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Objective:To investigate the correlation between Traditional Chinese medicine (TCM) constitution classification and clopidogrel-related CYP2C19 gene polymorphism of acute cerebral infarction patients.Methods:A cross-sectional method was used in this study. Patients with acute cerebral infarction were screened and enrolled according to our inclusion and exclusion criteria. TCM constitution were evaluated in patients with acute cerebral infarction. Digital fluorescence hybridization Technology was used to test genotypes of CYP2C19 and Hardy-Weinberg’s equilibrium was used to examine CYP2C19 gene polymorphism of the patients. Binary logistic regression analysis method was used to explore the relationship between TCM constitution types and clopidogrel-related CYP2C19 gene polymorphism in acute cerebral infarction.Results:Among the 100 patients with acute cerebral infarction, 18 belong to Yang-deficiency constitution (18 cases, 18%), 30 Yin-deficiency (30 cases, 30%), 18 belong to Qi-deficiency constitution (18 cases, 18%), 51 belong to phlegm-dampness constitution(51 cases, 51%), 14 belong to damp-heat constitution (14 cases, 14%), 2 belong to special constitution (2 cases, 2%), blood-stasis constitution (27 cases, 27%), Qi stagnation constitution (4 cases, 4%), and normal constitution (11 cases, 11%). The CYP2C19*2 gene polymorphism distribution: CYP2C19*2 (A/A, mutant homozygous) (21 cases, 21%), CYP2C19*2 (A/G, mutant heterozygote) (33 cases, 33%), CYP2C19*2 (G/G) (normal homozygous) (46 cases, 46%). The mutant allele frequency was 0.375. Binary logistic regression analysis showed that compared with A/A genotype, the G/G genotype of CYP2C19*2 in acute cerebral infarction was correlated with phlegm-dampness constitution ( OR=5.105, 95% CI: 1.308-19.928, P<0.05) and blood-stasis constitution ( OR=12.557, 95% CI: 1.741-90.558, P<0.05). Conclusions:The proportion of phlegm-dampness constitution is the highest in patients with acute cerebral infarction. The mutation rate of clopidogrel-related CYP2C19*2 was significantly higher than that of CYP2C19*3 clopidogrel-related CYP2C19*2 gene polymorphism might be related to phlegm-dampness constitution and blood-stasis constitution.
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Objective@#To investigate the correlation between Traditional Chinese medicine (TCM) constitution classification and clopidogrel-related CYP2C19 gene polymorphism of acute cerebral infarction patients.@*Methods@#A cross-sectional method was used in this study. Patients with acute cerebral infarction were screened and enrolled according to our inclusion and exclusion criteria. TCM constitution were evaluated in patients with acute cerebral infarction. Digital fluorescence hybridization Technology was used to test genotypes of CYP2C19 and Hardy-Weinberg’s equilibrium was used to examine CYP2C19 gene polymorphism of the patients. Binary logistic regression analysis method was used to explore the relationship between TCM constitution types and clopidogrel-related CYP2C19 gene polymorphism in acute cerebral infarction.@*Results@#Among the 100 patients with acute cerebral infarction, 18 belong to Yang-deficiency constitution (18 cases, 18%), 30 Yin-deficiency (30 cases, 30%), 18 belong to Qi-deficiency constitution (18 cases, 18%), 51 belong to phlegm-dampness constitution(51 cases, 51%), 14 belong to damp-heat constitution (14 cases, 14%), 2 belong to special constitution (2 cases, 2%), blood-stasis constitution (27 cases, 27%), Qi stagnation constitution (4 cases, 4%), and normal constitution (11 cases, 11%). The CYP2C19*2 gene polymorphism distribution: CYP2C19*2 (A/A, mutant homozygous) (21 cases, 21%), CYP2C19*2 (A/G, mutant heterozygote) (33 cases, 33%), CYP2C19*2 (G/G) (normal homozygous) (46 cases, 46%). The mutant allele frequency was 0.375. Binary logistic regression analysis showed that compared with A/A genotype, the G/G genotype of CYP2C19*2 in acute cerebral infarction was correlated with phlegm-dampness constitution (OR=5.105, 95% CI: 1.308-19.928, P<0.05) and blood-stasis constitution (OR=12.557, 95% CI: 1.741-90.558, P<0.05).@*Conclusions@#The proportion of phlegm-dampness constitution is the highest in patients with acute cerebral infarction. The mutation rate of clopidogrel-related CYP2C19*2 was significantly higher than that of CYP2C19*3 clopidogrel-related CYP2C19*2 gene polymorphism might be related to phlegm-dampness constitution and blood-stasis constitution.
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Objective To observe the relationship between Toll-like receptor 4 (TLR4) and the sensitivity of PANC1 cells to gemcitabine (GEM),and to analyze the potential mechanism.Methods PANC1 cells were divided into GEM group,lipopolysaccharide (LPS) + GEM group and TLR4-siRNA + GEM group.GEM group was treated by GEM alone.LPS + GEM group was pretreated with 1 mg/L LPS for 4 h and then treated by GEM.TLR4-siRNA + GEM group was transfected with 100 pmol/mL TLR4-siRNA for 4 h and then treated by GEM.The untreated cells were used as the control group.MTT method was used to detect the cell proliferation.Morphological changes and apoptosis rate of the cells were examined by Hoechst33258 staining and flow cytometry,respectively.The protein expression of TLR4,phosphorylated AKT (p-AKT) and activated Caspase-3 were detected by Western blot.Results The median inhibition concentration (ICs0) of GEM in the GEM group,LPS + GEM group and TLR4-siRNA + GEM group was (8.9 ± 0.32),(14.21 ±0.95),(3.96 ± 0.27) mg/L,respectively.The IC50 in LPS + GEM group was significantly higher than that in GEM group (P ＜ 0.01),and the IC50 of GEM in TLR4-siRNA + GEM group was significantly lower than that in GEM group (P ＜0.01).Compared with that in GEM group,the cells with typical apoptotic morphological changes were decreased in LPS + GEM group,which was increased in TLR4-siRNA + GEM group.The apoptotic rate in control group,GEM group,LPS + GEM group,TLR4-siRNA + GEM group was (2.1 ± 0.3) ％,(15.1 ± 2.3) ％,(9.8 ± 1.5) ％,(22.9 ± 3.1) ％,respectively.Compared with that in GEM group,the cells apoptotic rate was significantly reduced in LPS + GEM group (P ＜0.01),which was significantly increased in TLR4-siRNA + GEM group (P ＜0.01).TLR4 protein level in the 4 groups was 0.83 ±0.08,0.81 ±0.07,0.85 ±0.07 and 0.16 ±0.03;p-AKT protein level 0.61 ±0.05,0.36 ±0.03,0.73 ± 0.07 and 0.21 ± 0.02;activated Caspase-3 protein level was 0.66 ± 0.05,0.73 ± 0.07,0.45 ± 0.04 and 0.91 ± 0.07,respectively.The expression of TLR4 and p-AKT in TLR4-siRNA + GEM group was significantly lower than that in GEM group (P ＜0.01),while the expression of activated Caspase-3 protein was increased significantly (P ＜ 0.05).Compared with the GEM group,the expression of p-AKT protein in LPS + GEM group was significantly increased (P＜0.01),and the expression of activated Caspase-3 protein was significantly decreased (P＜0.01).Conclusions TLR4 can inhibit the sensitivity of pancreatic cancer PNAC1 cells to GEM,and the mechanism is related to the activation of PI3K/AKT pathway and downregulation of activated Caspase-3.
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Objectives To investigate the correlation between the level of peripheral blood inflammatory cytokines and kainic acid-induced seizure severity in rats.Methods 140 rats were divided into control and model group randomly,70 rats in each group.Model group rats were injected with kainic acid (10 mg/kg) by intraperitoneal,and the control rats were injected with sodium chloride.The change of their behaviors was observed and the concentrations of TNF-α,IL-1β,IL-4 and IL-10 were determined by ELISA in each group at different times.Results The rats showed epilepsy grand mal in 3 h-9 h after KA injection.The concentrations of TNF-α and IL-1β in model groups were significantly higher than those in control group in 6 h-12 h (6 h:(21.5±3.2) pg/ml vs (12.3±3.1)pg/ml;12 h:(20.6±4.2)pg/ml vs (11.5±3.8)pg/ml)(P＜0.05) and IL-4 in model group was higher at only 12 h ((53.55±3.08) pg/ml vs (33.26±4.16)pg/ml) (P＜0.05).The level of IL-10 in model groups was not statistically significant compared with control group (P＞0.05).Conclusion The proinflammatory cytokines (TNF-α and IL-l β) participate in the seizure procedure,meanwhile their levels and seizure severity have eminent correlations,but antiinflammatory cytokines (IL-4 and IL-10) had not.
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Objective To investigate the effect of Iodine 125 seeds short time low dose rate irradiation on perineural invasion (PNI) in pancreatic cancer Capan-2 cells,and explore its molecular mechanism.Methods The co-culture model was established by co-culturing the dorsal root ganglion (DRG) of SD rat and Capzn-2 cells line,while Capan-2 culture model and DRG culture model was also established.Iodine 125 seeds short time low dose rate irradiation tablet was used for the 3 models,and the model without irradiation was used as control.Cancer cell and DRG growth was observed under inverted microscopy,surface of neurite and cell colony growth was determined by image analysis software.The concentration of nerve growth factor (NGF),transforming growth factor-α (TGF-α) in cell culture supernatant and matrigel solution was tested by ELISA,and the expression of neurotrophin-3 (NT-3) mRNA was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR).Results In the co-culture model,neurite of DRG showed a direction to cancer cells and had a concentrated growth towards cancer cells.And Capan-2 cells formed more colonies towards neurite.However,in irradiation groups,the symbiotic phenomenon was inhibited to some degree.Increased surface of neurite in co-culture model at 5th day was 290.15 ± 12.08,which was significantly higher than that in DRG group (124.83 ± 6.96,P ＜ 0.01),but the surface of neurite was decreased to 201.53 ± 12.20 after irradiation (P ＜0.01).Increased surface of Capan-2 cell was 300.47 ± 12.99,which was significantly higher than that in Capan-2 group (199.30 ± 8.60,P ＜ 0.01),but the surface of Capan-2 was decreased to 202.35 ± 7.97 after irradiation (P ＜ 0.01).NT-3 mRNA was seldom or not expressed in supernatant of co-culture model,but it was strongly expressed (0.68 ± 0.04) after irradiation (P ＜ 0.05).The concentration of NGF and TGF-α in supernatant of co-culture model were (27.56 ± 13.73),(40.86 ± 20.73) ng/ml,after irradiation they were increased to (94.98 ± 33.80),(157.54 ± 83.76) ng,/ml,and the difference between the two groups was statistically significant (P＜0.05 or ＜0.01).The concentration of NGF and TGF-α in matrigel lysate of co-culture model were (60.42 ± 33.03),(64.39 ± 21.52)ng/ml,after irradiation they were increased to (132.52 ±53.01),(138.38 ±83.58)ng/ml,and the difference of NGF concentration between the two groups was statistically significant (P ＜ 0.05).Conclusions Iodine-125 seeds short-time low-dose rate irradiation could inhibit interactions between nerve and Capan-2 cells,and the mechanism may be related to up-regulation of cancer cells perineural invasion promoter NGF,TGF-α and NT-3.
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Objective To investigate the role of TLR4/NF-kB signaling pathway in inhibited invasion ability of pancreatic cancer cells caused by triptolide (TP).Methods PANC1 cells were divided into parental cells group,TP group,lipopolysaccharide (LPS) group and TP + LPS group.50 ng/ml of TP was added in culture medium in TP group,and 1 μg/ml of LPS was added in culture medium in LPS group,while 50 ng/ml of TP was pretreated for 2 h and 1 μg/ml of LPS was added in culture medium in TP + LPS group.All the ceils were cultured for 24 h.The TLR4 and matrix metalloproteinase-9 (MMP-9) mRNA and protein expression were evaluated by real-time PCR and Western blot.The NF-kB activity was determined by dual-luciferase reporter assay system.The invasion ability of pancreatic cancer cells was evaluated by transwell invasion chamberassay.Results The TLR4 mRNA expressions in parental cells group,TP group,LPS group and TP + LPS group were 0.41 ± 0.06,0.46 ± 0.10,0.20 ± 0.04,0.25 ± 0.06 ; the TLR4 protein expressions were 0.55 ±0.06,0.55 ±0.06,0.18 ±0.04,0.13 ±0.00; the activities of NF-kB were 13.0 ±3.0,31.6 ±4.3,7.3 ±1.5 and 10.8 ± 2.1,and the numbers of invasion cell were (56.8 ± 8.6),(104.5 ± 12.8),(32.0 ± 5.7) and (46.8 ± 7.0) ; the MMP-9 mRNA expressions were 0.36 ± 0.05,0.58 ± 0.07,0.18 ± 0.03,0.30 ± 0.004 ;the MMP-9 protein expressions were 0.31 ± 0.04,0.53 ± 0.08,0.11 ± 0.02,0.15 ± 0.00.In LPS group,TLR4 mRNA and protein expressions were not statistic significant when compared with those in parental cells group,but the activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions were statistically increased when compared with those in parental cells group (t =8.654,7.593,6.655,4.982,P ＜0.01).TLR4 mRNA and protein expressions,activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions in TP group were significantly lower than those in parental cells group (t =-7.609,-9.948,-4.176,-5.915,-8.179,-9.948,P＜ 0.01).TLR4 mRNA and protein expressions,activities of NF-kB,the numbers of invasion cell,MMP 9 mRNA and protein expressions in TP +LPS group were significantly lower than those in LPS group (t =-4.437,-14.805,-10.506,-9.700,-9.055,-8.932,P＜ 0.01).Conclusions TP can inhibit pancreatic cancer cell invasion,and the mechanism is related to the inhibition of TLR4/NF-kB signaling pathway and down-regulation of MMP-9 expression.
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Objective To establish the orthotopic human pancreatic cancer model in mice and study the method to detect the tumor growth. Methods Human pancreatic cancer cell lines SW1990 in logarithmic phase was made into cell suspension and in situ injected into the envelope of pancreatic tail of BALB/c nude mice. High-frequency endoscopy ultrasound was used to observe the growth of tumor mass and its imaging characteristics were studied. Results 20 nude mice were successfully implanted, and 1 died 25 h after implantation. 14 days after implantation, the tumor of pancreatic cancer on EUS was (8.09 ± 2.61) mm3, the tumor appeared as homogeneous hypoechoic mass with clear boundary, and envelope as well as sound halo was present, and the shape was regular; there was low speed circular vasculature signal in around 30% of tumor.The tumor size increased to (12.40 ± 3.51)mm3, and the shape of 70% tumor was irregular, and some appeared as lobulated, and the tumor appeared as heterogeneous hypoechoic mass, no necrosis or liquefaction was found 28 days after implantation. There was low speed circular vasculature signal in around 70% tumors.Conclusions The orthotopic pancreatic cancer model in nude mouse can be established by in situ injection and this method is relatively ideal because it is simple and effective. The high frequency probe of endoscopic ultrasonograph is a reliable method for monitoring implanted pancreatic cancer.
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Objective To investigate the role of the Wnt/LRPS/3-catenin signaling pathway in the pathogenesis of postmenopausal osteoporosis.Methods Fifty female Wistar rats aged 6-month-old,were randomly divided into control group ( NS,n =24 ) and ovariectomized group ( NOVX,n =26),NOVX underwent bilateral ovariectomy.At 0,4 and 8 weeks,all of rats were measured blood estrogen ( E2 ) and bone mineral density (BMD),4 and 8 weeks,low density lipoprotein receptor-related protein 5 (LRP5),3-catenin and Runx2 mRNA in bone were measured respectively by reverse transcription (RT)-PCR.Results In 4 and 8 weeks,compared with NS which had (117±29) and (114+15) pmol/L in E2 level,(0.098 ± 0.016 ) and (0.095 ± 0.028 ) g/cm2 in BMD,NOVX had significantly decreased to ( 92±15 ) and(95±22) pmol/L in E2 level ( P＜0.05 ),( 0.076 ± 0.016) and ( 0.052 ± 0.013 ) g/cm2 in BMD values ( P ＜ 0.01 ).And bone tissue LRP5,β-catenin and Runx2 mRNA expression was 1.02 ± 0.06,1.04 ±0.05,1.07 ±0.21 in NS,NOVX was significantly reduced to 0.97 ± 0.04,0.58 ± 0.05,0.86 ±0.03 (P＜0.05).Conclusion Wnt/LRP5/β-catenin signaling pathway may be important in the pathogenesis of postmenopausal osteoporosis.