This paper is aim to investigate the pharmacokinetics and absolute bioavailability of neoline in Beagle dogs, and provide a theoretical basis for further study. Ethyl acetate was used for liquid-liquid extracting after 10% ammonia alkalizing. The method of UPLC-Q-TOF-MS was established for the determination of neoline plasma concentrations. Beagle dogs were orally or intravenously administered with neoline for pharmacokinetic and absolute bioavailability study. Good linear relationship of neoline was found over the range of 0.1-4 mg x L(-1) (R2 = 0.9982) and 2-100 microg x L(-1) (R2 = 0.9945). Intra-and inter-day precision, expressed as the relativestandard (RSD) were less than 5.0%. Accuracy, expressed as the relative error (RE) was within 90.0%-115%. The recovery of neoline in dog plasma was more than 80%. After 6 mg x kg(-1) for ig and 1 mg x kg(-1) for iv administration of neoline, the main pharmacokinetic parameters were analyzed with Winnonlin software. t(1/2) were (313.88 +/- 63.18), (236.33 +/- 229.84) min, and AUC(0-infinity) were (58,027.40 +/- 14,132.69), (473,578.02 +/- 82,333.08) min x microg x L(-1) for ig and iv administration respectively. The absolute bioavail ability was (73.15 +/- 10.29) %. The method of UPLC-Q-TOF-MS described in the report was sensitive, reliable and specific, and suitable for pharmacokinetic study of neoline in Beagle dog. The high absolute bioavailability of neoline in dog suggested good absorption of neline which was worth of further investigation.
Aconitine ; analogs & derivatives ; chemistry ; pharmacokinetics ; Animals ; Biological Availability ; Dogs ; Drug Stability ; Female ; Male

Human hepatitis B virus surface antigen (HBsAg) binding protein(SBP) shows a specific binding ability to HBV surface antigen HBsAg. Previous work proved an ability of SBP to enhance the immune response of HBsAg vaccine. To investigate the function and mechanism of this protein, we constructed SBP-expression strains with Pichia pastoris expression system. We screened these strains and have got an expression strain with high protein expression quantity. Fermentation product was collected and purified to gain a large amount of purified protein. Identification of purified SBP with SDS-PAGE, High performance liquid chromatography, Western blotting and mass spectrometry suggested that the protein was highly purified and with a good integrity. ELISA test of purified SBP showed a significant binding ability to HBsAg, suggesting a good protein activity. This work offers a solid foundation to the research of SBP function and mechanism of immune enhancement.
Binding Sites ; Genetic Vectors ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Humans ; Pichia ; genetics ; metabolism ; Receptors, Virus ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification

Objective To study the FMS-like tyrosine kinase-3 (FLT3) gene, NPM1 gene and c-kit gene mutations in acute myeloid leukemia (AML) by extracting DNA from the storage of bone marrow slides, and to investigate the relationship between the three gene mutations and clinical features in AML. Methods The bone marrow slides of 55 patients diagnosed with AML were enrolled in this study. The PCR, DNA sequencing and molecular cloning were used to detect and analyse the FLT3-ITD, NPM1 and c-kit gene mutations. Patients' remission, progression and survival time were also recorded. Results The DNA was successfully extracted from the bone marrow slides with -20 ℃ frozen storage without Wright stained, chemically fixed, and room temperature storage Wright stained discoloured by phenol ∶ chloroform ∶ isoamyl alcohol method, which can be used in PCR, direct sequencing and molecular cloning sequencing analysis. 10 of the 55 cases (18.2 %) were FLT3-ITD positive, including 9 cases with heterozygous mutations and 1 case with homozygous mutation. FLT3-ITD positive group had lower complete remission (CR) rate, shorter event-free survival (EFS) time and overall survival (OS) time than the negative group (P< 0.05). 9 of the 55 cases (16.4 %) had NPM1 heterozygous gene mutations, all belonging to type A. The EFS rate of the patients with NPM1 mutation was higher in 10 months and the OS rate was higher in 19 months (P< 0.05). 3 of 9 NPM1 mutations patients were FLT3-ITD positive. The CR rates of the four groups after initial remission induction therapy in order were NPM1+FLT3-ITD-, NPM1-FLT3-ITD-, NPM1-FLT3-ITD+, NPM1+FLT3-ITD+(P<0.05). Besides, NPM1-FLT3-ITD+was a risk factor affecting the OS (RR=1.250, P=0.005). 2 of the 55 cases (3.6 %) had c-kit gene mutations, namely mutant D816H and mutant D816V. The c-kit gene mutations were not found in patients with FLT3-ITD and NPM1 mutations. Conclusions The FLT3-ITD mutation is a poor prognosis molecular marker in AML, and NPM1 mutation is a good factor for the prognosis. NPM1-FLT3-ITD+is a risk factor affecting OS. The rate of c-kit gene mutation is low in AML, without the overlap of FLT3 and NPM1 mutations.

OBJECTIVE: To observe the demethylation effect of demethylation inhibitor 5-azacytidine (5-Zac) on programmed death receptor 1 (PD-1) in Molt-4 cells (T lymphocyte cell line) and to investigate the relationship between DNA demethylation and expression of PD-1. METHODS: Molt-4 cells were cultured in the medium containing different concentrations of 5-Zac(0, 5, 10 μmol/L) for 72 h. According to the concentrations of 5-Zac, the Molt-4 cells were divided into a 0 μmol/L 5-Zac group, a 5 μmol/L 5-Zac group, and a 10 μmol/L 5-Zac group. The expression of PD-1 in Molt-4 cells was detected by flow cytometry and the apoptosis rate was calculated. The mRNA transcription level of PD-1 was detected by real-time polymerase chain reaction; Molt-4 cell DNA in all groups were treated by sodium bisulfite. The PD-1 promoter fragment was amplified by PCR, the amplification fragments were transformed into E. coli., the positive clones were selected for equencing, and the methylation status of the fragments of PD-1 promoter was examined. RESULTS Seventy-two hours after the 5-Zac treatment, the expression rate of PD-1 in the Molt-4 cells in the 0 μmol/L 5-Zac group, the 5 μmol/L 5-Zac group, and the 10 μmol/L 5-Zac group was (1.13 ± 0.01)%, (18.96 ± 1.87)%, and (63.09 ± 6.25)% respectively, in a low concentration-dependent way. The PD-1 mRNA expression level was increased significantly with the 5-Zac treatment. Cells apoptosis showed that:compared with the 0 μmol/L 5-Zac group, the apoptosid rate in the 5 μmol/L 5-Zac group and 10 μmol/L 5-Zac group was signficantly increased, which was (1.9 ± 0.06)%, (8.89 ± 1.36)%, and (24.50 ± 3.68)% in the 0 μmol/L 5-Zac group, the 5 μmol/L 5-Zac group, and the 10 μmol/L 5-Zac mol/L group respectively. The bisulfite genomic sequencing showed that the demethylation probability of CpG points on -601 bp and -553 bp was significantly increased in the 5-Zac treated cells compared with those untreated. CONCLUSION 5-Zac can result in the increase of PD-1 expression in the human lymphoid cell series Molt-4 in vitro, and the apoptosis rate increases, which is related to PD-1 gene promoter demethylation.
Apoptosis ; genetics ; Azacitidine ; pharmacology ; Cell Line ; CpG Islands ; genetics ; DNA Methylation ; drug effects ; genetics ; Enzyme Inhibitors ; pharmacology ; Humans ; Programmed Cell Death 1 Receptor ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; genetics ; metabolism ; T-Lymphocytes ; cytology ; metabolism

Objective:To compare the safety and effectiveness of two minimally invasive approaches for multi-vessel coronary revascularization.Methods:From August 2014 to February 2017,70 consecutive patients who underwent minimally invasive coronary artery bypass grafting in Peking University Third Hospital were randomly divided into two groups.In one group,40 patients underwent staged-hybrid coronary revascularization (staged-HCR) treatment;in the other group,30 patients underwent minimally invasive total arterial revascularization with bilateral internal thoracic artery (BITA).In staged-HCR group,the patients underwent minimally invasive direct coronary artery bypass grafting (MIDCAB) and percutaneous coronary intervention (PCI) procedure for treatment of multi-vessel disease.In BITA group,the patients underwent total arterial coronary artery bypass grafting with composite "Y" BITA graft.Preoperative and postoperative data of the two groups,including postoperative blood usage,mechanical ventilation time,domiciling duration in intensive care unit (ICU),major adverse cerebral and cardiovascular event (MACCE),and postoperative coronary angiography results were compared,in order to evaluate the safety and effectiveness of these surgical approaches.Results:The preoperative characteristics of 70 patients in the two groups showed no significant difference.All the patients underwent successfully,elective minimally invasive multi-vessel coronary artery bypass grafting as scheduled preoperatively.Postoperative result showed the patients in staged-HCR group took advantages in less postoperative mechanical ventilation time [Staged-HCR group (11.2 ± 8.7) h vs.BITA group (18.3 ± 9.1) h,P =0.013],shorter domiciling duration in ICU [Staged-HCR group (26.29 ± 4.05) h vs.BITA group (44.74 ± 28.75) h,P =0.022],and less total drainage [Staged-HCR group (695.57 ± 250.46) mL vs.BITA group (1 103.26±547.44) mL,P =0.03] than the patients in the group of minimally invasive total arterial revascularization with BITA.Postoperative in hospital coronary angiography showed satisfactory graft patency rates in both groups [97.5％ in Staged-HCR group vs.97.8％ in BITA group].No MACCE occurred in both groups during hospitalization.Conclusion:Staged-HCR is a feasible method for the treatment of multi-vessel revascularization involving right coronary artery.Minimally coronary revascularization with BITA is associated with superior long-term graft patency and it's recommended for patients who could not tolerate dual-antiplatelet therapy.This study shows that both minimally invasive surgical approaches are safe and effective for treatment of patients with multi-vessel coronary artery disease.

Objective To study the anti thrombotic effect of Ligustrazine injection combined with aspirin,clopidogrel in rabbits.Methods Forty-two healthy Japanese white rabbits were randomly divided into 7 groups:blank group,control A group,control B group,control-low dose herb group,control-high dose herb group,combined A group,combined B group.Blank group was given equal volume normal saline,control A group was given aspirin 5.13 mg· kg-1,control B group was given clopidogrel 3.85 mg · kg-1,control-low,high doses herb groups were given Ligustrazine injection 6.54,26.16 mg · kg-1,combined A group was given Ligustrazine injection 26.16 mg· kg-1 and aspirin 5.13 mg · kg-1,combined B group was given Ligustrazine injection 26.16 mg · kg-1 and clopidogrel 3.85 mg · kg-1.Ligustrazine injection was given for ten days through ear marginal vein and aspirin,clopidogrel were given for seven days through intragastric administration.On the last day of administration,heart blood was taken at 1 h after administration.Thrombus length and wet weight were measured by extracorporeal thrombus forming apparatus.The effect on the platelet aggregation rate induced by platelet activating factors (PAF) was measured by platelet aggregation instrument.And the content of platelet activating factors (PAF),cluster of differentiation 62 platelet (CD62p) and platelet factor-4 (PF-4) were detected by enzyme-linked immunosorbent assay.Results The throrobus length in combined B group was (7.4 ± 4.6) cm,which significantly reduced compared with that of blank group (22.8 ±2.3) cm (P ＜0.01).The wet weights of thrombus of control A,B group,control-high dose herb group and combined A,B group were (0.31 ± 0.04),(0.30 ± 0.05),(0.35 ±0.14),(0.28 ±0.08),(0.16 ±0.07) g,compared with that of blank group (0.55 ±0.10) g with significantly reduced(P ＜ 0.01).The platelet aggregation rates of control-low,-high doses herb groups and combined A,B groups induced by PAF were (43.3 ± 4.7) ％,(37.2 ± 4.7) ％,(35.5 ± 4.1) ％,(35.0 ± 3.2) ％,when compared with that of blank group (52.9 ± 4.4) ％,they reduced significantly (P ＜ 0.01).The platelet aggregation rates of combined A,B group were significantly lower than that of control-low dose herb group (P ＜ 0.05).The PAF contents of control-low,-high doses herb groups and combined A,B groups were (1043 ± 162),(1010 ± 189),(973 ± 159),(959 ± 178) ng · L-1,significantly lower than that of blank group (1510 ± 319) ng · L-1 (P ＜ 0.01).The CD62P contents of control A,B groups,control-low,-high doses herb groups and combined A,B groups were (156 ± 13),(130 ±21),(101 ±17),(85 ±8),(107 ± 18),(107 ± 19) ng · mL-1,significantly lower than that of blank group (234 ±69) ng · mL-1(P ＜ 0.01).The PF-4 content of control A,B groups,control-high dose herb group and combined A,B groups were (148 ± 14),(255 ± 14),(322 ±69),(242 ±26),(266 ±36) ng· mL-1,which significantly reduced compared with that of blank group (478 ±91) ng · mL-1 (P ＜0.05,P ＜0.01).Conclusion Ligustrazine injection combined with aspirin and clopidogrel can play a synergistic anti-thrombosis effect.

Objective: To evaluate the effectiveness of therapeutic massage (tuina) for treating knee osteoarthritis (KOA). Methods: Six English and Chinese databases, including Chinese National Knowledge Infrastructure (CNKI), Wanfang Academic Journal Full-text Database (Wanfang), Chongqing VIP Database (CQVIP), China Biology Medicine Disc (CBM), Cochrane Library and PubMed databases, were independently searched to identify appropriate randomized controlled trials (RCTs) studying therapeutic massage for KOA compared to oral non-steroidal anti-inflammatory drugs (NSAIDs) alone. The main outcome measures were total effectiveness and the Western Ontario and McMaster Universities osteoarthritis index (WOMAC) score. Results: A total of 8 RCTs were included and they were of average quality. The results showed that therapeutic massage was more effective than NSAIDs comparing total effectiveness [risk ratio (RR)=1.14, 95％ confidence interval (CI) (1.07, 1.21), P<0.0001]; compared with NSAIDs, therapeutic massage produced more significant improvements in pain [mean difference (MD)=-2.06, 95％CI (-2.75, -1.36), P<0.00001], stiffness intensity [MD=-0.90, 95％CI (-1.05, -0.75), P<0.00001] and joint function [MD=-12.48, 95％CI (-13.91, -11.05), P<0.00001]. Conclusion: Therapeutic massage was more effective than oral NSAIDs in treating KOA. In relieving pain and stiffness and improving the function of knee joint, therapeutic massage was superior to NSAIDs.

Aged ; Female ; Humans ; Hypertriglyceridemia ; blood ; epidemiology ; Male ; Middle Aged ; Prevalence ; Uric Acid ; blood

OBJECTIVETo explore a operative approach and its effect to the central protrusion of lumbar intervertebral disc.

METHODSFrom February 1999 to December 2005,34 patients with central protrusion of lumbar intervertebral disc were treated with an improved operative procedure. The study involved 25 males and 9 females with an average of 46.4 years (range, 35 to 63 years). The involved level of herniation were at L4,5 in 20 cases and L5S1 in 14 cases. Pains happened on one leg fixedly and seriously with another lightly in 21 cases, on one leg initially and lightly with another seriously later in 8 cases, on bilateral legs alike in 5 cases. Preoperative CT film showed central type in 8 cases and laterocentral type in 26 cases. The corresponding spinous process was resected on the basis of unilateral fenestration. The supraspinous ligament was retained and pulled to the opposite side for revealing spinal canal, and then diskectomy was done. The above procedure was named "fenestration with the spinous process resection".

RESULTSAll the 34 patients were followed up for 1 to 5 years. The outcome was evaluated according to the standard of HOU Shu-xun, 20 cases were excellent, 11 good and 3 fair. The total rate of excellent and good was 91.2%.

CONCLUSIONThe "fenestration with the spinous process resection" not only completed decompression of spinal canal and diskectomy, but also retained opposite lamina and supraspinous ligament and maintained the stability of posterior vertebral column, which are a new improved approach for the central protrusion of lumbar intervertebral disc.

Adult ; Female ; Humans ; Intervertebral Disc Displacement ; surgery ; Lumbar Vertebrae ; surgery ; Male ; Middle Aged

BACKGROUNDRecent studies have discovered that nuclear translocation of nerve growth factor (NGF) and its receptor fragments function differently from the traditional model. This study aimed to uncover the nuclear expression of NGF in astrocytoma and its biological significance.

METHODSNinety-four paraffin-embedded astrocytoma specimens were subjected to immunohistochemical (IHC) and hemotoxylin & eosin (HE) staining. Preoperative cerebrospinal fluid (CSF) specimens and intraoperative snap-frozen astrocytoma tissues were assayed for NGF expression by ELISA and Western blotting. The outcome of patients who contributed samples was tracked. Each ten tissue samples from patients with traumatic brain injury who had received decompression surgery and CSF samples from patients undergoing spinal anesthesia but with no history of nervous system disease were taken as control.

RESULTSNGF-positive immunoreactive products were distributed in both the cytoplasm and nucleus of astrocytoma, but were only located in the cytoplasm of traumatic brain injury (TBI) tissue. NGF nuclear-positive rate (NPR) of grades III - IV astrocytomas (70.0%) was higher than that of grades I - II astrocytoma (28.6%, P < 0.05). NGF-NP expression positively correlated with the NGF concentration in cerebrospinal fluid (CSF) (r = 0.755, P < 0.01). Kaplan-Meier survival analysis indicated that the median survival time was 25 months for NGF-NP astrocytoma grade I - II patients and 42 months in NGF nuclear negative (NGF-NN) astrocytoma grade I - II patients (P < 0.05). In astrocytoma III - IV patients, the median survival was 7 months for NGF-NP patients and 24 months for NGF-NN patients (P < 0.01). Two types of NGF with molecular weights of 13 and 36 kDa were present in astrocytoma, but only the 36 kDa NGF was found in the CSF. NGF expression elevated as the malignancy increased.

CONCLUSIONSNGF-NP expression and NGF level in CSF were significant prognostic factors in astrocytoma patients. Because of the easy access of CSF, it may be developed as an index for early diagnosis and surveillance of astrocytoma.

Astrocytoma ; metabolism ; Biomarkers ; metabolism ; Blotting, Western ; Cerebrospinal Fluid ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Nerve Growth Factor ; metabolism ; Prognosis