This study was designed to investigate inhibitory effects and possible mechanisms of snake venom tripeptide (pENW) on platelet adhesion in order to promote the development of a novel anti-platelet therapy. To study the inhibitory effects of pENW on platelet adhesion, washed platelets pre-incubated with pENW (116.5-466.2 μmol x L(-1)) were used to test the ability of platelet adhesion to fibrinogen. Effect of pENW on fibrin clot retraction was also tested. Effect of pENW on platelets viability was tested by MTT assay. Effect of pENW on reactive-oxygen species (ROS) levels of platelet was studied by flow cytometry assay. Calcium mobilization in Fura-2/AM-loaded platelets was monitored with a spectrofluorimeter. Cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), thromboxane A2 (determined as its metabolite thromboxane B2) were measured using enzyme immunoassay kits. Akt, ERK and p38 phosphorylation were tested by Western blot. The results showed that pENW inhibited platelet adhesion and fibrin clot retraction in a concentration-dependent manner without cytotoxicity. Intracellular cGMP and cAMP in both resting and thrombin-activated platelets were increased by pENW. In addition, pENW attenuated intracellular Ca2+ mobilization and TXA2 production in platelets stimulated by thrombin. As shown by Western blot assay, Akt, ERK and p38 phosphorylation in thrombin-induced platelet were attenuated by pENW. However, inhibitory effects of pENW had nothing to do with ROS. Thus, pENW exhibited a significant inhibition on platelet adhesion to fibrinogen, which means pENW could block the first step of thrombosis as while as retard the more stable clot formation. The mechanisms of pENW on inhibition platelet adhesion might be related to instant regulations, such as protein kinases.

AIM To establish an HPLC method for the simultaneous content determination of four constituents in Qingzhiyi Tablets (Puerariae lobatae Radix,Phyllanthi Fructus,Salviae miltiorrhizae Radix et Rhizoma,etc.).METHODS The analysis of 50％ methanol extract of this drug was performed on a 30 ℃ thermostatic Kromasil C18 column (4.6 mm × 250 mm,5 μm),with the mobile phase comprising of 0.1％ formic acid-methanol-acetonitrile flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 270 nm.RESULTS Gallic acid,puerarin,salvianolic acid B and tanshinone Ⅱ A showed good linear relationships within the ranges of 11.95-382.48,14.23-455.28,10.77-344.68 and 3.89-124.32 μg/mL,whose average recoveries were 99.96％,100.92％,98.87％ and 97.67％ with the RSDs of 1.09％,1.30％,1.11％ and 1.22％,respectively.CONCLUSION This sensitive,simple and accurate method can be used for the quality control of Qingzhiyi Tablets.

In order to study the therapeutic time window of dimethylaminoethyl ginkgolide B mesylate(XQ-1H) in the permanent focal ischemia of rat,we used the rat model of the permanent middle cerebral artery occlusion (pMCAO).Doses of 15.6,7.8 and 3.9 mg/kg of XQ-1 H were intravenously administered at 0.5,1,2,3 h after MCAO,respectively.Neurological scores,infarct sizes,water contents and pathological changes in each interval were determined at 72 h after MCAO.It was observed that XQ-1 H administered at 0.5 and 1 h after MCAO significantly reduced the cerebral infarct size and edema,and produced significant reductions in the neurological deficits.The protective effect of XQ-1H on the neuron cells was proved by pathological observations.In addition,the contents of MDA,lactate,and the activities of SOD were measured.Reduction in the contents of MDA and lactate and enhancement in the activities of SOD were attributed to the pretreatment of XQ-1H at 0.5 and 1 h.Our results showed that the therapeutic time window of XQ-1H extended for up to 1 h after MCAO.

Objective To analyze the regulatory of miRNA (miR)-149-5p for the expression of Aurora-B in esophageal squamous cell carcinoma (ESCC). Methods The pathologic histology paraffin blocks of 61 patients with ESCC in Shanxi Provincial People's Hospital from January 2010 to December 2017 were collected. The immunohistochemical staining and tissue section in situ hybridization method were used to observe the expressions of Aurora-B and miR-149-5p in the tumor tissues and adjacent mucosas of the patients, and their relationship with clinicopathological parameters was analyzed. miRNA was predicted by using software TargetScan and miR walk. The relationship between Aurora-B and miR-149-5p were verified by using Western blot in ESCC TE-1 cells. Results The result of immunohistochemical staining showed that in 61 patients, 37 (61％) tumor tissues showed higher expression of Aurora-B compared with adjacent mucosas, the Aurora-B expression in 15 (26％) tumor tissues were in line with benign tissues, the Aurora-B expression in 9 (13％) tumor tissues were inferior to benign tissues. The expression of Aurora-B were not correlated with age (χ2=0.008, P=0.929), gender (χ2=0.088, P=0.767), grade of differentiation (χ2=2.632, P=0.268), but correlated with TNM staging (χ2=15.153, P<0.01) and lymph node metastasis (χ2=5.979, P=0.014). The miR-149-5p was predicted to combine with Aurora-B 3'untraslated region (UTR) by using TargetScan and miRwalk software. The result of in situ hybridization showed that the miR-149-5p showed low expression in 22 (36％) tumor tissues. The expression of miR-149-5p was correlated with Aurora-B expression (χ2 = 5.622,P= 0.018), and not correlated with age (χ2= 2.617, P= 0.106), gender (χ2= 1.529, P= 0.216), grade of differentiation (χ2 = 2.854, P= 0.240), TNM staging (χ2 = 0.870, P= 0.351) and lymph node metastasis (χ2= 0.128, P= 0.351). The Western blot results of TE-1 cells showed that the expression of Aurora-B in simultaneous over-expression of miR-149-5p and Aurora-B group was higher than that in over-expression of miR-149-5p group, and lower than that in over-expression of Aurora-B group. Conclusion The miR-149-5p can be involved in ESCC progression through regulating the expressions of Aurora-B.