Humans ; Leukoplakia, Oral ; genetics ; Lichen Planus, Oral ; genetics ; Polymorphism, Single Nucleotide

OBJECTIVETo obtain the information on the glucuronidation of Ginkgo flavonoid and the interaction profile of Ginkgo flavones with other drugs in vitro.

METHODSGinkgo flavonoids (quercetin, isorhamnetin and keampferol) and other drugs were co-incubated with rat hepatic microsome at 25 degree; the residual concentrations of flavonoids were determined by HPLC. The enzymatic parameters of quercetin, isorhamnetin and keampferol metabolism were assessed. The interactions between flavonoids and these drugs on glucuronidation were observed.

RESULTThe K(m) values were ( 24+/-0.05), (148+/-0.09) and (110+/-0.03) micromol/L and the V(max) values were (60+/-0.21), (48+/-0.02) and (34+/-0.02) micromol x g(-1) x min(-1) for quercetin, isorhamnetin and kaempferol, respectively. The IC(50) of nifedipine propafenone ipriflavone and diphenytriazol on flavonoids metabolism were 54 - 70, 69 - 122, 85 - 98 and 210 - 362 micromol, respectively. The inhibition constants (Ki) of diphenytriazol propafenone and ipriflavone on quercetin, isorhamnetin and keampferol metabolism were (57.6, 50.5, 33.1) (33.6, 59.5, 45.2) and(13.7,24.0,15.7) microg/ml respectively. The ratio [I]/[Ki] of the plasma concentration and inhibition constant for propafenone was 0.002 - 0.003.

CONCLUSIONThe metabolic level of quercetin is the strongest among three Ginkgo flavonoids. Nifedipine propafenone and ipriflavone inhibit the metabolism of quercetin, isorhamnetin and keampferol at different levels. Because of the interaction between Ginkgo flavonoids with nifedipine, caution must be taken when two drugs are used together clinically.

Animals ; Drug Interactions ; Flavonoids ; metabolism ; Ginkgo biloba ; Glucuronides ; metabolism ; Microsomes, Liver ; metabolism ; Rats

Objective To explore the expression of osteopontin (OPN) in mucosal epithelium of oral local lesion in patients with oral lichen planus(OLP). Methods Forty patients with pathologically-confirmed OLP (erosive OLP,n=15; reticular OLP,n=25) were investigated,among whom 17 were complicated with mild dysplasia. Mucosal epithelium of oral local lesion was examined for the expression of OPN by immunohistochemical method. Forty healthy subjects were served as normal controls. Results The positive expression rates of OPN were 65.4% and 82.4%,respectively in patients with OLP and those complicated with mild dysplasia,and both were significantly higher than that in normal controls (10.0%) (P0.05),while both were significantly higher than that in normal controls (P

ObjectiveTo detect mRNA and protein expression of steroidogenic factor-1 ( SF-1 ) and DAX-1 in human adrenocortical tumors and normal adrenal cortex,and to investigate the effect of SF-1 and DAX-1 on the steroidogenesis and development of adrenocortical tumors.Methods Total RNA and protein was extracted from angiotensin Ⅱ unresponsive aldoterone-producing adenomas ( A Ⅱ -U-APA,n =12 ),angiotensin Ⅱ responsive aldoterone-producing adenomas ( AⅡ -R-APA,n =5 ),cortisol-producing adenomas ( CPA,n =10 ),adrenal nonfunctional adenomas ( NFA,n =10 ),aldosterone-producing carcinoma ( APC,n =2 ) and normal adrenal cortex ( NAC,n =8).To analyze gene expression of SF-1,DAX-1,ACTH receptor(ACTHR),and β-actin by real-time quantitative PCR in different tissues.The protein expression of SF-1,DAX-1,and β-actin in the same tissues by Western blot.To study the relationship of ACTHR,SF-1,and DAX-1 with clinical data in adrenocortical tumors.ResultsThe expression of SF-1,DAX-1 mRNA and protein was different in NAC,AⅡ -U-APA,A Ⅱ -R- APA,APC,CPA,and NFA tissues [ relative expression of SF-1 mRNA:24.58±2.45,23.89±3.17,21.59±3.00,(38.75,44.16),14.17±2.80,and 36.38±3.50; DAX-1 mRNA:0.57±0.06,0.37±0.05,0.43±0.05,( 1.52,1.21 ),0.39 ±0.04,and 0.83 ±0.08 ; SF-1 protein:0.76 ±0.11,0.76 ±0.10,0.73 ±0.07,(1.24,1.40),0.55±0.04,and0.98±0.10; DAX-1 protein:0.65±0.14,0.39±0.13,0.43±0.14,(1.18,1.02),0.56±0.04,and 1.03±0.13 ; all P＜0.05 or P＜0.01 ].There was negative correlation by higher SF-1/DAX-1 ratio and tumor size in AⅡ -U-APA tissues.The mRNA and protein expression of SF-1 was lower in CPA and there was the positive correlation with tumor size.Conclusion SF-1 and DAX-1 might play a key role in the development of the adrenocortical tumorigenesis and steroidogenic tissues.

Objective To investigate the expression of circulating tumor cells (CTC) in peripheral blood of patients with different stages of colorectal cancer (CRC), and to evaluate its significance in early diagnosis of colorectal cancer metastasis. Methods Sixty patients with CRC (42 inⅡ-Ⅲstage and 18 in IV stage ) and 30 patients with benign rectal disease were recruited from January 2014 to December 2015. The CTC in peripheral blood was purified with Immunomagnetic Separation Technologie, and detected by immunofluorescence in situ hybridization (imFISH). The serum levels of CEA were detected by electrochemiluminescence method meanwhile. The correlation between CTC and CEA was analyzed. Results CTC positive rates in CRC patients were significantly higher than those in benign rectal disease controls. CTC positive rates inⅣstage were significantly higher than those inⅡ-Ⅲ stage. The expression of CTC was significantly correlated with CEA (r = 0.6652, P < 0.01). Conclusions The expression of CTC in CRC patients is significantly higher than that in benign rectal disease control group. It is closely related to clinical stages. Detection of peripheral blood CTC has important clinical significance in the early diagnosis of CRC metastasis.

Objective To investiagate cell apoptosis and expressions of Bax,Bcl-2 andCaspase-3 in gambogic acid-treated colorectal cancer cells. Methods SW480/LOVO colorectal cancer cells were treated by gambogic acid. Cell Counting Kit-8 assay (CCK-8) was used to test cell proliferation. Microscopy was used to check the morphological changes. Immunofluorescence staining technique was used to detect cell apoptosis. Expressions of Bax,Bcl-2 and Caspase-3 protein were detected by Western blot assay. Results Gambogic acid inhibited the proliferation of SW480/LOVO in a dose and time-dependent manner. Gambogic acid could induce cell apoptosis. Gambogic acid increased expressions of Caspase-3 and Bax, increased the ratio of Bax/Bcl-2, and decreased Bcl-2 protein expression. Conclusion Gambogic acid can inhibit proliferation and induce apoptosis of SW480LOVO cells, with the mechanism of up-regulation of Bax/Bcl-2 and activation of Caspase-3.

Objective: To observe the regulatory effects of miRNA-31 (miR-31) on LATS2 and cardiomyocyte hypertrophy via down-regulating miR-31 expression in rat's cardiomyocytesin vitro. Methods: Rat's cardiomyocytes were isolated and cultured for 10 daysin vitro, according to different intervention methods, the cells were divided into 4 groups:①Blank control group,②AngII intervention group,③Lentivirus with miR-31 inhibitor infection group,④Negative lentivirus infection group. On day-8, gene expressions of MiR-31, LATS2, cardiac hypertrophy ANP and β-MHC were examined by qRT-PCR; on day-10, cell morphology was observed by fluorescence staining. LATS2 protein expression was examined by Western blot analysis. Dual luciferase reporter plasmids were transfected into 293T cells, then luciferase activity was detected to identify the targeting effect of miR-31 on LATS2. Results: Compared with Blank control group, AngII intervention group showed increased gene expressions of miR31, cardiac hypertrophy ANP and β-MHC,P<0.05, enlarged cardiomyocyte surface,P<0.05; while decreased gene and proteinexpressions of LATS2,P<0.05. Compared with AngII intervention group, Lentivirus with miR-31 inhibitor infection group had down-regulated expressions of miR31, cardiac hypertrophy ANP and β-MHC,P<0.05, reduced cardiomyocyte surface, P<0.05; while slightly increased LATS2 gene expression and obviously increased protein expression,P<0.05. Dual luciferase reporter assay presented that relative luciferase activity of TRAF6-3' UTR+miR-146b was significantly decreased than TRAF6-3' UTR+miR-NC,P<0.01 and relative luciferase activity of LATS2-3' UTR+ miR-31 was signiifcantly reduced than LATS2-3' UTR-NC+miR-31,P<0.01. Conclusion: Cardiomyocytes hypertrophy could be reversed at certain degree by down-regulating miR-31; the targeting effect of miR-31 on LATS2 was involved in cardiomyocyte hypertrophyregulation.

OBJECTIVETo obtain recombinant human CYP2E1 and to determine its activity by using the specific probe substrate.

METHODSCYP2E1 cDNA was obtained by RT-PCR using human liver RNA as template. The cloned CYP2E1 cDNA was ligated with pFastBac vector to generate recombinant pFastBac-CYP2E1, which was then transformed into E. coli DH 10 Bac. Recombinant Bacmid-CYP2E1 was generated by transposition. Then Spodoptera frugiperda (Sf9) insect cells was infected with Bacmid-CYP2E1 to generate recombinant baculoviruses carrying human CYP2E1 cDNA. Finally, Sf9 insect cells were triinfected with recombinant baculoviruses carrying human CYP2E1, CYPOR and CYPb5. The activity of the recombinant enzymes was determined using chlorzoxazone as the substrate.

RESULTThe Kmand Vmaxof recombinant CYP2E1 to chlorzoxazone was (72.4 +/-8.7) micromol. L(-1) and (2.41 +/-0.10) micromol.min(-1)?g(-1)protein, respectively.

CONCLUSIONActive recombinant CYP2E1 has been obtained by bac-to-bac expression system and its activity is similar to previous reports.

Animals ; Baculoviridae ; genetics ; metabolism ; Cytochrome P-450 CYP2E1 ; biosynthesis ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Spodoptera ; genetics ; metabolism ; Transfection

OBJECTIVE: To evaluate enantiomeric separation methods for beta-blocking agents and analogs. METHODS: Enantiomeric separation of racemates of 11 beta-blocking agents and their analogs was performed using chiral stationary phases and 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC). RESULTS: These beta -blocker racemates were separated into enantiomers in one or several chormatographic states such as propranolol, bisoprolol, metoprolol, celiprolol, carvedilol, sotalol, propafenone, ephedrine, and zomitriptan. Temperature had a significant effect on the resolution of the drugs when using chiralcel OD. Lower temperatures were associated with higher resolutions. CONCLUSION: When separating beta-blocking agents and their analogs, Chiralcel OD, Chiralpak AD, Chiral stationary phases and GITC chiral derivative reagents have complementary functions.

AIMTo develop a method for assaying Ginkgo flavones in rat hepatical microsome.

METHODSQuercetin, isorhamnetin and keampferol were added to microsome incubate and incubated for a given time then extracted with ether-acetone. After evaporated, the residue was reconstituted with 100 microL of phosphate buffer solution (pH 2.0)-tetrahydrofuran-methanol-isopropanol (60:15:10:20). An aliquot of 20 microL was injected into the HPLC system. According to the result of estimate by means of HPLC, the results of metabolism of Ginkgo flavones in different conditions was compared.

RESULTSThe assay was linear over the rang of 0.2-8 mg.L-1 for Ginkgo flavones. The limit of quantification was 0.1 mg.L-1 (n = 3). The recoveries of three components of Ginkgo flavones were 99.9%-113.8% for quercetin (RSD < 0.8%), 100.8%-117.3% for isorhamnetin (RSD < 1.9%) and 100.7%-116.5% for keampferol (RSD < 1.03%, n = 5).

CONCLUSIONThe method is simple, fast and accurate. It can be used for investigation of the metabolism of Ginkgo flavones.

Animals ; Chromatography, High Pressure Liquid ; Flavonols ; isolation & purification ; metabolism ; Ginkgo biloba ; chemistry ; In Vitro Techniques ; Kaempferols ; isolation & purification ; metabolism ; Microsomes, Liver ; metabolism ; Plant Leaves ; chemistry ; Plants, Medicinal ; chemistry ; Quercetin ; isolation & purification ; metabolism ; Rats