Expression Of Sec61Alpha And Its Mechanisms In Hepatic Steatosis Model

Objective:Non-alcoholic fatty liver disease (NAFLD) is the worldwide non-infectious liver disease. Insulin resistance (IR) and the genetic susceptibility are closed to NAFLD. It encompasses the disease spectrum ranging from simple fatty liver to non-alcoholic steatohepatitis (NASH), which can progress to NASH related cirrhosis and ultimately hepatocellular carcinoma. Although the "two-hit" theory has become the classical theory about NAFLD, but the pathogenesis has not been elucidated.The endoplasmic reticulum (ER) in eukaryotic cells is an important organelle. It is involved in protein synthesis, assembly, folding, transport, intracellular calcium store, as well as participation in lipid metabolism and the synthesis of steroid hormones. Under hypoxia, oxidative stress, abnormal glycosylation and Ca2+homeostasis, unfolded protein increased, while beyond the endoplasmic reticulum processing power can lead to endoplasmic reticulum stress (ERS). ERS is divided into three types:(1) the nuclear factor κB (NF-κB) induced endoplasmic reticulum overload response (EOR);(2) the sterol regulatory cascade reaction;(3) the unfolded protein response (UPR). In the process of liver cells steatosis, ERS regulation is very important, and Endoplasmic reticulum stress (ERS) is closely related to the occurrence and development of NAFLD, the unfolded protein response (UPR) in the ERS plays a key role in the progression of liver damage caused by NASH, diabetes and other metabolic diseases. However, NAFLD how to start, the unfolded protein how to be increased by lipid load of endoplasmic reticulum, hepatic lipid overloading what more direct link between the occurrence of ERS and that these issues are not get a clear answer.Sec61protein in the endoplasmic reticulum is important channel protein, is also unfolded protein transport and subsequent unfolded protein degradation pathway (ERAD) core structure to balance the ERS maintain ER plays an important role in the complex function. It comprises α, β, γ subunits, and its main function is to form a hydrophobic channel, so that the ribosome nascent polypeptides synthesized by and through the lumen of the endoplasmic reticulum to complete the correct for folding. As a channel protein, Sec61whether changes in the model of hepatic steatosis and is associated with liver steatosis, Sec61in the physiological function of NAFLD is unclear. Therefore, this study established a mixture of palmitic acid and oleic acid induced hepatic steatosis in vitro model to detect the expression of Sec61a, Ca2+regulatory proteins glucose-regulated protein94(GRP94), Bcl-2and ERS marker proteins glucose-regulated protein78(GRP78) to explore the possible mechanism of Sec61a in the hepatic steatosis.Method:1. L02liver cells were treated with palmitic acid and oleic acid mixture (PA:OA=1:2) to establish an in vitro cellular model of NAFLD, which divided the liver cells into normal group and steatosis group, were collected at0,2,4,8,12,16h after induction for.2. lipid droplets by oil red O staining in the cytoplasm of liver cells, the use of inverted phase contrast microscope to observe the degree of change in fat; liver cells in each group were randomly selected five horizons conduct mining maps, and application Image J software for image analysis, with the average area variable in evaluation of liver cells steatosis degree.3. According to L02cells steatosis process, whether to apply Sec61inhibitor Eeyarestatin I, we divided the liver cells into normal group, the steatosis group, the inhibitor group and DMSO group.4. Use of RT-PCR and Western blot to analysis the expression of Sec61α, GRP78, GRP94and Bcl-2.5. Using SPSS19.0software correlation statistical analysis.Results:1. The degree of steatosis assessed by Oil Red O staining.After the oil red O staining under the microscope, the lipid droplets in red, and with modeling prolonged lipid droplets within the cytoplasm increased significantly, gradual integration into the point sheet, and showed increasing trend. The average lipid droplets area per cell of2h group was3.36±0.32,4h was22.53±2.16,8h was85.27±13.82,12h was253.94±21.68,16h was427.56±29.73, which was significantly higher than the control group (P<0.01). 2. The expression of Sec61a and GRP78at mRNA level detected by Real-time quantitative PCR.At the mRNA level to0h is expressed as1, Sec61α mRNA relative expression levels in early steatosis model (2h,4h, P>0.05) declined, while in the latter part of the increase (8h,12h,16h, P<0.05); GRP78in the expression showed a gradual upward trend,8h increased significantly (P<0.05), which was significantly higher (P<0.01) at12h,24h compared to0h group (P<0.01).3. The expression of Sec61a and GRP78at protein level detected by western blot.At the protein level, the expression of Sec61a protein in early steatosis model (2h,4h, P>0.05) declined, while in the latter part of the increase (8h,12h,16h, P<0.05). The expression of GRP78protein was changed gradually rising (P<0.05).4. The degree of hepatic steatosis assessed which applied Sec61inhibitor treatment by Oil Red O staining.After the oil red O staining under microscope, it was not obvious difference between steatosis group and DMSO group in the lipid variable area (P>0.05). It was significant difference between steatosis group and inhibitor group in the lipid variable area (P<0.01).5. The expression of proteins level was detected with that applied Sec61inhibitor by western blot.It was not obvious difference between steatosis group and DMSO group in the protein level (P>0.05). The expression of Sec61α and GRP78protein level in inhibitor group was significantly decreased compared with steatosis group (P<0.01). The expression of GRP94and Bcl-2protein level in inhibitor group was obviously increased compared with steatosis group (P<0.01).Conclusions:1. Palmitic acid and oleic acid mixture applied successfully established in vitro model of liver cells steatosis, with the extension of time, the cytoplasm of hepatocytes lipid droplets in the area was increasing.2. Endoplasmic reticulum stress was involved in liver cells steatosis in vitro NAFLD model. The degree of ERS was increasing with the continuous extension of time.3. Sec61channel proteins in the endoplasmic reticulum decreased in the early stage (2h,4h) in the process of hepatic steatosis, although ERS happened, but to a lesser extent, Sec61may be an adaptive change. In the late stage hepatic steatosis (8h,12h,16h), Sec61with ERS further aggravated expression gradually increased, and after applying Sec61inhibitors, ERS significantly reduced, leading to reduce the degree of hepatic steatosis, these results indicated that Sec61a regulated endoplasmic reticulum GRP78protein-mediated ERS reactions involved in the development of hepatic steatosis.4. In the application of Sec61inhibitor, GRP94and Bcl-2expression in inhibitor group were significantly higher compared with steatosis group, which may regulate Ca2+concentration in the liver cells steatosis in vitro NAFLD model, it may involve in ERS, and the hepatic steatosis.