Study On The Effect And Mechanism Of Pure Total Flavonoids From Citrus Paradisi Against Non-alcoholic Fatty Liver Disease By Regulating Nrf2-ARE Signaling Pathway

Objective Nonalcoholic fatty liver disease(NAFLD)is a common chronic liver disease,which has become the second leading cause of end-stage liver disease and one of the main causes of liver cancer.Accumulating evidence have revealed that the occurrence and development of NAFLD is not only affected by obesity and lipid metabolism disorders,but also closely related to inflammation,bile acid metabolism and intestinal flora.So far,the pathogenesis of NAFLD has not been fully elucidated,and there is lack of effective prevention and control measures for NAFLD.Our previous studies have found that pure total flavonoids from Citrus Paradisi(PTFC)have antiinflammatory and antioxidant activities,as well as repairing intestinal mucosal barrier function.PTFC can effectively improve hepatic steatosis and inflammatory infiltration in NAFLD mice,but the specific mechanism of action is still unclear.Hence,by designing in vivo and in vitro NAFLD models,this study intends to investigate the effect of PTFC on Nrf2-ARE,a key signaling pathway of oxidative stress in NAFLD,and explore its influence on bile acid metabolism and intestinal flora.In addition,the role of PTFC in improving the symptoms of NAFLD and its inner mechanism are discussed,which might provide theoretical basis for its further development and application.Methods This study mainly consists of four parts.Part one:Literature mining-based summarization of traditional Chinese medicine(TCM)syndromes and therapeutic formulas of NAFLD.Through literature retrieval,the information of ancient literature on NAFLD was collected.The data concerning chemical composition and pharmacological action of classical prescriptions involved in NAFLD treatment were also mined and analyzed to interpret the internal mechanism of TCM in the treatment of NAFLD.Part two:Study on the protective effect of PTFC on hepatic steatosis in NAFLD model.The PTFC was extracted by alkali extraction and acid precipitation method,and purified by macroporous resin method.The content of each component in PTFC was determined by HPLC.The effect of PTFC on the proliferation of AML 12 cells was detected by CCK-8 assay.NAFLD cell model induced by FFA was established to explore the effects of PTFC on the levels of AST,LDH,and TG.NAFLD mice model were established by feeding C57BL/6J mice with high-fat diet,and randomly divided into high-dose(H-PTFC,200 mg/(kg·d)),medium-dose(M-PTFC,50 mg/(kg·d))and low-dose PTFC(L-PTFC,50 mg/(kg·d))groups.The histological changes of liver tissues after treatment with different doses of PTFC were observed by oil red O staining and H&E staining.The effects of different doses of PTFC on serum ALT,AST,TG,TC,LDL-C and HDL-C levels of mice were investigated.Moreover,serum levels of MDA,SOD,GSH-Px and 8-iso-PGF2α in mice treated with different doses of PTFC were analyzed.Part three:PTFC improves oxidative stress injury in NAFLD mice by regulating Nrf2-ARE signaling pathway.Firstly,the potential mechanism of PTFC in the treatment of NAFLD was explored by means of network pharmacology.Secondly,NAFLD cell model was established to explore the effects of PTFC on the levels of oxidative stress markers including MDA,SOD and GSH-Px.Moreover,the effects of PTFC on mRNA expression levels of Nrf2,Keapl,HO-1,γGCS,NQO1 and GST in hepatocytes were detected by RT-PCR.The effects of PTFC on the protein expression levels of Nrf2,Keap1 and MCU were investigated by western blot assay.The expression of Nrf2 protein in nucleus and cytoplasm before and after PTFC treatment was observed by cell immunofluorescence staining.Finally,NAFLD mice model was established,and the effect of different dose of PTFC on the protein expressions of Nrf2,Keapl,HO-1,NQO1,γ-GCS and GST in liver tissues was investigated by Western blot assay.The nuclear localization of Nrf2 in liver tissue was explored by immunohistochemistry.Part four:The effect of PTFC on bile acid metabolism and intestinal flora changes in NAPLD mice.The high-fat diet-induced NAFLD mice model were established,and the changes of intestinal flora composition and structure in mice treated with 50 mg/(kg·d)PTFC were analyzed by 16S rDNA gene sequencing.PICRUSt was used to predict KEGG metabolic pathways of intestinal flora after PTFC intervention.UPLC-MS/MS was applied to determine the serum bile acid profile of NAFLD mice treated with PTFC.The protective effect of PTFC on hepatic steatosis was investigated by H&E staining.The effect of PTFC on the mRNA and protein expression of FXR and TGR5 in liver tissues were detected by RT-PCR and western blot,respectively.Results Part one:Through a systematic review of the ancient and modern literature on NAFLD,it was found that TCM classified NAFLD into the categories of "liver fete","hypobilic distention",and "liver accumulation".The treatment of NAFLD mainly included "soothing the liver and regulating Qi,strengthening the spleen and eliminating turidity","regulating Qi and eliminating phlegm,promoting blood circulation and removing blood stasis","nourishing the liver and kidney,supplementing the marrow and essence".And the commonly used clinical prescription such as Zhizhu decoction,Zhishi Xiebai Guizhi decoction,and Zhishi Zhizi Temi decoction are.all use Fructus aurantii immaturus or Fructus aurantii as the main medicine.According to analysis and literature evidence,the main drug shared by the above formulas is Fructus Aurantii,and its flavonoid components may exert NAFLD therapeutic effects through antioxidant stress and regulating factors affecting the "gut-liver" axis.Part two:The total flavonoids content in PTFC obtained by separation and purification of Citrus Paradisi was 76.22%.The contents of narirutin,naringin,hesperidin,and neohesperidin were 12.08 ± 0.12 mg/g,243.86±2.67 mg/g,5.95±0.05 mg/g and 136.02±4.55 mg/g;respectively.The results of CCK-8 showed that PTFC inhibited the proliferation of AM 12 cells in a dose-and time-dependent manner.PTFC intervention can significantly reduce AST,LDH,and TG levels in NAFLD cell models(P<0.01;P<0.05).The results of oil red O staining and H&E staining confirmed that PTFC intervention could effectively improve hepatic steatosis and oxidative stress injury in NAFLD mice in a dose-dependent manner.The contents of HDL-C in NAFLD mice serum were increased,while the contents of ALT,AST,TG,TC and LDL-C were decreased(P<0.05;P<0.01)after M-PTFC or H-PTFC treatment.In contrast,there were no significant difference of the above biochemical indices with L-PTFC treatment.In addition,different doses of PTFC can significantly increase SOD and GSH-Px activities in NAFLD mice,while M-PTFC and H-PTFC can reduce MDA and 8-isoPGF2a levels in varying degrees(P<0.05;P<0.01).Part three:Network pharmacological studies have found that Nrf2 plays a key role in the intervention of flavonoids in QZQ to NAFLD target network.There are multiple targets overlap between Nrf2-ARE pathway and the common target of QZQ flavonoids and NAFLD,and the docking of its flavonoid components with their corresponding target molecules of Nrf2-ARE signaling pathway is basically stable.In the in vitro study,compared with model group,MDA content in PTFC group was significantly decreased(P<0.001),while SOD and GSH-Px contents were significantly increased(P<0.001),indicating that PTFC treatment could effectively improve the oxidative stress injury of NAFLD cells.Moreover,PTFC could promote the mRNA expression of Nrf2(P<0.05),but had no significant effect on the mRNA expression of Keapl,HO-1,NQO1,γ-GCS and GST.Furthermore,the protein expression of Keapl was decreased after PTFC intervention(P<0.05),while the protein expression of Nrf2 and MCU were increased(P<0.01).Western blot showed that both M-PTFC and H-PTFC could increase the protein expressions of Nrf2,Keapl,HO-1,NQO1,γ-GCS,and GST in liver tissue,while L-PTFC only promoted the protein expressions of HO-1 and γ-GCS.Similarly,immunohistochemical results showed that both M-PTFC and H-PTFC could promote Nrf2 protein expression(P<0.01),but there was no statistical difference on Nrf2 protein expression after L-PTFC treatment.Part four:The result of 16S rDNA gene sequencing revealed that,at the family level,PTFC intervention significantly increased the relative abundance of Christensenaceae and Erysipelotrichaceae(P<0.01),and significantly decreased the relative abundance of Porphyromonadaceae and Streptococcaceae(P<0.01;P<0.05).At the genus level,the relative abundance of Allobacullum was significantly increased after PTFC intervention(P<0.05),while that of Eubacterium was significantly decreased(P<0.05).PICRUSt analysis predicted that 12 of KEGG pathways in PTFC group were altered,including energy metabolism,amino acid metabolism,ethylbenzene degradation,peptidoglycan biosynthesis pathways,etc.In addition,PTFC intervention reduced the contents of TDCA,DC A,TCA,CA,and other toxic bile acids(P<0.05),and increased the ratio of secondary bile acids to primary bile acids.H&E staining showed that PTFC could reduce steatosis and inflammatory infiltration of liver tissue in NAFLD mice.Meanwhile,the protein expression of FXR and TGR5 in liver tissue was enhanced by PTFC intervention(P<0.01;P<0.05),and mRNA expression of TGR5 were significantly increased(P<0.01).Conclusion PTFC can activate Nrf2-ARE signaling pathway by promoting Nrf2 protein expression,regulate intestinal flora and bile acid metabolism,thus alleviating hepatic steatosis and oxidative stress injury of NAFLD.This study provides an important theoretical basis for the clinical application of PTFC,and puts forward a new idea for the application of natural products in the clinical treatment of NAFLD.