| ObjectiveThe liver is the most important organ of detoxification of the body. Hepatic necrosis even hepatic failure will occur when large amount of chemicals is uptak-en. In order to study molecular mechanism of acute hepatic injury, it is necessary to establish animal model of hepatic injury. Animal model of hepatic injury made by perfusion of carbon tetrachloride into the stomach of mouse has long been used in study of hepatic injury. But the mechanism of hepatic injury caused by this drug is still enigmatic. It is commonly regarded that hydrocarbon bond will be broken and free radicals will be produced when carbon tetrachloride catalysed by enzymes in the microbody. Free radicals initiate peroxidation of polyvalent fatty acids in lipids of biomembrane of the cells resulting in production of peroxide of lipids. Peroxidation of lipids is a chain reaction which happen continuously and large amount of lipids is consumed, large amount of lipid peroxide is produced resulting in injury of biomembrane even cell disintegration and cause hepatic injury. Pinghe et al. found a 30 - kDa protein in the cytoplasm of mouse hepatocytes in acute hepatic injury caused by carbon tetrachloride 24 - 72 hours. It was proved to be galectin -3 by Western blotting and can be phospho-rylated with tyrosine radical. At the same time, that p21 and PCNA protein were expressed in the cytoplasm and nucleus suggested that galectin - 3 might be involved in repair and survival of hepatocytes 24 -72 hours after hepatic injury. Galectin - 3 is a member of the super family of β - galactose agglutinin which exists in all embryo and tumor cells but is not expressed in normal body cells. It is now clear that it has many functions including cell adhesion, cell movement, and advanced glycation end product receptor etc.. But there is little study on itsfunction at the molecular level. There is even less study on the mechanism of the function of galectin - 3 in hepatic injury. It is very necessary to study it.In order to study on the mechanism of the function of galectin - 3 in hepatic injury gastric perfusion with carbon tetrachloride were practiced in gene -knocked out mice ( Gal -3( -/-)) established by UC Davis and Iiu et al and wild - typed mice ( Gal -3( +/ + )). At different times change of molecular -level phenotype were observed and shown in apoptotic expressed proteinase and molecular GRP78 to study antiapoptotic mechanism of galectin - 3 in CC14 induced hepatic injury.Large number of studies showed that galectin - 3 is involved in cell signal transduction pathway with its lactum guanidiaoacetate containing galactoside combining to target molecules. It is essential to find the high - molecule substance in studying the molecular biological significance of galectin - 3 in hepatic injury. In Western blot with anti - Gal -3 antibody, we found a 65 -kDa protein showing close relationship with the expression of Gal - 3 in wild - type mice perfused with CC14 i. e. hyper - expression of the gene occurred between 24 - 72 hours. In order to elucidate the nature of the 65 - kDa protein, we performed chromatography analysis and isolated Gal - 3 complex. This study will provide the basis for the further study.Materials and Methods1. Experimental animalICR healthy male mice of wild type(Gal -3( +/+)), 10 week - ages, obtained from The Japanese National Tumor Research Institute. ICR healthy male mice of the galectin - 3 gene knocked out, 10 week - ages, obtained from the first biochemics laboratory in Japanese Fu - shan Medical and Pharmacological University.2. Gastric lavage with CC14 caused the hepatic injury of miceThe ICR male healthy mice 10 -weeks old, Gal -3( +/ + ) type and Gal -3( -/ - ) type, divided into 0,10^24^48^2 set of hours feed, 10 per set. Fasting diet for 12 hours before intoxation, CC14 and edible olive oil were mixedto the poison venom, the mice were lavaged at 8ml/kg. The mice were feed normal feeding after intoxation.3. Preparation and observation of the pathological sections cutting from the hepatic tissue in the miceWe took blood from the hearts of the mice lavaged with CC14 which had been anesthetized by diethyl ether at 0^10^24^48^72 h. We took out the liver quickly and washed the blood using NS, the liver were divided into two parts with a small scissor.For the two parts, one was cut into the small lcm x lcm xO. 3cm pieces, fixed with lOOml/L neutral formalin for 32 hours immediately. These tissue pieces were dewatered at a series of ascending concentration alcohol, washed u-sing dimethyl benzene and embed into mineral wax, then the pieces were cut into 5 - jxm section with a section cutter. The sections were stained, with HE.The hepatic pathological change was examined under an optical microscope. The number of normal cell nucleus and abnormal cell nucleus was counted. The hepatic tissue sections were examined under a microscope enlarged 400 times, it could be discriminated to a normal cell nucleus and an abnormal cell nucleus including aggregated fragments of chromosome obviously. The quantity of the normal cell nucleus and abnormal cell nucleus was to be counted by two independent observers. Statisticed the number of the normal cell nucleus and abnormal cell nucleus using a software named " stereo investigate" which was installed in a computer connected a microscope. Each unit sampled 10 mice, each mouse sampled 10 sections to count the number of cell nucleus randomly. 9 100|xm x lOOjxm area count blocks were selected by software randomly and auto - optimizingly in each section. The percentage (the number of the abnormal cell nucleus/the number of total cell nucleus x 100% ) of the abnormal cell nucleus each unit was computed out immediately. The difference between the percentage of the abnormal cell nucleus in the hepatic tissue sections cutting from the wild type and the Gal - 3 gene knocked out type mice were examined using the chi - square criterion.4. Cell fractionation of the hepatic cell in the miceThe other part of hepatic tissue was placed on the flat plate(4X. ,all the fol-lowing manipulate would be ran in coolhouse at 4X.) , the hepatic tissues of the mice were cut into small pieces with a small scissor, transfered to the claviform tissue homogenates after weight, added in 0. 25M cane sugar buffer at 9ml/g, the homogenates were grinded with a electrical grind. The homogenate of each liver was fixed and filtered with an absorbent gauze, keeping the filtrates for preemergency. This liquid was hepatocellular grinds ( HO) , the liquid was transfered to the centrifuge tube, centrifuged with a centrifugal machine at 4X., 6000rpm for lOmin, the precipitate was compositions of cell nucleus( Nuc) , remove the supernatant to centrifuge(4X. ) , at 8000rpm for lOmin, the precipitate was composition of chondriosome (Mt) , remove the supernatant to ultracentri-fuge, at 4t , 105000rpm for 60min, the precipitate was composition of micro-some ( Mic) , the supernatant was composition of cell matrix ( Cyt). Store each of the compositions at - 80T!.5. Detection and protocol of the Western blottingThe protein BCA quantitate detecting agent was adapt to quantitate the total protein of grands of hepatic cell N chondriosome N cell matrix and microsome in the mice, each sample volumn(5 — 20|xl) was balanced according to the total protein, carried on SDS - PAGE, then the proteins were transfered from gel to a NC membrane, the membrane was blotted for 1 hour at room temperature in 5% non - fat dry milk, then incubated with the primary antibody for 1 h at room temperate , casted the antibody, washed by TBST three times for 5 min. The membrane was incubated with the secondary antibody for 1 h, casted the antibody, washed by TBST three times for 10 min. For the chemiluminescence reaction, protein ECL blotting detecting analytical reagents were added in, then the membranes were developed, photographed and recorded for 30min with a chemical cold light image analysator( LAS - lOOOplus). Immunoprotein blotting developing images were scanned, then the experimental results were analyzed from Band Leader V3.0 image analysis software, the average optical of the unit area representative the content of enzyme, we casted the average optical of backgroud, and compared the results to the Gal - 3 gene knocked out type mice with the wild type mice with an immunoprotein blotting using the t - test at the same time and with the same composition of cells.6. Active detection of enzyme ALT and AST in serumTo seperate the serum, we centrifuged the blood at 2000rpm for lOmin. The enzyme ALT and AST in the serum of the mice were detected using the a-gents Infinity TM ALT and Infinity TM AST (Sigma, St. Louis, MO, USA) respectively. The activity of enzyme ALT and AST in the serum extracting from the gene gal - 3 knocked out type and the wild type mice were tested using the t -test.7. Extract and purification of the galectin -3 complexWhen the wild type mice were lavaged with CC14 at 48 h, a kind of 65kDa weight protein in the galectin - 3 complex would be seperated and purified from the homogenate of the hepatic cell using a DE - 52 column chromatography, a Hydroxyapatite column chromatography, a salting - out, a desalinization, and a Sephacryl S -300 gel chromatography.Results1. Pathologic variation of the hepatic injury caused by CCl4in Gal -3 gene knocked out type and the wild type miceThe conspicious pathologic variation of the hepatic injury was found in the hepatic tissue of the Gal - 3 ( - / - ) type mice which were lavaged with CC14 at 10 h. It was found that hepatic cell cord arrayed in disorder, sinus hepaticus narrowed by compression, a large area of fat denaturated in the central of the central veins of hepatic lobules, and denaturated cytoplasm of hepatic cell appeared vacant and bright obviously, cell nucleus pressed, deviated to one side. Lots of small vacuoles were viewed in the cell matrix even with slight proliferation under a high power lens. The degree of hepatic injury in the Gal - 3 ( - / - ) type mice which were lavaged with CC14 was serious after 24 h, the hepatic cell appeared punctiform and lamellara necrosis on the base of the large area of fat denaturated seriously in the center of the central veins of hepatic lobules, we could view unorganized, homogeneous and peach cell circumsciptionNnuclear fragmentation ^ karyolysis and nucleus disappearance. There was no difference between the wild type mice in the 10 h unit and 0 h unit, the hepatic injury tothe mice in 24 h unit were not the same serious as the Gal - 3 ( - / - ) type mice in 10 h unit also. However, there were more hepatic cells fatty degenerated in peripheral of the hepatic lobules. When the mice were lavaged at 48 h, the circumscription of the hepatic lobules in the Gal - 3 ( - / - ) type mice began to disappear, the hepatic cells appeared a large area of necrosis, contrast to the wild type mice at 48 h, though hepatic cord was disordered, the circumscription of the hepatic lobules were still clear, the degree of the hepatocellular fatty degeneration was more serious, we could see lots of compensatory proliferation. When the wild type mice were lavaged with CC14 at 72 h, the hepatic cells began to necrosis, the degeneration became more serious, the hepatic cells became swell obviously, the hepatic cords broadened, the part of hepatic cord narrowed or disappeared, but there were still the circumscription of the hepatic lobules. And contrastly at the same time, the hepatic cords in the Gal - 3 ( - / - ) type mice dissociated, the hepatic cells dissolved, a large area of the liver necro-sised, and the hepatic cords obvious dirtended, congested, even bleed.2. The percentage of the abnormal cell nucleusCompared the Gal - 3 ( + / + ) type mice with the Gal - 3 ( - / - ) type mice at the same time when the mice lavaged with CC14 at 24 h, the percentage of the abnormal cell nucleus in the Gal - 3 ( -/ - ) type mice was 12. 33% , higher than that of the Gal -3( +/ + ) type mice(8. 19% ) , the difference had statistical significance ( P <0.01) . There was no obvious difference between that of the Gal - 3 ( + / + ) type mice and that of the Gal - 3 ( - / - ) type mice at other time (P>0.05).3. The levels of ALT and AST in the serum in wild type and the Gal - 3 ( - / - ) type miceWhen the mice were lavaged with CC14 at 10^24 h, the average ALT activity of the Gal - 3 ( - / - ) type mice was higher than the Gal - 3 ( + / + ) type mice at the same time( P < 0.01) , the peak was at 24 h, the average ALT activity was 2468U/L, then it descended, descended to 518U/L at 72 h. And there was no obvious difference between the ALT activity of the Gal - 3 ( + / + ) type mice and that of the Gal - 3 ( - / - ) type mice at other time (P > 0.05).When the mice were lavaged with CCl4at 10 h, the average AST activity ofthe Gal - 3 ( - / - ) type mice was higher than that of the Gal - 3 ( + / + ) type mice at the same time( P <0.01) , peak was at 48 h, the average AST activity was 1957U/L, then it descended, descended to 518U/L at 72 h. And'there was no obvious diflference between the AST activity of the Gal - 3 ( + / + ) type mice and that of the Gal - 3 ( - / - ) type mice at other time (P > 0.05).4. The expression of GRP78The average optical values from image analysis and protein blotting image were tested with the t - test, when the mice were lavaged with CC14, the expression of GRP78 in the microsomes of the Gal - 3 ( + / + ) type mice was higher than the Gal -3 gene knocked out type mice at the same time( P <0.01) , 0 h, 24 h, and the expression of GRF78 in the mitochondrion of the Gal - 3 ( + / + ) type mice was higher than the Gal - 3 gene knocked out type mice at the same time(P<0.01), 24 h.5. The expression of Caspase3 using the immunoprotein blottingThe expression of Caspase3 was located in hepatic cell matrix, there was no difference between the hepatic cell matrix of the Gal - 3 ( + / + ) type mice and that of the Gal - 3 ( - / - ) type mice at the same time ( P > 0.05) , we couldn 't find the expression of it in the other hepatocellular compositions.6. The expression of Caspase7 using the immunoprotein blottingThe expression of the Caspase7 was in hepatic cell matrix, there was no difference between the hepatic cell matrix and grinds of the Gal - 3 ( + / + ) type and the Gal - 3 ( -/ - ) type mice at the same time( P >0.05 ) (Table 2 ) , we couldn't find the expression of it in the other hepatocellular compositions.7. The expression of polytechnic ADP ribose pclymerase (PARP) using the immunoprotein blottingHigh expressing of polytechnic ADP ribose pclymerase (PARP) was found in hepatocellular nucleus, and the weight of the PARP detected was 116kDa, the fragments of weight 89kDa and 24kDa were undetected. There were no differences between the expression of the PARP in the hepatic cell nucleus of the Gal-3( +/ + ) type mice and the Gal - 3 ( -/-) type mice (P > 0.05) , we couldn't find the expression of it in the other hepatocellular compositions.8. A kind of galecin -3 complex protein weight 65kDa was dissociated andpurifiedConclusion1. Hepatic injury caused by CCl4in Gal -3( -/ - ) type mice appeared not only earlier but also more serious than that in wild type mice;the percentage of abnormal cell nucleus^the values of ALT and AST were higher in Gal -3( -/- ) type mice than in wild type mice, which indicates that Gal — 3 may have the function of relieving and protection of the hepatocellular injury caused by CC14.2. The expression of GRP78 in the microsomes of the injuried liver in wild type mice was much higher than that in Gal - 3 ( - / - ) type mice, it illustrates that Gal - 3 may be involved in the repair and protection of the injuried hepatic cells by up - regulating the expression of GRP78.3. The similar expression of Caspase3, Caspase7 and PARP in damaged liver cells of rat of Gal - 3 ( - / - ) and Gal - 3 ( + / + ) , indicates that galectin- 3 is involved in the regulation of apoptic mechanism to protect and repair the damaged hepatic cells caused by CC14, possibly through the other routes except the route related to Caspase3, Caspase7 and PARP.4. A new discovered 65kDa protein may be galecin -3 complex protein.
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