The Role And Significance Of UPR Combined With SREBP-1c Mediated By Endoplasmic Reticulum Stress In The Development Of NASH In Rats

Nonalcoholic fatty liver disease(NAFLD) is one of the most common hepatic disorders in China,which encompasses a disease spectrum ranging from simple hepatic steatosis to steatohepatitis(NASH),fibrosis and cirrhosis.NASH is a condition characterized by inflammation,hepatocellular necrosis and excessive deposition of fat in the liver.NASH usually has a poor prognosis,mainly because of fibrosis and progression to liver cirrhosis. So far,the mechanisms involved in the pathogenesis of NASH have not been thoroughly investigated.The "two-hit hypothesis" theory is the most widely accepted explanation for the pathogenesis of NASH,while insulin resistance,oxidative stress and lipid peroxidation are key factors.Hepatocytes contain abundant endoplasmic reticulum(ER) which is the main organelle for protein synthesis and modifications,calcium signaling and the biosynthesis of lipids. The ER is extremely sensitive to each kind of stimulation,the protein misfolding and unfolding caused by organism function disorder result in the accumulation of proteins within the ER and induced the endoplasmic reticulum stress(ERS) which is processed through a number of signaling pathways including the unfolded protein response(UPR),the ER overload response(EOR) and the sterol regulator element binding protein(SREBP) pathway.In recent years,activation of ER stress has been implicated in the pathogenesis of metabolic syndrome(MS) such as obesity,insulin resistance and type 2 diabetes in many studies.However,few researches were found the role of ER stress in the development of NASH which was strongly related to MS.Previous studies have shown that fatty acid metabolism generate reactive oxygen species(ROS) molecules in ER and the increased production of ROS is associated with known trigger of ER stress.Thus,we hypothesized that high fat diet could trigger ER stress via ROS and lipid peroxide which generated from overloaded fatty acid metabolism.ER stress causes the activation of UPR and SREBP pathway.Once activated,SREBP-1c act as transcription factors that regulate the genes that control the synthesis of fatty acids and cellular uptake of lipoproteins;when the hepatocytes cannot cope because of the great extent or duration of ER stress,the ER stress response unleashes pathological consequences including hepatic fat accumulation,inflammation and cell death which can lead to NASH. The aim of the present study was to detect the expression of the markers of UPR and SREBP signal pathway in ER stress in the pathogenesis of experimental fatty liver induced by high fat diet in rats.The main methods,results are as follows:Methods:Thirty male Wistar rats were randomly divided into two groups designated as control Group C and Group NASH.The former was fed with standard diet;while the latter was fed with high fat diet(82.5%standard diet,10%lard oil,2%cholesterin,5%sugar and 0.5%bile salt).Group NASH was divided into 4 subgroups(4,8,12,16weeks)(n=6 in each group).Observation on each group continuously:①The morphous and histopathology of liver was observed.②The levels of free fatty acid(FFA),triglyceride(TG),alanine aminotransferase(ALT),aspartate aminotrasferase(AST) in serum were measured.③The levels of free fatty acid(FFA),malondialdehyde(MDA) and ROS in liver tissue were detected.④The mRNA levels of ER stress marker glucose-regulated protein(GRP78), UPR marker activating transcription factor 6(ATF6) and SREBP pathway marker SREBP-1c in rat liver tissue were detected by reverse transcription polymerase chain reaction(RT-PCR).⑤The mRNA levels of GRP78 and ATF6 were detected by real-time quantitative RT-PCR(qRT-PCR).⑥The protein levels of GRP78,ATF6,Phospho-PERK and SREBP-1c in rat liver tissue were detected by Western blot.Results:1.Histological analysesThe results showed that the morphous and histopathology of liver in Group C were normal.But visible augmentation as well as was felt yellow and greasy in the morphous of liver were emerged after 4,8,12,16 weeks when the rats were fed with high fat diet;In contrast to Group C,the hepatic lesions in Group NASH was characterized by steatosis, ballooning and lobular inflammation,predominantly initially in zone 3 of the acinus,gradually into the pericentral region and relative sparing of portal areas.According to Guidelines for diagnosis and treatment of nonalcoholic fatty liver diseases,the rats with high fat diet for 4, 8,12 and 16 weeks were pathologically defined as NASH-F1G0 stage,F2G1 stage,F3G2 stage,F4G3 stage.2.Biochemical analysesThe levels of the FFA,TG,AST in the serum were raised gradually in Group G0 than those in Group C after the rats were fed with high fat diet 4 weeks.The levels of the FFA, TG,AST,ALT in the serum were significantly higher both in Group G1 and G2 than those in Group C with time prolong(P＜0.05,P＜0.01 respectively) and reached their peaks at week 16 in Group G3(P＜0.01).3.The changes of MDA,FFA and ROS in liver tissueThe level of the MDA in liver tissue showed obvious up-regulation in Group NASH than that in Group C(P＜0.01).The levels of the FFA,ROS showed elevated tendency after fed with high fat diet 4 weeks and reached their peaks at week 16 in Group G3(P＜0.01).4.The expression levels of GRP78,ATF6 and SREBP-1c genes were detected by RT-PCR on mRNAsThe mRNA expression levels of GRP78 and SREBP-1c increased gradually with the progression of hepatocellular steatosis and inflammation after the rats were fed with high fat diet 4 weeks(P＜0.05,P＜0.01 respectively),and reached their peaks in Group G3(P＜0.01).It was found that the mRNA expression level of ATF6 had significantly increased in 8 to 16 weeks(P＜0.01),while no significant difference was found in 4 weeks in Group G0 compared with Group C(P＞0.05).5.The mRNA levels of GRP78 and ATF6 were detected by qRT-PCRThe results indicated that the mRNA expression levels of GRP78 and ATF6 were obvious up-regulation in Group NASH than those in Group C(P＜0.05),which correlated with RT-PCR results.6.The expression levels of GRP78,ATF6,Phospho-PERK and SREBP-1c were detected by Western blot on proteinsThe protein levels of GRP78,ATF6,SREBP-1c started to increase after the rats were fed with high fat diet 4 weeks and reached their peaks at week 16 in Group G3(P＜0.01).The level of PERK phosphorylation showed no significant change in Group G0 compared with Group C but it reached its peak in Group G3(P＜0.01). Conclusion:1.High fat diet leads to an increase of FFA.Large amount of ROS,MDA were generated in the oxidation of FFA,which resulted in the excess accumulation of lipid peroxide in liver and the increase of stress factors related to ER stress.2.As the hepatocellular steatosis and inflammation aggravated in the development of NASH,the expression levels of GRP78,ATF6,Phospho-PERK and SREBP-1c increased.3.The increase of stress factors such as ROS resulted from high fat diet activated the UPR combined with SREBP-1c and then led to excessive ERS.It appeared to the elevation of expression in GRP78,ATF6,Phospho-PERK and SREBP-1c,then enhance the genetic transcription of enzymes related to fatty acid metabolism and finally led to the development of NASH.