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The Systemic Pharmacology Mechanisms Involved Into ErHuang Quzhi Recipe Treated Non-alcoholic Fatty Liver Disease Models

Posted on:2023-09-02Degree:MasterType:Thesis
Country:ChinaCandidate:J J HeFull Text:PDF
GTID:2544306848492594Subject:Clinical Pharmacy and Drug Evaluation (Professional Degree)
Abstract/Summary:PDF Full Text Request
Objective:Non-alcoholic fatty liver disease(NAFLD)affects about a quarter of the world’s population.At present,there are no FDA-approved therapeutic drugs.In clinical practice,diet and lifestyle changes are the main means of treatment,supplemented by drug therapy(including insulin sensitizers,antioxidants,lipid-lowering drugs,etc.).However,western medicine has a single target and many side effects,and its long-term safety and efficacy are still controversial.Er Huang Qu Zhi recipe(Er H)is an empirical recipe for NAFLD obtained by Professor Yuan Jinqi,a master of Chinese medicine from Xinjiang Corps,based on TCM theory and clinical experience.The clinical treatment effect is remarkable.At present,the mechanism of action of Er H in the treatment of NAFLD is still unclear,which is not conducive to the improvement of clinical efficacy and related new drug research.This project intends to evaluate the pharmacodynamics characteristics of Er H in the treatment of NAFLD from a mouse in vivo model,and to screen drug molecules and predict related target pathways by combining UHPLC-MS/MS analysis and systems pharmacology methods,preliminarily explore the antagonistic effect of Echinatin(Ech),the representative active component of Er H,on the apoptosis of Hep G2 cells induced by Free fatty acids(FFA)in the in vitro model,study the mechanism of Er H in the treatment of NAFLD from the perspective of traditional Chinese medicine compound pharmacology and to provide a preliminary theoretical basis.Method:1.Composition of Er H formula and prediction of drug forming molecular targets: Combined with the2020 edition of the Chinese Pharmacopoeia,multi-database and UHPLC-MS/MS analysis of drug meridian homing,active ingredients,action targets and disease-related targets in Er H,predict key druggable molecular targets and perform enrichment analysis;2.Establishment of NAFLD model in mice: Methionine and Choline-Deficient(MCD)diet fed C57BL/6J mice for 4 weeks to successfully establish an in vivo NAFLD model.The results of comprehensive tissue appearance observation,liver function detection and pathological section staining were analyzed to analyze the effect of Er H on lipid metabolism,inflammation and inflammation in mice.impact on the development of fibrosis;3.Establishment of FFA induced NAFLD in vitro model: Different concentrations of FFA induced Hep G2 cells,combined with cell viability assay,Oil Red O staining and Nile Red staining to judge lipid accumulation and observe cell morphological changes to establish the appropriate concentration of FFA for inducing Hep G2 cells to establish an in vitro NAFLD model;4.Pharmacodynamic study of representative components: Based on fluorescent probe staining,transmission electron microscope observation,flow cytometry and other techniques to explore the role of the key active component Ech in Er H on the in vitro NAFLD model;5.Molecular mechanism of Er H and its representative components: Using IHC staining,RCR technology,molecular docking technology and Western blotting experiments to detect the effects of the compound and its key active components on the protein expression of key pathways of inflammation,fibrosis and apoptosis.Results:1.Systematic pharmacology analysis showed that Er H in the treatment of NAFLD is mainly involved in biological processes such as inflammatory response,apoptosis and fibrosis response,and is closely related to MAPK signaling pathway,PI3K-AKT signaling pathway and other pathways,suggesting that Er H plays a more important role in the course of NAFLD.play a role in stages.2.The NAFLD model was successfully established by feeding C57BL/6J mice with MCD diet for 4weeks.The in vivo pharmacodynamic results showed that Er H intervention improved the liver status of MCD diet-induced mice and significantly decreased the level of hepatic triglyceride(TG).Oil red O staining showed that Er H alleviated lipid accumulation.Combined with HE staining NAS score and Masson staining results,Er H alleviated the development of MCD diet-induced inflammation and fibrosis.3.Er H treatment reduced the m RNA expressions of SREBP-1c and FATP2 in the liver tissue of MCD diet-induced mice,decreased the m RNA expressions of inflammatory factors IL-6,IL-1β,TNF-α and the expression of NF-κB p65 and i NOS,while positive expression of F4/80 IHC staining was reduced.Compared with the MCD group,Er H reduced the positive expression of α-SMA in liver tissue.Meanwhile,the protein expression of SMAD2/3 decreased,and the development of fibrosis alleviated.Er H decreased the protein expression of BAX and the activity of Caspase 3 and the protein expression of BCL-2 increased,which were mainly related to the inhibition of ASK1-JNK signaling pathway.4.It was confirmed that 0.75 m M FFA induced Hep G2 cells for 48 h to establish an in vitro NAFLD model.Based on present model,it was found that Ech could reduce the lipid accumulation in Hep G2 cells after FFA induction,increase the mitochondrial membrane potential and improve the damage to mitochondrial ultrastructure.The fluorescent staining results of DCFH-DA and CMF-DA showed that Ech could alleviate the oxidative stress in cells induced by FFA.Flow cytometry and Hoechst 33258 staining results showed that Ech could significantly reduce the apoptosis of Hep G2 cells induced by FFA;Ech can reduce the protein expression of NF-κB p65 and BAX in Hep G2 cells after FFA induction,mainly by reducing the protein expression of ASK1 and the phosphorylation of JNK.Conclusion:To sum up,this subject,combined with systems pharmacology technology and in vivo validation,clarified the mechanism of Er H in the treatment of NAFLD for the first time.Er H inhibited the ASK1-JNK signaling pathway in MCD-induced NAFLD mice,inhibited the phosphorylation of ASK1 and JNK,alleviated the apoptosis of the mouse liver,further affected the NF-κB pathway,alleviated the inflammation in the mice,and at the same time It is suggested that Er H alleviates the process of liver fibrosis at least in part by inhibiting the activation of hepatic stellate cells and the Smad 3 pathway;The key active component Ech in Er H can improve steatosis,mitochondrial dysfunction,oxidative stress and apoptosis in FFA-induced NAFLD model in vitro,and its protective effect on NAFLD is related to the inhibition of ASK1-JNK signaling pathway.
Keywords/Search Tags:ErHuang Quzhi Recipe, Non-alcoholic fatty liver disease, systems pharmacology, Free fatty acids, Echinatin
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