The Mechanism Of Tumor Necrosis Factor-α Up-regulation Of Rhcg Expression By Activating Transcription Factor 2 Resulting In Elevated Cerebral Ammonia And Cerebral Edema

Objective:The incidence of hepatic encephalopathy（HE）is up to 80%in patients with acute liver failure（ALF）,and HE is the most common cause of death in patients with ALF.At present,the theory of ammonia poisoning（ammonia generally refers to NH₃/NH₄⁺）has been the mainstream theory in the studies on the pathogenesis of HE.Clinical studies have confirmed that elevated blood ammonia is a risk factor for increased intracranial pressure,cerebral edema,cerebral hernia and even death in patients with acute hepatic encephalopathy（AHE）.Ammonia is a low molecular neutral gas.In the internal environment of the human body with p H 7.4,less than 2%exists in the form of NH₃ and more than 98%exists in the form of NH₄⁺.Free basic ammonia（NH₃）is lipid soluble and can pass through cell membrane and other structures in a free dispersion way,but the effect of free dispersion is limited.In addition to the passive diffusion along the concentration gradient,there may also be some special proteins used to complete the active transport of NH₃/NH₄⁺.Recent studies have shown that the Rh glycoprotein family（Rh AG/Rhag,Rh BG/Rhbg,Rh CG/Rhcg）may be related to the specific transport of NH₃/NH₄⁺.Rh CG/Rhcg is widely expressed in kidney,central nervous system,lung,liver,testis,gastrointestinal tract and other organs.Studies have confirmed that Rhcg plays an important role in renal ammonia metabolism.In our previous study,it was found that the expression of Rhcg in the brain tissue of mice with ALF and HE increased significantly,which was positively correlated with the increase of NH₃/NH₄⁺concentration in the brain tissue.The abnormally high expression of Rhcg was mainly distributed in cerebral microvascular endothelial cells and astrocytes.It is suggested that Rhcg with ammonia transport function is closely related to ammonia elevation in brain tissue.It has been found that the occurrence of HE is the result of synergistic action between inflammatory factors and ammonia,and the synergistic action between tumor necrosis factor-α（TNF-α）and ammonia is the most noticeable.Studies have shown that the deletion of TNF-αreceptor gene can delay the occurrence of HE.Serum TNF-αlevel was positively correlated with the occurrence and severity of HE in patients with cirrhosis.When the blood TNF-αlevel decreased,the severity of HE decreased.In our previous study found that block blood TNF-αALF mice,can make the brain tissue in mice Rhcg expression decreased obviously,brain tissue ammonia levels also declined obviously,explain ALF,TNF-αhave increased Rhcg expression effect,and participating in mechanism of brain tissue caused by ammonia increases.When HE occurs in ALF patients,cerebral edema and even cerebral hernia are often present,and the most prominent neuropathological feature is swelling of astrocytes in brain tissue.In ALF,a large amount of ammonia enters astrocytes,causing swelling of astrocytes and brain edema.In a study conducted by our research group,it was found that Rhcg knockout mice in astrocytes had reduced the degree of HE in the ALF model and significantly decreased the ammonia level in brain tissue,indicating that Rhcg knockout in astrocytes could significantly reduce the internal transport of ammonia in astrocytes.In cell experiments,it was also found that the internal transport of ammonia in astrocytes could be significantly reduced after Rh CG silencing,while the internal transport of ammonia in astrocytes could be significantly increased after Rh CG overexpression.At the same time,TNF-αcan up-regulate the expression of Rh CG in astrocytes and increase the transport of ammonia in astrocytes.Therefore,this study further confirmed the role of ammonia transporter Rhcg in ammonia transport in the HE mouse model caused by ALF through lentiviral vector mediated Rhcg gene silencing.In vitro,the blood-brain barrier（BBB）model was used to investigate whether TNF-αaffected ammonia transport by affecting Rh CG expression in the BBB.Meanwhile,nuclear factors downstream of TNF-αclassical pathway（c-Fos,ATF-2,ELK1,c-Jun,NF-κB）were screened on the in vitro culture model of astrocytes,and activation transcription factor 2,ATF-2),prepared ATF-2 gene silencing and overexpression lentivirus,and studied the effect of ATF-2 on Rh CG expression and ammonia transport in astrocytes by TNF-α.Methods:1.Rhcg gene silencing lentivirus vector was prepared,lentivirus transfection MOI values were screened,and the silencing efficiency of Rhcg gene silencing lentivirus in rat brain microvascular endothelial cells（BMECs）was detected by real-time PCR and Western blot.2.Mice were injected with Rhcg gene silencing lentivirus in the lateral ventricle to detect the silencing efficiency of Rhcg gene silencing lentivirus on mouse brain tissue.3.Four days after injection of Rhcg gene-silting lentivirus and negative control lentivirus in the lateral ventricle of mice,the mouse model of ALF was synthesized by intra-abdominal injection of D-galactosamine（D-Gal N）combined with lipopolysaccharide（LPS）.The grades of hepatic encephalopathy were observed at 5h,7h and 12h.The levels of serum ALT,serum TNF-α,blood ammonia and brain tissue ammonia were determined 12h after modeling,HE staining of liver and brain tissue and electron microscopy of brain tissue were determined.4.The in vitro BBB model was established with the non-contact co-culture method of human human microvascular endothelial cells（HBMEC）and human astrocytes（SVG p12）,and the 4-hour leakage test was conducted,and trans endothelial electric resistance（TEER）was measured.Verify the successful establishment of BBB model in vitro.5.Western blot and real-time PCR were used to detect the effect of different concentrations of TNF-αon RHCG expression in HBMECs.MTS assay was used to verify the cytotoxicity of TNF-αat different concentrations to HBMEC and SVG p12.In vitro BBB model was established.Different concentrations of TNF-αwere added to oxidase to determine the permeability of TEER and horse radish peroxidase（HRP）,and the effect of different concentrations of TNF-αon the permeability of BBB in vitro was studied.6.The minimum concentration of TNF-α,which can increase the expression of Rh CG but does not increase the permeability of BBB,was 50ng/m L.After adding 50ng/m L TNF-αto the BBB model in vitro for 12 hours,3m M NH₄Cl was added to the Transwell chamber.The indoor ammonia concentration was determined after 1 min,5 min and 15min,and the influence of TNF-αon ammonia permeability in vitro BBB model was studied.7.Astrocytes were added with 100ng/m L TNF-α,and the influence of TNF-αon the protein expression of nuclear factors（c-Fos,ATF-2,ELK1,c-Jun,NF-κB）in the downstream of TNF-αpathway was detected by Western blot.The effects of TNF-αon p-ATF-2 protein and ATF-2 transcription were detected by Western blot and real-time PCR.8.According to the TNF-αfound in the previous step,ATF-2 gene silencing lentivirus vector was prepared,and the silencing efficiency of ATF-2 gene silencing lentivirus in astrocytes was verified by real-time PCR and Western blot.9.After the ATF-2 gene was silenced in astrocytes,100ng/m L TNF-αwas added and treated for 12 hours.Western blot and real-time PCR were used to detect the effect of TNF-αon the expression of Rh CG.10.After ATF-2 gene silenced in astrocytes,100ng/m L TNF-αwas added for 12 hours,and 3m M NH₄Cl was added.After 0min,1min,5min,15min and 30min,the ammonia concentration in extracellular and intracellular fluid was determined.Effect of TNF-αon ammonia permeability.11.The ATF-2 gene overexpression lentivirus vector was prepared,and the overexpression efficiency of ATF-2 gene overexpression lentivirus in astrocytes was verified by real-time PCR and Western blot.12.After the ATF-2 gene was overexpressed in astrocytes,100ng/m L TNF-αwas added and treated for 12 hours.The effects of TNF-αon Rh CG protein and transcription levels were detected by Western blot and real-time PCR.13.After ATF-2 gene overexpression in astrocytes,100ng/m L TNF-αwas added for12 hours,and 3m M NH₄Cl was added.After 0min,1min,5min,15min and 30min,ammonia concentrations in extracellular and intracellular fluids were measured,and ATF-2 gene overexpression was verified.Effect of TNF-αon ammonia permeability.Results:1.Rhcg gene silencing lentivirus was prepared,and the fluorescence intensity of cells with different transfection concentrations was observed 72 h after transfection with lentivirus,and MOI=20+polybrene was selected as the optimal transfection concentration.In BMECs,Rhcg m RNA and protein expression in Rhcg gene silencing group were significantly lower than those in NC and NS groups（P<0.01）.2.The expression of Rhcg protein in the 2-day and 4-day groups injected with Rhcg gene silencing lentivirus in the lateral ventricle of mice was significantly lower than that in the normal saline group（P<0.01）.3.Effects of lateral ventricular injection of Rhcg gene silencing lentivirus on ammonia transport in the brain of ALF mouse.3.1 In the mouse model of ALF,the degree of HE in LV-Rhcg group was less than that in ALF group and LV-NC group（P<0.05）.3.2 There was no significant difference in the levels of serum ALT,serum TNF-αand blood ammonia in ALF group,LV-NC group and LV-Rhcg group.3.3 The ammonia level in brain tissue of ALF group,LV-NC group and LV-Rhcg group was significantly higher than that of NS group,and the ammonia level in brain tissue of LV-Rhcg group was significantly lower than that of ALF group and LV-NC group（P<0.05）.3.4 HE staining of liver in ALF group,LV-NC group and LV-Rhcg group indicated liver necrosis.3.5 HE staining of brain of mice indicated that brain edema of LV-Rhcg group was lighter than that of ALF group and LV-NC group.3.6 Electron microscopy of mouse brain tissue indicated that the swelling of astrocytes in the foot process of LV-Rhcg group was lighter than that of ALF group and LV-NC group.4.The non-contact coculture method of HBMEC and SVG p12 was used to establish the BBB model in vitro.The 4-hour leakage test showed that the BBB model could maintain the original level difference,while the blank control group could not.TEER was measured,and it was found that the TEER value reached the maximum at 72 h of co-culture,which was in line with the requirements of BBB model in vitro.Subsequently,HBMEC and SVG p12 were co-cultured for 72 hours and the experiment was carried out.5.Effects of different concentrations of TNF-αon Rh CG expression in HBMEC,MTS assay and permeability of BBB model in vitro5.1 m RNA and protein expression levels of Rh CG were significantly higher than those of NS group when HBMEC was added with TNF-α50 ng/m L,75 ng/m L and 100ng/m L（P<0.01）.There was no difference among the three doses of TNF-α.5.2 There was no difference in the MTS value of HBMEC and SVG p12 with different concentrations of TNF-αcompared with NS group.5.3 Different concentrations of TNF-αshowed no difference in the TEER difference and HRP transmittance of BBB model in vitro compared with NS group.6.In the BBB model in vitro,compared with the group without TNF-α,there was no significant difference in ammonia permeability in the group adding TNF-αat 1min,5min and 15min.7.The effect of TNF-αon the expression of downstream nuclear factors（c-Fos,ATF-2,ELK1,c-Jun,NF-κB p65）of the TNF-αpathway in astrocytes.7.1 After addition of TNF-α,ATF-2 protein was expressed in astrocytes at 8h（P<0.01）,12h（P<0.05）,24h（P<0.01）was significantly higher than that of control group.p-ATF-2protein expression at 4h,8h,12h and 24h was significantly higher than that of control group（P<0.01）.ATF-2 m RNA relative expression level at 4h,8h,12h and 24h was significantly higher than that of control group（P<0.01）.7.2 After addition of TNF-α,the expression of c-Fos protein in astrocytes was slightly increased at 8h and 12h compared with the control group,but there was no significant difference（P<0.05）.7.3 After adding TNF-α,the expression of ELK1 protein in astrocytes was slightly increased at 8h and 12h compared with the control group,but there was no significant difference（P<0.05）.7.4 After adding TNF-α,the expression of c-Jun protein in astrocytes was slightly decreased at 4h and 8h compared with the control group,but there was no significant difference（P<0.05）.7.5 After addition of TNF-α,the expression of NF-κB p65 protein in astrocytes had no significant changes compared with the control group（P<0.05）.8.The ATF-2 gene silencing lentivirus was prepared,and the Rh CG m RNA and protein expression in the ATF-2 gene silencing group were significantly lower than those in the NC and NS groups（P<0.01）.9.After ATF-2 gene silting in astrocytes,Rh CG protein and m RNA expression in LV-NC+TNF-αand LV-ATF-2+TNF-αgroups were significantly increased compared with NS and LV-NC groups（P<0.01）.Rh CG protein and m RNA expression in LV-ATF-2+TNF-αgroup were significantly lower than those in LV-NC+TNF-αgroup（P<0.01）..10.After ATF-2 gene silting in astrocytes and TNF-αaddition for 12 h,ammonia permeability in LV-ATF-2 group decreased significantly at 1min,5min,15min and 30min compared with that in LV-NC group at each time point of NH₄Cl addition（P<0.01）.11.The ATF-2 gene overexpression lentivirus was prepared.In astrocytes,the m RNA and protein expression of Rh CG in ATF-2 gene overexpression group were significantly increased compared with NC and NS groups（P<0.05）.12.After ATF-2 gene overexpression in astrocytes,Rh CG protein and m RNA expression in LV-NC+TNF-αand LV-ATF-2+TNF-αgroups were significantly increased compared with NS and LV-NC groups（P<0.05）.Rh CG protein and m RNA expressions in LV-ATF-2+TNF-αgroup were significantly higher than those in LV-NC+TNF-αgroup（P<0.05）.13.After the ATF-2 gene was overexpressed in astrocytes and treated with TNF-αfor12 h,the ammonia permeability of LV-ATF-2 group was significantly increased at 1min,5min,15min and 30min compared with that of LV-NC group after NH₄Cl addition（P<0.01）.Conclusion:1.In the mouse model of ALF combined with HE,after the silencing of Rhcg gene,the symptoms of hepatic encephalopathy in mice were alleviated,the degree of grading was reduced,brain tissue edema was improved,and the level of ammonia in brain tissue was significantly reduced.These results indicate that Rhcg promotes ammonia transport in brain tissue in the mouse model of ALF combined with HE.2.In the in vitro blood-brain barrier model,the addition of TNF-αhad no significant change in ammonia permeability,indicating that the main role of TNF-αin up-regulating Rh CG expression and thus affecting ammonia transport was not the blood-brain barrier.3.Through the screening of nuclear factors c-Fos,ATF-2,ELK1,c-Jun and NF-κB p65 in the downstream of TNF-αclassical pathway,it was found that TNF-αcould up-regulate the transcription and expression of ATF-2 in astrocytes.4.After ATF-2 gene silencing in astrocytes,Rh CG expression and ammonia permeability of TNF-αwere significantly decreased compared with the control group.However,after ATF-2 gene overexpression,TNF-αsignificantly increased Rh CG expression and ammonia permeability compared with the control group,indicating that TNF-αaffected Rh CG expression through ATF-2,thus affecting ammonia transport.