Xiaochaihutang Improves The Acute Hepatic Encephalopathy By Activating NRF2 Pathway In Rats

Objective:The therapeutic effect of xiaochaihutang（XCHT）on rat acute hepatic encephalopathy（AHE）induced by thioacetamide（TAA）was observed in vivo experiment.In vitro experiment,intervention effect of XCHT compound serum on astrocytes edema induced by high ammonia was further observed.The oxidative injury and NRF2 pathway were analyzed to explore the mechanism of XCHT on AHE.Methods:In vivo experiment,50 SD rats were randomly divided into vehicle group,model group,low,medium and high dose groups（1.0,2.0,4.0 g/kg）of XCHT.Rats were given 300mg/kg TAA for 3 days for AHE model.From the 4^th day,each group was given the same amount of double distilled water and different doses of XCHT for 2 weeks.24 h after the last administration,the behavior of rats was observed and materials were obtained.First,the degree of liver injury in rats was determined by alanine aminotransferase,aspartate aminotransferase and pathological sections of liver.Then,Blood ammonia level,brain water content,glutamine synthetase（GS）,malondialdehyde（MDA）and total superoxide dismutase（T-SOD）in cerebral cortex were measured.In addition,the expression of related genes and proteins of NRF2 pathway were detected by real-time PCR and Western Blot,including NRF2,KEAP1,HO-1,NQO1,GCLC,GCLM.The mRNA and protein expressions of glial fibrillary acidic protein（GFAP）and aquaporins 4（AQP4）were also detected.In order to investigate whether XCHT has the direct intervention on cerebral edema caused by AHE,we conducted in vitro experiments.The protective effect of XCHT compound serum normal astrocytes and astrocytes transfected with NRF2 siRNA（an inhibitor of NRF2）under high ammonia conditions through direct observation by microscope,immunofluorescence,real-time PCR and western Blot,and its mechanism was discussed.To further determined the direct intervention effect of XCHT on astrocytes edema and whether its intervention effect was activating NRF2 pathway.Result:1)In vivo experiment,we found that TAA successfully prepared for AHE rat,the behavior scoring of the rats has a significant increased,encephalopathy symptoms such as lethargy and ataxia occurred,and autonomic activities decreased.Meanwhile,liver damage occurred,the water content of brain,ammonia level（blood ammonia,brain ammonia）in TAA group increased,MDA in cerebral cortex increased,and T-SOD level decreased.After treatment with Xiaochaihutang,the score of rats decreased,the autonomic activities increased,and the blood ammonia level increased.The content of MDA in cerebral cortex decreased and the level of total superoxide dismutase increased.Mechanism study found that the expression of NRF2,HO-1,NQO1,GCLC and GCLM genes and proteins on NRF2pathway in TAA group decreased.In addition,GFAF expression decreased but the expression of AQP4 increased.However,after Xiaochaihutang treatment,the increased of NRF2 nuclear transfer activation,the expression of its downstream genes and proteins also increased,GFAP expression increased and the expression of AQP4 showed a downward trend.2)In vitro experiment,it showed that primary astrocytes treated with 5 mM ammonia had a significant increase in cell volume and refractive index.Immunofluorescence showed an increase in AQP4 expression of astrocytes.After the intervention of XCHT compound serum,the volume and refractive index of cells decreased significantly,and expression of AQP4 also decreased.The results of mechanism analysis showed that the gene and protein expressions of NRF2,HO-1,NQO1,GCLC,GCLM in primary astrocytes treated with ammonia decreased,while XCHT compound serum increased the nuclear transfer activation of NRF2,and increased the expression of downstream genes and proteins.Further experiments found that XCHT significantly weakened its intervention on ammonia-induced astrocytes edema in NRF2-siRNA treated astrocytes.Conclusions:1)Xiaochaihutang improves the AHE induced by TAA in rats.2)The mechanism of Xiaochaihutang on the treatment of AHE related to the inhibition of astrocytoedema by activated the NRF2 nuclear transfer and its downstream antioxidant damage signal pathway.