| Background:The rise of immunologic microenvironment provides new ideas for various studies at this stage,and gives us new insights on the treatment and development of diseases.In the process of repair and destruction of bone tissue,many scholars have found that ther e is bone immunity in osteogenesis and osteoclasis.The bone immune microenvironme nt provides a target for the formation of bone tissue.Lilalutide GLP-1 and GLP-1R ago nists,as a commonly used drug for diabetes,play a significant role in systemic applicati on.However,in recent years,many researchers have found that they also play a signific ant role in the differentiation of osteoblasts through a variety of ways,but the regulation of macrophage polarization in the microenvironment layer to promote osteogenesis und er pathological conditions has not yet been explored.Research purpose:This study mainly studies the regulation of the polarity of bone marrow-derived ma crophages and the effect of its polarity on the differentiation of bone marrow-derived m esenchymal stem cells in osteoporosis from the level of in vitro,and explores its osteoge nesis.Research methods:(1)The mouse osteoporosis model was established in vitro,and the success of the mode l was verified by CT;Extract bone marrow-derived mesenchymal stem cells(BMSCs)a nd bone marrow-derived macrophages(BMDMs)for treatment;Western blot was used t o analyze ALP protein markers in extracted osteoporosis and normal bone tissue.(2)After 8 days of primary culture of BMDMs under normal condition and BMDMs un der osteoporosis condition,the cells were stimulated with liraglutide for 6 hours.The m orphological changes of cells at different stages were observed under light microscope,and the supernatant was collected;The fluorescent identification of CD11 b was carried out by confocal method;Then RT-qPCR was performed on its cells to detect its inflammationrelated index: TNF-α 、 IL-4 、IL-10 、CD86 、CD206 、Arg-1 、INOS;And the related research of gene sequencing on its pathway expression.(3)After culturing BMSCs in normal state and BMSCs in osteoporosis state to the seco nd generation in vitro,the cell morphology of the first generation and the second genera tion was observed by light microscope;The fluorescence identification of its surface ma rker CD44 was performed by confocal microscope;The collected BMDM supernatant a nd osteogenic induction fluid were mixed in a ratio of 1:3 and added into the BMSCs of the second generation for indirect co-culture.After 7 days,alp quantitative analysis and RTqPCR were performed to detect the m RNA level of osteogenic related indicators.Research results:Compared with the micro-CT of the non-ovariectomized mice,the femurs of the ov ariectomized mice(ovx group)showed a significant decrease in the number of bone trab eculae,bone surface area,and bone trabecular junction density compared with the norm al mice(sham group).Ovariectomy directly destroyed the bone microstructure in the mi ce,and significantly reduced bone mass;At the same time,according to Western Bolt a nalysis,the early osteogenic protein of mice after ovariectomy was significantly reduce d;Bone marrow-derived macrophages(BMDMs)and bone marrow mesenchymal stem cells(BMSCs)were successfully extracted by L929 supernatant culture method and bon e marrow extraction method.The comparison showed that the BMSCs in the primary S HAM group were in the shape of fried eggs and arranged more closely,while the BMS Cs in OVX group were in irregular shape and arranged more loosely.However,the dev elopment of BMSCs in the second generation of Sham group showed a fusiform of mult ifunctional differentiation,while the cell differentiation in the ovx group was poor,sho wing an open fusiform and irregular shape;The results of immunofluorescence staining showed that the two extracted cells expressed the target surface markers CD11 b and CD 4 4,which were extracted cells for the target;QPCR results showed that liraglutide inhib ited inflammatory factors CD86 and TNF in macrophages-α The production of INOS c an also promote the production of IL-4,CD206,IL-10 and Arg-1 in macrophages under osteoporosis,which is consistent with the results of gene sequencing library;Under the i nfluence of liraglutide,the level of alkaline phosphatase activity(marker of early osteob lasts)in BMSCs and BMDM under the influence of liraglutide in osteoporosis increased,while the state in pathology was the worst.Research conclusion:(1)Osteoporosis model was successfully established by ovariectomy.(2)Lilarutide reduces the expression of inflammatory factors by inhibiting the M1 polar ization of macrophages.(3)Lilalutide can promote M2 polarization of BMDM.(4)Lilalotide can significantly regulate TNF signaling pathway,NOD-like receptor sign aling pathway and other pathways to promote bone formation.(5)The network pharmacology analysis found that liraglutide significantly up-regulated osteogenesis related genes.(6)The expression of alkaline phosphatase activity(ALP)was increased by liraglutide i n bone immune microenvironment. |
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