[Investigation of the inhibitory potential of caffeic acid phenethyl ester on prion replication, amplification, and fibril formation in vitro].

Objective: To investigate the effects and possible mechanisms of caffeic acid phenethyl ester (CAPE) on the replication, amplification, and fibre formation of prions (PrPSc). Methods: The CCK8 assay was used to detect the cell viability of the prion-infected cell model SMB-S15 after CAPE treatment for 3 days and 7 days and the maximum safe concentration of CAPE for SMB-S15 was obtained. The cells were treated with a concentration within a safe range, and the content of PrPSc in the cells before and after CAPE treatment was analyzed by western blot. Protein misfolding cycle amplification (PMCA) and western blot were used to assess changes in PrPSc level in amplification products following CAPE treatment. Real-time-quaking induced conversion assay (RT-QuIC) technology was employed to explore the changes in fibril formation before and after CAPE treatment. The binding affinity between CAPE and murine recombinant full-length prion protein was determined using a molecular interaction assay. Results: CCK8 cell viability assay results demonstrated that treatment with 1 µmol/L CAPE for 3 and 7 days did not exhibit statistically significant differences in cell viability compared to the control group (all P<0.05). However, when the concentration of CAPE exceeded 1 µmol/L, a significant reduction in cell viability was observed in cells treated with CAPE for 3 and 7 days, compared to the control group (all P<0.05). Thus, 1 µmol/L was determined as the maximum safe concentration of CAPE treatment for SMB-S15 cells. The western blot results revealed that treatment with CAPE for both 3 and 7 days led to a detectable reduction in the levels of PrPSc in SMB-S15 cells (all P<0.05). The products of PMCA experiments were assessed using western blot. The findings revealed a significant decrease in the levels of PrPSc (relative grey value) in the PMCA amplification products of adapted-strains SMB-S15, 139A, and ME7 following treatment with CAPE, as compared to the control group (all P<0.05). The RT-QuIC experimental results demonstrated a reduction in fibril formation (as indicated by ThT peak values) in CAPE-treated mouse-adapted strains 139A, ME7, and SMB-S15, as well as in SMB-S15 cells infected with prions. Furthermore, CAPE exhibited varying degrees of inhibition towards different seed fibrils formation, with statistically significant differences observed (all P<0.05). Notably, CAPE exhibited a more pronounced inhibitory effect on ME7 seed fibrils. Molecular interaction analyses demonstrated significant binding between CAPE and murine recombinant prion protein, and the association constant was (2.92±0.41)×10-6 mol/L. Conclusions: CAPE inhibits PrPSc replication, amplification, and fibril formation in vitro possibly due to specific interactions with the prion protein at the molecular level.

[Comparative study on the coronary artery anomalies between the Han and Tibetan nationalities living in middle and high altitude].

OBJECTIVE: To analyze the differences of coronary artery anomalies between Han and Tibetan nationality living in middle and high altitude. METHODS: A total of 7 028 adults living in the Qinghai Plateau(1 800-7 720 m altitude), who underwent coronary CT angiography in Qinghai Cardio-cerebro-vascular Disease Special Hospital between 2010 to 2015, were included in this study.There were 6 391 cases of the Han nationality and 637 cases of the Tibetan nationality. The differences of coronary artery anomalies between Han and Tibetan nationality were analyzed retrospectively. RESULTS: (1) Incidence of coronary artery anomalies was lower in Han nationality than in the Tibetan nationality (1.596%(102/6 391)vs. 4.239%(27/637), P<0.001). (2) There was 64.7%(66/102) male residents with coronary artery anomalies in Han nationality, and 74.1% (20/27) male residents with coronary artery anomalies in Tibetan nationality(P=0.359). (3) Left side coronary artery anomalies in Han nationality was similar as in Tibetan nationality (64.4%(67/104) vs. 55.6%(15/27), P=0.396). (4) Incidence of benign coronary artery anomalies was significantly lower in Han nationality than in Tibetan nationality (0.720%(46/6 391) vs. 2.200%(14/637), P<0.001). Incidence of potentially dangerous coronary artery anomalies was also significantly lower in Han nationality than in Tibetan nationality (0.876%(56/6 391) vs. 2.041%(13/637), P=0.004). (5)Ten kinds of coronary artery anomalies were found in this study. There were significant differences between Han and Tibetan nationality in left coronary artery originated from right coronary sinus(0.046%(3/6 391) vs. 0.471%(3/637), P=0.012), in left circumflex branch originated from right coronary sinus(0.046%(3/6 391)vs. 0.471%(3/637), P=0.012), and opening of right coronary artery in left coronary sinus or left anterior descending(0.704%(45/6 391)vs. 1.570%(10/637), P=0.018). CONCLUSION: The incidences of coronary artery anomalies and benign coronary artery anomalies were significantly lower in Han nationality residents than that of the in Tibetan nationality residents living in middle and high altitude.

[Effect of amoxicillin on the enamel mineralization of SD rats].

Objective: To determine whether amoxicillin had an effect on the enamel mineralization of SD rats. Methods: Eighteen SD rats were randomly divided into three groups. The rats in the control group were given distilled water. The rats in two experimental groups were administered 50 or 100 mg/kg amoxicillin by intragastric administration from day 3 to day 17 after birth. The general condition, the structure of liver and kidney, the enamel surface changes of mandibular first molars and incisors were observed. The changes of Ca/P ratio on enamel surface were analyzed by X-ray energy dispersive spectrometer (EDS). The surface morphology after phosphoric acid treatment was examined by scanning electron microscopy (SEM). Histological changes in the ameloblasts of mandibular incisors were analyzed by hematoxylin and eosin (HE) staining. Results: Compared with the control group, the general conditions as well as liver and kidney structures of SD rats in 50 and 100 mg amoxicillin groups had no significant differences. There was no obvious chalky changes on the first mandibular molars of SD rats in each group. All the incisors in 50 and 100 mg groups showed different degrees of chalkiness in the labial incisal 1/3 enamel. X-ray EDS analysis showed that the Ca/P ratios of occlusal and incisal 1/3 enamel in 50 and 100 mg groups (occlusal 1/3 of mandibular first molars: 1.51±0.03 and 1.52±0.02, incisal 1/3 of mandibular incisors: 1.46±0.01 and 1.43±0.01) was significantly lower than that in the control group (occlusal 1/3 of mandibular first molars: 1.67±0.41, incisal1/3 of mandibular incisors: 1.73±0.07) (P<0.05). However, there was no significant differences in the cervical 1/3 Ca/P ratio of mandibular first molars and incisors among the three groups (mandibular first molars: 1.56±0.04 for control group, 1.59±0.05 for 50 mg group and 1.57±0.04 for 100 mg group; incisors: 1.52±0.02 for control group, 1.47±0.01 for 50 mg group and 1.51±0.03 for 100 mg group) (P>0.05). SEM observation showed that the enamel rods of the first molars and incisors in the 50 and 100 mg group varied in size and arranged disorderly. The spaces between the enamel rods were larger than that in the control group and some areas even appeared large pits. HE staining showed that the gaps between ameloblasts in 50 and 100 mg groups were significantly wider than that in the control group. Conclusions: Intake of amoxicillin during the period of enamel development of SD rats might affect enamel mineralization.

MicroRNA-424-5p inhibits the development of non-small cell LCa by binding to ITGB1.

OBJECTIVE: The aim of this study was to explore the effect of microRNA-424-5p on the proliferation and apoptosis of non-small cell lung cancer (NSCLC) cells, and to investigate its influence on the expression of ITGB1 and potential regulatory mechanism. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the level of microRNA-424-5p in 44 paired NSCLC tissues and adjacent tissues. The relation between microRNA-424-5p expression and NSCLC clinical indicators was analyzed. Subsequently, microRNA-424-5p mimics and inhibitors were transfected into NSCLC cells to construct microRNA-424-5p overexpression or knockdown models, respectively. QRT-PCR was used to further verify the transfection efficiency. A series of experiments, including cell counting kit-8 (CCK-8) assay, colony formation, 5-Ethynyl-2'-deoxyuridine (EdU), and flow cytometry were used to analyze the effect of microRNA-424-5p on the biological function of NSCLC A549 and H358 cells. Finally, the potential association between microRNA-424-5p and its downstream gene ITGB1 was explored through luciferase reporter gene assay and cell recovery experiment. RESULTS: QRT-PCR results showed that microRNA-424-5p level was significantly lower in NSCLC tissues than that of adjacent normal tissues. Compared with patients with high expression of microRNA-424-5p, the pathological stage of those with low expression of microRNA-424-5p was significantly higher. In vitro experiments showed that microRNA-424-5p overexpression remarkably decreased cell proliferation and increased cell apoptosis, which were further validated in microRNA-424-5p inhibitor group. Subsequently, ITGB1 expression was found significantly up-regulated in NSCLC cell lines and tissues. Meanwhile, ITGB1 expression was negatively correlated with microRNA-424-5p level. In addition, a recovery experiment indicated that overexpression of ITGB1 could counteract the effect of microRNA-424-5p mimics on the proliferation and apoptosis of NSCLC cells. All these findings revealed that microRNA-424-5p and ITGB1 affected the malignant progression of NSCLC. CONCLUSIONS: MicroRNA-424-5p was closely correlated with the pathological stage and poor prognosis of NSCLC, thereby inhibiting the occurrence and development of NSCLC.

Antiapoptotic and antioxidant effects of rosmarinic acid in astrocytes.

The protective effects of rosmarinic acid (RA), a polyphenolic compound, on apoptosis induced by hydrogen peroxide in astrocytes were studied. Pretreating cells with RA significantly increased cell viability and decreased apoptosis rate induced by H2O2. The antiapoptotic effect of RA was further confirmed by increase of mitochondrial membrane potential and inhibition of caspase-3 activity. RA also attenuated cellular oxidative stress by decreasing the amount of reactive oxygen species and malondialdehyde. Results clearly show that RA was able to attenuate H2O2-induced cell injury by its antiapoptotic and antioxidant activity.

Synthesis, characterization and in vitro release of 5-aminosalicylic acid and 5-acetyl aminosalicylic acid of polyanhydride--P(CBFAS).

A novel polyanhydride, poly[(5-carboxybutyl formamide)-2-acetyl salicylic anhydride] (P(CBFAS)), with 5-aminosalicylic acid (5-ASA) incorporated into the polymer backbone was synthesized and characterized by infrared, (1)H-nuclear magnetic resonance, differential scanning calorimetry, vapor pressure osmometry, etc. The polyanhydride was subjected to degradation and simultaneously released 5-ASA and its derivative 5-acetyl aminosalicylic acid (5-acetyl ASA) in vitro under various conditions. The factors influencing the release profiles of 5-ASA and 5-acetyl ASA, including polymer molecular weights, pH value, enzyme and rat gastrointestinal contents, were examined. The results showed that the release rate of 5-ASA and 5-acetyl ASA increases with increasing pH value and with decreasing molecular weights. In PBS (pH 8.0, 37 degrees C) total ASA released was 8.0% for P(CBFAS)(1) (Mn 10770) in 13 h, but only 1.1 and 2.6% at pH 2.0 and 6.5, respectively. Enzymes including pepsin and trypsin, as well as rat gastric and jejunum contents had little effect on the release rate of 5-ASA and 5-acetyl ASA at pH 2.0 and 6.5 (less than 4% in 13 h). However, the release rate of 5-ASA and 5-acetyl ASA was much fast in PBS(pH 8.0) containing 5% of cecal contents, the total ASA released was 13.6% for the polymer in 13 h. Considering the high drug loading of the polymer (50.2% of 5-ASA moieties in the backbones) and the degradation characters, it is possible to reach high local concentration of 5-ASA in the colon site via oral administration. Therefore, P(CBFAS) may be potentially useful in the colon specific delivery of 5-ASA.