| Objective:To investigate the state change of the mitochondrial permeability transition pore in the development of nonalcoholic fatty liver disease in rats and in vitro cell model, and relation with the hepatocyte apoptosis and the proteins of Bcl-2, Bax, CytC.Methods:(1) A total of 24 S-D rats were randomly divided into basic diet control group (Group C) and high-fat diet group (Group F). The Group F was subdivided into 3 subgroups (4, 8, and 12 Weeks) (n = 6 in each group). (2) The following parameters in each group were observed dynamically:①The concentration of TG,FFA,ALT and AST in serum;②Pathological changes in the liver tissue observed by HE staining and light microscopy;③Ultrastructural changes in the hepatocyte mitochondrion observed by electron microscopy;④The state of mitochondrial permeability transition pore detected by spectrometer;⑤Hepatocyte apoptosis index detected by flow cytometry;⑥Expression of Bcl-2, Bax, CytC detected by Western-blot and immunohistochemistry.(2) A steatosis model of human hepatocyte in vitro was established by oleic acid incubated with L02 hepatocyte strain(Group S), according incubation time steatosis hepatocyte subdivided into four groups: 0hour, 24hours, 48hours, 72hours, normal hepatocyte as the control group(Group L). The parameters we detected were:①State change of the mitochondrial permeability transition pore. Ca++ and oleic acid applied as the inducer, the fluorescence decrease ratio of the fluorescence microscope was recorded as the state change of mitochondrial permeability transition pore;②Apoptosis ratio of Group S and Group L, by means of annexinV and propidium iodide (PI) double staining and flow cytometry;③Expression of Bcl-2, Bax and CytC by immunocytochemistry. Results:(1)①In Group F the serum TG, FFA, ALT and AST increased in a time-dependent manner and achieved pinnacle in the 12 W in Group F;②HE staining revealed that fatty degeneration at 4w, mild fatty liver at 8 W, moderate to severe fatty liver at 12 W in Group F;③Electron microscopy showed ultrastructural changes in hepatic mitochondrion in Group F (12W), mitochondrial changes were seen as condensation of the inner compartment accompanied by separation of inner and outer membranes and intracristal swelling;④After the stimulation of Ca++, the opening ratio of the mitochondria permeability transition pore in 4 W of Group F was significantly higher than Group C (P<0.05),the ratio turned higher as well as the steatosis lesion increased.⑤Apoptosis indexed in Group F at 8 W and 12 W were higher than those in Group C (P<0.05);⑥Immunohistochemistry showed that positive expressions of Bcl-2 was found in the normal group, but in Group F at 4, 8, and 12 W, the stained was much deeper at the sites with obvious fatty degeneration. The esprssion of Bax was lightly stained in the control Group,as the steatosis lesion increased ,the positive staining grown larger and deeper. Cyt C was in the form of lightly stained brown-yellow granules in the cell, and in Group F, the staining deepened and the stained area enlarged in a time-dependent manner, Western-blot showed that protein expressions of Bcl-2, Bax in Group F, particularly at 12 W increased in a time-dependent manner with significant difference as compared with those in Group C (P<0.05), Bcl-2/ Bax in Group F increased with the progression of fatty liver, Cyt C expression in Group F at 4, 8, and 12 W increased significantly as compared with those in the control group (P<0.05), but no difference was found between the subgroups in Group F (P> 0.05).(2)①A model of steatosis of human hepatocyte was established by oleic acid incubated with the cell type after 24 hours;②Oleic acid induced mitochondrial permeability transition pore opening, 24 hours later,steatosis of hepatocytes occurred. Ca++ could induce mitochondrial permeability transition pore open in steatosis hepatocyte and normal hepatocyte,but no significant difference was found between two groups (P> 0.05);③Hepatocyte apoptosis ratio of Group S in 24 hours made no difference to Group L,but in 48 hours and 72 hours , compared to Group L the ratio were higher (P<0.05);④The expression of Bcl-2 and CytC of Group L was lightly stained brown-yellow granules in the cell, and in Group S, the staining deepened and the stained area enlarged in a time-dependent manner. positive expression of Bax was found lightly stained in the Group L, but in Group S at 24hours,48hours, and 72hours, the cells were stained deeper than Group L (P<0.05).Conclusions:(1) Opening of the mitochondrial permeability transition pore and release of CytC may relate to hepatocyte apoptosis of NAFLD; (2) Protein of Bcl-2 family may participate in the regulation of hepatocyte apoptosis, increased Bcl-2/Bax ratio could not hold back the NAFLD hepatocyte from apoptosis;(3) Higher level of serum FFA may contribute to the mitochondrial permeability transition pore opening.
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