The Effect And Mechanism Of Lipid Droplet Associated Protein Perilipin 2 On Non-alcoholic Fatty Liver Disease And Its Influence On Blood Lipid Metabolism

It always causes a huge trouble that the morbidity of metabolic syndrome characterized by non-alcoholic fatty liver disease（NAFLD）,coronary heart disease and other manifestations shows a yearly increasing trend with the improvement of living standards and changes in eating habits^[1].The morbidity of NAFLD in the major western countries was 20%³0%^[2]while that in China was 15.5%³3.9%^[3].In recent years,it is found in basic and clinical studies that NAFLD and coronary heart disease,as the two manifestations of metabolic syndrome,are not only similar in risk factors^[4],but also the former as independent clinical risk factors is involved in the development and progression of coronary heart disease possibly through the secretion of inflammatory factors,affecting blood lipid metabolism,insulin resistance,etc.^[2,5-8].Furthermore,NAFLD is also associated with atrial fibrillation,QT interval prolongation and so on^[7,9-12].Therefore,the study in the prevention and treatment of NAFLD are able to help to treat patients with NAFLD,to improve blood lipid metabolism and correct insulin resistance,but also facilitate to reduce the incidence of coronary heart disease and other cardiovascular disease,and even provide a new prospective in the prevention and treatment of cardiovascular disease.CGI-58（Comparative Gene Indentification-58）,also named as Abhd5,is the fifth member of unfolded protein withα/βhydrolytic enzyme family^[13].It is widely expressed in various tissues,including human’s and mouse’s hepatocyte.CGI-58 mutations will lead to Chanarin-Dorfman syndrome in human beings^[14].Although CGI-58 itself does not have triglyceride hydrolase activity,it can act as a co-activator to enhance triglyceride hydrolysis by activating ATGL（Adipose triglyceride lipase）in vitro,a rate-limiting enzymes of enhydrolyzed by triglyceride hydrolysis^[14,15].The liver-specific CGI-58 knockout（LivKO）mice would display significant liver fibrosis after 42-weeks standard chow diet feeding.Histopathological manifestion includes microvesicular and macrovesicular panlobular steatosis,lobular inflammation,and centrilobular fibrosis over time^[16,17],which is highly similar to the pathological process of NAFLD.Therefore,this animal model well simulate NAFLD.We found that Perilipin 2（Plin2）was highly expressed in the liver of LivKO mice by proteomics,indicating that Plin2 may be involved in the development and progression of NAFLD.Plin2 is a member of the PAT protein family,in which five members have been found,namely Perilipin1（Plin1）,Plin2,Perilipin3（Plin3）,Perilipin4（Plin4）and Perilipin5（Plin5）^[18,19].This family is responsible for coating lipid droplets and their expression are different in tissues.Liver mainly expressed Plin2,Plin3 and Plin5,among which Plin2 are expressed the most highly^[20].Liver-specific deficiency of Plin2 can reduce hepatic steatosis and fibrosis caused by methoionine-choline-deficient diet（MCD）^[21].C57 mice showed the decrease in liver TG and the increase in hepatic insulin sensitivity after Plin2 antisense oligonucleotide treatment on high fat diet^[22-24].In the Leptin ob/ob mouse model with liver-specific Plin2 knockout,fed with,the liver steatosis induced by high-fat diet could be alleviated,and the insulin resistance was improved^[25].These suggest that Plin2 may be a potential target for the treatment of NAFLD.However,it is unclear that whether the inhibition of Plin2 expression in LivKO mice can alleviate hepatic steatosis and reduce fibrosis,and its impact on blood lipid metabolism have yet to be confirmed.In addition,there is a huge gap between the previous diet used in research with that used in actual human beings as without taking into account the effect of high cholesterol.Therefore,it is necessary to inhibit the expression of Plin2 in LivKO mice,observe the alteration of blood lipids and hepatic changes fed with long-term high fat and high cholesterol,and explore the mechanism of the Plin2-induced changes,so as to reinforce a research basis on the treatment of NAFLD,the improvement of dyslipidemia and the treatment of CGI-58-related diseases.Contents and Method1.Based on the LivKO mouse model,we screened the proteins that may be used to treat NAFLD and the diseases caused by the mutations of CGI-58.1.1 8 weeks old male LivKO mice and control mice were fed by Western Diet for 3weeks.After the samples of the liver were collected and pretreated,they were sent to the company for the proteomics iTRAQ.1.2 Find out the target protein according to the results of iTRAQ and the bioinformatics analysis,and test the target protein by Western Blot.2.Verify the effect of knocking out target protein on Huh7 cells,which would provide cell platform for the follow-up research on its mechanism.2.1 Crispr-cas9^[26]gene editing technology was adopted to knock out CGI-58 and Plin respectively.After that,Plin2 was further knocked out on the CGI-58^-/-cells.Then,pick up the monoclonal cells,and identify them by T7 Endonuclease I digestion analysis,Western Blot and DNA sequencing.2.2 those cells can be divided into four groups according to their gene background,namely WT group,Plin2 KO group,CGI-58 KO group and CGI-58/Plin2 KO group.After lipid extraction,the levels of triglyceride and cholesterol were tested.2.3 Observe the changes of lipid droplets’changes after fluorescent staining.the 4groups of cells were stained with BODIPY,and then we observed their lipid droplets under fluorescence microscope.3.In LivKO mice model,AAV-mediated shRNA was applied to interfere with the mRNA of Plin2 in liver.Then,we observed the morphological changes of liver,test lipid levels and blood biochemical indexes,evaluate the influence on glycometabolism and hepatic fibrosis,and explore the possible mechanisms.3.1 The six-week-old LivkO mice were divided into 4 groups（6 mice in each group）,namely Control（AAV-）,Control（AAV+）,KO（AAV-）and KO（AAV+）.Both male and female were grouped according to this method.We would inject AAV-ShRNA to the groups marked AAV+,while inject AAV-control to those marked AAV-.All of the mice were fed with Western Diet for 14 weeks.3.2 Body weight were measured weekly.The amount of food intake was measured at11 weeks of feeding.Body fat ratios were measured at the 2th week and the 12th week.GTT and ITT tests were carried out at 3th week and the 12th week.PPT was tested at the13th week.3.3 The mice were sacrificed at the 14th week after fed with Western dietn and the specimen were collected.the interference efficiency of Plin2 was tested by Western Blot and Q-PCR.the level of CGI-58 was tested by Western Blot to confirm the genotype.3.4 the lipid extraction from liver was applied to measure the levels of TG,TC,FC,CE and PL.The levels of plasma TG,TC,FC,CE,PL,ALT and AST were measured as well.3.5 HE staining was adopted to observe the morphological changes of liver,while Picro-Sirius Red staining was employed to evaluate the hepatic fibrosis.3.6 The protein levels of Plin1,Plin3,Plin4,ATGL,HSL,MTTP were tested by Western Blot,while the mRNA levels of Plin1,Plin3,Plin4,Plin5 and ATGL were measured by Q-PCR.3.7 Q-PCR was applied to test the mRNA levels of Procollagen,Col.a1,a-SMA and TGF-β.Results1.Proteomics iTRAQ and Western BlotA total of 3404 proteins were detected by iTRAQ,amongst which 3020 proteins were reliable.Plin2 was highly expressed in the liver of LivKO mice.Compared with the control group,131 proteins were up-regulated,while 51 proteins were down-regulated protein with significant differences.Thereinto,the protein level of Plin2 were increased by 1.67 times（P<0.01）and western blot confirmed the above-mentioned changes in the mice fed by western diet and chow diet.2.Test the effect of Plin2 knock-out on Huh7 cells2.1 We confirmed by sequencing the point mutation in the knockout cells edited by Crispr-as9.Meanwhile,western blot verified the the disappearance of the bands for CGI-58and Plin2.2.2 We measured the concentrations of cellular TG.The levels of Cellular TG in WT group,Plin2 KO group,CGI-58 KO group and CGI-58/Plin2 KO group were respectively49.77±2.67ug/mg protein,38.76±3.82ug/mg protein,176.31±7.52ug/mg protein and110.78±6.49ug/mg protein.There were all significant differences between different groups in pair.2.3 We measured the concentration of cellular TC.The levels of Cellular TC in WT group,Plin2 KO group,CGI-58 KO group and CGI-58/Plin2 KO group were respectively32.97±1.75ug/mg protein,40.58±3.95ug/mg protein,53.20±3.44ug/mg protein and56.90±3.58ug/mg protein.There were significant differences in WT group vs.CGI-58 KO group and Plin2 KO group vs.CGI-58/Plin2 KO group（P<0.01）.2.4 It was found that the differences existed in the cellular lipid droplets between each group by fluorescence microscope.3.The knock-down of Plin2 in liver had influence on the hepatic morphology,the levels of lipids,the blood biochemical indexes,the glycometabolism and the hepatic fibrosis in LivKO mice3.1 AAV-shRNA reduced the mRNA expression by 68.18%-72.52%and the protein expression by 52.15%-67.45%of Plin2 with statistical significance.3.2 The liver weight of male mice in Control（AAV-）,Control（AAV+）,KO（AAV-）and KO（AAV+）were 1.89±0.18g,1.66±0.15g,3.34±0.27g and 2.95±0.22g respectively,while the corresponding female mice were 1.71±0.14g,1.60±0.16g,3.14±0.26g and 2.75±0.36g respectively.For both male and female,there were significant differences in Control（AAV-）vs.KO（AAV-）and Control（AAV+）vs.KO（AAV+）.3.3 Regardless of the gender of mice,the body weight between each groups had no significant differences at either checking points.There were no obvious differences in body fat ratio in 2nd and 12th week after western diet feeding.There was only gender difference in food intake amount with 2.95±0.06g for male and 2.63±0.06g for female.3.4 There was no difference in GTT or ITT at 3rd week for male,while at 12th week,GTT,ITT and PTT（fasting 16h or 6h）all displayed differences that mainly occurred between Control（AAV-）and KO（AAV-）and between Control（AAV+）and KO（AAV+）.However,no differences existed in female.3.5 The hepatic TG of the male mice in Control（AAV-）,Control（AAV+）,KO（AAV-）and KO（AAV+）were respectively 1257.53±187.19ug/mg protein,903.64±218.24ug/mg protein,3871.94±325.52ug/mg protein and 2882.21±276.56ug/mg protein,while the corresponding value of female mice were 1187.15±231.68ug/mg protein,959.73±159.00ug/mg protein,3450.44±266.75ug/mg protein and 2543.39±270.70ug/mg protein respectively.Regardless of gender,there were significant differences between Control（AAV-）and KO（AAV-）（P<0.01）and between KO（AAV-）and KO（AAV+）（P<0.05）.The significant differences of TC,FC,CE and PL mainly existed between Control（AAV-）and KO（AAV-）and between Control（AAV+）and KO（AAV+）.3.6 There were no significant changes in plasma TG,TC,FC,CE and PL of each group irrespective of gender.3.7 The levels of ALT and AST were significantly different between Control（AAV-）and KO（AAV-）（P<0.01）and between KO（AAV-）and KO（AAV+）（P<0.01）,regardless of gender.3.8 Regardless of gender,no differences existed in the protein expression of Plin1,Plin3 and Plin4 between Control（AAV-）and Control（AAV+）and between KO（AAV-）and KO（AAV+）.There was a significant difference（P<0.05）between Control（AAV-）and KO（AAV-）in the protein and mRNA expression of Plin3.There were no differences in the mRNA expresssion of Plin5 between each group.3.9 HE staining showed that the accumulation of lipid droplets was obvious in KO（AAV-）and KO（AAV+）,and that the lipid droplets in KO（AAV+）group were less than those in KO（AAV-）group.Picro-Sirius red staining showed that hepatic fibrosis appeared in KO（AAV-）and KO（AAV+）group,and that the fibrosis in KO（AAV+）group was obviously less than that in KO（AAV-）.3.10 There were significant differences in the mRNA expression of Procollagen,Col.a1,a-SMA and TGF-βfor male.Compared with Control（AAV-）,those expression in KO（AAV-）respectively increased by 12.58,15.47,6.99 and 2.84 folds（P<0.01）,while compared with KO（AAV-）,those expression in KO（AAV+）decreased by 66.63%,58.56%,47.78 and 50.89%（P<0.01）resprestivly。Conclusions1.Hepatocyte knockout CGI-58 gene in mice can lead to the increase in the protein expression of Plin2.2.Plin2 knockout can effectively reduce the triglyceride in Huh7 cells,which may be related to the reduced lipid droplets after Plin2 knock-down,no matter whether or not CGI-58 was knocked out.3.Knockdown of Plin2 reduced the hepatic triglyceride effectively,and reduce the liver injury and fibrosis induced by high-fat and high-cholesterol diet after CGI-58knockout,which is relevant to the reduced expression of fibrosis factor after knockout.Plin2 is expected to become a potential target for the prevention and treatment of NAFLD and NAFLD related diseases.4.Combined with literature results^[22,23],Plin2 knockdown neither significantly affect blood triglyceride,total cholesterol,free cholesterol,cholesterol ester and phospholipids,nor glucose metabolism,body weight,and body fat ratio,indicating that it was safe as a potential target.5.In LivKO mice model,the phenotype of hepatic fibrosis can be simulated earlier by Western Diet feeding,which will facilitate to the relevant research.Meanwhile,it suggests that high-fat and high-cholesterol diet has greater damage on the patients with the diseases caused by CGI-58 mutations.