Aim To observe the effect of ramipril on ischemia-reperfusion induced apoptosis of cardiomyocytes and expression of apoptosis-related genes.Method Male Wistar rats were subjected to 30 min of myocardial ischemia and 120 min of reperfusion.Rats were randomized to receive vehicle or ramipril(1 mg?kg~(-1)) orally 24 h before surgery.Myocardial infarct size was measured using the staining agent 2,3,5-triphenyl tetrazolium chloride(TTC).Cardiomyocyte apoptosis was assessed using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL) assay and confirmed with detection of DNA ladder formation.The expression of antiapoptotic gene(Bcl-2) and proapoptotic gene(Bax) was determined by immunohistochemistry.Results At the end of reperfusion,infarct size was reduced by ramipril.TUNEL assay and DNA-laddering study demonstrated that cardiomyocyte apoptosis was attenuated in ramipril-treated hearts,correlating with increased expression of Bcl-2 and ratio of Bcl-2/Bax.Conclusion Ramipril exerts cardioprotection by decreasing the ischemia-reperfusion induced necrosis and apoptosis of cardiomyocyte and increasing the expression of Bcl-2 and ratio of Bcl-2/Bax.

Aim To investigate the protection of ramipril,BQ-123 and their combination against myocardial ischemia/reperfusion(I/R)injury in vivo in anesthetized rats,and to explore the mechanism of action of drugs on myocardial oxidation-antioxidation system.Methods Healthy male Wistar rats were divided into 5 groups randomly,sham operated(Sham)group,I/R group,ramipril(RAM)group,BQ-123(BQ)group and ramipril and BQ-123(R&B)group.All groups but not sham were subjected to I/R procedure.Twenty four hours before ligation,ramipril(1 mg·kg~(-1))was intragastrically administered to rats in RAM and R&B groups.The equal volume of normal saline was given to rats in other groups.BQ-123(10 μg·kg~(-1)· min-1)was infused intravenously from 10 min before ligation to the end of 30 min ischemia to rats in BQ and R&B groups.The equal volume of normal saline was given to other groups.HR,MAP and the change of ST-segment were observed;ventricular arrhythmias were monitored during ischemia;the infarct size was examined by TTC staining;the activity of myocardial T-SOD,Mn-SOD,CAT and the content of MDA were detected by spectrophotometer.Results Compared with I/R group,the elevation of ST-segment was decreased,onset of VPC and VT was delayed,duration of VPC and VT was shortened,incidence of VPC,VT and VF was decreased,IS and IS/AAR were improved,activity of T-SOD,Mn-SOD and CAT was increased,the content of MDA was decreased in RAM,BQ and R&B groups.Compared with RAM and BQ alone group,onset of VPC and VT,duration of VPC and VT,size,activity of T-SOD and Mn-SOD and content of MDA were changed dramatically in R&B group.Conclusions Ramipril,BQ-123 and the combined use of these two agents protected myocardium from I/R injury in vivo.The protective effects of the combination on delaying onset of VA,shortening duration of VA,decreasing infarct size and content of MDA,and increasing activity of SOD are better than those of using ramipril or BQ-123 alone.

Objective To observe the efficacy and safety of intravitreal injection of ranibizumab in the treatment of retinopathy of prematurity (ROP).Methods Data from 49 consecutive ROP patients (95 eyes) including type Ⅰ pre-threshold,threshold and aggressive posterior ROP who had received anti-VEGF treatment for the first time in our hospital from June 2014 to August 2015 were collected.60 eyes from the 95 eyes were confined as the zone Ⅰ disease group,while the remaining 35 eyes as zone Ⅱ disease group.The difference of birth weight,gestational age,corrected gestational age,treatment effects,recurrence and re-treatment time between two groups were compared.0.025 mL ranibizumab (10 mg · mL-1) was injected through 1.5 mm puncture after corneal limbus by using 30G 1 mL injection syringe.At the end of the injection,tobramycin and dexamethasone ophthalmic ointment eye bag was used.After the injection of 3 days,the portable slit lamp and tonometer were used to observe the intraocular pressure,intraocular hemorrhage and endophthalmitis.The indirect ophthalmoscope was used to observe the retinal vascular tortuosity and ridge regression of lesion expansion at 1 week after treatment.At the same time,the systemic adverse reactions related to treatment were observed.Results After receiving ranibizumab treatment for the first time,93 eyes (95.9％) exhibited ROP regression after single injection,including 58 eyes in zone Ⅰ disease group,35 eyes in zone Ⅱ disease group.There was no statistical difference between two groups (P ＞ 0.05).22 eyes required additional anti-VEGF injection or laser treatment for ROP recurrence,including 17 eyes in zone Ⅰ disease group,5 eyes in zone Ⅱ disease group.There was statistical difference between two groups (P ＜0.05).The time from recurrence to re-treatment was (6.50 ±2.54) weeks,which in zone Ⅰ disease group was (6.44 ± 2.74) weeks and in zone Ⅱ disease group was (6.67 ± 2.31)weeks,there was no statistical difference between two groups (P ＞ 0.05).No local or systemic adverse events associated with the treatment or drug was observed within the following period.Conclusion Intravitreal injection of ranibizumab is an effective and well tolerated method for zone Ⅰ and zone Ⅱ ROP,but the recurrence rate is high.There Is no local or systemic adverse events associated with the treatment or drug.

Objective To establish a flow-cytometric method to detect microvesicles (MVs) in rat peripheral blood, and to detect platelets-derived MVs (PMVs) and endothelial cell-derived MVs (EMVs) in blood from ischemic precondition-ing (IPC) treated rats. Methods Blood was withdrawn from rat abdominal aorta and anticoagulated with sodium citrate. Platelets-free plasma (PFP) was isolated through two centrifugations at room temperature. PFP was incubated with FITC-conjugated mouse anti-rat CD61 or PE-conjugated mouse anti-rat CD144. Standard beads in diameter of 1 and 2μm were used for calibration and absolute counting, respectively. Analysis was performed on flow cytometer. Results When 3.5%so-dium citrate was mixed with blood at volume ratio of 1∶4, clear supernatant was collected after centrifugation. Signals of parti-cles smaller than 1μm accounted for more than 99%of overall signals. PMVs and EMVs were CD61 positive and CD144 positive, respectively. Their diameters were both smaller than 1 μm. The concentration of PMVs and EMVs in peripheral blood from IPC treated rats was (4 053±1 987)/μL and (4 870±825)/μL, respectively. Conclusion The method for MVs de-tection by flow cytometry was successfully established and optimized, and verified through detecting PMVs and EMVs in pe-ripheral blood from IPC treated rats.

Cinobufacino injection is purified from water extraction of the skin of Bufo bufo gargarizans, which has been widely used for various cancers in clinic with significant anti-tumor effects. Bufadienolides were regarded as the main active constituents of cinobufacino injection in previous reports. In present study, 6 bufadienolides were isolated and purified from Cinobufacino injection. Their structures were identified as 3-epi-ψ-bufarenogin (1), ψ-bufarenogin (2), 3-epi-arenobufagin (3), arenobufagin (4), 3-epi-gamabufotalin (5), and 3-oxo-arenobufagin (6), separately. Among them, 1 and 3 were new compounds, 5 and 6 were new natural products. Compounds 1, 2 and compounds 3, 4 were two pairs configuration isomers at C-3, separately.

Cinobufacino injection is a significant anti-tumor medicine for the treatment of various tumors in clinic, which was made from water extraction of the skin of Bufo bufo gargarizans. In present paper, HPLC-DAD-FT-ICR-MS method was used to identify the major bufadienolides in cinobufacino for the first time. Solid-phase extraction with dichloromethane and silica was used to enrich the total bufadienolides in cinobufacino. Based on the UV and high resolution MS/MS data, 33 bufadienolides were analyzed and characterized. Among them, eight compounds were identified by comparing with standard references unambiguously. This study elucidated the major bufadienolides in cinobufacino, which provided material foundation of cinobufacino and will be benefit for the further pharmacological research.

Aim To explore the effects of salvianolic acid B(SAB) and tanshinone ⅡA(TA) alone or the compatibility of these two effective components on the proliferation of rat vascular adventitial fibroblasts, and to observe the effects of the compatibility of the two on cell proliferation with the stimuation of angiotensin Ⅱ(Ang Ⅱ).Methods The effects of SAB and TA alone or the compatibility of the two on cell proliferation were detected by methyl thiazolyl tetrazolium(MTT) method, and flow cytometry was adopted to detect cell cycle with or without the induction of Ang Ⅱ.Results It was shown that SAB and TA alone could inhibit fibroblast proliferation in different degree.Through a series of concentration screening, three effective concentrations were obtained respectively and then the inhibition of cell growth was detected by Pairwise compatibilities.The results showed that the compatibility of TA(10-8 mol·L-1) and SAB(10-5 mol·L-1) had the most significant inhibitory effect(P<0.01), and they could inhibit cell proliferation, further flow cytometry was adopted to detect drug effects on cell cycle, the results indicated that the compatibility of SAB and TA mainly blocked the cells in G0/G1 phase.Induced by Ang Ⅱ, the compatibility of SAB and TA group, compared with Ang Ⅱ group, blocked thee cells in G0/G1 phase;and compared with combination of SAB and TA group, it mainly blocked cell cycle in S phase.Conclusion SAB and TA have certain inhibitory effect on fibroblast proliferation, also they could inhibit the proliferation induced by Ang Ⅱ, mainly by blocking the cells in G0/G1 phase.

Objective: To investigate the expression of gamma-aminobutyric acid type A receptor beta3 subunit (GABRB3) on cleft palate in C57BL/6J mice induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Methods: Sixty C57BL/6J pregnant mice on gestation day (GD) 10.5 were divided into two groups: one group was administered through gastric tubes one dose of 28 μg/kg TCDD (experimental group) and the other group was administered through gastric tubes one dose of 5.6 ml/kg corn oil (control group). Embryos were removed by cesarean section from pregnant mice during the palatal formation stage (GD 13.5-17.5) and the palatal tissue studied in morphological and histological observation. The relative mRNA and protein expression of GABRB3 was measured by real-time quantitative PCR and Western blotting. Localization of GABRB3 protein was measured by immunohistochemistry or immunofluorescence. Results: The incidence of cleft palate at GD17.5 was 100% in experimental group and there was no cleft palate occurred in the control group (0); elevation of palatine processes in experimental group was completed on GD15.5 which was clearly delayed by a day compared with that in control group. On GD14.5-GD17.5, the mRNA expression (0.561±0.073, 0.728±0.104, 0.782±0.137, 0.686±0.145) and protein expression (0.288±0.013, 0.404±0.017, 0.399±0.012, 0.307±0.010) in the experimental group were significantly lower than the control group mRNA expression (0.818±0.088, 0.865±0.086, 1.021±0.054, 1.163±0.179) and protein expression (0.481±0.017, 0.456±0.009, 0.474±0.016, 0.529±0.015)(P<0.05). Immunohistochemistry and immunofluorescence showed that GABRB3 was mainly expressed in the mesenchymal cells and medial edge epithelium. Conclusions TCDD delayed palatal shelf elevation and eventually led to cleft palate may be associated with a decrease in GABRB3. GABRB3 may play an important role in the elevation and fusion phases of the palate development.

Defining and visualizing the three-dimensional (3D) structures of pharmaceuticals provides a new and important tool to elucidate the phenomenal behavior and underlying mechanisms of drug delivery systems. The mechanism of drug release from complex structured dosage forms, such as bilayer osmotic pump tablets, has not been investigated widely for most solid 3D structures. In this study, bilayer osmotic pump tablets undergoing dissolution, as well as after dissolution in a desiccated solid state were examined, and visualized by synchrotron radiation micro-computed tomography (SR-μCT). In situ formed 3D structures at different in vitro drug release states were characterized comprehensively. A distinct movement pattern of NaCl crystals from the push layer to the drug layer was observed, beneath the semi-permeable coating in the desiccated tablet samples. The 3D structures at different dissolution time revealed that the pushing upsurge in the bilayer osmotic pump tablet was directed via peripheral "roadways". Typically, different regions of the osmotic front, infiltration region, and dormant region were classified in the push layer during the dissolution of drug from tablet samples. According to the observed 3D microstructures, a "subterranean river model" for the drug release mechanism has been defined to explain the drug release mechanism.

Currently, there is still no effective curative treatment for the development of late-stage liver fibrosis. Here, we have illustrated that TB001, a dual glucagon-like peptide-1 receptor/glucagon receptor (GLP-1R/GCGR) agonist with higher affinity towards GCGR, could retard the progression of liver fibrosis in various rodent models, with remarkable potency, selectivity, extended half-life and low toxicity. Four types of liver fibrosis animal models which were induced by CCl₄, α-naphthyl-isothiocyanate (ANIT), bile duct ligation (BDL) and Schistosoma japonicum were used in our study. We found that TB001 treatment dose-dependently significantly attenuated liver injury and collagen accumulation in these animal models. In addition to decreased levels of extracellular matrix (ECM) accumulation during hepatic injury, activation of hepatic stellate cells was also inhibited via suppression of TGF-β expression as well as downstream Smad signaling pathways particularly in CCl₄-and S. japonicum-induced liver fibrosis. Moreover, TB001 attenuated liver fibrosis through blocking downstream activation of pro-inflammatory nuclear factor kappa B/NF-kappa-B inhibitor alpha (NFκB/IKBα) pathways as well as c-Jun N-terminal kinase (JNK)-dependent induction of hepatocyte apoptosis. Furthermore, GLP-1R and/or GCGR knock-down results represented GCGR played an important role in ameliorating CCl₄-induced hepatic fibrosis. Therefore, TB001 can be used as a promising therapeutic candidate for the treatment of multiple causes of hepatic fibrosis demonstrated by our extensive pre-clinical evaluation of TB001.