Based On Gut-liver Axis To Study The Pharmacological Mechanisms Of Semen Cassia Extract On Non-alcoholic Fatty Liver Disease

Chapter I Network pharmacology investigation into the mechanisms of the semen cassia on NAFLDObjective:To construct the interaction network between semen cassia and treatment targets,and predict the possible mechanism of semen cassiae on non-alcoholic fatty liver disease(NAFLD).Methods:1.Collection of active ingredients and targets of semen cassia: To identify the active ingredient and targets of semen cassia,the TCMSP,Pub Chem,Swiss Target Prediction,and Pharm Mapper databases were used.2.Collection of NAFLD-related targets: Gene Cards,OMIM,Therapeutic target(TTD),and Drug Bank databases were used to download NAFLD-related targets3.Protein-protein interaction(PPI)network construction: Semen cassiaNAFLD intersection targets were filtered out using R software and then imported into the String database.4.Topology analysis of PPI network: The core targets of the PPI network were selected based on the parameters calculated by Cytoscape3.7.2 software such as degree value,betweenness centrality,and closeness centrality.5.GO functional enrichment and KEGG pathway enrichment analysis: Go function enrichment analysis and KEGG pathway enrichment analysis were carried out through DAVID database,and the results were visualized by R software.Results:1.A total of 14 active components and 481 targets were collected.Among 426 potential NAFLD targets,there were 76 targets overlapped with those of semen cassia.2.The topology analysis of the PPI network shows that the network consists of76 nodes and 154 edges,and the average degree value is 4.05.Topological analysis with Cytoscape revealed that Toll-like receptor 4(TLR4),Tumor necrosis factor-α(TNF-α),Occludin(OCLN),Tight junction protein 1(TJP1),Interleukin-6(IL-6)and Interleukin-1β(IL-1β)were considered as the hub gene.3.The molecular functions results suggested that these targets mainly involved in steroid hormone receptor activity,enzyme binding,and protease binding.Cellular components results included the cytosol,extracellular space,and membrane raft.For biological processes,the targets were mostly enrich in cellular response to lipopolysaccharide(LPS),positive regulation of smooth muscle cell proliferation and negative regulation of apoptotic process.4.KEGG pathway enrichment analysis showed that these common targets were enriched in multiple pathways known to participate in the progression of NAFLD,such as the NAFLD,TLR,TNF,Peroxisome proliferators-activated receptor(PPAR)and Insulin resistance.Conclusion:The main active ingredients of semen cassia(anthraquinone and naphthapyrone)exert beneficial actions on NAFLD by interfering with LPS-mediated chronic inflammation,participating in the transcriptional expression of TLR,TNF,OCLN,TJP1 J genes and signaling pathways,therefore interfering with the pathophysiological processes of inflammatory response and glucolipid metabolism.Chapter II Aurantio obtusin from semen cassia improves NAFLD by regulates enterohepatic circulation of bile acidsObjective:To evaluate the therapeutic effect of semen cassia extract of Aurantio Obtusin(AO)on NAFLD mice,and further explore the effect of AO on enterohepatic circulation of bile acids.Methods:1.24-28 g male mice were randomized into six groups: Control group(CON)administered a standard chow diet,High-fat diet group(HFD)administered an HFD,AO 1.5g/kg/d group,AO 3g/kg/d group,AO 5g/kg/d group,AO 8g/kg/d group.Mice in CON and HFD groups received an equal volume of physiological saline.Animals were euthanized at 4 weeks after AO or saline administration,and odyweight was monitored once a week.2.Liver triglyceride(TG)and serum total bile acid(TBA)levels were measured by biochemical assay kits.3.The histopathological changes of liver tissues were observed by H&E staining.4.The m RNA expressions of farnesoid X receptor(FXR),small heterodimer partner(SHP),fibroblast growth factor receptor 4(FGFR4),cholesterol 7-alpha hydroxylase(CYP7A1),CYP7B1,CYP8B1 in the liver,and FXR,Fibroblast growth factor 15(FGF15)in the ileum were examined by RT-PCR.Results:1.Mice fed with HFD showed a significantly increase in body weight and liver index in comparison with CON group(P<0.001),which were significantly reduced by AO treatment(P<0.001).2.Liver TG and serum TBAlevels of HFD group were greater than in CON group(P < 0.001),while the TG and TBA levels of AO group were significantly decreased in comparison with HFD group(P < 0.001).3.H&E staining results revealed that the obvious fat degeneration,swelling of the liver cells,uneven lipid droplets,and inflammatory cells were observed in the liver tissue of the HFD group.AO treatment were found to relieve hepatic steatotic,fat degeneration and inflammatory of liver cells.4.The relative m RNA expression of SHP,FGFR4 and FGF15 was increased in HFD group compared to CON group(P<0.05),while AO supplementation reverted SHP,FGFR4 and FGF15 gene overexpression(P<0.05).The treatment of mice with AO significantly increased the m RNA expressions of FXR,SHP,FGFR4 in the liver,and FXR,FGF15 in the ileum(P<0.05),while the m RNA expressions of CYP7A1,CYP7B1,CYP8B1 significantly decreased in comparison with HFD group(P <0.01).Conclusion:Our results suggested that AO may be the protective compound on the liver lipid deposition in NAFLD rats.The changes of gene expression related to the enterohepatic circulation of bile acids in ileum and liver showed that AO intervention activated the natural antagonistic mechanism of FXR,thus improving enterohepatic circulation of bile acids.Chapter III Protective effect of aurantio obtusin from semen cassia on biological barrier in NAFLD miceObjective:The present chapter aimed to explore the correlation between the therapeutic effect of AO on NAFLD and the abundance and diversity of gut microbiota,and to evaluate the protective effect of AO on the intestinal barrier of NAFLD mice.Methods:1.Collection of fecal samples: Fecal samples of mice were collected using EP tube and then placed to ice box immediately to prevent DNA degradation.After collecting all the samples,transfer it to-80 refrigerator for storage.2.DNA extraction and PCR amplification: DNA was extracted from fecal samples according to the protocol.Final DNA concentration and purification were determined by Nano Drop2000 and DNA quality was detected by 1% agarose gel electrophoresis.The v3-v4 hypervariable region of bacterial 16 Sr RNA Gene was amplified by thermal cycle PCR system with primers 338 F and 806 R.3.Illumina Mi Seq sequencing: Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina Mi Seq platform according to the protocols.4.Bioinformatics analysis: The analysis was conducted by following the "Atacama soil microbiome tutorial" of Qiime2 docs along with customized program.Results:1.The results of taxon-based analysis showed showed that the faecal intestinal flora was mainly composed of Bacteroidetes,Firmicutes and Proteobacteria at phylum level.Compared with CON group,the abundance of Firmicutes in HFD group was significantly increased and the abundance of Bacteroidetes was significantly decreased(P<0.001).AO intervention could significantly reduce the abundance of Firmicutes,and significantly increased the abundance of Bacteroidetes(P<0.01).The ratio of Firmicutes/Bacteroidetes in HFD group was significantly higher than that in CON group(P<0.001),while the ratio of Firmicutes/Bacteroidetes in AO group was significantly lower than that in HFD group(P < 0.001).2.At the general level,Akkermansia abundance in HFD group was significantly lower than that in CON group(P<0.001),and AO intervention significantly increased Akkermansia abundance(P<0.001),suggesting that AO has the function of maintaining the integrity of intestinal mucus layer,reducing intestinal permeability and regulating liver immune response.Compared with CON group,the abundance of Bifidobacterium in HFD group was significantly decreased(P<0.05),and the abundance of Bifidobacterium in AO group was significantly increased(P<0.05).3.Compared with CON group,Chao1,Faith’s PD,Observed OTUs and Shannon indexes in HFD group were significantly decreased(P<0.05);Compared with HFD group,Chao1,Faith’s PD,Observed OTUs and Shannon index of mice treated by AO were significantly increased(P<0.05).4.The comparison of microbial composition between HFD and AO groups based on the β diversity index showed that the Principal components analysis(PCA),Principal co-ordinates analysis(PCo A),and Partial least squares Discriminant Analysis(PLS-DA)analysis divided the intestinal flora into three different communities,suggesting differences between groups.These results indicate that AO treatment can significantly affect the β diversity of intestinal flora.Conclusion:AO treatment corrects gut microbiota dysbiosis,while increased Akkermansia and Bifidobacteriun level,thus protect biological barrier in NAFLD mice.Chapter IV Protective effect of aurantio obtusin on mechanical and immune barrier in NAFLD miceObjective:To investigate whether the therapeutic effect of AO on NAFLD is related to repairing intestinal mechanical barrier,reducing intestinal permeability,and alleviating LPS-induced liver inflammation.Methods:1.H&E staining were used to observe the histopathological changes of intestinal tissues.2.The expression of the intestinal tight junction proteins occludin and zonula occludens 1(ZO-1)was measured by q-PCR and western blot.3.Plasma lipopolysaccharide(LPS)and hepatic inflammatory factors(TNF-α,IL-1β,IL-6)were detected by ELISA kit.4.The expression of toll-like receptor 4(TLR4)and nuclear factor kappa-B(NF-κB),p-NF-κB in liver was measured by q-PCR and western blot.5.RDA was performed to reveal the association of microbial communities in relation to environmental factors based on the relative abundance of microbial species at different taxon levels,the R software package“vegan” was used.Results:1.The intestinal mucosal villi of CON group were arranged tightly and the structure was integrated.The intestinal mucosal villi of HFD group became shorter and sparser,and partial intestinal mucosal villi were lost.The intestinal mucosal villi of group AO were mildly edema and arranged relatively neatly and tightly in comparison with HFD group.2.Compared with CON group,m RNA and protein expressions of Occludin andZO-1 in ileum of HFD group were significantly decreased(P<0.05);The m RNA and protein expressions of Occludin and ZO-1 in the ileum of mice in the AO group were significantly up-regulated compared with those in the HFD group(P<0.001),suggesting that AO could repair the tight junctions between epithelial cells,thus protecting the intestinal mechanical barrier and reducing intestinal permeability.3.The levels of plasma LPS and liver TNF-α,IL-6 and IL-1β in HFD mice were significantly higher than those in CON group(P<0.001).AO significantly reduced the levels of plasma LPS and liver TNF-α,IL-6 and IL-1β(P<0.001).4.Compared with CON group,m RNA and protein expressions of TLR4 and NF-κ B in liver tissues of HFD mice were significantly up-regulated(P<0.001),while m RNA and protein expressions of TLR4 and NF-κB in liver tissues of AO groups were significantly down-regulated compared with HFD group(P<0.001).It was confirmed that AO can reduce endotoxemia,possibly reduce LPS invasion from portal vein to liver,inhibit LPS/TLR4/NF-κB pathway mediated liver inflammation,and repair liver immune barrier.5.RDA correlation analysis showed that TG,LPS and inflammatory factors levels were significantly correlated with community distribution and species distribution(P<0.05).The correlation heat map showed that the level of environmental factors was significantly correlated with the level of most bacteria at phylum level,suggesting that liver lipid deposition and LPS-induced inflammatory response were significantly correlated with intestinal microbial changes.Conclusion:AO treatment strengthening the intestinal barrier by restoring the widened tight junctions.Thus,alleviates hepatic inflammation by disrupting the loop of LPSmediated inflammatory responses.