Study On Mechanism Of Huanglian's Anti-inflammatory Effect And Comparison Between Huanglian And Huangbai,Fuzi,Ganjiang's Pharmacological Effects

Objective: To investigate the characteristics of cold or hot medicine's effects and provide basis for finding the microcosmic essence of cold or hot nature of chinese medicine by studying on the mechanism of Huanglian's anti-inflammatory effect and the comparison between Huanglian and Huangbai,Fuzi,Ganjiang's pharmacological effects.Methods: We detect Huanglian's influence on the transcriptional activity of STATs,NF?B and E2 F by double luciferase reporter gene test,Huanglian's influence on the mRNA level of STAT4,TNF,IL-12 in Jurkat cell and rat thymus by RT-qPCR.We compare Huanglian,Huangbai,Fuzi,Ganjiang's influence on cell multiplication and migration respectively by MTS test and migration test with 293 T cell,rats' differential leukocyte count,weight,spleen index and thymus index after intragastric administration.In the part of network pharmacology,we use TCMSP database to find and screen drug ingredients and targets,the Uniprot website to find genes of targets,the Metascape website to carry gene enrichment analysis,the CooLGeN website to estimate Huanglian's target of inflammation,the Gephi software to build Huanglian' s ingredient-target network.Results: In vivtro experiments: Huanglian down-regulates the transcriptional activity of E2 F,restrains cell multiplication and migration,up-regulates the transcriptional activity of STAT4,NF?B,down-regulates the transcriptional activity of STAT5,STAT6,up-regulates the mRNA level of STAT4,TNF,IL-12 in Jurkat cell,decreases the up-regulational effect of LIF on STAT4,demonstrates an effect of dual-directional regulation on STAT4 signaling.In vivo experiments: Huanglian down-regulates the transcrpition of mRNA level of STAT4,TNF,IL-12 in the thymus of rats,decreases the lymphocyte count and the weight,spleen index,thymus index of rats.Huanglian or Huangbai down-regulates the transcriptional activity of E2 F,restrains cell multiplication and migration,decreases the lymphocyte count,leukocyte count and the weight,spleen index,thymus index of ratsbut.Fuzi or Ganjiang has no obviouseffect on E2 F,cell multiplication or cell migration,up-regulate spleen index of rats slightly,but the weight,thymus index have no identical trend.Fuzi or Ganjiang increases the neutrophile granulocyte count,monocyte count and leukocyte count of rats,peripheral blood,but has no obvious effect on lymphocyte count.In the part of network pharmacology: we retrieve 9 drug molecules of Huanglian,26 drug molecules of Huangbai,and their common42 targets,the gene enrich analysis displays that Huanglian or Huangbai mainly has connection with Amine ligand-binding receptors,regulation of body fluid levels,regulation of lipolysis in adipocytes,PID AR TF pathway,rhythmic process,negative regulation of synaptic transmission,response to temperature stimulus,behavior,interleukin-4 and 13 signaling,cyclic nucleotide metabolic process,serotonergic synapse,neurotransmitter biosynthetic process,metal ion homeostasis,cellular response to organic cyclic compound,cellular response to nitrogen compound,catecholamine transport,estrogen signaling pathway,regulation of ion transport,response to ammonium ion,blood circulation.We retrieve 7 drug molecules of Fuzi,9 drug molecules of Ganjiang,and their common 22 targets,the gene enrich analysis displays that Fuzi or Ganjiang mainly has connection with regulation of muscle contraction,lipid modification,protein tetramerization,regulation of postsynaptic membrane potential,salivary secretion,phase I-functionalization of compounds,heat generation,GABA-A receptor activation,neuroactive ligand-receptor interaction,cellular response to organic cyclic compound.The study of network pharmacology on Huanglian shows that the molecules of Huanglian's drug ingredients have 15 targets connected with inflammation,among which PTGS2 has the biggest degree 8.The average degree of the targets is 4.3,The average degree of the molecule is 8.1.Conclusions: First,Huanglian performs an effect of anti-inflammation through down-regulating E2 F,inhibiting the growth and migration of cells,and dual-directional regulation on STAT4 signaling,which can provide further basis for studying the mechanism of cold or hot medicine,s effects.Second,Huanglian and the cold medicine Huangbai have simple effects of inhibiting the transcription factor E2 F,the growth and migration of cells,which may be one of their mechanisms of effects of anti-inflammation and anti-tumor.Third,the hot medicine Fuzi or Ganjiang has an up-regulational effect on leukocyte,and theirpharmalogical effects and mechanisms of anti-inflammation are different from cold medicines like Huanglian.Fourth,cold or hot medicines,drug effects have close relations with their chemical molecules and biological targets,and Huanglian acts on multiple targets of inflammation through chemical molecules like berberine,concerned with STAT4 and E2 F signaling.