Andrographolide Ameliorates Hepatic Steatosis By Suppressing FATP2-Mediated Fatty Acid Uptake In Mice With Nonalcoholic Fatty Liver Disease

Objective: Excess lipid accumulation or steatosis in hepatocytes is caused by abnormal lipid metabolism,a common feature in the development of non-alcoholic fatty liver disease(NAFLD),which can progress to cirrhosis and hepatocellular carcinoma(HC)in the later stages of the disease.With the progressive globalisation of the epidemic of obesity and its associated metabolic syndrome,its high prevalence and complications are attracting increasing attention,and NAFLD is evolving into an increasingly serious global public health burden problem.However,the choice of drugs and treatments available for NAFLD is limited and expensive,and it’s necessary to develop and find new drugs to reduce the cost of care for patients and slow the progression of the disease.However,due to its limitations,including the complex and diverse composition of drugs,the uncertainty of the specific active ingredients,and the lack of internationally accepted principles of drug use,we need to further explore its small molecule components and specific therapeutic mechanisms.In this study,a high fat diet(HFD)-induced in vivo mouse model of non-alcoholic fatty liver and an in vitro model of fatty liver stimulated by a BSA-oleic acid mixture were constructed,and serum biochemistry,histopathological staining,Real-time PCR,western blotting,CCK8 viability assay,fatty acid uptake,overexpression plasmids and other experimental assays,aiming to investigate the role of andrographolide(Andro),a major monomeric limited component of the Chinese medicine Andrographis paniculata,in the treatment of NAFLD and the potential molecular mechanisms mediating its role in inhibiting lipid accumulation in NAFLD through the regulation of fatty acid transporter protein 2(FATP2).Methods: The cells were treated with 20 u M Andro for 48 h and the supernatant was collected and the protein and RNA were extracted and frozen for subsequent experiments.The lipid accumulation in hepatocytes was observed by oil red o staining,the expression of genes related to lipid anabolism and catabolism was screened by Realtime-PCR,the expression of FATP2 in cells was detected by Western blotting,and the fatty acid uptake in cells was analysed by fatty acid uptake assay;further in LO2 cells,the expression of FATP2 was verified by in vitro FATP2 was transfected in vitro in LO2 cells to verify the protective role of FATP2 in Andro against lipid accumulation.In vivo NAFLD model was established in C57BL/6 mice.After one week of acclimatization feeding,10 mice were randomly selected as control group and fed with normal chow,while the rest were given HFD with 60% fat content for 8 weeks to induce NAFLD model.after 8 weeks of high-fat diet,except for the control group fed with normal chow,the HFD mice were randomly divided into(1)normal diet group;(2)high-fat diet group(HFD);(3)HFD +pioglitazone capsules(PIO)6.83mg/kg.d;(4)HFD + andrographolide(Andro)(50mg/kg.d);(5)HFD + Andro(100mg/kg.d);(6)HFD + Andro(200mg/kg.d);After 8 weeks,the mice were fasted without water for 12 h and subjected to oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)on the following day;After the OGTT and ITT experiments,blood was taken from the hearts of anesthetized mice,serum was separated,some liver tissues were fixed for paraffin embedding and OCT embedding for pathological analysis,and some livers were frozen for RNA and protein analysis.Serum glucose,total cholesterol(TC),triglyceride(TG)and mouse insulin and C-peptide levels were measured.Tissue sections were stained with HE and Oil Red O to observe liver pathology and lipid analysis;Realtime-PCR was performed to detect the expression of FATP-related genes in the liver,and Western blotting and immunohistochemistry were performed to detect the expression of FATP2 in the liver.Results: In this study,we screened Andro for suitable concentrations(20u M)for use at the cellular level based on CCK8 assays,and in vitro,Andro also reduced the accumulation of oleic acid-induced intracellular lipid droplets in LO2 cells.Realtime-PCR revealed that treatment with Andro inhibited lipogenesis and the expression of fatty acid synthesis-related factors,while the expression of fatty acid transport proteins(FATPs)was suppressed.Oleic acid-induced increased uptake of fluorochrome-labelled fatty acids by LO2 cells was inhibited by Andro treatment.The administration of Andro(50,100 and 200 mg/kg/d)for 8 weeks reduced obesity and metabolic syndrome in HFD-fed mice,thereby improving impaired glucose tolerance,increasing insulin sensitivity,and reducing hyperglycemia and hyperlipidemia.liver steatosis was significantly reduced in HFD-fed mice.In addition,we found that Andro consistently reduced the expression of fatty acid transporter protein 2(FATP2)in the liver tissues of both oleic acid-induced LO2 cells and HFD-fed mice.Overexpression of FATP2 in oleic acid-induced LO2 cells disrupted the hypolipidemic effect of Andro.Conclusion: 1.Andro can improve the metabolic syndrome in mice on a HFD by inhibiting disruption of glucolipid metabolism;2.Andro reduces lipid accumulation in hepatocytes by inhibiting lipid anabolism without promoting lipid catabolism;3.Andro improves hepatic steatosis in NAFLD by inhibiting FATP2-mediated fatty acid uptake.In conclusion,Andro may achieve its anti-statogenic effect by inhibiting FATP2-mediated fatty acid uptake and supports its potential application in the treatment of NAFLD.