Mechanism Of Astrocyte Swelling In Acute Hepatic Encephalopathy

Objective:The incidence of hepatic encephalopathy（HE）in patients with acute liver failure（ALF）is 80%.HE is the most common cause of death in ALF patients.Patients with HE often have brain edema even brain hernia,and the swelling of brain astrocyte（AS）is the most prominent neuropathological feature.Ammonia poisoning theory has always been the mainstream theory of the pathogenesis of HE.When ALF occurs,a large amount of ammonia enters into AS and is catalyzed to glutamine（Gln）by glutamine synthase（GS）.On one hand,the enrichment of Gln lead to a sharp increase in the internal osmotic pressure of AS,resulting in the inflow of extracellular fluid and swelling.Gln is transported to mitochondria and metabolized into ammonia and glutamate again,cause secondary mitochondrial damage.The main route of ammonia entering AS is active transport,but its transporter has not been found.Recently,Rhglycoprotein family has been found involved into NH₃/NH₄⁺transportation,with RhAG/Rhag,RhBG/Rhbg,Rhcg/RhCG as the main transporters.RhCG/Rhcg is widely expressed in ammonia transport organs,including kidney,central nervous system,testis,lung,liver,stomach and intestine.The previous research of our team found that the expression of Rhcg in cerebral microvascular endothelial cells and AS in ALF complicated with HE mice increased significantly,which was positively correlated with the increase of NH₃/NH₄⁺concentration in the brain tissue.Therefore,this study speculates that RhCG may play a key role in the transport of ammonia and swelling of AS.TNF-αalso plays an important role in the AS swelling.Previous studies have shown that TNF-αmay promote AS ammonia absorption.Anti TNF-α-Ig G antibody and TNF-α-R1 antibody block TNF-α.It can reduce the incidence rate and mortality of HE in ALF mice.In conclusion,our study took SVG p12 astrocyte as the research object,and constructed a lentivirus transfected RhCG gene silencing and overexpression stable transformation cell line to verify whether RhCG protein is the main ammonia transporter on AS.At the same time,to explore whether the inflammatory factor TNF-αaffects the expression of RhCG and ammonia transport on AS.In vivo experiment,AS Rhcg gene c KO mice were obtained by CRISPR/cas9 technology to confirm the effect of Rhcg gene on brain edema in ALF.The elaboration of these problems will help us to find better therapy for HE.Methods:1.Two stable cell lines were established with lentivirus,in which RhCG was knock-out or overexpressed.The ammonia permeability,GS activity in AS,Gln concentration in AS,mitochondrial dehydrogenase activity（MTS method）and transmission microscope morphology in two cell lines were studied in high blood ammonia environment.2.The effect of TNF-αon RhCG expression was investigated,and the above indexes and mitochondrial morphology were tested with or without TNF-α.3.As Rhcg gene c KO mice were obtained by CRISPR/cas9 technology.Rhcg^-/-group（knockout group）and Rhcg^flox/flox（control group）mice underwent ¹³N ammonia micro PET imaging.Then the ALF model was prepared by D-Gal N/LPS,and the blood ammonia,brain ammonia,serum ALT and serum TNF-αlevel were detected.HE staining of liver and brain tissue were performed.Gln was detected by immunohistochemistry（IHC）and GS was detected by immunofluorescence assays（IFA）.Results:1.Establishment of stable RhCG-knock-out cell line.1.1 The expression of RhCG protein was significantly lower in shRNA 1355 than NC group,so it was selected as the sequence of synthetic lentivirus.1.2 MOI=20+P as the optimal transfection concentration for fluorescence intensity investigation after 72 h of incubation.1.3 A concentration of 0.5μg/m L was selected for puromycin screening.1.4 It was found that the expression of RhCG mRNA and protein were significantly decreased with RhCG silencing compared with negative control（NC）and normal saline（NS）control groups.The RhCG mRNA transcription levels was decreased by 89%and89%compared with NC and NS,respectively,while the expression of RhCG protein was decreased by 69%and 55%.2.Establishment of stable RhCG-overexpressed cell line.2.1 MOI=20+P as the optimal transfection concentration for fluorescence intensity investigation after 72 h of incubation.2.2 The expression of RhCG protein after lentivirus RhCG（28252-1）transfection was significantly higher than that in NC and NS control groups.3.The effect of RhCG gene silencing on astrocyte swelling in high ammonia environment.3.1 The ammonia permeability of lentivirus RhCG gene silencing group was 33.99%,46.29%and 55.75%lower than that in NC group at 1,5 and 15 min,respectively（P<0.05）.3.2 The GS activity of lentivirus RhCG gene silencing group was 48.35%,35.85%and18.07%lower than that of NC group at 1,5 and 15 min,respectively（P<0.05）.3.3 The concentration of Gln in lentivirus RhCG gene silencing group was 51.23%and45.61%lower than that in NC group at 1 and 5 min,respectively（P<0.05）.3.4 The mitochondrial MTS values of lentivirus RhCG gene silencing group were24.56%,22.15%,22.40%and 27.35%higher than those of NC group at 1,5,15 and 30min,respectively（P<0.05）.3.5 Observation of cell morphology by transmission electron microscope3.5.1 SVG p12+NH₄Cl:The astrocytes exhibited features of edema,the cell membrane was complete and continuous.Most mitochondria were oval and swollen.The matrix was slightly shallower and a small amount of cristae were broken and reduced.A small amount of mitochondria were seriously swollen and exhibited local vacuolization,deficient or disordered cristae,which is consistent with the performance of HE.3.5.2 Lentivirus silencing RhCG+NH₄Cl:The cell membrane was complete and continuous.There was no obvious edema in the cytoplasm,but more organelle vacuoles could be seen.The nuclear membrane was clear,and the perinuclear space was not significantly widened.Most mitochondria remained in normal shape.The matrix was uniform,and a small part was partially dissolved.The ridge was missing,and the membrane was damaged.The observation suggested AS swelling and mitochondrial damage,but the damage was significantly reduced compared with AS cells that normally expressing RhCG.4.Effect of ammonia on RhCG-overexpressed astrocyte.4.1 The ammonia permeability of lentivirus RhCG gene overexpression group was101.30%,60.06%and 83.62%higher than that of NC group at 1,5 and 15 minutes,respectively（P<0.05）.4.2 The GS activity of lentivirus RhCG gene overexpression group was 40.30%,63.49%and 48.85%higher than that in NC group at 1,5 and 15 minutes,respectively（P<0.05）.4.3 The concentration of Gln in lentivirus RhCG gene overexpression group was 75.87%,83.17%and 166.89%higher than that in NC group at 1,5 and 15 min respectively（P<0.05）.4.4 The mitochondrial MTS values of lentivirus RhCG gene overexpression group was14.55%,14.06%,25.75%and 12.61%lower than that of NC group at 1,5,15 and 30minutes,respectively（P<0.05）.4.5 Observation of cell morphology by transmission electron microscope4.5.1 SVG p12+NH₄Cl:The cells showed features of edema and cell membrane was complete and continuous.Most mitochondria were oval and swollen.The matri was slightly shallower,and a small amount of cristae were broken and reduced.A small amount of mitochondria were seriously swollen,and exhibited partial vacuolization,cristae loss and disorder.The observation suggested AS swelling and mitochondrial damage,which was consistent with the performance of HE.4.5.2 Lentivirus overexpression of RhCG+NH₄Cl:The cells were oval and edema.The cell membrane was relatively complete,and no obvious damage was found.Mitochondria were swollen,the matrix was uneven,and some cristae were broken.The observation suggested AS swelling and mitochondrial damage,but the damage was significantly worse than that of AS normally expressing RhCG.5.The effect of TNF-αon RhCG gene expression and ammonia transport in AS.5.1 TNF-αcould significantly increase the transcription and expression levels of RhCG in AS compared with NS control group at 4 h,8 h,12 h and 24 h.The increase was significant at 12 h.5.2 TNF-αsignificantly increased the ammonia permeability of AS by 263.79%,149.96%and 148.88%,respectively compared with control group at 1,5 and 15 min（P<0.05）.5.3 Compared with control group,TNF-αsignificantly increased GS activity in AS by59.49%,54.95%and 72.70%,respectively,at 1,5 and 15 min（P<0.05）in high ammonia environment.5.4 With TNF-α,the Gln concentration of AS in high ammonia environment was remarkedly decreased by 39.76%and 53.21%at 5 and 15 min,respectively compared with control group（P<0.05）.5.5 With TNF-α,the MTS value of AS in high ammonia environment was decreased by12.15%,11.60%,11.95%and 12.80%at 1,5,15 and 30 min,respectively,compared with control group（P<0.05）.5.6 Observation of cell morphology by transmission electron microscope5.6.1 SVG p12:The cell membrane was complete and continuous,no obvious edema was observed in the cytoplasm.Most mitochondria were observed with even matrix and clear cristae structure,indicating that the status of AS is normal.5.6.2 SVG p12+NH₄Cl:The cells exhibited features of edema,cell membrane was complete and continuous.Most mitochondria was oval and swollen.The matrix was slightly shallower and a small amount of cristae were broken and reduced.A small amount of mitochondria were seriously swollen and partially vacuolization,and cristae was deficient and disordered.It is suggested that AS swelling and mitochondrial damage are consistent with the performance of HE.5.6.3 SVG p12+NH₄Cl+TNF-α:Cell edema and organelle swelling were obvious.Many organelle vacuoles could be seen in the cell,and the cell membrane was complete and continuous.The number of mitochondria was decreased.The mitochondria were seriously swollen and the volume was increased.The matrix was uneven,and the cristae was missing and disordered.The observation suggested that AS swollen and mitochondrial damage is aggravated with the present of TNF-α.6.The role of Rhcg in brain ammonia poisoning was studied by using AS Rhcg c KO mice of ALF model.6.1 The head SUV value of ¹³N ammonia micro PET imaging in Rhcg^-/-group（knockout group）was significantly lower than that in Rhcg^flox/flox group（control group）.6.2 ALF model was established with D-Gal N+LPS.The degree of HE in Rhcg^-/-group was lighter than that in Rhcg^flox/flox group.6.3 Serum ALT,serum TNF-αand blood ammonia level between Rhcg^-/-and Rhcg^flox/floxgroups had no significant difference.6.4 The level of brain ammonia in Rhcg^-/-group was significantly lower than that in Rhcg^flox/flox group（P<0.05）.6.5 HE staining of liver in both two groups showed liver necrosis.6.6 HE staining of brain showed brain edema and AS swelling of Rhcg^-/-group was lighter than Rhcg^flox/flox group.6.7 The contents of GS and Gln in the brain of mice in Rhcg^-/-group were significantly lower than Rhcg^flox/flox group（P<0.05）.Conclusion:1.Astrocyte SVG p12 cell line transcribes and expresses RhCG gene on cell membrane.2.RhCG is one of the main proteins involved in the ammonia active transport of SVG p12.It could transport ammonia into cells,which enhances GS activity,producing a large amount of Gln,and cause mitochondrial damage and cell edema.3.TNF-αcould significantly promote the transcription and expression of RhCG gene in SVG p12,promoting ammonia transport into astrocyte SVG p12 and finally aggravate mitochondrial damage and cell edema.4.Vivo experiments confirmed that specific knockout of Rhcg on AS can significantly reduce the internal transport of ammonia to AS,so as to reduce the accumulation of Gln in AS and improve the swelling of AS.