The Influences Of Modified Bugan Powder Medicated Serum On The Mitochondrial Energy Metabolism In Injured Hepatic Cells

Objectives:To provide experimental basis for the clinical application of Bugan Powder, studying on the injury of human normal hepatic cells LO2 cultured in serum from hepatitis-B cirrhosis patients and the protective effects of Bugan Powder medicated serum to hepatic cells as well as its mitochondrial mechanism systematically, by researching the mitochondrial energy metabolism via starting from peripheral benzodiazepine receptor (PBR) observing the functional hepatic cells injury, apoptosis rate, mitochondrial transmembrane potential (MTP), calcium concentration within cells, and mitochondrial regulatory protein, for conducting the study in layers of "efficacy-mechanism-target".Methods:1. LO2, subjected to subculture, were put to 3 groups stimulating environment of liver cirrhosis after hepatitis B by composing the nutrient solution in different proportions (in volume) of serum from patients and that from fetal bovin:5% and 10%,10% and 10%, 10% and 5%. Optimal serum concentration for culturing were selected by MTT assay at 8h, 12h and 24 for determining hepatic cell proliferation.2. After determining the optimal serum concentration,3 groups were divided for the study: normal group (cultured in 10% FBS contained nutrient solution), blank group (contained no hepatic cells, subjected to 10% patient serum and 5% FBS), and model group (cultured in 10% patient serum and 5% FBS). LO2 cells morphological changes in optical microscope, qualitative measurement for HBV-M, quantitative detection of liver cell function were observed for assessment for the in vitro model of hepatic cells injury.3. After confirming the model, the experiment were conducted in 6 groups:normal group, model group, control group (GSH), high, medium, low doses groups of Bugan Powder. The concentration of GSH was set to 100μg/ml according to literatures. The volume proportion of Bugan Powder medicated serum in the nutrient solutions were respectively 20%,10% and 5%. Indexes were observed at 24h and 48h, as follows:(1) morphological changes of LO2 by optical microscope, (2) hepatic cell function indexes by automatic biochemical analyzer, (3) cell apopotosis in groups with FCM, (4) mitochondrial transmembrane potential and calcium concentration in cells were determined by FCM, (5) the quantity of PBR expression in hepatic cells with ELISA.Results:1. Cultured cells were observed in fragmentation, enlarged volume, nucleolus shrinking and cell count decreasing, with the most obvious in group 3 (containing 10% patient serum and 5% FBS) and being more obvious with the patient serum volume proportion increased and time prolonged. MTT showed the light absorption in group 3 was significantly lower than that in normal group (P<0.05), the proliferation was obviously restrained, implying that serum proportion in group 3 was suitable for modeling.2. Morphological changes as shrinking volume, chromatic agglutination, karyopyknosis at 24h after cultured respectively, with higher HBsAg positive rate that that in normal group (P<0.01), and ALT, AST and TBil higher than that in normal group, were observed. ALT, AST, TBil in model group were all higher than that in normal group of significant difference (P<0.05). This implied that this model could reflect cell injury in patient liver.3. For the liver cell function, at 24h and 48h, the ALT, AST, TBil were higher than that in normal group with significant differences (P<0.05). These indexes in GSH group and the Bugan Powder groups were all lower than that in model group of statistical significance (P<0.05).4. Model group showed increase in apoptosis when compared with normal group (P<0.05), while the GSH group and Bugan Powder groups were observed lower than that in model group (P<0.05), both of the two differences were of statistical significance.5. On MTP, Rh123 OD value in the model group was declining when compared with normal group(P<0.01), with which compared higher values were observed in Bugan Powder groups. There were significant differences in the above comparisons (P<0.05, P<0.01).6. The fluorescence value of Ca2+ in model group was higher than in normal group(P<0.01), while the opposite was observed in GSH group and Bugan Powder groups. The differences were of statistical significance (P<0.05,P<0.01).7. The model group showed an increase in PBR comparing with normal group (P<0.05), also significantly higher that than in GSH group and Bugan Powder groups, of significant differences (P<0.05).Conclusions:1. The environment where hepatic cells injury occurs could be simulated in vitro by putting the cells in the nutrient solution composed of 5% FCS and 10% hepatitis-B cirrhosis serum, which is suitable for reflecting the damage to hepatic cells caused by hepatitis B virus.2. The serum proportion mentioned above can be used for in vitro stimulating hepatic cells injury in hepatitis-B cirrhosis patients by putting LO2 into this proportioned nutrient solution for 24h.3. Hepatic cells appeared morphological changes in patient serum and functional damage was observed. The increase of hepatic cells apoptosis may be related to mitochondria, and PBR may play a role in its regulation, which may also serve the mechanism of the obvious protective effects of Bugan Powder medicated serum on hepatic cell injury.