Objective To investigate the role of nerve growth factor (NGF) in the pathogenesis of diabetic neuropathy (DNP) and the correlation of NGF level with,sensory nerve conduction velocity (SNCV) and duration of DNP.MethodsThe 41 patients with DNP,35 diabetics without DNP and 33 healthy controls were enrolled.And the serum level of NGF was measured with ELISA,the SNCV of left medial nerve,ulnar nerve,peroneal nerve and tibial nerve were measured too. Then the correlation analysis was completed. ResultsThe serum level of NGF was significantly lower in DNP patients [(665.18±188.32) ng/L] than in healthy controls [(976.44±159.07)ng/L] and in diabetics without DNP [(943.32±167.33) ng/L,F=9.316,P<0.001].The NGF level of DNP patients was positively correlated with SNCV of left medial nerve (r=0.810,P<0.001 ),left peroneal nerve (r=0.760,P<0.001) and duration of DNP (r=0.542,P<0.001).Conclusions The serum level of NGF is lower in DNP patients than in healthy controls.The decrease of the production of NGF may play a role in the pathogenesis of DNP.

Objective To establish an animal model to study endometriosis. Methods Six female Bama miniature pigs that were sexually mature were chosen and their endometrial tissues were acquired surgically. The endometrial tissues were implanted to abdominal cavity and subcutaneous tissues. Results The ectopic endometria in the implant location formed mass or cyst that was verified pathologically to be similar to normal endometrial tissues. Conclusion The establishment of ectopic endometriosis in Bama miniature pigs is successful, which provides an ideal model for further therapeutic study of endometriosis.

Objective To investigate the effects of nordihydroguaiaretic acid(NDGA)on the endometriosis established in Bama miniature pigs.Methods Six Bama pigs that had been successfully established into animal model of endometriosis subcutaneously received 3% NDGA at dose of 20 mg/kg(n=4)or PBS solution(n=2)for 20 d.The grafts in abdominal cavity and subcutaneous tissues were observed and the serum concentrations of hormone,were measured by chemiluminoimmunoassay before and after the period of NDGA or PBS injection.The expressions of FⅧAg and VEGF in the endometriotic tissues were observed.Results The endometriotic masses were reduced to some degree after the time period of NDGA injection,even partially disappeared,while those treated with PBS showed no obvious changes.Histoimmunochemical analysis showed NDGA decreased the number of endometrial glands,the proliferation rate of the glandular epithelial cells,the endometrial stroma cells and the vessels,and inhibited the microvascular density and VEGF in the endometriotic tissues(P

A chiral high-performance liquid chromatography method was developed for the simultaneous determination of ibuprofen enantiomers in dog plasma. It was used to study the pharmacokinetics in the Beagle dog after intravenous administration of racemic-ibuprofen, S-ibuprofen and R-ibuprofen. Ketoprofen was chosen as the internal standard. After a simple precipitation using methanol as the precipitating solvent, both analytes and IS were separated on a Kromasil 100-5CHI-TBB chiral column (250 mm x4.6 mm, 5 μm) with isocratic elution using acetonitrile - 20 mmol x L(-1) phosphate buffer (pH 3.0, containing 5% methanol) (6 : 4) as the mobile phase. The detection wavelength was 220 nm. Liner calibration curves for both of the ibuprofen enantiomers were over the concentration range from 0.5 to 50 μg x mL(-1) with a lower limit of quantification of 0.5 μg x mL(-1), the accuracies were all in standard ranges. The intra- and inter- assay precisions were all below 7%. The recovery rate was 93.1% to 100.4%. The experiments proved that the method was simple, rapid and sensitive. It can be used in the quantitative determination of ibuprofen enantiomers in dog plasma. The method was used to determine the concentration of ibuprofen enantiomers in Beagle dog plasma after a single intravenous administration of racemic-ibuprofen, S-ibuprofen and R-ibuprofen (9 mg x kg(-1)) and the pharmacokinetics parameters were calculated based on the concentration-time curves. The C(max) of S-ibuprofen in Beagle dog plasma after a single intravenous administration of racemic-ibuprofen, S-ibuprofen and R-ibuprofen were 30.8 ± 4.7, 46.1 ± 5.9 and 20.0 ± 2.6 μg x mL(-1), respectively. In terms of the exposure of active ingredient, it revealed a significant difference between the administration of S-ibuprofen and the other two groups. The systematical R- to S- chiral inversion was discussed. Comparing the pharmacokinetic parameters at different doses, chiral inversion were 70.1% ± 36.6% and 76.4% ± 36.2%, respectively, after intravenous administration of racemic- and R-ibuprofen. This study provides a theoretical basis for the safety of ibuprofen formula of injection drug.
Animals ; Chromatography, High Pressure Liquid ; Dogs ; Ibuprofen ; blood ; pharmacokinetics ; Stereoisomerism

Aim To investigate the effects of neurotro-phin-3 ( NT-3 ) gene overexpression on the differentia-tion into cholinergic neuron of neural stem cells ( NSCs) in vitro and its underlying mechanism. Meth-ods Brain-derived NSCs from newborn mice were iso-lated and cultured in vitro and determined by immuno-fluorescence. The NSCs were divided into three groups: NSCs, GFP-NSCs and NT-3-NSCs groups. The expression of NT-3 was detected by immunofluo-rescence and ELISA. Then, the ability of NSCs on dif-ferentiation into cholinergic neuron was detected by im-munofluorescence and RT-PCR, and the Acetylcholine Assay Kit was used for acetylcholine ( ACh) , and the expression of Hes1 , Mash1 and Ngn1 mRNA was de-termined by RT-PCR. Results The neurosphere dis-played Nestin and Sox 2-postive by immunofluores-cence, suggesting that the cultured cells were NSCs. The proportion of ChAT immunopositive cells was sig-nificantly higher in the NT-3-NSCs group than that in the other two groups ( P <0. 01 ) . Ach secretion in NT-3-NSCs was significantly elevated compared with the other two groups ( P <0. 01 ) . NSCs transfected with NT-3 increased the levels of Mash1 and Ngn1 mR-NA, and decreased the level of Hes1 mRNA ( P <0. 05 ) . Conclusion NT-3 can significantly promote the in vitro differentiation of NSCs into cholinergic neu-rons via probablly inhibiting Notch signaling pathway.

BACKGROUND:The number of hematopoietic stem cel s in the peripheral blood is very low at normal physiological state. So it is critical to mobilize hematopoietic stem cel s from donor’s bone marrow into the peripheral blood.
OBJECTIVE:To study the combination effect of AMD3100 and dexamethasone on the mobilization of hematopoietic stem cel s in mice, thereby laying the foundation for clinical application.
METHODS:C57BL/6 mice were injected with AMD3100 and dexamethasone alone or in combination. Then, hematopoietic stem cel s in the peripheral blood and bone marrow were col ected. CD34+cel concentration in the peripheral blood and bone marrow was determined by flow cytometer and the content of leucocytes in the peripheral blood was counted by a normal method. The CFU-Mix colony formation ability of hematopoietic stem cel s was identified by cel colony forming assay.
RESULTS AND CONCLUSION:The concentration of CD34+cel s in the peripheral blood and bone marrow, the content of leukocytes in the peripheral blood and the CFU-Mix colony formation ability of hematopoietic stem cel s were al improved by both AMD3100 and dexamethasone and especial y their combined use. This indicates that both AMD3100 and dexamethasone could mobilize hematopoietic stem cel s to migrate from the bone marrow to the peripheral blood, and when used in combination, the mobilization effect is better than that of single drug.

AIM:To explore the protective effect of osthole on the SH-SY5Y cells transfected with APP595/596 gene, and to investigate the molecular mechanism.METHODS:The SH-SY5Y cells were transfected with APP595/596 gene in vitro for establishing a cell model to study the pathogenic role of amyloid β-protein ( Aβ) .The cell viability was detected by CCK-8 assay.The release of lactate dehydrogenase ( LDH) was determined by the colour reaction of dia-phorase-INT.The cell apoptotic rate was analyzed by flow cytometry.The expression of β-site APP cleaving enzyme 1 ( BACE1) at mRNA and protein levels was detected by RT-PCR and Western blot.The expression of Aβwas measured by the technique of immunofluorescence cytochemistry and Western blot.RESULTS: Treatment with osthole inhibited the LDH release, and increased the viability of the cells.The percentage of apoptotic cells was also significantly decreased. Osthole also inhibited the expression of BACE1 at mRNA and protein levels and the protein expression of Aβ.CONCLU-SION:Osthole has protective effect on SH-SY5Y cells transfected with APP595/596 gene.The mechanism may be associ-ation with inhibiting the mRNA and protein expression of BACE1.

Aim To investigate the effects of osthole ( Ost) on the ability of proliferation and differentiation in APP transduced neural stem cells( NSCs) , and neu-ronal apoptosis, in order to find related mechanism. Methods A model of Alzheimer′s disease( AD) cells was successfully established by transducing APP gene into NSCs in vitro. The ability of proliferation and dif-ferentiation was tested by staining. The viability of NSCs was determined by using CCK-8 assay. The cell apoptosis was tested by Hoechst 33258 staining. The expression of GSK-3β and β-catenin mRNA was deter-mined by RT-PCR. The expression of GSK-3β and β-catenin protein was determined by Western blot. Re-sults The ability of proliferation had increased by 10 . 24% with Ost treatment, compared with APP group. The ability of differentiation had increased by 6 . 74%with Ost treatment, compared with APP group. The vi-ability of NSCs had increased and cell apoptotic rate had decreased significantly. From the results of RT-PCR and Western blot, we could find the expression of GSK-3βmRNA and protein had decreased, and the ex-pression of β-catenin mRNA and protein had increased significantly, compared with APP group. Conclusion Ost could enhance the ability of proliferation and dif-ferentiation into more neurons of NSCs transducing APP gene, and reduce neuronal apoptosis. It might be relat-ed with activiting Wnt/β-catenin signaling pathway.

Aim To investigate the effects of osthol on cell apoptosis and inflammatory cell infiltration after brain stab wound injury in mice. Methods The mice underwent the stab wound injury by a needle, then were randomly divided into sham operation group, model group, osthol 10, 20, 30 mg · kg-1 treatment group. The main examinations included mice brain wa-ter content; the apoptotic cytokines Bax, Bcl-2, Caspase-3 mRNA expression were assessed by PT-PCR; immunohistochemistry staining was used to de-tect neutrophils (MPO) and microglia (Iba-1) infiltra-tion and Caspase-3 positive cell expression around in-jured lesions. Results Treatment with osthole 20, 30 mg·kg-1 group significantly reduced the water content in injured brain, improved the ratio of Bax/Bcl-2, and reduced the expression of apoptosis cytokine Caspase-3 mRNA. Osthole 30 mg·kg-1 treatment group obvious-ly reduced the infiltration of neutrophils and microglial cells and significantly reduced the number of apoptotic cells around the injured cerebral cortex. Conclusion Osthole has therapeutic effect on stab wound injury in mice, and the possible mechanism may be by reducing the infiltration of inflammatory cells and reducing apop-totic cells.

Aim To investigate the effect of osthole on neuron synapses infected APP gene and its underlying mechanism. Methods The neurons were divided into three groups:GFP, APP, APP+Ost groups. The neu-rons were infected APP gene with containing mutational site in vitro for mimicking the characterstics of Alzhei-mer’ s disease ( AD) . The cell viability was assessed by CCK-8 , the expression of synapsin-1 was deter-mined by immunofluorescence, and the concentration of PSD-95 and SYP were detected by ELISA. The ex-pressions of Aβ1-42 , CAMKK2 , phoshorylated AMPKα1 , AMPKα1 protein were determined by West-ern blot. Results Strong APP staining was visible in neurons infected with APP and abundant expression of Aβ1-42 , a neurotoxic oligomer. Compared with APP group, APP+Ost group significantly increased cell vi-ability, promoted the expression of synapsin-1, up-reg-ulated the concentration of PSD-95 and SYP, and de-creased the expressions of CAMKK2 and p-AMPKα1 . Conclusions Ost can protect the neuron synapses a-gainst infected with APP gene. Its neuroprotective effect may be related to inhibiting the CAMKK2/AMPK signal pathway.