Role Of Store Operated Ca²⁺ Channels In Ethanol-Induced Intracellular Ca²⁺ Increase And Cell Injury In Hepatocytes

Alcohol abuse has been a primary factor in chronic liver disease in western countries for many years. As the proportion of alcoholics keeps on increasing in China, alcohol has become the second largest etiological factor of liver disease while the first one is viral hepatitis. The pathogenesy of alcoholic liver disease (ALD) still remains obscure so far. Ethanol induced hepatocyte injury is based on individual genetic susceptibility and results from multiple overlapping mechanisms including the direct and indirect toxicity of alcohol and its'metabolic products, oxidative stress, cytokines, inflammatory/immune responses, endotoxin, altered intracellular signaling, and endoplasmic reticulum (ER) stress.By establishing rat model of chronic alcoholic liver disease, our previous study revealed that chronic alcohol significantly increased cytoplasmic free Ca2+concentration ([Ca2+]i) in rat primary hepatocytes, leaded to sustained opening of mitochondrial permeability transition pore (MPTP) and decrease of mitochondria transmembrane potential (Δψm). These pathological changes finally resulted in ethanol induced hepatocyte apoptosis and necrosis. Disturbed intracellular Ca2+homeostasis, usually present as prolonged elevation of cytosolic Ca2+concentration, has been implicated as one of the main mechanisms underlying ethanol induced liver damage. The source of the ethanol increased [Ca2+]i content is debated.Pathological [Ca2+]i increase usually originates from increased plasmalemmal Ca2+entry from extracellular medium, enhanced Ca2+release from intracellular stores such as mitochondria and endoplasmic reticulum (ER) or both. Some evidence suggested that the enhanced agonist induced Ca2+6release from ER the observed in ethanol treated hepatocytes results from increased inositol trisphosphate receptor (IP3R) expression caused by an inhibition of IP3R degradation pathways by alcohol. Chronic ethanol induced oxidative stress perturbs membrane permeability and fluidity of hepatocytes and leads to membrane ion transport protein dysfunction, which eventually increased extracellular Ca2+influx. Recent studies have found MPTP opening in sustained high conductive mode is the one of the important cause for aggravated hepatocyte calcium overload induced by ethanol.But there are still some following issues remain unresolved. The increased [Ca2+]i as a result of Ca2+release from intracellular stores mediated by IP3R is transient, the durative source of Ca2+to maintain calcium overload status in hepatocytes is unclear. Which membrane ion transport proteins are implicated in ethanol induced extracellular Ca+influx still can not determine. Some research indicated that compensatory accumulation of Ca2+in mitochondria caused by sustained enhanced [Ca2+]i played an critical role in high conductive opening of MPTP. So the previous source of increased [Ca+]i in hepatocytes still need to be identified.Store operated of Ca2+channels (SOCs) are the principal mechanism for Ca2+influx in non excitable cells, including hepatocytes. SOCs play an irreplaceable role in a variety of Ca2+medicated physiological functions such as signal transduction, cell proliferation and apoptosis. As the patch clamp experiments revealed, there is only one type of SOC, a highly Ca2+selective channel could be detected in liver cells. Hepatocytes SOCs are activated by reduced Ca2+content in a subregion of the ER enriched in type1IP3receptors. SOCs have an essential role in refilling depleted Ca2+stores in ER. The molecular components of SOCs in hepatocytes identified so far are stromal interacting molecule1(STIM1) acting as a sensor for level of Ca2+stored in ER and calcium release activated calcium channel protein1(Orial) proteins constituting pore forming subunits of the SOCs.Based on the observation that Ca2+release from the ER through the phospholipase C (PLC)-inositol1,4,5-triphosphate (IP3)-protein kinase C (PKC) pathway was involved in ethanol-induced [Ca2+]i elevation, and SOCs are plasma membrane Ca+channels activated by a decrease in Ca+content in the ER lumen, the following assumptions was proposed that SOCs may be the missing Ca2+influx route implicated in ethanol induced sustained [Ca2+]i elevation. SOCs may play an essential role in maintaining durative extracellular Ca2+influx in liver cells after the transient Ca2+release from endoplasmic reticulum induced by chronic ethanol. The store operated calcium entry (SOCE) finally increased [Ca2+]i in hepatocytes to an extent of calcium overload leading to cell damage.In this study, the in vitro model of chronic ethanol induced hepatocyte injury and the in vivo rat model of alcoholic liver disease were established first. Then the possible role of SOCs in the ethanol induced extracellular Ca2+influx and the related liver cell injury was determined by using different channel blockers. To further determine the mechanism responsible for probably involveed SOCs in ethanol induced [Ca2+]i rise and hepatotoxicity, we detected the effect of chronic ethanol on the expression quantity and location of STIM1and Orail in hepatocytes both in vitro and in vivo. To further clarify the specific role of upregulated SOC proteins in the alcohol induced [Ca2+]i increase and liver cell damage, siRNA against STIM1(Si-STIM1), Orail (Si-Orail), or both (Si-STIM1/Orail) were utilized to detect the effect of SOCs siRNA hnockdown on ethanol-induced [Ca2+]i increase in hepatocytes and the related cell injuries. This study was divided into three parts:PART Ⅰ Store Operated Ca2+Channels are Involved in Ethanol Induced Hepatocytes [Ca2+]i Increase and Liver Cell DamageAimsTo observed the effect of chronic ethanol treatment on [Ca2+]i increase, cell viability and cell damage in HepG2cells. To clarify the role of extracellular Ca2+influx and store operated calcium entry in chronic ethanol induced [Ca2+]i rise and the related cell damage in hepatocytes by determining the intervention effect of EGTA,2-APB,La3+on such process. Methods1. Cell culture:The human HepG2hepatoma cell lines were purchased from ATCC Company and routinely cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose) supplemented with10%fetal bovine serum (FBS). When grown to80～90%confluence, cells were passaged and those at passage4-8were selected for further study.2. Experimental grouping:HepG2cells were divided into the following groups:normal control group (PBS treatment), chronic ethanol treated group (HepG2cells were treated with25mM,50mM,100mM,200mM,400mM and800mM ethanol respectively for24hours); ethanol+EGTA group (HepG2cells were treated with200mM ethanol and0.25mM,0.5mM,1mM EGTA respectively), ethanol+2-APB group (HepG2cells were treated with200mM ethanol and25μM,50μM,75μM,100μM2-APB respectively), ethanol+La3+group (HepG2cells were treated with200mM ethanol and0.1μM,1μM,10μM La3+respectively).3. Measurement of [Ca2+]i in liver cells:HepG2cells were loaded with Fluo-3/AM or Fura-2/AM. The elevated levels of [Ca2+]i were detected by flow cytometry which determines levels in an average single cell and fluorescence spectrophotometer which determines levels in all cells.4. Cell viability assays:The effect of ethanol treatment and EGTA,2-APB, La3+intervention on the cell viability of HepG2cells was estimated by both trypan blue exclusion and CCK-8kit.5. Biochemical assessment of hepatocytes injuryThe culture supernatant was collected from differently treated cells. Damage of HepG2cells induced by ethanol treatment and the protect effect of EGTA,2-APB, and La3+was evaluated by alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) leakage in such culture supernatant by use of an Automatic Biochemistry Analyzer.Result1. Ethanol-induced cytotoxicity in HepG2cells:Both trypan blue exclusion and CCK8assay revealed that24hours'of0-800mM ethanol reduced cell viability of HepG2cells in a concentration dependent manner. Ethanol treatment markedly increased ALT, AST, ALP and LDH leakage from cells. Cell viability was65.28±7.38%(trypan blue exclusion) and67.83±4.41%(CCK8assay) at200mM ethanol. So200mM ethanol was chosed as an appropriate concentration in the subsequent research.2. Effect of ethanol on intracellular calcium levels in HepG2cells:Both flow cytometry and fluorescence spectrophotometer confirmed that0-400mM ethanol dose-dependently increased [Ca2+]j in HepG2cells. The elevated level of [Ca2+]i was higher with fluorescence spectrophotometer which determines levels in total cells than by flow cytometry which determines levels in average single cell.3. Effect of EGTA,2-APB and La3+intervention on ethanol induced intracellular calcium rise in HepG2cells:EGTA,2-APB and La3+concentration-dependently decreased [Ca2+]i levels in HepG2cells increased by200mM ethanol. No statistically significant difference could be found among these three regents.4. Effect of EGTA,2-APB and La3+intervention on ethanol induced cytotoxicity in HepG2cells:The cell viability of HepG2cells significantly increased in EGTA group,2-APB group and La3+group in a dose dependent manner compared with200mM ethanol group. The ALT and AST leakage level remarkably decreased in all intervention groups. Among three intervention groups, no statistically significant difference could be found.Conclusion1. The chronic ethanol treatment in vitro concentration dependently increased [Ca2+]i content and hepatocyte damage in HepG2cells. This can be used as an in vitro model of ethanol induced hepatocytes damage.2. Extracellular Ca2+influx is implicated in chronic ethanol induced [Ca2+]i increase in HepG2cells and store operated calcium entry may be the major source of it. Further more, store operated calcium entry is involved in ethanol-induced liver cell injury.3. In addition to extracellular Ca2+influx, Ca2+release from intracellular calcium stores may be another part of the pathological Ca2+signal contributing to ethanol-induced liver cell damage. PART Ⅱ Chronic Ethanol Treatment Increased Expression Level of Store Operated Ca2+ChannelsAimsSTIM1and Orail are two major molecular components of SOCs in hepatocytes. To further clarify the exact mechanism responsible for probably involved SOCs in ethanol induced [Ca2+]i increase and hepatotoxicity, the effect of chronic ethanol on STIM1and Orail expression was determined in HepG2cells. Chronic alcoholic liver disease rat model was also used to investigate the expression and location changes of STIM1and Orail in liver tissue in order to analyze the relationship between SOCs and ethanol induced damage of liver tissue.Methods1. Chronic ethanol treatment in vitro:HepG2cells were routinely cultured in DMEM supplemented with10%FBS. When grown to80-90%confluence, cells were passaged and then seeded into6-well plates. Cells were treated with200mM ethanol after adherence. According to the different time of ethanol treatment, cells were divided into normal control group (PBS treatment), chronic ethanol treated24h group,48h group, and72h group.2. Rat model of chronic alcoholic liver disease:After fed normally for a week, forty adult male Wistar rats were divided into control group and ALD group randomly. The rats in the ALD model group were given intragastric administration of alcohol twice daily base on olive oil diet. The alcohol was administrated to rat in distillate spirit form. The quantity and concentration of ethanol converted from distillate spirit were increased every two weeks. At the same time the rats in control group were given equal volume of normal sodium twice daily by intragastric administration. All the rats were sacrificed at the end of16th week. The pathologic changes of liver were observed by using HE stains.3. The expression of STIM1and Orail:Total RNA and total protein were extracted from both HepG2cells and liver tissue. Quantitative real-time PCR analysis was performed to determine the mRNA expression of STIM1and Orail gene. The protein expression was measured by Western blot. The location of STIM1and Orail were investigated by immunohistochemisty.Results1. Effect of ethanol on STIM1and Orail expression in HepG2cells:Compared with control group,200mM ethanol treatment significantly increased the expression of STIM1and Orail in both gene and protein level at all times examined, suggesting that the ethanol increased expression of STIM1and Orail could last for at least72h.2. Rat model of chronic alcoholic liver disease:The rats in ALD model group were more depressed. The weight gain significantly decreased in ALD group while the liver weight/body weight ratio remarkably increased compared with control group. Hepatic steatosis, ballooning degeneration, focal necrosis and infiltration of inflammatory cells could be observed in liver tissue by using HE staining.3. Effect of chronic ethanol on STIM1and Orail expression in hepatic tissue of ALD ratsThe gene and protein expression of STIM1and Orail in liver tissue was much higher in ALD model group than that in control group. As shown in immunohistochemistry staining, the small amount positive particles of STIM1and Orail were distributed around the central vein and portal area in normal group. STIM1was expressed in cytoplasm while Orail was expressed on membrane. The positive particles of STIM1and Orail increased significantly in ALD model group. A similar distribution of STIM1and Orail could be observed. They both mainly distribute in the susceptible domain of liver acinus in alcoholic liver injury. STIM1had an membrane gathering trend. STIM1and Orail expression and liver pathology score was positively correlated.ConclusionBoth in vitro and in vivo studies showed that chronic ethanol treatment can increase the expression of STIM1and Orail in liver cells in a parallel mode, suggesting that ethanol may increase extracellular calcium influx by up-regulating expression of SOCs protein molecule, finally aggravate liver cell damage. PART Ⅲ Effect of knockdown of SOCs proteins on ethanol induced [Ca2+]i rise and hepatotoxicityAimsTo further clarify the specific role of upregulated SOC proteins in the ethanol-induced [Ca2+]i rise and hepatotoxicity, the effect of RNA interference of STIM1and Orail were determined.Methods1. Design, synthesis and screening of small interfering RNA(siRNA):Based on gene sequences of STIM1and Orail retrieved from Genebank,3pairs of siRNA sequence were designed targeting human STIM1and Orail, respectively. HepG2cells were transfected with siRNA using lipofectamine2000. The efficacy of RNA interference was determined with western blot and the strongest siRNA was chose for following experiments.2. Effect of STIM1and Orail knockdown on ethanol induced [Ca2+]i rise and hepatotoxicity:HepG2cells were divided into ethanol treated group, STIM1interference group, Orail interference group and STIM1/Orail co-interference group. Intracellular calcium concentration, cell viability and cell damage were detected in accordance with the methods in the first part.Results1. Screening of SiRNA targeting STIM1and Orail:One pair of siRNA sequence targeting human STIM1or Orail was chosen. The inhibition efficiency vertified by Western Blot was60-70%.2. Effect of STIM1and Orail knockdown on ethanol induced [Ca2+]i rise:SiRNA knockdown of STIM1resulted in a significant decrease in ethanol-increased [Ca2+]i content, whereas siRNA knockdown of Orail had almost no effect. Down-regulation of both STIM1and Oril inhibited ethanol-increased [Ca2+]i level to a degree a little lower than that with siRNA knockdown of STIM1.3. Effect of STIM1and Orail knockdown on ethanol induced liver cell injury:Both trypan blue exclusion and WST-8assay revealed that a marked increase in cell viability only with siRNA knockdown of STIM1and STIM1/Orail. Consistently, ethanol-induced ALT, AST, ALP and LDH leakage was effectively decreased only with siRNA knockdown of STIM1and STIM1/Orail. No significant effect could be detected with siRNA knockdown of Orail alone.ConclusionAlthough chronic ethanol could upregulate the protein levels of both STIM1and Orail, ethanol-induced [Ca+]i rise may depend more on increased frequency of SOCs opening mediated by STIMl. STIM1plays a major role in ethanol-induced [Ca2+]i rise and hepatotoxicity. Ethanol can aggravate hepatocytes injury by increasing STIM1expression and up-regulating frequency of SOCs channels opening. The increased numbers of opening SOCs mediated by up-regulated Orail may play an assistant role in this process.