Study On Spontaneous Lipid Deposition And Multi-tissue Inflammation Induced By Atp Binding Cassette Transporter A1 Gene Knockout In Hamsters

Objective:Cardiovascular and cerebrovascular diseases are the primary fatal diseases of global population.Atherosclerosis（AS）is the most important pathological basis.Low levels of high density lipoprotein cholesterol（HDL-C）and dysfunction are one of the independent risk factors of AS.HDL protects artery form AS via many mechanisms including reverse cholesterol transport（RCT）.Adenosine triphosphate binding cassette transporter A1（ABCA1）,a very important membrane protein maintaing intracellular cholesterol homeostasis,initiates the free cholesterol efflux via active transportmanner.The mutantion or dysfunction of ABCA1 could cause systemic diseases related to cholesterol metabolism disorder,such as Tangier disease（TD）,Alzheimer disease（AD）,coronary artery disease（CAD）,type 2 diabetes mellitus（T2DM）.To clarify the molecular mechanism of ABCA1mutation or dysfunction related diseases,a human like lipid metabolism animal model is needed.After cooperation with Hebei Medical University and Peking University,an ABCA1 knockout golden hamster model was established.By studying the spontaneous arterial lipid deposition and multi-tissue inflammation in the ABCA1 knockout hamster model,we explored the possible mechanism of ABCA1 gene mutation-related diseases.Materials and methods:Hamster strain is Syrian golden hamster.ABCA1 heterozygote(ABCA1^+/-)hamsters came from Professor Liu Guoqing,Department of Medical Sciences,Peking University.The following experimental hamsters ABCA1^-/-,wild type（WT）hamsters came from the Institute of Atherosclerosis,Shandong First Medical University.The temperature and humidity of the hamster room is well controlled,and the light and darkness cycle was set as 14/10 hours,respectively.The hamsters were used in the experiment after genotyping at the age of 6 weeks.Thirty WT and ABCA1^-/-male hamsters were selected.At the age of16 weeks,twenty hamsters were fed by Chow diet（CD）,10 hamsters were fed by high cholesterol high fat（HCHF）（0.2%cholesterol+15%lard）.At the age of 20-week,the hamsters were sacrifieced.All animal tests were approved by the Ethics Committee of Experimental Animals of Shandong First Medical University and followed the National Guidelines for Animal Care and Use.1.The body weight of ABCA1^-/-,WT hamsters was recorded every two weeks,and the weight of organs was recorded after sacrifieced.2.Routine blood test:Hamster blood was collected from medial canthal venous plexus after anesthesia,and blood routine was detected by Hematology analyzer.3.Oral glucose tolerance test（OGTT）:At 20 weeks of age,hamsters fasting for 12hours,blood glucose test paper was used to detect the impaired glucose tolerance of hamsters after oral glucose for 0 min,30 min,60 min and 120 min.4.³H-cholesterol reverse transport experiment:Acetylated low density lipoprotein（ac-LDL）loaded RAW264.7 macrophages were injected into two kinds of hamsters,respectively.The ³H-cholesterol content in serum,liver tissue and feces was measured by liquid scintillation counter,and the cholesterol transport efficiency of each group was measured,respectively..5.Determination of blood lipid and lipid profile:taking blood,separating plasma,testing total cholesterol（TC）,triglyceride（TG）,high Density Lipoprotein-Cholesterol（HDL-C）,low density lipoprotein-cholesterol（LDL-C）and other blood lipids by kit;using fast protein solution;The lipid profile was measured by Fast protein liquid chromatography（FPLC）following the cholesterol content determination.6.Protein isolation,electrophoresis and Western blot（WB）:ABCA1^-/-,WT-type hamster plasma and liver were harvested to detect the levels of interleukin-1 beta（IL-1beta）,interleukin-6（IL-6）,Tumor necrosis factor-alpha（TNF-alpha）,monocyte chemoat tractant protein-1（MCP-1）,Matrix metalloproteinase-9（MMP-9）content.Hamster plasma were harvested to detect the levels of apolipoprotein A1（apo A-1）,apolipoprotein B（apo B）,and Coomassie brilliant blue was used to detect apolipoprotein E（apo E）,HDL protein.7.RNA extraction and real-time polymerase chain reaction（rt-PCR）:RNA was extracted from spleen and the transcriptional level expression of interleukin-4（IL-4）and interleukin-10（IL-10）m ENA were detected.8.HE staining:After paraffin section,HE staining was used to observe the hi-stomorphology of liver,spleen and kidney,respectively.9.Oil red O staining:The aortic root was frozen and the lipid plaques on the inner surface of aortic root and aorta were observed by oil red O staining.Statistical analysis:The experiment was repeated three times or more,and the results were analyzed by Graph Pad software.The data are representation by mean±Standard error.The data between the two groups were analyzed by t-test,and the data between multiple groups were analyzed by one-way ANOVA.P<0.05 had statistical significance.Result:1.The body weight of WT hamsters increased with animal growing,and the weight of ABCA1^-/-hamsters decreased after 12 weeks.The weight of ABCA1^-/-hamster was lower than WT hamster.The weight of liver,back fat,abdominal fat and perigonadal fat of WT hamster was significantly higher than that of ABCA1^-/-hamster.2.Mean Corpuscular Hemoglobin Contention（MCHC）（g/L）of ABCA1^-/-hamster erythrocyte in blood routine was significantly higher than that of WT hamster.3.ABCA1^-/-hamsters took oral glucose for 30 minutes and the blood sugar concentration for 60 minutes was higher than that of WT hamsters.4.³H-cholesterol reverse transport experiment:The plasma ³H-cholesterol content of WT hamsters was significantly higher than that of ABCA1^-/-hamsters at all time points.The ³H-cholesterol in the feces and livers of WT hamsters was significantly higher than that of ABCA1^-/-hamsters.5.Blood lipid and lipid profile results:ABCA1^-/-hamster plasma TC and HDL-C were significantly lower than WT,TG was significantly higher than WT,LDL-C decreased,but there was no statistical difference.The above trends were not related to diet.The results of lipoprotein spectrum showed that ABCA1^-/-hamster VLDL-C increased,LDL-C decreased slightly,but HDL-C could not be detectedd.6.WB results:Whether in CD diet or HCHF diet,the expression of pro-inflammatory factors（IL-1beta,IL-6,TNF-alpha）and the expression of MCP-1 and MMP-9 in ABCA1^-/-hamsters were increased,respectively.ABCA1^-/-hamster HDL and apo A1 disappeared.ABCA1^-/-hamster apo B100 content remained unchanged,apo B48content increased,and ABCA1^-/-hamster apo E level remained unchanged.7.RNA results:The expression of ABCA1^-/-hamster anti-inflammatory factor（IL-4,IL-10）was decreased.8.HE staining results:Compared with WT hamsters,ABCA1^-/-hamsters had disordered hepatic lobule structure and hepatic cord arrangement,a large number of inflammatory cells infiltration and adipogenic cells were observed in portal area,decreased white pulp composition,decreased lymphocyte density and a large amount of hemosiderin deposition in spleen,structural disorder of kidney,enlargement of glomerular saccular cavity and infiltration of inflammatory cells in interstitium.After HCHF diet,ABCA1^-/-hamster liver,spleen and kidney sections aggravated the inflammatory infiltration and pathological changes.9.Oil red O staining:Arterial plaque deposition was found in the whole aorta of WT hamsters and ABCA1^-/-hamsters,but there was no statistical difference.After CD diet,ABCA1^-/-hamsters had more lipid deposition in aortic valve than WT hamsters.After HCHF diet,the development of AS in the aortic root of ABCA1^-/-hamster was aggravated.Conclusion:For the first time,we found that:（1）ABCA1^-/-hamster apo A1 and HDL-C almost disappeared,cholesterol reverse transport efficiency was low,but the level of TG increased significantly.（2）The expression of ABCA1^-/-hamster pro-inflammatory factor（IL-1beta,IL-6,TNF-alpha）increased,the expression of MCP-1 and MMP-9 increased,while the expression of anti-inflammatory factor（IL-4,IL-10）decreased.（3）Inflammatory infiltration of ABCA1^-/-hamster liver,spleen and kidney increased significantly,and the area of lipid plaque in aortic root increased.These results suggest that ABCA1 gene knockout in human-like cholesterol metabolic hamsters can alter the reverse cholesterol transport and the distribution of inflammatory cells,showing similar characteristics to human dangil disease.