| Objective:Multiple myeloma(MM)is the second most common haematological malignancy around the world.The main clinical manifestations of MM are unexplained bone pain,renal insufficiency.Studies have shown that the bone marrow microenvironment is inextricably linked with the occurrence and development of MM,which is composed of stromal cells,osteoblasts(OBs),osteoclasts(OCs)and growth factors and cytokines.At present,the commonly used therapeutic drugs are bortezomib,thalidomide,etc.,but after treatment,a series of adverse reactions are accompanied and the recurrence rate is high.Therefore,we are eager to find drugs that are more safe and effective for the treatment of MM and the regulation of MM bone marrow microenvironment.Huangkui Capsule is commonly used in the treatment of chronic nephritis.It is used as a medicine for the flowers of the Abelmoschus manihot(Linn.)Medic.Studies have shown that its flavonoids can promote the differentiation of osteoblasts.Therefore,we separately explored the effect of Huangkui Capsule on MM,the effect of Astragalus membranaceus on MM and its regulation on bone marrow microenvironment.Methods:We first examined the regulation of the survival time of 5TMM3VT myeloma model mice by Huangkui capsule and the proteasome inhibitor bortezomib(BTZ)at the animal level.Secondly,the flavonoids of Abelmoschus manihot(Linn.)Medic were isolated by the method of modern Chinese medicine chemical separation and purification.A total of 9 compounds were obtained and verified from MM cells,osteoblast induction and osteoclast induction.The effects of 9 compounds on the proliferation of MM cells were detected by MTT assay.The effect of compounds on the differentiation of mouse embryonic osteogenic precursor MC3T3-E1 cells into osteoblasts was detected by alizarin red staining.The effect of compounds on the differentiation of mouse macrophage leukemia cell Raw264.7 cells into osteoclasts was detected by the tartrate-resistant acid phosphatase kit.Based on transcriptome sequencing,the mechanism of some compounds on MM cells,osteoclasts and osteoblasts was studied.The differential genes were verified by Western blot.Results:We first found that Huangkui Capsule has significant anti-tumor activity against myeloma mice.Compared with BTZ alone,Huangkui Capsule can significantly prolong the survival time of mice.Secondly,we got 9 flavonoids compounds from the flowers of the Abelmoschus manihot(Linn.)Medic,which are respectively hyperoside(HK-2),cannabiscitrin(HK-3),quercetin-3’-glucoside(HK-4),Floramaroside F(HK-7),isomyricitrin(HK-8),dihydromyricetin(HK-9),3-O-kaempferol-3-O-acetyl-6-O-(p-coumaroyl)-a-D-glucopyranoside(HK-11),8-(2"-pyrrolidione-5-yl)-quercetin(HK-B10),8-(2"-pyrrolidione-5-yl)-3’-O-β-D-glucopyranoside(HK-E3).In vitro studies,we found that HK-11 has a certain inhibitory effect on the proliferation of MM cells(ARP1,H929);HK-2,HK-3,HK-B10,HK-11 can promote the differentiation of osteoblast precursor cells MC3T3-E1 into osteoblasts.HK-8 and HK-E3 also significantly inhibited the differentiation of osteoclast precursor cell Raw264.7 into osteoclasts.Through transcriptome sequencing,we found some differential genes,and found that HK-11 inhibited of β-catenin expression in MM cells;HK-8,HK-E3 can significantly inhibit the expression of NFATC1 and BTK during osteoclast differentiation.Conclusion:In view of the multi-dimensional study,we found that HK-11 can inhibit the proliferation of MM cells,and had a significant promoting effect on osteoblast differentiation.This indicates that the monomer component of Abelmoschus manihot(Linn.)Medic has good selectivity and anti-tumor activity in vitro and in vivo regulation of MM,which is expected to improve a new choice for anti-multiple myeloma drugs. |
