Gene Biomarker-Based Phenotypic Screening For The Identification Of A Series Of 3,4-Disubstituted Piperidine Derivatives As Macrophages M2 Polarization Modulators For The Treatment Of EAE

Objectives:Multiple sclerosis(MS)is a clinically common demyelinating disease of the central nervous system.Its pathological process can cause damage to the brain and spinal cord and seriously affect the quality of life of patients.At present,the clinically adopted treatments such as the immunomodulator fingolimod can only delay the progression of the disease to a certain extent,and cannot fundamentally repair the damaged myelin and prevent inflammation.Macrophage M2 type polarization is beneficial to the repair of myelin and axons,and has the effect of inhibiting inflammation,which has both myelin repair and immune regulation.In this study,the skeletal structure of a novel compound that promotes M2 type polarization of macrophages was screened by a compound library in the laboratory,and the structure of this compound was optimized to study its therapeutic effect and specific mechanism on multiple sclerosis.Methods:1.Compounds screening for promoting macophage M2 polarization:1)RAW264.7 mouse macrophage strain were given compounds with different skeletons,and qRT-PCR was used to detect macrophages polarization mRNA levels of M2 type gene Argl and M1 type gene Mcpl for 24 hours;2)RAW264.7 mouse macrophage strain was stimulated with LPS for 24h,and then treated with compounds with different skeletons for 24h.qRT-PCR was used to detect the mRNA level of M1 gene of macrophage Mcpl;3)Bone Marrow Derived Macrophages(BMDMs)were treated with D11 for 24h,qRT-PCR was used to detect mRNA levels of M2 polarization related gene:Argl,Mrcl and Fizzl;and M1 polarization related gene:Inos,Mcpl and Cd86;M2 macrophages(CD206+F4/80+)proportion in total macrophages(F4/80+)was detected by flow cytometry.4)BMDMs were pre-stimulated with LPS for 24 h,then treated with D11 for 24 h,qRT-PCR was used to detect mRBNA levels of M1 polarization related gene:Inos,Mcpl and Cd86;The proportion of M1 macrophages(CD86+F4/80+)in total macrophages(F4/80+)was detected by flow cytometry.2.Effect of D11 on dendritic cell maturation and proliferation or differentiation of CD4+T cells:1)Flow cytometry was used to detect the proportion of mature dendritic cells(MHC-II+CD11C+)and(CD80+CD11C+)in total dendritic cells(CD 11C+)treated with D11 for 24 h and control group.2)IL-6 and IL-12 ELISA kits were used to detect IL-6 and IL-12 levels in BMDCs treated with D11 for 24 h and in control group.3)Use naive CD4+ T cell isolation kit to isolate naive CD4+ T cells from C57/BL6 murine lymph nodes,addition of anti-CD3(1 9g/ml),anti-CD28(5 ?g/ml),incubation in 1640 medium + 10%FBS for 72 h,and addition of D11 in the last 24 h.Flow cytometry was used to detect the ratio of Th1 cells(CD4+ IFN-?+)in total CD4+ T cells under Thl induction(IL-121?g/ml)condition and the ratio of Th17 cells(CD4+IL-17+)in total CD4+ T cells under Th17 induction(IL-6 2.5 ng/ml,TGF-?1 ng/ml)condition.4)BMDMs were co-cultured with CFSE-labeled CD4+ T cells for 48 h after D11 treatment,and the ratio of proliferating CD4+ T cells in total CD4+ cells was detected by flow cytometry.3*Dll pharmacokinetic study:Eight quarantine SD rats(male and female)were selected and divided into two groups,namely D11 gavage group and D11 intravenous group,4 SD rats in each group,male and female.In the gavage group,blood was taken from the tail vein before and after gavage,10 min,30 min,1 h,2 h,3 h,4 h,8 h,24 h and 48 h.No more than 0.2 ml.The intravenous injection group took blood before administration and 5 min,15 min,30 min,1 h,2 h,4 h,8 h,24 h and 48 h after administration,and each animal collected a small amount of blood each time.At 0.2 ml.The plasma samples were taken,and the protein was precipitated by adding 3 times of methanol containing 0.20 ?g/ml of internal standard,vortexed for 10 min,centrifuged at 10000 r/min for 10 min,and the supernatant was taken for injection analysis.LC-MS analysis was detected by positive ion method.The obtained concentration-time data were calculated by statistical moment method using DAS 3.0 pharmacokinetic software,and the main absorption kinetic parameters AUC(0-t),AUC(0-?)3 Cmax,Tmax,etc.were calculated after rat D11 administration by intragastric administration and intravenous injection.4.Therapeutic effect and mechanism of Dll on MS mice model EAE:The quarantined C57BL/6 mice were selected and the classical MS mice model EAE was induced with MOG35-55.The mice were given different doses of D11 for every day from the 8th day of modeling.1)The clinical symptoms of each group of EAE model mice were scored daily by using Kono's evaluation standard,and the body weight changes of each group of mice were recorded daily;2)Hematoxylin&Erosin(HE)and Fast Blue(Luxol Fast Blue)were used.Staining evaluation of the protective effect of D11 on inflammatory and myelin injury in mice;3)Detect Th1 cells(CD4+ IFN-?+)and Th17 cells(CD4+ IL-17+)ratio in CD4+ T cells in EAE mice by flow cytometry;4)Flow cytometry was used to detect M1(CD86+F4/80+)and M2(CD206+F4/80+)macrophages in total macrophages(F4/80+)in mice.The proportion of mature dendritic cells(MHC-?+ CD11C+)in total dendritic cells(CD11C+)was detected by flow cytometry;6)The proportion of Treg cells(CD4+ FOXP3+)in CD4+T cells in mice by flow cytometry.5.The mechanism of Dll promoting macrophages M2 polarization:1)RNA-seq technology was used to analyze the expression level of macrophage genome of D11 and control group in RAW264.7;2)Western Blot was used to detect the activation of STAT3,AKT and STAT6 after D11 administration;3)Flow cytometry was used to detect the proportion of M1 and M2 polarization macrophages in total macrophages in wild-type and STAT3fl/fl mice-derived BMDMs respectively.4)Flow cytometry was used to detect the proportion of mature dendritic cells in total dendritic cells derived from wild-type and STAT3fl/fl mice-derived BMDCs.5)Detection of IL-6 and IL-12 levels in wild-type and STAT3fl/fl mice-derived BMDCs cell culture media by using IL-6 and IL-12 ELISA kits;6)The EAE model was induced in myeloid specific knockdown STAT3 mice and wild type mice,clinical symptoms were scored daily,and the body weight changes of the mice in the two groups were recorded daily.7)Flow cytometry was used to detect the proportion of M1 and M2 macrophages in total macrophages mice and the proportion of mature dendritic cells in total dendritic cells in each group of mice;8)Detection of the proportion of M1 type and M2 type polarization macrophages in peripheral infiltrating macrophages(CD45hiCD11bhi)and microglial(CD45intCD11bhi)in central immune organs of mice by flow cytometry.Results:1.Compounds screening for promoting macophage M2 polarization28 representative structural backbone compounds(S-1?S-28)were given to Raw264.7 for 24h,respectively.S-2,S-9,S-11,and S-28 significantly promoted macrophage M2 polarization marker Arg1 mRNA expression(p<0.05,n=3,up-regulation level:S-2:7.3 times,S-9:8.1 times,S-11:7.0 times,S-28:10.1 times),S-9,S-13,S-16,S-18 can significantly promote the expression of Mcp1 mRNA of macrophage polarization M1 marker(p<0.05,n=3,up-regulation level:S-9:3.3 times,S-13:3.0 times,S-16:2.7 times,S-18:3.5 times).Based on the similarity,the S-28 structural analogs(A1?A10)obtained from the Molport database were given to Raw264.7 for 24h,respectively,and A2 significantly promoted the expression of macrophages M2 marker gene Arg1 mRNA in macrophages(p<0.05,n=3,Up-regulation level:A2:4.9 times)but the effect was weaker than S-28,and the expression of Mcp1 mRNA of macrophages M1 marker gene did not change significantly(p>0.05,n=3).After the chiral compound B1 of S-28 was given to Raw264.7 for 24h,the expression of Arg1 and Mcp1 mRNA in macrophage cells did not change significantly(p>0.05,n=3).After ab ring and cd ring structural analogs(C1?C11)of S-28 were given to Raw264.7 for 24h,there was no significant change in the expression of Arg1 and Mcp1 mRNA in macrophages(p>0.05,n=3).The different substituent compounds of S-28(D1?D21)were given to Raw264.7 for 24h,respectively.D8,D9,D10,D11,D12,D13 and D15 could significantly promote the expression of macrophages M2 polarization marker Argl mRNA(p<0.05,n=3,up-regulation level:D8:10.1 times,D9:8.4 times,D10:6.5 times,D11:14.0 times,D12:10.1 times,D13:5.1 times,D15:10.3 times),D14,D15,D16 can Significantly promoted macrophages M1 polarization marker Mcp1 mRNA expression(p<0.05,n=3,up-regulation level:D14:4.85 times,D15:3.54 times,D16:5.08 times).2.D11 effects on dendritic cell maturation and proliferation and differentiation of CD4+T cellsThe results of flow cytometry showed that there was no obvious change in the proportion of differentiated mature dendritic cells(CD80+CD11C+)and(MHC-II+CD11C+)in total dendritic cells(CD11C+)after compound D11 was given to primary dendritic cells BMDCs for 24 h(p>0.05,n=3).ELISA results showed that the cytokines IL-6 and IL-12 secreted by mature dendritic cells did not change significantly after D11 administration(p>0.05,n=3).CD4+ T cells was treated with compound D11 for 24 h,and CD4+ T cells differentiated into Thl(CD4+ IFN-y+)and Th17(CD4+ IL-17+)were not influenced.The BMDMs were co-cultured with CFSE-labeled CD4+ T cells for 48 h after D11 treatment,and the proportion of proliferating CD4+ T cells in total CD4+ cells was significantly decreased(p<0.05,n=3).The concentration of inflammatory factors IFN-y and IL-17 in the supernatant of CD4+ T cells and BMDMs co-culture medium were significantly decreased(p<0.05,n=3).3.D11 pharmacokinetic studyD11 single-dose pharmacokinetic studies were performed in rats,and D11 showed good absolute oral bioavailability in rats with an F value of 50.63%.In addition,D11 showed proper drug half-life and oral absorbance indicating that D11 has good oral drug-forming properties(t1/2 3.3 h,Cmax = 313 ng/mL,AUC0-t ?4669 ng/mL h).4.D11 relieves disease progression in EAE miceOn the 8th day after mice EAE modeling,20%of the mice began to develop early EAE symptom of weak in the tail,and the mice were randomly divided into groups and the dexamethasone group(10 mg/kg)and D11 group(100 mg/kg,200 mg/kg)were administrated from this day.All groups of mice were scored and weighed daily.On the 11th day after modeling,the clinical symptoms of the EAE model group reached 1.7 ± 0.2,the clinical symptoms scores of the positive drug dexamethasone group and the D11 200 mg/kg group were 0.3 ±0.1 and 0.5 ±0.1,respectively,which were significantly lower than the EAE model group(p<0.01,n=5),and D11 100 mg/kg group scored 1.1,which was lower than EAE model group,but no significant difference(p>0.05,n=5).On the 17th day after modeling,the clinical symptom scores of the EAE model group reached a peak of 3.3 ± 0.1.At this time,the clinical symptoms scores of the positive drug dexamethasone group and the D11 200 mg/kg group were 0.7 ± 0.2 and 1.3±0.1,respectively,were lower than the EAE model group(p<0.001,n=5),while the D11 100 mg/kg group scored 3.1±0.1,which was not significantly different from the EAE model group(p>0.05,n=5).On the other hand,in the EAE model group and the D11 100 mg/kg group,the body weight gradually decreased after modeling,and the body weight rose after the disease peak period,while the positive drug dexamethasone group and the D11 200 mg/kg group mice were relatively stable.These results suggest that D11(200 mg/kg)treatment can alleviate the pathogenesis of EAE,improve motor dysfunction in EAE modeling mice,and maintain the weight stability of EAE mice.5.Dll attenuates inflammatory response and myelin injury in the spinal cord of EAE miceThe results of HE staining showed that in the peak of disease of EAE mice(17th day after modeling),a large amount of inflammatory cell infiltration was observed in the white matter region of the model group,but there was no obvious inflammation in the spinal cord of the D11(200 mg/kg)treatment group.Flow cytometry results showed that the proportion of inflammatory Th1(CD4+ IFN-y+)and Th17(CD4+IL-17+)cells in total helper T cells(CD4+)infiltrated in the central nervous system(brain and spinal cord)in the D11(200 mg/kg)treated group compared with the EAE model group was significantly decreased(p<0.05,n=3).The results of LFB staining showed that in the peak of disease of EAE mice(17th day after modeling),the white matter area of the model group was lightly stained and the vacuole-like changes were more,indicating the loss of myelin.There was no vacuole-like change and obvious damage in the white matter area of the spinal cord of mice treated with D11(200 mg/kg).The above results indicate that D11(200 mg/kg)therapeutic administration can reduce the inflammatory response and myelin injury in the spinal cord of EAE mice.6.D11 promotes macrophage M2 polarization in EAE miceFlow cytometry results showed that The proportion of M2 macrophages(F4/80+CD206+)in total macrophages(F4/80+)in the peripheral immune organs(spleen and lymph nodes)and central nervous system(spinal cord and brain)increased significantly(p<0.05,n=3)in D11(200 mg/kg)treatment group mice comparing with EAE model group mice at the peak of disease(13th day to 17th day after modeling).While the proportion of M1 macrophages(F4/80+CD86+)decreased significantly(p<0.05,n=3).7.Dll has no effect on mature dendritic cells and Treg cells in EAE miceAt the peak of disease(13th day to 17th day after modeling)in the peripheral and peripheral immune organs(spleen and lymph nodes)and central nervous system(spinal cord and brain),Flow cytometry results showed that there was no significant difference in the proportion of mature dendritic cells(MHC-?+CD11C+)account for total dendritic cells(CD11C+)of D11(200 mg/kg)treatment group mice comparing with EAE model group mice(p>0.05,n=3).and there was no significant difference in the proportion of Treg cells(CD4+Foxp3+)account for helper T cells(CD4+)(p>0.05,n=3).8.D11 promotes macrophages M2 polarization gene expressionAfter compound D11 was added to the macrophage cell line Raw264.7 for 24h,the RNA-seq results indicated that D11 could activate many macrophages M2 polarization related genes and inhibit many M1 polarization related genes.Primary macrophage BMDMs were treated with compound D11.After 24h,the mRNA level of macrophage M2 related genes:Argl,Mrcl and Fizz1 were significantly up-regulated(p<0.05,n=3),western blot results showed macrophage M2 marker protein Arg11 Yml expression was up-regulated;the proportion of M2 macrophage(CD206+ F4/80+)account for total macrophages(F4/80+)(p<0.05,n=3)increased.The mRNA level of M1 related genes:Mcp1,Inos,Cd86 in BMDMs administered D11 for 24 h after pre-stimulation of LPS was significantly down-regulated(p<0.05,n=3);the proportion of M1 macrophages(CD86+F4/80+)in total macrophages decreased(p<0.05,nF=3).9.D11 promotes activation of AKT/STAT3 signaling pathway in macrophagesAfter compound D11 added to the macrophage cell line Raw264.7 for 24h,the KEGG signaling pathway analysis showed that D11 mainly affected the cellular immune and signal transduction-related pathways,and further analysis revealed significant differentially expressed genes(SDE)such as Ikk,Pim-1 and Amli-etc mainly belonged to JAK-STAT and PI3K-AKT signaling pathways.Western blot analysis showed that after treated primary macrophages BMDMs with D11 for 24 h,the phosphorylated AKT and phosphorylated STAT3 at both 308 and 473 sites were significantly enhanced,while the phosphorylation of STAT6 was not significantly changed.10.The effect of knocking out STAT3 on macrophages and dendritic cellsBMDC cells and BMDM cells were extracted and cultured from wild-type(WT)and myeloid STAT3 knockout C57BL/6 mice(lysm cre STAT3fl/fl),respectively.Flow cytometry results showed that the proportion of mature dendritic cells(CD80+CD11C+),(CD86+ CD11C+),(MHC-?+ CD11C+)in total dendritic cells(CD11C+)was not significantly different(p>0.05,n=3).The proportion of M1 macrophages(CD86+F4/80+)and M2 macrophages(CD206+F4/80+)in total macrophages(F4/80+)was not significant different(p>0.05,n=3).The results of ELISA showed that the cytokines IL-6 and IL-12 secreted by mature dendritic cells did not have significant difference(p>0.05,n=3).11.Lysm ere STAT3fl/fl mice get relieved clinical symptoms of EAEWT and lysm cre STAT3fl/fl C57BL/6 mice were induced EAE model,respectively.The EAE clinical symptom of lysm cre STAT3fl/fl mice was significantly relieved comparing with WT group from 10th day to 18th day after modeling(p<0.05,n=5).And the average body weight of mice in WT group was significantly lower than the lysm cre STAT3fl/fl group from 12th day to 16th day after modeling(p<0.05,n=5).The results of flow cytometry showed that the proportion of inflammatory Thl(CD4+ IFN-?+)cells infiltrating the central nervous system(brain and spinal cord)in total Th cells(CD4+)of lysm cre STAT3l/fl group were less than WT model group(p<0.05,n=3).12.The mechanism of rellieved EAE in Lysm ere STATSfl/fl miceThe results of flow cytometry showed that in the peak period of EAE(from 13 th day to 16th day after modeling),the peripheral immune organs in the lysm cre STAT3fl/fl group were compared with the EAE model group.There was no significant difference in the proportion of Ml(F4/80+CD86+)and M2(F4/80+CD206+)macrophages in total macrophages(F4/80+)(p>0.05,n=3),and there was no significant difference in the proportion of mature dendritic cells(MHC-?+CD11C+)in total dendritic cells(CD11C+)both the peripheral immune organs and the central nervous system(brain and spinal cord)(p>0.05,n= 3).13.Lysm ere STAT3fl/fl increased central infiltration of M2 macrophagesFlow cytometry results showed that during the peak disease period(from 13th day to 16th day after modeling),in central nervous system(brain and spinal cord)compared with WT model group mice,lysm cre STAT3fl/fl group of mice had a significant increase of M2 macrophages(F4/80+CDS206+)in peripherally infiltrated macrophages(CD11b+CD454+)(p<0.05,n=3).However,there was no significant difference of M1 macrophages(F4/80+CD86+)in peripherally infiltrated macrophages(CD11b+CD45+)(p>0.05,n=3).But M1(F4/80+CD86+)and M2(F4/80+CD206+)macrophages in microglials(CD11b+C45int)inherent in the mouse central nervous system had no significant difference(p>0.05,n=3).Conclusion:This study found and confirmed that 3,4-disubstituted piperidine derivatives can promote the expression of macrophage M2 marker Argl,and its structural optimization D11 as a candidate compound can finally promote macrophage M2 polarization.Compound D11 showed good oral bioavailability,and administration of D11 on the MS mice model EAE significantly attenuated its clinical symptoms with an increase in M2 macrophages polarization in mice.Administration of D11 on primary macrophages BMDMs significantly inhibited its activity in promoting CD4+T cell proliferation.This mechanism of action may be through enhanced phosphorylation of AKT and STAT3.In summary,the small molecule compound D11 obtained by compound library screening and structural optimization can inhibit the occurrence of inflammation by promoting M2 polarization of macrophages,so D11 is expected to be an effective therapeutic drug for multiple sclerosis.