Objective To examine the effect and mechanism of Duhuo Jisheng Decoction on lumbar intervertebral disc degeneration.Methods In total,105 Sprague-Dawley(SD)rats were randomly assigned to seven groups:sham operation group,model group,Duhuo Jisheng Decoction 1,rapamycin group,rapamycin+Duhuo Jisheng Decoction group,MHY1485 group,and MHY1485+Duhuo Jisheng Decoction group.Except for the sham operation group,the other groups underwent annulus fibrosus puncture to establish a lumbar intervertebral disc degeneration(IDD)animal model.After successful model establishment,a six-week drug intervention was performed,followed by MRI evaluation of the degree of disc degeneration.Samples of the L5-6 intervertebral disc were collected for HE and Alcian blue-nuclear fast red staining,and the degeneration of the nucleus pulposus and changes in the extracellular matrix were observed using light microscopy.Simultaneously,the expression levels of the downstream proteins of the mTOR pathway,p70S6K and 4E-BP1,along with the autophagy-related genes Beclin-1 and LC3,were assessed at both the protein and mRNA levels.Autolysosomes in nucleus pulposus cells were visualized using transmission electron microscopy(TEM).Results ①MRI showed disc Pfirrmann grade I in the sham group,and disc Pfirrmann grade Ⅲ-Ⅳ in the remaining 6 groups in L5-6 segments.②The degree of lumbar disc degeneration with histomorphological observation:MHY1485 group>model group>MHY1485 group+Duhuo Jisheng Decoction group>rapamycin group>Duhuo Jisheng Decoction group>rapamycin+Duhuo Jisheng Decoction group>sham group.Allan-nuclear red staining extracellular matrix proteoglycan content:sham group>rapamycin+Duhuo Jisheng Decoction group>rapamycin group>MHY1485 group+Duhuo Jisheng Decoction group>model group>MHY1485 group.③Relative expression of p-mTOR,p70S6K and 4E-BP1 downstream of mTOR signaling pathway:MHY1485 group>MHY1485 group+Duhuo Jisheng Decoction group>model group>rapamycin group>Duhuo Jisheng Decoction group>rapamycin+Duhuo Jisheng Decoction group>sham group,each experimental group varied significantly from the model group(P<0.01).④Autophagy-related protein Beclin-1 and LC3 expression levels:rapamycin+Duhuo Jisheng Decoction group>Duhuo Jisheng Decoction group>rapamycin>MHY1485 group+Duhuo Jisheng Decoction group>model group>MHY1485>sham group,and the content of each group was significantly(P<0.01).⑤TEM observation of autophagy levels in the cells of each group showed the formation of autolysosomes in Duhuo Jisheng Decoction group,rapamycin group andrapamycin+Duhuo Jisheng Decoction group.Conclusion Duhuo Jisheng Decoction can slow down the degenerative process of lumbar intervertebral discs in rats,and its effect may be linked to the suppression of the mTOR signaling pathway and the promotion of autophagy in nucleus pulposus cells.

BACKGROUND:The incidence of fragility fractures caused by osteoporosis is increasing year by year,and it is urgent to explore its pathophysiology,potential biomarkers,therapeutic targets and effective drugs.There are multiple cells in the bone microenvironment,which are crucial for maintaining bone metabolism.In recent years,single-cell RNA sequencing technology has been developed to characterize the transcriptome of single cells,and has been gradually applied to the study of osteoporosis prevention and treatment.OBJECTIVE:To review the application and progress of single-cell transcriptome sequencing technology in osteoporosis research.METHODS:The first author used a computer search of Web of Science,PubMed database,and CNKI from 2009 to 2023.Chinese and English search terms were "single-cell RNA sequencing,bone marrow derived stromal cells,osteoblasts,osteocytes,osteoclasts,immune cells,bone marrow vascular endothelial cells." Through reading and screening of relevant literature,89 articles were finally included in the review analysis.RESULTS AND CONCLUSION:(1) Single-cell transcriptome sequencing technology can gain an in-depth understanding of the trajectory and regulatory mechanism of cell development,proliferation,differentiation.(2) Candidate genes affecting bone mineral density are mainly concentrated in bone marrow mesenchymal stem cell subpopulation,in which Sox9 is a key transcription factor.(3) The differential expression of Runx2 in different subsets of osteoblasts controls the differentiation of bone marrow mesenchymal stem cells into osteoblasts.(4) RAB38 is related to histone modification and transcriptional regulation during osteoclast differentiation.(5) Studies at the single-cell level have confirmed that there are many kinds of intercellular communication in the bone microenvironment,and osteoclast may conduct intercellular communication through CD160-TNFRSF14 ligand-receptor binding.The neutrocyte/monocyte may interact with osteoblasts through the RESISTIN pathway.Plasma dendritic cells communicate with osteoblast cell lines through the epidermal growth factor pathway.Both can provide directions for target prediction and drug development.(6) An unrecognized capillary subset is called S-type endothelial cells,which originates entirely from the secondary ossification center of the epiphysis,and is even more malleable than H-type blood vessels.(7) This paper reviews the progress of single-cell transcriptome sequencing in characterizing the differentiation fate and differentiation trajectory of different bone tissue cells,identifying new cell subsets,identifying cell regulatory factors,and clarifying intercellular communication from a cellular perspective,so as to provide cell-level information for exploring the pathogenesis of osteoporosis,screening potential diagnostic markers,predicting therapeutic targets,and exploring the mechanism of drug action.(8) In the future,single-cell sequencing technology will provide more valuable information for changes in the bone microenvironment in the direction of single-cell multiomics (genomics,proteomics,and metabolomics) and other technologies such as spatial transcriptome technology.

BACKGROUND:The incidence of fragility fractures caused by osteoporosis is increasing year by year,and it is urgent to explore its pathophysiology,potential biomarkers,therapeutic targets and effective drugs.There are multiple cells in the bone microenvironment,which are crucial for maintaining bone metabolism.In recent years,single-cell RNA sequencing technology has been developed to characterize the transcriptome of single cells,and has been gradually applied to the study of osteoporosis prevention and treatment.OBJECTIVE:To review the application and progress of single-cell transcriptome sequencing technology in osteoporosis research.METHODS:The first author used a computer search of Web of Science,PubMed database,and CNKI from 2009 to 2023.Chinese and English search terms were "single-cell RNA sequencing,bone marrow derived stromal cells,osteoblasts,osteocytes,osteoclasts,immune cells,bone marrow vascular endothelial cells." Through reading and screening of relevant literature,89 articles were finally included in the review analysis.RESULTS AND CONCLUSION:(1) Single-cell transcriptome sequencing technology can gain an in-depth understanding of the trajectory and regulatory mechanism of cell development,proliferation,differentiation.(2) Candidate genes affecting bone mineral density are mainly concentrated in bone marrow mesenchymal stem cell subpopulation,in which Sox9 is a key transcription factor.(3) The differential expression of Runx2 in different subsets of osteoblasts controls the differentiation of bone marrow mesenchymal stem cells into osteoblasts.(4) RAB38 is related to histone modification and transcriptional regulation during osteoclast differentiation.(5) Studies at the single-cell level have confirmed that there are many kinds of intercellular communication in the bone microenvironment,and osteoclast may conduct intercellular communication through CD160-TNFRSF14 ligand-receptor binding.The neutrocyte/monocyte may interact with osteoblasts through the RESISTIN pathway.Plasma dendritic cells communicate with osteoblast cell lines through the epidermal growth factor pathway.Both can provide directions for target prediction and drug development.(6) An unrecognized capillary subset is called S-type endothelial cells,which originates entirely from the secondary ossification center of the epiphysis,and is even more malleable than H-type blood vessels.(7) This paper reviews the progress of single-cell transcriptome sequencing in characterizing the differentiation fate and differentiation trajectory of different bone tissue cells,identifying new cell subsets,identifying cell regulatory factors,and clarifying intercellular communication from a cellular perspective,so as to provide cell-level information for exploring the pathogenesis of osteoporosis,screening potential diagnostic markers,predicting therapeutic targets,and exploring the mechanism of drug action.(8) In the future,single-cell sequencing technology will provide more valuable information for changes in the bone microenvironment in the direction of single-cell multiomics (genomics,proteomics,and metabolomics) and other technologies such as spatial transcriptome technology.

Objective To examine the effect and mechanism of Duhuo Jisheng Decoction on lumbar intervertebral disc degeneration.Methods In total,105 Sprague-Dawley(SD)rats were randomly assigned to seven groups:sham operation group,model group,Duhuo Jisheng Decoction 1,rapamycin group,rapamycin+Duhuo Jisheng Decoction group,MHY1485 group,and MHY1485+Duhuo Jisheng Decoction group.Except for the sham operation group,the other groups underwent annulus fibrosus puncture to establish a lumbar intervertebral disc degeneration(IDD)animal model.After successful model establishment,a six-week drug intervention was performed,followed by MRI evaluation of the degree of disc degeneration.Samples of the L5-6 intervertebral disc were collected for HE and Alcian blue-nuclear fast red staining,and the degeneration of the nucleus pulposus and changes in the extracellular matrix were observed using light microscopy.Simultaneously,the expression levels of the downstream proteins of the mTOR pathway,p70S6K and 4E-BP1,along with the autophagy-related genes Beclin-1 and LC3,were assessed at both the protein and mRNA levels.Autolysosomes in nucleus pulposus cells were visualized using transmission electron microscopy(TEM).Results ①MRI showed disc Pfirrmann grade I in the sham group,and disc Pfirrmann grade Ⅲ-Ⅳ in the remaining 6 groups in L5-6 segments.②The degree of lumbar disc degeneration with histomorphological observation:MHY1485 group>model group>MHY1485 group+Duhuo Jisheng Decoction group>rapamycin group>Duhuo Jisheng Decoction group>rapamycin+Duhuo Jisheng Decoction group>sham group.Allan-nuclear red staining extracellular matrix proteoglycan content:sham group>rapamycin+Duhuo Jisheng Decoction group>rapamycin group>MHY1485 group+Duhuo Jisheng Decoction group>model group>MHY1485 group.③Relative expression of p-mTOR,p70S6K and 4E-BP1 downstream of mTOR signaling pathway:MHY1485 group>MHY1485 group+Duhuo Jisheng Decoction group>model group>rapamycin group>Duhuo Jisheng Decoction group>rapamycin+Duhuo Jisheng Decoction group>sham group,each experimental group varied significantly from the model group(P<0.01).④Autophagy-related protein Beclin-1 and LC3 expression levels:rapamycin+Duhuo Jisheng Decoction group>Duhuo Jisheng Decoction group>rapamycin>MHY1485 group+Duhuo Jisheng Decoction group>model group>MHY1485>sham group,and the content of each group was significantly(P<0.01).⑤TEM observation of autophagy levels in the cells of each group showed the formation of autolysosomes in Duhuo Jisheng Decoction group,rapamycin group andrapamycin+Duhuo Jisheng Decoction group.Conclusion Duhuo Jisheng Decoction can slow down the degenerative process of lumbar intervertebral discs in rats,and its effect may be linked to the suppression of the mTOR signaling pathway and the promotion of autophagy in nucleus pulposus cells.