Emotional Stress Increases The Susceptibility Of Nonalcoholic Fatty Liver Disease(NAFLD) And Theacrine Protects Against NAFLD

Background: Huang di’s Canon of Medicine,a medical works from acient China,indicated that two people with comparable age,living standards,gave sick or not under a bad condition.This theory showed that there are differences in the incidence and severity of diseases among the individuals,which is the disease susceptibility.Although two individuals possessed the same genotypes,they would also show differences in disease susceptibility under the same pathogenic factors when the two people were in different emotional state.Recently,comsumming high fat diets has become an inevitable lifestyle,therefore nonalcoholic fatty liver disease(NAFLD)has become one of the main diseases influencing the public health.Traditional Chinese medicine indicates that Liver-Qi Stagnation leads to Spleen Deficiency Syndrome.It demonstrates that emotional stress disturbs normal metabolic function,therefore increases the susceptibility of NAFLD.Pervious epidemiological investigations indicated that occupational stress increased the incidence of NAFLD in high-fat diet(HFD)-comsumed individuals while non-clinical studies also demonstrated restraint stress aggravated liver phenotypes of NAFLD in mice.Those studies highlighted that emotional stress increased the susceptibility of NAFLD.However,the mechanism of stress-susceptible NAFLD remains unclear.Objective: The mechanism of stress-susceptible NAFLD was investigated by appling in vivo and in vitro models induced by two factors,emotional stress and high fat diet feeding.In addition,the stress-susceptible NAFLD models were employed to evaluate the positive effects and probable mechanism of theacrine,a rare purine alkaloid from Camellia assamica Var.kucha.Method: ApoE-/-and C57BL/6J mice were fed with HFDs for 14 weeks.At the 8th week,HFD-fed Apo E-/-and C57BL/6J mice received restraint stress or corticosterone(CORT,2 mg/kg,S.C.)injection,respectively.In addition,cortisol(CS)-treated Hep G2 cells were employed to establish an emotional stress-susceptible NAFLD in vitro model.Thses models were regarded as emotional stress-susceptible NAFLD models.Liver phenotypes were investigated,and acylcarnitine profiles and fatty acid metabolism-related gene and protein expression levels were determined.Subsequently,the emotional stress-susceptible NAFLD mice models were treated with theacrine and the pharmacological effects of theacrine were evaluated.Moreover,HFD-fed Apo E-/-mice and oleate-treated hepatocytes were applied as NAFLD models to explore the pharmacological effects and mechanism of theacrine.Herein,fatty acid flux analysis and gene silencing methods were used to confirm the underlying mechanism of theacrine.Results: Restraint stress aggravates hepatic steatosis,inflammation fibrosis and acycarnitine metabolism disoreder in HFD-fed Apo E-/-mice.These results indicated that emotional stress increased the susceptibility of NAFLD,which may be related to excess plasma CORT scrected by hypothalamic-pituitary-adrenal axis activation.When mice were given with 2 mg/kg of CORT,the dosages of which induced equivalent plasma CORT in mice compared to that of the restraint stress treatment,CORT-treated mice were characterized by similar liver phenotypes such as macrovesicular steatosis,excess triglycerides and fatty acids accumulation,and hyperacetylation of mitochondrial proteins.Moreover,emotional stress-induced CORT inhibited the function of Sirtuin 3(SIRT3)/long-chain acyl coenzyme A dehydrogenase(LCAD)signaling pathway and upregulated PLIN2 gene and adipophilin(ADFP)protein expressions through glucocorticoid receptor(GR)translocation into nuclei.In addition,CORT increased cytoplasm GR translocation into lipid droplet membranes and recruitd ADFP by increasing interaction of GR and ADFP.Blockade of GR reversed macrovesicular steatosis and increased the function of SIRT3/LCAD signaling pathway;and inhibited excess ADFP expressions and ADFP translocation into lipid droplet membranes.In addition,CS inhibited cellular acylcarnitine metabolism.After Hep G2 cells were treated with CS and oleate,cellular lipid accumulation was increased and SIRT3 activity was decreased.The oleate and CS-induced hepatocytes model was considered as an in vitro model of emotional stress-susceptible NAFLD.Increasing cellular SIRT3 expressions rescued steatosis in CS-susceptible hepatosteatotic cells.The results indicated that SIRT3 was a key regulator on the mechanism of emotional stress-susceptible NAFLD.Moreover,the results indicated that theacrine improved acylcarnitine metabolism and activated the SIRT3/LCAD signaling pathway.Theacrine decreased macrovesicular steatosis,inflammation or fibrosis in the 3 emotional stress-susceptible NAFLD models.When HFD-fed NAFLD mice and oleate-treated Hep G2 cells were treated with theacrine,hepatic steatosis and inflammation were reduced.The obtained data also confirmed that theacrine decreased long-chain acylcarnitines accumulation via improving fatty acid oxidation and inhibiting fatty acid elongation and desaturation.In particular,the results demonstrated that theacrine enhanced SIRT3 activity through targeting on SIRT3 and increasing SIRT3 expression,thereby decreasing the acetylation of LCAD and increasing LCAD activity,thereby improving acylcarnitine metabolism.Conclusion: This study indicated that the mechanism of emotional stress-susceptible NAFLD was involved in decreased SIRT3/LCAD activity and acylcarnitine metabolism disorder and increased ADFP accumulation through GR translocation into nuclei or lipid droplet membranes,respectively.Theacrine protected against stress-susceptible NAFLD mice and HFD-induced NAFLD mice.The findings above provided a therapy basis of theacrine on stress-susceptible NAFLD and those NAFLD related to Liver-Qi Stagnation and Spleen Deficiency Syndrome.