Synergistic action on hypnosia: Yinao capsules with pentobarbital sodium of threshold and sub-threshold dosages

BACKGROUND： Sedative and hypnotic drugs could cure insomnia in a dependent manner, and traditional Chinese medicine has some superiority in treating insomnia. OBJECTIVE： To observe the synergistic action of yinao capsules with pentobarbital sodium in threshold and sub-threshold dosages in hypnosia and sedation. DESIGN： A completely randomized grouping design and control experiment. SETTING： Pharmacological laboratory, College of Pharmacy, Henan College of Traditional Chinese Medicine. MATERIALS： Totally 200 grade II Kunming mice of 18 - 21 g, either male or female, were used. Yinao capsules, main ingredients of which were turtleback glue, thinleaf milkwort root, Chinese magnoliavine fruit, mythic fungus, tangshen, ginseng and grassleaf sweetflag rhizome, were offered by Chinese-American Huayi Pharmacy, Co.,Ltd. （ratified number： 040901）; Kangnaoshuai capsules, main ingredients of which were prepared rehmannia root, tuber fleeceflower root, ginseng, membranous milkvetch root, thinleaf milkwort root, Fushen, grassleaf sweetflag rhizome, spine date seed, lecithin, barbary wolfberry fruit, pueraria root, vitamin E, etc., were produced by Shijiazhuang Siyao, Co.,Ltd. （ratified number： 040964）; Pentobarbital sodium was produced by China Medicine （Group） Shanghai Chemical Reagent, Co,.Ltd. （Ratified number： 030816）. ZZ-6 mice spontaneous activity apparatus was produced by Chengdu Taimeng Science and Technology, Co.,Ltd. METHODS： The experiment was carried out in the Animal Experimental Center, Henan College of Traditional Chinese Medicine from October to December in 2005. （1） Influence of Yinao capsules on the spontaneous activity of mice： Fifty mice were randomly divided into five groups with 10 mice in each group： Mice in the large, middle and small dosages of Yinao capsules groups were intragastrically infused with Yinao capsules suspension （1.36, 0.68, 0.34 g/kg）; Those in the Kangnaoshuai capsules group were infused with Kangnaoshuai suspension （1.12 g/kg）; Those in the control group were given physiologic saline of the same volume. The mice were administrated once a day for 7 days continuously, and they were placed into the mice spontaneous activity apparatus after 60 minutes from the last administration, the times of spontaneous activities and the times of arising within 10 minutes were recorded after adaptation for 5 minutes. （2） Synergistic action in hypnosia by Finao capsules with pentobarbital sodium of threshold dosage： Seventy mice were randomly divided into 5 groups as above-mentioned with 14 mice in each group, and they were treated the same as above. They were intraperitoneally injected with 50 mg/kg pentobarbital sodium after 60 minutes from the last administration, then conditions of falling asleep were observed. The disappearance of righting reflex was taken as the index of falling asleep, the latency of falling asleep was the duration from intraperitoneal injection of pentobarbital sodium to fall asleep, the sleeping time was from falling asleep to the disappearance of righting reflex. （3） Synergistic action in hypnosia by Yinao capsules with pentobarbital sodium of sub-threshold dosage： Eighty mice were randomly divided into 5 groups as above-mentioned with 16 mice in each group, and they were treated the same as above. They were intraperitoneally injected with 27 mg/kg pentobarbital sodium after 60 minutes from the last administration. Mice whose righting reflex disappeared for at least 1 minute within 30 minutes were taken as falling asleep, the number of sleeping animals in each group was recorded, and the rate of falling asleep was calculated. MAIN OUTCOME MEASURES： Synergistic action of Yinao capsules with pentobarbital sodium of threshold and sub-threshold dosages in hypnosia and sedation. RESULTS： All the 200 mice were involved in the analysis of results. （1） Influence of Yinao capsules on the spontaneous activity of mice： The times of spontaneous activities within 10 minutes in the large and middle dosage of Yinao capsules groups and Kangnaoshuai capsules group [（138.0±37.0）, （156.8±28.3）, （133.3±46.1） times] were obviously fewer than those in the control group [（204.3±61.3） times, P 〈 0.05- 0.01]. The arising times within 10 minutes in the middle and small dosages of Yinao capsules groups and Kangnaoshuai capsules group [（30.7 ± 18.3）, （26.5± 11.2）, （24.2±11.6） times] were obviously fewer than those in the control [（71.7±38.6） times, P 〈 0.01 ]. （2） Synergistic action in hypnosia by Yinao capsules with pentobarbital sodium of threshold dosage： The sleeping latencies in the large and middle dosages of Yinao capsules groups and Kangnaoshuai capsules group [（4.49±1.84）, （4.83±1.72）, （3.85± 1.94） minutes] were obviously shorter than that in the control group [（6.73 ±2.75） minutes, P 〈 0.05 - 0.01 ]. The sleeping time in the large, middle and small dosages of Yinao capsules groups and Kangnaoshuai capsules group [（89.0± 38.42）, （67.21 ±24.07）, （66.28±18.94）, （84.36±29.81） minutes] were obviously longer than that in the control group [（45.78±20.78） minutes, P 〈 0.05- 0.01]. （3） Synergistic action in hypnosia by Yinao capsules with pentobarbital sodium of sub-threshold dosage： The rates of falling asleep in the large and middle dosages of Yinao capsules groups were higher than that in the control group [56% （9/1 6）, 38% （6/1 6）, 6% （6/16）, P 〈 0.01, 0.05], whereas Kangnaoshuai capsules and small dosage of Yinao capsules had no synergistic action with pentobarbital sodium of sub-threshold dosage （P 〉 0.05）. CONCLUSION： Yinao capsules have synergistic action with pentobarbital sodium in hypnosia. Yinao capsules possess obvious sedative and hypnotic effects in a dosage-dependent manner.

Adeno-associated virus mediated endostatin gene therapy in combination with topoisomerase inhibitor effectively controls liver tumor in mouse model

AIM:rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer.However, a sustained and high protein delivery is required to achieve the desired therapeutic effects.We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model. METHODS:rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines.To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin,Western blotting and ELISA were performed.The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays. The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor,etoposide,were evaluated in a mouse liver tumor model. RESULTS:Topoisomerase inhibitors,including camptothecin and etoposide,were found to increase the endostatin exPression level in vitro.The over-expressed endostatin, as a result of pretreatment with a topoisomerase inhibitor, was also biologically active.In animal experiments,the combined therapy of topoisomerase inhibitor,etoposide with the rAAV-endostatin vector had the best tumor- suppressive effect and tumor foci were barely observed in livers of the treated mice.Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice.Finally,the mice treated With rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models. CONCLUSION:rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.

The Effect of calmodulin antagonist berbaminederivative-EBB on hepatoma in vitro and in vivo

OBJECTIVE: To evaluate the anti-hepatoma effect of Calmodulin antagonist 0 - 4-ethoxyl-butyl-Berbamine (EBB), one of the berbamine derivatives. METHODS: Monotetrazolium (MTT) method was used to analyze the effect of EBB on the proliferation and growth inhibition effect. Of a hepatoma cell line in vitro. A mouse hepatoma model was induced by injection of hepatoma cells (H22) in the abdominal cavity. The effect of EBB on survival at different concentrations as well as in combination with 5-FU were investigated in vivo. Flow cytometry analysis, dot blot hybridization, western blot, immunochemistry, enzyme-linked lectin assay (ELISA), trifluoperazine (TFP) and electron microscopic observation were used to study the effect of EBB on cell cycle process, P53 mRNA and protein levels, calmodulin content and ultrastractural changes of hepatoma cells. RESULTS: EBB exerts a very strong inhibitory effect on human hepatoma cell line 7402 and mouse hepatoma cell line H22 in vitro. The IC(50) value of EBB for the two cell lines are 3.312 microg/ml and 1.167 microg/ml, respectively. The sensitivity of H22 cells to 5-FU can be markedly enhanced: The IC(50) dosage of 5-Fu can be decreased from 0.75 microg/ml down to 0.15 microg/ml, when jointly administered with nontoxic dosages of EBB (IC(10)). In vivo, EBB can prolong the lifespan of mice with ascites H22 to more than three months. 64% of mice survived, while all animals in the control group died by the 18th day. When EBB (5 mg x kg(-1) x d(-1)) is jointly used with 5-FU (25 mg x ml(-1) x d(-1)), 73% of mice with ascites H22 survived, much higher than 27% in the 5-FU treated group. EBB can enhance the anti-hepatoma ability of 5-Fu treatment. EBB mechanism against hepatoma: P53 expression in the EBB treated group is substantially higher than that in the control group. EBB increased the translation of P53. As a calmodulin antagonist, EBB decreases amount of the CaM in hepatoma cells and blocked the hepatoma cell proliferation cycle at the G(2)M phase. Before the G(0)/G(1) phase, a diploid peak and apoptic cells in the treated groups were observed. CONCLUSIONS: The CaM antagonist, EBB, has a strong anti-hepatoma effect and enhances the effect of 5-FU, induces hepatoma cell to apoptosis, promotes the P53 protein expression and decreases the amount of CaM in the cytoplasm. All these results demonstrate that EBB is a new and potentially useful drug against hepatoma and should be researched further.