[Identification of effective components from Naoxintong Capsules intestinal absorption liquid in inhibiting ADP-induced platelet aggregation].

Intestinal absorption liquid was prepared by using everted intestinal sac method; meanwhile, its recipes were decomposed or restructured. Platelet aggregation activity was examined by biochemical tests and a microplate reader. One or more kinds of Chinese medicines which displayed inhibiting activity in Naoxintong Capsules were screened through separation and combination of prescription. The results showed that Naoxintong Capsules could inhibit ADP-induced platelet aggregation. Recipe decomposition and restructuring results showed that Salviae Miltiorrhizae Radix et Rhizoma, Paeoniae Radix Rubra, Cinnamomi Ramulus and Hirudo were the main effective medicines in inhibiting platelet aggregation. Furthermore, Cinnamomi Ramulus played a vital role in inhibiting activity among those four kinds of Chinese medicines. Coumarin derived from intestinal absorption liquid of Cinnamomi Ramulus had inhibiting activity in the range of 50-200 µmol·L⁻¹, and other ingredients such as cinnamyl alcohol and cinnamaldehyde also had inhibiting activities. In conclusion, Salviae Miltiorrhizae Radix et Rhizoma, Paeoniae Radix Rubra, Cinnamomi Ramulus and Hirudo are the main components for inhibiting ADP-induced platelet aggregation, and Cinnamomi Ramulus has the most strongest inhibiting activity in Naoxintong Capsules.

[Analysis on composition regularity of carthami flos-containing traditional Chinese medicines].

To analyze the composition regularity of Carthami Flos-containing prescriptions of the Drug Standards of Ministry of Health of People's Republic of China-Traditional Chinese Medicine Preparations (the ministerial standards for Traditional Chinese Medicine) based on the traditional Chinese medicine inheritance support system (TCMISS, RZDZ No. 0389952). Efforts were made to identify 331 prescriptions containing Carthami Flos and summarize 16 attending functions and 10 commonly used drug combinations. Three commonly used drug combinations were selected for an in-depth analysis on Carthami Flos's combined administration regularity. Based on Carthami Flos's attending functions, its effects in paralysis, traumatic injuries and dysmenorrheal were compared to analyze Carthami Flos's core drug combinations for treating different diseases. The regularity of clinical administration and the characteristics of commonly used drug combinations were summarized to provide reference for Carthami Flos's clinical application and new ideas for new drug R&D. Carthami Flos prescriptions was mainly used to treat blood stasis and pain and mostly combined with drugs that could activate blood, promote the circulation of qi and dispel pathogenic wind to treat Qi-stagnation and blood stasis caused by various pathogenic factors such as wind, cold and dampness.

[Study on absorption location of four components from Naoxintong capsule].

OBJECTIVE: To study the absorption characteristics of four components from Naoxintong capsule in intestines. METHOD: In vitro everted gut sac method was adopted for preparing the intestinal absorption solution of Naoxintong capsule. UPLC was used to detect the content of chemical components in different intestinal segments, and comparing the results with the absorption of chemical components of Naoxintong capsule in each intestinal segment. The time-accumulative absorption curve was drawn to observe the changes in the accumulative absorption concentration with time. RESULT: Four ingredients of Naoxintong capsule can be detected in intestinal absorption solution, they are ferulic acid, paeoniflorin, salvianolic acid B and hydroxysafflor yellow A. Specifically, the accumulative absorption concentrations of ferulic acid, salvianolic acid B and paeoniflorin in ileum and rear jejunum segments were higher than that in front and middle jejunum segments; the absorption of ferulic acid, paeoniflorin, salvianolic acid B and hydroxysafflor yellow A did not reach saturated conditions in 3 hours. CONCLUSION: Ferulic acid, paeoniflorin, salvianolic acid B and hydroxysafflor yellow A are absorbed in the whole intestine. Ferulic acid, paeoniflorin and salvianolic acid B may be absorbed in specific segments.

Therapeutic effects of Chinese medicine Di-Long (Pheretima vulgaris) on rheumatoid arthritis through inhibiting NF-κB activation and regulating Th1/Th2 balance.

Chinese medicine Di-Long, the dried body of Pheretima vulgaris (Chen) has been used for the treatment of joint inflammation, arthralgia and numbness of limbs for many years. This study was to investigate the anti-rheumatoid arthritis (RA) effects of Di-Long and to explore its possible mechanisms. The identification and quantification of representative components in Di-Long extracts (DL) were carried out by HPLC analysis. The anti-RA effects and mechanisms of DL were studied in CIA mice, RAW 264.7 macrophages and spleen T lymphocytes. The Th1/Th2 cell ratio in CIA mice spleens were determined by Flow cytometry. The cytokine levels were determined by ELISA method. The expressions of p-NF-κB p65 in ankle joints of CIA mice were detected by Immunohistochemistry analysis. The phosphorylation of NF-κB signaling pathway in RAW 264.7 macrophages and expressions of T-bet and GATA-3 in CIA mice spleens were determined by Western blots. The treatment with DL significantly decreased the paw thickness, arthritis scores and inflammatory cells infiltration in CIA mice. The TNF-α, IL-6 concentrations in both mice serum and macrophages secretion were markedly reduced with the treatment of DL, as well as the phosphorylation of NF-κB pathway. DL inhibited the expressions of T-bet and GATA-3 and decreased Th1/Th2 cells ratio in CIA mice spleens. DL reduced IFN-γ, IL-2 levels in mice serum and spleen T lymphocytes, and increased IL-4 levels in CIA mice serum. Chinese medicine Di-Long have significant anti-RA effects. The mechanisms might be inhibiting the activation of NF-κB signaling pathway and regulating the balance of Th1/Th2 cells.

[Clinical efficacy and action mechanism of mild moxibustion combined with salt-separated moxibustion for gastrointestinal response in breast-cancer chemotherapy patients].

OBJECTIVE: To evaluate the clinical efficacy and partial action mechanism of mild moxibustion combined with salt-separated moxibustion for gastrointestinal discomfort caused by chemotherapy for breast cancer. METHODS: A total of 48 patients were randomly divided into an observation group and a control group, 24 cases in each group. The patients in the control group were treated with intravenous infusion of tropisetron hydrochloride (5 mg), once a day for three days; the patients in the observation group were additionally treated with mild moxibustion at Zusanli (ST 36), Zhongwan (CV 12), Guanyuan (CV 4), Qihai (CV 6) and salt-separated moxibustion at Shenque (CV 8), 15 min per treatment, once a day for 7 days. Before treatment and on the 7th day of chemotherapy, the levels of pepsinogenⅠ(PGⅠ), pepsinogenⅡ (PGⅡ), the ratio of PGⅠto PGⅡ (PGR) and gastrin 17 (G-17) in serum were measured. Before treatment and on the 3rd, 5th, 7th day of chemotherapy, the gastrointestinal reactions (nausea, vomiting, constipation, diarrhea) were compared between the two groups. RESULTS: On the 7th day of chemotherapy, the serum levels of PGⅠ, PGⅡand G-17 in the observation group were lower than those in the control group (P<0.05), and the difference in the level of PGR in serum between the observation group and the control group was not statistically significant (P>0.05). The total scores of nausea, vomiting and constipation during chemotherapy in the observation group were significantly lower than those in the control group (all P<0.05). CONCLUSION: The mild moxibustion combined with salt-separated moxibustion could effectively improve the symptoms of nausea, vomiting and constipation caused by chemotherapy in patients with breast cancer, and its mechanism may be related to the down-regulation of the levels of PGⅠ, PGⅡ and G-17 in serum.

Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of Goose parvovirus in vivo.

BACKGROUND: Goose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, it severely affects geese production worldwide. The objective of this study was to develop a fluorescent quantitative real-time polymerase chain reaction (PCR) (FQ-PCR) assay for fast and accurate quantification of GPV DNA in infected goslings, which can aid in the understanding of the regular distribution pattern and the nosogenesis of GPV in vivo. RESULTS: The detection limit of the assay was 2.8 x 10(1) standard DNA copies, with a sensitivity of 3 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intraassay and interassay coefficients of variation. CONCLUSION: The high sensitivity, specificity, simplicity, and reproducibility of the GPV fluorogenic PCR assay, combined with a high throughput, make this method suitable for a broad spectrum of GPV etiology-related applications.

Identification and characterization of the duck enteritis virus UL51 gene.

Compared to the UL51 gene of other alphaherpesviruses, the duck enteritis virus (DEV) UL51 gene contains ten conserved motifs and has a close evolutionary relationship with members of the genus Mardivirus. The DEV UL51 gene product was identified using a rabbit polyclonal antiserum raised against a 6-His-UL51 fusion protein expressed in Escherichia coli as a 34-kDa protein. Western blotting and RT-(real time) PCR analysis of DEV-infected cells showed that the protein was produced at the late stage of infection and that its production was highly dependent on viral DNA synthesis, suggesting that the gene should be classified as gamma2 class. Analysis of extracellular virions revealed that the protein was a component of extracellular mature DEV virions. Indirect immunofluorescence studies localized most of the protein to the juxtanuclear region. These results will provide a basis for further functional analysis of the gene.

Viral load in 1-day-old ducklings acutely infected with duck hepatitis B virus by different doses and routes of inoculation.

In order to define clearly the conditions leading to the outcome of acute duck hepatitis B virus (DHBV) infection, 1-day-old Pekin ducklings were infected with DHBV by different routes and given different doses of inoculum. Groups of 24 ducklings were inoculated either intravenously via the vena cruralis, or intraperitoneally with pooled serum containing either 1.6 x 10(7) or 1.6 x 10(4) DHBV genomes. One control duck from each group was inoculated with an equal volume of normal duck serum. A sensitive and reproducible real-time polymerase chain reaction assay based on TaqMan technology was developed for the detection and quantitation of DHBV DNA in the serum and liver. DHBAg was observed in the hepatocytes by immunohistochemistry. Histological changes in the liver tissue were also observed. The results demonstrate that ducklings at each time point and in all groups developed detectable viraemia. In each group, DHBV DNA in the liver was at a lower level than in serum and the peak DNA titre was found in serum earlier than in the liver. In the low-dose groups it was always at a lower level than in the high-dose groups. The DHBV replication levels appeared to be directly related to the number of DHBAg-positive hepatocytes. The variation trends of DHBAg-positive hepatocytes were similar in the high-dose groups. Histological changes were associated with liver viral DNA levels. We suggest that this dose and route of inoculation can be used as a model to study acute DHBV infections.

[Studies on the propagation characteristics of duck plague virulent virus in duck embryo fibroblasts].

The propagation characteristics of virulent duck plague virus (DPV) in duck embryo fibroblast (DEF) were studied by the method of light microscopy observation of DEF cell culture monolayer, electron microscopy observation of infected DEF cell culture, real-time PCR detecting virus propagation. The results demonstrated that on duck embryo fibroblast a number of plaques were formed by DPV 42 h postinfection. Electron microscopy of the ultrathin section of infected duck embryo fibroblasts demonstrated that the nucleic acid of DPV was round in shape with diameter of 35-45 nm and was often in a cluster in the nucleus of DEF. The nucleocapsid of DPV was round in shape with diameter of 90-100 nm and could be observed both in nucleus and cytoplasm of DEF. The mature DPV which had the structures of envelop and tegument was spherical in shape with diameter of 150-300 nm and was located in cytoplasmic vacuoles. DPV penetrated the DEF cell membrane by direct fusion between the viral envelop and the plasma membrane. Progeny viral nucleic acid was produced in the nucleus and the assembled nucleocapsids obtained the structure of tegument in the cytoplasm and obtained the structure of envelop by budding into the cytoplasmic vesicles. The mature DPV particles were released out of the cell through exocytosis of the cytoplasmic vesicles. Detection of DPV by real-time PCR demonstrated that virus in DEF began its obvious propagation 10 h postinfection and virus amount tended to increase until 30 h postinfection. DPV began to be released into the supernatant 22 h postinfection and the DPV amount peaked 50 h postinfection, when the virus content in DEF and supernatant both underwent approximately 10(3) fold increase. DPV mainly existed in the DEF and the virus content in DEF was 10(2)-10(3) fold than the supernatant.

[Studies on porcine reproductive and respiratory syndrome virus morphogenesis in Marc-145 cell and the ultrastructural changes of the infected cells].

Electron microscopy was employed for ultrastructural observation of Marc-145 cells infected with porcine reproductive and respiratory syndrome virus (PRRSV) SC1 strain and studied the virus morphogenesis in infected cells. The results demonstrated that PRRSV was spherical and enveloped. The virion is 45-65 nm in diameter and its nucleocapsid was approximately 25-30 nm. PRRSV entered Marc-145 cells by endocytosis, and replicated in the cytoplasm. The mature viruses were released from infected cells by budding or exocytosis. The main ultrastructural changes of the infected cells were as follows: increased number of cytoplasmic vacuoles, dilated endoplasmic reticulum, mitochondria underwent hyperplasia with its ridges swollen, sloughed, and eventually vacuolated. Typical apoptosis was also observed in the infected Marc-145 cells, which included microvilli sloughing off the cell, appearance of apoptotic bodies and cell fragmentation.