AIM To investigate the role of Trp3 in the Ca 2+ influx induced by ? 1B AR in HEK293 cells and the effect of tyrosine kinase on it. METHODS With lipofect AMINE2000 reagent, hTrp3 cDNA was transfected to HEK293 cells and ? 1B HEK293 cells respectively. The expression of Trp3 was examined by Western blot. With Fura 2/AM spectrophoto fluorometry, Ca 2+ influx was determined. RESULTS HTrp3 was expressed endogenously in HEK293 cells, and the expression increased in hTrp3 transfected cells. Compared with untransfected cells, transfection of hTrp3 cDNA increased Ca 2+ influx induced by ? 1B AR ( P 0 05). 5～30 ?mol?L -1 genistein inhibited Ca 2+ influx induced by ? 1B AR in hTrp3 cDNA transfected cells and the maximum inhibitory rate was (75 2?12 6)% . CONCLUSION Transfection of hTrp3 cDNA increased Ca 2+ influx induced by ? 1B AR in HEK293 cells. This process was regulated by tyrosine kinase.

Objective To study the effective composition of Aster ageratoides Turcz for relieving asthma and its toxicity . Methods Seventy‐two qualified male guinea pigs ,body mass(300 ± 20)g ,were randomly divided into the control group ,aminophyl‐line group(0 .047 g/kg) and different ratio group of total saponins and total flavonoids (8∶1 ,4∶1 ,2∶1 ,1∶1 ,1∶2 ,1∶4 ,1∶8) . Every group received at dose of 0 .38 g/kg once a day for 3 days by intragastric administration .The effect of relieving asthma was taken as the indicators for the optimization of compositions ,which was detected by observing the latent period of asthma induced by acetylcholine‐histamine in guinea rats .The relaxation percentage of smooth muscle retracted by acetylcholine was detected by Pow‐erlab biological signal collecting system when aminophylline(1 .25 mg/mL) and different matching drugs(0 .02 g/mL) were added in Krebs′solution .The acute toxicities of total saponins ,total flavonoids and mixture of total saponins with total flavonoids (ratio of 1∶2) were detected by classical acute toxicity test of mice .Results Compared with the control group ,aminophylline group ,each group latent period of inducing asthma significantly or very significantly prolonged (P<0 .05) ,among them ,1∶1 ,1∶2 ,1∶4 group and other matching group were significantly different(P<0 .05) ,but 1∶2 group cited the longest incubation period .Aminophylline group and the ratio could significantly relaxe guinea pig tracheal smooth muscle retracted by acetylcholine .Aminophylline group di‐astolic percentage was the largest(24 .12 ± 1 .20)% ,all the groups were less than the percentage of diastolic aminophylline group (P<0 .05) ,but in 1∶2 group with the closest .Aster ageratoides Turcz ,tatarian aster total saponins ,total flavonoids and the ratio of 1∶2 to mice maximum tolerance was respectively 300 ,345 ,325 g/kg .Conclusion For the same total dosage ,the relieving asth‐ma effects of proportions of 1∶2 Aster ageratoides Turcz were obvious .There are no abnormal changes in the acute toxicity trila with total flavonoids ,total saponins ,as well as the ratio of 1∶2 ,safety is relatively good .

Objective To clone the cDNA encoding human HMGB1, express it in E. coli, and identify its biological activity. Methods Human HMGB1 cDNA was amplified by RT-PCR and cloned into vector pUC19. After sequence analysis, the cDNA was ligated into prokaryotic expression vector pQE-80L and induced by IPTG to express HMGB1. The protein was purified with Ni~(2+)-NTA chromatography and polymyxin B affinity column. To identify the function of purified protein, the product was co-cultured with THP1 cells. Results Recombinant expression plasmid pQE-80L/HMGB1 was constructed successfully. After purification, the protein purity reached 96%. The recombinant HMGB1 stimulated THP1 to secrete TNF-? . Conclusion The highly purified HMGB1 was obtained successfully, which showed biological activity. These results lay the foundation for further research on the function of human HMGB1.

OBJECTIVETo conduct an experimental study on in vitro transdermal absorption of prepared Shangshi Zhitong cataplasm.

METHODFranz diffusing cells and mice were adopted for the percutaneous penetration study. The accumulative percutaneous permeation of total alkaloids, strychnine and atropine in certain time was determined by acid dye colorimetry and HPLC.

RESULTThe accumulative permeation of alkaloids (Q) increased with time (t), with a linear relation between them.

CONCLUSIONThe in vitro percutaneous penetration of Shangshi Zhitong cataplasm complies with the zero-order kinetics.

Administration, Cutaneous ; Alkaloids ; pharmacokinetics ; Animals ; Atropine ; pharmacokinetics ; Drugs, Chinese Herbal ; pharmacokinetics ; Male ; Mice ; Rats ; Rats, Wistar ; Skin Absorption ; Strychnine ; pharmacokinetics

Purpose: We aimed to investigate whether obtaining a higher level of education was causally associated with lower breast cancer risk and to identify the causal mechanism linking them. Methods: The main data analysis used publicly available summary-level data from 2 large genome-wide association study consortia. Mendelian randomization (MR) analysis used 65 genetic variants derived from the Social Science Genetic Association Consortium as instrumental variables for years of schooling. The outcomes from the Breast Cancer Association Consortium (BCAC) were the overall breast cancer risk (122,977 cases/105,974 controls in women) and the two subtypes: estrogen receptor (ER)-positive breast cancer and ER-negative breast cancer. Fixed and random effects inverse variance weighted methods were used to estimate the causal effects, along with other additional MR methods for sensitivity analyses. Results: Results showed that each additional standard deviation of 4.2 years of education was causally associated with a 27% lower risk of ER-negative breast cancer (odds ratio, 0.73; 95% confidence interval, 0.64–0.84; p-value < 0.001). This finding was consistent with the results of the sensitivity analyses. Physical activities can help improve the protective effect of education against breast cancer, with relatively large mediation proportions. Education increases the risk of ER-positive breast cancer due to alterations in high-density lipoprotein level, triglyceride level, height, waist-to-hip ratio, body mass index, and smoking status, with relative medium mediation proportions. Other mediators including low-density lipoprotein, hip circumference, number of cigarettes smoked per day, time spent performing light physical activity, and performing vigorous physical activity for > 10 minutes explain a small part of the causal effect of education on the risk of developing breast cancer, and their mediation proportion is approximately 1%. Conclusion A low level of education is a causal risk factor in the development of breast cancer as it is associated with poor lipid profile, obesity, smoking, and types of physical activity.

ObjectiveTo explore the effect of tetramethylpyrazine （TMP） on the vascular mimicry （VM） of non-small cell lung cancer A549 stem cell-like cells （CSLCs） in hypoxic state， and on the expression of hepatocyte growth factor （HGF）/mesenchymal-epithelial transition factor （c-Met）. MethodSerum-free sphere culture method was used to separate and enrich A549 CSLCs， and flow cytometry to detect the expression of stem cell marker CD44⁺/CD24^-/low. CoCl₂ was employed to induce hypoxia model. Cell counting kit-8 （CCK-8） assay was employed to examine the influence of 100， 200， 400， 800， 1 600， and 3 200 μmol·L^-1 TMP on the viability of A549 CSLCs. The low， medium， and high concentration （100， 200， 400 μmol·L^-1） of TMP that did not significantly affect the viability of A549 CSLCs was selected for subsequent experiments. Tube formation assay was used to detect the effect of different concentration of TMP on the formation of A549 CSLCs VM under hypoxia condition， and Western blot was applied to measure the expression of HGF/c-Met. ResultThe CD44⁺/CD24^-/low expression ratio of the isolated and enriched CSLCs was （80.3±0.21）%， which was significantly higher than that of the A549 group （P<0.01）. Compared with the control group， TMP groups showed increase in the inhibition rate of CSLCs， particularly the 24 h TMP （800， 3 200 μmol·L^-1） groups and the 48 h TMP （1 600， 3 200 μmol·L^-1） groups （P<0.05）. Compared with blank group and Bevacizumab （Bev） group， each concentration of TMP decreased the number of tubes formed and intersections （P<0.01）. The number of tubes formed and intersections decreased in TMP groups compared with that in the SU11274 group， particularly the 200 μmol·L^-1 TMP group （P<0.05） and 400 μmol·L^-1 TMP group （P<0.01）. Levels of HGF and c-Met in all TMP groups were down-regulated compared with those in the blank group （P<0.05， P<0.01）. ConclusionTMP can inhibit the formation of VM in A549 CSLCs in vitro under hypoxia condition which may act by regulating HGF/c-Met related signaling pathways.