Study On Sey Protecting Function To Iron Overload Caused By Mice Liver Damage

BackgroundThe main physiology function of the iron builds blood, when human lack of iron,will take place anemic,the lowing immunity,dynamia,headache,stomatitis,sufferfrom a cold and liver cancer etc.But when iron loaded, then will because of can'tdiscard the outside of the body in time thus deposing in the liver,pancreas,heart andthe skin.Accordingly, causing hematochromatosis(haematochrome deposition),liverfunction abnormality, myocardial damage and diabetes.People can because of takein great amount iron enrichment food, especially because of the change of dietstructure and industry pollution etc.,thus making in vivo iron increase through variouspath .Especially, the liver damage caused by iron load have already been value bymany scholars, relevant research also increases increasingly.Because the liver is themain organ of iron deposite, the iron as a kind of powerful catalyst, promoting theoxygen free radicals produce, causing the liver cell deuto-cell membranephospholipid lead to peroxidation harm, especially in mitochondriaon andmicrosomal membrane damage,through the interference electronics to make the celldeath to succeed formation of hepatic fibrosis.The trace element selenium regulationbe subjected to the liver restrict in vitro absorption,metabolize,revolve,distribute,etc., the selenium is GSH-Px essential constitute part, the GSH-Px can catalyst deoxidize GSH become oxidize GSH, making noxious peroxide(ROOH) deoxidizeharmless hydroxy compound,and make H₂O₂ resolve, as a result can protect the thestructure and function of the cell membrane was free from oxide damage andinterfere.ObjectivesThis experiment chooses to use Sey as antagon to replace deuto-sodium selenateto start the research of experiment because of Sey is the most wide microorganism ofmankind utilization,its protein content is up to 50% above, also contain abundantvitaminB family and various mineral matter, be called the source of the high-qualitynourishment, and have a high rich selenium ability and the ability of the transformingchemistry selenium to creature selenium, removed deuto-sodium selenate consumedlybe a kind of chemistry selenium to biosystem produce of the poisonous vice-reactionand stomach intestine stimulation. Contrary, it can more effective,more safelyabsorbed exploitation by the organism. The data and result of the experiment aremore accurate, can more reflect trace element selenium to the protection function thatthe iron causes the mice liver damage. To people because of various iron overloadthat the reason cause then result in of the liver injure, the Sey leaven conduct andactions a kind of health care article can drive increase to our daily food in the middle,have protective function to the liver that gets this disease crowd with this.MethodsThe whole experiment is divided into two, parts.The first part: The establishment of the mice iron overload model.The experimental mice are Kunming mice, adopt by feeding the mice withfull-rate diet pellets and added with regular and quantitate intraperitoneal injection toestablish high iron mice model and observe the ratio of iron,the changes of organcoefficient inside the mice,the pathological damage of each organ and the level of lipid peroxidatio in vivo. Cocretely build the model method as follows: The micewere randomly divided into two groups:control group(n=10) and high-iron modelgroup(n=30), among them, high-iron model group is divided into low,medium andhigh three dosage groups. Mice in each group were raised in plastic stainless steelcages respectively,indoor temperature(23±3)℃and day and night clear,50%～60%,they were free to the access of food and deionized water. Beforeexperiment,adapt-this environment for three days, and weigh each group mice weight,measure serum iron density. Start experiment in the fourth day, the low dosesgroup,the medium amount of group and the high amount of group continue to drink tothe deionized water, and treated with intraperitoneal injection of differentconcentration iron dextran once every other day,0.8 mL for each time(the ironcontentthe of iron dilution liquid distinguish for the 6.25 mg/ml, 12.5 mg/ml and 25mg/ml); Control group changes to use to drink tap water and treated withintraperitoneal injection of normal saline once every other day,0.8 mL for eachtime,inject continuously for 6 weeks. Measurese the serum iron concentrationevery week and measures weight, observe the general condition of the mice.All themice were killed and dissected at the end of the 6th week. The iron contents of everyorgans and serum were determined with atomic absorption spectrophotometer andautomatic biochemical analyzer respectively, pathohistological examination of liverwere performed,the organ coefficients of liver and spleen were calculated.MDAcontent and SOD activity in serum were determined.The second part: Study on Sey protecting function to iron load leaven to miceliver damage.The 50 mice were randomly divided into five groups: control group(n=10) andtreated with intraperitoneal injection of normal saline once every other day,0.8 mL foreach time; high-iron model group(n=40), and treated with intraperitoneal injection of iron dextran once every other day,0.8 mL for each time(the iron contentthe of irondilution liquid distinguish for the 12.5 mg/ml), inject continuously for 6 weeks.After6 weeks,the high-iron model groups randomly were divided into four groups,respectively is: high-iron model groups(n=10), Fe+20mgSey(n=10), Fe+40mgSey(n=10), Fe+60mgSey(n=10).At this time, control group treated withintragastric administration of distilled water and intraperitoneal injection of normalsaline once every other day,0.8 mL for each time; model groups treated withintragastric administration of distilled water and intraperitoneal injection of irondextran once every other day,0.8 mL for each time(the iron contentthe of iron dilutionliquid distinguish for the 12.5 mg/ml);the rest three treatment groups respectivelywith the concentration of 20mg/(kg·d), 40 mg/(kg·d), 60 mg/(kg·d) intragastricadministration of Sey suspil, everyday and intraperitoneal injection of irondextran once every other day,0.8 mL for each time(the iron contentthe of iron dilutionliquid distinguish for the 12.5 mg/ml), inject continuously for 4 weeks. All the micewere killed and dissected at the end of the 4th week. Measurese the enzyme activityof ALT,AST in serum respectively; The level of lipid peroxidation in liver; Thepathological change and the variety of apoptosis and reverse transcription-polymerasechain reaction (RT-PCR) technique was used to examine the expression of IRP2mRNA and TfR mRNA in liver.ResultsThe first part: The establishment of the mice iron overload model.1. The general condition of the high iron mice:Skin color: Compare with the control group,the high iron model mice begin skinand hair crude,sparseness,the hair color loses sheen, the ear, feet claw,tail all appearsan obvious color and luster change, presenting a gradually deep rust color, the tooth present tan in the third week of the experiment,and prolong above-mentionedsymptom along with time of the experiment more obvious.The behavior change: Along with experiment of carry on, the high iron modelmice growth is slow-moving, the body is emaciated. Express languid, the activityreduce, responding muff, fear cold love to sleep, conglobation of scrunch, frighten tofrighten easily, the food reduces obviously.2. Each comparison of mice liver and spleen organ coefficient:The high iron model mice liver,spleen organ coefficient compaared with controlgroup go up obviously, have significance difference (P＜0.01).3. The comparison of the content of iron in every organ and blood-serum:The content of iron in every organ and blood-serum of high iron mice modelgroup increased distinctly (P＜0.01) compared with control group,and grows by theincreasing of dosage,but the content of iron in heart have no significance difference inlow dosage and midum dosage(p＞0.05). The amount of deposition of iron in the liveris the biggest in each organs, the heart is the least, the condition of deposition arrangeone by one as follows:Liver＞spleen＞kidney＞lung＞heart.4. The comparison of SOD content and MDA activity in serum.The high iron mice model group of the activity of SOD in the blood-serumsdecreased significantly compared with control group (P＜0.001), and decrease by theincreasing of dosage. The high iron mice model group of the MDA content in theblood-serums rose significantly compared with control group (P＜0.001).5. Pathohistological examination of liver of each group:The pathohistological examination of liver indicates overload iron can injurycells,make cells arrangement disorder, have no boundary,to happen hydropicdegeneration,necrosis,etc, pathohistological changes, furthermore, the degree moreheavy in header area, this has something to do with iron of distribute probably.The iron staining analysis indicates,the ferrugination material in the liver of treatmentgroup increase obviously,the blue iron by stained existence under light microscope,and distribute extensive by the increasing of dosage.The iron distribute uniformity inthe liver in low dosage group,but the content of iron in header area obviously higherin central veins area in midum, high dosage group.The second part: Study on Sey protecting function to iron load leaven to mice liverdamage.1. The ALT, AST activeness change situation of the different concentration Seyexcessive causes the mice liver damage by overload iron:The change of ALT content:compared with model group, middle,high dosagegroup have significance difference(P＜0.01),low dosage group have no significancedifference(P＞0.05); compared with normal group,low and high dosage group bothhave significance difference.The results indicate, add low dosage Sey, can haveimprovement effect to ALT which the liver damage caused by overload iron.but thedifference is not remarkable;add midum and high dosage Sey ,ALT have significantimprovement effect,among them,add midum dosage Sey, ALT have the mostimprovement effect.2. The examination of change circumstance of MDA content,SOD vitality, theCAT vitality and the GSH-PX activity of different concentration Sey to liver tissue:The research result of this experiment indicate:in the setting concentration scopeof this experiment,along with the Sey concentration goes up,the liver homogenategenerating content of MDA present decrease tendency compare to high irongroup,have significant difference (P＜0.05).Among them,the generating content ofMDA in low concentration Sey group decrease more obviously compare to themiddle,high dosage group. The liver homogenate variety trend of the SOD activity evenly together the MDA content variety trend is contrary, go up along with the Seyconcentration, the SOD activity present significant rise tendency(P＜0.05) compare tohigh iron group.Among them,the SOD activity of low Sey concentration is thehighest,low,midum dosage group are second, three groups of improvementcomparison detection,between low and midum dosage group have significantdifference(P＜0.05),but between low and midum dosage group,between midum andhigh dosage group have no significant difference(P＞0.05),indicates the improvementeffect of low dosage Sey is the best. The CAT activity of liver homogenate along withthe Sey concentration goes up,compares to the high iron group present rise tendencygradually,have significant difference(P＜0.01).Among them,the CAT activity ofmidum concentration Sey group is the highest,low and high dosage group aresecond,indicates ability of antioxygen begin to weaken at the time of Sey reach tohigh concentration,CAT activity present decrease tendency gradually. The GSH-PXactivity of liver homogenate after along with the Sey concentration goes up,low andmidum dosage group present rise tendency compare to the high iron group,amongthem,the GSH-PX activity of low dosage group rise more obviously,have significantdefference(P＜0.05);but between high dosage group and high iron group have nosignificant defference(P＞0.05).3. The pathology slice light mirror observation of liver caused by differentconcentration Sey:In the course of liver damage caused by supply excess iron,the most significantcharacter is many abnormal brown stain(hemosiderin deposition) existence in theliver cells,after Schmeltzer staining,present obvious blue colour, and distribution extensive,besides have increasingconnective tissue. Under the light mirror, can find that the cell nucleus disappear,some cell appear punctiform necrosis, phlegrnonosis cell infiltrate in necrosis focus,many coagulability necrosis focus in header, appear karyopycnosis,dissolve,massive cell hydropic degeneration. After supply Sey, it is thus clear that theiron of the blue deposite obvious decrease,, among them, in the phenomenon ofdecrease the iron deposition,the midum dosage group's effect is the most obvious,low,high dosage are the second.4. The apoptosis influence of liver tissue caused by different concentration Sey:AO/EB fluorescence double staining show: The high iron group present massivemiddle, late period apoptosis cell and manipulus death cell. But the many of nucleusof midum dosage Sey group present uniform green fluorescence,have thimblefulsalmon pink apoptosis cell,near to the normal group,but high dosage Sey group isnear to the high iron group.5. The IRP-2 mRNA and TfR mRNA express level of liver tissue caused bydifferent concentration Sey:Carry on an examination to the liver tissue mRNAs IRP-2 and the TfR mRNAexpression levels in foundation built up mice model and improvement better mice ofhigh iron, the result indicates along with the increment of the iron dosage, theexpression of IRP-2 mRNA and TfR mRNA descend, after the Sey improvement, theexpression of IRP-2 mRNA and TtR mRNA reinforce to some extent, the mice livergene expression that after midum dosage improvement have significant up-regulationcompare with the model group(P＜0.01).It shows that the adjustment of TfR mRNAexpression after IRP2 transcription influence the iron deposited in liver, it is importantadjuster of iron metabolism in the body.