The Effect And Mechanism Of Dangguiliuhuang Decoction Against Insulin Resistance,Fatty Liver,and Hepatic Fibrosis

PART I Exploring the Mechanism of Dangguiliuhuang Decoction against Hepatic Fibrosis by Network Pharmacology and Experimental ValidationBackground Liver fibrosis is a common pathological process for the majority of liver diseases,the characteristics of which is extracellular matrix deposition and collagen fibers proliferation induced by a variety of pathogenic factors.If liver fibrosis is not treated in time,it will develop into end-stage liver disease,cirrhosis,liver failure and/or hepatocellular carcinoma.However,many therapies are still in the preclinical research stage except for liver transplantation,lacking of effective therapies to cure the disease thoroughly.Therefore,it is urgent for us to discover effective anti-fibrosis treatments.Traditional Chinese medicine(TCM),as an important part of the complementary and alternative medicine system,has a clinical application history for more than 2000 years in China.Dangguiliuhuang decoction(DGLHD)is a kind of classical formula for the treatment of diabetes,infection,and myocarditis,which consists of angelica,radix rehmanniae,rehmanniae radix praeparata,scutellaria baicalensis,coptis chinensis,golden cypress,and astragalus membranaceus.A large number of studies have shown that the components of DGLHD such as astragalus membranaceus,angelica,and coptis chinensis have anti-inflammatory,anti-fibrotic and hepatoprotective effects.However,it remains unclear whether DGLHD has anti-hepatic fibrosis effect,what active ingredients and underlying mechanism are.Objective To explore the active ingredients contained in DGLHD and its anti-liver fibrotic targets by network pharmacology.Then,we verified the theoretical predictions using animal and cell experiments,clarifying the mechanisms of DGLHD against hepatic fibrosis and providing new approach for the treatment of liver fibrosis.Methods Prediction of active ingredients and targets in DGLHD Chemical ingredients and targets of each herb in DGLHD were obtained from TCMSP,TCM Database@Taiwan,Pub Med database,and associated literatures.We then analyzed the similarities and differences of physiochemical characteristics of compounds in seven herbs.In order to discover active components and targets of DGLHD,we selected ingredients meeting the requirements of OB≥30% and/or DL≥0.1.Network analysis Liver fibrosis-associated targets were collected from OMIM,Genecards,and KEGG databases.To explore the interaction of DGLHD and hepatic fibrosis,we performed GO function and pathway enrichment analysis for targets with degree value over 3.We then established an interactive network among active ingredients,putative targets of DGLHD,and hepatic fibrosis-associated targets by Cytoscape 2.8.3 software.Experimental validation C57BL/6J mice were randomly divided into 3 groups: control group,model group,DGLHD(5.0g/kg)group.Liver fibrosis was induced by intraperitoneally injection of 4%(v/v)carbon tetrachloride(CCl4)dissolved in olive oil twice a week for 6 weeks.After treatment with DGLHD for 6 weeks,the liver index and activity of ALT and AST were examined,pathological change of liver was assessed by HE and Masson staining,the levels of TNF-α,IL-1β and TGF-β1 cytokines in serum and liver were detected by CBA or q RT-PCR method,the expressions of TGF-β1,α-SMA,PPAR-γ and NF-κB in liver and activated HSC-T6 cells were evaluated by Western blot and q RT-PCR.,the proliferation of HSC-T6 cells was determined by MTT assay.Results We collected 545 compounds in DGLHD,among which 75 compounds met the requirements of OB ≥30% and/or DL ≥0.1 and were recognized as the potential active ingredients of DGLHD.Afterwards,we collected 286 targets corresponding to 75 active ingredients in DGLHD.And there are 71 target genes with degree value more than 3.Functional enrichment analysis data revealed that these active targets not only modulated cell proliferation,apoptosis,growth,and signal transduction,but also tuned inflammatory response,and the activity of PPAR and NF-κB.Additionally,pathway enrichment analysis suggested that DGLHD mainly regulated PI3K/Akt,NF-κB,MAPK,NAFLD,and hepatitis-related signaling pathways.There are 51 direct targets for hepatic fibrosis among 71 putative targets modulated by DGLHD,like PTGS2,NF-κBp65,PPAR-γ,PI3 K,and AKT1.Moreover,there are plenty of components contained in DGLHD regulating the activity of PTGS2,PI3 K,Akt,PPAR-γ,NF-κBp65,and TGF-β1,such as berberine,baicalein,ferulic acid,coptisine,baicalin,and catalpol.Therefore,some ingredients in DGLHD interfere with liver fibrosis probably through these active targets.Experimental results showed that DGLHD obviously attenuated liver injury and hepatic fibrosis,reduced ALT and AST levels,decreased the synthesis and deposition of extracellular matrix,relieved the secretion of proinflammatory cytokines,lowered the activation of NF-κBp65 and TGF-β1,and enhanced protein and gene expression of PPAR-γ,consequently exerting hepatoprotective and anti-liver fibrotic effects.Conclusion DGLHD inhibited the occurrence and development of hepatic fibrosis through attenuating proinflammatory signaling,decreasing HSC cells activation and extracellular matrix accumulation with multiple components,multiple targets,and multiple pathways.PART II The Effect and Mechanism of Dangguiliuhaung Decoction against Obesity-related Insulin Resistance and Hepatic SteatosisBackground Obesity is a pathological condition associated with many diseases,such as diabetes,fatty liver,hypertension,and atherosclerosis,which often accompanies with inflammatory cells infiltration,abnormal glucose and lipid metabolism,and decreased insulin sensitivity.Increasing evidences have shown that impaired insulin signaling pathway,together with inflammatory responses and abnormal glucolipid metabolism,synergistically promotes hepatic lipid biosynthesis and steatosis,which in turn contribute to the development of fatty liver and insulin resistance.Hence,inflammation,abnormal metabolism,insulin resistance,and hepatic steatosis are intertwined pathological events that are commonly presented in obese individuals.Various types of immune cells are involved in the development of obesity-related inflammatory and metabolic diseases,like DCs,Tregs,CD8+ T cells,and B cells.Boosted Tregs number in adipose tissue of obese mice ameliorates insulin sensitivity and fat accumulation.It has been confirmed that depletion of DCs by using CD11 cDTR transgenic mice improves obesity-related inflammation,insulin resistance,and decreases hepatic steatosis and lipid metabolism-related gene expression.Thus,Tregs and DCs execute important roles in the development of inflammation,insulin resistance,and hepatic steatosis.The incidence and mortality of obesity-related diseases increased year by year as the change of modern lifestyles.However,the current therapeutic drugs have the characteristics of single target,high failure rate,and significant adverse effects,which cannot effectively prevent the development of insulin resistance and hepatic steatosis.Modern pharmacological studies have shown that DGLHD has hypoglycemia,insulin sensitization,anti-inflammation,and anti-infection.Whether DGLHD inhibits the progression of obesity-related insulin resistance and fatty liver,and the mechanism of DGLHD exerting these actions has not been elucidated.Objective To investigate the influences and mechanisms of DGLHD on immune cells,metabolites,and glucolipid metabolism during the progression of obesity-associated insulin resistance and hepatic fibrosis,providing basis for the clinical application of DGLHD and new ideas for the research of traditional Chinese medicine.Methods Ob/ob mice were randomly divided into four groups: control group,DGLHD(1.5g/kg)group,DGLHD(3.0g/kg)group,DGLHD(6.0g/kg)group.Mice were administrated with equal volume of three distilled water or DGLHD for eight weeks.OGTT and ITT experiments were used to measure glucose tolerance and insulin sensitivity of mice,respectively.Liver weight index and visceral adipose index were examined;pathological changes of liver and adipose tissues were evaluated by HE and oil red O staining;the level of insulin secreted from pancreatic islets was detected by immunohistochemistry.The contents of TC,TG,and FFA in serum and liver were determined by microplate method,and glucose consumption was assessed by glucose oxidase method.The levels of cytokines,insulin,and adiponectin in serum were measured by ELISA.The percentage,phenotype,and function of immune cells were detected by flow cytometry;mixed lymphocyte reaction and cell proliferation were evaluated by MTT assay;the content of metabolites in liver and adipose tissues was determined by targeted metabonomics.Western blot and q RT-PCR methods were adopted to analyze the expression levels of related proteins and genes,respectively.In order to investigate the direct effects of DGLHD on DCs,T cells and insulin resistant 3T3-L1 cells,our in vitro experiment was performed to detect cytokines,phenotype,glucose uptake,and protein and gene levels according to the above methods.Results DGLHD,especially at the dosage of 6.0g/kg,attenuated fatty aggregation and degeneration of liver tissues,downregulated the expression levels of FFA,TC,and TG in serum and liver,decreased the weight of liver and visceral adipose tissues,and maintained normal morphological structure of liver and adipose tissues.Additionally,DGLHD markedly improved oral glucose tolerance and insulin resistance in ob/ob mice,reduced insulin secretion,and enhanced adiponectin expression.DGLHD also significantly decreased the expression levels of pro-inflammatory cytokines TNF-α and IFN-γ in serum,spleen,liver and visceral adipose tissues,and mitigated splenic T lymphocytes proliferation.We further found that DGLHD reduced isoleucine,adenosine,and cholesterol levels,and increased glutamine level in fat and liver tissues of ob/ob mice mainly by regulating amino acid and purine metabolism.DGLHD promoted the differentiation of Tregs and enhanced Foxp3 gene expression in spleen,lymph node,liver,and adipose tissues of ob/ob mice,and upregulated the level of negative regulator PD-1 in T cell surface.After depletion of Tregs in ob/ob mice,fatty liver,insulin resistance,and inflammatory status of mice were aggravated.Interestingly,administration of DGLHD reversed these associated changes.In vitro experiment showed that DGLHD inhibited the proliferation of T cells,promoted the differentiation of T cells into Tregs,and facilated the conversion of Th1 type cytokines to Th2 type cytokines.DGLHD reduced the expression levels of MHC-II and CD86 in DCs surface,enhanced the expression of negative costimulatory PD-L1,weakened the ability of DCs stimulating T cells proliferation,upregulated the levels of IDO and ILT-3 genes,and inhibited DCs secreting IL-12p70 cytokine.In addition,DGLHD also inhibited the maturation of DCs from Tregs-neutralized mice.Our in vitro experiment also demonstrated that DGLHD repressed DCs maturation,antigen presentation ability,and IL-12p70 secretion.Metabolism-related indicators suggested that DGLHD reduced glucose uptake of DCs and T cells,suppressed the activation of PI3K/Akt signaling pathway,increased the expression of PPAR-γ protein and gene,and promoted the formation of Tregs and tolerant DCs in vitro and in vivo.At the same time,DGLHD also attenuated the expression of FAS,ACC-1,CD36,and SREBP-1c genes,activated PI3K/Akt signaling pathway in liver and adipose tissues,and ameliorated the glucolipid metabolism of liver and adipose tissues in ob/ob mice.In insulin resistant 3T3-L1 adipocytes,DGLHD also elevated glucose consumption,adiponectin level,and activation of PI3K/Akt pathway,and reduced lipid biosynthesis.Conclusion DGLHD targeted PI3K/Akt signaling pathway or PPAR-γ to exert beneficial effects on immune and metabolic balance,playing anti-inflammatory,anti-insulin resistant,and anti-steatotic effects in ob/ob mice.