Levo-tetrahydropalmatine Attenuates FFAs-induced NAFLD Through AMPK Activation

Nonalcoholic fatty liver disease(NAFLD)is metabolic stress liver injury caused by other factors except alcohol,which the main characteristic of NAFLD is too much fat stores in liver cell on the basis of not excessive drinking.NAFLD is a common pathological disease of the liver,which is recognized as the most common liver disease in the world affecting 25%-30% of the global population,and its incidence is rising and the age of onset is getting younger.Meanwhile,in 2018,the prevalence of NAFLD in China has reached 32.9%,becoming the leading cause of chronic liver disease.So far,there has been no effective drugs or treatment in western medicine approved by FDA because of complex pathological mechanism of NAFLD,and the treatments to reverse NAFLD only focus on behavior modification,diet,exercise and weight loss.In recent years,with the development of National Traditional Chinese Medicine Health Industry,traditional Chinese medicine(TCM)has made some progress in the study of syndromes differentiation treatment of NAFLD and the treatment of NAFLD has been effective and diversified,which may provide new ideas for NAFLD.Natural products have been proven significant pharmacological effects to NAFLD,for example,including polygonin,emodin,curcumin,ganoderma triterpenes.As one of the most commonly used TCM,Corydalis Yanhusuo W.T.Wang has good effects on the nervous,the cardiovascular and the endocrine.Levo-tetrahydropalmatine(THP)is a common clinical drug on the nervous system diseases which is one of the main components of Corydalis Yanhusuo and belongs to alkaloids,its pharmacodynamics research mainly focusing on analgesia,sedation,hypnosis;however,the effect of THP on NAFLD remains poorly understood and reports are rare.Therefore,we explored the potential mechanism of THP in NAFLD in vitro and in vivo.Objective: This study aims to target NAFLD based on AMPK-dependent signaling pathways to clarify THP "how to regulate lipid metabolism and activation of oxidative stress","how to evaluate the effectiveness and safety of the treatment of NAFLD",which provides new ideas and strategies for NAFLD treatment.Methods: Hepatic steatosis-mimetic conditions were induced by treating the cells with a mixture of free fatty acids [sodium oleate : sodium palmitate 2:1,w/w],dissolved in th culture media;simultaneously,a more comprehensive medium composition is created to form a microenvironment of excess nutrition so that the steatosis degree of the cell model is higher,and a cell model of NAFLD with increased volume and number close to the clinic is established.The traditional Chinese medicine THP with good effectiveness and strong safety was selected using rational drug screening strategy;the cytotoxic effects of drug molecules on cell viability and colony formation of different NAFLD cell models were studied.The x CELLigence real time cellular analysis system was used to screen the appropriate concentrations of drug-treated NAFLD cell models,ensuring and the lipid-alleviated activity and safety.q PCR assays explore the mechanism by which drug molecules play a role in regulating m RNA of NAFLD to improve lipid accumulation.The expression level changes of key proteins in the process of lipid synthesis and oxidation were detected by Western blot to clarify the pathway network of AMPK-based to regulate lipid metabolism and oxidation.Using ATP synthase inhibitors,uncoupler FCCP,respiratory chain inhibitors Rotenone/Antimycin A,glucose and glycolysis inhibitors 2-DG to stimulate NAFLD cell models,it was in order to determined that THP affected effects of OXPHOS and glycolysis,revealing energy mechanism of NAFLD cell models THP-alleviated.Establishment and grouping of HFD animal model: thirty-two 6-week-old SPF C57BL/6J male mice were fed adaptively for 7 days and were randomly divided into control group,model group,40 mg/kg of L-THP group and 80 mg/kg of L-THP,respectively with 8 mice in each group;the control group was fed with normal diet,and the other three groups were fed with high-fat and-sugar diet for 12 weeks to establish the animal model of NAFLD;assessment of medicinal properties and pharmacological mechanism of L-THP in HFD animal models were explored to complete pre-clinical evaluation of pharmacology and toxicology through data analysis of biochemical and vital indexes.The metabolites with important biological significance and statistically significant differences in the occurrence and development of NAFLD were detected and screened using metabolomics,which provided an important strategy for the diagnosis and treatment of NAFLD.Results: Cell steatosis model of SMMC-7721 and BEL-7402 cells was successfully established in vitro according to Oil red O staining lipid droplets of sodium oleate and sodium palmitate combined,determining the optimal cell steatosis model that in SMMC-7721 cells were 1000 μM sodium oleate and 500 μM sodium palmitate(2:1),in SMMC-7721 cells were 400 μM sodium oleate and 200 μM sodium palmitate(2:1).The effective and safe concentrations of THP were screened based on the lipid accumulation model in vitro;the concentrations were respectively 75,100 μM and 50,75 μM which could be used for subsequent experiments.It was further verified that the concentrations screened of L-THP were highly protective and low toxicity,which could reduce the damage to the cell steatosis model through colony formations and x CLEEigence real-time label-free system.Relative quantitative PCR results showed that the m RNAs of AMPK,ACC,FAS,HMGCR,SREBP-1c,TBC1D1,PGC-1α and CPT1 showed significant physiological changes in L-THP-treated cell steatosis models,participating in lipid metabolism and sugar metabolism related transcription factors,compared with HFD group.Similarly,in western blot assays,the expression levels of SREBP-1c,FAS and HIF-1α in HFD group were increased compared with control group;the expression levels of SREBP-1c,FAS and HIF-1α in L-THP group were decreased compared with HFD group;the expression levels of p-AMPK,SIRT1 and p-ACC in HFD group were decreased compared with control group;the expression levels of p-AMPK,SIRT1 and p-ACC in L-THP group were increased compared with HFD group.OCR and ECAR in cell steatosis models were measured that basal respiration,ATP production,and maximal respiration were promoted by OCR,and glycolysis and glycolytic capacity were inhibited by ECAR;the results indicated that THP reduced the activity of oxidative stress in vivo and further interfered with the energy requirement for lipid synthesis,in this process,verifying that THP played a role in inhibiting OCR and ECAR depended on AMPK activation.The results in vivo showed that the body weight and liver weight of HFD mice reduced because of the treatment of THP;biochemical indexes analysis showed that THP effectively decreased the levels of triglyceride,cholesterol and low density lipoprotein,and increased the level of high density lipoprotein;the results of protein expression in liver tissue showed the phosphorylation of AMPK and ACC promoted to oxidate fatty acids and the SIRT1 was also increased in THP group,on the other hand,the FAS and SREBP-1c were inhibited leading to suppression of fat synthesis,consistent with liver tissue immunohistochemistry of FAS and AMPK.The pathological sections of liver tissue showed that the liver cells with bulbous steatosis and inflammatory cells infiltration were improved to be orderly,and the lipid accumulation and steatosis were significantly reduced after treatment with THP,therefore,other organs including heart,lung,kidney and spleen,showed no significant pathological damage compared to HFD group.Comprehensive metabolome information were obstained through liver lipidomics analysis and studied the metabolomics differences between HFD group and THP-treated group in vivo models;a total of 1371 metabolites were detected,including glycerides,glycerophospholipids,fatty acyls,sterolipids,sphingolipids,etc.,and 8 different metabolites were screened out.Importantly,the metabolic process and change mechanism of NAFLD were elucidated by analysis of PCA,OPLS-DA/S-Plot,Cluster Analysis,VIP,Heatmap,KEGG,and KEGG enrichment.Conclusion: In the SMMC-7721 and BEL-7402 cell steatosis models induced by free fatty acids and C57BL/6 HFD mice models fed with high-fat and-sugar diet,THP effectively alleviated excessive oxidative stress and excessive fat accumulation in liver cells by targeting AMPK activity,enhancing SIRT1 activity,and regulating the SREBP-1c/FAS pathway,which further inhibited lipid synthesis and promotes lipid oxidation,and ultimately improved NAFLD.