AIM: To test the effect of ethanol precipitation on the purifying technology of chlorogenic acid (CGA). METHODS: Using HPLC, the effect of the extraction of CGA from the crude Flos Lonicerae Japonica powder with three concentrations of ethanol was compared. RESULTS: Ethanol with concentration of 60% was optimal. CGA content was raised from 5.5% to 37.72% by use of ethanol precipitation and extraction in combination. CONCLUSION: The ethanol precipitation can get rid of the impurity in the crude powder of Flos Lonicerae Japonica effectively and the yield of CGA can reach 90.16%.

Objective To investigate the biological functions of HBeAg-binding protein 1(HBEBP1), suppression subtractive hybridization(SSH) technique was used to screen genes regulated by HBEBP1. Methods HBEBP1(GenBank number:AF529372) was screened and identified by yeast two-hybrid system 3 and co-immunoprecipitation technique. The HBEBP1 coding DNA fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) technique from HepG2 cell genome. The expressive vector of pcDNA3.1(-)-HBEBP1 was constructed by routine molecular biological methods. The HepG2 cells were transfected with pcDNA3.1(-) and pcDNA3.1(-)-HBEBP1, respectively by using FuGENE6 transfection reagent, then the mRNA was isolated. SSH method was employed to analyze the differentially expressed DNA sequences between the two groups. After restriction enzyme Rsa I digestion, small sized cDNAs were obtained. Then tester cDNAs were divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNAs were hybridized with driver cDNAs twice and underwent polymerase chain reaction (PCR) twice, they were then subcloned into pGEM-Teasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5?. The cDNAs were sequenced and analyzed in GenBank with Blast search after PCR. Results The subtractive library of genes up-regulated by HBEBP1 was constructed successfully. The amplified library contained 85 positive clones. Colony PCR showed that these clones contained 200-1 000bp inserts. Sequence analysis was performed in 26 clones at random, and the full length sequences were obtained with bioinformatics method. Altogether 15 coding sequences were obtained. Conclusions The obtained sequences may be target genes up-regulated by HBEBP1, among which some genes coding proteins were involved in cell cycle regulation, metabolism, immunity and cell apoptosis. This finding brought some new clues for the study of the biological functions of HBEBP1 and HBeAg.

Objective TostudytheextrahepaticinfectionandreplicationofhepatitisCvirusinthe patientswithhepatitisC .Methods HCVRNA ,intermediateofHCVreplication(minus strandofHCV RNA) ,andHCVantigensweredetectedbyreverse transcriptasepolymerasechainreaction(RT PCR) ,in situhybridization(ISH)andimmunohistochemistryin 38autopsyextrahepatictissuespecimens(including kidney ,heart,pancreas ,intestine ,adrenalgland ,spleen ,lymphnode,andgallbladder)from 9hepatitisC patients.Results ByRT PCR ,all 9patientswerepositiveforHCVRNAinkidney ,heart ,pancreas , andintestine .However,only 6 (6 6 .7% ) patientswerepositiveforintermediateofHCVreplication .The positiveratesofHCVRNAandHCVantigensinextrahepaticorgansotherthanspleenwere 5 5 .6 % (5 pa tients)and 6 6 .7% (6 patients)respectively .HCVRNAandHCVantigenswerepositiveinthefollowing cells:myocardialcells,epithelialcellsofintestine ,interstitialcellsofkidney ,epithelialcellsoftubulesand glomerulus ,pancreasacinarcellsandepithelialcellsofpancreaticduct,epithelialcellsofmucousmembrane sinusofgallbladder ,cortexandmedullacellsinadrenalgland ,andmononuclearcellsinlymphnode .Con clusion TheseresultsshowthatHCVreplicationexistsinvariousextraheptictissuecellsatlow level, whichmayhavecertainpathogeneticandclinicalsignificanceinthepatientswithHCVinfection .

Objective To explore epirubicin hydrochloride combined with capecitabine tablets on serum levels of osteopontin (OPN), vascular endothelial growth factor C ( VEGF-C) and its efficacy in the treatment of patients with triple-negative breast cancer.Methods 80 cases triple-negative breast cancer were selected, randomly divided into two groups, 40 cases in each group.The control group were given epirubicin treatment, joint group on the basis of the control group were combined with capecitabine, 21 days for a course of treatment, four courses of treatment.The serum levels of OPN and VEGF-C pre-and post-treatment were detected, the efficacy and adverse reactions were observed.Results Compared with before treatment, the serum levels of OPN and VEGF-C in two groups were lower post-treatment(P<0.05).Compared with control group, the OPN and VEGF-C levels in joint group were lower (P<0.05), the efficacy was higher and the adverse reaction was lower (P<0.05).Conclusion The epirubicin hydrochloride combined with capecitabine tablets could reduce serum OPN and VEGF-C levels in the treatment of patients with triple-negative breast cancer, with high security.

Objective To analyze the effects of Fuzheng Xiaoai decoction joint tamoxifen on serum sex hormone and endometrial thickness with breast cancer.Methods 124 patients with breast cancer were divided into control group and research group by lot drawing method, all as 62 cases, treated with the same surgery, control group was treated with tamoxifen, research group was treated with Fuzheng Xiaoai decoction based on control group, the sex hormones, endometrial thickness, tumor markers, immune function and complications were compared between two groups.Results The prolactin (PRL), progesterone (P), estradiol (E2) of research group were all lower than control group, the difference were statistically significant (P<0.05).The endometrial thickness of research group [(8.61+1.07) mm]was lower than the control group [(9.74+1.21) mm](P<0.05).The tumor markers, immune function of research group were better than that of control group (P<0.05).The complications was no difference between two groups. Conclusion Fuzheng Xiaoai decoction joint tamoxifen can regulation the serum levels of sex hormone, relieve tamoxifen-induced endometrial thickening, can improve tumor markers and immune function .

Objective To explore the caregiver burden and psychological resilience of patients with dilated cardiomyopathy and the correlation between the two factors.Methods Totally 96 caregivers of dilated cardiomyopathy patients were selected by convenience sampling and investigated for their level of burden and psychological resilience with the Caregiver Burden Inventory (CBI) and Conor-Davidson Resilience Scale (CD-RISC). The correlation between the two factors was then analyzed.Results The CBI score of these caregivers was (47.19±10.38), significantly higher than the average level nationwide (24.2±5.8). Their CD-RISC score was (63.07±13.18), lower than (65.4±13.9) of China's community population. There was apparent negative correlation between caregiver burden and psychological resilience (r=-0.62,P<0.01). The total score of resilience was negatively related to the burden of development constraint, social burden and emotional burden (r=-0.72, -0.65, -0.51;P<0.01).Conclusions The caregivers of dilated cardiomyopathy patients have a heavy burden, which is negatively correlated with their psychological resilience. Therefore, measures should be taken in clinical work to optimize the caregivers' psychological resilience, reduce their burden, and improve their quality of care.

Objective To investigate the effect of tigecycline on coagulation function and to provide a reference for the clinical rational use of tigecycline.Methods The data of patients treated with tigecycline in Shanghai Tenth People's Hospital between June 2019 and December 2023 by retrospective analysis.Statistical analysis was performed by collecting data on patients'basic information,routine coagulation parameters and thromboelastogram(TEG)parameters before and after the use of tigecycline.Results Activated partial thromboplastin time,prothrombin time and thrombin time were prolonged and fibrinogen levels were decreased with the use of tigecycline in 41 patients,the differences were statistically significant(P＜0.05).There was no significant difference in levels of coagulation factor activation time,clot formation rate parameter,maximum angle of tangency,maximum amplitude of elastography and coagulation index after treatment with tigecycline(P＞0.05).Conclusion For patients with suspected coagulation abnormalities after tigecycline administration,a comprehensive assessment of coagulation should be made by combining routine coagulation indexes with TEG.

Objective:To construct a microfluidic organ-on-a-chip and evaluate its capability in simulating subchondral bone remodeling during the progression of osteoarthritis.Methods:The chip′s main body was designed based on the microfluidic technology and cell co-culture technique. MC3T3-E1 cells were cultured adherently within the cell seeding micro-chamber, with the culture medium perfused at a flow rate of 0.5 ml/min at the bottom of the micro-chamber. Evaluation metrics were as follows: (1) Assessment of the microfluidic organ-on-a-chip: The growth culture medium was perfused and simulation experiments were conducted to test the concentration differences and equilibrium times of the fluid inside and at the bottom of the cell seeding micro-chamber at various time points; live-dead staining was performed to observe the biocompatibility of cells cultured continuously for 3 days and 7 days at a set flow rate, which was divided into 3-day and 7-day groups. (2) Osteogenic potential of the microfluidic organ-on-a-chip: The osteogenic induction medium was perfused, and ALP staining and PCR were performed to compare the number of the black alkaline phosphatase (ALP)-positive cells and the expression levels of osteogenesis-related marker genes including osteoblast-specific transcription factor 2 (RUNX2), type I collagen (COL1A1), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN) under static, 3-day and 7-day perfusion conditions, which was divided into static non-induced, static-induced and perfusion-induced groups. (3) Characterization of morphology and size, and biocompatibility of extracellular vesicles (EVs) of three osteoblast subtypes: Three different subtypes of osteoblasts were obtained [endothelial-type osteoblasts (EnOB)-EVs, stromal-type osteoblasts (StOB)-EVs and mineralizing-type osteoblasts (MinOB)-EVs]. Their morphology and size were obtained through transmission electron microscopy and particle size analysis. Growth medium containing EVs of three different cell subtypes was perfused, and cell proliferation/apoptosis assay was performed to compare the biocompatibility of the addition of different EVs concentrations (1, 1.25, 2.5, and 5 μg/ml) for 24 hours, which was categorized into the EnOB-EVs group, StOB-EVs group and MinOB-EVs group. (4) Osteogenic effect of EVs from three subtypes of osteoblasts: Osteogenic induction media containing EVs from three different osteoblast subtypes were perfused for 3 days, and ALP staining and PCR were performed to compare the number of black ALP-positive cells and the expression levels of osteogenesis-related marker genes including RUNX2, COL1A1, BMP-2, and OCN, which was divided into non-EVs group, EnOB-EVs group, StOB-EVs group and MinOB-EVs group.Results:(1) Evaluation of the microfluidic organ-on-a-chip: Simulation results showed that the concentration in the top layer of the upper chamber reached more than 95% of that in the lower chamber and that the concentration in the bottom layer was about 96.5% of that in the lower chamber after 12 hours of continuous perfusion, reaching an equilibrium state of the concentration difference between the upper and lower chambers. The results of live-dead staining showed that the chip was biocompatible at a flow rate of 0.5 ml/min, and the cell survival rate at 3 and 7 days of perfusion was (99.48±0.12)% and (97.07±1.05)% ( P<0.01). (2) ALP staining results showed that at 3 days, the perfusion-induced group showed the highest number of black ALP-positive cells, followed by the static-induced group, and the least in the static non-induced group. At 7 days, the static-induced group had the highest number of black ALP-positive cells, followed by the perfusion-induced group, and the least in the static non-induced group. PCR results indicated that at 3 days, the expression levels of RUNX2, COL1A1, BMP-2, and OCN were 1.00±0.03, 1.00±0.12, 1.00±0.01, and 1.00±0.02 respectively in the static non-induced group; 1.80±0.04, 4.05±0.37, 9.80±1.94, and 4.38±0.89 respectively in the static-induced group, and 2.45±0.23, 5.48±0.42, 91.50±4.56, and 10.82±4.96 respectively in the perfusion-induced group ( P<0.01). At 7 days, the expression levels of RUNX2 was 1.00±0.01 in the static non-induced group, 1.46±0.46 in the static-induced group, and 1.11±0.08 in the perfusion-induced group ( P>0.05); the expression levels of COL1A1, BMP-2, and OCN were 1.00±0.03, 1.00±0.13, and 1.00±0.09 respectively in the static non-induced group, 9.38±0.25, 14.27±4.35, and 84.01±4.02 respectviely in the static-induced group, and 2.39±0.08, 133.64±8.87, and 86.64±8.36 respectively in the perfusion-induced group ( P<0.01). When comparing the static non-induced, static-induced, and perfusion-induced groups at both 3 and 7 days, the perfusion-induced group demonstrated the strongest osteogenic capability. (3) Characterization of morphology and size and biocompatibility of EVs from three osteoblast subtypes: Under the transmission electron microscope, EVs from EnOB-EVs, StOB-EVs, and MinOB-EVs all exhibited a typical saucer-shaped morphology. The particle sizes of EnOB-EVs, StOB-EVs, and MinOB-EVs were (91.3±14.7)nm, (106.0±16.0)nm, and (68.1±10.7)nm, respectively. Cell proliferation/apoptosis assay results indicated that the optimal administration concentration of EnOB-EVs, StOB-EVs, and MinOB-EVs was all 1.25 μg/mL. (4) Validation of osteogenic effect of the microfluidic organ-on-a-chip on three types of EVs: ALP staining results showed that the non-EVs group had the fewest black ALP-positive cells, followed by the EnOB-EVs group, then the StOB-EVs group, and the MinOB-EVs group had the most. PCR results showed that the expression levels of RUNX2, COL1A1, BMP-2, and OCN were 1.00±0.01, 1.00±0.03, 1.00±0.02, and 1.00±0.02 respectively in the non-EVs group, 1.95±0.11, 6.78±2.04, 7.99±0.57, and 6.93±3.83 repectively in the EnOB-EVs group, 0.79±0.12, 5.68±1.53, 12.59±3.15, and 25.59±0.95 respectively in the StOB-EVs group, and 0.68±0.10, 4.36±0.69, 18.75±3.21, and 34.74±3.98 repectively in the MinOB-EVs group ( P<0.01). Compared with the non-EVs group, EnOB-EVs group, StOB-EVs group, and MinOB-EVs group, the MinOB-EVs group showed the most significant osteogenic effect. Conclusions:The microfluidic organ-on-a-chip constructed using microfluidic technology and cell co-culture techniques is capable of maintaining the normal growth of MC3T3-E1 cells, enhancing their proliferation and osteogenic induction differentiation. EVs released by osteoblasts at different stages possess osteogenic effects and can accelerate the bone sclerosis in the remodeling of subchondral bone during the progression of osteoarthritis.