The Mechanism Of Soce In Alcohol Induced Liver Injury In Vitro And In Vivo

Alcoholic liver diseases, including alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis and alcoholic cirrhosis, are the most common liver diseases. The mortality of alcoholic liver diseases accounted for 40% of liver cirrhosis,28% of all liver diseases. The pathogenesis of alcoholic liver disease was complicated, which comprised direct liver toxicity of alcohol and acetaldehyde, alcohol-induced ROS production and metabolism, tissue hypoxia, endoplasmic reticulum stress, iron overload, apoptosis and immune mechanisms. Those all play important roles in the development of alcohol-induced liver injury. However, the specific pathogenesis of alcoholic liver disease remains unknown.Calcium ion (Ca+) as a common second messenger is involved in many cellular functions, including energy metabolism, cell death and so on. Store-operated calcium channels (SOCs) are the main calcium influx channels in non-excitable cells. SOCs include two main mediators, stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (CRAR, also called Orail). Store-operated calcium channels entry (SOCE) has close relationship with calcium homeostasis, which can sense calcium ion level in the endoplasmic reticulum, then mediate extracellular calcium influx to increase the cytoplasmic calcium concentration to refill calcium stores.Previous studies have shown that chronic ethanol significantly increased extracellular calcium influx by increasing the expression of SOCE associated proteins in HepG2 cells. However, although the HepG2 cells are widely used in liver toxicity research in vitro, because it remains a lot of expressions of Ⅰ, Ⅱ phase enzyme of the human stem cells, HepG2 cells still cannot completely represent the human primary hepatocytes. Moreover, the role and mechanism of SOCs in ethanol-induced liver cell injury is still poorly understood.Therefore, the present study was aimed to investigate the role and mechanism of SOCE in alcohol-induced liver cell injury and the effect of blocking SOCE in decreaing alcoholic liver damage in vitro and in vivo, which may provide a new target for clinical therapy of alcoholic liver diseases.PART I:THE ROLE OF CALCIUM IONS IN ALCOHOL-INDUCED LIVER INJURYObjectives:To observe the morphology change in liver cells, evaluate cells damage and the calcium concentration in the cytoplasm after exposure of ethanol. To evaluate cytoplasmic calcium concentration and liver cells injury after giving intracellular and extracellular calcium chelators, in order to analysis the role of calcium ion in ethanol-induced liver cell injury.Methods:1. Cell culture:Rat hepatocytes cell lines BRL, cultured in DMEM+10% FBS. When BRL cells reached 80%-90% confluence, cells were passaged, then we selected the passage 4-6 to do the next experiment.2. Cell treatment groups:BRL cells were seeded into six-well plates. After adherent, cells were treated with different stimulis. The cells were divided into the groups as followed:0,25mM,50mM, 100mM,200mM,400mM different concentrations of ethanol stimulation (24 hours) group; normal control group, ethanol stimulation group, ethanol added EDTA group and ethanol added BAPTA-AM stimulation group.3. Hepatocyte morphology was observed:After BRL cells were stimulated with different concentrations of ethanol (0,50mM, 100mM,200mM) for 24 hours, changes in cell morphology were observed under the microscope.4. Detection of cytosolic calcium concentration:The calcium ions in the cytoplasm of BRL cells were marked by Fluo-3/AM, which was tested by flow cytometry to detect the concentration of calcium ions in hepatocytes.5. Assessment of liver cells injury:We detected the survival rates of liver cells by MTT assay, and collected cell supernatant from each group to do the biochemical test by automatic biochemical analyzer.Results:1. The effects of ethanol on the morphology of BRL cells:Compared with hepatocytes from normal control group, after stimulations of 0,50mM, 100mM, 200mM ethanol for 24 hours, most of liver cells became significantly swelled and the cells were polygonal, sometimes apoptotic body existed.2. The impacts of ethanol on the damage of BRL cells:The ethanol of 0,25mM, 50mM, 100mM,200mM,400mM concentration-dependently reduced BRL cell viability, while concentration-dependently increased aspartate aminotransferase (AST) content and alanine aminotransferase (ALT) content in cell culture supernatant, and AST increased more than ALT significantly. The cell viability of BRL cells after stimulation of 100mM ethanol was 57.58%±2.08%, so we selected the concentration of 100mM ethanol as the next experimental stimulus.3. The effects of ethanol on cytosolic calcium ions concentration:The ethanol of 0, 25mM,50mM, 100mM,200mM,400mM concentration-dependently increased cytosolic calcium ions concentration in rats liver cells BRL cells.4.The effects of calcium ions chelators on cytosolic calcium ions concentration:The extracellular calcium chelator EDTA and intracellular calcium chelator BAPTA-AM can decreased cytosolic calcium ions concertration elevated by ethanol. There was no significant difference between the extracellular and intracellular calcium chelator.5. The effects of calcium ions chelators on liver cells injury:The extracellular calcium chelator EDTA and intracellular calcium chelator BAPTA-AM can increase the cell viability reduced by ethanol, and reduce ALT, AST levels elevated due to ethanol. There was no significant difference between the extracellular and intracellular calcium chelator.Conclusions:1. Ethanol can concentration-dependently decrease cell viability of BRL cells, elevated transaminase levels in culture supernatant, and increased the concentration of calcium ions in the cytoplasm, indicating that calcium may be involved in alcohol-induced liver cells damage.2. The extracellular and intracellular calcium chelator can decrease the concentration of calcium ions in the cytoplasm, reduce liver cells damage, which may provide further evidence that calcium ions may be involved in alcohol-induced liver injury.PART II:THE ROLE AND MECHANISM OF SOCE IN ETHANOL INDUCED BRL LIVER CELL INJURYObjectives:To detect the genes and proteins expressions of SOCE associated proteins STIM1, Orail after ethanol stimulation. To detect liver cells injury by MTT and biochemical testing; cytosolic calcium ion concentration, apoptosis rate, cell cycle and mitochondrial membrane potential by flow cytometry; cytochrome C in hepatic cell cytoplasm and mitochondria by Elisa; changes of the apoptotic proteins by western blot after blocking SOCE by giving SOCE inhibitors and sh-RNA knockdown to analysis the role and mechanism of SOCE in ethanol-induced liver cell injury in vitro.Methods:1. Cell transfection and efficiency of transfection:Synthesis sh-RNA sequences for SOCE associated proteins STIM1 and Orai1. The synthesized sh-RNA STIM1 and Orail were transfected into BRL cells by using lipo2000. After 24 hours we extracted proteins from transfected cells to verify the efficiency of transfection by western blot.2. Cell treatment groups:PBS group, stimulation of 100mM ethanol 24 hours group, 48 hours group,72 hours group; 100mM ethanol stimulation group, ethanol+ corosolic acid group, ethanol+La3+, ethanol+2-APB group, ethanol+GSK-7975A group, ethanol+RO2959 group; ethanol+sh-RNA control interference group, ethanol+sh-RNA STIM1 interference group, ethanol+sh-RNA Orai1 interference group, ethanol+sh-RNA STIM1+sh-RNA Orai1 interference group.3. Detection of the genes and proteins expressions of SOCE associated proteins STIM1 and Orai1:After 24-hour stimulation, RNA and proteins were extracted from each group. We detected the gene and protein expression of STIM1, Orai1 by qPCR and western blot after ethanol stimulation.4. Detecting the change of cytoplasmic calcium ion concentration:We used flow cytometry to detect the calcium ions in cells cytoplasm marked by Fluo-3/AM. And hepatocytes cytoplasmic calcium ions concentration were detected after blocking SOCE by inhibitors and shRNA interference5. Detection of liver cells injury:We detected the cell viability by MTT assay, and supernatant transaminases by automatic biochemical analyzer after adding SOCE inhibitors and SOCE associated proteins shRNA interference.6. Detection of apoptosis rate, cell cycle, mitochondrial membrane potential from each group:After collection of cells from each group in vitro, we used Annexin-V/PI staining, PI staining and Rh123 to detect apoptosis rate, cell cycle and mitochondrial membrane potential by flow cytometry.7. Detection of the content of the cytosolic and mitochondrial cytochrome C and the expression of various apoptosis proteins from each group:The cytosolic and mitochondrial portions were collected respectively from each group. We detected cytochrome C content in cytoplasm and mitochondria of liver cells, and detected the expression of SOCE associated apoptosis proteins by western blotResults:1. The efficienct transfection of shRNA STIM1, shRNA Orai1 into liver cells:We designed and synthesised shRNA of STIM1, Orai1, and found that shRNA can significantly inhibit protein expression of STIM1, Orail by western blot.2. Ethanol increased the gene and protein expressions of SOCE associated proteins STIM1, Orai1:100mM ethanol increased the genes and proteins expressions of STIM1, Orai1 compared with control group, and the effect last up to 72 hours.3. The change of cytosolic calcium ions concentration after blockade of SOCE: Corosolic acid, La3+,2-APB, GSK-7975A, RO2959, sh-RNA STIM1, sh-RNA Orai1, sh-RNA STIM1+sh-RNA Orai1 could significantly decrease the ethanol-increased cytosolic calcium ions concentration. There were no statistical differences among the blockers.4. The effect of SOCE blockers and shRNA interference on liver cells injury: Corosolic acid, La3+,2-APB, GSK-7975A, RO2959, sh-RNA STIM1, sh-RNA Orai1, sh-RNA STIM1+sh-RNA Orai1 could significantly increase the 100mM ethanol-decreased cell viability and reduced alcohol-elevated transaminases. There were no statistical differences among the blockers.5. The impact on cell apoptosis, cell cycle and mitochondrial membrane potential after different stimulus:Compared with normal control group, 100mM ethanol stimulation significantly increased apoptosis rate of of liver cells; increased the numbers of cells in S phase, reduced the numbers of cells in G2/M phase, indicating that the ethanol increase S phase arrest. 100mM ethanol stimulation also reduced concentration of Rh123 fluorescence, meaning reduction of the mitochondrial membrane potential. Compared with 100mM ethanol stimulation group, blockers and shRNA interference can decrease the apoptosis rate of liver cells, reduce the S phase arrest, and increase mitochondrial membrane potential.6. The impact on the cytochrome C content after each group stimulus:Compared with ethanol stimulation group, the blockade of SOCE can significantly reduce the Cytochrome C content in cytoplasm, while relatively increase the Cytochrome C content in the mitochondria, which indicated that blocking SOCE can significantly reduce the mitochondrial cytochrome C released into the cytoplasm.7. The change of proteins expression of STIM1, Orai1 and various apoptotic proteins after blocking SOCE:Compared with ethanol stimulation group, blocking SOCE can inhibit the protein expression of SOCE associated proteins STIM1, Orai1, and reduced Bax/Bcl-2 ratio and caspase3 activation increased by the ethanol stimulation.Conclusions:1. Ethanol significantly increased the genes and proteins expressions of SOCE associated proteins STIM1, Orai1, and the impact can sustain up to 72 hours, which indicated that SOCE may be involved in alcohol-induced liver cell injury.2. Compared with ethanol stimulation, SOCE blockers and shRNA interference significantly reduced the concentration of calcium ions in the cytoplasm, increased cell viability and decreased the levels of transaminase. There were no significant differences among the various blockers. These further illustrated SOCE may play an important role in the ethanol induced liver cell injury.3. Ethanol stimulation can significantly increase the apoptosis rate, S phase arrest of liver cells, while blocking SOCE can significantly reduce apoptosis rate and S-phase arrest, indicating that SOCE may lead to alcohol related liver cell damage by inducing apoptosis.4. Ethanol stimulation can reduce cell mitochondrial membrane potential, increase cytochrome C from mitochondria into the cytosol, elevate Bax/Bcl-2 ratio and the activation of cleavage caspase 3. Whereas blocking SOCE can significantly reduce the above process, further indicating apoptosis played a role in SOCE induced liver cells damage in alcoholic liver disease.PART III:THE ROLE AND MECHANISM OF SOCE IN ALCOHOLIC LIVER DISEASES IN RATSObjective:To observe the proteins expressions and positions of SOCE associated proteins STIM1, Orail in alcohol feeded rats.To observe the liver damage and detect the changes of blood transaminases after giving SOCE blocker corosolic acid. To detect the cytochrome C contents in the cytoplasm and mitochondria of liver tissue by Elisa and the apoptotic proteins expression by western blot. To analysis the role and mechanism of SOCE in rat liver tissues injury caused by alcohol.Methods:1. Division of rats experiments:40 adult male Sprague-Dawley rats were divided into four groups:control group, alcoholic liver disease group, alcohol+corosolic acid (4 weeks) group, alcohol+corosolic acid (8 weeks) group. Alcoholic liver disease group: 60% of Beijing Red Star Erguotou was diluted with water. Alcohol was given intragastrically 4.5,6.5,9g/Kg/day for 1-4 weeks,5-8 weeks,9-12 weeks.The daily dose of alcohol was administrated in 2 divided dose,12h apart. Normal group was given normal saline orally twice a day. Corosolic acid Group 1:At the beginning of the eighth week, daily oral 4mL 20% of corosolic acid 10 minutes before alcohol administration. Corosolic acid group 2:The same methods as corosolic acid Group 1, began at the fourth week. Rats were weighed before and after the experiments, and blood specimens and liver tissues were stored in liquid nitrogen or 4% formaldehyde.2. Detection of the expressions and localizations of SOCE associated proteins STIM1, Orail after alcohol administration:We detected the expressions and localizations of STIM1 and Orail by western blot and immunohistochemistry after alcohol administration.3. Observation of liver tissue damage after alcohol feeded:We observed pathological changes in the liver after ethanol feeded and corosolic acid treatment by HE staining, and detected rats’blood transaminases by automatic biochemical analyzer.4. Detection of the cytosolic and mitochondrial cytochrome C in each group:The cytoplasm and mitochondria portions of the liver tissue were collected from each group. The content of cytosolic and mitochondrial cytochrome C were detected by Elisa.5. Detection of the expressions of apoptosis proteins in each group in vivo:The proteins of liver tissue were collected from each group, and the expressions of SOCE associated proteins and apoptosis proteins were detected by western blot.Results:1. Intragastric alcohol caused liver tissue injury:The body weight of rats in alcoholic liver disease model increased more slowly than the control group. HE staining found that compared with the control group, severe liver injury in alcoholic liver diseases model group:hepatic steatosis, inflammatory cell infiltration and hepatic cell necrosis increased significantly. The levels of serum ALT, AST increased higher than the control group, and the ratio of AST/ALT was significantly more than 1.2. Alcohol influenced the protein expression of SOCs:Compared with normal saline group, alcohol feeded significantly increased the proteins expressions of SOCE associated proteins STIM1, Orai1.3. The effect of Corosolic acid for liver injury in rats:Compared with ethanol group, liver tissue injury in corosolic acid group, hepatic steatosis, inflammatory cell infiltration, and necrosis was alleviated; meanwhile transaminase levels were also significantly reduced.4. The effect of blockade of SOCE on cytochrome C in vivo:Compared with ethanol feeded group, after administration of corosolic acid cytoplasmic cytochrome C content decreased, while mitochondrial cytochrome C relatively increased, indicating that blocking SOCE can significantly reduce the release of cytochrome C from mitochondria into the cytoplasm.5. Blockade of SOCE influenced the expressions of STIM1, Orai1 and various apoptotic proteins:Compared with alcoholic liver disease group, corosolic acid reduced the protein expressions of SOCE associated proteins STIM1, Orai1, and decreased Bax/Bcl-2 ratio and caspase3 activation increased by alcohol stimulation.Conclusions:1. The expressions of SOCE associated proteins STIM1, Orail in alcohol feeded group significantly increased. Administration of SOCE inhibitor corosolic acid can significantly alleviate alcohol caused liver tissue injury, decrease the levels of serum transaminases, indicating that SOCE may be involved in SD rat liver tissue damage caused by alcohol.2. Ethanol feeded rats released more cytochrome C from mitochondria into the cytosol, increased Bax/Bcl-2 ratio and the activation of cleavage caspase3. The SOCE inhibitor corosolic acid can significantly alleviate the above process, which further indicated that apoptosis may be involved in SOCE caused liver injury in alcoholic liver diseases.