Induction Of M1 Type Polarization In Macrophages Derived From THP-1 Cells And The Regulation Of Kaponane Diterpenoids PV006

Macrophage as an important immune cell in the body,involved in the pathogenesis of chronic inflammatory diseases.According to the different polarization phenotype,macrophages are divided into M1 and M2 polarization state.M1 type is the classic activated macrophages,involved in acute proinflammatory response,released of a large number of proinflammatory factors,regulating and promoting Thl type immune response.With the role of killing pathogens and tumor cells,M1 type macrophages also lead to the body's normal tissue inflammatory response,which may constitute the basis of chronic inflammatory disease.M2 type macrophages are selectively activated macrophages,regulate wound healing and inhibit the occurrence of inflammatory responses.The variability and functional diversity of macrophage phenotype are important features,and macrophages,differentiation is induced and regulated by various signals in microenvironment.Intracellular signal transduction and transcription activator(STAT)1 and nuclear transcription factor ?B(NF-?B)are the key factors to promote the macrophage polarization of Ml.Activation of these two signaling pathways involved in the regulation of the occurrence and development of inflammatory response.The disorder of macrophage polarization can reflect inflammatory state of the local tissue microenvironment,this is important for understanding the occurrence and development of the disease.Because macrophages have a high degree of plasticity,changes in the balance of M1 and M2 subtypes in the inflammatory site also indicate the reversal of various inflammatory processes and the potential for therapeutic interventions.Design strategies for macrophage polarization may be a new target for infectious diseases,autoimmune diseases,and cancer therapy.Kaurane Diterpenoids have been found to have anti-tumor and anti-inflammatory effects,which are the main active constituents of the labiaceae plant.In this study,human monocyte cell line THP-1 cells were used as target cells,and the classical phorbol ester(PMA)-induced THP-1 cell differentiation model was used to simulate the differentiation of monocytes into macrophages in vitro.LPS and IFN-?,were given to induce the differentiation of macrophages to Ml subtype.On this basis,we investigated the effect of Kauri Diterpenoids PV006 on the key factors and downstream inflammatory factors of signal transduction pathway in THP-1 cell-derived macrophages M1 polarization.Chapter ? Establishment of M1 polarizational model in THP-1 cell-derived macrophagesObjective:To investigate the induction conditions of THP-1 cells differentiated into M1 macrophages in vitro and to detect the activation of intracellular signaling pathways and cytokines expression in Ml polarizational THP-1 cell-derived macrophages.Methods:THP-1 cells were firstly induced into macrophages with PMA,and then cells were stimulated by different concentrations of LPS combined with 20ng/ml rhINF-? or 1000ng/ml LPS+20ng/ml rhINF-? for different stimulating times.Morphological changes of THP-1 macrophages differentiated into M1 type were observed by inverted microscope.The expression of IL-1?,TNF-?,IL-6 and IL-8 mRNA and the production of IL-8 protein in Ml-polarized THP-1 cells were detected by qRT-PCR and ELISA respectively.The activation of STAT-1 and NF-?B signaling pathways in Ml-polarized THP-1 cells were analyzed with Western Blot.Results:After THP-1 cells were induced with 200ng/ml PMA for 6h into MO and then 1000ng/ml LPS +20 ng/ml rhIFN-y for 24h,suspended cells changed into adherent cells,and the morphology of THP-1 cells changed from round to long spindle or spindle shape.The expression of IL-1? TNF-?,IL-6,IL-8 mRNA and IL-8 protein in Ml polarizational THP-1 cells were significantly up-regulated.The expression of STAT1 and NF-?B p-p65 in Ml polarizational THP-1 cells were significantly increased.Conclusion:THP-1 cells could be induced and polarizatd into Ml macrophages by 200 ng/ml PMA combined with 1000ng/ml LPS+20 ng/ml rhIFN-?.The changs of in the cell morphology?the expression of inflammatory factors,as well as the activation of intracellular STAT1 and NF-?B signaling pathways,suggest that this induction pattern is viable as an simulation systerm of M1 polarizational macrophages in vitro.Chapter ? Effects of Kaponane Diterpenoid PV006 on Imiquimod Induced Psoriasis-like Dermatitis in MiceObjective:A model of psoriasis-like dermatitis induced by imiquimod in mice was established and the effect of compound PV006 on the inflammation model was studied.Methods:Psoriasis-like dermatitis were induced by stimulated skin of back in mice with imiquimod.According to lesion inflammation score(PASI),and histopathological analysis the effect of compound PV006 on imiquimod induced psoriasis-like dermatitis in mice was preliminarily evaluated.Results:The PASI score of PV006-100mg/kg group was significantly lower than that of the vehicle control group at d3,d5 and d6(P<0.01).The difference of PASI score between 12.5mg/kg-PV006 group with vehicle control group was not significant.Histopathological analysis showed that compared with the vehicle control group,100mg/kg-PV006 group appaered partial keratosis and partial hyperkeratosis,spine thickness and dermal inflammatory cell infiltration partially alleviated,Munro micro abscess rare.The improvement in epidermis of 12.5mg/kg-PV006 group was weaker than PV006-100mg/kg group.There was dermal inflammatory cells moderate infiltration in this group.Conclusion:Comprehensive PASI score and histopathological examination results,the 100 mg/kg-PV006 compound by intragastric administration has a certain degree of therapeutic effect on imiquimod-induced mouse psoriasis-like dermatitis.Chapter ? Regulation of Kaponane Diterpenoid Compound PV006 on M1 Polarization of THP-1 MacrophagesObjective:Through the established THP-1 cell-derived macrophage M1 polarization model in vitro,the regulating effect of compound PV006 on Ml polarization of macrophage was studied,and the aim is to provide the experimental basis for further study of the anti-inflammatory mechanism of the compound.Methods:MTT assay was used to detect the effect of compound PV006 on the proliferation of THP-1 cells.After compound PV006 acted on M1 polarized THP-1 cells for 24 h,morphological changes were observed under a microscope,the expression of IL-1?,TNF-?,IL-6 and IL-8 mRNA and the production of IL-8 protein were detected by qRT-PCR and ELISA respectively,and the activation of STAT-1 and NF-?B signaling pathways were analyzed with Western Blot.Results:Compound PV006 had no significant inhibition of THP-1 cells growth and proliferation at 0.78125?g/ml and less than 0.78125?g/ml concentrations.It could be observed by microscope that most THP-1 cells still appeared round instead of long spindle or spindle shape after PMA+LPS+rhIFN-? stimulating and different concentrations of PV006 treating cells for 24 hours.IL-1?,TNF-?,IL-6 and IL-8 mRNA and IL-8 protein levels in cells were significantly down-regulated by compound PV006 in a dose-dependent pattern.,and the expression of p-STATl and NF-p-p65 in the cells decreased.Conclusion:Kaponane Diterpenoid compound PV006 plays a role in preventing the differentiation of macrophages into M1 by affecting cell morphology,down-regulating genes and protein expression of inflammatory factors and inhibiting phosphorylation of STAT-1 Tyr701 site and NF-?B p65 Ser536 site.Inhibiting Ml polarization of macrophages may be one of anti-inflammatory mechanisms of compound PV006.