Effect Of Ammonium Chloride On Gene Expression Of RHCG And AQP8in LO2Cells

BackgroundThe liver is a vital organ of the human body metabolism, which has the important functions of metabolism, detoxification, excretion.When a serious liverdisea-se lesions,such as liver cirrhosis, liver cancer and severe hepatitis, it will seriouslyaffect the body’s normal physiological function. In clinical, the indication of bloodammonia (BA) to the hepatic metabolism receives significant attentions, which not only reflect the severity of liver disease,but also diagnose hepatic encephalopathy. The normal concentration of blood ammonia is18-72umol/L, mainly from the kidney excretion, intestinal production, muscle production.In common conditions,the main metabolic pathway of blood ammonia in vivo isthe synthesis of urea in the liver and the excretion by the kidneys.While itsmetabolic pathway in the brain, heart, skeletal muscle, is the synthesis of glutamine in the glutamine synthetase enzyme catalysis, and the transport of glutamine to the liver via the blood circulation, afterwards the decomposition of ammonia by glutamine enzymatic, and then the decomposed ammonia involved inthe ornithine cycle which produces urea in the liver. When the liver appearsmetabolic lesions, it can not effectively metabolize the produced ammonia, resulting in the accumulation of it, and furth-er aggravate the liver lesions, bringing out more severe diseases, such as the hep-atic encephalopathy, and even the liver failure;At the same time hepatic injury will cause the accumulation of ammonia, which lead to many influences on the body, including the following aspects:1. Ammonia causes the astrocytes swelling;ammonia can cause the astrocytes swelling in vivo and vitro. Clinical studies have found that the astrocyte swelling is also an important indicator of cellular pathology in brain edema of acute liver failure.2.the Metabolic disorder of energycaused by the ammonia;3.the mitochondrial permeability transition caused by the a-mmonia.4. TheOxidative stress caused by the ammonia.New researches found that a class of RH protein obligated to the ammonia-mediated transportation, mythylamine permese (MEP) and ammonium transporter (AMT)together constitute the super protein family which participate ammonia tran-sportation.However, the main position of three kinds of proteins are found differe-nt,where the RH glycoprotein is mainly expressed in animals, MEP is mainly exp-ressed in yeast, and AMT is mainly expressed in plants.RH glycoproteins include RHAG, RHBG and RHCG. RHAG is an erythroid-related protein, but RHBG and RHCG are non-erythroid-related proteins.However,the main ammonia transport proteins which exist in various organs of human body are RHBG and RHCG. Stu-dies have found RHBG and RHCG play a key rolein ammonia metabolism and transportation of the kidney and liver.They also found a kind of protein-aquaporin (AQP),which is highly selective for water, and mainly responsible for the secreti-on of water absorption and maintaining thewater balance on both sides of cells.The study also showed that in some special conditions, AQP is also permeable to ammonia, CO2, Cl-, pyrimidine, purine, polyols,etc.So far in mammals13species of AQP have been found,that from AQP0to AQPl2. AQP differ in monomer si-ze, whose molecular weight arebetween25000to34000. But each AQP mole-cule contains six hydrophobic transmembrane protein and5connecting ring(A-E)，whose primary structure is binded with the amidogen of peptide chain and the carboxy-terminaland all have an asparagine-proline-chrysamminic acid(NPA)sequence.Dimensional structure of the tetramer is formed on the cell membrane by AQP, and each monomer contains an aqueous pore.Current Studies have demonstrated that there were at least seven kinds of AQP (0,1,3,4,8,9,11) play an important role in the expression of liver cells, biliary system and physiological effects of the gallbladder.T here are only four kinds of AQP (0,8,9,11)expressing in rodents, but AQP3also expresses in human liver cells.In the liver cells of animals, AQP8’s expressio-n is most abundant, and has a variety of subcellular localization, including the smooth endoplasmic reticulum, mitochondrial inner membrane, plasma membrane vesicles and tubules. In the liver cells of rodents, there are two types ofAQP8,one type is AQP8shuttling between a class near the top of the plasmamembrane vesicles and tubules plasma membrane, and this activity is to promote the secreti-on of bile-stimulating hormone control;The other is AQP8persisting on the mitochondria and smooth endoplasmic of the liver cells.While the AQP8from the intracellular smooth endoplasmic reticulum may be involved inmaintaining the osmotic concentration of glycogen synthesis and decompositionprocess in the cytoplasm of liver tissue.Daily research shows that the AQP8from mitochondrial has two po-tential roles: on the one hand to absorb NH4+for urea cycle use; on the other hand to release H2O2from the mitochondrial matrix in the process of reactive oxy-gen species generated.There are many studies on severe hyper ammonemia induced by liver injury, mainly about the ammonia poisoning theory, the theory of plasma aminoacid metabolism, neurotransmitter false doctrine, and a variety of toxins synergism reinforce each other, rely on each other, and jointly promote the occurrence and development of the hepatic-encephalopathy, and then lead to the liver failure; Therefore,investigating and clarifying the interaction between risk factors have an important significance.While according to the ammonia position theory, there are few studies on the ammonia transport mechanism, but it has avital role to the ammonia toxicity.This study constructed a ammonia-poisoned cell model, and by detecting the level of protein RHCG and AQP8,preliminarily explored the mechanisms of ammonia transportation and toxicity in the liver,in order to offer a theoretical basis for further study on the mechanisms of ammonia toxicity, and provide theoretical basis for the clinical treatment of liver failure.Objective:To inspect whether ammonium chloride can specifically stimulate liver cell to changethe internal RHCG and AQP8gene expression. Methods:1.The human liver cell line (LO2)、thyroid cancer cell line(TPC-1)、ovarian cancercell line(SKOV-3) and esophageal cancer cell line(9706) were cultured.2.MTT method was used to check the proliferation ability of those4cell lines aftercells were treated with NH4Cl（work concentration:2.5、5、10、20、40、50mmol/L,respectively）for24h.3.RT-PCR and Western blot were used to detect the RHCG and AQP8expression ofLO2cells after treated with NH4Cl（5、10、20mmol/L）for6、12and24h.4.TheRHCG and AQP8gene expression of TPC-1、SKOV-3and9706cells was detectedafter treated with NH4Cl（10mmol/L）the same method above.Results:1.MTT method finds the growth inhibitory rate in LO2cells increase with ammoniaconcentration increasing, which is significantly greater than that in other3cell lines.2.RT-PCR finds that RHCG mRNA in LO2cells decreased when treated with10mmol/L ammonium chloride for6hours (p<0.05). while AQP8mRNA in LO2cellsincreased when treated with10mmol/l ammonium chloride for12hours (p<0.05);10mmol/L.3.Western blot finds that the AQP8protein in LO2cells increased whentreated with10mmol/L ammonium chloride for24hours (p<0.05), while the RHCGprotein expression has no change.4.No obvious change is found in the AQP8and RHCG expression in TPC-1、SKOV-3and9706cells when treated with ammonia of the same concentration within the sametime.Conclusion:NH4Cl could specifically stimulates the liver cells, leading to the gene expressionchanges of RHCG and AQP8that are involved in inner transport of ammonia, thedetails are as follows:1. liver cells can be damaged specifically in high ammonia environment..2.In the environment with ammonia of high concentration, human liver cells canmaintain the balance of absorbing ammonia by changing RHCG and AQP8expression to reduce the harm of ammonia to liver cells.3.The expression of the proteins that are involved in ammonia transport in liverchanges in high ammonia environment while TPC-1、SKOV-3and9706cells have no such function.