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.

Objective To observe the clinical efficacy of Zishen Tongyang Huoxue Formula combined with western medicine in treating angina pectoris caused by coronary heart disease (CHD).Methods Ninety patients were randomly divided into control group, TCM group and combining group, each group of 30 cases. The control group was given western medicine standardized treatment;the TCM group was treated with Zishen Tongyang Huoxue Formula only;the combining group took Zishen Tongyang Huoxue Formula and western medicine standardized treatment. The treatment course for the three groups lasted for four weeks. The clinical presentations, angina pectoris, ECG and serum biomarkers of patients in the three groups were assessed and evaluated before and after treatment.Results The clinical symptoms of TCM were significantly improved;the duration of angina was shortened;the dosage of nitroglycerin reduced after the treatment (P<0.05). The effects in the combining group was more evident compared with the other two groups (P<0.05), but there was no statistical difference between the other two groups (P>0.05). The total TCM effective rates of three groups were 83.33% (25/30), 83.33% (25/30) and 96.67% (29/30), respectively, among which the combining group was more significant (P<0.05). The total ECG effective rates of three groups were 70.00% (21/30), 76.67% (23/30) and 76.67% (23/30), respectively. Hs-CRP, IL-6 and HCY in the three groups decreased after the treatment (P<0.05). The combining group was more significant than the other two groups (P<0.05), but there was no statistical difference between the other two groups (P>0.05). Blood routine test and hepatorenal function of the three groups were all in normal range before and after treatment (P>0.05).Conclusion Using Zishen Tongyang Huoxue Formula combined with western medicinein to treat angina pectoris is effective, safe, and reliable.

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.

AIM: To explore the effect of pyrrolidine dithiocarbamate (PDTC), the specific inhibitor of NF-?B, on anti-thymocyte serum nephritis (ATSN) in rats. METHODS: The rat model of ATSN was reproduced with rabbit anti-thymocyte serum (ATS). The rats were divided into ATSN group, ATSN+PDTC group and control group. The expression of NF-?B p65 and the apoptosis, lysis as well as proliferation of mesangial cells (MC) were examined by immunohistochemical staining, Tdt-mediated X-dUTP nick end labeling (TUNEL), light microscope and electron microscope at 40 minutes, 24 hours and 7 days after injection of ATS or normal serum. RESULTS: The expression of glomerular NF-?B p65 in the ATSN group was observed with significant difference compared to controls at 40 min (P

Dbait is a small double-stranded DNA molecule that has been utilized as a radiosensitizer to enhance the sensitivity of glioma to radiotherapy (RT). However, there is no effective drug delivery system to effectively overcome the blood-brain barrier (BBB). The aim of this study was to develop a gene delivery system by using the BBB and glioma dual-targeting and microenvironment-responsive micelles (ch-K(s-s)R8-An) to deliver Dbait into glioma for RT. Angiopep-2 can target the low-density lipoprotein receptor-related protein-1 (LRP1) that is overexpressed on brain capillary endothelial cells (BCECs) and glioma cells. In particular, due to upregulated matrix metalloproteinase 2 (MMP-2) in the tumor microenvironment, we utilized MMP-2-responsive peptides as the enzymatically degradable linkers to conjugate angiopep-2. The results showed that ch-K(s-s)R8-An micelles maintained a reasonable size (80-160 nm) with a moderate distribution and a decreased mean diameter from the cross-linking as well as exhibited low critical micelle concentration (CMC) with positive surface charge, ranging from 15 to 40 mV. The ch-K5(s-s)R8-An/pEGFP showed high gene transfection efficiency , improved uptake in glioma cells and good biocompatibility and . In addition, the combination of ch-K5(s-s)R8-An/Dbait with RT significantly inhibited the growth of U251 cells . Thus, ch-K5(s-s)R8-An/Dbait may prove to be a promising gene delivery system to target glioma and enhance the efficacy of RT on U251 cells.

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.