Effect Of The Glycyrrhiza Glabra L.extract To Alleviate Lipid Accumulation In Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease(NAFLD)is a disease of fatty liver accumulation not caused by excessive alcohol intake and has become the most common cause of chronic liver disease worldwide.In animals,NAFLD can cause loss of appetite and depression in pets or livestock,and liver damage can lead to liver failure when aggravated,eventually leading to animal death.NAFLD is caused by multiple factors,and the widely accepted “two hits” theory suggests that the first hit is the milder and less harmful hepatic steatosis,and the second hit is the oxidative stress that brings inflammation and damage.Glycyrrhiza glabra L.is a common traditional herb with triterpene saponins and flavonoids as its main bioactive components.Glycyrrhiza glabra L.has the ability to suppress cough and expectorant,clear heat and detoxify,tonic the spleen and stomach,anti-inflammatory,anti-viral,hepatoprotective as well as improve the body’s ability to resist tumors.Compared with other types of licorice,the unique component of Glycyrrhiza glabra L.is Glabridin,which has strong antioxidant,anti-atherosclerotic,lipid regulating and hypotensive effects.In this experiment,the effect of the Glycyrrhiza glabra L.extract(TGGE)on lipid accumulation in NAFLD was systematically investigated by establishing a lipid accumulation model in Hep G2 cells with free fatty acid(Oleic acid + palmitic acid,OA + PA)and an in vivo NAFLD model in mice fed with high fat diet(HFD).In this study,TGGE was obtained by grinding and sieving the powder,dissolving it in methanol for ultrasonic extraction,and evaporating the filtrate to obtain TGGE.We determined the contents of Liquiritin,Isoliquiritin,Liquiritigenin,Glycyrrhizic acid,Isoliquiritigenin,Glycyrrhetnic acid and Glabridin as the main active ingredients in TGGE by using high performance liquid chromatography(HPLC).To predict the potential targets of TGGE,the most important active components of TGGE,Glabridin,Liquiritin and Isoliquiritin were compared with KEAP1 and ATG5,respectively.We predict that TGGE can bind to KEAP1 to affect the Nrf2 signaling pathway and thus counteract oxidative stress in NAFLD.To clarify the role of TGGE in NAFLD and its mechanism,we examined the effects of TGGE on antioxidant signaling pathway and autophagic signaling pathway in OA + PA-induced lipid accumulation model in Hep G2 cells and HFD-induced NAFLD model in mice.In vitro cellular Oil red and BODIPY staining assay and in vivo mice liver H&E staining and Oil red assay showed that TGGE alleviated lipid accumulation in NAFLD.In vitro cellular reactive oxygen species(ROS)flow assay and in vivo biochemical indexes of malondialdehyde(MDA),glutathione(GSH),ROS and superoxide dismutase(SOD)levels indicated that TGGE could enhance the antioxidant stress ability of the body.To further investigate its potential mechanism of action,we used western blot to detect the expression of antioxidant-related proteins Nrf2,Lamin B1,KEAP1,NQO1,GCLC and GCLM and autophagy-related proteins PI3 K,Akt,m TOR,Beclin1,ATG5,ATG7,LC3 Ⅰ/Ⅱ and P62.Meanwhile,we examined the expression of Nrf2,LC3 and P62 using cellular immunofluorescence staining and animal immunohistochemical staining.Finally,we also determined the expression of Nrf2,LC3 and P62 genes in cells as well as mouse liver using fluorescence quantification.Based on the experimental results,we clarified that TGGE could alleviate the lipid accumulation in NAFLD by activating the antioxidant signaling pathway and promoting the autophagic signaling pathway.In summary,we obtained TGGE by extracting the active ingredients of Glycyrrhiza glabra L.and identified its most important active components as Glabridin,Liquiritin and Isoliquiritin.The TGGE obtained in this study could alleviate lipid accumulation in NAFLD by activating antioxidant and promoting autophagy.Therefore,TGGE has the potential to become an effective drug for the treatment of NAFLD.