Protective Effect Of Patchouli Alcohol Against Nonalcoholic Fatty Liver Disease By Suppressing Endoplasmic Reticulum Stress

ObjectiveEndoplasmic reticulum stress(ERS)has been reported to work as a key factor in nonalcoholic fatty liver disease(NAFLD)progression,which can trigger very low-density lipoprotein(VLDL)metabolism disruption and induce nod-like receptor protein 3(NLRP3)inflammasome activation.In our previous study,we found that patchouli oil can alleviate NAFLD by reducing hepatic steatosis.Therefore,we speculated that patchouli alcohol(PA,the main component of patchouli oil)may also exert a protective effect against NAFLD.In this study,we aimed to investigate the effect of PA in ERS and VLDL metabolism as well as NLRP3 activation,to elucidate the mechanism of PA against NAFLD.Methods1.The effect of PA on nonalcoholic fatty liverForty-eight male SD rats were randomly assigned into six groups(n=8):model group(high-fat diet,HFD),normal group(normal diet,ND),positive group(vitamin E,VE,100 mg/kg),different doses of PA(10,20,and 40 mg/kg)group,respectively.Except for the ND group(fed with ND),all rats were fed with HFD for 4 weeks to establish nonalcoholic fatty liver(NAFL)model.Meanwhile,rats were received relative medical treatment once daily.After the experiment,the serum contents of alanine aminotransferase(ALT)and aspartate transaminase(AST)were measured to evaluate the injury degree of liver.Serum and hepatic levels of total cholesterol(TC)and triglyceride(TG)were measured to evaluate lipid index.Liver tissues were stained with hematoxylin and eosin(H&E)and Oil red O to analyse histopathological alteration.Levels of free fatty acid(FFA),VLDL,apolipoprotein B100(apoB 100)and oxidative stress-related factors,such as reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),glutathione(GSH)and catalase(CAT)were also measured.The protein expressions of hepatic ERS-related factors such as inositol-requiring transmembrane kinase/endoribonuclease 1α(IRE1α),activating transcription factor 6(ATF6),protein kinase-like ER kinase(PERK)and glucose-regulated protein 78 kDa(GRP78)were measured by western blot.We also measured the protein expressions of hepatic VLDL metabolism-related factors like apoB 100,microsomal triglyceride-transfer protein(MTP),very low-density lipoprotein receptor(VLDLR)and their upstream proteins.2.The effect of PA on nonalcoholic steatohepatitisForty-eight male SD rats were randomly assigned into six groups(n=8):HFD group,ND group,positive group(VE,100 mg/kg),different doses of PA(20,40,and 80 mg/kg)group,respectively.Except for the ND group(fed with normal diet),all rats were fed with HFD for 8 weeks to establish nonalcoholic steatohepatitis(NASH)model.Meanwhile,rats were received relative medical treatment once daily.After the experiment,we measured the contents of AST and ALT,evaluated lipid index,analysed histopathological alteration and measured levels of FFA,oxidative stress-related factors and protein expressions of ERS-related factors as well as NLRP3 inflammasome-related factors like apoptosis-associated speck-like protein containing CARD(ASC),NLRP3 and caspase-1.Besides,we measured levels of Interleukin-1β(IL-1β),IL-18,IL-6 and tumor necrosis factor-a(TNF-α).Results1.The effect of PA on nonalcoholic fatty liverCompared to ND-fed rats,visceral obesity,elevated AST and ALT level as well as ERS,and reduced VLDL level were presented in HFD-induced NAFL rats.PA treatment can decrease serum levels of AST and ALT,reduce accumulation of visceral fat,reduce levels of TG and TC,and restore histopathological alteration of liver tissue.Our results suggested that PA can alleviate oxidative stress by reducing levels of FFA,ROS and MDA,and increasing levels of SOD,CAT and GSH.Reduced oxidative stress can alleviate ERS,thereby suppressing protein expression of GRP78,and activations of PERK,IRE1α as well as ATF6.PA can also increase protein expression and secretion of apoB 100,increase protein expressions of MTP and its related factors.In addition,PA can lower the protein expressions of VLDLR and its related factors,thereby restoring VLDL metabolism,reducing fat accumulation and steatosis in liver.2.The effect of PA on nonalcoholic steatohepatitisApart from visceral obesity,fat accumulation,elevated AST and ALT levels,as well as ERS occur in HFD-fed rats compared to ND-fed rats,the hepatic NLRP3 inflammasome was also activated in HFD-fed rats.Our results suggested that PA can reduce protein expression of C/EBP homologous protein(CHOP)by suppressing activation of PERK signaling pathway.CHOP is an important mediator of NLRP3 inflammasome activation.Overexpression of CHOP can promote the activation of NLRP3 inflammasome.PA can lower the protein expressions of ASC and NLRP3,reduce the ratio of cleaved-caspase-1/pro-caspase-1,and decrease the secretions of its downstream cytokines:IL-1β and IL-18.Additionally,the secretions of pro-inflammatory cytokines:TNF-α and IL-6 were also decreased by PA treatment.ConclusionIn this study,PA was proved to effectively ameliorate NAFLD,and exert hepatoprotective effect against NAFLD by inhibiting ERS in rats.The mechanism of PA against NAFL and hinder its progress may associate with restored VLDL metabolism,reduced fat accumulation and steatosis.In NASH model,PA can reduce inflammatory response by inhibiting NLRP3 inflammasome activation.