The Protection And Mechanism Of Lithium For Steatotic Liver Ischemia/Reperfusion Injury In Rats

PART ONE MCD+HF Diets Induced S-D Rats Hepatic Steatotic Models.[Objective] Many efforts have been spent to develop animal models reflecting the pathogenesis and progression of NAFLD precisely as the basis to develop novel therapeutic strategies.However,all of the existing models didn’t reflect the NAFLD in all its facets.Generally,nutritional models of hepatic steatosis are methionine-and choline-deficient(MCD)models and high fat models(HF).Models induced by MCD didn’t develop obesity,and it’s body weight got loss severely,while the models induced by HF was unstable.This study was designed to investigate compound nutritional models(MCD+HF)of hepatic steatosis in respect to the time course and intensity of hepatic steatosis,which would be ready for next research.[Methods] Two diets feed the Sprague-Dawley(S-D)rats to induce hepatic steatosis.Rats were described as two groups: normal diets for control group,and MCD+HF diets for NAFLD group.Before rats were entered into experiment,all rats were fed with the normal diet up until reaching a body weight of 250-320 g,then 6 rats/group were fed for 1,2,4,6 weeks and 3 months with MCD+HF and normal diet respectively.The weight of rats was recorded every day individually.The weight of each liver lobe wasdetermined using a precision balance after hepatectomy.Blood of rats were collected and analysed by Automated Chemical Analyzer.The assessments of hepatic steatosis were three aspects as follows:(1)assessment of descriptive macroscopic appearance,calculated liver-to-body weight ratio,histological estimation of the distribution of fatty changes,(2)hepatic damage was assessed qualitatively based on determination of hepatic enzymes,(3)assessment of hepatic metabolic disorder through determining LPO and GSH levels.[Result] None of the rats died in experiment,and different severity of hepatic steatosis were induced in NAFLD groups.MCD+HF diet didn’t impair the rat regular growth and age associated body weight gain.At the end of the 3 months feeding period,both two groups rats reached a body weight of about 400-450 gr,which was no significant.NAFLD group rats appeared larger,greasy,and yellow in color compared with control group after 1-2 weeks of feeding,and the edge of liver became rounded and the consistence appeared slightly softer.Macroscopic changes became more apparent over time with a maximum after 4-6 weeks of feeding and seemed to decrease slightly thereafter.Absolute resected liver weight of NAFLD group increased at 4th,6th and 3m,which is significant compared with the control group(13.80±1.79 vs 8.76±0.92,14.20±1.15 vs 8.75±0.91,12.56±1.03 vs 8.78±0.82,p<0.05,unit: g).In the rats fed with MCD+HF diet for 1 week,mild to moderate microvesicular steatosis and mild macrovesicular steatosis,and severity of steatosis is about 15%,which is mild hepatic steatosis.The microvesicular steatosis localized predominantly in the periportal zone(zone 1)and zone 3 while mixed-vesicular steatosis localized in zone 2.After 2 weeks of feeding,livers showed moderate mixed-vesicular steatosis in all zones,and moderate steatosis was determined according the 32% steatotic changes.Macrovesicular steatosis localized in zone 1 and 2,and the microvesicular steatosis localized predominantly in zone 3 and mixed-vesicular changes localized in zone 1 and 2.At the end of 4 weeks feeding,severe mixed-vesicular steatosis appeared,which were determined with severe hepatic steatosis according the 63% steatotic changes.Macrovesicular steatosis localized in the zone 1 and zone 2 predominantly whereas microvesicular changes were more confined to zone 3.At the end of 6 weeks feeding,the type and distribution of hepatic steatosis were similar to the changes observed at 4 weeks group,but appeared less severe.At the end of 3 months feeding,severe mixed-vesicular steatosis,macrovesicular steatosis in zone 1 and zone2 predominantly and mixed-vesicular steatosis localized in zone 3.Hepatic enzyme release was observed according to NAFLD group.Enzyme release started within 1-2 weeks of feeding and reached a maximum after 4 weeks of feeding,and predominantly an elevation of the AST.ALT levels remained below 100 U/L at all time points.AST levels were higher than control group at 6th week significantly(89.50±29.83 vs 42.50±5.51,p<0.05,unit: IU/L),while the AST levels higher than the control group at 4th,6th and 3m significantly(210.17±95.28 vs 92.03±23.70,199.67±54.06 vs 90.03±21.70,140.01±22.73 vs 90.03±21.70,p<0.05,unit: IU/L).LPO levels elevated in NAFLD group significantly in all points(68.7±14.0 vs 53.2±2.8,120.6±18.3 vs 53.1±2.7,150.3±43.0 vs 53.3±2.5,168.7±44.5 vs 52.8±3.0,148.1±26.7 vs 53.3±2.7,p<0.05,unit: μg/g),while the GSH levels decresed from 1st week,and a mild restored at 3rd month slightly(2476.4±67.7 vs 3698.3±122.8,2371.8±127.3 vs 3720.2±117.5,2237.9±118.1 vs 3710.7±102.8,2399.1±60.8 vs 3701.4±98.8,2426.6±113.5 vs 3710.2±103.4,p<0.05,unit: μg/g).[Conclusion] None of the rats died in experiment,and different severity of hepatic steatosis were induced by MCD+HF diets.This compound nutritional model of hepatic steatosis is stable,which is suitable for next research.Respect to steatotic hepatic ischemis-reperfusion injury,feeding with MCD+HF for 2~4weeks is suitable.Part Two The Protective Properties of Lithium Treatment Liver Ischemia/Reperfusion Injury in Rats[Objective] Lithium has been used widely as the treatment of mental disorders.Recent studies suggest that lithium has protective properties against IRI in several organs.The precise mechanisms by which lithium confers protection against hepatic IRI are still understood poorly,which will be investigate in present research,that base for mechanism of lithium protection against steatotic hepatic IRI.[Methods] Before enter experiment,all the S-D rats were feed to standardized body weight by normal diet.There were two groups,lithium group and saline group.Rats started treatment with lithium 3 days before warm I/R performed,and they were continuously injected to a daily lithium injection for up to 2 days.Then all rats were given 60 min selective warm ischemia,continuously harvested after 0.5h,6h,24 h,48h reperfusion respectively.Left lateral lobe and right superior lobe were collected for histological analysis.Right median lobe and serum were collected for molecular biological analysis.Rehydrated sections were stained by using the naphthol AS-D chloroacetate esterase technique to investigate the neutrophil infiltration in the liver after reperfusion.10 high-power fields(HPF,400×)were selected randomly for quantitative analysis.ASDCL-positive neutrophils were counted manually.HMGB1 translocations were determined by HMGB1 negative staining in hepatocellular cytoplasm and nuceus,and the volume of HMGB1 in serum were tested by ELISA.The expression of GSK3β,p-GSK3β Ser9,ERK,p-ERK,p38,p-p38,JNK,p-JNK,caspase-3,LC3,p-62 were tested by western blotting respect to various reperfusion times.Then,the influence of lithium subject to inflammatory,autophagy and the signal pathway of GSK3β and MAPK will be analysed.[Result] Liver function,include ALT and AST serum level,were tested after 0.5h,6h,24 h,48h reperfusion.After 60 minutes of warm ischemia injury,both serum ALT and AST level was increased as early as 0.5 hours after reperfusion,reached a peak at 6 hours,and decreased thereafter.Lithium treatment did significantly decrease I/R injury,as indicated by the lower liver enzyme levels compared with saline group at different reperfusion time respectively.(ALT levels: 596±171 vs 392±126,823±155 vs 604±119,221±34 vs 147±34,73±5 vs 62±9,all p<0.05,unit: IU/L;AST levels: 574±140 vs 371±101,892±160 vs 594±127,426±35 vs 363±45,110±20 vs 98±11,all p<0.05,unit: IU/L).The number of ASDCL staining-positive neutrophils was increased after I/R injury.Neutrophil infiltration was significantly lower in the rats subjected to lithium treatment versus the saline-injected rats(lithium: 9±1/HPF;saline: 14±4/HPF,p<0.05).The HMGB1 immunohistochemical analysis indicated that HMGB1 translocated from nucleus and cellular cytoplasm.Lithium treatment group showed lower serum HMGB1 levels by ELISA than saline group,there was an approximately 3-fold decrease 0.5 hours after reperfusion.Phosphorylation of GSK3β were determined after 60 minutes of warm ischemia followed by various periods of reperfusion.In comparison with the levels in normal control,phospho GSK3b/Ser9 levels had rapidly decreased at 0.5 hours,and they remained dephosphorylated in reperfusion phase.In the lithium treatment group phospho GSK3b/ Ser9 levels were higher than the saline group.The total cellular levels of GSK3 b were not affected.To determine whether chronic lithium pretreatment could influence MAPK activation,the phosphorylation of ERK,JNK,and p38 were assessed by western blotting after I/R.the phosphorylation of ERK,JNK,and p38 was increased 0.5 hours after reperfusion in the saline group.In contrast,the phosphorylation of ERK in the lithium group was greater than the saline group,while JNK and p38 activation was decreased in the lithium group.Treatment with lithium did not affect the total cellular levels of ERK,JNK,and p38.Apoptotic protein caspase-3 were determined by western blotting in present study.Compared with saline group,caspase-3 cleavage in the lithium group was inhibited in 0.5h,6h after reperfusion.The expression of LC3 and p62 were determined by western blotting,which relative with autophagy.Both groups of LC3 increased after reperfusion,while higher in the lithium group in reperfusion phase compared with the saline group.Respectively,the expression of p62 decreased in both groups,while lower in the lithium.[Conclusion] Lithium protect the liver from IRI.The protective mechanism of lithium is inhibition of inflammatory,activation of GSK3β and hepatocellular apoptosis,modulation of MAPK,and induction of autophagy.PART THREE The Protection and Mechanism of Lithium for Steatotic Liver Ischemia/Reperfusion Injury in Rats[Objective] NAFLD are particularly vulnerable to ischemia/reperfusion injury(IRI),which affects liver function after hepatic surgery.In this study,the protection and mechanism of Lithium for steatotic liver ischemia/reperfusion injury in rat will be investigated.[Mothods] Feeding S-D rats with MCD+HF for 2weeks induce moderate hepatic steatosis.There were two groups,lithium group and saline group.Rats started treatment with lithium 3 days before warm IRI performed,and they were continuously injected to a daily lithium injection for up to 2 days.Then all rats were given 60 min selective warm ischemia,continuously harvested after 0.5h,6h,24 h,48h reperfusion respectively.Left lateral lobe and right superior lobe were collected for histological analysis.Right median lobe and serum were collected for molecular biological analysis.Right median lobe and serum were collected for molecular biological analysis.Rehydrated sections were stained by using the naphthol AS-D chloroacetate esterase technique to investigate the neutrophil infiltration in the liver after reperfusion.10 high-power fields(HPF,400×)were selected randomly for quantitative analysis.ASDCL-positive neutrophils were counted manually.HMGB1 translocations were determined by HMGB1 negative staining in hepatocellular cytoplasm and nuceus.The expression of GSK3β,p-GSK3β/Ser9,ERK,p-ERK,p38,p-p38,JNK,p-JNK,caspase-3,LC3,p-62 were tested by western blotting respect to various reperfusion times.Then,the influence of lithium subject to inflammatory,autophagy and the signal pathway of GSK3β and MAPK will be analysed.[Result] Feeding S-D rats with MCD+HF for 2weeks induced moderate hepatic steatosis successfully.Liver function,include ALT and AST serum levels,were tested after 0.5h,6h,24 h,48h reperfusion.After 60 minutes of warm ischemia injury,both serum ALT and AST level was increased as early as 0.5 hours after reperfusion,reached a peak at 6 hours,and decreased thereafter.Lithium treatment did significantly decrease I/R injury,as indicated by the lower liver enzyme levels compared with saline group at different reperfusion time respectively(ALT: 402±120 vs 616±171,p<0.05;450±102 vs 860±134,p<0.05;310±52 vs 402±44,p<0.05;198±34 vs 210±35,p>0.05;AST: 391±127 vs 598±139,p<0.05;657±178 vs 1123±234,p<0.05;610±56 vs 178±18,p<0.05;788±98 vs 245±36,p<0.05;unit: IU/L).The expression of LC3 and p62 were determined by western blotting,which relative with autophagy.Before warm ischemia,the expression of LC3-II decreased in moderate steatotic liver,and p62 increased respectively.Both groups of LC3 increased after reperfusion,while higher in the lithium group in reperfusion phase compared with the saline group.Respectively,the expression of p62 decreased in both groups,while lower in the lithium.Phosphorylation of GSK3β were determined after 60 minutes of warm ischemia followed by various periods of reperfusion in moderate steatotic liver.Phospho GSK3b/Ser9 levels had rapidly decreased at 0.5 hours,and reached the peak at 24 h.In the lithium treatment group phospho GSK3b/ Ser9 levels were higher than the saline group.The total cellular levels of GSK3 b were not affected.The HMGB1 immunohistochemical analysis indicated that HMGB1 translocated from nucleus and cellular cytoplasm.HMGB1 negtive nuclear(%),lithium group: 3.42±1.04%,saline group: 6.18±1.21%,p<0.01.Lithium treatment group showed lower serum HMGB1 levels by ELISA than saline group in 0.5h,6h after reperfusion significantly.The number of ASDCL staining-positive neutrophils infiltrating the liver was determined 24 hours after reperfusion neutrophil infiltration was significantly higher in the saline-injected rats versus the rats subjected to lithium treatment,p<0.05.The phosphorylation of ERK,JNK were assessed by western blotting after 60 min I/R.The phosphorylation of ERK,JNK was increased 0.5 hours after reperfusion in the saline group.In contrast,the phosphorylation of ERK in the lithium group was greater than the saline group,while JNK activation was inhibited in the lithium group.Treatment with lithium did not affect the total cellular levels of ERK,JNK.Apoptotic protein caspase-3 were determined by western blotting in present study.Compared with saline group,caspase-3 cleavage in the lithium group was inhibited in 0.5h after reperfusion significantly.[Conclusion] Lithium pretreatment protect the steatotic liver from IRI.The protective mechanism of lithium is inhibition of inflammatory,activation of GSK3β and hepatocellular apoptosis,modulation of MAPK,and induction of autophagy.Lithium pretreatment may a simple and applicable strategy for protecting against liver IRI in steatosis.It may be promising in the setting of transplantation to expand the choice of donors and to reduce the risk of postoperative morbidity and mortality after surgery.