Aβ1-42 in the hippocampus of CT group were significantly increased as compared with those in the CC group (P＜0.05).The expressions of p-eIF2α,p-PERK,Grp78,BACE1,Aβ1-40 and Aβ1-42 in the hippocampus of TT group were further significantly increased as compared with those in the CT group (P＜0.05).There was no significant difference in BACE1 activity among the different groups (P＞0.05).Conclusion The prenatal stress can promote the formation of hippocampal Aβ protein induced by chronic stress in 6-month-old male offspring mice,whose mechanism may be that prenatal stress aggravates hippocampal neurons endoplasmic reticulum stress,activates the PERK,then causes eIF2 alpha phosphorylation,and finally promotes BACE1 expression.

OBJECTIVETo observe the effect of Rgl treatment on prognosis of alcoholic hepatitis using a rat model.

METHODSFemale Sprague-Dawley rats were radomly divided into four groups:unmodeled control, untreated model, Rgl-treated model, and dexamethasone (DXM)-treated model. The model groups were generated by intragastric injection of alcohol. The unmodeled control group was given an equal dosage of normal saline by the same route. After model establishment, the Rg1 treatment group and the DXM treatment group were administered a 120-hour treatment of Rgl or DXM; the unmodeled controls were administered normal saline on the same schedule. All rats were then fasted for 120 hours and venous blood samples were collected for detection of serum aspartate aminotransferase (AST), alanine transaminase (ALT), total bilirubin (TBil), albumin (Alb), tumor necrosis factor-alpha (TNFat) and interleukin 6 (IL-6). Markers of liver inflammation were measured by immunohistochemistry, western blotting, and real-time quantitative reverse transcription PCR. Fat and apoptosis indices were assessed by hematoxylin-eosin staining and TUNEL assay, respectively. The t-test and F test were used for statistical analyses.

RESULTSThe model group showed remarkably more liver steatosis (over one-third of the tissue) than the unmodeled control group, indicating proper establishment of alcoholic liver disease in the modeled rats. The AST, ALT, TBil, and IL-6 levels were significantly higher in the untreated model group than in the Rgl-treated group and the DXM-treated group. The values were significantly different between the Rg1-treated group and the DXM-treated group:ALT, 69.19+/-8.00 U/L vs.102.88+/-5.16 U/L; TBil, 0.36+/-0.07 µmol/L vs.1.20+/-0.18 µmol/L; IL-6, 126.50+/-6.50 U/ml vs.169.19+/-7.68 U/ml; TNFa, 268.31+/-13.19 µg/L vs.318.94+/-7.87 µg/L (all P less than 0.05). Expression of caspase3 and caspase8 was significantly higher in the model group than in the Rgltreated group and the DXM-treated group (both P<0.05). The apoptosis index was significantly lower in the Rgltreated group and the DXM-treated group than in the model group (both P<0.05). The mRNA and protein expression of caspase3, caspase8 and NF-kB were significantly lower in the Rgl-treated group and the DXM-treated group than in the model group (allP less than 0.05), and the levels of all were significantly lower in the Rgl-treated group cornered to the DXM-treated group (all P<0.05). Conehision In rats with alcoholic hepatitis, Rg1 can significantly relieve pathological injury, improve liver function by blocking the apoptotic pathway, and inhibit release of inflammatory cytokines.

Alanine Transaminase ; Animals ; Aspartate Aminotransferases ; Bilirubin ; Cytokines ; Disease Models, Animal ; Ethanol ; Female ; Ginsenosides ; Hepatitis, Alcoholic ; NF-kappa B ; Rats ; Rats, Sprague-Dawley

ObjectiveTo investigate the changes in gut microbiota after transjugular intrahepatic portosystemic shunt (TIPS) in cirrhotic patients with mild hepatic encephalopathy (MHE) in different prognosis groups. MethodsA total of 28 MHE cirrhotic patients who were hospitalized and underwent TIPS in Xijing Hospital of Digestive Diseases from July 2016 to July 2017 were enrolled. Fecal samples and related clinical data were collected on days 1-3 before surgery and at 1 month after surgery. According to the prognosis after surgery, the patients were divided into none-hepatic encephalopathy (HE) group with 8 patients, MHE group with 12 patients, and overt hepatic encephalopathy (OHE) group with 8 patients. Fecal samples were analyzed by 16S rRNA sequencing to obtain the relative abundance of gut microbiota, and SPSS and R packages were used to analyze the biodiversity, postoperative changes, and differences in such changes of gut microbiota at the genus level between groups. The chi-square test was used for comparison of categorical data between groups; the Kruskal-Wallis H test was used for comparison of continuous data between three groups; the Bonferroni method was used for multiple comparisons of multiple samples; the Wilcoxon signed-rank test was used for comparison before and after surgery within each group. For microbiome beta-diversity analyses, a principal coordinate analysis (PCoA) was performed based on Bray-Curtis distance matrix, and the Adonis method (PerMANOVA) was used for comparison between groups. ResultsPCoA based on Bray-Curtis distance matrix showed that only the MHE group had a significant change in beta diversity after surgery (F=2.71, P=0.049). After surgery, the non-HE group had significant increases in the abundance of the native flora Dialister, Coprococcus, Ruminococcaceae_uncultured, Flavonifractor, and Clostridium_sensu_stricto_1 (Z=2.521, 2.1, 2.1, 2.1, and 1.96, all P＜0.05); the MHE group had significant reductions in the abundance of the harmful flora Granulicatella（Z=2.521,P=0.012）, Enterococcus（Z=2.51,P=0.012）, Streptococcus（Z=2.432,P=0.015）, and Rothia（Z=2.001,P=0.045） and significant increases in the abundance of Veillonella（Z=2.353,P=0.019） and Megasphaera（Z=1.955,P=0.05）; the OHE group only had a significant increase in the abundance of Veillonella after surgery (Z=2.38, P=0.017). There was a significant difference in the change in gut microbiota (postoperative abundance/preoperative abundance) between the non-HE group, the MHE group, and the OHE group ［2.00 (1.11-91.61) vs 1.21 (0.26-679) vs 0.09 (0.01-0.92), χ2=6.249, P=0.043］. ConclusionThere is a significant difference in the change in gut microbiota after TIPS between patients with different prognoses, and the increase in the abundance of native flora may have a certain influence on the remission of MHE.