The Coexistence Of Hypcrlipidemia And Acute Cerebral Ischemia/Reperfusion Induces Severe Liver Damage In A Rat Model

Objective: To investigate the effect of acute cerebral ischemia/reperfusion injury （I/R） onhyperlipemia （HL） induced liver damage and its mechanism.Methods:1. The rats were divided into four groups:（1） control group,（2） hyperlipidemia （HL） group,（3）cerebral ischemia/reperfusion （I/R） group,（4） hyperlipidemia combined with cerebralischemia/reperfusion （HL+I/R） group.1.1Animals in control group and cerebral I/R group were given normal diet, while rats in HLgroup and HL+I/R group were fed with a high-fat diet（HFD） for18weeks. During18weeksof HFD, blood was withdrawn from the tail vein of overnight fasted rats. The serumtriglycerides （TG）, cholesterol （TC）, high-density lipoprotein cholesterol （HDL-C） andlow-density lipoprotein cholesterol （LDL-C） were detected using commercial kits by anautoanalyzer weekly in order to evaluate the model of HL.1.2After the establishment of HL model, the qualified rats were randomly divided into HL andHL+I/R group. The rats in I/R and HL+I/R group were subjected to middle cerebral arteryocclusion （MCAO） for2h and followed by24h reperfusion to induce focal cerebral I/Rinjury. The method of triphenyltetrazolium chloride （TTC） staining and the Longa’s scorewere employed to detect the infarct volume and the neurological deficit, in order to evaluatethe model of I/R. The rats whose infarction volume and neurological deficit had statisticalsignificance compared with the sham control group were used in the following experiments.2. Blood samples were obtained from the common carotid artery for detecting serum alanineaminotransferase （ALT）, aspartate aminotransferase （AST） to assess the liver function whenrats were sacrificed at24h after reperfusion. HE staining was used to analyze the extent ofliver injury.3. After high-fat diet treatment for18weeks and/or2h transient focal cerebral ischemiafollowed by24h reperfusion, serum tumor necrosis factor-α（TNF-α） and interleukin-1（IL-1）were detected to reflect inflammatory status. Liver subcellular fractions were prepared bydifferential centrifugation. Liver mitochondrial superoxide dismutase （SOD）, glutathioneperoxidase （GSH-Px）, malondialdehyde （MDA） and Ca²⁺levels were measured to reflectoxidative/antioxidative status. And CYP2E1-dependent aniline hydroxylase activity inmicrosome was measured; the expression of CYP2E1in liver tissue was determined byimmunohistochemistry and Western blot.4. Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-endlabeling （TUNEL） assay. The expression of genes related to apoptosis （caspase-3, bcl-2） inliver was assayed by immunohistochemistry and Western blot.Results:1. After18weeks of HFD and prior to ischemia, serum levels of TC, TG and LDL-C in HL orHL+I/R group were significantly higher than those in control or I/R group, whereas HDL-Cwas lower （p<0.05or p<0.01）. And there was no significant difference between HL group and HL+I/R group. And after24h reperfusion, rats in I/R or HL+I/R group appearedsignificantly higher neurological deficit score and brain infarct volume than those in controlor HL group （p<0.01）. These results indicated that the animal model of HL combined withI/R was established successfully.2. As compared to control group, plasma activities of ALT and AST were elevated in HL group（p<0.05or p<0.01） but not affected significantly by I/R alone; marked changes in serumALT and AST activities suggesting hepatic damage were shown in HL+I/R group （p<0.01）.The most severe liver damage was created in HL+I/R group compared to HL or I/R alone（p<0.01）. HE staining also showed that no apparent damage was found in liver sections fromthe control or I/R group. Hepatocytes of HL group showed fatty infiltration changes, lightvacuolar change, scattered inflammatory cells and mild hepatocytes necrosis. However,extensive damage was detected in liver sections from HL+I/R rats.These results suggestedthat acute cerebral I/R injury can aggravate the liver injury caused by HL.3. SOD and GSH-Px in liver mitochondria were lower, MDA and Ca²⁺levels were higher in HLgroup than control （p<0.01）. Liver mitochondrial SOD also decreased and Ca²⁺levelincreased in I/R group （p<0.01vs control）. HL plus I/R enhanced serum TNF-α, IL-1, livermitochondrial MDA and Ca²⁺levels, suppressed SOD and GSH-Px in liver mitochondria,markedly up-regulated the activity and expression of CYP2E1in liver when compared to HLor I/R alone（p<0.05or p<0.01）, suggesting cerebral I/R can aggravate HL-triggeredsystemic inflammation and the imbalance in oxidant production and antioxidant defensesystems.4. TUNEL-positive cells appeared occasionally in liver sections of control or I/R group,diffused widely in those of HL group, and were most frequently observed in those of HL+I/Rgroup. Protein expression of caspase-3in livers of HL and HL+I/R rats had a markedelevation, and the anti-apoptosis gene bcl-2was dramatically decreased. The changes inHL+I/R group had statistical difference when compared to HL or I/R alone（p<0.05orp<0.01）,suggesting cerebral I/R can enhance HL-activated hepatocyte apoptosis.Conclusions:The coexistence of HL and acute cerebral I/R induced severe liver damage in experimental HL ratmodel with cerebral I/R, and this effect is possibly due to enhanced CYP2E1induction whichfurther promotes oxidative damage, inflammation and hepatocyte apoptosis.