Different Model of Acute Liver Injury in Oval Cell of Rat Biological Differences and Autophagy Change
|School||Hebei Medical University|
|Keywords||Model Hepatic oval cells Proliferation Differentiation Apoptosis Autophagy|
A variety of reasons can cause acute liver injury occurred, the highmortality and poor prognosis after acute liver injury has been a problem of themedical profession. At home and abroad in the treatment of liver injury is stilldifficile. Severe acute liver injury is a common cause of acute liver failure,caused irreversible liver function and life-threatening. End-stage liver diseaseis the best treatment for orthotopic liver transplantation; donor is limited andimmune rejection after two major obstacles. Oval cells are the stem cells ofthe liver, they play an important role in the repair of liver injury. The studyfound that the proliferation rate of oval cells is different at different time points.So, Oval cell proliferation rate is differences at each time point. Oval cellmaker EpCAM and PCNA reflect the Oval cell proliferation rate. There aremany kinds for Oval cell differentiation, Differentiate into liver cells, bile ductcells and liver cancer cells. Expression of CK19was bile duct cells,Expression of AFP were hepatoblastoma. It can judgment that direction of theoval cell differentiation. It is advantageous that the study of oval cellsdifferentiation contribute to the treatment of liver disease.In recent years, the study of autophagy and apoptosis is a hot issue.Autophagy is an essential degradation process of the lysosome to conservativecontrol the cytoplasm nature, by eliminating the protein aggregation anddestruction of organelles. There is a close relationship between autophagy andapoptosis, both mutual synergistic engender impact on cell outcome. The exactrelationship between autophagy and apoptosis remains controversial. Ovalcells as liver stem cells involved in liver regeneration repair and diseaseevolution. Whether autophagy existed in oval cells, and whether it isassociated with the differentiation and proliferation of oval cells? In theexperiment, we make different acute liver injury models, and studied whether the oval cells proliferation and differentiation different in different cases. Wealso studied autophagy and apoptosis in the process of liver injury preliminary.Objective: To establish the different oval cells proliferation models,investigate the differences of proliferation and differentiation and changes ofautophagy.Methods:①SD rats were randomly divided into five groups: Controlgroup, sham operation group, CCL4-treated group, D-gal-treated group,2-AAF/PH group. The animals were killed and take out the liver tissue at thefirst day,7th day,14th day and21st day.②Hematoxylin&eosin staining (H&E staining) observed in rat liver tissue histopathology performance.③Immunofluorescence technique oval cells expression at different time points,quantitative analysis of positive cells.④Oval cell proliferation power wasobserved by double immunofluorescent labeling.⑤Oval cell differentiationmarkers AFP, CK-19and C-kit were observed by immunohistochemicalmethods, and quantitative analysis to determine its expression difference.⑥Autophagosome ultrastructure in oval cells was observed by Electronmicroscopy.⑦The expression of autophagy markers, LC3-II protein andBeclin-1protein, were observed by Western Blotting.⑧TUNEL was used todetect the apoptosis in the cell.Results:①The livers of rats by application of carbon tetrachloride weredark red in color, crisp Compared with the control group, it got back to normalabout at14th days; D-gal-treated group rats’ livers were edema, dark red color,crisp, it got back to normal about at14th days; Sham-operated group rats wereno significant difference in the liver;2-AAF/PH group rats’ livers was pale red,crisp at first days compared with the control group, the liver got back tonormal of size and color about at21st days.②Results showed that hepatocyteswelling, disorganized, and cell morphology visible around the portal area wasless than the liver cells, nuclear quality darkly stained nucleus was oval cellsin CCL4-treated group, D-gal-treated group and2-AAF/PH group comparedwith the control group. Inflammatory cell infiltration of carbon tetrachloridegroup and D-gal group less than2-AAF/PH group, oval cells are less than 2-AAF/PH group.③Liver function tests: there is no significant difference inSham-operated group. AST，ALT and ALP were significantly increased inCCL4-treated group, D-gal-treated group and2-AAF/PH group.(AST:133.97±14.35U/L vs82.47±21.86U/L, P <0.05;346.17±9.75U/L vs82.47±21.86U/L, P <0.05;557.97±12.85U/L vs82.47±21.86U/L, P <0.05;ALT:47±9.35U/L vs41.97±6.64U/L, P <0.05;102.44±5.04U/L vs41.97±6.64U/L, P <0.05;249.1±19.71U/L vs41.97±6.64U/L, P <0.05;ALP:270.33±36.09U vs53.27±8.50U, P<0.05;379.33±21.55Uvs53.27±8.50U, P <0.05;520.67±33.02U vs53.27±8.50U, P <0.05); Albumin(ALB) levels were significantly decreased (23.37±2.21g/L vs32.13±3.73g/L,P <0.05;22.9±2.50g/L vs32.13±3.73g/L, P <0.05;23.9±1.06g/L vs32.13±3.73g/L, P <0.05). The liver function change of2-AAF/PH group islarge, compared with CCL4-treated group and D-gal-treated group.④Mouseanti-rat OV6antibody expression detected by immunofluorescence showed:oval cell marker OV6antibody was upward trend at1st to14th days,downward trend at14th to21st days, and was high at14th days. It showed nosignificant regularity in Sham-operated group and control group. The ovalcells marker OV6antibody was significantly higher at first day,7th day,14thday and21st day after the operation, and was obvious at14th day especially.(109.67±4.04vs5.67±1.53, P <0.05;154.67±8.02vs5.67±1.53, P <0.05;292.67±8.50vs5.67±1.53, P <0.05), Sham-operated group showed nosignificant changes (2.67±0.58vs5.67±1.53, P>0.05). The oval cells markerOV6antibody was significantly high in2-AAF/PH group, compared withCCL4-treated group and D-gal-treated group.⑤Oval cell proliferation powerwas investigated by double immunofluorescent staining with EpCAM andPCNA: oval cell proliferation rate showed downward trend, at first day,7thday,14th day and21st day after the operation, it significantly decreased at7to14days, the oval cell proliferation rate was（82.8±2.60）%at the7th day,dropped to (51.90±2.01)%at the14th day, P <0.05was statisticallysignificant. Sham-operated group showed no significant changes, comparedwith control group.⑥Immunohistochemical staining showed: the oval cells marker AFP, CK19and C-kit antibody were significantly increased inCCL4-treated group, D-gal-treated group and2-AAF/PH group, comparedwith control group. Oval cell marker AFP, CK19and C-kit antibody wereupward trend at1st to14th days, downward trend at14th to21st days, ovalcell marker AFP, CK19and C-kit were maximum at the14th day,(AFP、CK19and C-kit in CCL4-treated group：116.67±3.51vs4.33±2.31, P <0.05;108.67±6.51vs2.67±0.89, P <0.05;105.33±4.16vs4.00±1.00, P <0.05;D-gal-treated group：155.33±5.69vs4.33±2.31, P <0.05;148.67±6.03vs2.67±0.89, P <0.05;157.67±3.22vs7.67±2.08, P <0.05;2-AAF/PH group:265.33±8.08vs4.33±2.31, P <0.05;261.0±18.68vs2.67±0.89, P <0.05;236.33±6.51vs4.00±1.00, P <0.05). Sham-operated group showed nosignificant changes, compared with control group (4.67±1.15vs4.33±2.31, P>0.05;3.00±1.00vs2.67±0.89, P>0.05;3.67±0.58vs4.00±1.00, P>0.05). Theoval cells marker AFP was different in CCL4-treated group, D-gal-treatedgroup and2-AAF/PH group.(F=260.421, P <0.01); the oval cells markerCK19was different in CCL4-treated group, D-gal-treated group and2-AAF/PH group.(F=144.632，P <0.05); The oval cells marker C-kit wasdifferent in CCL4-treated group, D-gal-treated group and2-AAF/PH group.(F=273.875，P <0.05); The oval cells marker AFP, CK19and C-kit antibodywere significantly increased in2-AAF/PH group, compared with CCL4-treatedgroup and D-gal-treated group.⑦The Autophagosome was not found by thetransmission electron microscopy at first day,7th day,14th day and21st day.Volume of oval cell is small at the first day, the ratio of the nucleus and thecytoplasm is higher, the organelle composed of microfilament, manyribosomes and a little rough endoplasmic reticulum. Volume of the oval cell isbig compare with volume of the oval cell at first day, and there are many therough endoplasmic reticulum and glycogen and glycogen, we could see asmall amount of tension microfilament from7th days to21st days.⑧Westernblot analysis showed: The liver markers Beclin-1and LC3-Ⅱproteinsignificantly increased in CCL4-treated group, D-gal-treated group and2-AAF/PH group. Beclin-1protein expression was differences between the groups,(F=7.811, P <0.05); LC3-Ⅱ protein expression was differencesbetween the groups,(F=27.592, P <0.05); Sham-operated group showed nosignificant changes, compared with control group,(Beclin-1: F=0.413, P＞0.05; LC3-Ⅱ: F=1.173, P＞0.05).⑨TUNEL assay results showed: Theapoptotic cells population was upward trend at the1st to14th days, downwardtrend at14th to21st days, in2-AAF/PH group.Conclusions: It is different that the degree of acute liver injury indifferent model of acute liver injury. Acute liver injury is severe at2-AAF/PHgroup. Acute liver injury is light at CCl4group and D-gal group compare with2-AAF/PH group. Oval cell activation repair acute liver injury in2-AAF/PHgroup. Acute liver injury is moderate at CCl4group and D-gal group, theyrepair mainly through the regeneration of liver cells and a small amount ofoval cell activation. The number of oval cells is proliferation activation morethan other models in2-AAF/PH group. The peak of oval cell proliferationappeared at7th to14th days; proliferation power of oval cell began to declinewhen the number of oval cells reached a peak. The differentiation of oval cellsare different in different stages, oval cells are the different stages ofdifferentiation at different times; Autophagy strengthened could promote therepair of liver injury; Autophagy may inhibit liver cell apoptosis.