Several factors influencing the extraction of tetrandrine and demethyltetrandrine from Chinese medicinal herbs, Stephania tetrandra S. Moore were studied by means of Ortogonal method. The optimum Condition was eight hours steeping, in 33.3% ammonia in absolute alcohol for four hours under ultrasonic agitation. Tctrandrine aud demethyltetrandrino were detected by HPLC. The contents were 0.678% and 0.616%, the detection limits were 3.87?10-3and 0.48?10-3 mmol/L respectively.

Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that ADP-ribosylation factor (ARF) can regulate the growth of tumor cells,and inhibiting ARF will decrease angiogenesis.However,whether ARF antagonist plays an action on CNV is unclear.Objective The aim of this study was to explore the effect and mechanism of ARF inhibitor on alkali-burn induced CNV.Methods Sixty clean male BABL/c mice aged 7-8 weeks were divided into PBS group and ARF antagonist group according to randomized number table.CNV models were induced by NaOH burn method in all the mice.ARF at the concentration of 0.5 g/L(0.5 ml) was intraperitoneally injected 3 times per week for 1 week followed the induction of CNV in the ARF antagonist group,and 0.5 ml PBS was used in the PBS group.CNV was examined 2,4,7,14 days after injection by the slit lamp microscope and the CNV related area in the cornea was calculated.Betore modeling(0 day) and 4,7,14 days after modeling,real-time PCR and Western blot were used to analyze the expressions of ARF mRNA and protein in the corneas.Forteen days after modeling,the expression of the CD31 in the CNV was detected using immnofluorescence of corneal whole mount;the expression of vascular endothelial growth factor(VEGF) in the cornea was assayed by Western blot.Cellular wound scratch test was employed to evaluate the effects of ARF antagonist on proliferation and migration of human retinal vascular endothelial cells (RECs).All animal experiments were done in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and Guideline for the Care and Use of Laboratory Animals on the the Soochow University Animal Care Committee.Results ARF mRNA and protein were expressed in the mice corneas in both the PBS group and the ARF antagonist group at various time points.The expression of ARF mRNA in mice corneas was enhanced with the lapse of the time (Ftime =65.17,P =0.00),but no significant difference was found among the groups (Fsroup =1.98,P=0.18).There was also significant difference in the expression of ARF protein in mice corneas at different time points in the ARF antagonist group (F =10.77,P =0.00).The related CNV area was 0.45±0.05 in the ARF antagonist group,and that in the PBS group was 0.72±0.11,with significant difference between them (t =-3.87,P ＜ 0.05).The green fluorescence area of C D31 expression in the cornea was smaller in the ARF antagonist group than that of the PBS group.Expression level of VEGF in the ARF antagonist group was 1.20±0.21,and that in the PBS group was 2.47±0.33,showing a significant difference (t =-5.62,P ＜ 0.05).As the increase of ARF antagonist concentration,the inhibiting rate of cell proliferation was reinforced among 10,100 and 1 000 μg/L ARF antagonist groups (F=8.47,P =0.02).Twenty-four hours after scratch test,the migrating distance of human RECs was (5.46±1.32) μm and (5.04±1.68) μm in the 100 μg/L and 1 000 μg/L ARF antagonist groups,respectively,which were shorter than (8.49± 1.18) μm of the PBS group (t=-2.94,-2.91,both at P＜0.05).Conclusions ARF inhibitor can reduce CNV by down-regulating the expression of VEGF in alkaline burn cornea and inhibiting the proliferation and migration of vascular endothelial cells.

Background The pathogenesis and mechanism research of corneal neovascularization is of important significance for the prevention and management of corneal neovascularization.Some relative researches are being performed on non-corneal neovascularization-derived vascular endothelial cells, so the results are affected to a certain extent.Objective This study was to isolate and culture vascular endothelial cells from experimental corneal neovascularization tissue and detect the expression of chemokine receptors in vitro.Methods Corneal neovascularization models were established on 10 SPF male BALB/c mice with the age of 7-8 weeks by sticking the filter papers with NaOH on the central corneas, and then the immunofluorescence technique was use to assay the CD31 expression in corneal flatmount 2 weeks after modeling.Corneal pieces were made in 2 weeks after alkali burn and then were digested by collagenase type D.Vascular endothelial cells were isolated from neovascularized tissue by affinity purification using magnetic beads coated with anti-CD31.The cells were cultured on fibronectin-coated walls and then identified by immunocytochemistry.Reverse transcription-PCR was employed to detect the expressions of chemokine receptors in the cells.The use and care of the animals complied with ARVO Statement and this experimental procedure was approved by Soochow University Animal Care Committee.Results Corneal neovascularization occurred at 7 days and peaked at 2 weeks after modeling, and immunofluorescence exhibited the green network-like fluorescence for CD31 antibody in corneas.The cells grew against the wall 2 hours after culture with the polygon shape and large dimension, and the growth obviously quickened after passage.The cultured cells showed the positive response for CD31 antibody, showing the brown dye in cytoplasm,in contrast,the expression of CD31 was absent in corneal stromal cells.Chemokine receptors were positively expressed in the cells with the strongest expression levels in CCR1 ,CCR2,CCR3 and CCR4 mRNA and the weakest expression levels in CCR9,CXCR4 and CXCR5 mRNA,while CXCR3, CCR6, CCR10 and CX3CR1 mRNA were expressed with the moderate intensity.Conclusions Vascular endothelial cells can be obtained from experimental neovascularized corneas by affinity purification and express chemokine receptors,which facilitate the study of their biological properties.

Objective To observe the clinical efficacy of wrist-ankle acupuncture in treating pain due to laparoscopic cholecystectomy.Method Totally 150 patients who were going to receive laparoscopic cholecystectomy were randomized into group A, group B, and group C, 50 cases in each group. Group A was intervened by wrist-ankle acupuncture prior to anesthesia, with the needles retained for 12 h; group B was by subcutaneous needling at the area nearby the points prior to anesthesia, with the needles retained for 12 h; group C didn’t receive any intervention before anesthesia. For the three groups, general inhalational and intravenous anesthesia was adopted for surgery, and patient-controlled intravenous analgesia for post-operation analgesia. The incision pain and visceral pain in the three groups were recorded by using Visual Analogue Scale (VAS) respectively 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h after the operation. The total effective rate, analgesics consumption after operation, and incidence rate of adverse reaction were compared.Result There were significant differences in comparing the VAS scores of incision pain and visceral pain between group A and group C 4 h, 8 h, 12 h, 24 h, and 36 h after the operation (P<0.01,P<0.05). Between group A and group B, there were significant differences in comparing the VAS score of incision pain 8 h, 12 h, 24 h, and 36 h after the operation and the VAS score of visceral pain 12 h, 24 h, and 36 h after the operation (P<0.05). The total effective rate was 96.0% in group A, which was significantly different from 84.0% in group B and 86.0% in group C (P<0.05). The consumption of Fentaneyl citrate injection was (52.4±10.8)μg in group A, which was significantly different from (92.2±11.0)μg in group B and (107.2±11.5)μg in group C (P<0.05,P<0.01). The incidence rate of adverse reactions was 12.0% in group A, which was significantly different from 58.0% in group B and 66.0% in group C (P<0.01).Conclusion Wrist-ankle acupuncture plus patient-controlled intravenous analgesia can mitigate pain after laparoscopic cholecystectomy, and thus it can be taken as one of the post-operational analgesic approaches.

Objective To study the phase transformations and chemical composition changes of Pyritum Preparata (PP) influenced by different processing. Methods To analyse the changes of microstructure, morphology, mineral phase, and chemical compositions in different PP by ore microscope, electron probe microanalyzer technique. Results Greater changes have taken place in chemical compositions of PP after calcination: pyrite (FeS_2) changed into pyrrhotite (Fe_(1-x)S), even changed into magnetite (Fe_3O_4); and chalcopyrite generated mutation to be low-copper chalcopyrite, the contents of Cu reduced, the rate of Cu/Fe changed. At the same time, in the pyrrhotite around the mutative chalcopyrite, the contents of Cu increased. That is to say, the Cu got into pyrrhotite and generated solid-solution with high dispersion, and then ore became loose and porous. In the hard-burned spot of calcined quenched samples by vinegar, the contents of Pb and S are far lower than non-burned spot, the minimum estimate of Pb is less than 1/10 of crude ore. Conclusion There is a co-relativity between phase transformations and dissolution of chemical compositions before and after calcining PP.

Background Intracorneal macrophages play a critical role in corneal neovascularization (CNV)by secreting relative chemokines.But macrophages are characteristic by heterogeneity which has different biologic functions under different induction or stimulation from microenvironment.Objective This study was to detect the effects of chemokine (C-X3-C motif) ligand 1 (CX3CL1) and chemokine (C-C motif) ligand 2 (CCL2) on macrophages in vitro.Methods CNV was induced by corneal alkali burn in the left eyes of 20 male BALB/c mice aged 7-8 weeks.The CNV was evaluated under the slit lamp microscope 4 days after alkali burn,and then the corneal sections were prepared after mice were sacrificed.The expressions of CCR2 and CX3CR1 in the corneal specimens were detected by histo-fluorescence staining.Human peripheral blood mononuclear cells were separated using density gradient centrifugation and incubated in RPMI-1640 medium containing 10％ fetal bovine seruml(FBS) with 30 μg/L granulocyte-macrophage colony-stimulating factor (GM-CSF).The cells were divided into CD68 +CCR2 group and CD68+CX3CR1 group,and the percentage of the CX3CR1 and CCR2 expressions in the infiltrated macrophages of corneal specimens and human monocyte-derived macrophages was assayed by flow cytometry.The cultured cells were stimulated using human recombinant CX3CL1 and CCL2 proteins,and real-time PCR was used to detect the relative expressions of angiogenesis-related factors in macrophages.Results CNV was found in corneas 4 days after alkali burn and the CNV onsets from corneal limbus to central zone observed by a slit lamp.CCR2 and CX3CR1 were expressed in the F4/80-positive macrophages in alikali burned corneas.The macrophages grew for two weeks and appeared more dead cells in without GM-CSF group,but in GM-CSF induced group,the number of macrophages was increased.The percentage of CX3CR1-positive cells was 75％ and that of CCR2-positive cells was 45％.Real-time PCR showed that expression level of vascular endothelial growth factor (VEGF) mRNA increased and that ADAMTS-1 mRNA or TSP-1 mRNA decreased on macrophages after CCL2 stimulation,with significant differences in the 150 mg/L CCL2 group compared with the control group (t =-5.09,P =0.03 ; t =3.01,P =0.04 ; t =4.27,P =0.02).However,the VEGF mRNA expression decreased and ADAMTS-1 mRNA and TSP-1 mRNA increased after CX3CL1 stimulation,showing significant differences between the 150 mg/L CX3CL1 group and the control group (t=6.35,P=0.O2;t=-2.92,P=0.04; t=-3.81,P=0.03).Conclusions These results suggest that the macrophages have high heterogeneity.CCL2-and CX3CL1-expressing macrophages can regulate the expressions of angiogenesis-related factors.Macrophage chemokine signal may be a good target for treatment of neovascular ocular disease.

Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that zonula occludens-1 (ZO-1) can inhibit pathological angiogenesis through physical barrier formed by tight junction structure.However,whether ZO-1 plays a role in CNV is unclear.Objective The aim of this study was to explore the effect of ZO-1,a tight junction protein on experimental CNV.Methods The CNV models were established in the left eyes of 24 clear male BALB/c mice aged 7-8 weeks by putting NaOH filter paper in the center of corneas for 15 seconds (15 s group) or 40 seconds (40 s group).CNV was examined and evaluated under the slit lamp microscope,and the expression of ZO-1 mRNA in the corneas were detected and compared by reverse transcription PCR (RT-PCR) between the two groups 2 weeks after modeling.In addition,54 models created by the same method were assigned to 3 groups according to randomized number table,0.2％ hyaluronic acid (HA),antiZO-1 neutralizing antibody (10 mg/L) +0.2％ HA and mouse hypoxia inducible factor-1α (HIF-1α) recombinant protein (5 mg/L)+0.2％ HA were topically administrated in the mice three times a day for 1 week after modeling respectively.The corneas were extracted 2 weeks after application of the drugs.Expression of CD31 in the CNV was assayed to calculate the number and the area of CNV by immunohistochemistry.The expression of VEGF mRNA in the corneas was detected by RT-PCR.The percentages of macrophage-specific F4/80 positive cells and neutrophilsspecific Ly-6G positive cells were calculated to evaluate the infiltrations of inflammatory cells in the corneas by flow cytometry.Results In 2 weeks after alkali burn of corneas,the number of severe CNV was more in the 40 s group than that in the 15 s group (x2 =6.032,P=0.049),and the expression level of ZO-1 mRNA was lower in the 40 s group than that in the 15 s group (1.15±0.08 versus 1.53±0.04) (t=4.157,P=0.014).CD31 positive cell number was more and the staining area was larger in the ZO-1 antibody group and HIF-1α positive control group than those in the 0.2％ HA group (cells:t=-129.590,-226.820,both at P=0.000;area:t =-5.310,-8.840,both at P=0.000).The relative expressions level of vascular endothelial growth factor (VEGF) mRNA was 1.33±0.10 and 1.46±0.11 in the ZO-1 antibody group and HIF-1 α positive control group respectively,which were significantly higher than 0.93±0.06 of the 0.2％ HA group (t =-5.820,-7.284,both at P =0.000).The percentages of positive cells in the ZO-1 antibody group and HIF-1α positive control group were significantly increased in comparison with the 0.2％ HA group for F4/80 (t =-16.750,-17.480,both at P =0.000) and for Ly-6G (t =-21.450,-27.680,both at P=0.000).Conclusions Alkali burn induced CNV downregulates the expression of ZO-1 mRNA.Administration of ZO-1 antibody causes the rise of VEGF mRNA in CNV and the infiltration inflammation cells,which suggests that the influence of ZO-1 on CNV is associated with the expression of VEGF.

Background Researches showed that ADP-ribosylation factor (ARF) promotes intracorneal secretion of multiple angiogenesis-related factors,such as vascular endothelial growth factor (VEGF) and nitricoxide synthase (NOS) etc., and therefore results in corneal neovascularization.However, whether ARF affects the tube formation of human retinal endothelial cells(HRECs) is unelucidated.Understanding the effect of ARF tube formation of HRECs is important for the target treatment of retinal vascular diseases.Objective The aim of this study was to explore the effect and mechanism of ARF inhibitor on tube formation of HRECs in vitro.Methods HRECs (HREC line) were cultured and passaged.The growth-well cells were harvested and divided into two groups.The cells were regularly cultured in the control group,and ARF antagonist (lml) was added in the culture medium in the ARF antagonist group.The expression levels of ARFmRNA and protein in the cells were examined by reverse transcription (RT)-PCR and Western blot.The morphology and number of HREC tube formation were detected by using three-dimensional Matrigel assay.The relative expression levels of VEGF, NOS, focal adhesion kinase (FAK) and heat shock protein 90 (HSP90) at gene level and protein level were examined by RT-PCR and Western blot in vitro.Results The relative expression levels of ARFmRNA in the cells were 0.65 ±0.14 and 0.32±0.10, and those of ARF protein were 0.85±0.15 and 0.24±0.17 in the control group and ARF antagonist group,showing significant differnces between the two groups (t =7.32, P =0.00;t =5.15, P =0.00).The number of HREC tube formation was (34.66±8.57)/field in the ARF antagonist group, which was significantly lower than (51.46±7.12)/field in the control group (t=2.99 ,P=0.04).The relative expression levels of VEGF mRNA, NOSmRNA and their proteins in the cells were significantly lower than those of the control group (t =3.02, P =0.04;t =3.68, P =0.02;t =3.33,P=0.03;t=2.89 ,P=0.04).The relative expression levels of FAKmRNA and HSP90mRNA in the ARF antagonist group were 0.65±0.18 and 0.28±0.05 ,which were significantly lower than 0.76±0.25 and 0.46±0.09 in the control group (all at P＜0.05).Conclusions ARF antagonist appears to have an inhibitory effect on the tube formation ability of HRECs propably by down-regulating the expressions of VEGF, NOS and the downstream signal transduction factors FAK and HSP90 in HRECs in vitro.

Objective:To compare the differences in incidences of insomnia,anxiety(irritability)and manic ep- isode induced by fluoxetine and amitriptyline in treating depression.Method:CBM discs were selected for the data sources. The rates of insomnia,anxiety(irritability)and manic episode from published clinical control trials on depression treated by fluoxetine and amitriptyline were analyzed by applying fixed effect model(FEM)of evidence-based medicine(EBM). Result:Of 1205 cases in 15 studies,the rates of insomnia induced by fluoxetine or amitriptyline were 21.71% and 1.80% ,OR 9.39(95 %CI 5.37-16.44),P0.05.Conclusion:Fluoxetine induces in- somnia and anxiety(irritability)more easily than amitriptyline when treating depression.

Objective To evaluate the diagnostic utilities of CD64,CDllb,sICAM-1 and sE-selectin in early identification of neonatal sepsis related to bacterial infection. Methods The group of sepsis consisted of 36 newboms and the control group included 26 healthy newboms. The blood samples were collected right after being admitted to hospital and at recovery stage in the group of sepsis,as for the control the blood samples were collected only once in the study. In the sepsis group,blood samples were also taken in bacterial culture before treatment CD64 and CDllb were quantified with direct immunofluorescence staining and the whole blood cell flow cytometry analysis. sICAM-1 and sE-selectin level were determined by ELJSA assay,along with CRP. Results The expression level of CD64 in neonates with sepsis was (60. 37 22. 70) .shown in MFI,which was significantly higher than that in the group of control (23. 14 ±5. 10) MFI(P <0. 01). The expression level of CDllb in neonates with sepsis was (1645. 14 ±463. 68) MFI,which was significantly higher than that in the group of control (1041.48 ±260. 34) MFI (P < 0. 01). The concentration of blood sICAM-1 in neonates with sepsis was (240. 20 ± 83.46) μg/L, which was significantly higher than that in the group of control (100. 24 ±51.03)μg/L(P <0. 01). The concentration of blood sE-selectin in neonates with sepsis was (29. 63 ±9. 88μg/L,which was significantly higher than that in the group of control (14. 12 ±5. 33)μg/L(P <0. 01). In the sepsis group,the level of CD64,CDllb,sICAM-l and sE-selectin in the primary stage was higher than the recovery stage significantly (P <0. 01). The sensitivity of above-mentioned molecular markers were 95. 7% , 82. 6% , 81. 8% and 87. 0% , respectively, and the specificity were 95. 8% , 79. 2% ,86.9% and 79.2% . CD64 was the best one. Conclusions CD64 may serve as one of the reliable biomarkers in the early diagnosis of neonatal sepsis, and it may play important role in the treatment of neonatal sepsis.