| Objective: Non-alcoholic fatty liver disease (NAFLD) isa disease whose incidence is increased year by year, posing a seriousthreat on human health in rencent years,Its pathological changes is similarto that of alcoholic liver disease (ALD), but NAFLD patients has nohistory of excessive alcohol consumption.its pathological changes areliver cell inflammation, necrosis or apoptosis,even steatohepatitis,liverfibrosis and liver cirrhosis. Studies suggest that non-alcoholic liverdisease is a stress-induced metabolic liver injury, which is closely relatedto insulin resistance and genetic susceptibility.The purpose of thisexperiment was aimed to establish model of nonalcoholic fatty liverdisease rat model through an improved high-fat diet in which continuousdynamic time points of liver cell apoptosis, inflammation, fibrosis, and itsexpression of related factors BCL-2, NF-KB, angiotensin Ⅱ-1receptorwere observed,and expression of BCL-2,, angiotensin Ⅱ-1receptor werestudied after inhibition of NF-KB. so that it might reveal the roles and itsmechanism of hepatocytic apoptosis in the pathogenesis of NAFLD inorder to provide new evidences for studying the pathogenesis andtherapy management of NAFLD. Methods:fotrty-two healthy adult maleSpague-Dawlay (SD) rats were divided into threes groups randomedly,: normal group (normal diet), model group, the intervene group (10weeksafter high-fat diet feeding.then the PDTC intraperitoneal injection),6ratsin each group were sacrificed respectively at6th,10th,14th weekend.Blood was collected through heart and serum lipids and serumaminopherase were determined, in order to observe the progress ofhepatic steatosis of NAFLD model.After liver tissue were taken, liverindex was calculated as follows: liver wet weight/body weight100%,and paraffin sections of liver tissue specimens were prepared,hematoxylin-eosin (HE) staining was made, pathological changes inliver tissue, and liver fibrosis were observed by light microscope;thepercentage of hepatocyte apoptosis was measured by TUNEL method andBcl-2, NF-KB expressions in the liver tissue were detected withImmunohistochemical method;the expression of angiotensin Ⅱ-1receptor in the liver tissue was detected by Reverse Transcription-Polymerase Chain Reaction(RT-PCR) method. The measurement datawere expressed as mean±standard deviation (ˉx±S), two sample t testwas used to compare the normal group and model group group ofdifferent time periods; paired t test was used to compare the differencebetween the intervention group and the model group, analysis of variancewas used to compare the model group with the normal group.Homogeneity of variance was analysized with single factor analysis ofvariance, H variance analysis was used to compare the variance. P <0.05 was statistically significant.Results：None of Rats had deaths, all datawere analysized.（1）With the modeling time extending, the model ofNAFLD were constructed successfully after6weeks and10weeks. liverfibrosis models in four rats were made in the model group, fibrosismodel in one rat was made in the intervention group at14weeks.（2）With time of the model extending, body weight, liver index, serum lipidand serum transaminase level in the model group rats was increasedsignificantly, liver steatosis, inflammation and fibrosis were aggravatedgradually.While in the intervention group,the body mass, rat liver index,serum lipid and transaminase levels were not incrased obviously thanthose in the model group.（3）In the model group animal liver tissuesteatosis degrees were aggrevated at6,10,14weeks with the modelingtime increasing, it was significantly higher than in normal group(P<0.01);in the model group,different degree of necrosis of liver cellswas visible and small leaves, punctate inflammation, focal necrosis withobvious ballooning degeneration,Partial necrosis and confluent necrosiswere observed,in model group liver inflammatory activity scores at6,10,14week were higher than that in normal group (P<0.01). Activeinflammation in liver tissue in the intervention group were lower thanthose in the model group (P<0.05).HE staining showed that only at14weeks, liver fibrosis in4/6rats of model group and in1/6rats ofintervene group were observed.(4)in Model group liver cell apoptosis percentages were increased significantly than in normal group at6weeks,10weeks,14weeks(11.15±0.82、16.19±1.23、19.23±2.31vs1.11±0.15、1.26±0.11、1.15±0.20),(P<0.01);Immunohistochemical staining showedthat BCL-2protein was brown in the cell membrane or cytoplasmicexpression in model group with time prolonged, fatty degeneration andinflammatory degree were aggravated and staining was deepened,patchy distribution was expanded;Expression of Bcl-2in normal groupwere scattered in weakly positive,.In model group at6,10,14weeks thenumber of positive cells was gradually increased.In the interventiongroup the expression of Bcl-2was lower than in model group at14weeks(P<0.05).NF-kappa B cells stained yellow or ensemble yellow aspositive, localized in the cytoplasm and (or) nucleus，The model groupshowed NF-kappa B was activated6,10,14weeks in liver cells of rats,compared to the same period the difference between the normal groupwas statistically significant (P<0.01），But with the model with time, itsexpression increased, the difference was statistically significant(P<0.05)，14weeks of intervention group expressed by over14weeksmodel group was inhibited, but the expression is also compared with thenormal group significantly increased (P<0.05).(5)Detection of AT1RmRNA in liver with RT-PCR:AT1R mRNA expression in the modelgroup was increased gradually with the model time prolonged, and at14weeks the expression was significantly different compared with at10 weeks,and at10weeks it also significantly different compared with at6weeks(p<0.05).In module group AT1R mRNA expression wassignificantly higher than in the same period of time of the normal group(p <0.01).Conclusion:1.In NAFLD model group, apoptosis,inflammation and fibrosis were more obvious, the expression of the Bcl-2,NF-KB, angiotensin II-1receptor were increased.2.After NF-KB indevelopment of NAFLD is mediated, hepatic inflammation, fibrosis isinhibited.At the same time, expression of Bcl-2, angiotensin Ⅱ-1receptor expression are decreased.NF-KB can regulate the Bcl-2,angiotensin II receptor expression in NAFLD, play a role in thepathogenesis of NAFLD. |
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