Iron Overload Induced Rat Liver Injury And The Effect Of Baicalin

With the improving of living standards,the proportion of the meat in daily meal isincreasing,and the iron overload in our body should be deserved our attention.Ironoverload was defined that a series of pathological changes in the body which induced bygenetic factors or pathological conditions.In recent years,the deleteriousness of ironoverload has been given more and more attention.Ironcan catalyze the generation of freeradicals which are able to cause lipid peroxidation and protein oxidation.Meanwhile,itcan promote the generation of reactive nitrogen or even directly catalyze the reaction ofreactive nitrogen and protein which resulted in protein tyrosine nitration.It also has animpact on the metabolism of other trace elements in the body at the same time.However,little was known about the role of tyrosine nitration and the effect of excessive iron onother trace elements in iron overload induced hepatic injury.In this dissertation,theimpacts of excessive iron on other trace elements and oxidative/nitrosative injury of ratliver which induced by iron overload were studied through aimimal model,cellular modeland biochemical analysis.Moreover,the role ofbaicalin was also studied.1) The male rats were used to induce chronic iron overload by peritoneal injection ofiron-dextran five times in two weeks,and supplemented with baicalin containing diet(0.3% and/or 1% w/w) at the same time.Some basic biological indicators,nitrate contentin urine and protein nitration in liver were assayed and the effect of baicalin was studied.The results showed that iron stimulated liver injury in a dose-dependent manner,the levelsof serum non-heme iron,transferin saturation,aspartate aminotransferase (AST) andalanine aminotransferase (ALT) contents were increased and total iron binding capacitywas decreased;the contents of iron,malondialdehyde (MDA),protein nitration in liverand nitrite content in urine were elevated,whereas liver glutathione (GSH) content wasreduced.Supplemented with baicalin could significantly reduce the increased transferin saturation,ALT,AST,liver iron content,MDA content,nitrate content and proteinnitration caused by iron overload.It meant that iron overload could induce proteinnitration besides of changes of some conventional indicators;and baicalinsupplementation could attenuate liver injury caused by iron overload.2) The mice were peritoneal injection with iron-dextran (100 mg/kg each) was used toinduce chronic iron overload,and supplemented with baicalin containing diet (1% w/w) atthe same time,to study its intervention effects.The high performance liquidchromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) wasused to analyze the subcellular distribution of metal-containing proteins in mouse liver.The results showed that Fe was present only in high-molecular-weight (HMW) protein;Cu,Zn and Cd were found in different MW proteins.And excessive iron accumulation invivo could increase the contents of Zn and Cd,and also affect the distribution of Cu andCd,which demonstrated that iron overload has an impact on the metabolism of other traceelements (such as Cu,Zn and Cd).Moreover,the contents of Fe,Zn and Cd decreasedafter supplemented with baicalin.3) Glutamate dehydrogenase (GDH),which is an important enzyme in liver,waschosen to study the difference between the effects of protein nitration and oxidation onprotein function which caused by ONOO^- and/or heme peroxidases (hemin)-H₂O₂-NO₂^-.The results showed that (1) Both hemin-H₂O₂-NO₂^- and SIN-1 could catalyze proteinoxidation and tyrosine nitration of GDH at physiological conditions,and the effect ofprotein oxidation on enzyme activity was stronger than that of tyrosine nitration.(2)Protein oxidation and tyrosine nitration of GDH induced by hemin-H₂O₂-NO₂^- weredifferent from those induced by SIN-1.Mass spectrometric analysis indicated that nitratedtyrosine residues by hemin-H₂O₂-NO₂^- were Tyr²⁶² and Tyr⁴⁷¹ while by SIN-1 were Tyr⁴⁰¹and Tyr⁴⁹³.4) HepG2 cell was incubated with exogenous nitrating agents (hemin-nitrite-H₂O₂ andferric citrate-H₂O₂-NO₂^-),and then glutathione S-transferase (GSTs) extracted from it was used to study the differences between ferric citrate and heme in catalyting H₂O₂-NO₂^- tocause protein oxidation and tyrosine nitration.Moreover,the effects of baicalin on bothmodels have also been discussed.It was found that there were some differences betweenhemin-H₂O₂-NO₂^- and ferric citrate-H₂O₂-NO₂^- in inducing protein oxidation and nitrationof GSTs of HepG2 cell.Hemin has a strong catalytic role in oxidation and nitration,andferric citrate only has a strong catalytic role of protein oxidation.GSTs expression wassignificantly decreased in the heme-H₂O₂-NO₂^-treated HepG2 cell,while the proteinoxidation and tyrosine nitration was increased.However,ferric citrate-H₂O₂-NO₂^- had nosignificant effect on GSTs expression and tyrosine nitration,and only significantlyincreased GSTs oxidation.Moreover,after supplemented with baicalin,both oxidative andnitrative injuries of GSTs of HepG2 caused by Fe(â…¢)- H₂O₂-NO₂^- were reduced.