| Objective:Acute liver failure is a rare but life-threatening disease.For patients with no history of liver disease,the function of a huge number of hepatocytes may lose quickly.Acute and chronic liver failure is different from acute decompensated liver failure.It generally occurs on the basis of chronic liver dysfunction,accompanied by many complications of decompensation and it is relevant to a higher rate of organ failure.They are all accompanied by many serious neurological complications,including brain edema and hepatic encephalopathy,and mental diseases characterized by severe cognitive and mental injury,from consciousness to coma.For decades,ammonia is believed to play an important role in the pathophysiological basis of acute liver failure,neurological complications,but in a recent study,both in person and in animal models strongly suggest that inflammatory reaction is involved in terms of either being alone with ammonia or working together with ammonia.Inflammatory response is an important feature of acute liver failure,and the increase of proinflammatory cytokines is also a risk factor independent of potential liver diseases.Tumor necrosis factor TNF-alpha plays a strong role in these proinflammatory cytokines.It can stimulate a variety of inflammatory responses and trigger immune functions by triggering downstream targets such as interleukin-6 synthesis.In hepatic encephalopathy ammonia poisoning theory,Free NH3 functions as poison in different ways,like reducing energy supply in brain cells by disturbing tricarboxylic acid cycle,absorbing neutral amino acid,which restain brain function,leading to brain edema by making astrocytes swell,disturbing the neural electrical activity,all of which increase to incidence of HE.The ammonia transporter family Rh glycoprotein provide the way and convenience for transcellular membrane to transport of NH3 and NH4+.Among them,RhCG exists in non red blood cells,expressed in various tissues,including kidney,liver,skin and gastrointestinal tract,and is considered to be able to transport ammonia.The purpose of this article is to study the mechanism of the action of ammonia transporter RhCG and tumor necrosis factor TNF-alpha.Methods:After 5 times of recovery,culture and passage of HBMEC cells,two 6orifice plates were paved with 2 x 105 cells/holes for 2 days.Add the TNF-alpha at different time points respectively?0.1?g/mL ECM?into 37 degree centigrade,5%CO2incubator in cultured cells and collect cells,inspect RhCG RNA and protein with PCR and western blot.And analyse the trend of change with the change of TNF-alpha at different time of action.PKC inhibitors according to different time points,the treatment group was divided into:TNF-alpha treatment group 0h;TNF-alpha treatment group 24h;Safingol alone treatment group;TNF-alpha+inhibitor group:pretreatment HBMEC cells 1H,then TNF-alpha stimulation 24h.The expression level of RhCG mRNA and protein in HBMEC cells was detected by Western blot and PCR.The change trend of different groups of RhCG RNA and protein was also analyzed.Results:Successful recovery,cultivation of HBMEC,and stable passages.After adding human TNF-alpha at different time points and collecting cells,the expression of RhCG RNA increased with the prolongation of the time of TNF-alpha by using Real-time PCR,and the highest expression was observed at 24 hours.Western Blot was used to detect the increase in the expression of RhCG protein with the prolongation of the time of TNF-alpha action,and the highest expression was observed at 24 hours.On the basis of proving that tumor necrosis factor TNF-alpha can increase RhCG mRNA and protein expression in HBMEC cells add PKC-alpha specific inhibitor of Safingol,and the expression of protein decrease.Conclusin:Tumor necrosis factor TNF-alpha can increase the expression of RhCG mRNA and protein in HBMEC cells.PKC-alpha participates in the effect on TNF-alpha up regulation of RhCG expression in HBMEC cell. |
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