Effect Of Ranunculi Ternati Radix On Bone Marrow Derived Macrophages Polarization

ObjectiveMacrophage polarization is closely related to the occurrence and development of infection and tumor.The current research shows that macrophage polarization will become the significant intervene spat in the treatment of disease.Because of Ranunculus ternate has the effect of anti-tumor,anti-tuberculosis and antiinflammatory,and is used to assist the treatment of diseases such as tuberculosis,lung cancer,and malignant lymphoma.Report display that Ranunculus ternate can enhance macrophage phagocytosis but the effect on macrophage polarization and molecular mecha is not clear.We intends to use Ranunculi Ternati Radix stimulated mouse bone marrow-derived macrophages,explore the main ingredients of Ranunculus ternate Radix can stimulate macrophage polarization,and to probe its possible mechanism.It provides a scientific basis for the further development and utilization of Ranunculus ternate,and lays the foundation for further in-depth discussion on the prevention and treatment of tuberculosis and tumor.Methods1.Effect of Ranunculi Ternati Radix alcohols extracts on M0 type macrophages polarizationDifferent concentrations of Ranunculi Ternati Radix(RTR)induced bone marrow derived M0 macrophages in mouse,and CCK8 technique was used to detect macrophage activity.Exploring the best concentration of RTR acting on macrophages.M0 macrophages stimulated by the best concentration RTR.The morphology of macrophages were observed at different activation time points such as 0h,12 h,24h,36 h,48h and 72 h.The expression of Arg-1,IL-10,TGF-β,i NOS,IL-6 and TNF-α were detected by Real-time PCR.The leveal of TNF-α,IL-10,TGF-β1 in cell culture supernatant were examined by ELISA assay.And expression level of F4/80,CD206,NOS2 molecular were analyzed by FCM.2.Screening of effective components of RanunculiHigh performance liquid equality technique was used to isolate and identify all components of the alcohol extract of ranunculi,and the possible monomer components that RTR could induce the M1 polarization of macrophages were screened.Were used to stimulate mouse bone marrow macrophages 24 h,detection of M1/M2 type Real-time PCR relative expression levels of m RNA cytokines in macrophages,screened stimulation mouse bone marrow macrophages and RTR M0 type to M1 type polarization may be a monomer compound of palmitic acid(Palmitic Acid,PA),ternatolide(Ternatlide,Tern).3.Effect of PA/Tern on polarization of mouse bone marrow source M0 macrophagesCCK8 was used to detect the activity of macrophages,the optimum concentration for PA/Tern macrophages.The best concentration were induced respectively by M0 macrophages incubated with 24 h,observe the change of macrophage morphology.Real-time PCR text Arg-1,IL-10,TGF-β,i NOS,IL-6 and TNF-α cytokine m RNA expression level.ELISA cells detected at different time points the supernatant of TNF-α,IL-10 TGF-β1 cytokine expression changes.FCM was used to detect F4/80,CD206,NOS2.4.The effect of the Ranunculi induced the process polarization of M0 macrophages by PA/TernAccording to the proportion of Ranunculi components in PA/Tern,respectively in the concentration of mouse bone marrow souce macrophages induced by M0 0-72 h,Real-time PCR,Arg-1,IL-10,TGF-β,i NOS,IL-6 and TNF-α cytokine m RNA expression level.ELISA detected at different time points of cell culture supernatant of TNF-α,IL-10 TGF-β1 cytokine expression changes.Flow cytometry was used to detect F4/80,CD206,NOS2.5.Interaction of PA and Tern during the polarization of M0 macrophages induced by RanunculiThe mouse bone marrow souce macrophages used by PA/Tern working concentration,and incubation 0-72 h and text Arg-1,IL-10,TGF-β,i NOS,IL-6 and TNF-α cell factor m RNA expression level by Real-time PCR.Different time to text the cell culture fluid supernatant TNF-α,IL-10,TGF-β1changes in the level of cytokine secretion by ELISA.And text the F4/80,CD206,NOS2 expression level by flow cytometry6.Possible mechanism of RTR induced polarization of mouse bone marrow source macrophages.After 24 h or 48 h,M0 mouse bone marrow macrophages were stimulated by RTR,PA and Tern.Text the p38-MAPK,p-p38-MAPK,NF-κB and p-NF-κB expression level by western blot.Result1.RTR stimulated M0 type mouse bone marrow source macrophages after 24 h,CCK8 detected that the best concentration of RTR is 100 μg/m L to induced macrophages.Observe the macrophage morphology under light microscope.Cells showed long fusiform,long pseudopodia,consistent with the M1 type macrophage morphology.Real-time PCR results showed that: RTR stimulated M0 mouse bone marrow source macrophages 0-72 h,high expression of i NOS,IL-6 and TNF-α began after 12 h,low expression of Arg-1,IL-10 and TGF-β.ELISA showed that : after RTR stimulation,starting from 12 h TNF-αand i NOS secretion increased,the serum level of TGF-βdecreased;FCM results showed that: after RTR stimulation,the expression level of 36 h from the beginning macrophage NOS2 surface film molecules began to increase,the expression level of CD206 decreased.Suggest that RTR can promote bone marrow source macrophage polarization from type M0 to type M1.2.HPLC technology showed that: RTR content higher than 0.01% of more than 50 compounds.combined with the literature was screened with inflammation related compounds of palmitic acid,ternatolide,stigmasterol,2-deoxy-D-RNA-1,4-lactone.Real-time PCR showed that: after induced by palmitic acid,ternatolide M1 type cytokine m RNA relatively high expression level,showed the upregulated expression.Suggest that palmitic acid and ternatolide may be take part in RTR induced macrophage polarization process.3.Different concentrations were induced in mouse bone marrow source macrophages M0 24 h by ternatlide and palmitic acid,CCK8 showed that PA had no effect on macrophage activity concentration is less than 400μmol/L,had no effect on the activity of macrophage Tern concentration is higher than 150mmol/L.Under light microscope observation,found that 400μmol/L PA,150mg/L Tern after induction of macrophage were fusiform.Respectively with 400 mol/L PA/150mg/L Tern M0 induced by macrophage 0-72 h for subsequent experiments.The results of Real-time PCR detection showed that the relative expression level of M1 related cytokine m RNA was higher,and all showed up-regulated expression.ELISA results showed that after PA stimulation,TNF-αand i NOS secretion level was higher than the control group,the serum level of TGF-βwas lower than control group;FCM results showed that after PA stimulation,the expression level of NOS2 macrophage surface molecules began to increase,the expression level of CD86 macrophage surface molecules of Tern stimulation,the expression level of CD206 decreased;note: PA/Tern can induce mouse bone marrow-derived macrophages M1 polarization type.4.According to the PA/Tern composition ratio,respectively with 9.5 μmol/L PA and 75mmol/L Tern were used to induce mouse bone marrow source macrophages M0 0-72 h.The results showed that 75mmol/L induced by Tern Real-time PCR found group: Arg-1,IL-10,TGF-β low expression;high expression of i NOS,IL-6 and TNF-α,the results showed that the high expression of TNF-α,TGF-β1;IL-10 low expression;flow cytometry showed that high expression of CD86;the low expression of CD206;and there were no significant changes in PA induced group;tip: RTR induced murine bone marrow-derived macrophages M1 polarization process.Tern plays a leading role.5.The interaction of 9.5μmol/L PA and 75mmol/L Tern in mouse bone marrow source M0 macrophages 0-72 h.Real-time PCR text Arg-1,IL-10,TGF-β detection of low expression and high expression of i NOS,IL-6 and TNF-α.Flow cytometry showed: high expression of NOS2,the low expression of CD206.Tip: RTR induced mouse bone marrow source macrophages M1 polarization probably is a result of interaction between PA and Tern.6.The expression level of signal pathway related protein was detected by Western blot.The expression of NF-κB,p-p38-MAPK increased significantly after RTR and induced M0 macrophages 24 h and 48 h.The expression level was higher than that induced by PA and Tern,suggesting that RTR induced M1 polarization in mouse bone marrow source macrophages may be related to MAPK and NF-κB signaling pathway.Conclusion:1.Ranunculi ternati radix can promot the source of mouse bone marrow macrophages from M0 type macrophages to M1 type macrophages polarization.2.In the ranunculi ternati radix stimulated macrophages M1 type polarization that main element is palmitic acid and ternatlide,and induced polarization through the common action of PA/Tern.3.Ranunculi ternati radix,palmitic acid and ternatlide induced mouse bone marrow source from M0 macrophages to M1 polarization maybe related with NF-κB/p38-MAPK signaling pathway.