Role Of Ammonia For Brain Abnormal Protein Glycosylation During The Development Of Hepatitis B Virus-related Liver Diseases

Background:End-stage liver diseases caused by hepatitis B were often complicated with hepatic encephalopathy（HE）.The theory of ammonia poisoning is one of the main pathophysiology,but its exact mechanism is not completely clear.Glycosylation of proteins is a kind of post-translational modification that exists widely in the vertebrate nervous system.It is of great significance to elucidate the alternation and function of glycans to reveal its biological roles in the development of diseases.However,the underlying pathophysiology between ammonia and aberrant glycosylation in HE remains unknown.Here,we used HBV transgenic mice and astrocytes to present a systems-based study of glycosylation changes and corresponding enzymes associated with the key factors of ammonia.This study will provide a theoretical basis for clarifying the potential pathophysiological mechanism between abnormal glycosylation and ammonia toxicity in HE.Methods:1.H&E staining was used to reveal the progress of liver disease and astrocyte swelling of HBV transgenic mice,and serum ammonia level was analyzed by ELISA.2.The glycan profiles of glycoproteins of olfactory bulb,left hemisphere,right hemisphere,midbrain and cerebellum from 8-18 months old HBV transgenic mice and control mice were analyzed by lectin microarrays,protein microarrays and lectin blotting,and the localization of glycans were studied by fluorescence-based lectin histochemistry.3.Human astrocytes and NH₄Cl were used to simulate a high ammonia concentration environment in vitro.The effects of ammonia on the specific glycoprotein glycans were detected by lectin microarrays and confirmed by fluorescence-based lectin cytochemistry.The expression of glycotransferase responsible for the synthesis of specific glycan was analyzed by Real-time PCR and Western Blotting.4.The effects of specific glycotransferase on intracellular calcium homeostasis and the key molecule involved in regulation were studied by si RNA interference,calcium ion detection and rescue experiments.Results:1.The H&E staining of mice liver tissues showed severe and persistent hepatocellular injury during HBV accumulation in HBV transgenic mice.The serum ammonia concentration was up-regulated,and astrocytes were swollen with the development of hepatic disease in HBV transgenic mice.2.The glycan profiles of glycoproteins from HBV transgenic mice and control mice were compared by lectin microarrays,and the results indicated that the expression levels of glycopatterns recognized by MPL,PSA,SJA,LCA,PHA-E,DSA,LEL,SBA,LTL,NPA,HHL and WFA exhibited significantly changed.The results of fluorescence-based lectin histochemistry indicated that the distribution of specific glycans were different in mice.3.In vitro studies,the increase of Galβ1-3Gal NAc related to the upgrade of ammonia concentration.Ammonia up-regulated the expression of core 1β1,3-galactosyltransferase（C1GALT1）,which was associated with the synthesis of Galβ1-3Gal NAc.4.Ammonia-induced up-regulation of C1GALT1 resulted in deregulates Ca²⁺homeostasis in astrocytes.After C1GALT1 was interfered with si RNA,the expression of inositol 1,4,5-trisphosphate receptor type 1（IP3R1）was restrained and intracellular Ca²⁺homeostasis was disturbed.The results of rescue experiments showed that down-regulation of C1GALT1 could reverse the up-regulation of Ca²⁺induced by ammonia.Moreover,IP3R1agonist could restore intracellular Ca²⁺imbalance induced by C1GALT1.5.The expression levels of C1GALT,IP3R1 and Ca²⁺in the brains of 18 months old HBV transgenic mice were higher than those in the control mice,which was consistent with the results observed in astrocytes.Conclusion:Together our data point toward that increased Galβ1-3Gal NAc mediated by C1GALT1in hyperammonemia and C1GALT1 facilitated Ca²⁺concentration due to the overexpression of IP3R1.Moreover,we demonstrated that interfering with C1GALT1 could depress the IP3R1 expression,an effective strategy to inhibit the ammonia-induced upregulation of Ca²⁺activity,thereby C1GALT1 and IP3R1 may serve as therapeutic targets in hyperammonemia of HE.Better understanding of the connection between glycosyltransferase and ammonia may provide more targeted options for future treatments.