Objective To establish a method to simultaneously determine contents of alantolactone and isoalantolactone in Sanwei Ganlusan. Methods Contents of alantolactone and isoalantolactone was determined by HPLC was used with PDAD detector;the column was CAPCELL PAK MG C18 (4.6 mm×250 mm, 5μm);the mobile phase consisted of acetonitrile-0.4%phosphoric (58∶42);the flow rate was 1.0 mL/min;the detection wavelength was set at 225 nm;the column temperature was maintained at 30 ℃. Results The linear range of alantolactone was 0.083-0.517 μg (r=0.999 9), and isoalantolactone was 0.108-0.672 μg (r=0.999 9). The mean recovery of alantolactone was 97.95%, RSD=1.31%. The mean recovery of isoalantolactone was 97.69%, RSD=1.24%. Conclusion The method is accurate and simple in operation, which can be used to simultaneously determine contents of alantolactone and isoalantolactone in Sanwei Ganlusan.

Objective To explore the efficacy of intraductal chilled saline perfusion (ICSP) to reduce the thermal bile duct injury during the treatment of radiofrequency ablation (RFA) associating with occlusion of hepatic blood supply in rabbits.Methods 16 healthy New Zealand white rabbits were divided into 2 groups.Rabbits of the ICSP group were placed tubes in the common bile duct after laparotomy,and ICSP was performed during the RFA procedure accompanied with hepatic blood occlusion.While for rabbits of the non-ICSP group,hepatic blood occlusion and RFA were performed without ICSP.RFA electrodes were placed about 5 mm away from the hilus hepatis approximately.Contrast-enhanced ultrasonography (CEUS) was administrated to evaluate the sizes of the ablative zones after the procedure.On post-procedure 6 week,ultrasonography was prerformed to evaluate the changes of the biliary structure,and liver specimens of rabbits wcrc obtained for histopathologic observation of main bile ducts.Results Post-procedure CEUS examination showed that there was no significant difference in the size of the ablative zone between the groups (P ＞0.05).On post-procedure 6 week,rabbits of the ICSP group appeared with biliary dilatation more frequently by ultrasonography (P ＜0.05),and a higher degree of the injury of main bile duct by histopathologic observation (P ＜0.05).Conclusions In treatment of RFA accompanied with hepatic blood occlusion,RFA-induced bile duct injury may be decreased significantly with ICSP.

Objective To observe the changes of the intercellular spaces of squamous epithelium of lower esophagus in gastroesophageal reflux disease(GERD).Methods Eleven outpatients with GERD symptoms more than 3 months [6 with nonerosive reflex disease(NERD)and 5 with erosive esophagitis(EE)]and 5 healthy volunteers were recruited.All of them underwent endoscopy and 24-hr ambulatory pH monitoring.Biopsies were taken in lower esophagus(2 cm above Z-line)for electron microscope examination.Results Intercellular spaces of esophageal epithelial cell in volun teers,NERD patients and EE patients were (0.374?0.073)?m,(1.308?0.079)?m and (1.332?0.144)?m respectively,with significant differences between the control group and the NERD or EE group.There was no difference between NERD group and EE group.Conclusions Dilated intercellular spaces were seen in both NERD and EE cases,which was significantly different from the control cases.

OBJECTIVE To investigate the effect of Radix Isatidis and its constituents indigo and in?dirubin on two principal subtypes of organic cation transporters(OCT)OCT1,OCT2 in vivo in mice. METHODS Decoction of Radix Isatidis (DRI) 1.6 and 6.4 g · kg-1,granules of Radix Isatidis (GRI) 0.615 and 2.460 g·kg-1,indigo 0.008 and 0.640 mg·kg-1 and indirubin 0.0192 and 1.536 mg·kg-1 were ig given to NIH mice(60 mice per group),twice a day for 5 d. Four control groups were set up,including the vehicle of water,0.5% sodium carboxymethyl cellulose(CMC),additives of sucrose plus dextrin (1.5 g · kg-1)and positive control quinidine(0.025 g · kg-1). Sixty minutes after the last dosing,all the mice were iv given metformin(Met)5 mg·kg-1,and at 1.0,2.5,5.0,7.5,10.0 and 20.0 min after Met iv,10 mice in each group were sacrificed to collect whole blood and kidneys respectively. The right kidney was homogenized for Met accumulation test and the left one used to extract total RNA for analysis of OCT1 and OCT2 mRNA expressions by real-time PCR. The contents of Met in sera and kidneys were quantified by HPLC. Major pharmacokinetic parameters of Met in sera were analyzed by pharmacokinetic software(DAS 2.0). RESULTS There was no significant difference between water control group,0.5%CMC group and sucrose plus dextrin group in any examined item. Compared with vehicle control group (water and 0.5%CMC group),all the related pharmacokinetic parameters in DRI 6.4 g · kg- 1,GRI 2.46 g · kg-1,indigo 0.640 mg · kg-1 and indirubin 1.536 mg · kg-1 groups were changed significantly (P<0.05,P<0.01). The elimination half time (t1/2β) was prolonged 13%-97%,volume of distribution reduced by 13%-72%,clearance(Cl)reduced by 9%-65%,and the area under the concentration-time curve (AUC0-20 min) increased by 13%-135%. AUC0-20 min obtained from renal Met accumulations was significantly increased(P<0.01)while Met uptake by kidney slices was reduced(P<0.05,P<0.01). The expressions of OCT1 and OCT2 mRNA were obviously down-regulated(P<0.05,P<0.01). CONCLUSION The mouse renal OCT1 and OCT2 are significantly inhibited by DRI,GRI,indigo and indirubin. The inhibitory effect of Radix Isatidis on OCT1 and OCT2 probably arises from indigo and indirubin contained.

Objective To discuss the clinical significance of serum neutrophil gelatinase associated lipocalin(NGAL)detection in the diagnosis of contrast induced nephropathy .Methods A total of 299 inpatients with contrast medium in department of inten‐sive care unit(ICU) ,department of cardiovascular disease ,department of urology from Guangdong Province Hospital of Traditional Chinese Medicine were enrolled ,and the fasting blood glucose and lipids were detected .Sera were collected before examination of contrast medium and 1 ,2 and 6 d after examination of contrast medium respectively .Serum creatinine(SCr)and cystatin C(CysC) were detected ,the concentration of NGAL was determined .CIN was defined as of increased by 44 μmol/L or 25% from baseline in SCr within 24-48 h .The NGAL detection variation tendency was analyzed in different time point .Results In 299 cases ,CIN oc‐curred in 28 patients ,the incidence rate was 9 .36% .The diabetes patients in the incidence of CIN was 16 .21% (12/74) ,no inci‐dence of diabetic patients was 7 .11% (16/225) .When compared with that of before contrast ,CIN in serum of patients with Cr and CysC coronary angiography in 2 d significantly increased after contrast medium ,returned to the level before angiography on 6 d .But the serum NGAL in angiography 1 d after it was significantly increased (P<0 .05) ,statistically significant differences compared with pre contrast ,and was still at a high level on sixth day .Conclusion Prediction of CIN level of serum NGAL in contrast after 1 d could be a good predictor for CIN ,and the prediction time is earlier than the serum levels of Cr and CysC .The level of serum NGAL can be used for early diagnosis of CIN .

Objective To construct a recombinant fusion protein with pneumococcal surface pro-tein A (PspA) of Stretococcus pneumonia (SPN) familyⅠclade 1 and 2, and to analyze the immunogenici-ty of the fusion protein.Methods The gene fragments encoding theα-helix of PspA of the two clades were amplified by PCR and then inserted into the expression vector pET-27b(+) to construct the recombinant ex-pression plasmid.The transformed Escherichia coli BL21 strains carrying expression plasmid were induced by IPTG to express the recombinant protein.The titers and affinity of antibodies against PspA protein were measured by ELISA.An opsonophagocytic assay and an animal experiment were performed to evaluate the immunogenicity of the recombinant protein.Results Double enzyme cutting and gene sequencing confirmed the two purpose gene fragments were correctly expressed in the expression vector pET-27b(+).The titers of anti-PspA antibody in the serum of Kunming ( KM) mice immunized with the fusion protein were 1 ×104 . The affinity of anti-PspA antibody reached to 2×105 .The rates of recombinant PspA6B-PspA05 protein me-diated phagocytosis for SPN6B, SPN05 and SPN01 strains were 20%, 15% and 8.8%, respectively.No SPN23F strain was engulfed by macrophages upon the stimulation with PspA6B-PspA05 protein.The survival rates of mice injected with SPN05, SPN6B, SPN01 and SPN23F strains were respectively 75%, 92%, 75%and 33%upon the immunization of PspA6B-PspA05 protein.Conclusion The recombinant fusion protein PspA6B-PspA05, constructed with the PspA proteins of Stretococcus pneumonia familyⅠclade 1 and 2, was successfully expressed in the E.coli prokaryotic system with the advantage of high immunogenicity.High ti-ters of anti-PspA antibodies with high specificity were induced in KM mice upon the stimulation with Ps-pA6B-PspA05 protein.Moreover, a cross-protective immunity was induced in KM mice upon the immuniza-tion with PspA6B-PspA05 protein.

OBJECTIVE:To study the effects of 10 kinds of nephrotoxic TCM on three main subtypes(Oat1,Oat2 and Oat3) of kidney organic anion transporter(Oats)in mice. METHODS:A total of 1 840 SPF NIH mice were randomly divided into nor-mal control group(isovolumic pure water),probenecid group(30 kg/mg),sodium carboxymethyl cellulose(CMC)group,Pulsa-tillae radix,Corydalis rhizoma,Aconiti kusnezoffii radix,Aconiti radix,Angelicae pubescentis radix,Gleditsiae spina,Polygo-num cuspidatum,Kansui radix,Platycladi cacumen,Aucklandiae radix high and low dose groups. Mice were treated twice a day for 5 d,ig. After 1 h of the last dosing,they were iv given PAH in tail(30 mg/kg). The PAH pharmacokinetic parameters of the kidney homogenate were determined and the PAH intake in kidney tissue at the time point of 1,5,10,15 and 20 min was detect-ed. The PAH in blood was analyzed by DAS 2.0 software. The grouping and dosing were the same as before,after 1 h of the last dosing,kidney slices were made and put into PAH-buffer. The PAH intake of kidney slices was determined. RESULTS:Compared with normal control group,the t1/2β in C. rhizoma high dose group,A. kusnezoffii high and low dose groups,A. pubescentis high dose group,P. cuspidatum high and low dose groups and P. cacumen group were increased;Vd were all decreased in 10 kinds of TCM high and low dose groups;except for A. pubescentis low dose group,G. spian low dose group and K. radix low dose group, the CL was decreased and AUC0-20 min was increased in all other groups,with significant difference (P<0.01 or P<0.05). Com-pared with normal control group,the content of PAH in kidney tissue in P. radix high dose group,C. rhizoma high dose group,A. kusnezoffii high dose group,A. radix high and low dose groups,A. pubescentis high and low dose groups,G. spina high and low dose groups,P. cuspidatum high and low dose groups,K. radix high and low dose groups,P. cacumen high and low dose groups and A. radix high and low dose groups were increased,with significant difference (P<0.01 or P<0.05). Compared with normal control group,the intake of PAH in kidney slices in C. rhizoma high dose group,A. kusnezoffii high and low dose groups,G. spi-na high and low dose groups,K. radix high dose group,P. ca-cumen high and low dose groups and A. radix high dose group were decreased,with significant difference (P<0.01 or P<0.05). CONCLUSIONS:The 10 kinds of nephrotoxic TCM probably induced kidney injury through inhibiting the Oat1,Oat2 and Oat3 of Oats.

Dengue virus (DENV) envelope [E] protein is the major surface protein of the virions that indued neutralizing antibodies. The domain III of envelope protein (EDIII) is an immunogenic region that holds potential for the development of vaccines; however, the epitopes of DENV EDIII, especially neutralizing B-cell linear epitopes, have not been comprehensively mapped. We mapped neutralizing B-cell linear epitopes on DENV-1 EDIII using 27 monoclonal antibodies against DENV-1 EDIII proteins from mice immunized with the DENV-1 EDIII. Epitope recognition analysis was performed using two set of sequential overlapping peptides (16m and 12m) that spanned the entire EDIII protein from DENV-1, respectively. This strategy identified a DENV-1 type- specific and a group-specific neutralizing epitope, which were highly conserved among isolates of DENV-1 and the four DENV serotypes and located at two regions from DENV-1 E, namely amino acid residues 309-320 and 381-392(aa 309-320 and 381-392), respectively. aa310 -319(310KEVAETQHGT319)was similar among the four DENV serotypes and contact residues on aa 309 -320 from E protein were defined and found that substitution of residues E309 , V312, A313 and V320 in DENV-2, -3, -4 isolates were antigenically silent. We also identified a DENV-1 type-specific strain-restricted neutralizing epitope, which was located at the region from DENV-1 E, namely amino acid residues 329-348 . These novel type- and group-specific B-cell epitopes of DENV EDIII may aid help us elucidate the dengue pathogenesis and accelerate vaccine design.
Amino Acid Sequence ; Animals ; Antibodies, Neutralizing ; immunology ; Dengue ; virology ; Dengue Virus ; chemistry ; genetics ; immunology ; Epitope Mapping ; Epitopes, B-Lymphocyte ; chemistry ; genetics ; immunology ; Humans ; Mice ; Molecular Sequence Data ; Viral Envelope Proteins ; chemistry ; genetics ; immunology