OBJECTIVE:To find the active ingredient of on antithrombotic chuanxiong rhizoma using computer aided drug de-sign. METHODS:Usingthrombosisas keyword,thrombosis related proteins were searched and screened in therapeutic target da-tabase;target proteins'three-dimensional structure were downloaded in protein database,then the protein preparing tool were used to determine the coordinates of the active area center. PyRx software and Discovery Studio Visualizer were used to match the 247 small molecules of chuanxiong rhizoma with target protein that downloaded from Taiwan traditional Chinese medicine database. The active molecules were screened and binding force was analyzed. RESULTS:Active molecules of neochlorogenic acid,1-H-benz-imidazole-2-amine,3,8-dihydrodiligustilide,chuanxiongterpene were selected by blinding energy,and there were high binding ac-tivity among these active molecules,thrombin,antithrombinⅢ,coagulation factorⅩa and thrombomodulin,and the binding ener-gy were -6.1,-4.5,-7.7,-8.6 kJ/mol. Analysis results showed van Edward force and electrostatic interactions played an im-portant role in their respective docking. CONCLUSIONS:Neochlorogenic acid,1-H-benzimidazole-2-amine,3,8-dihydrodiligusti-lide,chuanxiongterpene may be the antithrombotic activity ingredients of Chuanxiong rhizoma.

Objective To assess the therapeutic effect of plum-blossom needle tapping combined with topical imiquimod immunotherapy on cutaneous squamous cell carcinoma (SCC) in SKH-1 mice,and to explore the immunological mechanism.Methods A total of 40 SKH-1 mice with ultraviolet light-induced cutaneous SCC were randomly and equally divided into 4 groups:control group receiving no treatment,plum-blossom needle group receiving plum-blossom needle tapping on all the tumors once a day,imiquimod group topically treated with imiquimod 5％ cream at a dose of 1.2 g/kg once a day,combination group firstly treated with plum-blossom needle tapping on all the tumors,and after the stop of bleeding topically treated with imiquimod 5％ cream at the same dose as the imiquimod group once a day.All the mice were treated for 30 days.Morphological changes of tumors in all groups were photographed and recorded every day.The tumor size was measured once every three days,and changes of total tumor volume and survival rate of the mice were compared among the 4 groups.At the end of treatment,tunor tissues were resected,and histopathological changes were compared among the 4 groups.Real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to measure the mRNA expression of interferon-α (IFN-α),IFN-β,interleukin-1β (IL-1β),tumor necrosis factor-α (TNF-o) and IL-12 in tumor tissues.Results In the combination group,tumors on the back of mice grew slowly,and some even regressed.However,tumors grew fast in the control group,plum-blossom needle group and imiquimod group,and grew more slowly in the plum-blossom needle group and imiquimod group than in the control group.Before the treatment,there was no significant difference in the total tumor volume among the 4 groups (F =0.90,P ＞ 0.05).After 24-day treatment,the total tumor volume significantly differed among the 4 groups (F =5.16,P ＜ 0.05).The LSD-t test showed that the total tumor volume significantly decreased in the combination group compared with the control group (P ＜ 0.01),but no significant difference was observed among the other groups (P ＞ 0.05).Log-rank test revealed that survival curves significantly differed among the 4 groups (x2 =8.32,P ＜ 0.05).The survival rate was significantly higher in the combination group than in the control group (x2 =4.62,P =0.03),but did not differ between the plum-blossom needle group or imiquimod group and the control group or combination group (all P ＞ 0.05).Histopathological examination showed atypical cells arranged closely,a large number of tumor cells and some keratin pearls in the control group and plumblossom needle group,few dead tumor cells in the imiquimod group,and plenty of dead tumor cells,mild nuclear atypia and increased keratinization in the combination group.qRT-PCR revealed that the relative mRNA expression levels of IFN-α,IFN-β,IL-12,IL-1β and TNF-α were significantly higher in the combination group than those in the control group,plum-blossom needle group and imiquimod group (P ＜ 0.05).The imiquimod group showed significantly higher mRNA expression of IL-1β than the control group (P ＜ 0.01),but no significant differences were observed among the other groups (P ＞ 0.05).Conclusion Plum-blossom needle tapping can effectively enhance the anti-SCC activity and immunological effects of imiquimod in SKH-1 mice.

Objective:To establish SKH-1 mouse models of subcutaneously transplanted B16F10 melanoma and of lung-metastasized B16F10 melanoma.Methods:Seven SKH-1 mice and seven C57BL/6 mice were subcutaneously inoculated with 5 × 10 6 B16F10 cells on the back, and the survival duration of mice was observed and recorded. The tumor volume was measured by using a precision vernier caliper every 3 days. Mice were considered as ethically dead when the tumor length was more than 15 mm, or when cachexia or ulceration occurred. In addition, 5 SKH-1 mice were injected with 5 × 10 6 B16F10 cells via the tail vein, and the activity, nutritional status and survival duration of the mice were observed. The mice were sacrificed after the observation, and the lungs were weighed after dissection. Histopathological examination was performed on the lungs of all mice. All the experiments were repeated 3 times. Results:On day 6 after the subcutaneous inoculation, black spots appeared at the skin inoculation site in the SKH-1 mice, and gradually developed into round black nodules, then progressed into ulcerative and hemorrhagic tumors until the death of mice, and the time to ethical death ranged from 20 to 33 days. In the C57BL/6 mice, small black nodules measuring 2 - 3 mm in length appeared at the skin inoculation site on day 4 after the subcutaneous inoculation, and the time to ethical death ranged from 12 to 18 days. The survival duration of SKH-1 mice was 26.57 ± 4.03, 27.86 ± 4.53, and 27.43 ± 5.32 days in the 3 times of experiments respectively, and there was no significant difference ( F = 0.14, P = 0.87) ; on day 27 after the subcutaneous inoculation, the tumor volume was 1 367.9 ± 150.2, 1 452.0 ± 50.1, and 1 490.3 ± 69.0 mm 3 in the 3 times of experiments respectively, and there was also no significant difference ( F = 0.92, P = 0.46). SKH-1 mice had shown decreased activity and anorexia since day 25 after tail vein injections of B16F10 cells, and dullness, emaciation, ascites and death had been observed since day 31, and the time to ethical death ranged from 31 to 40 days; multiple black nodules were observed in the lung after dissection, and the survival duration was 34.20 ± 2.58, 36.40 ± 2.60, and 34.80 ± 2.38 days in the 3 times of experiments respectively, which did not differ among the 3 times of experiments ( F = 1.01, P = 0.39) ; there was also no significant difference in the lung weight among the 3 times of experiments (156.1 ± 18.5, 164.0 ± 19.6, and 172.0 ± 17.2 mg, respectively, F = 3.18, P = 0.72). All the mice developed tumors, and histopathological examinations of subcutaneous tumor masses and lung tissues confirmed the diagnosis of melanoma. Conclusion:In this experiment, the SKH-1 mouse models of subcutaneously transplanted B16F10 melanoma and of lung-metastasized B16F10 melanoma were successfully established, which showed high tumor formation rates and favorable stability in tumor formation.