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Objective To optimize the use of non-anticoagulant pig blood preparation protoporphyrin sodium (NAPP) process NAPP acute liver injury induced by carbon tetrachloride (CCl4) protective effect and its mechanism in vitro and on people, on the basis of research role in the inhibition of hepatoma cell line SMMC-7721 cells and in vitro anti-hepatitis B virus (HBV). The first non-anticoagulant pig blood as raw material extracted heme by the heme protoporphyrin dimethyl directly obtained NAPP; using UV - visible absorption and infrared spectra of heme extracted and prepared NAPP qualitative detection by UV spectrophotometric detection on both quantitative and calculated purity; through the single factor experiment and orthogonal experiment method to optimize the use of non-anticoagulated the pig blood preparation NAPP process, to determine the optimum process parameters. 60 ICR mice were randomly divided into normal control group, CCl4 model group, DDB Group, NAPP low, in the high-dose group (n = 10); each treatment group daily oral administration Bifendate (200 NAPP, of mg / kg) or different doses (30,60,120 mg / kg) for 10 d after a single intraperitoneal injection of CCl4 (60 mg / kg) of acute liver injury model; 16 h after the mice in each group eyeballs to take blood serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity; laparotomy liver, weighing calculated liver index; prepare liver homogenates Determination of catalase enzyme (CAT), glutathione peroxidase ( GSH-Px), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content, and to observe the changes in liver histopathology. QSG-7701 cells in normal human liver cell line drug toxicity experimental screening NAPP normal human hepatocytes without apparent toxicity The maximum non-toxic concentration (TC0); TC0 is the initial concentration, 10-fold serial dilutions into five different NAPP concentration, the role of in vitro cultured human hepatoma cell line SMMC-7721 cells by MTT assay the NAPP inhibited the proliferation of SMMC-7721 cells; morphology was observed by an inverted microscope and Hoechst 33258 staining inverted fluorescence microscope counts apoptosis cells, and calculated the apoptotic index (AI); measured by flow cytometry cell cycle distribution and apoptosis, necrosis rate of SMMC-7721 cells. HepG2.2.15 cells cultured in vitro with different concentrations of NAPP role 4d, MTT, time-resolved fluorescence immunoassay (TRFIA)-linked immunosorbent assay (ELISA) to explore NAPP HepG2.2.15 cell cytotoxicity secretion HBsAg.HBeAg. before S1 antigen (Pre-S1Ag); HepG2.2.15 cell culture supernatant HBV-DNA replication inhibition by real-time quantitative PCR (FQ-PCR) detection NAPP. Results to optimize the process of non-anticoagulant pig's blood as a raw material preparation NAPP the average, 1 kg nonanticoagulant pig blood extract 10.6 g heme 5g per heme average was 3.46 gNAPP; detection, extraction of heme a purity of 90.8% and the preparation of NAPP purity of 88.8%; extract heme single factor test and orthogonal test, to determine the best extraction process: homogenate 5 min homogenate after adding an equal volume of double distilled water, the V hydrochloric acetone: V dissolved blood = 5:1, and stirring the extract for 10 min; prepared NAPP, the determined optimum preparation process is: an amount of 70 mL methanol esterification, saponification, the amount of methanol solution of sodium hydroxide of 80 mL , 90 ℃ heated under reflux for 2.0h. Compared with CCl4 group, reduced to varying degrees in each treatment group mice liver index, serum ALT and AST activity (P lt; 0.05, P lt; 0.01), liver homogenates CAT, GSH-Px, SOD activity varying degrees liter high (P lt; 0.05, P lt; 0.01), and MDA content in varying degrees to reduce (P lt; 0.05, P lt; 0.01); biopsy also showed that, NAPP treatment group liver injury in mice varying degrees to alleviate, including high-dose lightest group degree of liver injury. Different after the concentration NAPP role of SMMC-7721 cell proliferation was significantly suppressed OD value was significantly lower (P lt; 0.05 or P lt; 0.01), and the inhibition rate was a certain level of concentration and time dependent; under an optical microscope see the cell number was significantly reduced cell density was significantly thinning undergone significant changes in cell morphology, the disappearance of many connections between cells, cell rounding, shrinkage or see cell shape change long fusiform; cells showed a typical fluorescence microscope morphological changes of apoptosis, AI was significantly higher (P lt; 0.05 or P lt; 0.01); visible SMMC-7721 cell apoptosis rate increased to varying degrees by flow cytometry, cell cycle arrest at G2 / M phase. NAPP in the concentration range of 0.01 ~ 100μg/mL HepG2.2.15 cells without significant cytotoxicity, the the half toxic concentration (TC50) 4654μg/mL; NAPP in 0.01-100μg/mL concentration range within HepG2.2.15 cell culture supernatant HBsAg, HBeAg and the Pre-S1Ag and of HBV-DNA have a certain inhibition and the four therapeutic index (TI) 119,27,19 and 83, respectively. Conclusion to optimize the process from non-anticoagulated the pig blood preparation NAPP, the preparation process is feasible, is simple, easy to get raw materials, preparation of a short cycle, low-cost, high yield, it is an ideal preparation NAPP method for the make full use of our rich pig's blood resources provide practical technical route. The one hand, NAPP may be reduced by blocking the activity of antioxidant enzymes and anti-lipid peroxidation way has a significant protective effect on CCl4-induced acute liver injury in mice. On the other hand, NAPP inhibit proliferation and promote apoptosis of human hepatoma cell line SMMC-7721 cells. In addition, NAPP also has some anti-HBV effects in vitro. [25] see [80] [27] Table
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