| Objective:To optimize the preparation technology of protoporphyrin disodium (NAPP) from non-anticoagulant porcine blood. On the basis of NAPP was obtained, then to study the hepatoprotective effect and it's mechanisms of NAPP on carbon tetrachloride (CCl4) induced acute hepatic injury in mice and the inhibitory effect of NAPP on human hepatoma carcinoma cell strain of SMMC-7721 cells in vitro, and to study the anti-hepatitis B virus (HBV) effect of NAPP in vitro besides.Methods:Heme was prepared from non-anticoagulant porcine blood first, then NAPP was directly prepared from the heme through the intermediate of protoporphyrin-dimethyleser. Ultraviolet-visible absorption spectrum and infrared spectrum were applied for qualitative test and ultraviolet spectrophotometry was utilized for quantitative detection of heme and NAPP, and the purity quotient was calculated. Two methods of monofactorial test and orthogonal experiment were used to optimize the extration and preparation technology of heme and NAPP to determine the optimal parameters.60 ICR mice were randomly divided into six groups of normal control group, CCl4 model group, bifendate pills group, and NAPP low, medium, high dose groups with 10 animals in each group. Bifendate pills (200 mg/kg) or different dosages (30,60,120 mg/kg) of NAPP were injected to each treatment group by intragastric administration for 10 days and then a single dose of CCl4 at 60 mg/kg was given by intraperitoneal injection to establish acute hepatic injury model.16 hours later, blood was collected by picking eyeballs to detect the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. Liver was taken out by laparotomy and weighed to calculate liver index (LI), and liver homogenate was prepared to determine the activities of catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA). The observation of liver histopathological change was made at the same time. The maximum atoxic concentration (TC0) of NAPP was determined by drug toxicity test on normal human hepatocyte strain of QSG-7701 cells. Took TC0 as the initial concentration, five different concentrations of NAPP were diluted by 10 times gradient dilution, and applied to treat the SMMC-7721 cells cultured in vitro. MTT assay was used for evaluating the proliferation inhibitory effect of NAPP. Morphology observation of SMMC-7721 cells was undertaken via the inversion optical microscope and inversion fluorescence microscope after Hoechst 33258 staining. Apoptotic cells were counted and apoptosis index (AI) was calculated besides. Flow cytometry was applied to detect the cell cycle distribution and to measure apoptosis and necrosis rates of SMMC-7721 cells. HepG2.2.15 cells were administrated with different concentrations of NAPP which were cultured in vitro for four days. The MTT assay, time-resolved fluoreimmunoassay (TRFIA) and enzyme-linked immunosorbent assay (ELISA) were applied to explore the cytotoxic effect of NAPP on HepG2.2.15 cells and the inhibitory effects of NAPP on HBsAg, HBeAg and Pre-S1Ag secreted by HepG2.2.15 cells. The real-time fluorescence quantitative PCR (FQ-PCR) was employed to evaluate the inhibitory effects of NAPP on the content of HBV-DNA in the cell culture supernatant solution of HepG2.2.15 cells.Results:The preparation technology of NAPP from non-anticoagulant porcine blood was optimized. On the average,10.6 g heme could be harvested from per kilogram non-anticoagulant porcine blood and 3.46 g NAPP could be obtained from per 5 g heme. The purity quotients of the extracted heme and the prepared NAPP was 90.8% and 88.8% respectively. Through monofactorial test and orthogonal experiment, the determined optimum extraction technology of heme which was extracted from non-anticoagulant porcine blood was that homogenating for 5 min, adding equal volume double-distilled water, Vhydrochloric acid acetone solution Vhemolysate at 5:1 and agitating/extracting for 10 min, and the optimal preparation technology of NAPP which was prepared from heme was as followed:adding 70 mL methanol in estrification, adding 80 mL sodium hydroxide-methanol solution in saponification, and heating and distilling at 90℃for 2.0 h. Compared with CCl4 model group, the liver indices and the activities of ALT and AST in serum of each treatment group were decreased significantly at different level (P<0.05,P<0.01), the activities of CAT, GSH-Px and SOD in liver homogenate of each treatment group were increased dramatically at different extent (P<0.05, P<0.01), and the content of MDA of each treatment group in liver homogenate was reduced remarkably at different degree (P<0.05,P<0.01). The pathological sections showed that hepatic injury of each treatment gourp was relieved to different extent clearly, and the slightest gourp was NAPP high dose group. The proliferation of SMMC-7721 cells was inhibited dramatically after the treatment of different concentrations of NAPP, and the OD values were decreased significantly (P<0.05 or P<0.01). The inhibition rate was depended on the concentration of NAPP and the treatment time to some extent. These were visible in the optical microscope that cell number reduced obviously, cell density was thinning markedly, the appearance of SMMC-7721 cells was changed evidently, many conjunctions between cells disappeared, and cells became round and corrugativus or cellular shape turned slender like fusiform shape. The SMMC-7721 cell nucleus showed the typical changes of apoptosis morphology in fluorescence microscope, and AI increased significantly (P<0.05 or P<0.01). Though the detection of flow cytometry, apoptosis rates of SMMC-7721 cells was raised at different level, and the cell cycle was arrested at G2/M phase. The MTT results showed that NAPP had no obvious cytotoxic effect in the concentrations from 0.01μg/mL to 100μg/mL and the TC50 of NAPP was 4654μg/mL. There were some inhibitory effects to a certain extent of NAPP in the concentrations from 0.01μg/mL to 100μg/mL on HBsAg, HBeAg, Pre-SlAg and HBV-DNA in the cell culture supernatant solution of HepG2.2.15 cells. The therapeutic indices (TI) of NAPP on HBsAg, HBeAg, Pre-S1Ag and HBV-DNA were 119,27,19 and 83 respectively.Conclusions:The optimized preparation technology of NAPP from non-anti-coagulant porcine blood is an ideal approach with simple operation, abundant raw material, short preparation cycle, low cost and high yield. The preparation technology in this study is practicable and it provides a practical technical route for taking full advantage of the rich porcine blood resources in our country. On the one hand, NAPP has a good protective effect on the acute hepatic injury in mice induced by CCl4, and the mechanism is probably related with the anti-lipid-peroxidation effect and the prevention of the decrease of liver anti-oxidative enzyme activities. On the other hand, NAPP also can inhibit the proliferation of human hepatoma carcinoma cell strain of SMMC-7721 cells and promote its apoptosis. In addition, NAPP also has no obvious cytotoxic effect on HepG2.2.15 cells and shows an inhibitory effect on HBV in vitro to some extent. |
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