AIM To explore the let-7a-mediated anti-cancer effect of Yangzheng Sanjie decoction

AIM To explore the let-7a-mediated anti-cancer effect of Yangzheng Sanjie decoction (YZSJD) in gastric malignancy (GC) cells. g. All the medicinal materials used to prepare formulae were purchased from Kangmei Pharmaceutical Co., Ltd. (Guangzhou, China) and were recognized by two pharmacognosy experts. The natural herbs (100 g) were soaked in distilled water (1000 mL) and boiled for 30 min twice, and then the extracts were filtered, mixed and centrifuged. The upper layer was concentrated SB-277011 to 0.9 g primitive extract per millilitre in a rotary evaporator (SENCO, China) and stored at -20 C for future use. To establish quality control requirements for YZSJD, the crude draw out preparation and subsequent high-performance liquid chromatography (HPLC) determination were repeated ten occasions. The similarity of the HPLC fingerprints of 10 batches of YZSJD samples was assessed using the Computer-Aided Similarity Evaluation System for Chromatographic Fingerprint of TCM (Chinese Pharmacopoeia Commission rate, version 2004A). Preparation of YCS Forty male SD rats (SPF grade, weighing 250 20 g) were purchased from the laboratory animal centre of Southern Medical University or college. The animals were acclimatized to laboratory conditions (23 C, 12 h/12 h light/dark, 50% humidity, access to food and water) for two weeks prior to experimentation. Subsequently, the rats were randomly and equally divided into a YZSJD group and a Control group. Animals in the YZSJD group were gavaged with an comparative dose of YZSJD (9 g/kg), while those in the Control group were given the same volume of normal saline once daily. On the seventh day, blood was drawn from the abdominal aorta 1 h after feeding, and the serum was isolated. Each group of sera was mixed, sterilized by filtration and Mmp9 inactivated at 56 C before being stored at -20 C. These experiments were approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital of Guangzhou University or college of Chinese Medicine, and efforts were made to minimize animal suffering. Cell culture The human GC cell lines AGS and HS-746T were purchased from the American Type Culture SB-277011 Collection (Manassas, VA, United Says), the cell lines MKN-45 and SGC-7901 were obtained from the Type Culture Collection of Chinese Academy SB-277011 of Sciences (Shanghai, China), and the human immortalized gastric mucosa cell collection GES-1 was provided by SB-277011 the Beijing Institute for Malignancy Research. The cells were cultured in RPMI-1640 medium (HyClone, United Says) supplemented with 10% foetal bovine serum (HyClone, United Says) and maintained in a humidified incubator with 5% CO2 at 37 C. Cell groups and cell treatments AGS and HS-746T cells were hanging and seeded in 96-well dishes at a density of 6000 cells/well or in 6-well dishes at a density of 20000 cells/well. The cells were divided into a YZSJD group and a Control group. After 12 h of culture, the cells in the YZSJD group were treated with 10% YCS, while those in the SB-277011 Control group were treated with 10% normal rat serum. Cell proliferation assay The effects of YZSJD on AGS and HS-746T cell proliferation were estimated using the Cell Counting Kit-8 (CCK-8) assay (Jingxin, China). After 24, 48 or 72 h of incubation with YCS or normal rat serum, 10 T of CCK-8 answer was added to each well of a 96-well plate, followed by a 2-h incubation in the dark. Cell proliferation was evaluated by the absorbance of each well at 450 nm, which was assessed with a VICTOR Times5 Multilabel Plate Reader (PerkinElmer, United Says). Cell apoptosis assay The effects of YZSJD on apoptosis were decided by circulation cytometry using an Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, United Says). After 48 h of incubation with YCS or normal rat serum, cells in 6-well dishes were gathered and resuspended in 1 binding buffer at a concentration of 1 106 cells/mL. Then, 5 T of Annexin V-FITC and 10 T of propidium iodide were added to 100 T of the cell suspension. The cells were incubated for 15 min in the dark before 400 T of 1 binding buffer was added. The samples were analysed by circulation cytometry within 1 h. MiRNA PCR array Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, United Says) following the manufacturers instructions. Contaminating DNA in the RNA preparations was removed with DNase I, and the RNA was purified using an RNeasy MinElute Clean-up Kit (Qiagen, Germany). The.

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