Integrated network pharmacology analysis and in vitro cell experiments to reveal the mechanisms of Ligusticum chuanxiong Hort.in the treatment of non-alcoholic fatty liver disease

Background:Non-alcoholic fatty liver disease(NAFLD)is a liver disease of unknown etiology.A traditional Chinese medicine Ligusticum chuanxiong Hort.(CX),it has been used about 2,000 years.Until now,the mechanism of action of CX on NAFLD remains unclear.Method:We first tested the toxicity of CX to AML12 cells with CCK-8.In vitro cell models of NAFLD were made using free fatty acid,and used Oil Red O staining tested lipid droplets.Then the active compounds of CX were collected from TCMSP and literatures,and SwissTargetPrediction,Search Tool for Interacting Chemicals,Encyclopedia of Traditional Chinese Medicin,Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine database were used to predict the targets of the compounds.DRUGBANK,Online Mendelian Inheritance in Man,Therapeutic Target Database,DisGeNET and GeneCards database were used to predict the targets of NAFLD.Use Venn diagram to obtained the intersection targets by,and analyzed the protein-protein intersection network.Use Kyoto Encyclopedia of Genes and Genomes and Gene Ontology to forecast the function of intersection genes.Molecular docking was used to evaluate the interaction between hub gene and active ingredients.Finally,use western blotting to determine the effects of CX on PPARA,PPARG,IL1B and TNF proteins.Result:CX can reduce the production of AML12 cell lipid droplets.A total of 15 chemical components were identified from CX.Folic acid,chrysophanol and sitosterol were the main components of CX against NAFLD.ALB,TNF,PPARG and PPARA proteins were the main targets of CX in the treatment of NAFLD.PPAR signaling pathway and fatty acid degradation were closely related to anti-NAFLD.Molecular docking results shows that folic acid was the main active ingredient of CX for NAFLD treatment,and TNF is the main potential target.The cellular NAFLD model showed that CX up-regulated the expression of PPARA and PPARG protein and down-regulated inflammatory factor IL-1B and TNF expression.Conclusion:Our study suggests that CX has a therapeutic effect on NAFLDA,which may be related to the PPAR pathway and the reduction of inflammatory cytokines.

Integrating network pharmacology and molecular docking to explore the potential mechanism of Zanthoxylum bungeanum against gout

Background:Gout is an inflammatory joint disease.Modern pharmacological studies have shown that Zanthoxylum bungeanum(Z.bungeanum)has significant anti-inflammatory and osteoprotective effects.This study is aimed to explore the therapeutic effect of Z.bungeanum on gouty arthritis.Methods:Firstly,the compounds in the water extract of Z.bungeanum(WZB)were analyzed by UPLC-QE-Orbitrap-MS/MS.The compounds and disease targets were predicted using SwissTargetPrediction platform and GeneCards database,and the candidate targets were acquired by taking the intersection.Next,the protein interaction network was constructed and the hub genes were screened using String platform and Cytoscape software.Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes analysis of hub genes were performed using Metascape platform.Finally,the disease-pathway-target-drug-component network diagram was constructed to obtain the active components and targets for treating disease,and the affinity between components and targets were predicted by applying Discovery Studio software.In vitro,using an acute gout model of RAW 264.7 macrophages induced by Monosodium Urate to evaluate the anti-inflammatory effect of WZB on Gout.Meanwhile,we established a mice model of hyperuricemia and measured the serum xanthine oxidase(XOD)activity and uric acid(UA)contents.Results:A total of 20 chemical components were identified from WZB,mainly alkaloid components.The network pharmacology results showed that Dihydrobungeanool,Quercetin,Hydroxy-α-sanshool and Hydroxy-ɛ-sanshool were the main active components of WZB against gout.MAPK1,PIK3CA,EGFR and TLR1 proteins were the main targets of WZB in the treatment of gout.Pathways in cancer,PI3K/Akt signaling pathway,Th17 cell differentiation and MAPK signaling pathway were closely related to anti-gout.Molecular docking results have shown that Dihydrobungeanool may be the main active ingredient of WZB for gout treatment,and PIK3CA is the main potential target.The cellular gout model showed that WZB significantly reduced IL-1βand TNF-αinflammatory factor levels and influenced the expression of PI3K protein.In addition,WZB administration effectively reduced UA levels and XOD activity in the serum of mice with hyperuricemia.Conclusion:Our study results suggest that WZB could treat gout by acting on PIK3CA gene target to reduce the level of inflammatory factors,and reduce XOD activity to lower UA levels.

Fructus Zanthoxyli extract improves glycolipid metabolism disorder of type 2 diabetes mellitus via activation of AMPK/PI3K/Akt pathway:Network pharmacology and experimental validation

Objective:This study investigated the potential mechanisms behind the beneficial effects of Fructus Zanthoxyli(FZ)against type 2 diabetes mellitus(T2DM)based on network pharmacology and experimental validation.Methods:Ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry,and gas chromatography-mass spectrometry were used to identify the constituents of FZ.Next,the differentially expressed genes linked to the treatment of diabetes with FZ were screened using online databases(including Gene Expression Omnibus database and Swiss Target Prediction online database),and the overlapping genes and their enrichment were analyzed by Kyoto Encyclopedia of Genes and Genomes(KEGG).Finally,the pathway was verified by in vitro experiments,and cell staining with oil red and Nile red showed that the extract of FZ had a therapeutic effect on T2DM.Results:A total of 43 components were identified from FZ,and 39 differentially expressed overlapping genes were screened as the possible targets of FZ in T2DM.The dug component-target network indicated that PPARA,PPARG,PIK3R3,JAK2 and GPR88 might be the core genes targeted by FZ in the treatment of T2DM.Interestingly,the enrichment analysis of KEGG showed that effects of FZ against T2DM were closely correlated with the adenosine monophosphate-activated protein kinase(AMPK)and phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)signaling pathways.In vitro experiments further confirmed that FZ significantly inhibited palmitic acid-induced lipid formation in Hep G2 cells.Moreover,FZ treatment was able to promote the AMPK and PI3K/Akt expressions in Hep G2 cells.Conclusion:Network pharmacology combined with experimental validation revealed that FZ extract can improve the glycolipid metabolism disorder of T2DM via activation of the AMPK/PI3K/Akt pathway.

Processing methods and mechanisms for alkaloid-rich Chinese herbal medicines:A review

The processing of Chinese herbal medicine is a form of pharmaceutical technology developed over thousands of years,in order to increase efficiency and decrease toxicity of herbs in traditional Chinese medicine(TCM).Herbal processing is essential for safe and effective application of TCM in clinical practice,as it alters the active chemical components and therefore the functions of herbal medicines.Alkaloid-rich herbal medicines in TCM are commonly processed by cleansing,cutting,processing by dry stir-frying,stir-frying with liquid adjuvants,and processing by water decoction.In addition,commonly used adjuvants for processing alkaloid-rich herbal medicines are river sand,wine,vinegar,brine,honey and herbal juice.For alkaloid-rich herbal medicines,the main chemical reactions that occur during processing include hydrolysis,oxidation,replacement,decomposition and condensation.This paper aimed to summarize the processing methods and mechanisms for alkaloid-rich Chinese herbal medicines,and provide much-needed theoretical support and scientific evidence for understanding those mechanisms and effects.Information on processing methods for alkaloid-rich herbal medicines was collected from classic books of herbal medicine,PhD and MSc dissertations,online scientific databases including PubMed,SciFinder,Scopus,Web of Science,Baidu Scholar and Google Scholar.This paper should help to advance our knowledge of the processing mechanisms and aid in the development of processing methods for alkaloid-rich Chinese herbal medicines.