β-patchoulene Alleviates Non-alcoholic Fatty Liver Disease:Involvement Of Activiating AMPK Signaling Pathway

ObjectivesPatchouli is a folk traditional Chinese medicine which has been known for its efficacy of anti-gastrointestinal diseases.However,it has been documented that its effect is not limited to the gastrointestinal tract,but also the liver.Based on our previous study,the effective part of patchouli,patchouli oil and its characteristic ingredient,patchouli alcohol(PA)can be able to prevent non-alcoholic fatty liver disease(NAFLD).β-Patchoulene(β-PAE),as one of the representative components separated from patchouli oil and the metabolite of PA,has multiple activities,but its effect and mechanism against NAFLD is still unknown.NAFLD is a disease of excess energy and metabolic disturbance associated with hepatic tissue storing lipids or energy exceeding its storage capacity.It has become a health issue that public is very concerned and worried about due to its rising incidence and limited interventions.As a significant target for the prevention of NAFLD,AMPK,an AMP-dependent protein kinase,controls energy homeostasis,which can availably regulate lipid metabolism.And our earlier study found that patchouli displayed AMPK-activating potential.Thus,this study was to examine the positive effect of β-PAE on NAFLD based on AMPK signaling pathway.Methods1.The pharmacodynamics study of β-PAE against non-alcoholic fatty liver diseaseIn cellular experiments,when the number of L02 cells increases exponentially,they were divided into 3 groups:normal control group(NC),model group(Model),and β-PAE group.In addition to the complete medium for the NC group,the Model and β-PAE group were added with 1 mM FFA high-fat medium without or with β-PAE for 24 h to establish a NAFLD cell model.We performed CCK-8 assay to identify a suitable concentration ofβ-PAE for subsequent experiments on L02 cells.The cellular lipid was stained by oil red O for assessment,and the level of TG was also measured.In animal experiments,SD rats were split up into 7 groups(n=8)after adaptively reared:normal control group(NC),model group(Model),vitamin E positive group(VE,100 mg/kg),rosuvastatin positive group(RSV,20 mg/kg),three doses of β-PAE group(10,20,40 mg/kg).All rats were subjected to a HFD for 4 weeks to construct a NAFLD animal model,with the exception of those in the NC group ad normal diet feeding.Meanwhile,groups of rats were perfused with saline or corresponding drugs.The serum ALT,AST,HDL-C,LDL-C,TG and TC as well as the liver TG,TC and NEFA were measured.Besides,H&E and Oil Red O staining were used for pathological analysis of hepatic tissue.2.The mechanism study of β-PAE against non-alcoholic fatty liver diseaseBased on the pharmacodynamic research method,PA group(20 mg/kg)was added for comparison with the 20 mg/kg β-PAE group,and the expression of key proteins related to hepatic lipid systhesis,fatty acid oxidation and endoplasmic reticulum stress including SREBP-1c,HMG-CR,CPT-1a and GRP78 were measured by western-blot to explore the crucial mechanism of β-PAE on NAFLD.According to the results,the expression of the proteins correlated with lipid systhesis and fatty acid oxidation such as p-AMPKα,AMPKα,SREBP-1c,p-ACC1,ACC1,FASN,SCD1,HMG-CR,SIRT1,PGC-1α,PPARα,FGF21,CPT-1a and ACOX1 were determined by western-blot,and the mRNA expression of the pivotal proteins like AMPKα,SREBP-1c,HMG-CR,SIRT1,PPARα,CPT-1a and ACOX1 were observed by qRT-PCR,in order to further explore the mechanism.Besides,hepatic MDA,GSH-Px and SOD levels were measured for antioxidant analysis.3.The mechanism verification of β-PAE against non-alcoholic fatty liver diseaseBased on the pharmacodynamic research method,the administration group of Compound C(AMPK inhibitor)and Compound C combined with β-PAE was added for verifying the mechanism of β-PAE on NAFLD.Except for the NC,Model and β-PAE group with complete medium,other groups were pretreated with 10 μM Compound C(an AMPK inhibitor)for 2 h to inhibit AMPK activity.The cellular lipid was stained by oil red O for assessment,and the level of TG was also measured.Besides,the protein expression of p-AMPKα,AMPKα,SREBP-1c,HMG-CR,and SIRT1 were measured by western-blot.Results1.The pharmacodynamics study of β-PAE against non-alcoholic fatty liver diseaseIn cellular experiments,it was found that β-PAE could markedly improve red lipid drops stacking and reduce the cellular TG levels,suggesting that it can ameliorate lipid deposition and alleviate steatosis.In animal experiments,β-PAE significantly relieved the body weight gain and visceral obesity,reduced the levels of liver injury indexes(ALT and AST),regulated serum lipid profiles(HDL-C,LDL-C,TG and TC),decreased hepatic lipid parameters(TG,TC and NEFA),and improved hepatic pathological abnormalities,suggesting that it can relieve hepatic steatosis and injury to prevent NAFLD.2.The mechanism study of β-PAE against non-alcoholic fatty liver diseaseContrasted with PA treatment,β-PAE had a stronger capacity to regulate hepatic lipid synthesis and fatty acid oxidation while it had a weaker ability to improve hepatic endoplasmic reticulum stress.Thus,its mechanism was further analyzed from the perspective of "regulating liver lipid synthesis and oxidation".β-PAE dramatically reduced the hepatic lipid synthesis-related protein expression like SREBP-lc,p-ACC1，ACC1,FASN,SCD1,HMG-CR and mRNA expression of pivotal proteins such as SREBP-lc,HMG-CR,up-regulated fatty acid oxidation-related protein expression like SIRT1,PGC-1α,PPARα,FGF21,CPT-1a and mRNA expression of key proteins such as SIRT1,PPARα,CPT-1a,nevertheless,there was no effect on either expression of ACOX1.β-PAE also restored antioxidant factors MDA,GSH-Px and SOD levels.Meanwhile,it significantly enhanced AMPK expression to improve hepatic lipid synthesis and oxidation.Overall,it strongly suggested that β-PAE is likely to regulate hepatic lipid synthesis and mitochondrial fatty acid β-oxidation via activating AMPK signaling pathway to improve hepatic lipid metabolism,and attenuate NAFLD.3.The mechanism verification of β-PAE against non-alcoholic fatty liver diseaseβ-PAE improved the protein expression of AMPK and its regulatory factors like SREBP-1c,HMG-CR and SIRT1.Nevertheless,Compound C abrogated the benefits derived from β-PAE in L02 cells,suggesting that β-PAE can play the role of AMPK agonist to prevent NAFLD by regulating hepatic lipid metabolism.Conclusionβ-PAE exerts AMPK agonist-like effect to regulate hepatic lipid synthesis and oxidation,improve hepatic lipid metabolism,eventually prevent NAFLD progression.This study illustrates the mechanism of β-PAE on NAFLD prevention,provides a scientific basis for the research on pharmacodynamic substance basis and mechanism of patchouli against NAFLD.