The Mechanism Of Aqueous Extract Of Cimicifuga Dahurica Reprogramming TAMs In The Treatment Of Multiple Myeloma Via Activating TLR4-NF-κB Signaling Pathway

ObjectiveMultiple myeloma(MM)is a malignant disease with abnormal proliferation of clonal plasma cells.At present,it is still an incurable disease.There is an urgent need to develop new drugs with different mechanisms and optimize the treatment effect,so as to reduce the risk of disease recurrence and deepen the persistence of response.Natural products are the main resources of modern drug development.Finding and developing natural plant drugs with high efficiency and low toxicity is one of the directions of MM related clinical drug research and development.A large number of studies have shown that the survival and proliferation of malignant plasma cells in bone marrow microenvironment depend on nonmalignant stromal cells.Among them,tumor associated macrophages(TAMs)are mainly characterized by M2 like macrophages,which are closely related to the biological characteristics of tumor survival,recurrence and metastasis.Cimicifuga dahurica has a wide range of clinical applications in China.In the early stage,we took Shengma Biejia Decoction with Cimicifuga dahurica as the main drug as the research object,and found that it could inhibit the growth of myeloma.However,the effect of Cimicifuga dahurica on MM and its molecular biological mechanism have not been clarified.Based on the single-cell sequencing analysis of the public database,we carried out a series of studies in vivo and in vitro,in order to clearly reveal the anti-tumor mechanism of aqueous extract of Cimicifuga dahurica(CRAE)on MM,and provide a theoretical basis for the clinical application of Cimicifuga and the traditional Chinese medicine compound with Cimicifuga as the main component.Methods1.Bioinformatics analysis of clinical samples in public databaseThe scRNA-seq data information of bone marrow samples from 11 MM patients in GSE 163278 data set was obtained from GEO database.The data quality was controlled.The UMI counting matrix was integrated by RPCA analysis method.The gene expression of each cell was standardized by R software package Seurat,and then logarithmic transformation was carried out.Then the data were analyzed by dimension reduction analysis and cluster analysis,and then the gene enrichment characteristics of M1 like and M2 like macrophages were analyzed.We identified the characteristic expression genes of M1 like and M2 like macrophages from the published literature,calculated the enrichment fraction in the macrophage population by using the Addmodulscore function in Seurat,and compared it in the heat map and violin map.2.In vivo experiments1)The effect of CRAE combined with bortezomib on tumor growth in nude mice.After 7 days of adaptive feeding,8 BALB/C nude mice were randomly selected as the blank group and the other 32 as the experimental group.The nude mice in the experimental group were modeled by SP2/0 cell transplantation tumor model,and then the nude mice in the experimental group were randomly divided into 4 groups:Vehicle group,CRAE group,Bortezomib group and Combo group.A total of 5 groups were intervened for 14 days.On the 53rd day of modeling,the nude mice were euthanized,the tumor tissues were obtained and weighed,and the liver,spleen and kidney were dissected and separated for WB,IHC staining and HE staining.2)The effect of CRAE on TAMs in MM transplantation tumor model.The tumor tissues of SP2/0 cell transplanted tumor model of nude mice were obtained.We detected the expression of macrophage related proteins in tumor tissue by IF and IHC,isolated macrophages in tumor tissues by magnetic bead sorting technology,and detected the level of macrophage related proteins using WB.3)The effect of CRAE on polarization of M2-BMDM macrophages.Bone marrow derived macrophages(BMDM)were extracted from the femur of C57BL/6 healthy mice.After directional differentiation using M-CSF,IL-4 and IL-13,BMDM derived M2 like macrophages(M2-BMDM)were obtained,and M2-BMDM was treated with different concentrations of CRAE.CD86+/CD206+cells were detected by flow cytometry,and the levels of TNF-α and IL-12 were detected by ELISA.4)Depleting macrophages in vivo to verify the antitumor effect mediated by CRAE.After 7 days of adaptive feeding,20 BALB/C nude mice were selected as the experimental group and the SP2/0 transplanted tumor model was established.The macrophages in nude mice were removed by liposome clodronate liposomes(CL),and the negative PBS liposome(PL)was used as the control.The nude mice were randomly divided into 4 groups:Vehicle group,CRAE group,CRAE+PL group and CRAE+CL group for 14 days.On the 31rd day of modeling,the nude mice were euthanized,and the tumor tissues were obtained and weighed.3.In vitro experiments1)The effect of CRAE on differentiation of TAMs in co-culture system constructed in vitro.The in vitro co-culture system of PMA differentiated human THP-1 cells and human myeloma cell line H929 was constructed using Transwell co-culture method.The effects of CRAE on the differentiation of TAMs in the co-culture system were observed by flow cytometry,WB and IF.2)The effect of CRAE on inflammation related signaling pathway of TAMs in co-culture system constructed in vitro.WB was used to detect the levels of main effector proteins of CRAE on TAMs inflammation related signal pathway TLR4-MyD88-NF-κB signal pathway in vitro co-culture system,and TLR4 protein inhibitors TAK-242 and NF-κB inhibitor Bay 11-7085 were used to verified further,and the nuclear transcription of p65 in each group was observed.3)The effect of macrophages treated with CRAE on the growth of myeloma cells in co-culture system constructed in vitro.The effect of macrophages treated with CRAE on the apoptosis of myeloma cell line H929 in vitro co-culture system was detected by flow cytometry and TUNEL.4)Identification of chemical components in CRAE and analysis of CRAE-TLR4 binding sites.The chemical components of CRAE were analyzed by UHPLC-ESI-Q-Orbitrap-MS,and the compounds that may bind to TLR4 and binding sites were predicted by molecular docking technology.Results1.Bioinformatics analysis of clinical samples in public database.The top 10 differentially expressed genes in macrophages of the 11 MM patient samples were MS4A7,SERPINA1,IFITM3,CFD,LST1,AIF1,FCGR3A,COTL1,SAT1 and FIH1.They are mainly involved in neutrophil activation and immune response,neutrophil degranulation and T cell activation.Enrichment pathway shows that these genes are mainly involved in osteoclast differentiation,antigen processing and presentation,phagosome related pathway and so on.The macrophages in the samples showed high M2-like macrophage activity,and the expression of M2 like characteristic genes was higher than that of M1 like characteristic genes.2.In vivo experiments1)The tumor volume and tumor weight in CRAE group were significantly lower than those in Vehicle group.The antitumor effect of bortezomib was better than that of CRAE,while the antitumor effect of the Combo group was stronger.Kaplan Meier survival curve showed that CRAE could prolong the survival time of tumor bearing nude mice to a certain extent.There was no significant difference in body weight of nude mice in each group,and CRAE did not cause significant damage to liver,spleen and kidney.2)The percentage of F4/80+CD86+cells in CRAE group and Combo group increased significantly,the expression level of M2 like macrophage markers(Argl and CD163)decreased significantly,while the expression level of M1 like macrophage markers(iNOS and CD86)increased significantly.3)CRAE could significantly increase the proportion of CD86+cells and reduce the proportion of CD206+cells in M2-BMDM.Compared with M2-BMDM,the levels of TNF-α and IL-12 in medium of M2-BMDM treated with CRAE increased significantly.4)When macrophages were depleted,the inhibitory effect of CRAE on tumor growth was significantly weakened.Compared with the Vehicle group,the body weight of CRAE group and CRAE+PL group was slightly higher.3.In vitro experiments1)After CRAE treatment,the morphology of TAMs changed significantly,pseudopodia was prolonged,and the cells were long spindle shaped.In CRAE group,the proportion of CD86+cells increased,while the proportion of CD 163+cells decreased.In CRAE group,the levels of M1 like macrophage characteristic proteins iNOS and CD86 increased,while the levels of M2 like characteristic proteins CD163 and Argl decreased.2)In CRAE group,the phosphorylation levels of TLR4,MyD88 and TAK1 increased,and the downstream phosphorylated protein p-IKKβ,p-IκB and p-p65 levels increased.After CRAE intervention,the red fluorescence intensity of p65 increased,and the expression in the nucleus increased significantly in a dose-dependent manner.The above effects of CRAE were significantly weakened after the application of TAK-242 and BAY 11-7085.3)The percentage of apoptosis of H929 cells co-cultured with TAMs increased in a dose-dependent manner.4)Molecular docking results showed that Cimicifugic acid B in CRAE could bind to TLR4 and form three hydrogen bonds with the residues GLN 333,SER 352 and LEU 283 of TLR4,which may affect TLR4 activity.ConclusionThe active component of CRAE could cause the activation of TLR4 and cause the activation of NF-κB through TLR4-MyD88-NF-κB axis,promoted the reprogramming of macrophages,improved the tumor microenvironment and then induced the apoptosis of myeloma cells.