The Mechanism Of Activating PPAR? To Inhibit The Occurrence And Development Of Cholesterol Stones And Non-alcoholic Fatty Liver

Background:Gallstone disease is one of the most prevalent and expensive digestive diseases.In western countries,the prevalence of gallstone disease is about 10%-20%.According to statistics,while in our country the incidence is 8%-10%,and the incidence increases fast annually due to the lifestyle change.Gallstone disease burdens more to our health care system.CGD is a multifactorial disease caused by the interaction of several poorly defined factors.Among these factors,dysfunction of metabolism of liver,gallbladder and intestine play great parts in cholesterol gallstone formation.Liver is the major place for bile synthesis.Bile is consist of water,bile acid,phospholipids,cholesterol,sodiun,kalium,calcium,phosphate,carbonate and protein.Cholesterol is only slightly soluble in water and its hydrophilicity increases only when it is mixed in appropriate proportions with BA and phospholipids and consequence,forms a mixture in micelle status.Cholesterol crystals form when the balance between cholesterol,bile acid(BA),and phospholipids in bile is disrupted.These crystals grow and finally aggregate to form gallstones.As previous studies established,five conditions promote the formation of cholesterol crystals:(1)biliary cholesterol supersaturation due to hypersecretion of cholesterol by the liver;(2)enhanced intestinal cholesterol absorption;(3)relative reduction of BA and phospholipid content in biliary bile,with resultant decrease in bile hydrophilicity;(4)biliary stasis due to impaired gallbladder motility,accompanied by iumunomediated gallbladder inflammation;and(5)genetic defects.The bile salt export pump(BSEP),is the major canalicular BA transporter,while ABCB4 is responsible for the transportation of phospholipids.Gene defects on ABCB4 or BSEP could attribute to abnormal secretion of either bile acid or phospholipids,resulting in cholesterol crystal or cholesterol gallstone formation.All these transporters are subject to the transcriptional regulation of several hepatic nuclear receptors(such as LXR and FXR).These nuclear receptors also play great roles in hepatic lipid metabolism.Further study on these nuclear receptors may provide a new approach to the pathophysiological progress of cholesterol gallstone formation.Peroxisome proliferator-activated receptor-gamma(PPAR?),is one of the PPAR family,which are members of the nuclear receptor superfamily of ligand-inducible transcription factors.By regulating with several relative genes,the PPARs control the expression of networks of genes involved in adipogenesis,lipid metabolism,inflammation,and maintenance of metabolic homeostasis.Since these metabolic dysfunction share the same high risk pathogenic factors with cholesterol gallstone,we hypothesize that PPAR? may play great role in all these metabolic dysfunction including cholesterol gallstone disease.Objective:To explore the relationship between PPAR? and cholesterol gallstone incidence.High fat high cholesterol diet induced CGD will be used for the detection of CGD incidence and several metabolic parameters through activation of PPAR?.Key enzymes like ABCG5/ABCG,BSEP,NPCIL1 involved in enterohepatic circulation and cholesterol transportation will be detected for the exploration of PPAR?regulative effect and the potential mechanism.The mechanism involved in PPAR?regulation on enterohepatic circulation through PPAR ?-LXR-FXR and their downstream target genes will be identified.Methods:1.In vitro,Real-Time PCR was used to measure transcriptional expression of LXRa,FXR,ABCG5/ABCG8,ABCG1,CYP7A1,BSEP in human hepatocytes cell line L02 followed by overexpressing PPAR?(LO2pre).In addition,transcriptional expression of ABCG5/ABCG8 and BSEP were also measure followed by downregulating LXRa or FXR in LO2pre cell line.2.In vivo,Twenty-eight male C57/B6 mice were randomly divided into 4 groups,normal diet group(NC group,n=7),normal diet and medicine group(CM group,n=7),lithogenic diet group(L group,n=7),lithogenic diet and medicine group(LM group,n=7).Mice in NC group were fed on normal diet,while mice in CM group were fed on normal diet and Pioglitazone.Cholesterol gallstones were induced in mice of the latter two groups by feeding lithogenic diet.In addition,mice in LM group were also intragastricly administered Pioglitazone as that in CM group.After 10-week treatment,all mice were killed and sampled to calculate the incidence of stone formation and measure metabolic parameters including TC,TG,HDL,LDL,ALT,AST.3.Bile was collected to caculate bile cholesterol saturation index(CSI).Gallbladders were collected for hematoxylin-eosin staining.4.Fecal was collected to measure bile acid salt and cholesterol concentration.5.Real-Time PCR was used to measure transcription of PPAR Y,LXRa,FXR,ABCG5/ABCG8,ABCG1,CYP7A1,BSEP in liver and PPAR ?,ABCG5/ABCG8,NPC1L1 in intestine.6.Imnunohistochemistry was used to detect expression of PPAR ?,ABCG5/ABCG8,BESP in liver and PPAR ?,ABCG5/ABCG8,ASBT5 NPC1L1 in intestine.Results:1.In vitro,transcription levels of ABCG5/ABCG8,LXRa,FXR,ABCG1,CYP7A1,and BSEP were significantly increased in L02 cells parallel to overexpression of PPAR ?.In LO2pre,transcription levels of ABCG5/ABCG8,ABCG1 and CYP7A1 were reduced by downregulating LXR?,and transcription levels of BSEP were also reduced by downregulating FXR;2.Gallbladder inflammation of mice in L group was more severe than that in LM group,and there was no gallbladder inflammation in both NC and CM groups;3.Liver histological examination of mice in L group showed significant symptoms of NAFLD(nonalcoholic fatty liver disease)compared with the other groups.Mice in L group gained larger body weight,liver weight,higher levels of serum TC,TG and lower levels of serum HDL,blood bile acid,and the metabolism profile could be obviously improved by pioglitazone treatment in LM group;4.Instead of higher concentrations of bile cholesterol and biliary CSI index,mice in L group showed lower bile acid content than that in LM group.;5.There were decreased transcription levels of PPAR y,LXRa,FXR,ABCG5/ABCG8,ABCG1,CYP7A1,BSEP in liver in L group compared with NC group,while those transcription levels were enhanced by pioglitazone treatment in LM group.There were decreased transcription levels of PPAR ?,ABCG5/ABCG8 and increased transcription levels of NPC1L1 in intestine in L group compared with NC group,and this trend could also be reversed by pioglitazone treatment in LM group.Mice in L group showed decreased expression of PPAR ?,ABCG5/ABCG8,BSEP in liver as well as decreased expression of PPAR ?,ABCG5/ABCG8 and increased ASBT and NPC1L1 in intestine compared with LM group.6.The fecal BA content in L group mice was significantly lower than L group,wheras the fecal cholesterol content was higher in comparison with L group mice.Conclusion:The anti-lithogenic effect of PPAR ? was mainly based on the regulation through the PPAR ?-LXRa-ABCG/NPC1L1 and PPAR ?-FXR-BSEP/ASBT pathways,resulting in enhanced BA synthesis and enterohepatic circulation which benefit the high content of BA in group LM mice bile.Intestinal cholesterol absorption was prevented through the PPAR ?-LXR?-ABCG/NPC1L1 regulation route.The anti-NAFLD effect was mainly due to an enhanced hepatic cholesterol clearance though PPAR ?-LXR?-ABCG5/G8 pathways..